Obesity and brain cancer: unveiling molecular ... - Investigo

International Doctoral School

Joana Filipa Correia de Almeida

OBESIT Y AND BRAIN CA NCER:

U NVEILING MOLEC U LAR MECHANISMS OF

INF LAMMATORY AND ANG IOGENIC PATHWAYS IN G LIOMA

Supervised by:

Maria Cristina Prudêncio Pereira Soares, PhD, MD

Rúben Miguel Pereira Fernandes, PhD

2019


Joana Filipa Correia de Almeida

OBESIT Y AND BRAIN CA NCER:

U NVEILING MOLEC U LAR MECHANISMS OF

INF LAMMATORY AND ANG IOGENIC PATHWAYS IN G LIOMA

Supervised by:

Maria Cristina Prudêncio Pereira Soares, PhD, MD

Rúben Miguel Pereira Fernandes, PhD

2019


Cristina Prudêncio y Rúben Fernandes,

DECLARES that the present work, entitled “Obesity and brain cancer: unveiling

molecular mechanisms of inflammatory and angiogenic pathways in glioma”,

submitted by Joana Filipa Correia de Almeida to obtain the title of Doctor, was carried

out under their supervision in the PhD programme “Biotecnoloxía Avanzada”. This is a

joint PhD programme integrating the University of Vigo and Escola Superior de Saúde

– Politécnico do Porto.

Vigo, 12 de Julio de 2019

The supervisors,

Dr. Cristina Prudêncio Dr. Rúben Fernandes

“Nothing in life is to be feared, it is only to be understood.

Now is the time to understand more, so that we can fear less.”

Marie Curie

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TABLE OF CONTENTS

Table of contents ...........................................................................................................................1

Acknowledgements ........................................................................................................................5

Abbreviations .................................................................................................................................7

Abstract ....................................................................................................................................... 11

Chapter One ................................................................................................................................ 13

Brain tumors .................................................................................................................................. 14

Adipose tissue as an endocrine organ ......................................................................................... 17

An epidemic called obesity, inflammasome and adipocytokines .................................................. 23

Sphingolipids in obesity: ceramide and sphingosine-1-phosphate ............................................... 26

Aims of the study ......................................................................................................................... 33

Framework .................................................................................................................................... 34

Chapter Two ................................................................................................................................ 35

2.1. Material and Methods......................................................................................................... 37

Cell culture and in vitro treatment .......................................................................................... 37

Adipocyte cell culture and conditioning ................................................................................. 37

2

Proteomics analysis by 2D-PAGE and MS-MALDI-TOF Spectrometry ................................ 38

Chemicals .............................................................................................................................. 38

Sample preparation for 2D-PAGE ......................................................................................... 39

In-Gel Protein Digestion ........................................................................................................ 40

MALDI-TOF-MS analysis ....................................................................................................... 41

Data analysis and database searching ................................................................................. 42

Proteome Profiler™ Array Assay .......................................................................................... 42

Statistical analysis ................................................................................................................. 43

2.2. Results ........................................................................................................................... 44

Differentially Expressed Proteins .......................................................................................... 44

Expression proteomic profile of GL261 conditioned medium ................................................ 45

2.3. Discussion ...................................................................................................................... 49

Chapter Three .............................................................................................................................. 53

3.1. Material and Methods ..................................................................................................... 54

Cell culture ............................................................................................................................. 54

Syngeneic mouse model of glioma ....................................................................................... 54

Protein microarrays ............................................................................................................... 55

Immunohistochemistry on FFPE tissue sections for IL1β, IL6, Ob, PDGFA and TNFα ........ 56


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3.2. Results ........................................................................................................................... 57

Proteomic alterations in glioma exposed to adipocyte secretome ........................................ 57

3.3. Discussion ...................................................................................................................... 61

Inflammatory and angiogenic pathways in glioma: a cellular and an animal model ............. 61

Expression proteomic profile of GL261 conditioned medium ................................................ 61

Morphologic analysis ............................................................................................................. 62

Immunohistochemical evaluation of IL1β, IL6, Ob, PDGFA and TNFα on adipose and tumoral

tissue of syngeneic C57Bl/6J mice ........................................................................................ 63

Chapter Four................................................................................................................................ 67

4.1. Material and Methods ..................................................................................................... 68

Tandem Mass Spectrometry for Ceramide and S1P ............................................................ 68

Extraction of sphingolipids from mice plasma ....................................................................... 69

Quantification of sphingolipids by UPLC-MS / MS of mouse plasma.................................... 69

Clinical biochemical markers ................................................................................................. 70

Histochemistry of hepatic tissue: Haematoxylin-Eosin and Sirius Red ................................. 70


4.2. Results ........................................................................................................................... 71

Sphingolipids alterations in obese mice ................................................................................ 71

4.3. Discussion ...................................................................................................................... 76

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High-fat diet decreases sphingosine-1-phosphate ................................................................ 76

Hepatic enzymes and lipoproteins levels increase with obesity............................................ 79

Morphologic analysis ............................................................................................................. 81

Chapter Five ................................................................................................................................ 83

Bibliographic references ............................................................................................................... 89

Appendixes ................................................................................................................................ 117

I. Summary ...................................................................................................................................i

II. Resumen ................................................................................................................................ vii

III. Publication I ....................................................................................................................... xiii

IV. Publication II .................................................................................................................... xlvii

V. Awards ........................................................................................................................... lxxxv

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ACKNOWLEDGEMENTS

I am thankful to Professora Doutora Cristina Prudêncio for the tutoring and careful review, the sharing

of knowledge, enlightenment in the most stalling moments, for always having a cheerful word and

patience through all these years.

I would like to thank Professor Doutor Rúben Fernandes for the tutoring and careful review, the

sharing of knowledge, enlightenment in the most stalling moments, for always having a new idea to

pursue and patience through all these years.

I am thankful to Professor Doutor Pedro Coelho, a friend and a mentor, for all the teaching and

companionship through all these years, for all of the personal time spent on the laboratory helping

with this project and many others in the meanwhile.

Thank you, Professor Doutora Raquel Soares, for always being available to give new insights and

advice. I am also thankful for always having supported all my studies at Department of Biochemistry

and at the Animal Facilities of Faculty of Medicine of University of Porto.

To all my colleagues in Escola Superior de Saúde and Faculty of Medicine, thank you for the

companionship and support through the most insane of laboratory and lecturing times, and all the

scientific support and after-hours company on the laboratory.

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I am thankful to Professora Doutora Carmen Sieiro and Diana Valverde of University of Vigo for the

scientific and pedagogic guidance through this doctorate.

To all my friends, here and abroad, thank you for all the great moments, understanding and

acceptance of all the denied time. I am also thankful for all scientific and mental help by my

everlasting friends and PhD partners Luísa Marinho e Raquel Pinho.

Most of all, I will always be thankful to my family for all the unconditional support through, not all

these years, but all my life. I am most thankful to my mother for all the enlightenment in the darkest

moments. It would not have been possible any other way.

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ABBREVIATIONS

AKT Protein Kinase B

ANXA-2 Annexin A2

AT Adipose Tissue

BAT Brown Adipose Tissue

CGl Conditioned medium from Glioma

CHOL Cholesterol

DNA Deoxyribonucleic Acid

DTT di-dithiothreitol

ECM Endothelial Cell Matrix

EGF Endothelial Growth Factor

ERK Extracellular signal-Regulated Kinase

FFA Free-Fatty Acids

FGF Fibroblast Growth Factor

GWAS Genome-Wide Association Studies

HDL High Density Lipoprotein

HFD High-Fat Diet

HGF Hepatocyte Growth Factor

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HIF-1α Hypoxia-Induced Factor one alpha

hnRNP L Heterogeneous nuclear ribonucleoprotein L

IAA Iodoacetamide

IARC International Agency for Research on Cancer

IGF Insulin Growth Factor

IL Interleukin

KIF5C Kinesin Family Member 5C

LDL Low Density Lipoprotein

LIF Leukocyte Inhibitor Factor

MALDI-TOF-MS

Matrix Assisted Laser Desorption/Ionization – Time Of Flight – Mass

Spectrometry

MMP Matrix Metalloproteinase

n.s. Non-significant

NAFLD Non-alcoholic fatty liver disease

NASH Non-alcoholic steatohepatitis

NCGl Non-Conditioned medium from Glioma

ND Normal Diet

NF-Kβ Necrosis Factor kappa beta

N-RAP Nebulin-related-anchoring protein (NRAP)

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PAGE Poly-Acrylamide Gel Electrophoresis

PAI-1 Plasminogen Activator Inhibitor one

PDGF Platelet-Derived Growth Factor

PGK1 Phosphoglycerate Kinase 1 (PGK1)

PMF Peptide Mass Fingerprint

RACK1 Guanine nucleotide-binding protein subunit beta-2-like 1

RANTES Regulated on Activation, Normal T Cell Expressed and Secreted

RBP-4 Retinol Binding Protein 4

RFC1 Replication factor C subunit

ROS Reactive Oxidative Species

S1P Sphingosine-1-Phosphate

S1PR Sphingosine-1-Phosphate Receptor

SAT Subcutaneous Adipose Tissue

SGPP Sphingosine-1-Phosphate Phosphatase

SpK Sphingosine Kinase

ST1 Stress-induced phosphoprotein 1

TCA Tri-Carboxylic Acid

TGA Triglycerides

TIMP-1 Tissue Inhibitor Metalloproteinases

10

TNFα Tumor Necrosis Factor alpha

VAT Visceral Adipose Tissue

VEGF Vascular Endothelial Growth Factor

VLDL Very-Low Density Lipoprotein

WAT White Adipose Tissue

WHO World Health Organization

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ABSTRACT

Gliomas represent the most common primary malignant brain tumors in adults, with an extremely

poor prognosis. Despite its high morbidity, the etiology of glioma remains largely unknown. Obesity,

favoured by the modern lifestyle, acquired epidemic proportions nowadays, and has been associated

with various major causes of death and morbidity including malignant neoplasms. So, there might be

a relationship between the increased incidence of gliomas and dysfunction of adipose tissue. The

present study begins with 3T3-L1 adipocytes in culture, and its conditioned medium (adipokines-

enriched medium), then it is disclosed adipocyte-released factors related with glioma growth,

hormones, metabolic and angiogenic behavior by MALDI-TOF-MS and proteomic array analysis.

Formerly, it is studied some modified cytokines on adipose tissue from diet-induced obese C57Bl/6J

mice. To conclude, it is analysed the sphingolipid profile, namely ceramides and sphingosine-1-

phosphate levels on normal and obese diet-induced mice serum, as well as liver function, lipid profile

and uric acid and glucose. Adipocyte cells overexpressed several pro-inflammatory and angiogenic

factors, such as IL6, IL11, LIF, TNFα, endocan, HGF, IGF1 and VEGF. Nevertheless, they

underexpressed TIMP-1, SerpinE1, coagulation factor III, endostatin, PDGFA and ADAMTS1. On

adipose tissue, IL6, Ob (Y-20) and TNFα were overexpressed in HFD group, while IL1β and PDGFA

were underexpressed. On tumoral tissue, IL6 did not present significant variation, though IL1β and

Ob tended to be overexpressed while TNFα and PDGFA disposed to be underexpressed.

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Sphingolipid analysis, as well as liver function, reinforces the premise that excessive dietary intake

worsens metabolic syndrome in general and, more precisely, chronic low-grade inflammation.

Relationship between obesity and cancer is paradoxical, however it is strengthened the idea on

obesity protecting against glioma only at initiation and proliferation phases.

Keywords: brain cancer, C57Bl/6J, obesity, obesomics.

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CHAPTER ONE

State of the art

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Brain tumors

Gliomas, the most common primary malignant brain tumors in adults, represent 30% of all brain

tumors and 80% of all malignant brain tumors [1]. Prognosis is extremely poor, with a median survival

time of approximately 12 to 15 months and is almost invariably fatal, and its aftermath on cognitive

function and life quality is increasingly degrading over time [2,3]. Gliomas increase in frequency with

age, and affect preferentially men [2,4,5]. Genetic factors in glioma etiology are poorly understood;

less than 5% of glioma cases are familial in origin, with only a few described by rare genetic

syndromes [6]. Gliomas are heterogeneous and are typically classified according to World Health

Organization (WHO) tumor grade, and also classified on the basis of cellular lineage: astrocytic,

oligodendroglial, and mixed tumors [7]. Histologically, gliomas are tumors of the central nervous

system, classified by WHO in four groups, according to progressively increasing degree of

aggressiveness, anaplasia and undifferentiation. WHO grade I (pilocytic astrocytoma, subependymal

giant cell astrocytoma, angiocentric glioma) and grade II (diffuse astrocytoma, oligodendrogliomas

and oligoastrocytomas) tumors represent the more differentiated neoplasms with better prognosis,

when diagnosed in a precocial moment, without which, unvaryingly progress to a higher grade

overtime; WHO grade III tumors (anaplastic variants of astrocytoma, oligodendroglioma and

oligoastrocytoma), and the most aggressive of all, WHO grade IV tumors (diffuse midline glioma and

glioblastoma and its variants) [8,9].

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Malignant gliomas represent a complex process of cumulative genetic alterations due to intrinsic and

environmental factors. However, genome-wide association studies (GWAS) reveal that a myriad of

genes is only scarcely related to glioma development, reflecting molecular heterogeneity. Regarding

classic glioblastoma, the most studied malignant glioma, there are some chromosome mutations,

namely in EGFR, NF-κβ, PDGFRA, among many others. Glioma behavioral and biological

processes are complex and poorly understood. Its microenvironment and interactions with adjacent

cells and tissue proteins (inflammatory, immunological and others) has recently began to be closely

studied. Even though, due to its high heterogeneity, available therapies only benefit a small subgroup

of tumors [1,3,10,11].

In 2001, different studies from the International Agency for Research into Cancer (IARC) and the

World Cancer Research Fund (WCRF) have reported a relationship and established a link between

obesity and cancer risk [12,13]. Excess adiposity is related to an increased the incidence and/or

death rates from a wide variety of human cancers, being the most common colon, rectum,

esophagus, kidney, pancreas, gallbladder, ovary, cervix, liver, prostate and certain hematopoietic

cancers [14,15]. Overweight and obesity are important risk factors for developing cancer and also

for cancer related mortality [16,17]. It is important to understand the pathophysiological mechanisms

involved in the link between obesity and cancer, in order to target future preventive and therapeutic

strategies for cancer in obese people [16].

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Despite its high morbidity, the etiology of glioma remains largely unknown. Among several risk

factors, lifestyle was also recently identified as a major risk factor for the development of primary

glioma [18]. Consequently, there might be a relationship between its increasing and adipose tissue

dysfunction. Obesity, favored by the modern lifestyle, acquired epidemic proportions nowadays,

being associated and recognized as determinant in the development of different tumors. Concerning

central nervous system (CNS) tumors there has been an increasing association between obesity and

its contribution for their development, however glioma data has been inconsistent [19]. There is even

a disparity regarding diabetes as some studies found a protective status of diabetes against glioma.

Metabolic variations derived from obesity and its relationship with glioma development may be

complex and heterogeneous. It has been suggested that it may include insulin-growth-factor (IGF),

chronic inflammation and adipokines signaling [20]. Some studies analysed body weight in relation

to survival in glioma demonstrating higher death rates in patients with an excessive body weight [21].

Obesity, and in particular visceral obesity, plays a major role in the pathogenesis of several metabolic

disorders [22]. Genes that influence obesity are highly expressed in the brain and could also mediate

glioma susceptibility [18,23].

At present, the median patient survival in glioma cases are diminished (less than two years), even

with adequate care and safe resection following radio-chemotherapy with Temozolomide, an

alkylating agent approved. As previously mentioned, prognosis and treatments depend on the glioma

grade. Therefore, low-grade gliomas have better prognosis, being more sensitive to therapy (radio

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and/or chemo) directed by molecular type, and after resection. On the other hand, most of the high-

grade gliomas are recognized by its resistance to therapy, namely additional therapies commonly

used against biological hallmarks of cancer [1,3].

Adipose tissue as an endocrine organ

Adipose tissue is now considered a dynamic endocrine organ, secreting fatty acids, hormones,

proteins, peptides, complement factors, and many bioactive substances referred as adipokines -

cytokines, chemokines and tissue products of enzyme activity - which act in an autocrine, paracrine

and/or endocrine mode [24]. The white adipose tissue (WAT) is constituted of approximately 50%

adipocytes, 10% macrophages, and 40% of preadipocytes, fibroblasts and endothelial and epithelial

cells [25]. All these cell types are responsible for adipokines’ secretion. All these factors are involved

in important physiological processes, particularly in the appetite regulation, energetic metabolism,

lipid and glucose metabolism, adipocyte differentiation, blood flow regulation and vascular and

lymphatic immune function. In case of fat increment due to a fatty diet, or metabolic dysfunction,

adipocytes can trigger an inflammatory reaction leading to lymphatic drainage failure by primary

capillaries. Thus, there are neutrophils, macrophages and other inflammatory mediators’

accumulation, as well as unregulated synthesis of adipokines (in particular pro-inflammatory) in the

tissue, which enhance inflammatory reactions and increasing of interstitial vessels. Macrophage

infiltration in obesity is directly related to the size of adipocytes, suggesting a modification of a non-

inflammatory phenotype to foam cells. In addition to secretion of inflammatory mediators such as

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Interleukin-6 (IL6) or Tumor Necrosis Factor-α (TNFα), it is observed an infiltration with large

numbers of macrophages (Figure 1), suggesting that pro-inflammatory cytokines are also produced

by them, and not only by adipocytes [26–28].

Adipose tissue is structurally and functionally divided into two types of tissue: white and brown

adipose tissue. Each of them with distinct, and even opposite functions: WAT is majorly recognized

by its single vacuole triglyceride reservoir and secretory/endocrine function, while brown adipose

tissue (BAT) has a highly oxidative and catabolic role, producing heat. BAT secretory potential has

been studied but information is still scarce.

First adipokine described regarding secretory properties of WAT was leptin, by Kennedy in 1953, as

a “lipostatic” factor exerting control on energy expenditure and food intake [29]. Afterwards, and with

the discovery of many other molecules, secretory role of WAT was consolidated as autocrine,

paracrine and endocrine by its factors named as adipokines (cytokines secreted by adipose tissue).

macrophage

HFD

adipocyte capillary

LEAN OBESE

Figure 1 - Increased infiltration of macrophages in high fat diet induced obesity. Hypertrophy of adipose tissue contributes to

the accumulation of neutrophils, macrophages and other inflammatory mediators in the tissue, potentiating inflammatory

reactions in the capillaries and the instertitium. Adapted from Galic et al. 2010.

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Even if the term adipokine respects to molecules secreted by adipocytes, nowadays this term is used

not only for factors secreted by adipocytes, but also by macrophages and adipose stromal cells

(ASC).

Besides to adiponectin, an anti-inflammatory adipokine, WAT releases a myriad of molecules with

anti and pro-inflammatory effects. These molecules are often involved in insulin resistance and,

almost consequently, obesity and lipodystrophy through activation or inhibition of inflammation in

adipose tissue. Regarding this inflammatory state proportioned by obesity there are some

established adipokines of reference, namely hepatocyte growth factor (HGF), IGF, IL6, IL10, MCP1,

plasminogen activator inhibitor (PAI-1), RANTES, TGF-β, TNF-α, among many others with

predominant relevance in angiogenesis, such as leptin, vascular endothelial growth factor (VEGF),

fibroblast growth factor (FGF), IL1β, PlGF, visfatin, resistin, neuropeptide Y, leptin and angiopoietins,

as well as anti-angiogenic factors thrombospondin-1, or adiponectin. Some of the pro-inflammatory

factors act not only as insulin-resistance inducers, but also as endothelial enhancers of inflammation,

remodeling and proliferation of endothelial cells through inflammatory cell extravasation and

interaction with immune cells [30,31].

Regarding this inflammatory process, it is suggest by some authors that the number of dead

adipocytes and macrophages in adipose tissue increases with the increment in fat mass, being

related to inflammation due to obesity, since this is considered a low-grade inflammatory chronic

condition [26,32,33]. So, it is observed increasing in pro-inflammatory plasmatic cytokines in

20

response to adipocyte death and hypoxic areas of adipose tissue. Along with this increase, it is

possible that blood supply is compromised, making it insufficient. Thus, there are areas of adipocyte

hypoxia and subsequent inflammatory response that stimulates angiogenesis, in order to ensure the

maintenance of tissue fluid homeostasis. The cellular response to hypoxia is the production of the

transcription factor hypoxia-inducing factor-1 (HIF-1), which regulates several genes whose

expression is stimulated by cytokines such as TNFα and IL6. Some of target genes for HIF-1 are

VEGF-A, VEGF-C and VEGF-D, which play essential roles in the formation of new blood and lymph

vessels. Additionally, there is a strong association between the adipose tissue increase observed in

obesity, adipocyte hypoxia and stimulation of angiogenesis [34–36]. However, this topic will be

closely approached later on this chapter.

Concerning WAT anatomic distribution, it is classified in visceral adipose tissue (VAT) and

subcutaneous adipose tissue (SAT). Referring to healthy individuals, approximately 80% of the total

adipose tissue is located in subcutaneous depots, whereas VAT is accounted for 5-20%, depending

on gender (men are accountable for higher percentages of VAT when compared to women). More

than gender differences, this distribution also varies depending in age, race, genetics and disease

status [29]. Undeniably, the risk for metabolic dysfunction relies much more on its location than on

the total amount of fat. This is straightforward related to morphologic and functional differences

between the both. Regarding metabolic profile, and particularly adipokine expression, it was noted

that the patterns of secretion differ from VAT to SAT. This is, VAT is responsible for an increased

secretion of pro-inflammatory cytokines such as VEGF and IL6. This secretory differential expresses

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the harmful potential of VAT on different organs/tissues, which is higher than the effects caused by

SAT [25,37]. The differences in adipokine metabolites expression contribute to changes in flow

systems, which may cause structural changes observed in different pathological cases. Out of these,

we highlight lipid disorders, diabetes, hypertension, atherosclerosis and hepatic disorders, which are

highly correlated with VAT increasing. Even hepatic disturbances are directly related to free fatty

acids (FFA) and pro-inflammatory adipokines released by this specific type of tissue [29,38].

Studies in mice have revealed a high correlation between increased adiposity and expression of a

significant number of genes typically expressed by macrophages. In these cases, both adipocyte

size and total body weight proved to be predictive about the number of macrophages found in

adipose tissue (ATM), which is bigger in comparison to that for VAT/SAT. It has also been established

an association between mice weight gain and infiltration of adipocytes by macrophages, and

subsequent production of pro-inflammatory factors by the AT. These inflammatory changes were

more visible in VAT, which is associated with a higher metabolic risk [39–41]. On the other hand,

according to these studies, it was suggested that SAT is associated with less physical stress and

pressure variations so that the inflammatory response in subcutaneous obesity state is smaller when

compared with a state visceral obesity [37,40]. It should be noted that the relationship between

visceral adipose tissue and the low-grade inflammation was also observed in humans.

Obesity is also associated with substantial modulation of adipose tissue structure, involving

adipogenesis, angiogenesis, and extracellular matrix remodeling [42]. Thus, AT can trigger blood

vessel formation, and in turn AT endothelial cells promote pre-adipocyte differentiation. Under

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physiological circumstances, angiogenesis is important and natural to wound healing and

cellular/tissue growth. However, tumors depend on angiogenic processes to proliferate from benign

to malignant status [43–46]. Anti-angiogenic therapy has been a widely used strategy to fight tumor

growth, and has also been recently acknowledged as a potential therapeutic intervention for obesity

[11,32,47–49].

Furthermore, and concerning lipid diet content, evidence demonstrates that lipid metabolism is

dysregulated in obesity and there might be a common pathway between increased metabolic risk

and excessive nutritional intake and inflammation [50]. Increase in lipolysis in AT leads to higher

levels of FFA in plasma and contribute to fat deposition in liver and skeletal muscle, which may

promote systemic insulin resistance through several mechanisms, such as induction of oxidative

stress through incomplete oxidation and inflammatory responses [51]. AT is also recognized by its

endocrine function as producer of biological mediators. Enlargement of adipose tissue due to obesity

is characterized by a boosted inflammation and secretion of a myriad of adipokines and cytokines,

rising the search for potential biomarkers. It has been reported that high-fat diet induced obesity in

humans induce epigenetic modifications, which has a repercussion on protein expression and

cellular/tissue response [16,17,50]. There are several cytokines regarding cell proliferation,

angiogenesis, cytoskeletal rearrangement and survival. Some receptors and signaling pathways

have revealed to stimulate growth and invasiveness capacity of glioma cells, whereas others have

been correlated to blockage of glioma cell proliferation [12,16]. The mechanisms underlying the

associations between energy balance and cancer risk should be clarified.

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The pathways involved in cell death and proliferation, angiogenic process in obesity and lipid profile

involve multiple transcription factors, receptors and signaling mediators. Accordingly, the approaches

for the study of lipid metabolism should be varied and complementary.

An epidemic called obesity, inflammasome and adipocytokines

Obesity has tripled since 1975 being already considered an epidemic of our days. According to WHO,

in 2016, more than 1,6 billion adults (18 and older) were overweight and more than 650 million were

obese; over 340 million children and adolescents were overweight or obese, as well as 41 million

children under 5 years old. Prevalence is continuously increasing, which has a great impact on global

health mainly through metabolic complications. Even further than these metabolic alterations, it is a

major cause for dyslipidemia due to increasing circulating lipids, as triglycerides and free fatty acids,

released from liver and adipose tissue and subsequent accumulation in non-fatty depots, leading to

a lipotoxicity owing to the exceeding capacity of oxidative and storage in these depots. This process

carries to major concerns regarding metabolic and increased risk of heart disease [20,52].

As previously described, both WAT and BAT reveal secretory function and potential, releasing

numerous angiogenic modulators, whose expression balance will determine the triggering or halting

of the angiogenic switch [53–56]. In addition to adipocytes, adipose tissue contains diverse cells

types including ASC and ATM that also contribute to angiogenesis. ASC also produce a myriad of

angiogenic factors, VEGF, HGF, GM-CSF, FGF-2, and TGF while macrophages also produce VEGF

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and the angiogenic cytokines TNF-α, FGF-2, IL1β, IL6, and IL-8. Adipose tissue produces several

matrix matalloproteinases (MMPs) including MMP-2 and -9, which could potentially affect

preadipocyte differentiation and microvessel maturation by modulating ECM. Moreover, MMPs

release the matrix-bound VEGF and indirectly induces angiogenesis [57].

Adipose tissue expansion due to adipocytes’ accentuated hypertrophy disrupts tissue oxygenation

because vascular network do not effectively accompany this progress [58]. Therefore, in the obese

status hypoxia is responsible for triggering inflammatory process and subsequent angiogenesis, in

order to equilibrate adipose tissue expansion [58–62]. In this hypoxic state, adipocytes express a

varied number of factors, HIFs, a family of well characterized heterodimeric factors that establish the

main hypoxia-responsive cellular sensors and molecular mediators [63]. Under a normal O2

interstitial pressure HIF-α subunit is quickly degraded but then, under low O2 pressures HIF-α is

stabilized. In this situation, HIF-α is translocated to the nucleus, dimerizes with the constitutively

expressed HIF-β promoting expression of angiogenic and pro-inflammatory factors [64].

HIF-1α is a major pro-angiogenic factor, accountable for vasculature homeostasis (together with

many other factors) not only in disorders involving physiological processes, but also in

carcinogenesis [46,65]. Due to HIF1-α increased levels in adipose tissue, it has been presumed a

relevant role in adipocyte differentiation and function [61,66,67]. Moreover, studies have

demonstrated that silencing HIF expression resulted in diet-induced obese mice regulation of insulin

sensitivity, dyslipidemia and resistance to weight gain, as well as reduced immune cell tissue

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infiltration [68–71]. These data made drug-induced inhibition of HIF-1α an appealing approach to

adipose metabolic dysfunction [72]. However, other studies have reported that HIF-1α expression

has benefits on metabolism by protecting from high fat diet-induced obesity, insulin resistance, as

well as by increasing energy expenditure through mitochondrial biogenesis and thermogenesis [73].

Unexpectedly, a deeper analysis of HIF-1α role in adipose tissue secretion did not determine

categorically the modified (over)expression of proangiogenic factors, such as VEGF family

[58,69,74,75]. Actually, HIF-1α is upregulated and overexpressed by adipogenesis and

hyperinsulinemia, not by adipose tissue hypoxic status, which may be due to independent pathways

in tissue angiogenesis by VEGF regulation, and hypoxia-induced inflammassome [76,77]. Studies

involving human adipose tissue presented high levels of pro-angiogenic factors, such as VEGF-A,

VEGF-C and many others, but no suitable vascular response to increasing in HIF-1α expression.

Additionally, even though adipocyte hypertrophy due to diet-induced obesity logically lead us to

believe in hypoxic-state increment, in humans, adipose tissue expansion is slower, so demanding of

O2 from adipocytes is lower than in an animal model, and vasculature reorganization may happen,

even if partially adequate [78–80]. Nevertheless, in a mouse model the expected hypoxia-triggered

angiogenesis is conceivable and adipose tissue secretory response to obese state should not be

neglectable, and its complex process must be pursued, as we intended to.

Thus, adipose tissue and obesity is much more than hypertrophic adipocyte. There is a myriad

angiogenic factors such as growth inducers, HGF, PDGFB and FGF-2 by dependent or independent

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pathways of VEGFR. There are many different growth factors and pathways linked to endothelium

and involved in adipose tissue expansion, even more in carcinogenesis, which contribute to

molecular heterogeneity of which much is still unknown [81,82]. In this context, when considering the

adipose tissue and the inflammatory state described in obesity, many more unknowns and

controversial issues arise for discussion.

Sphingolipids in obesity: ceramide and sphingosine-1-phosphate

Adipogenesis consists of pre-adipocytes differentiation throughout complex mechanisms and

modifications in physiology and morphology. Obesity can be a result of hypertrophy by adipogenesis

and hyperplasia by adipocyte differentiation, and its prevalence is continuously increasing, which has

a great impact on global health mainly through metabolic complications. Fat deposition occurs mainly

due to triglycerides storage in fat cells [83]. Furthermore, overdue of storage and oxidative capacities

due to excessive accumulation of FFA, sphingolipids and ceramides (Cer), in non-adipose tissues,

also contributes to lipotoxicity.

Lipidomic studies on twins with different body types (lean versus obese) suggested that increased

levels of poly-unsaturated fatty-acids containing phospholipids occur in early stages of obesity [84].

So, circulating lipids have become an indicator and biomarker for disease development. Increasing

concentration of FFA and inflammatory cytokines activate receptors associated with signaling

pathways involved in insulin signaling in muscle and liver. Furthermore, there is evidence that

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Ceramide may result from different processes: hydrolysis of membrane sphingomyelin by

sphingomyelinase (Figure 3), or de novo synthesis from long-chain saturated fatty acids via serine-

palmitoyl transferase. Subsequently, by the action of two sphingosine kinase isoforms (SpK1 and

SpK2), ceramide can be metabolized into sphingosine, converting it into S1P [86–89]. Studies carried

out to study the bioactivity of these molecules describe them as involved in various cell signaling

pathways, namely in the regulation of cell growth, motility, survival, senescence and cellular

apoptosis. Additionally, some pathophysiological changes, such as obesity and diabetes, in adipose

tissue and plasma, are associated with anomalies in the metabolism of these sphingolipids, among

others [89]. It should be noted that, in relation to the described processes, functions antagonistic to

ceramide and S1P are proposed (Figure 3), and the activation of a certain signaling pathway over

another is due to the balance between both biomolecules [88,89].

Figure 3 - Schematic diagram of the metabolism of sphingolipids, and sphingomyelin targets as well as metabolites and

sphingolipid metabolism. It is worth noting the reversible degradation of ceramide in S1P, maintaining an equilibrium

associated with a situation of apoptosis or cell proliferation. Adapted from Alewijnse & Peters, 2008.

29

Ceramide and sphingosine are precursors of S1P, being part of apoptosis, cell death and anti-

inflammatory signaling pathways. Ceramide is considered the building block to all sphingolipids,

namely sphingolipid and sphingomyelin biosynthesis, as well as acting as signaling molecule in

signal transduction, cellular differentiation, proliferation and degeneration. Ceramide involvement in

apoptotic pathways occurs via different targets as protein kinase C, protein kinase suppressor of

Ras, Rac, c-Jun N-terminal kinase, ceramide-activated protein phosphatase and inducible NO

synthase. ROS intracellular increasing is associated with mitochondrial dysfunction and releasing of

cytochrome C, inducing apoptosis. Ceramide varied, and even contradictory, cell functions and

responses may be related with different levels, targets and cell-specific localization [90,91]. These

intracellular levels may arise from a variety of extracellular stimuli, such as oxidative stress, nitric

oxide, pro-inflammatory cytokines (such as TNF-α, IL1), low-density lipoproteins, high levels of FFA

and/ or endotoxins, conditions that characterize adipose tissue in obesity. Short-chain ceramides (2

to 6 carbons) have been related with cell senescence, cytotoxicity, insulin resistance, inflammation

and apoptosis, while long-chain ceramides (16 to 24 carbons) are associated with cell growth,

proliferation and differentiation, and subsequently with motility and adhesion. They have been

associated with complications such as diabetes and insulin resistance, inflammation, atherosclerosis

and neurodegenerative disorders. Some studies already prove that ceramide is relevant in the

initiation and activation of inflammasome in obesity and some other pathological alterations, through

defective autophagy and excessive ROS. Moreover, in the context of obesity, metabolic tissues as

30

adipose tissue, liver as skeletal muscle, reveal altered sphingolipid metabolism with increasing

ceramide levels, inflammatory adipocytokines and inflammation [92–94].

Sphingosine-1-phosphate is uniquely produced by ceramidases and is the major lipid present in

mammalians. Once it is produced by ceramidase, sphingosine can follow two different pathways: the

“salvage” pathway in which is reacylated to renew ceramide, or being phosphorylated by sphingosine

kinase to produce S1P. Another less frequent substrate to sphingosine kinases is sphinganine

through de novo pathway or by the action of enzymes that act on sphinganine-containing substrates.

It is a bioactive sphingolipid involved in a myriad of pathophysiological processes. S1P regulates a

large number of cellular processes including inflammation, development of microvascular structures

essential to oncogenesis, metastasis and proliferation. Besides, it can regulate intracellularly on gene

expression and cell function regulation, as it can be secreted and extracellularly act as an

autocrine/paracrine mediator [85]. Its regulation as a secondary messenger is mediated by catabolic

and anabolic enzymes, namely sphingosine-1-phosphate phosphatase (SGPP) one and two, and

SpK1 and SpK2, respectively. S1P cell functions are dependent on its receptors S1PR1 to S1PR5,

belonging to G-protein family. These receptors are involved in cell growth, apoptosis, proliferation,

angiogenesis, chemoresistance, or vascular stability. Moreover, it is a chemotactic factor for immune

cell trafficking as well as a regulator of microenvironmental cancer development and proliferation. As

an oncogenic factor it has already been reported to be involved in breast, lung, melanoma, kidney,

colorectal and glioblastoma multiform cancers [95]. Glioblastoma Multiforme has revealed higher

31

levels of S1P compared with non-malignant brain tissue, and the poorer prognostic on these patients

is correlated with higher expression of SpK1 and S1PR2. However, S1PR1 has been correlated with

better prognosis [95].

In the last years, studies have been conducted to unveil the complete role of sphingolipids in obesity

and related pathologies with the perspective to discover new therapeutic approaches to disease.

S1P relationship with proliferation and oncogenesis depends on S1P receptors (S1PRs), as well as

its enzymes sphingosine kinases, SpK1 and SpK2 and phosphatases. The difficulty in understanding

S1P mechanisms in carcinogenesis process is due to the complexity of the signaling trigger and

subsequent response by the specific receptor and pathway activated. When exported by S1P

specific transporters (for example, ATP-binding cassette) it binds to its receptors developing

autocrine, paracrine or endocrine functions. SpK1 is present in almost every cell, close to the cell

membrane in the cytosol. SpK2 is mainly expressed in specific organs like brain, liver and kidney,

involved in regulation of lipidic metabolism as well as in mitochondrial β-oxidation [90,96].

S1P receptors are expressed on cancer cells as well as other cells in tumor microenvironment. There

are five S1P receptors, each of them activating a signaling pathway.

Some of the studies performed on adipose tissue from mice with genetically induced obesity, found

simultaneous decrease in ceramide content and increase in S1P, compared with control group.

Additionally, balance of sphingolipid metabolism in adipose tissue tends to move towards

sphingosine formation, thereby reducing the expression of ceramide in adipose tissue. Regarding

32

angiogenesis, some authors observed in vitro migration and formation of capillary structures in

HUVEC cultures by stimulation of migration and differentiation of endothelial cells via the signaling

pathway S1P1 / Gi / PLC / Ca2 +. S1P induced cell migration observed in vitro was superior to that

induced by VEGF-C, and it was more closely observed in in vivo studies. These studies also revealed

increased plasma levels of S1P in pathological conditions. However, S1P levels on different tissues,

specifically on adipose tissue, remain unknown [96,97].

33

AIMS OF THE STUDY

Research conducted in the last decades provided scarce insights into the pathways involved in

gliomagenesis. However, the exact mechanisms involved in glioma development and progression

are unclear. In this thesis it is hypothesized that adipose tissue dysregulation could be a relevant

event in disease progression. Therefore, a better understanding of the molecular mechanisms

leading to abnormalities in adipose tissue, metabolic dysfunction and cancer development and

progression could open novel avenues for early therapeutic intervention and the development of

biomarkers for early disease diagnostics.

In this context, the major aims of this study were to:

o develop an in vitro rodent model for the study of the influence of obesity role in glioma, based

on 3T3-L1 pre-adipocytes differentiation and subsequent exposition to GL261 glioma cell

culture from mouse in order to also study obesity influence on carcinogenesis by proteomic

analysis of inflammatory adipokines and cytokines expressed by glioma cells to the 3T3-L1

medium;

o study cytokines relationship within inflammation and angiogenesis by analysis of the cellular

model of brain cancer GL261 previously inoculated on C57Bl/6J mice, as well as

immunohistochemical expression on adipose and tumoral tissue;

34

o approach sphingolipid rheostat on diet-induced obesity, through ceramide and S1P analysis

on the C57Bl/6J model serum and correlate with tissue response.

Framework

This thesis is organized in five chapters in order to present the reader with the purpose of the work/

state of the art on the subject followed by a segmented presentation of the methodology used to fulfill

each of the objectives, as well as the results obtained and due discussion.

The first experimental chapter consist of already published work. Then, the following two chapters

present results submitted and under revision. Lastly, and in order to properly complete and close this

thesis, there is a final chapter with a general discussion and concluding remarks.

35

CHAPTER TWO

Adipocyte proteome and secretome influence

inflammatory and hormone pathways in glioma

establishment of 3T3-L1 conditioned medium

36

In this chapter it is approached the development of the in vitro cell model for the study of rodent

obesity. This model is based on 3T3-L1 pre-adipocytes differentiation and subsequent exposition to

B16 melanoma cell line from mouse. This melanoma model was developed in order to also study

obesity influence on carcinogenesis and its results helped to enlighten the progress on glioma and

obesity in vitro model. Also, it is loomed the influence of obesity role in glioma by proteomic analysis

of inflammatory adipokines and cytokines expressed by glioma cells to the 3T3-L1 medium.

3T3-L1 preadipocytes are a commercially available cell line for studying adipogenesis [28,98]. Their

differentiation, in culture, into mature adipocytes is well widespread and can be achieved by

established protocols. It was used conditioned medium from fully differentiated 3T3-L1 cells to

explore the potential effects of adipocytes secretome over glioma GL261 cells proliferation and

apoptosis.

Presented data have already been published in two papers, as it follows:

P. Coelho, J. Almeida, C. Prudêncio, R. Fernandes, R. Soares, Effect of Adipocyte Secretome in

Melanoma Progression and Vasculogenic Mimicry, J. Cell. Biochem. 117 (2016) 1697–1706.

doi:10.1002/jcb.25463.

V. Cea, P. Coelho, C. Sala, J. Costa, M.S. Diniz, C. Prudêncio, R. Fernandes, J.P. Noronha, M.

Galesio, J. Almeida, R. Soares, Adipocyte proteome and secretome influence inflammatory and

hormone pathways in glioma, Metab. Brain Dis. 34 (2018) 141–152. doi:10.1007/s11011-018-0327-

y.

37

2.1. Material and Methods

Cell culture and in vitro treatment

GL-261 (gently given by Prof. Pedroso Lima, CNC, Coimbra, Portugal) mouse cell lines were used.

3T3-L1 and GL-261 cells were cultured in DMEM (Dulbecco´s Modified Eagle´s Medium with 4.5g/L

Glucose & L-Glutamine, without Sodium Pyruvate. Santa Cruz Biotechnology, Inc.), supplemented

with 10% FBS, and 1000 units/mL gentamycin solution, maintained in T-25 tissue culture flasks in

5% CO2/95% air at 37oC in a humidified incubator.

Adipocyte cell culture and conditioning

3T3-L1 pre-adipocytes were propagated and allowed to reach confluence. After 2 days (day 0), the

differentiation was initiated by addition of a hormonal mixture composed of 2 μM insulin, 1 μM

dexamethasone and 0.25 mM isobutylmethylxanthine. Three days after (day 3), the induction

medium was replaced by complete medium supplemented with insulin only. At day 6 cultures were

washed twice in phosphate buffered saline and incubated in serum-free medium. After 24h (day 7),

medium was harvested from the adipocyte’s cultures, spun for 3000 g for 5 minutes and the

supernatant (mature adipocytes conditioned medium) was stored at -80ºC for the subsequent

treatments. This conditioned medium was rich in adipokines which are globally referred as

secretome.

38

Afterwards, glioma cells were divided in two distinct groups. On one hand, glioma cells GL-261 were

grown under the influence of the mature adipocytes secretome were designated conditioned glioma

cells (CGl). On the other hand, the control group consisted in the glioma cells cultured in the absence

of 3T3-L1 conditioned medium and was designated as non-conditioned glioma cells (NCGl).

Proteomics analysis by 2D-PAGE and MS-MALDI-TOF Spectrometry

The protein expression was accessed by means of bidimensional polyacrylamide gel electrophoresis

under denatured conditions (2D-PAGE) followed by mass spectrometry analysis of the interest spot.

The spots were analysed by matrix assisted laser desorption ionization time-of-flight/mass

spectrometry (MALDI-TOF-MS). The non-conditioned glioma cells served as the control group for

the conditioned glioma cells. We have used the mature adipocytes secreted adipokines (secretome)

and enriched the GL261 medium, followed by analysis of the proteins on GL261 on a 2-dimensional

proteomic gel approach, so different spots could be studied by means of mass spectroscopy. Also It

was analysed by microarrays-based proteomics.

Chemicals

The reagents acetonitrile, iodoacetamide (IAA), dl-dithiothreitol (DTT) (99% w/w) and trypsin from

porcine pancreas (proteomics grade) were purchased from Sigma (Steinheim, Germany). Formic

acid puriss for mass spectrometry (≥98%), ammonium bicarbonate (>99.5% w/w) and the matrix

assisted laser desorption ionization MALDI matrix α-Cyano-4-hydroxycinnamic acid (α-CHCA) puriss

for MALDI-MS were from Fluka (Buchs, Switzerland). Trifluoroacetic acid (TFA, 99% v/v) was from

39

Riedel-de-Haën (Seelze, Germany). ProteoMass Peptide MALDI-MS Calibration Kit (MSCAL2) from

Sigma was used as mass calibration standard for MALDI-TOF-MS.

Sample preparation for 2D-PAGE

Sample preparation for 2D-PAGE was performed according to published procedures with minor

modifications [99]. The cells were manual detached from the flask and were mechanically lysed at

4oC in lysis solution [CHAPS 4%, 5 mM Tris, pH 8.8, 0.05% protease inhibitor cocktail (Sigma)] using

a glass potter, and the samples were centrifuged at 2000 g for 15 minutes at 4oC in order to eliminate

aggregates. The protein concentrations in each sample were measured by means of a DC Bio-Rad

assay. A total of 900 mg of protein from each sample was precipitated with cold acetone and

resuspended in thiourea buffer (7M urea, 2M thiourea 2% CHAPS, 2% ASB14, 5% glycerol, 40 mM

DTT, 4 mM TCEP, 1% 3–10 IPG buffer, Amersham), and the samples were mixed overnight at 4oC

in the dark and clarified by centrifugation at 16000 g for 15 minutes at 4oC. The supernatant was first

separated by isoelectric focusing over a pH range of 3–10 using precast first-dimension drystrip 3–

10 NL 11 cm (Bio-Rad) following a multi-step protocol for 90,000 Vht (Protean IEF cell, Biorad). The

first dimension strip was equilibrated in 50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 2% SDS plus

16 mM DTT for 20 minutes, and then plus 25 mM iodioacetamide for 15 minutes, and loaded on a

midi format (11 cm) 9–16% acrylamide gel to separate the proteins by molecular weight. Second-

dimension runs were performed using Biorad midi cells at 30 V 1 h, 300 V 4 h at a constant

temperature of 18oC. Protein spots were revealed using home-made blue Coomassie staining, and

40

the gel images were acquired by means of an Image scanner at 300 DPI resolution and analysed

using Image2D Master Platinum software (both from Amersham).

At least three gels per condition were included in the analysis. Normalized spot volume values were

studied using SPSS software version 13.0 for statistical analysis (SPSS Inc.). In brief, the data for

each spot match set were analysed using a box-plot test in order to eliminate outliers, a K-S test to

check normal distribution, an F test to analyze the variance, and finally Student’s T test to compare

the mean values, which were considered significant for a P-value cut off of 0.01.

In-Gel Protein Digestion

In-gel digestion of the excised proteins with trypsin was performed according to published procedures

with minor modifications [100–102]. Protein spots were destained for 10 min with a solution of

acetonitrile 50% (v/v) in 25nM NH4HCO3 and mixed vigorously using a vortex. After removal of the

destaining solution, gel pieces were washed with water for 10 min using vigorous vortexing. This step

was repeated twice. Afterwards, the gel pieces were dehydrated in acetonitrile for 10 min, which was

then removed, and the gel pieces were placed to dry in a vacuum centrifuge. Reduction of protein

disulfide bonds was performed with a solution 10mM of dithiotreitol (DTT) in 25mM NH4HCO3, for 15

min at 60 ºC. After cooling to room temperature for about 15 min, the DTT solution was replaced with

a solution 110mM of iodoacetamide (IAA) in 25mM NH4HCO3. Incubation with the alkylation agent

was performed for 35 min at room temperature in the dark. After reduction and alkylation steps, the

gel pieces were submitted once more to the washing and dehydration procedure with water and

41

acetonitrile, in the same way as described above. Subsequently, the gel pieces were completely

dried in a vacuum centrifuge. The dried protein spots were incubated with a solution of trypsin 25

ng/µL in 12.5 mM NH4HCO3 in an ice bath for 30 min to rehydrate the gel and to allow enzyme

penetration into it. In-gel protein digestion was performed at 37ºC overnight. After collecting the

supernatant to clean vials, further peptide extraction was performed by addiction of a solution of

trifluoroacetic acid 0.1% (v/v) in acetonitrile 50% (v/v) to the gel pieces and incubation at room

temperature with shaking for 10 min. This step was repeated twice. All extracts were pooled and

evaporated to dryness. The samples were re-suspended with 10 µL of trifluoroacetic acid 0.1% (v/v).

MALDI-TOF-MS analysis

Prior to MALDI analysis, the sample was mixed with an equal volume of the MALDI matrix solution,

10 mg/ml α–CHCA in trifluoroacetic acid 0.1% (v/v) and acetonitrile 50% (v/v). An aliquot of the

sample/matrix solution (0.5 µL) was hand-spotted onto the MALDI sample plate and the sample was

allowed to dry. The mass spectrometric analyses were performed using the Applied Biosystems

MALDI-TOF-MS system model Voyager-DE PRO Biospectrometry Workstation equipped with a

nitrogen laser radiating at 337 nm (Applied Biosystems, Foster City, USA) and the laser intensity was

set just above the threshold for ion production. MALDI mass spectra were acquired in positive ion

reflectron mode, with an accelerating voltage of 20 kV, a grid voltage of 15 kV, 0.4 V of guide wire

and an ion extraction delay of 100 ns. The MS spectra for each sample were based on the average

42

of 700 laser shots per spot with an acquisition rate of 2 ns. MS acquisition data was calibrated

externally using the ProteoMass Peptide MALDI-MS Calibration Kit.

Data analysis and database searching

All data was processed using DataExplorer 4.5 software from Applied Biosystems. Peptide Mass

Fingerprint (PMF) data was used to search for candidate proteins using the MASCOT database

search (http://www.matrixscience.com) engine. SwissProt database was selected by default for all

Mascot searches. NCBInr database was used each time no significant identification was obtained

with SwissProt. Database searches were, by default, performed with no taxonomy restriction and

allowing up to a maximum peptide mass tolerance of 100 ppm. The number of allowed missed

cleavages for trypsin was set to one. Carbamidomethylation of cysteine and methionine oxidation

were selected as fixed and variable modifications, respectively. In order to provide accurate results,

protein identification was considered positive for MASCOT protein scores higher than 77 (p<0.01)

that present a minimum of 4 peptides matching.

Proteome Profiler™ Array Assay

Determination of relative levels of pre-selected mouse adipokines in conditioned and non-conditioned

GL261 cell cultures was performed by using Mouse Adipokine Array Kit (R&D Systems Europe, UK).

Cells were rinsed with PBS before Lysis Buffer solubilization at, approximately, 1 x 107 cells/mL.

Then, they were ressuspended and rocked at 4°C for 30 minutes, following a microcentrifugation at

14,000 x g for 5 minutes. After supernatant transfer into a clean test tube, proteins were quantified a

43

total protein assay. Array Procedure was performed according to manufacture instructions

(Catalogue Number ARY024). Data analysis was based on pixel densities on developed X-ray film

collected and analysed using Gel-Doc XR (Bio-Rad© Laboratories), acquired by Image Lab Software

5.2.1 (Bio-Rad© Laboratories) then quantified by ImageJ 1.49 (NIH, USA).

Statistical analysis

The differences in expression of proteins between glioma medium and CGl by Proteome Profile

Microarray were analysed by 2-way ANOVA for multiple comparisons with Prism Graphpad. P-values

of <0.05 were considered statistically significant. MALDI-TOF-MS was conducted by T-test at P-

value cut off of 0.1 using default parameters with Prism Graphpad.

44

2.2. Results

In order to understand which adipocyte secreted proteins potentially influenced glioma tumor

behavior, it was analysed the protein expression of glioma cells conditioned medium retrieved from

adipocytes cultures, by means of bidimensional polyacrylamide gel electrophoresis under denatured

conditions (2D-PAGE) followed by mass spectrometry analysis of the selected spots. These spots

were analysed by matrix assisted laser desorption ionization time-of-flight/mass spectrometry

(MALDI-TOF-MS). The non-conditioned glioma cells (NCGl) were used as the control group for the

conditioned glioma cells (CGl).

Differentially Expressed Proteins

The proteomic analysis has allowed the identification of eleven well differentiated proteins (among

the 1192 matched protein spots) that are secreted by 3T3-L1 adipocytes with a potential role in

cancer biology. So, as it can be seen in table 1, there are over, under and partially (only in one

condition) expressed proteins.

The 5 over-expressed proteins found on the CG1 cells. RFC1 corresponds to spot-1 with 9.38

isoelectric point, 126705 relative molecular mass and presenting a 2.32 ratio. KIF5C corresponds to

spot-2, with 5.86 isoelectric point, 109777 relative molecular mass and 2.91 ratio. ANXA2

corresponds to a spot-3 protein spot, with 7.55 isoelectric point, 38937 relative molecular mass and

2.23 ratio. NRAP, corresponds to spot-4, with 9.34 isoelectric point, 196716 relative molecular mass

45

and 2.82 ratio. RACK 1, corresponds to spot-5, with 7.60 isoelectric point, 35511 relative molecular

mass and 2.07 ratio (Table 1). The 3 spots significantly under-expressed in CGl corresponding to 3

proteins identified as follows: STI1, corresponds to spot-6 and it presents a 6.4 isoelectric point,

63170 relative molecular mass and 0.33 ratio; hnRNP L, corresponds to spot-7 spot, with 8.33

isoelectric point, 64550 relative molecular mass and 0.66 ratio; PGK1, corresponding to spot-8, with

8.02 isoelectric point, 44921 relative molecular mass and 0.4 ratio.

Only 2 spots were present in NCGl control group were aldose reductase, corresponds to spot-9, with

6.71 isoelectric point, 36052 relative molecular mass, and carbonic anhydrase, corresponds to spot-

10, with 6.45 isoelectric point, 30124 relative molecular mass.

The only spot present in the CGl was Citrate Synthase that corresponds to spot-11, with 8.72

isoelectric point and 51988 of relative molecular mass.

Expression proteomic profile of GL261 conditioned medium

Intending to further elucidate the involvement of adipose secretome in glioma, we evaluated the

profile of GL261 under conditioned medium (CGL). The medium proteome is a desirable sample due

to their accessibility and representability due, in part, to the wide dynamic range of protein

concentrations, which lead to the discovery of new protein markers (Figure 1). As illustrated in Figure

1 different secretion patterns of numerous growth-factors, adipokines, cytokines and angiogenesis-

related molecules, were found between the different media. Endocan, IGFBPs, FGF acidic, vascular

endothelial growth factor (VEGF), among many others were found upregulated in GL-261

46

conditioned medium. Nevertheless, a significant reduction in adiponectin, serpin E1 and TIMP-1

levels were observed in CGl (p≤ 0.01).

Figure 4 - Expression proteomic profile of GL261 with and without the effect of the secretome of 3T3-L1 mature

adipocyte cells [160].

47

Table 1 - Differential protein expression (> 2.0 fold up-regulated and < 0.5-fold down-regulated in CGl/NCGl) of a total of 192

protein spots analysed by MALDI-TOF-MS/PMF queries in Mascot database search engine [103].

Protein

spot

Mascot

MOWSE

score 1

N matches

(n queries)

% sequence

coverage2 Ratio3 Protein ID Function

Spot-14 69 14 (43) 14 2.32

(↑)

Replication factor C

subunit (RFC1)

Clamp loader in eurcarioric replication.

Role in maintaining the stability and the

integrity of the genome [103].

Spot-2 80 25 (122) 25 2.91

(↑)

Kinesin heavy chain

isoform 5C (KIF5C)

Kinesin motor protein present in motor

neurons and associated with trafficking of

mitochondria and vesicles [104,105].

Spot-3 102 20 (107) 52 2.23

(↑) Annexin A2 (ANXA2)

Phospholipid-binding intracellular protein.

Involved in anti-inflammatory and

anticoagulation, cell proliferation and

differentiation and apoptosis [106–108].

Spot-4 90 37 (109) 24 2.82

(↑)

Nebulin-related-anchoring

protein (NRAP)

Known for cytoskeletal stability, cell

migration and protein scaffolding [109].

Spot-5 125 14 (67) 53 2.07

(↑)

Guanine nucleotide-

binding protein subunit

beta-2-like 1 (RACK1)

Intracellular adapter protein in signal-

tranuction pathways. Influences cell

proliferation, migration, adhesion and

spreading and protein synthesis

[110,111].

Spot-6 100 18 (68) 35 0.33

(↓)

Stress-induced

phosphoprotein 1 (STI1)

Co-chaperone in Hsp70 and Hsp90

folding and protein transcription, signal

transduction and cell division. Associated

with cell proliferation and invasion [112].

Spot-7 93 17 (65) 35 0.66

(↓)

Heterogeneous nuclear

ribonucleoprotein L

(hnRNP L)

Central role in RNA metabolism. Also

implicated in cell proliferation and invasion

[113,114].

Spot-8 93 21 (128) 61 0.4 (↓) Phosphoglycerate Kinase

1 (PGK1)

Catayzes production one molecule of ATP

during glicose breackdown [115].

Spot-9 110 14 (72) 48 Only in

NCGl Aldose reductase (AR)

Metabolic reduction of glucose to sorbitol

and catalyzes reduction of ROS and lipid

peroxidation, increasing mutagenesis risk

[116,117].

Spot-

105 79 10 (54) 40

Only in

NCGl Carbonic anhydrase (CA)

Metalloenzyme participating in

respiration, calcification and acid-base

balance. Product of HIF-1α,

overexpressed in hypoxic solid tumors

[118,119].

Spot-

114 64 8 (58) 17

Only in

CGl

Citrate synthase,

mitochondrial (CS)

Catalyzes mytochondiral reactions and it

is considered to play role in cancer cells

metabolism regarding metabolic

reprogramming [120,121].

1 scores greater than 77 are significant for p<0.01

48

2 represents the percentage of the protein's sequence represented by the peptides identified in the MS run

3 Ratio = CGl/NCGl (CGl – conditioned medium from glioma, NCGl – non-conditioned medium from glioma)

4 results obtained by limiting the research to entries from Mus musculus. In this case, scores greater than 62 are significant

for p<0.01.

5 result obtained allowing up to 2 missed cleavages.

49

2.3. Discussion

In order to understand which adipocyte secreted proteins potentially influenced glioma tumor

behavior, it was analysed the protein expression of glioma cells conditioned medium obtained from

adipocytes cultures, by means of bidimensional polyacrylamide gel electrophoresis under denatured

conditions (2D-PAGE) followed by mass spectrometry analysis of the selected spots. These spots

were analysed by matrix assisted laser desorption ionization time-of-flight/mass spectrometry

(MALDI-TOF-MS). The non-conditioned glioma cells (NCGl) were used as the control. Findings

revealed lower expression of STI1, hnRNP L and PGK1 when glioma cells were exposed to mature

adipocytes conditioned medium. Regarding multiple effects of STI1, hnRNPs and PGK1 this under

expression might reflect that the adipokines secreted by mature adipocytes might be able to stop

some important pathways in carcinogenesis, complemented with remodeling the oxidative

metabolism toward the utilization of fatty acids substrates instead of glucose. This interpretation may

be further supported by the increased expression of mitochondrial citrate synthase. Also, proteome

array profiling reveals under expression of TIMP-1, an inhibitor of ECM degradation, Serpin E1 and

adiponectin significant downregulation in conditioned medium, supporting adipokine results of

carcinogenesis development by adipocytes.

Additionally, data revealed that aldose reductase and carbonic anhydrase were present only in

glioma cells and that their expression was completely blocked when glioma cells were exposed to

mature adipocytes conditioned medium. These results suggested that this blockage may reflect a

50

total inhibition of aldose reductase and carbonic anhydrase, possibly associated to oxidative stress

response prevention, inhibition of inflammation and cancer proliferation, angiogenesis and

carcinogenesis, which may suggest some sort of protective mechanism related to obesity.

Considering overexpressed proteins in conditioned medium, RFC1, KIF5C, ANXA2, NRAP, RACK1

results were supported by overexpression of almost all the proteins profiled by arrays, comprising

angiogenesis, growth, metabolic and hormonal functions. This analysis revealed that ANXA2

expression (2.23 fold) was more pronounced when glioma cells were exposed to CGl. This finding is

in agreement with previous studies that also revealed that ANXA2 is elevated in glioma, in which it

plays a central role in cell motility, migration, invasion and angiogenesis [106–108].

Moreover, and considering proteome array profiling, endocan, which is a proteoglycan secreted by

endothelial cells and upregulated by proangiogenic factors, appears to be overexpressed in tumour

vessels [122,123]. Considering its overexpression in CGl, it may explain the involvement of adipose

tissues in tumor aggressiveness. In addition, adipose tissue contains diverse cells types including

adipose stromal cells, which produce a myriad of angiogenic factors, VEGF, HGF, FGF-2, and TGF,

contributing to the increment of released angiogenic and growth factor cytokines.

Thru microarray analysis it were further identified factors released by adipocyte cells, and revealed

that several pro-inflammatory factors (IL6, IL11, LIF, PAI-1, TNF-α), metabolic markers (FGF-21,

IGFBPs), angiogenic growth factors (endocan, HGF, VEGF IGF-I), and hormones (leptin, resistin,

RBP-4) were secreted to the medium into a high extent, whereas TIMP-1, an inhibitor of ECM

51

degradation, SerpinE1, wich encodes Plasminogen Activator Inhibitor 1 (PAI-1), were

underexpressed on CGl. Adiponectin, as it was expectable, was significantly downregulated in CGl

medium when compared with NCGl. Adiponectin modulates inflammatory responses, energy

expenditure in the CNS and periphery, glucose regulation and fatty acid catabolism. Its circulating

levels inversely correlate with insulin resistance, metabolic syndrome, obesity and cardiovascular

diseases. Curiously, different hormones related with insulin resistance and obesity, including TNF-α

and IL6, downregulate adiponectin expression and secretion in fat cells in vitro [48,124]. Our results

on TNF-α (adipocytokine implicated in inflammation, apoptosis and cell survival), IL6 (related to

proliferation in human glioblastoma cases, leading to poor prognosis) and IL11, corroborate these

inverse relationship with adiponectin [125].

Endocan, as well as FGF-21, are extensively present along with pro-inflammatory and proangiogenic

molecules. It is associated with a vast number of pathological conditions where endothelium gets

highly vascularized cancers, making it a likely marker of the vascular growth and angiogenesis during

cancer evolution [126,127]. These data support our findings in which endocan is overexpressed in

CGl. Moreover, and regarding other growth factors, FGF-21, HGF and IGFBPs, they all were

overexpressed in CGl. Hepatocyte Growth Factor (HGF), a multifunctional trophic factor activates a

tyrosine kinase signaling cascade, is elevated in obese adults and adolescents. In vitro, HGF

secretion from adipocytes of obese compared to lean individuals is greater [48,128].

52

Development and basic functions of organs and tissues relay on angiogenesis, and angiogenesis

role in adipose tissue development and obesity is crucial. Additionally, a great number of studies

point that an insufficiency in adipose tissue angiogenesis contributes to metabolic disease in obesity

[56,129]. These data from the literature reinforce our findings concerning the effect of conditioned

medium from adipose tissue in augmenting GL261 cell proliferation, migration, ability to invade and

decreasing apoptosis.

Figure 5 - Proposed classification of molecules analysed by microarrays considering its cellular and molecular functions.

according to over and underexpression in different contexts [160].

53

CHAPTER THREE

Role of inflammation and angiogenesis in

gliomagenesis in obesity:

a proteomic and immunhistochemical approach

54

Adipose tissue is defined as a lipid store depot, inflammatory and endocrine organ. Obesity chronic

low-grade inflammation is potentiated by the presence of multiple cytokines and hormones. Then, in

this chapter is presented proteomic analysis of angiogenic cytokines expressed by in vitro model

followed by immunohistochemical analysis on the expression of some relevant (and available)

angiogenic proteins on the in vivo C57Bl/6J diet-induced obesity adipose and tumoral tissue.

Presented data have already been submitted and is under revision as it follows:

J. Almeida, P. Coelho, R. Soares, C. Prudêncio, R. Fernandes, Proteomic alterations in xenografted

mice with GL261 tumor cells reveal obesity-associated inflammation in gliomagenesis.


Cell culture

3T3-L1 pre-adipocytes were propagated and allowed to reach confluence. After differentiation, the

conditioned medium rich in adipokines which are globally referred as secretome. Afterwards, glioma

cells were divided in two distinct groups: conditioned glioma cells (CGl) and non-conditioned glioma

cells (NCGl).

Syngeneic mouse model of glioma

GL261 cells were harvested after trypsinization, washed in DMEM without FBS and implanted in

male C57Bl/6J (Jackson Laboratories, Bar Harbor, ME, USA). The experiment consisted of four

groups of a total amount of 32 male mice, organized in controls and glioma induced for ND and HFD,

55

equally distributed. The ND group, which consisted of wild-type male C57BL/6J mice fed a

standardized low-fat diet (4% by weight, D12450B-I, Research Diets, New Brunswick, NJ, USA) from

age 3 weeks onward; HFD group, which consisted of wild-type male C57BL/6J mice fed a

standardized high-fat diet (35% fat by weight, D12451-i, Research Diets) from age 3 weeks onward.

Mice were grown in rooms with controlled temperature and humidity under a 12-hour light-dark cycle,

with water and food ad libitum, and weighed daily to ensure that all the animals were gaining weight

in an equivalent manner. Animals received a subcutaneous cell injection, approximately 107 per

animal, in dorsal region and 14 days after that, they were euthanized at the whole timepoint of 182

weeks. This study was approved by the Portuguese National Authority for Animal Health.

Protein microarrays

Determination of relative levels of pre-selected mouse adipokines in conditioned and non-conditioned

GL261 cell cultures was performed by using Mouse Adipokine Array Kit (R&D Systems Europe, UK).

Cells were rinsed with PBS before Lysis Buffer solubilization at, approximately, 1 x 107 cells/mL.

Then, they were ressuspended and rocked at 4°C for 30 minutes, following a microcentrifugation at

14,000 x g for 5 minutes. After supernatant transfer into a clean test tube, proteins were quantified a

total protein assay. Array Procedure was performed according to manufacture instructions

(Catalogue Number ARY024). Data analysis was based on pixel densities on developed X-ray film

collected and analysed using Gel-Doc XR (Bio-Rad© Laboratories), acquired by Image Lab Software

5.2.1 (Bio-Rad© Laboratories) then quantified by ImageJ 1.49 (NIH, USA).

56

Immunohistochemistry on FFPE tissue sections for IL1β, IL6, Ob, PDGFA and TNFα

Immunohistochemistry of inflammatory citokines previously identified on microarray profiling we

selected IL1β, IL6, Ob, PDGFA and TNFα. Immunohistochemistry was performed with TNFα: ab6671

rabbit anti-mouse polyclonal antibody (Abcam plc, Cambridge, UK) and IL6: ab6672 rabbit anti-

mouse polyclonal antibody. IL1β (sc-52012), Ob (sc-842) and PDGFA (sc-7958) rabbit anti-mouse

polyclonal antibody (Santa Cruz Biotechnology, Inc., EUA). They were incubated with biotinylated

goat anti-rabbit secondary antibody IgG-B: sc-2040 (Santa Cruz Biotechnology, Inc., EUA).

VECTASTAIN Elite ABC Kit (Vector Laboratories, Inc., EUA) and 3,3′-diaminobenzidine (Abcam plc,

UK) were used for signal amplification. Quantification was performed through pixel density evaluation

of 7–10 randomly selected tissue sections per mouse, acquired using Nikon Microscope.


The differences in expression of proteins between glioma medium and CGl by Proteome Profile

Microarray and IHC data was analysed by 2-way ANOVA for multiple comparisons with Prism

Graphpad. P-values of <0.05 were considered statistically significant.

57

3.2. Results

In order to further enlighten this study, it was analysed the proteome profile of a cellular model of

brain cancer GL261 conditioned by adipocyte secretome, as well as immunohistochemical

expression on adipose and tumoral tissue. So, it will be approached by focusing cytokines

relationship within inflammation and angiogenesis.

Proteomic alterations in glioma exposed to adipocyte secretome

Figure 7 - Angiogenesis microarray expression on 3T3-L1 conditioned glioma cells: cytokines with relevant functions but no

statistical significance, regarding tumor development and migration. NCGl in white and CGl in black.

* * * * * * *

R e fere

n c e Sp o ts

A D A MT S 1

C o a g u lat io

n Fa c to

r II I

R e fere

n c e Sp o ts

E n d o s tat in

/Co lla

g e n XV III

P D GF -A

A

P A I-1

N e g a t ive C

o n trol

0

1 0 0

2 0 0

3 0 0

Pro

tein

de

ns

ity

(p

ixe

ls)

Figure 6 - Angiogenesis microarray expression on 3T3-L1 conditioned glioma cells: cytokines over and under-expressed

significantly (p<0.05) on CGl. NCGl in white and CGl in black. There is a significant decreasing (p<0.05) in ADAMTS1,

Coagulation Factor III, Endostatin and PDGF-AA in CGl. PAI-1 levels increased significantly in CGl.

F GF a

c idic

F GF b

a s ic

IGF B P -1

IGF B P -2

IGF B P -3

IL1 a

IL1 ß

IL1 0

L e p t in

MM

P -9 (p

ro a

n d ac t iv

e form

)

Os te

o p o n tin

T h rom

b o s p o n d in-2

T IMP -1

T IMP -4

V E GF -A

V E GF -B

0

1 0

2 0

3 0

Pro

tein

de

ns

ity

(p

ixe

ls)

60

IL 1 *A T

D ie t

Pro

tein

den

sity

(p

ixel

s)

N DH F D

0

5

1 0

1 5

2 0

2 5C o n tro l

G liom a

IL 1 * T u m o r a l

D ie t

Pro

tein

den

sity

(p

ixel

s)

N DH F D

0

1 0

2 0

3 0

4 0C o n tro l

G liom a

IL 6 * T u m o r a l

D ie t

Pro

tein

den

sity

(p

ixel

s)

N DH F D

0

2 0

4 0

6 0

8 0

1 0 0C o n tro l

G liom a

IL 6 *A T

D ie t

Pro

tein

den

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(p

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s)

N DH F D

0

2 0

4 0

6 0

8 0C o n tro l

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P D G F A *A T

D ie t

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(p

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N DH F D

0

2 0

4 0

6 0

8 0C o n tro l

G liom a*

*

O b * T u m o r a l

D ie t

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tein

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(p

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N DH F D

0

1 0

2 0

3 0

4 0C o n tro l

G liom a

O b *A T

D ie t

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tein

den

sity

(p

ixel

s)

N DH F D

0

5

1 0

1 5

2 0

2 5C o n tro l

G liom a

P D G F A * T u m o r a l

D ie t

Pro

tein

den

sity

(p

ixel

s)

N DH F D

0

2 0

4 0

6 0C o n tro l

G liom a

*

*

T N F * T u m o r a l

D ie t

Pro

tein

den

sity

(p

ixel

s)

N DH F D

0

2 0

4 0

6 0

8 0C o n tro l

G liom a

T N F *A T

D ie t

Pro

tein

den

sity

(p

ixel

s)

N DH F D

0

2 0

4 0

6 0

8 0C o n tro l

G liom a

Figure 7 - Immunohistochemistry semi-quantitative data analysis for IL1β, IL6, Ob (Y-20), PDGFA and TNF-α, retrieved from

C57Bl/6J mice according to diet and tumoral or non-tumoral grouping. ND – normal diet; HFD – high-fat diet. Black column

regards control group, while grey column respects experimental group.

61

3.3. Discussion

Inflammatory and angiogenic pathways in glioma: a cellular and an animal model

Adipose tissue is defined as a lipid store depot, inflammatory and endocrine organ [130]. Thus, it

provides a chronic low-grade inflammatory condition potentiated by the presence of multiple

cytokines and hormones, sometimes associated with disease. Most part of these factors are known

to play a role in cell proliferation, apoptosis, migration and invasiveness, as well as adhesion

capacity, becoming expected to be overexpressed in conditioned medium. Recent findings point that

inflammatory, angiogenic and metabolic factors, significantly influence tumor behavior [131,132].

This study analysed the proteome profile of a cellular model of brain cancer GL261 under the

presence of adipocyte secretome, as well as immunohistochemical expression on adipose and

tumoral tissue. This will be approached by focusing cytokines relationship within inflammation and

angiogenesis.


Intending to further elucidate the involvement of angiogenic secretome in glioma, it was evaluated

the profile of GL261 under conditioned medium (CGl). The medium proteome is a desirable sample

due to their accessibility and representability due, in part, to the wide dynamic range of protein

concentrations, which lead to the discovery of new protein markers (Figure 6).

62

As illustrated, numerous proteins with many distinct roles presented different secretion patterns in

the two media. Plasminogen Activator Inhibitor 1 (PAI-1) was found upregulated (p<0,05) in GL-261

conditioned medium, supporting the pro-inflammatory status in HFD condition. Nevertheless, a

significant reduction in coagulation factor III, endostatin, an anti-angiogenic adipokine, PDGFA,

biomarker for glioblastoma multiforme and metastazation, as well as ADAMTS1, inflammatory in

CNS and anti-angiogenic adipokine, were observed in CGl (p≤ 0,05).

It was also observed non-significant underexpression of CYR61, a pro-inflammatory and cell

adhesion involved molecule, as well as CXCL16, an angiogenic adipokine. However, it was also

observed non-significant overexpression on CGl of TIMP-1 and thrombospondin-2, both anti-

angiogenic adipokines. These data suggest a leaning for an angiogenesis inhibition in GL-261

conditioned medium, at least in an early stage.

Morphologic analysis

On adipose tissue, it can be observed bigger adipocytes on HFD tissue sections when compared to

ND samples. On tumoral tissue, it can be observed a more inflammatory cellular response when

comparing with adipose tissue, with fibrotic cellular compounds, as well as a necrotic response.

Furthermore, glioma tumoral tissue reveals a morphologic structure that mimics lung tissue, which

can be explained by the shared embryonic origin. This phenomenon has already been described in

melanoma cells under adipocytes influence [133].

63

Immunohistochemical evaluation of IL1β, IL6, Ob, PDGFA and TNFα on adipose

and tumoral tissue of syngeneic C57Bl/6J mice

In immunohistochemistry evaluation it was studied previously evaluated and significantly upregulated

proteins with angiogenic and metabolic relevance- IL1β, IL6, Ob (Y-20), PDGFA and TNFα, in

adipose and tumoral tissue (Figure 7 and 8). On adipose tissue, it was observed overexpression of

IL6 (non-significant), Ob (n.s.) and TNFα (p<0,05) in glioma HFD when compared with glioma ND.

The opposite was observed regarding IL1β (n. s.) and PDGFA (p<0,05).

However, on tumoral tissue results were quite different. It was not observed a significant variation in

glioma group in IL6, however it was perceived a tendency to underexpression of TNFα in glioma

HFD, and a significant underexpression of PDGFA. Contrariwise, IL1β and Ob (Y-20) tend to be

overexpressed in glioma HFD.

IL1β is underexpressed in glioma HFD adipose tissue but overexpressed in glioma HFD tumoral

tissue. The same behaviour was observed in IL6 analysis, though almost imperceptibly. In the

cellular assay it was also observed both proteins overexpressed in CGl conditioned medium. The

first finding may be related with the similar result regarding IL6, as far as it is known that IL1β is

secreted by microglial cells post Il6 secretion contributing for tumour development [10,134]. So, as

far as it can be seen, IL1β behaviour could be related to IL6 one. Nonetheless, on tumoral tissue it

may be observed non-significant overexpression of IL1β in glioma HFD group, which may be related

with tumoral capacity for proliferation and invasion. Even though IL1β role in glioblastoma is

64

controversial, its overexpression was reported to upregulate MMP-9 overexpression and subsequent

enhancement of glioblastoma cell lines invasiveness [10].

Regarding Ob (Y-20), it was expected an overexpression on both tissue depots as far as it contributes

to glioma development by enhancing cell growth and survival through activation of anti-apoptotic and

migration pathways [135]. Furthermore, Ob receptor is associated with tumour malignancy degree.

PDGFA, involved in proliferation and metastasisation, as well as being a VEGF regulator, was

significantly underexpressed in CGl and both tissue depots, in HFD glioma group, corroborating

cellular array assay data. This data suggests that it may be underexpressed in some initial phase,

supporting the theory of delayed tumour proliferation due to potential hypoxia and pro-inflammatory

response. Regarding VEGFs, in CGl a slightly underexpression of VEGF-A and VEGF-B was

observed, data colluding with PDGFA levels [81,136].

Concerning TNFα, its underexpression in the tissue under HFD may be an unexpected result looking

at the literature. Thus, there is published data reporting different adipokines patterns according to

adipose depots in humans versus rodents [47,137]. In this study it was analysed subcutaneous and

visceral adipose tissue were analysed as a whole, because C57Bl6/J are mice with a reduced body

volume when compared with other rodents, and the main source of adipose tissue in HFD group is

visceral. This observation leads us to the first hypothesis for these results: in diet-induced obesity it

may be observed a cell response in the first place, however hypertrophy and hypoxia may condition

pro-inflammatory state. Supporting this idea, it is seen PAI and IL6 overexpression in CGl, both

65

described as the first inflammatory adipokines modified in the cancer model. So, maybe only in a

later stage of proliferation and metastasisation it is observed overexpression of TNFα.

Furthermore, there is the hypothesis based on Mathias Blüher theory that TNFα effect may be mainly

paracrine, which may explain why this adipokine may not be ideal target for metabolic marker in

obesity [30].

Moreover, it is known that adipocytes in obese state secrete IL6, HGF and MMPs envisaging

proliferation and metastasisation. Even though not significantly, it was observed underexpression of

these proteins in CGl, which supports ongoing theory of delayed proliferation. Only osteopontin,

which is associated with invasive behavior in breast cancer, is slightly overexpressed in CGl [136].

In conclusion, results obtained from proteomic cellular and tissue response under obese and tumoral

environment, can be roughly summarized as it follows.

Table 2 - Concluding remarks of cellular and histological common findings. ↗ meaning increasing, ↘ meaning decreasing of

reported levels. CGl – conditioned medium from glioma; GL261 – glioma cells; HFD – high-fat diet.

IL1β IL6 Y-20 PDGFA TNFα

GL261 CGl ↗ ↗ ↗ ↘ ↗

Tumoral tissue HFD ↗ ↘ ↗ ↘ ↘

66

67

CHAPTER FOUR

Sphingolipid rheostat dysfunction:

a biochemistry and sphingolipidomic approach

68

In the last years, sphingolipids, especially sphingosine-1-phosphate and ceramides, have been more

thoroughly studied due to its bioactive functions. They can act as regulatory molecules in

inflammation, angiogenesis, apoptosis, among many other processes related to metabolic syndrome

and/or cancer, either as signal transducers or therapeutic targets.

In this chapter it is approached the sphingolipid rheostat by establishment of the relationship of

relevant sphingolipids, as S1P and ceramide, with C57Bl/6J diet-induced obesity tissue response.

Presented data have already been submitted and is under revision as it follows:

J. Almeida, B.Macedo, R. Soares, C. Prudêncio, R. Fernandes, Sphingolipid rheostat dysfunction in

obese mice: a lipidomic approach.


Tandem Mass Spectrometry for Ceramide and S1P

Quantification of the individual species (C14, C16, C18, C18: 1, C20, C20: 1, C24, C24: 1) is

achieved by multiple reaction monitoring (MRM). In MRM, the first mass analyzer is configured to

pass a specific mass / charge ion precursor (m / z), while the second analyzer is configured to allow

the passage of a particular ionic product m / z. Thereafter, only ions simultaneously encountering

both the precursor and ionic product m / z conditions are transmitted to the detector. Additionally,

and in conjunction with liquid chromatography and suitable internal standards, MRM provides more

accurate quantitative results by answering critical questions regarding ionization suppression, ion

69

dissociation kinetics and sensitivity. Calibration curves are created by adding varying amounts of

sphingolipid patterns and a constant amount of internal sphingolipid standard to the lipid matrix

control extracts, which were then transported throughout the isolation and purification procedure.

The sphingolipid response rate to the internal standard is calculated, and the sphingolipid

concentration is determined by comparison with the calibration curve. The areas under quantification

peaks are determined by the ion chromatograms obtained.

Extraction of sphingolipids from mice plasma

The plasma homogenate was fortified with 50 μL of standard internal solution, and the supernatant

corresponding to the lipid organic fraction was evaporated to dryness using nitrogen gas.

Subsequently, the residue was resuspended in 150 μL of Mobile Phase A and transferred to the

UPLC auto-sampler vial.

Quantification of sphingolipids by UPLC-MS / MS of mouse plasma

Separations were obtained with the ACQUITY UPLC BEH C18 1.7 micrometer 2.1 x 150 mm column.

All analysis were performed using the Waters ACQUITY UPLC ™ System with a Quattro Premier ™

XE Triple Quadrupole (Waters Corporation, Milford, MA) equipped with Electrospray Ionization (ESI)

probe. Succinctly, 10 μL of the resuspended extracts were injected onto a reverse phase C18 column

and eluted at 0.250 mL per minute using a linear gradient of 30% methanol solution (70% H2O) at

100% methanol for 3 minutes.

70

Clinical biochemical markers

Data related to uric acid, glucose, lipoproteins (triglycerides, total cholesterol, HDL, VLDL, LDL) and

hepatic enzymes (AST, ALT and ALP) on C57Bl/6J serum was analysed by means of

spectrophotometric assay using CORMAY Liquick Cor Kits (PZ CORMAY S. A., Polska). Uric acid,

glucose, total cholesterol, HDL, VLDL and LDL levels were accessed by sequential enzymatic

reactions with development of a colored product, which intensity is proportional to serum

concentration. Hepatic enzymes activity (AST, ALT and ALP) were measured by the rate of

absorvance at a reference wavelength, which is directly proportional to enzymatic activity.

Histochemistry of hepatic tissue: Haematoxylin-Eosin and Sirius Red

Liver samples were embedded in paraffin after standard processing and cross-sectioned into 3 μm-

thick sections. Afterwards, they were stained for routine Haematoxylin-Eosin and Sirius Red staining.

Sirius Red was performed with Sirius Red solution dissolved in acqueous saturated picric acid.

Photos were acquired with Nikon Microscope.


Data was analysed for Spearman correlation test by SPSS and t-test. Spearman correlation test rank

variables between -1 and 1, considering it a negative or positive correlation, respectively, and with 0

as reference point. P-values p<0.05 or p<0,01 were considered statistically significant.

71

4.2. Results

Sphingolipids alterations in obese mice

Regarding adipose tissue buildup, due to a fat diet or metabolic dysfunction, adipocytes can trigger

an inflammatory reaction. In addition to adipokines’ expression, it is of great interest to study the type

of circulating lipids, being obesity a suitable model for this effect. Sphingolipids constitute an

important class of bioactive compounds. Lipids such as ceramides, sphingosines and sphingosines-

1-phosphate (S1P) are important in molecular signaling and therefore play a key role in the regulation

of various cellular events, such as cell growth, differentiation, stress responses and apoptosis.

Therefore, it was proposed to study the involvement of the Cer / S1P signaling pathway in obesity.

Figure 8 - C57Bl/6J serum ratio levels of ceramide (stripes) and sphingosine-1-phosphate (points) depending on diet: normal

diet (ND) and high-fat diet, on 3 timepoints: 12, 16 and 20 weeks old. * significant for p<0,05.

0,0

0,5

1,0

1,5

2,0

2,5

3,0

12 wk 16 wk 20 wk

HF

D / N

D (

x fo

ld)

*

*

72

Table 3 - Correlation between uric acid, glucose and lipoproteins levels and ceramides and sphingosine-1-phosphate serum

levels on C57Bl/6J at 20 weeks.

* significant for p<0,05.

** significant for p<0,01.

Presented results highlight a positive correlation between uric acid levels and diet, as well as a

negative correlation with ceramide C22:0 and C24:0. Glucose correlates positively with S1P, but also

correlates negatively with C20:0, C22:0 and C24:0. Regarding lipid profile, it can be observed a

positive correlation between TAGs and age, diet and C16:0 and C24:0. On the other hand, it

correlates negatively with C18:0 and S1P. VLDL correlates positively with diet and C16:0. LDL has

Uric acid Glucose

Lipid profile

TAGs CholT VLDLc LDLc HDLc

Age rho -,184 -,267 ,597

** ,122 ,044 -,309

* ,192

p-value ,210 ,066 ,000 ,410 ,765 ,032 ,190

Diet

rho ,352* ,280 ,457

** ,866

** ,854

** ,806

** ,866

**

p-value ,014 ,054 ,001 ,000 ,000 ,000 ,000

Ceramides

Total

rho -,262 -,718** ,214 ,157 ,013 -,102 ,120

p-value ,072 ,000 ,144 ,287 ,931 ,490 ,417

C16:0 rho ,221 ,118 ,687

** ,819

** ,771

** ,560

** ,853

**

p-value ,131 ,424 ,000 ,000 ,000 ,000 ,000

C18:0 rho ,261 ,174 -,432

** ,014 ,037 ,374

** -,092

p-value ,074 ,236 ,002 ,924 ,801 ,009 ,532

C20:0 rho -,213 -,791

** ,240 ,039 -,149 -,231 -,041

p-value ,146 ,000 ,100 ,794 ,313 ,115 ,785

C22:0 rho -,290

* -,342

* ,086 ,217 ,201 ,057 ,276

p-value ,046 ,017 ,560 ,139 ,170 ,699 ,058

C24:0 rho -,305

* -,643

** -,018 ,088 -,006 -,043 ,065

p-value ,035 ,000 ,901 ,552 ,969 ,771 ,660

C24:1 rho -,121 -,225 ,375

** ,301

* ,254 ,030 ,342

*

p-value ,414 ,123 ,009 ,038 ,082 ,840 ,017

Sphingosine 1-phosphate rho ,266 ,579

** -,323

* -,162 -,049 ,159 -,169

p-value ,068 ,000 ,025 ,271 ,739 ,279 ,252

73

a positive correlation with diet, C16:0 and C18:0, and correlates negatively with age. HDL reveals

positive correlation with diet, as well as C16:0 and C24:1.

As it can be observed in the graphic below, all the above-mentioned parameters’ serum levels

increase in a progressive, but not always statistically significant.

Figure 9 - Lipid profile analysis. C57Bl/6J serum ratio levels of triglicerydes (white), total cholesterol (black), HDL (stripes),

VLDL (dots) and LDL (squares) depending on diet: normal diet (ND) and high-fat diet (HFD), on 3 timepoints: 12, 16 and 20

weeks old. * significant for p<0,05.

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

12 wk 16 wk 20 wk

HF

D / N

D (

x fo

ld)

*

*

74

Table 4 – Correlation between different hepatic enzymes activity levels and ceramides and sphingosine-1-phosphate serum

levels on C57Bl/6J at 20 weeks.

* significant for p<0,05.

** significant for p<0,01.

Hepatic function analysis presents a strong positive correlation between AST and diet, C16:0 and

C24:1. The same correlation can be observed regarding ALT levels. It can also be observed a

positive correlation between ageing and S1P serum levels.

As it can be observed in the graphic, AST and ALT reveals a progressively increasing through time.

ALP has no significant variation, not only with the aforementioned parameters, as also with ageing.

Age Diet AST ALT ALP

Age rho

,000 ,155 ,251 -,267

p-value 1,000 ,294 ,085 ,066

Diet rho ,000

,863

** ,866

** -,053

p-value 1,000 ,000 ,000 ,722

Ceramides

Total rho ,239 ,098 -,012 ,186 ,022

p-value ,102 ,509 ,934 ,205 ,880

C16:0 rho ,478

** ,878

** ,832

** ,881

** -,174

p-value ,001 ,000 ,000 ,000 ,237

C18:0 rho -,956

** ,098 -,043 -,097 ,213

p-value ,000 ,509 ,771 ,511 ,147

C20:0 rho ,120 -,098 -,157 ,054 -,053

p-value ,418 ,509 ,287 ,715 ,721

C22:0 rho ,359

* ,293

* ,150 ,248 ,109

p-value ,012 ,043 ,310 ,089 ,460

C24:0 rho ,000 ,098 -,089 ,074 ,137

p-value 1,000 ,509 ,548 ,619 ,353

C24:1 rho ,717

** ,293

* ,320

* ,417

** -,047

p-value ,000 ,043 ,027 ,003 ,751

Sphingosine 1-phosphate

rho -,598**

,000

-,098

,509

-,078

,596

-,250

,086

,033

,826 p-value

76

4.3. Discussion

High-fat diet decreases sphingosine-1-phosphate

Metabolism of sphingolipids can be activated in different situations, such as by pro-inflammatory

cytokines, growth factors and oxidative stress, which characterize the environment in which the

adipose tissue is found in obesity. Lipidomic studies of ceramide and S1P sphingolipids in C57Bl/6J

mice plasma, displayed agreement with some other of the analysed parameters.

Ceramide is characterized by being a biomolecule which, in certain intracellular levels, presents anti-

proliferative and pro-apoptotic power. Its pro-apoptotic function is related to the activation of signaling

cascades involving activation of caspase by cytochrome C [87,88]. According to presented data,

ceramide serum levels ratio increased progressively over time. Giving existing information on this

sphingolipid, the secretion of pro-inflammatory cytokines and the increase of fatty acids in obesity

may justify the increase of the ceramide levels observed up to 20 weeks. That is, the inflammatory

state observed at this stage triggered anti-proliferative and anti-apoptotic effects by increasing the

levels of plasma ceramide. Moreover, HFD promotes an increase of body weight and a myriad of

systemic cytokines levels, as well as an almost completely suppression of ASMase activities on

adipose tissue. In this sequence, ASMase inhibition reduces insulin receptor sensitivity, which

constitutes plausible evidence of ceramide biosynthesis playing an important role in metabolic

syndrome, as well as in obesity development. Furthermore, clinical studies reveal that reduction of

77

ceramide levels correlate with weight loss, insulin sensitivity improvement as well as decreasing of

TNF-α concentration [138,139]. This data correlates not only with results obtained from serum

ceramide, but also with increased expression of TNF-α in adipose tissue from adipose tissue of

glioma induced mice. Other studies involving mice also reported an increase, although not very high,

in total serum and tissue ceramide levels. They also observed that the consistently elevated

concentration of TNF-α in obesity increases the biosynthesis of ceramide, which contributes to

several distinctive changes of the metabolic syndrome [140]. Contributing to these contradictory

results, it has been described an increase in pro-inflammatory cytokines, such as TNF-α and IL6, by

induction of S1P and not by ceramide [89]. Although not significantly, it was observed a minor

decreasing in TNF-α tumoral tissue expression, supporting the theory of these cytokines being

induced by S1P and not ceramide, as well as that sphingolipids produced at different locations may

have different functions. This may be a plausible explanation for the observed differences between

adipose tissue and blood plasma results [141].

S1P reveals a slight decrease in serum levels over time, and correlates negatively with age (table

3), which may be due to the advanced inflammatory state and subsequent hypoxia, in which the

proliferative and angiogenic capacity of the adipocytes tries to overlap the pro-apoptotic effect of

ceramide. S1P is involved in cell proliferation and survival, angiogenesis and vascular maturation,

tumorigenesis and inhibition of apoptosis induced by ceramide, TNF-α and others toxic agents

[87,97,142,143]. Its relationship with proliferation also explains the strong negative correlation with

78

age. S1P is secreted by a myriad of cells, some of them present in adipose tissue stroma. Cellular

proliferation promoted by S1P occurs through effectors such as phospholipase C, adenylate cyclase,

extracellular signal regulation kinase (ERK), among others [88]. However, some studies performed

with cells transfected with S1P phosphatase revealed increased cell death simultaneously with

increased levels of ceramide and sphingosine [143]. So, data here presented, in which mice

undergoing ND have higher S1P levels than HFD mice may be related to the advanced hypoxic state

previously mentioned, not allowing proliferative and anti-apoptotic purposes of S1P. Thus, perhaps

in a later evaluation, the inflammatory process could already have been triggered, promoting the

angiogenic process and, consequently, increasing S1P levels in a low-grade inflammatory state that

is obesity. Regarding the regulation of cell migration, the S1P2 receptor is a powerful inhibitor of

S1P-induced cell migration, as well as tyrosine kinase receptors, such as IGF-1 and PDGF. Analysis

unveiled on this thesis until this point revealed underexpression in PDGF-α in adipose and tumoral

tissues, adding to decreasing in S1P serum levels. These may suggest activation of different

receptors in obesity and would also require further studies for clarification.

The observed inverse tendency of both ceramide and S1P is in line with previously and widely

described information on the antagonistic functions of these sphingolipids. Moreover, obtained data

manifestly supports the concept that clinical animal model of obesity should be older than 20 weeks

years old for undoubtful observation of relevant clinical features.

79

Hepatic enzymes and lipoproteins levels increase with obesity

The lipid profile and hepatic function analysis displayed a predictable behavior of these factors

according to obesity low-grade inflammatory state and metabolic syndrome in general.

On a broader analysis, and regarding uric acid, glucose and lipoproteins (Figure 11) – triglycerides

and cholesterol -, it is noticeable a continuously increment in its levels throughout time, except for

HDL, which suffers a decrease characteristic from the negative impact of obesity progression. As it

has been already described, obesity and possible development of obesity related problems regarding

the liver, namely non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver disease (NAFLD)

promote an increase in FFA circulating levels, either in children/ adolescents and adults [144,145].

These FFA stimulate β-oxidation and mitochondrial excessive ROS production. These sequence of

events not only contributes to hepatic accumulation of TAGs, as well as LDL and VLDL vascular

deposition. Concerning uric acid, it is a product from cell death resulting from nuclear degradation or

cellular destruction, and a final product of purine metabolism, exerting pro-inflammatory and pro-

oxidant effects on adipose tissue. Furthermore, cumulative levels of FFA in the liver contributes to

de novo synthesis of purines, which increases its serum levels, as well as activity of hepatic enzymes,

namely AST, ALT. On the opposite, ALP expresses a slightly, but not significant, decrease on the

last timepoint (Figure 12). These enzymes, among many others, are related with metabolic

syndrome, type 2 diabetes, insulin resistance and cardiovascular diseases, acting as metabolic

syndrome predictive factors. Despite AST not being a specific marker for liver function it is present

80

on other relevant tissues under the metabolic and cardiovascular point of view, such as

musculoskeletal tissue [146,147].

On a closer analysis, it can be observed a positive correlation between uric acid and diet, and a

negative correlation with C22:0 and C24:0. Also glucose presents a positive correlation with S1P,

but negatively with C20:0, C22:0 and C24:0. Taken together, these data reinforces the previously

mentioned relationship between excessive lipids’ intake and FFA circulating levels, which contribute

not only to mitochondrial dysfunction, but also act as a signal that tumoral tissue (and surrounding

cells) consume high levels of glucose (as its main fuel), decreasing its serum levels (table 2).

Moreover, and regarding ageing and lipid profile, TAG, VLDL and LDL levels correlate positively with

diet and C16:0 as these parameters relate directly with dietary excessive intake of lipids.

Furthermore, hepatic function analysis presents a strong positive correlation between AST and ALT

(in a lesser extent) and diet, C16:0 and C24:1, which emphasizes the idea of hepatic activity overload

due to excessive dietary intake. Taken together, and considering published results, these data

discloses a potential for C16:0 and C24:1 to be used as early clinical biomarkers for low-grade

chronic inflammation [148]. Last, but not least, ageing and S1P serum levels correlation supports the

fact that proliferation and cellular activity in general decreases along time.

Data retrieved from sphingolipid analysis, as well as hepatic function supports the idea of worsening

state of general metabolic condition along with the progression of obese low-grade inflammatory

state. This response is more visible and immediate than tissue response, which may be related to

81

the fact that serum levels increase almost directly in response to FFA from a diet rich in saturated

fats, largely exceeding adipocytes storage capacity and accumulating in hepatic and other

metabolically active tissues.

Morphologic analysis

On hepatic tissue (Figure 13) from ND group, it can be observed regular hepatocytes morphology,

as well as absent fibrosis. On the contrary, on hepatic tissue from HFD group it is observed extensive

fat necrosis, also designated as hepatic steatosis, which is compatible with our enzymatic (increased

ALT and AST serum levels) and lipid profile (increased TAGs and VLDL, among others).

Furthermore, it is visible fibrosis around blood vessels, which is expected according with increased

FGF expression observed in proteomic assays.

82

83

CHAPTER FIVE

General discussion and Future perspectives

84

Expansion and elementary purposes of organs and tissues relay on angiogenesis, and angiogenesis

role in adipose tissue development and obesity is crucial. Besides, a great number of studies point

that an insufficiency in adipose tissue angiogenesis contributes to metabolic disease in obesity.

Understanding and defining the mechanisms involved in glioma development and progression, even

more in obesity epidemics, may enable the identification of novel therapeutics targets for an earlier

intervention, as long as modifications are likely to take place at the gene, and then, at the protein

level. The results presented in this thesis provide further insights into this matter, but also unclutter

intriguing questions worth to be explored in the future.

In a first approach, to understand which adipocyte secreted proteins influence glioma tumor behavior,

conditioned medium of glioma cells was analysed by bidimensional polyacrylamide gel

electrophoresis under denatured conditions (2D-PAGE) followed by matrix assisted laser desorption

ionization time-of-flight/mass spectrometry (MALDI-TOF-MS). MALDI-TOF and proteomic array

results suggest that, on an initial phase, obesity contributes for diminishing the tumor power to initiate

its development. Even though, as time goes by, studied growth factors positively influenced

proliferation and restrict tumorigenic growth. However, decreased levels of growth and hypoxic

factors, and different metalloproteases, suggest that previous tumor proliferation due to obesity is

restricted and confined, not metastizing. Thus, presented data reinforce the idea on obesity

protecting against glioma initiation and metastization. Moreover, it is defended the hypothesis that

85

under an obesity environment, glioma cells may reshuffle their metabolic patterns towards increased

lipid consumption for aerobic ATP production over glucose and other simple sugars.

Then, microarray analysis allowed identification of several factors released by adipocytes with

characteristic functions, such as pro-inflammatory, metabolic, angiogenic growth factors and

hormones, which are secreted to the medium into a higher or lesser extent. Whereas PAI-1 was

overexpressed on CGl, ADAMTS1, Coagulation Factor and PDGFA were underexpressed on CGl.

In situ evaluation by immunohistochemistry reveals some controversial results as it was observed

Il1β and IL6 underexpression in glioma HFD adipose tissue but overexpression in glioma HFD

tumoral tissue; PDGFA significant underexpression in CGl and both tissue depots, in HFD glioma

group; TNFα underexpression in the tissue under HFD, and Ob (Y-20) overexpression on both tissue

depots, this one, an expected result. So, data from the literature reinforce these findings on the effect

of conditioned medium from adipose tissue in GL261, as long as it was observed an early impediment

to cell proliferation and migration, due to a delayed pro-inflammatory response. Similar behavior was

observed on adipose tissue (in vivo), suggesting that initial hypertrophy delays the angiogenic and

inflammatory major response. Nevertheless, tumoral tissue had a slightly opposite behavior,

displaying an augmentation on inflammatory response and cell proliferation, suggesting a more

efficient local angiogenic response to hypoxia and evasion to apoptosis.

Moreover, lipidomic studies in the obesity mice model allowed to detect some significant differences

in the Cer and S1P serum levels, within agreement with some other of the analysed parameters.

86

According to these results, ceramide serum levels in normal diet mice decreased progressively over

time, whereas that HFD mice increased through time. These data, along with lipidic and enzymatic

activity profile and pro-inflammatory cytokines secretion may justify the increase of the ceramide

levels observed up to 20 weeks. That is, the inflammatory state observed at this stage triggered anti-

proliferative and anti-apoptotic effects by increasing the concentration of plasma ceramide. Literature

have already described a correlation between weight loss, insulin sensibility and TNF-α decreasing

levels, which relate not only with evaluated serum ceramide levels, but also with elevated expression

of TNF-α in adipose tissue from adipose tissue of glioma induced mice. Additionally, S1P analysis

reveals a slight decrease in serum levels over time, which may be due to the advanced inflammatory

state and subsequent hypoxia, in which the proliferative and angiogenic power of adipocytes tries to

overlap with the pro-apoptotic effect of ceramide. Regarding different serum levels and tissue

expression considering its localizations, it would be of utmost interest and pertinence to

simultaneously evaluate the expression of sphingolipids in plasma serum and target tissues, thus

enabling an understanding of sphingolipid behavior in obesity and its contribution to gliomagenesis.

So, the analysis of ceramide and S1P levels sustain the concept of a prevailing inflammatory process

in the obese mice contributing for a hypoxic state, albeit at a very early stage, delaying the apoptotic

process conducted by ceramides and other pro-inflammatory adipokines.

This study presents a paradoxical relationship between obesity and cancer, with results reinforcing

the idea on obesity protecting against glioma initiation and proliferation, and undoubtedly requires

88

Research keypoints:

〤 Glioma cells conditioned medium release a myriad of adipocytokines related to

angiogenic, growth, hormone and metabolic behavior, mentioned as secretome.

〤 Adipocytes overexpressed several pro-inflammatory and angiogenic factors, such

as IL6, IL11, LIF, TNFα, endocan, HGF, IGF1 and VEGF.

〤 Nevertheless, they underexpressed TIMP-1, SerpinE1, coagulation factor III,

endostatin, PDGFA and ADAMTS1.

〤 Tissue response may be delayed, at an early phase, due to hypoxic state.

〤 On adipose tissue, IL6, Ob (Y-20) and TNFα were overexpressed in HFD group,

while IL1β and PDGFA were underexpressed.

〤 On tumoral tissue, IL6 did not display significant variation, though IL1β and Ob

tended to be overexpressed while TNFα and PDGFA disposed to undexpression.

〤 Sphingolipid analysis, as well as liver function, reinforced the premise that

excessive dietary intake worsens metabolic syndrome in general and, more

precisely, chronic low-grade inflammation.

〤 Relationship between obesity and cancer is paradoxical, however it is strengthened

the theory of obesity protecting against glioma only at initiation and proliferation

phases.

89

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117

APPENDIXES

I. Abstract

II. Resumen

III. Publication I

P. Coelho, J. Almeida, C. Prudêncio, R. Fernandes, R. Soares, Effect of Adipocyte Secretome in

Melanoma Progression and Vasculogenic Mimicry., J. Cell. Biochem. 117 (2015) 1697–706.

doi:10.1002/jcb.25463.

IV. Publication II

J. Almeida, J. Costa, P. Coelho, V. Cea, M. Galesio, J.P. Noronha, M.S. Diniz, C. Prudêncio, R.

Soares, C. Sala, R. Fernandes, Adipocyte proteome and secretome influence inflammatory and

hormone pathways in glioma, Metab. Brain Dis. 34 (2019) 141–152. doi:10.1007/s11011-018-0327-

y.

V. Awards

Obesity and brain cancer: proteomic analysis of the influence of the adipocyte secretome on glioma

GL261 cells. R. Fernandes, J. Costa, J. Almeida, P. Coelho, V. Cea, M. Galesio, M.S. Diniz, C.

Prudêncio, C. Sala. Prémio de Investigação Básica. 17º Congresso Português de Obesidade (2013)

i

I. Summary

Gliomas represent the most common primary malignant brain tumors in adults, with an extremely

poor prognosis. Despite its high morbidity, the etiology of glioma remains largely unknown. Obesity,

favoured by the modern lifestyle, acquired epidemic proportions nowadays, and has been associated

with many major causes of death and morbidity including malignant neoplasms. In fact, excessive

adiposity is related with increased incidence and/or death rates from a wide variety of human

cancers. Among several risk factors, lifestyle was also recently identified as a major risk factor for

the development of primary glioma. So, there might be a relationship between the increased

incidence of gliomas and dysfunction of adipose tissue. In the present study, it is explored this

relationship between the influence of adipocytes secretome on glioma cell line GL261 (CGl),

sphingolipid rheostat (sphingosine-1-phosphate/ceramide) and compare it in a similar animal model.

Using the 3T3-L1 adipocytes in culture, and its conditioned medium (adipokines-enriched medium),

it is disclosed adipocyte-released factors related to glioma behavior regarding angiogenesis, growth,

hormones and metabolism by MALDI-TOF-MS and proteomic array analysis. Then, it is studied some

modified cytokines on adipose tissue from diet-induced obese C57Bl/6J mice. To conclude, it was

analysed the sphingolipid profile, namely ceramides and sphingosine-1-phosphate levels on normal

and obese diet-induced mice serum, as well as hepatic function – AST, ALT and ALP –, cholesterol

– total, VLDL, LDL and HDL –, uric acid and glucose.

ii

In a first approach, it is was used a microarray analysis of adipocytokines released by glioma

secretome under the influence of adipocytes, by which it was observed some over and under-

expressed, inexistent or without variation proteins. STI1, hnRNPs and PGK1 are underexpressed on

CGl. Similarly, both carbonic anhydrase and aldose reductase were suppressed in glioma cells that

grown under adipokines-enriched environment. Contrariwise, RFC1, KIF5C, ANXA2, N-RAP and

RACK1 were overexpressed in GL261 cell the in the presence of the adipokines-enriched medium.

Intending to further elucidate the involvement of angiogenic secretome in glioma, it was evaluated

the profile of GL261 under conditioned medium. It was further identified factors released by adipocyte

cells, and revealed that several pro-inflammatory and angiogenic factors, such as IL6, IL11, LIF, PAI-

1, TNF-α, endocan, HGF, VEGF IGF-I, were secreted to the medium into a high extent, whereas

ADAMTS, coagulation factor III, endostatin and PDGFAA, TIMP-1 and SerpinE1 were downregulated

on CGl. The medium proteome is a desirable sample due to their accessibility and representability

due, in part, to the wide dynamic range of protein levels, which lead to the discovery of new protein

markers.

Then, it followed immunohistochemical expression analysis on adipose and tumoral tissue. This was

addressed by focusing cytokines relationship within inflammation and angiogenesis. In

immunohistochemistry evaluation it was studied previously evaluated and significantly upregulated

proteins with angiogenic and metabolic relevance- IL1β, IL6, Ob (Y-20), PDGFA and TNFα, in

adipose and tumoral tissue. On adipose tissue, it was observed overexpression of IL6 (non-

iii

significant), Ob (n.s.) and TNFα (p<0,05) in glioma HFD when compared with glioma ND. The

opposite was observed regarding IL1β (n. s.) and PDGFA (p<0,05). Nevertheless, on tumoral tissue

results were quite different, as it was not observed a significant variation in glioma group in IL6,

however it was perceived a tendency to downregulation of TNFα in glioma HFD, and a significant

underexpression of PDGFA. Contrariwise, IL1β and Ob (Y-20) tended to be overexpressed in glioma

HFD. Although, on the in situ analysis of adipose tissue, it was observed overexpression of IL6 and

underexpression of TNFα in glioma HFD when compared with glioma ND. On tumoral tissue, neither

of the proteins varied significantly, however it was observed a tendency to downregulation of TNFα

in glioma HFD.

Last of all, it was analysed serum levels of sphingolipid profile – ceramide and sphingosine-1-

phosphate – in serum of C57Bl/6J obese mice. Metabolism of sphingolipids can be activated in

different situations, such as by pro-inflammatory cytokines, growth factors and oxidative stress, which

characterize the environment in which the adipose tissue is found in obesity. So, presented data

demonstrated an overtime increased levels of ceramide and decrease of S1P. These results

reinforce the hypothesis of delayed tissue response due to hypoxic state in an earlier phase. Besides,

it was approached the main clinical and metabolic features compatible with obese animal model:

dyslipidemia and weight. So, a myriad of clinical features, such as uric acid, glucose, triglycerides,

cholesterol and hepatic enzymes (AST, ALT and ASP) were evaluated on animal serum, by a

spectrophotometric assay. The relevance of these parameters relates with their potential as

iv

metabolic biomarkers to chronic low-grade inflammation. The lipid profile and hepatic function

analysis displayed a predictable behavior of these factors according to obesity low-grade

inflammatory state and metabolic syndrome in general. On a broader analysis, and regarding uric

acid, glucose and lipoproteins – triglycerides and cholesterol -, it is noticeable a continuously

increment in its levels throughout time, except for HDL, which suffers a decrease characteristic from

the negative impact of obesity progression. Furthermore, cumulative levels of FFA in the liver,

attained through excessive dietary lipids intake, contributes to de novo synthesis of purines, which

increases its serum levels, as well as activity of hepatic enzymes, namely AST, ALT. On the opposite,

ALP expresses a slightly, but not significant, decrease on the last timepoint (20 weeks).

The present thesis discloses an interesting in vitro model for the study of glioma biology under an

“obesity” resembling environment, that can be explored for the understanding of cancer cells biology,

for the search of biomarkers, prognostic markers and therapeutic approaches. Furthermore, in vivo

model reveals some debatable data which may contribute for another approach to adipose tissue in

tumoral environment.

So, in a conclusion mode, it will be summarized the main points of this thesis:

o Adipocytes overexpressed several pro-inflammatory and angiogenic factors, such as IL6,

IL11, LIF, TNFα, endocan, HGF, IGF1 and VEGF.

o Nevertheless, they underexpressed TIMP-1, SerpinE1, coagulation factor III, endostatin,

PDGFA and ADAMTS1.

v

o Tissue response may be delayed, at an early phase, due to hypoxic state.

o On adipose tissue, IL6, Ob (Y-20) and TNFα were overexpressed in HFD group, while IL1β

and PDGFA were underexpressed.

o On tumoral tissue, IL6 did not show significant variation, though IL1β and Ob tended to be

overexpressed while TNFα and PDGFA disposed to underexpression.

o Sphingolipid analysis, as well as liver function, reinforced the premise that excessive dietary

intake worsens metabolic syndrome in general and, more precisely, chronic low-grade

inflammation.

o Relationship between obesity and cancer is paradoxical, however it is strengthened the

theory of obesity protecting against glioma only at initiation and proliferation phases.

vi

vii

II. Resumen

Los gliomas representan los tumores cerebrales malignos primarios más comunes en adultos, con

un pronóstico muy desfavorable. Pese su alta morbilidad, la etiología del glioma sigue siendo en

gran parte desconocida. La obesidad, favorecida por el estilo de vida moderno, adquirió

proporciones epidémicas en la actualidad y se ha asociado con varias causas importantes de muerte

y morbilidad, incluidas las neoplasias malignas. De hecho, la adiposidad excesiva se relaciona con

un aumento en la incidencia y / o las tasas de mortalidad de una amplia variedad de enfermedades

oncológicas humanas. Entre los varios factores de riesgo, el estilo de vida también se identificó

recientemente como un factor importante para el desarrollo de glioma primario. Por lo tanto, podría

haber una relación entre el aumento de la incidencia de gliomas y la disfunción del tejido adiposo.

En el presente estudio, se explora esta relación entre la influencia del secretoma de los adipocitos

en la línea celular de glioma GL261 (CGl), el reostato de esfingolípidos (esfingosina-1-fosfato /

ceramida) y se compara en un modelo animal similar.

Utilizando los adipocitos 3T3-L1 en cultivo y su medio condicionado (medio enriquecido con

adipocinas), se describen los factores liberados de adipocitos relacionados con el glioma, la

angiogénesis, el crecimiento, las hormonas y el comportamiento metabólico mediante MALDI-TOF-

MS y el análisis de la matriz proteómica. En seguida, se estudiaron algunas citoquinas en el tejido

adiposo de ratones C57Bl / 6J obesos inducidos por la dieta. Para concluir, se analizó el perfil de

esfingolípidos, es decir, los niveles de ceramidas y esfingosina-1-fosfato en suero de ratones

viii

inducidos por dieta normal y obesa, así como la función hepática (AST, ALT y ALP), colesterol, total,

VLDL, LDL y HDL, ácido úrico y glucosa.

En un primer enfoque, se aplica un análisis de micromatrices de las adipocitocinas liberadas por el

secretoma de glioma bajo la influencia de los adipocitos, mediante el cual se observaron algunas

proteínas sobreexpresadas y poco expresadas, inexistentes o sin variaciones. STI1, hnRNPs y

PGK1 están subexpresados en CGl. De manera similar, tanto la anhidrasa carbónica como la aldosa

reductasa se suprimen incluso en las células de glioma que crecen en un ambiente enriquecido con

adipocinas. Por el contrario, RFC1, KIF5C, ANXA2, N-RAP y RACK1 se sobreexpresan en células

GL261 en presencia del medio enriquecido con adipocinas. Con la intención de dilucidar más la

implicación del secretoma angiogénico en el glioma, se evaluó el perfil de GL261 en medio

condicionado. Se identificaron además los factores que son liberados por las células adipocitarias y

revelaron que varios factores proinflamatorios y angiogénicos, como IL6, IL11, LIF, PAI-1, TNF-α,

endocan, HGF, VEGF IGF-I, fueron secretados al medio en gran medida, mientras que ADAMTS,

factor de coagulación III, endostatina y PDGFAA, TIMP-1 y SerpinE1 no se expresaron en exceso

en CGl. El proteoma del secretoma es una muestra deseable debido a su accesibilidad y

representabilidad debido, en parte, al amplio rango dinámico de las concentraciones de proteínas,

lo que lleva al descubrimiento de nuevos marcadores proteicos.

Luego, se siguió el análisis de la expresión inmunohistoquímica en tejido adiposo y tumoral. Esto se

abordó enfocando la relación de las citoquinas dentro de la inflamación y la angiogénesis. En la

ix

evaluación de inmunohistoquímica, se estudió previamente, evaluó y aumentó significativamente las

proteínas con relevancia angiogénica y metabólica: IL1β, IL6, Ob (Y-20), PDGFA y TNFα, en tejido

adiposo y tumoral. En el tejido adiposo, se observó una sobreexpresión de IL6 (no significativa), Ob

(n.s.) y TNFα (p <0,05) en glioma HFD en comparación con glioma ND. Se observó lo contrario con

respecto a IL1β (n. S.) Y PDGFA (p <0,05). Sin embargo, en el tejido tumoral los resultados fueron

bastante diferentes, ya que no se observó una variación significativa en el grupo de glioma en IL6,

sin embargo, se percibió una tendencia a la subexpresión de TNFα en la HFD de glioma, y una

subexpresión significativa de PDGFA. Por el contrario, IL1β y Ob (Y-20) tendieron a expresarse en

exceso en glioma HFD. Aunque, en el análisis in situ en tejido adiposo, se observó sobreexpresión

de IL6 y subexpresión de TNFα en glioma HFD en comparación con glioma ND. En el tejido tumoral,

ninguna de las proteínas varió significativamente, sin embargo, se observó una tendencia a la

subexpresión de TNFα en el glioma HFD.

Por último, se analizaron los niveles séricos de perfil de esfingolípidos ceramida y esfingosina-1-

fosfato en suero de ratones obesos C57Bl / 6J. El metabolismo de los esfingolípidos se puede activar

en diferentes situaciones, como las citocinas proinflamatorias, los factores de crecimiento y el estrés

oxidativo, que caracterizan el entorno en el que se encuentra el tejido adiposo en la obesidad. Por

lo tanto, los datos presentados demostraron un aumento de los niveles de ceramida a lo largo del

tiempo y una disminución en S1P. Estos datos refuerzan la hipótesis de la respuesta tardía del tejido

debido al estado hipóxico en una fase anterior. Además, se abordaron las principales características

x

clínicas y metabólicas compatibles con el modelo animal obeso: dislipidemia y peso. Por lo tanto,

una gran cantidad de características clínicas, como ácido úrico, glucosa, triglicéridos, colesterol y

enzimas hepáticas (AST, ALT y ASP) Se evaluaron en suero animal, mediante un ensayo

espectrofotométrico. La relevancia de estos parámetros se relaciona con su potencial como

biomarcadores metabólicos para la inflamación crónica de bajo grado. El perfil lipídico y el análisis

de la función hepática mostraron un comportamiento predecible de estos factores según el estado

inflamatorio de bajo grado de la obesidad y el síndrome metabólico en general. En un análisis más

amplio, y con respecto al ácido úrico, la glucosa y las lipoproteínas (triglicéridos y colesterol), es

notable un incremento continuo en sus niveles a lo largo del tiempo, excepto el HDL, que sufre una

disminución característica del impacto negativo de la progresión de la obesidad. Además, los niveles

acumulativos de FFA en el hígado, alcanzados a través de una ingesta excesiva de lípidos en la

dieta, contribuyen a la síntesis de novo de purinas, lo que aumenta sus niveles séricos, así como la

actividad de las enzimas hepáticas, a saber, AST, ALT. Por el contrario, la ALP expresa una

disminución leve, pero no significativa, en el último punto de tiempo (20 semanas).

La presente tesis describe un interesante modelo in vitro para el estudio de la biología del glioma en

un entorno similar a la "obesidad", que se puede explorar para la comprensión de la biología de las

células cancerosas, para la búsqueda de biomarcadores, marcadores de pronóstico y enfoques

terapéuticos. Además, el modelo in vivo revela algunos datos discutibles que pueden contribuir para

otro enfoque del tejido adiposo en el entorno tumoral.

xi

Entonces, en un modo de conclusión, se resumirán los puntos principales de esta tesis:

o Las células adipocitarias sobreexpresan varios factores proinflamatorios y angiogénicos,

como IL6, IL11, LIF, TNFα, endocan, HGF, IGF1 y VEGF.

o Sin embargo, subexpresaron TIMP-1, SerpinE1, factor de coagulación III, endostatina,

PDGFA y ADAMTS1.

o La respuesta del tejido puede demorarse, en una fase temprana, debido al estado hipóxico.

o En el tejido adiposo, se sobreexpresaron IL6, Ob (Y-20) y TNFα en el grupo HFD, mientras

que IL1β y PDGFA no se expresaron.

o En el tejido tumoral, la IL6 no presentó una variación significativa, aunque IL1β y Ob

tendieron a expresarse en exceso, mientras que el TNFα y el PDGFA estaban dispuestos a

la no expresión.

o El análisis esfingolipídico, así como la función hepática, refuerzan la premisa de que la

ingesta excesiva de la dieta empeora el síndrome metabólico en general y, más

precisamente, la inflamación crónica de bajo grado.

o La relación entre la obesidad y el cáncer es paradójica, sin embargo, se refuerza la idea de

que la obesidad protege contra el glioma solo en las fases de iniciación y proliferación.

xii

xiii

III. Publication I

Title: Effect of Adipocyte Secretome in Melanoma Progression and Vasculogenic Mimicry

Pedro Coelho1,2,3, Joana Almeida2,3, Cristina Prudêncio2,3, Rúben Fernandes2,3, Raquel Soares1,3

1 Department of Biochemistry, Faculty of Medicine, University of Porto, Portugal

2 Ciências Químicas e Biomoléculas, Escola Superior de Tecnologia da Saúde do Porto, Instituto

Politécnico do Porto, Portugal

3 Instituto de Inovação e Investigação em Saúde, Universidade do Porto, Portugal

Correspondence to:

Raquel Soares

Departamento de Bioquímica, Faculdade de Medicina da Universidade do Porto,

Al. Prof. Hernâni Monteiro,

4200-319 Porto, Portugal.

Tel/Fax: (+351) 22 551 36 24

E-mail: [email protected]

Short Title: Adipocyte Secretome in Melanoma Progression

mailto:[email protected]

xiv

Abbreviations used: AT: adipose tissue; CM: conditioned medium; DMEM: Dulbecco's modified

eagle's medium; FBS: foetal bovine serum; FGF: fibroblast growth factor; HGF: hepatocyte growth

factor; HMEC-1: human microvascular endothelial cells; IGF: insulin-like growth factor; IGFBP:

insulin-like growth factor binding protein; IL: interleukin; LIF: leukaemia inhibitory factor; MCP-1:

monocyte chemotactic protein 1; PAI-1: plasminogen activator inhibitor-1; PBS: phosphate buffered

saline; RBP-4: Retinol binding protein-4, SAT: subcutaneous adipose tissue; SEM: standard error of

the mean; TIMP: tissue inhibitor of metalloproteinases; VAT: visceral adipose tissue; VEGF: vascular

endothelial growth factor;

xv

Abstract

Obesity, favoured by the modern lifestyle, acquired epidemic proportions nowadays. Obesity has

been associated with various major causes of death and morbidity including malignant neoplasms.

This increased prevalence has been accompanied by a worldwide increase in cutaneous melanoma

incidence rates during the last decades. Obesity involvement in melanoma aetiology has been

recognized, but the implicated mechanisms remain unclear. In the present study, it was address this

relationship and investigate the influence of adipocytes secretome on B16-F10 (B16) and MeWo

melanoma cell lines. Using the 3T3-L1 adipocyte cell line, as well as ex vivo subcutaneous (SAT)

and visceral (VAT) adipose tissue conditioned medium, it was able to show that adipocyte-released

factors play a dual role in increasing melanoma cell overall survival, both by enhancing proliferation

and decreasing apoptosis. B16 cell migration and cell-cell and cell-matrix adhesion capacity were

predominantly enhanced in the presence of SAT and VAT released factors. Melanocytes morphology

and melanin content were also altered by exposure to adipocyte conditioned medium disclosing a

more dedifferentiated phenotype of melanocytes. In addition, exposure to adipocyte-secreted

molecules induced melanocytes to rearrange, on 3D cultures, into vessel-like structures and

generate characteristic vasculogenic mimicry patterns. These findings are corroborated by the

released factors profile of 3T3-L1, SAT and VAT assessed by microarrays, and led us to highlight

the mechanisms by which adipose secretome from sub-cutaneous or visceral depots promote

melanoma progression.

xvi

Keywords: obesity, melanoma, vasculogenic mimicry, adipose tissue

xvii

Introduction

Obesity prevalence has significantly increased worldwide [Rubenstein, 2005], leading to a public

health concern and branded as “the modern epidemic” [Haffner and Taegtmeyer, 2003; Rubenstein,

2005]. The prevalence of obesity in Europe has increased by approximately 30% over the past 10

years and this phenomenon is corroborated by data from several other countries [Berghöfer et al.,

2008]. It has long been recognized that excess adipose tissue (AT) increases the risk of

cardiovascular disease, type 2 diabetes and metabolic syndrome [Haffner and Taegtmeyer, 2003;

Rubenstein, 2005], but only in the past few decades it became widely accepted that augmented body

adiposity is a risk factor for several types of malignancies [Taubes, 2012; Vucenik and Stains, 2012].

Additionally, obesity can lead to worsened prognosis, poorer treatment outcome and increased

cancer-related deaths [Parekh et al., 2012].

Cutaneous melanoma incidence rates have increased in the last decades worldwide from 3% to 7%

annually [Tuong et al., 2012]. These statistics suggest a doubling of rates every 10 to 20 years

[Marks, 2000; Garbe and Leiter, 2009], raising melanoma to the most rapidly increasing cancer in

Caucasians [Garbe and Leiter, 2009]. Several reports showed positive associations between

increased body fat and the risk of cutaneous melanoma later in life [Dennis et al., 2008; Morpurgo et

al., 2012; Nagel et al., 2012; Sergentanis et al., 2013], suggesting that the increasing incidence of

melanoma may be related to the enlarged obesity prevalence.

xviii

In vivo adiposity-related stimulation of melanoma growth has been demonstrated [Brandon et al.,

2009; Pandey et al., 2012; Wagner et al., 2012; Jung et al., 2015]. Tumour-associated macrophages

[Wagner et al., 2012; Jung et al., 2015] and endothelial cells [Jung et al., 2015] have been pointed

out as possible mediators in the growth-promoter effect of adipose tissue towards melanomas. In

fact, tumour stroma comprises many different cell types, including fibroblasts, adipocytes, immune

and endothelial cells that, along with the extracellular matrix, are key players in cancer development

and progression [Brychtova et al., 2011; Friedl and Alexander, 2011]. However, we hypothesize that

adiposity might also exert a direct effect over melanocytes without the involvement of stromal cells

in a paracrine or endocrine manner. Herein, we explored the biological role of adipocytes secretome

in B16 and MeWo melanoma cell survival and plasticity.

xix

Material and Methods

Cell Culture and In vitro treatments

B16-F10 (B16) murine melanoma cell line (ATCC CRL-6475), MeWo human melanoma cells (ATCC

HTB-65) and 3T3-L1 pre-adipocytes (ATCC CL-173) were maintained in Dulbecco's modified eagle's

medium (DMEM; Sigma-Aldrich), Human dermal microvascular endothelial cells (HMEC-1, ATTC

CRL-3243) were cultured in RPMI-1640 (Sigma-Aldrich) containing 10ng/mL epidermal growth factor

(BD Biosciences) and 1 µg/mL cortisone (Sigma-Aldrich). Both media formulations were

supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 1%

penicillin/streptomycin/amphotericin B (Sigma-Aldrich). Cells were grown at 37ºC under a humidified

5% CO2 atmosphere. Cells were serum-deprived during 16h before incubation with each treatment

for every experiment. Unless otherwise specified, all treatments and controls were carried out in

serum-free conditions.

Adipocyte Differentiation and Conditioned Medium collection

3T3-L1 pre-adipocytes were harvested and allowed to reach confluence. After 2 days (day 0), the

differentiation was initiated by addition of a hormonal mixture composed of 2 μM insulin (Sigma-

Aldrich), 1 μM dexamethasone (Sigma-Aldrich) and 0.25 mM isobutylmethylxanthine (Fluka) in

complete medium. Three days later (day 3), the induction medium was replaced by complete medium

supplemented with insulin only. At day 7, cultures with a differentiation yield higher than 80% were

washed with phosphate buffered saline (PBS) and incubated in serum-free DMEM. After 24h (day

xx

8), the conditioned medium (CM) was harvested from the adipocyte’s cultures, spun for 5 minutes at

300g and the supernatant was stored at -80ºC for the subsequent treatments.

Adipose Tissue Organ Culture

Fragments (8–10 mg) from visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT)

depots were collected from 8 week male C57BL/6J mice. The fragments were then washed with 1%

penicillin/streptomycin/amphotericin B (Sigma-Aldrich) in PBS and cultured afterwards in 1 mL of

DMEM supplemented with 10% FBS. The fragments were serum-deprived and 24h later the CM

harvested and centrifuged at 300g for 5 minutes.

Mouse Adipokine Array Analysis

Mouse adipokine antibody arrays (-# ARY-013; R&D systems) were performed using 1 mL of 3T3-

L1, SAT and VAT CM and following the manufacturer’s protocol. The pixel density of each spot was

calculated with the microarray profiler plugin of the Image J software (NIH). The relative adipokine

levels were calculated upon normalization with the adipokine levels of B16 CM.

Cell Viability Assay

To measure B16 metabolic activity, after a 24 h incubation of 1x104 cells/mL with the different

treatments, 20 μL of the MTS reagent, from CellTiter 96 Aqueous assay (Promega), was added into

each 96-plate well followed by a 3 hour incubation period. Colour development was determined by

measuring absorbance at 490 nm.

xxi

Apoptosis Assay

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling

(TUNEL) assay was performed in B16 cells (1x104 cells/mL), after 24h incubation with the different

treatments using the in situ Cell Death Detection Kit (Roche Diagnostics) following the manufacturers

protocol. Nuclei were counter-stained with DAPI (Roche Diagnostics) and immunofluorescence was

visualized under a fluorescence microscope (Nikon). The percentage of TUNEL-positive cells was

evaluated by counting the cells stained with TUNEL divided by the total number of DAPI-stained

nuclei at a 200x magnification field. One thousand nuclei were evaluated. The results are presented

as mean ± standard error of the mean (SEM).

BrdU Proliferation Assay

B16 cells (1x104 cells/mL) were cultured with standard treatments in serum-free conditions for 20h.

Then bromodeoxyuridine (BrdU; Roche Diagnostics), at a final concentration of 0.01 mM, was added

to each well for 4h. The detection was performed using the colorimetric Cell Proliferation ELISA,

BrdU (Roche Diagnostics), according to the manufacturer’s instructions.

Cell Spreading and Adhesion determination

To determine cell spreading and adhesion [Humphries, 2001], B16 cells were re-suspended in the

different treatments at a final concentration of 5x104 cells/mL, seeded in a 24-well microplate and

allowed to adhere and spread for 3 hours and then washed with PBS to remove non-adherent cells.

Fixation was performed with 4% (w/v) p-formaldehyde in PBS. Afterwards, 200 μL of crystal violet

xxii

0.1% (w/v) was added to each well for 20 minutes and later washed with H2O. To evaluate the

morphologic parameters (cell area, sphericity, size and perimeter), 1000 cells were analysed by

CellProfiler software (Broad Institute) on photographs of random fields, for each treatment, captured

under an inverted microscope (Nikon) at a 200x magnification. To determine cell adhesion, the above

protocol was reproduced but only allowing cells to adhere for 30 minutes. Afterwards, crystal violet

dye was solubilized in 100 μL 10% (v/v) acetic acid (Fluka) and the absorbance measured at 570 nm

using a plate reader.

Adhesion to endothelium evaluation

HMEC-1 cells were cultured in a 24-well microplate with glass coverslips and allowed to reach

confluence. Media was aspirated and the monolayers were washed with PBS. Afterwards, 1x105

cells/mL B16 cells, in the different treatments, were added to the wells for 30 minutes. To remove

non-adherent cells, the wells were washed with PBS. Fixation was performed with 4% (w/v) p-

formaldehyde for 10 minutes. Coverslips were then stained with Giemsa’s stain, thoroughly washed

in Sorensen's phosphate buffer and mounted on a microscope slide. Using a light microscope (Nikon)

the number of cells adherent to the endothelial monolayer were counted in 5 random fields.

Melanin content determination

B16 cells (5x105 cells/mL), were incubated with the different treatments for 24h. Following

detachment with 0,5% trypsin, cell density was assessed on a haemocytometer. After 300g

centrifugation for 10 min, the pellet was washed twice with PBS and the melanin was solubilized in

xxiii

1.0 mL of 1M NaOH (Panreac) containing 10% (v/v) dimethyl sulfoxide (Merck). Next, the absorbance

at 475 nm was recorded. Results represent the mean absorbance (±SEM) of 1x106 cells.

Transwell migration

Migration capacity was quantified on 24-well plates with 8 μm-pore transwell inserts (BD

Biosciences). B16 cells (5x104 cells/ml) were harvested on inserts in serum-free medium and placed

on wells containing the standard treatments plus 10% FBS for 24h. Membranes were then stained

with crystal violet 0.1% (w/v) and visualized under a light microscope (Nikon). Five random fields of

each membrane were counted on the microscope (Nikon) at a 100x magnification.

Injury assay

To perform the injury assay, melanoma cells at confluence were scrapped from the culture dish using

a pipette tip, which left a void space. Cells were then incubated for 24h following the standard

treatments. After this period, damage recovery was then visualized and photographed under an

inverted microscope (Nikon) at a 200x magnification. The wound closure was determined by

measuring the wound area and by subtracting this value from the initial void space (CellProfiler

software; Broad Institute). Shown are the means values (±SEM) of nine measurements for each time

point and condition.

Matrigel cultures

To perform Matrigel cultures, 24-well plates were coated with 250 µL per well of Matrigel Basement

Membrane Matrix (Corning). Afterwards, B16 (5x104 cells/mL) or MeWo (20x104 cells/mL)

xxiv

melanocytes harvested in the respective treatments were fed on top of the Matrigel layer. Cell growth

was then monitored and photographed under an inverted microscope (Nikon) at a 200x magnification

for 24h.

Soft-agar colony formation

The bottom of each 12-well plate was coated with 2 ml of 0.5% agarose and DMEM 2X. After

polymerization the wells were fed with 5x103 B16 cells in 2 ml of 0.2% agar and DMEM 2X. The

medium was changed every 2-3 days. After 15 days, the wells were stained with 0.01% crystal violet

and photographed. The number and area of the colonies was assessed with ImageJ software (NIH).

Hanging-drop cell Cultures

Generation of melanoma spheroids was conducted as previously described [Foty, 2011]. Briefly,

2,5x106 B16 or MeWo cells/mL were collected in the respective treatments and pippeted to the lid of

a 60mm non-adhesive petri dish leaving 10 μL drops onto the bottom of the lid. The lids were inverted,

placed on top of the PBS-filled bottom chamber of the dish and incubated for 3 days.

Statistical Analysis

Results are expressed as mean±SEM. Data was analysed with GraphPad Prism 6.0 (GraphPad

Software Inc.). Differences between samples and parameters among two experimental groups were

evaluated by Student’s t-test. When three or more conditions were evaluated, statistical analysis was

conducted through one-way ANOVA with Sidak post-hoc test. Significance was set at p<0.05.

xxv

Results

3T3-L1 secretome increases melanocytes survival, proliferation and melanin content, and decreases

apoptosis

3T3-L1 preadipocytes are a commercially available cell line for studying adipogenesis [Green and

Kehinde, 1975; Poulos et al., 2010]. Their differentiation, in culture, into mature adipocytes is well

widespread and can be achieved by established protocols. We used CM from fully differentiated 3T3-

L1 cells to explore the potential effects of adipocytes secretome over malignant melanoma B16 cells

viability, proliferation and apoptosis.

We first analysed the effects of this conditioned medium in melanoma cells viability by the MTS

assay. After a 24-hour exposure to adipocyte CM, melanoma cells showed a 48% increase in their

metabolic activity (Fig. 1A). We found as well that the fraction of proliferating B16 cells was

significantly increased, as shown by the higher BrdU incorporation, in the presence of these

adipocyte-released factors (Fig. 1B). The effect of 3T3-L1 secretome on apoptosis was examined

using TUNEL analysis. Concomitantly, 3T3-L1 CM medium decreased B16 programmed cell death

by approximately 50% (Fig. 1C). These effects were further accompanied by a significant reduction

in melanin content per cell (Fig. 1D).

Adipocyte secretome profile characterization

xxvi

The previous findings imply that adipocytes released factors stimulate melanoma cell viability and

aggressiveness. Therefore, we then assessed the secretion profile of adipocytes to further elucidate

the involvement of adipose tissue in melanoma behaviour (see Supplemental data). Given the well-

established distinct biological and metabolic roles of sub-cutaneous (SAT) and visceral (VAT)

adipose tissue, microarray assay was performed not only in 3T3-L1 cells, but also in organ cultures

of SAT and VAT fragments.

As illustrated in Fig. 2, the secretion patterns of numerous growth-factors, adipokines, cytokines and

angiogenesis-related molecules are different among the different CM. Greater levels of vascular

endothelial growth factor (VEGF) and endocan were found in SAT and VAT CM, whereas tissue

inhibitor of metalloproteinases-1 (TIMP-1) levels were significantly reduced in these two CM (Fig 2A).

On the other hand, 3T3-L1 CM exhibited higher levels of VEGF, TIMP-1 and endocan than B16-CM

(Fig 2A). The levels of the interleukin (IL) family of cytokines: IL6, IL11 and leukaemia inhibitory factor

(LIF) were also significantly overexpressed within SAT and VAT, although no substantial differences

were found in the 3T3-L1 CM (Fig 2B). Nonetheless, monocyte chemotactic protein 1 (MCP-1) and

plasminogen activator inhibitor-1 (PAI-1) expression was upregulated in every CM (Fig 2B). Adipose

tissue hormones were also identified. Resistin and leptin relative expression levels were higher in

3T3-L1, SAT and VAT CM. In addition, Retinol binding protein-4 (RBP-4) was significantly released

in SAT and VAT, but not in 3T3-L1 CM. Interestingly enough, a significant reduction in adiponectin

levels was observed in all CM (Fig 2. C). The levels of several growth factors involved both in

metabolism and in cell behaviour were also analysed. VAT CM revealed the higher expression of

xxvii

fibroblast growth factor (FGF)-21, insulin-like growth factor binding protein (IGFBP)-5, whereas

hepatocyte growth factor (HGF), insulin-like growth factor(IGF)-I, IGFBP-2, IGFBP-3 and IGFBP-6

levels were significantly higher in SAT than VAT CM (Fig 2. D).

SAT and VAT distinctively modulate melanocyte migration and cell spreading

According to the previous secretome profile, TIMP-1, an inhibitor of extracellular matrix degradation

was significantly decreased, whereas several inflammatory cytokines and angiogenic growth factors

were upregulated particularly in SAT and VAT. These findings led us to evaluate the influence of CM

in melanocytes motility. We first analysed whether 3T3-L1 CM mechanically induced a wound to B16

confluent cultures by injury assay. As illustrated in Figure 3A, the resulting void area was promptly

occupied by melanocytes upon 3T3-L1 CM incubation in comparison to untreated B16 cultures.

Following a 24h incubation with CM, the migrated distance is almost twice the distance of the control

treatment as seen by the microscopic examination of the wounds (Fig. 3B). We then examined the

effect of SAT and VAT fat depots secretome in B16 cells motility. In the double-chamber migration

assay, the number of migrating cells following a chemotactic gradient was significantly enhanced in

every treatment as compared to untreated B16 cultures (Fig. 3C). However, SAT CM exerted this

effect into a much larger extent. Cell spreading evaluation revealed that VAT and 3T3-L1 secreted

molecules, but not SAT, induced spreading dynamic of B16 cells (Fig. 3D). In addition, VAT CM-

treated B16 cells exhibited significantly higher areas and perimeter, whereas a smaller number of

round shaped cells were observed. In contrast, SAT secreted factors did not significantly alter B16

xxviii

spread plasticity (Fig. 3E). These findings highlight a distinct paracrine role of AT depots regarding

B16 locomotive behaviour: while SAT-released factors boost melanoma cell chemotaxis, VAT CM

signalling enhance cell morphological plasticity and haptotactic spreading.

AT secretome modulates melanoma cells adhesion and tumorigenesis

Decreased cell-cell adhesion and anchorage-independent growth are prominent features necessary

for successful metastization [Chiang and Massagué, 2008; Friedl and Alexander, 2011]. Therefore,

we evaluated the adhesion of CM-treated B16 cells to standard cell culture plates. Interestingly, B16

cells adhesion was significantly increased after incubation with every CM, being higher upon VAT

medium exposure (Fig 4A).

Identical findings were observed in B16 cells adhesion to endothelial cells (Fig. 4C). The number of

B16 cells adherent to HMEC-1 monolayers was significantly enhanced both after SAT and VAT CM

treatments as compared to untreated cells or to cells treated with 3T3-L1 CM (Fig. 4B), being the

highest increase found upon VAT CM incubation. These findings confirm that VAT improves B16

adhesive properties. Conversely, in the soft-agar clonogenic assay, SAT soluble factors promoted

the anchorage-independent proliferation of B16 cells upon culture in non-adherent conditions (Fig.

4D and 4E), whereas exposure to VAT CM did not exert any effect. SAT released factors increased

the number of colonies formed, but not in a statistically significant manner (Fig. 4F). These results

further emphasize the divergent roles of SAT and VAT towards melanocytes malignancy.

Adipocytes secreted factors induce malignant melanocytes vasculogenic mimicry

xxix

Given that malignant melanomas are highly vascularized tumours [Pirraco et al., 2010; Chung and

Mahalingam, 2014] and metastatic melanoma cells actively participate in tumour vascularization

[Hendrix et al., 2003; Chung and Mahalingam, 2014], we next addressed whether CM from 3T3-L1

adipocytes prompted B16 melanoma cells toward vasculogenic mimicry.

In comparison to untreated cells, 3T3-L1 CM-treated B16 cells rapidly assembled into capillary-like

structures when cultured in Matrigel basement matrix (Fig. 5A). To further confirm the three-

dimensional growth and spatial arrangement of the melanoma cells, B16 cells were cultured in

hanging-drops for 72 hours. Microscopic observation of the generated spheroids upon incubation

with 3T3-L1 CM revealed the formation of large and diffused aggregates of B16 cells, whereas in the

control treatment, a spherical and compact mass of melanocytes was observed (Fig. 5B). Next, we

used MeWo cell line to additionally examine the vasculogenic mimicry inducer effects of 3T3-L1 CM

in human melanoma cells. MeWo cells, when cultured on top of Matrigel and in the presence of 3T3-

L1 CM displayed the same vessel-like rearrangement already observed for B16 melanocytes (Fig.

5A). MeWo spheroids further revealed a loophole growth pattern when exposed to 3T3-L1-released

factors (Fig. 5B). Altogether, these findings regarding cell-cell adhesion and reorganization into

vessel-like structures formation provide additional support and reinforce the deleterious effects of fat-

secreted molecules on melanoma tumour progression.

Discussion

xxx

Emerging evidence indicates that systemic factors, including inflammatory, angiogenic or metabolic

markers, significantly influence tumour behaviour [Hanahan and Weinberg, 2011].

The current study reveals that adipocyte-released factors increase B16 melanoma cells viability,

proliferation, and reduces apoptosis and melanin content. Exposure to adipose tissue CM of these

cells further resulted in increased motility, enhancing the capacity to migrate and spread. These

findings were accompanied by an augmented adhesion capacity, in particularly to endothelial cells,

and anchorage-independent proliferation as assessed by soft agar assays. We further identified the

factors that are released by adipocyte cells of different depots, and revealed that several pro-

inflammatory factors (IL6, IL11, LIF and PAI-1), metabolic markers (IGFBPs, FGF-21), angiogenic

growth factors (endocan, HGF, VEGF IGF-I), and hormones (leptin, resistin, RBP-4) were secreted

to the medium into a high extent, whereas TIMP-1, an inhibitor of ECM degradation, and adiponectin

were significantly downregulated.

Adipose tissue is no longer considered a mere lipid store depot, but also an inflammatory and

endocrine organ [Monteiro, 2009; Mendonça and Soares, 2015]. Accordingly, obesity provides a

chronic low-grade inflammatory condition, further potentiated by the presence of hormone cues and

cytokines, which are strongly associated with disease. Therefore, the presence of high amounts of

pro-inflammatory cytokines and hormones in adipocyte CM is expected. Remarkably, the majority of

these factors are known to play a role in cell proliferation, apoptosis, migration and invasiveness, as

well as adhesion capacity. Recently, it has been reported that both leptin and resistin, two adipocyte-

xxxi

released hormones, were able to stimulate melanoma cell growth and proliferation through Akt and

fatty acid synthase modulation [Malvi et al., 2015]. In addition to these hormones, FGF-21, a

metabolic regulator also increases cell proliferation in vitro and increased tumorigenesis and invasive

potential in vivo [Osawa et al., 2009]. Likewise, FGF and IL6 are known to be produced by melanoma

cells and stimulate proliferation in a autocrine manner, while other paracrine growth factors (HGF,

IGF-I and VEGF) modulate the microenvironment and potentiate tumour growth and invasion

[149,150]. We have found that adipose tissues CM have significant higher levels of the above growth

factors, reinforcing our findings concerning the effect of CM from the distinct adipose tissue depots

studied in augmenting B16 cell proliferation, migration, ability to invade and decreasing apoptosis.

Along with the progression of melanoma, malignant melanocytes dedifferentiate losing most of their

epithelial characteristics through a process similar to epithelial-mesenchymal transition [Fenouille et

al., 2012], leading thus to more aggressive phenotypes [Thiery, 2002]. Melanogenesis is an inherent

process in melanocytes. Melanocytes respond to the inflammatory cytokines, namely IL6, with a

dose-dependent inhibition of melanogenesis [151]. Nevertheless, whenever these cells exhibit a

more malignant phenotype, melanocytes decrease melanin synthesis [Jimbow et al., 1993]. Our

findings show that when treated with CM from adipocytes, the melanin content per B16 melanocyte

was significantly reduced, further enlightening the growth-promoter and dedifferentiation paracrine

effects of adipocytes.

xxxii

Furthermore, active locomotion of tumour cells is fundamental to malignant invasive and metastatic

capacity [Zhao et al., 2001; Fidler, 2003; Friedl and Wolf, 2003]. By crossing tissue stroma and the

vascular bed, tumour cells interact with both extracellular matrix and soluble tropic factors. Our

findings showed that chemotactic migration of B16 cells was enhanced upon exposure to SAT-

released factors while cell spreading and haptotactic migration of tumour cells were positively

modulated by visceral-fat secretome. Intercellular adhesion is reduced in many aggressive tumours

[Chiang and Massagué, 2008], allowing cells to detach from the primary lesion and metastasise to

distant organs. Again, we found discrete effects among subcutaneous and visceral fat depots on B16

adhesive properties. In fact, several biological, metabolic and secretory differences distinguish intra-

abdominal visceral-adipocytes from peripheral subcutaneous adipose tissue [Ibrahim, 2010; Ali et

al., 2013]. In agreement, our results unveiled distinct effects of SAT and VAT upon melanocytes

aggressiveness. SAT CM enhances B16 locomotion and anchorage-independent proliferation. In

turn, VAT prominently improves melanocytes cell-substratum adhesion. Despite the higher release

of visceral-fat pro-inflammatory cytokines [Ibrahim, 2010], subcutaneous fat depots, which are

located close to melanoma lesions, influence the malignant transformation of melanocytes.

Nevertheless, our microarray assay revealed that the secretome profile of SAT and VAT is

significantly different regarding some of the inflammatory, hormonal and metabolic molecules

addressed. TIMP-1 and IGF-I are chemotactic agents for human melanoma cells that mediate

motility [150,152]. IGF-I was significantly higher in SAT CM while TIMP-1 was under expressed in

the adipose tissues CM. PAI-1 is also known to modulate cell adhesive properties and its

xxxiii

overexpression is correlated with the metastatic capacity of melanomas [153]. Antagonistically,

IGFBPs inhibit melanoma migratory and invasive behaviour, and induce melanocytic differentiation

[154] counterbalancing the IGF-I effects and most likely contribute to the discrete differences

observed between subcutaneous and visceral CM. The effect of the different AT depot secretome in

tumour cells behaviour is beyond the scope of this study, however our results reinforce the need for

additional molecular studies to investigate how SAT and VAT affect tumour progression.

Accumulating data point out mechanisms associating obesity to melanoma [Dennis et al., 2008;

Brandon et al., 2009; Meeran et al., 2009; Morpurgo et al., 2012; Pandey et al., 2012; Chen et al.,

2013]. Recent reports unveiled the involvement of high-fat diet-induced increased cytokines and

angiogenic factors in the crosstalk between tumour cells and macrophages [Costa et al., 2007;

Wagner et al., 2012; Jung et al., 2015; Mendonça et al., 2015]. Recruitment of the immune system

cells, which release factors that enhance tumour plasticity and the maintenance of stromal

microenvironment, has already been described for other malignancies [Fridman et al., 2012;

Hanahan and Coussens, 2012]. In contrast, our in vitro results disclose a direct effect of adiposity on

melanoma cells, without the influence of either tumour-associated cell-mediated immunity or

endothelium.

Vascularization plays a central role in tumour development and progression. Besides angiogenesis,

vasculogenic mimicry provides an alternative, angiogenic-independent tumour microcirculation

[Maniotis et al., 1999; Hendrix et al., 2003; Ribatti et al., 2013]. Melanoma cell-lined vascular

xxxiv

networks sustain a redundant blood supply required for both growth and metastasis [Maniotis et al.,

1999; Ribatti et al., 2013]. The presence of these functional vascular channels by the tumour itself is

a predictor of poor prognosis in human melanoma patients [Thies et al., 2001; Cao et al., 2013] and

might circumvent the effectiveness of anti-vascular drugs and anti-angiogenic therapies [Loges et

al., 2010]. Fully differentiated 3T3-L1 adipocytes CM were able to induce malignant melanocytes to

rearrange on Matrigel cultures into vessel-like structures typically reported for endothelial cells

[Kleinman and Martin, 2005]. Moreover, microscopic inspection of B16 and MeWo spheroids,

obtained by hanging-drop cultures, revealed the same pattern of vasculogenic mimicry observed in

cultures of human melanoma cell lines [Maniotis et al., 1999; van der Schaft et al., 2004]. Data

confirming melanoma angiogenesis stimulation by AT tropic factors has recently been highlighted

[Wagner et al., 2012; Jung et al., 2015]. However, we report for the first time supporting evidence

endorsing the potential adipocyte secretome inducer effects of tumour vasculogenic mimicry.

Accordingly, several angiogenic growth factors were significantly higher in CM, including HGF and

VEGF. HGF was involved in vascular mimicry of hepatocellular carcinoma [Lirdprapamongkol et al.,

2012]. Additionally, VEGF signalling was found to be involved in vascular mimicry through activation

of VEGFR1 expression in melanoma cells [Frank et al., 2011], explaining the increased capacity of

melanoma cells to form vasculogenic structures after treatment with adipocyte CM. Endocan, in turn,

is a new tumour invasion and angiogenic marker, being overexpressed in tumour vessels [Matano

et al., 2014]. The fact that it is upregulated in both SAT and VAT conditioned medium may also

explain the involvement of adipose tissues in tumour aggressiveness.

xxxv

Altogether, our results indicate that adipocytes secretome induce malignant melanocytes

aggressiveness. The synergic increase in melanocyte survival, adhesion, motility and plasticity allied

to the stimuli for cell-cell networks of vasculogenic mimicry patterns, support the deleterious effects

that adiposity partakes directly in melanoma progression.

Acknowledgements:

We would like to thank Professor Conceição Calhau (Faculty of Medicine of the University of Porto)

and Professor Paula Soares (Instituto de Inovação e Investigação em Saúde, Universidade do Porto)

for kindly providing the 3T3-L1 and MeWo cell lines, respectively. We are also in debt to the

“Fundação para a Ciência e a Tecnologia” (FCT) and FEDER-COMPETE for the financial support

through the research unit PEst-OE/SAU/UI0038/2011, UID/BIM/04293/2013 and the PhD grant

attributed to PC (SFRH/BD/80434/2011).

There is no conflict of interests regarding the present paper.

xxxvi

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Figure Legends

Figure 1. 3T3-L1 secretome increases melanoma cell survival. B16 cells were incubated with CM

from differentiated 3T3-L1 cells cultures (3T3-L1 CM) or untreated (Control) for 24h. A) An increase

in the percentage of viable melanoma cells was observed when compared with control. Results

represent the percentage of viable cells normalized by the absorbance of control. B) The number of

proliferating cells, incorporating BrdU, increased in the presence 3T3-L1 CM. Results represent the

percentage of proliferating cells normalized over the absorbance of control. C) A significant decrease

in the percentage of apoptotic cells was observed when melanocytes were treated with 3T3-L1 CM.

Results represent the percentage of apoptotic cells evaluated by the ratios of TUNEL-positive cells

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versus total DAPI-counterstained nuclei. D) The melanin content per cell was significantly decreased

when melanocytes were cultured in presence of 3T3-L1 CM. Melanin concentration was calculated

by determination of OD 475. Results represent the absorbance of 1x106 cells. (*p <0.05 vs. Control;

n=9)

Figure 2: Secretion profiles of 3T3-L1 cells and subcutaneous and visceral AT organ cultures. A)

Both SAT and VAT CM exhibit greater levels of VEGF and endocan with a concomitant reduction in

the expression of TIMP-1. B) Distinct relative levels of IL6, IL11 and LIF were found among SAT and

VAT CM C) Adipokines leptin and resistin are overexpressed in all CM, whereas diponectin levels

are significantly reduced D) Numerous cellular growth factors were present at considerably higher

concentrations in both VAT and SAT CM (*p<0.05 vs Control; #p<0.05 SAT CM vs VAT CM; n=2)

Figure 3. In vitro motility, migration and spreading analysis of B16 melanocytes. A) A rapid B16 cell

migration was observed after incubation with 3T3-L1 CM in comparison to untreated (Control) for

24h. Cell cultures were visualized under an inverted microscope at a 200x magnification. B) When

exposed to 3T3-L1 CM, melanocytes rapidly occupied the injury-created void area. The wound

closure was determined by measuring cultures void area and by subtracting it from the initial area of

the wound. Bars show the means values (±SEM) of nine measurements for each time point/condition

C) The effects of AT secretome on cell migration were quantified in a double-chamber assay using

FBS as a chemoattractant. The number of cells that invaded the membrane was significantly higher

when treated with 3T3-L1, and particularly with SAT and VAT CM. Mean values (±SEM) of three

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independent measurements are shown (*p<0.05 vs Control; #p<0.05 SAT CM vs VAT CM; n=3). D)

B16 cells were allowed to adhere and spread for 3 hours in the standard treatments. Representative

images of crystal-violet stained melanoma cells are shown (200X). E) Morphologic analysis of B16

cells revealed that VAT and 3T3-L1 CM significantly enhanced cell spreading. Bars are the means

values ± SEM, represented as percentage of Control, of 1000 evaluated cells (*p<0.05 vs Control;

n=9)

Figure 4. Melanoma cells adhesion and anchorage-independent growth were increased after

incubation with AT CM. (A) Adhesion to culture plates for 30 min; (B) Adhesion to HMEC-1

monolayers for 30 min. VAT-released factors were prominent in enhancing melanocytes adhesive

potential both to culture plates and endothelial cells. Results are expressed as percentage of Control

(±SEM). C) Representative images (800X) of Giemsa stained slides showing B16 cells (dark blue)

adherent to HMEC-1 monolayers (pink). (D) Evaluation of anchorage-independent proliferation of

B16 melanocytes was conducted by soft-agar colony formation assay. (E) The number of colonies

formed was significantly higher in both 3T3-L1 and SAT CM treatments but not in VAT CM.(F) Mean

B16 colony area was significantly increased after 3T3-L1 CM incubation, but not after VAT or SAT

CM treatment. Bars represent means±SEM (*p<0.05 vs Control; #p<0.05 SAT CM vs VAT CM; n=3-

9).

Figure 5. Vasculogenic mimicry by melanoma cells was stimulated by AT CM. (A) B16 and MeWo

cells were cultured on top of Matrigel layers. Upon treatment with 3T3-L1 CM, melanocytes acquired

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a vessel-like tubular phenotype. Images were captured under an inverted microscope (200x) at 12

and 24 hours post treatment. (B) Microscopic examination of B16 and MeWo spheroids in hanging-

drop cultures. The presence of 3T3-L1 secreted factors inhibited the formation of a compact spheroid

cellular mass. Shown are representative light-microscopy photographs (80x) of the spheroids.

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IV. Publication II

Adipocyte secretome influences inflammatory and angiogenic pathways in glioma

Almeida J1,2,*, Costa J1,*, Coelho P1,2, Cea V3, Galesio M4, Noronha JP4, Diniz MS4, Prudêncio C1,2,

Soares R2,5, Sala C3, Fernandes R1,2,#

1- CISA, Centre for Environmental and Health Research, School of Health, Polytechnic of Porto

2- i3S, Instituto de Investigação e Inovação em Saúde, University of Porto

3- CNR Institute of Neuroscience and Department of Biotechnology and Translational Medicine,

University of Milan, Milan, Italy

4- REQUIMTE, Department of Chemistry, Faculty of Sciences and Technology, Centre for Fine

Chemistry and Biotechnology, NOVA University

5- Department of Biomedicine, Unit of Biochemistry, Faculty of Medicine, University of Porto

* equal contribution of both authors

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#Corresponding author:

Rúben Fernandes, PhD

[email protected]

Key words:

Adipo-proteomics, 2D-PAGE-MALDI-TOF, Glioma, Obesity

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Abstract

Aims: Gliomas represent the most common primary malignant brain tumors in adults, with an

extremely poor prognosis. Despite its high morbidity, the etiology of glioma remains largely unknown.

Excess adiposity is related with increased incidence and/or death rates from a wide variety of human

cancers. Among several risk factors, lifestyle was also recently identified as a major risk factor for

the development of primary glioma. So, there might be a relationship between the increased

incidence of gliomas and dysfunction of adipose tissue. In the present study, we explore this

relationship between the influence of adipocytes secretome on glioma cell line GL261.

Methods: Using the 3T3-L1 adipocyte cell line, and its conditioned medium (adipokines-enriched

medium), we showed that adipocyte-released factors relate with glioma angiogenic, growth,

hormones and metabolic behavior by MALDI-TOF-MS and proteomic array analysis.

Results: In a first view, STI1, hnRNPs and PGK1 are underexpressed on CGl. Similarly, both

carbonic anhydrase and aldose reductase are even suppressed in glioma cells that grown under

adipokines-enriched environment. Contrariwise, RFC1, KIF5C, ANXA2, N-RAP and RACK1 are

overexpressed in GL261 cell the in the presence of the adipokines-enriched medium. We further

identified the factors that are released by adipocyte cells, and revealed that several pro-inflammatory

and angiogenic factors, such as IL6, IL11, LIF, PAI-1, TNF-α, endocan, HGF, VEGF IGF-I, were

secreted to the medium into a high extent, whereas TIMP-1 and SerpinE1 were underexpressed on

CGl.

l

Conclusions: This study discloses an interesting in vitro model for the study of glioma biology under

a “obesity” environment, that can be explored for the understanding of cancer cells biology, for the

search of biomarkers, prognostic markers and therapeutic approaches.

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Introduction

Gliomas, a wide term which comprises all tumors arising from the supportive tissue of the brain,

represent 30% of all brain tumors and 80% of all malignant brain tumors. They are the most common

primary malignant brain tumors in adults 1,2. Prognosis is extremely poor, with a median survival

time of approximately 12 to 15 months and is almost invariably fatal 3. This tumor represents about

12-15% of all primary brain tumors and about 60-75% of all astrocytomas. Gliomas increase in

frequency with age, and affect preferentially men 3–5. Genetic factors in glioma etiology are poorly

understood; less than 5% of glioma cases are familial in origin, with only a few described by rare

genetic syndromes 6. Gliomas are heterogeneous and are typically classified according to World

Health Organization tumor grade, and also classified on the basis of cellular lineage: astrocytic,

oligodendroglial, and mixed tumors 7.

In 2001, different studies from the International Agency for Research into Cancer (IARC) and the

World Cancer Research Fund (WCRF) have reported a relationship and established a link between

obesity and cancer risk 8,9. Excess adiposity is related with an increase the incidence and/or death

rates from a wide variety of human cancers, being the most common colon, rectum, esophagus,

kidney, pancreas, gallbladder, ovary, cervix, liver, prostate and certain hematopoietic cancers 8–12.

Overweight and obesity are important risk factors for developing cancer and also for cancer related

mortality 13,14. It is important to understand the pathophysiological mechanisms involved in the link

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between obesity and cancer, in order to target future preventive and therapeutic strategies for cancer

in obese people 13.

Despite its high morbidity, the etiology of glioma remains largely unknown. Among several risk

factors, lifestyle was also recently identified as a major risk factor for the development of primary

glioma 15. Consequently, there might be a relationship between the increase and dysfunction of

adipose tissue. Obesity, favored by the modern lifestyle, acquired epidemic proportions nowadays,

and accelerated weight gain, in adults, is associated with increasing incidence of all central nervous

system tumors, mostly glioma 16. At the present time, it is not clear which factors might be involved

in this relation. Some studies analysed body weight in relation to survival in glioma demonstrating

higher death rates in patients with an excess body weight 17. Obesity, and in particular visceral

obesity, plays a major role in the pathogenesis of several metabolic disorders 18. Genes that influence

obesity are highly expressed in the brain and could also mediate glioma susceptibility 15,19.

Development of obesity is also associated with substantial modulation of adipose tissue (AT)

structure, involving adipogenesis, angiogenesis, and extracellular matrix remodeling 20. Thus, AT can

trigger blood vessel formation, and in turn AT endothelial cells promote pre-adipocyte differentiation.

Angiogenesis, the physiological process involving the growth of new blood vessels from existing

vasculature, plays a central role in growth and development of both normal and malignant tissues

21,22. Angiogenesis is required for AT expansion, as well as for cancer progression and metastization

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21,23. Anti-angiogenic therapy has been a widely used strategy to fight tumor growth, and recently has

also been acknowledged as a potential therapeutic intervention for obesity 23,24.

Furthermore, and concerning lipid diet content, evidence demonstrates that lipid metabolism is

dysregulated in obesity and there might be a common pathway between excess nutrients and

inflammation to increased metabolic risk 16,25. Increase in lipolysis in AT leads to elevation in free

fatty acids (FFA) in plasma and contribute to fat deposition in liver and skeletal muscle, which may

promote systemic insulin resistance through several mechanisms, such as induction of oxidative

stress through incomplete oxidation and inflammatory responses. However, the cause of the

adipocyte malfunction remains to be identified in obesity25.

AT is also recognized by its endocrine function as producer of biological mediators13,14. There are

several cytokines regarding cell proliferation, angiogenesis, cytoskeletal rearrangement and survival.

Some receptors and signaling pathways associated have revealed to stimulate growth and

invasiveness capacity of glioma cells, whereas others have been correlated to glioma cell

proliferation blockage 8,13,16. The mechanisms underlying the associations betwwen energy balance

and cancer risk should be clarified.

The present study aims to develop an in vitro rodent model for the study of the influence of obesity

role in glioma. We propose a model in which GL261 cells, a mouse glioma cell line, is cultured in the

presence or absence of 3T3-L1 mature adipocytes conditioned medium. The 3T3-L1 pre-adipocytes

were differentiated under controlled experiments and the adypokines and inflammation chemokines

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pattern expression were examined by proteome microarrays in the mature adipocytes conditioned

medium were grown.

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Materials and Methods

The protein expression was accessed by means of bidimensional polyacrylamide gel electrophoresis under

denatured conditions (2D-PAGE) followed by mass spectrometry analysis of the interest spot. The spots were

analysed by matrix assisted laser desorption ionization time-of-flight/mass spectrometry (MALDI-TOF-MS). The

non-conditioned glioma cells served as the control group for the conditioned glioma cells. We have used the

mature adipocytes secreted adipokines (secretome) and enriched the GL261 medium, followed by analysis of

the proteins on GL261 on a 2-dimensional proteomic gel approach, so different spots could be studied by means

of mass spectroscopy.

Chemicals

The reagents acetonitrile, iodoacetamide (IAA), dl-dithiothreitol (DTT) (99% w/w) and trypsin from porcine

pancreas (proteomics grade) were purchased from Sigma (Steinheim, Germany). Formic acid puriss for mass

spectrometry (≥98%), ammonium bicarbonate (>99.5% w/w) and the matrix assisted laser desorption ionization

MALDI matrix α-Cyano-4-hydroxycinnamic acid (α-CHCA) puriss for MALDI-MS were from Fluka (Buchs,

Switzerland). Trifluoroacetic acid (TFA, 99% v/v) was from Riedel-de-Haën (Seelze, Germany). ProteoMass

Peptide MALDI-MS Calibration Kit (MSCAL2) from Sigma was used as mass calibration standard for MALDI-

TOF-MS.

Cell culture

For the current work 3T3-L1 (purchased from American Type Culture Collection) and GL-261 (gently given by

Prof. Pedroso Lima, CNC, Coimbra, Portugal) mouse cell lines were used. 3T3-L1 and GL-261 cells were

lvi

cultured in DMEM (Dulbecco´s Modified Eagle´s Medium with 4.5g/L Glucose & L-Glutamine, without Sodium

Pyruvate. Santa Cruz Biotechnology, Inc.), supplemented with 10% FBS, and 1000 units/mL gentamycin

solution, maintained in T-25 tissue culture flasks in 5% CO2/95% air at 37oC in a humidified incubator.

Adipocyte differentiation and conditioned medium collection

3T3-L1 pre-adipocytes were propagated and allowed to reach confluence. After 2 days (day 0), the

differentiation was initiated by addition of a hormonal mixture composed of 2 μM insulin, 1 μM dexamethasone

and 0.25 mM isobutylmethylxanthine. Three days after (day 3), the induction medium was replaced by complete

medium supplemented with insulin only. At day 6 cultures were washed twice in phosphate buffered saline and

incubated in serum-free medium. After 24h (day 7), medium was harvested from the adipocyte’s cultures, spun

for 3000 g for 5 minutes and the supernatant (mature adipocytes conditioned medium) was stored at -80ºC for

the subsequent treatments. This conditioned medium is rich in adipokines which are globally refered as

secretome.

Afterwards, Glioma cells were divided in two distinct groups. On one hand, glioma cells GL-261 were grown

under the influence of the mature adipocytes secretome were designated conditioned glioma cells (CGl). On

the other hand, the control group consisted in the glioma cells cultured in the absence of 3T3-L1 conditioned

medium and was designated as non-conditioned glioma cells (NCGl).

Sample preparation for 2D-PAGE

Sample preparation for 2D-PAGE was performed according to published procedures with minor modifications

26. The cells were manual detached from the flask and were mechanically lysed at 4oC in lysis solution [CHAPS

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4%, 5 mM Tris, pH 8.8, 0.05% protease inhibitor cocktail (Sigma)] using a glass potter, and the samples were

centrifuged at 2000 g for 15 minutes at 4oC in order to eliminate aggregates. The protein concentrations in each

sample were measured by means of a DC Bio-Rad assay. A total of 900 mg of protein from each sample was

precipitated with cold acetone and resuspended in thiourea buffer (7M urea, 2M thiourea 2% CHAPS, 2%

ASB14, 5% glycerol, 40 mM DTT, 4 mM TCEP, 1% 3–10 IPG buffer, Amersham), and the samples were mixed

overnight at 4oC in the dark and clarified by centrifugation at 16000 g for 15 minutes at 4oC. The supernatant

was first separated by isoelectric focusing over a pH range of 3–10 using precast first-dimension drystrip 3–10

NL 11 cm (Bio-Rad) following a multi-step protocol for 90,000 Vht (Protean IEF cell, Biorad). The first dimension

strip was equilibrated in 50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 2% SDS plus 16 mM DTT for 20 minutes,

and then plus 25 mM iodioacetamide for 15 minutes, and loaded on a midi format (11 cm) 9–16% acrylamide

gel to separate the proteins by molecular weight. Second-dimension runs were performed using Biorad midi

cells at 30 V 1 h, 300 V 4 h at a constant temperature of 18oC. Protein spots were revealed using home-made

blue Coomassie staining, and the gel images were acquired by means of an Image scanner at 300 DPI

resolution and analysed using Image2D Master Platinum software (both from Amersham).

At least three gels per condition were included in the analysis. Normalized spot volume values were studied

using SPSS software version 13.0 for statistical analysis (SPSS Inc.). In brief, the data for each spot match set

were analysed using a box-plot test in order to eliminate outliers, a K-S test to check normal distribution, an F

test to analyze the variance, and finally Student’s T test to compare the mean values, which were considered

significant for a P-value cut off of 0.01.

In-Gel Protein Digestion

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In-gel digestion of the excised proteins with trypsin was performed according to published procedures with minor

modifications 27–29. Protein spots were destained for 10 min with a solution of acetonitrile 50% (v/v) in 25nM

NH4HCO3 and mixed vigorously using a vortex. After removal of the destaining solution, gel pieces were washed

with water for 10 min using vigorous vortexing. This step was repeated twice. Afterwards, the gel pieces were

dehydrated in acetonitrile for 10 min, which was then removed and the gel pieces were placed to dry in a vacuum

centrifuge. Reduction of protein disulfide bonds was performed with a solution 10mM of dithiotreitol (DTT) in

25mM NH4HCO3, for 15 min at 60 ºC. After cooling to room temperature for about 15 min, the DTT solution was

replaced with a solution 110mM of iodoacetamide (IAA) in 25mM NH4HCO3. Incubation with the alkylation agent

was performed for 35 min at room temperature in the dark. After reduction and alkylation steps, the gel pieces

were submitted once more to the washing and dehydration procedure with water and acetonitrile, in the same

way as described above. Subsequently, the gel pieces were completely dried in a vacuum centrifuge. The dried

protein spots were incubated with a solution of trypsin 25 ng/µL in 12.5 mM NH4HCO3 in an ice bath for 30 min

to rehydrate the gel and to allow enzyme penetration into it. In-gel protein digestion was performed at 37ºC

overnight. After collecting the supernatant to clean vials, further peptide extraction was performed by addiction

of a solution of trifluoroacetic acid 0.1% (v/v) in acetonitrile 50% (v/v) to the gel pieces and incubation at room

temperature with shaking for 10 min. This step was repeated twice. All extracts were pooled and evaporated to

dryness. The samples were re-suspended with 10 µL of trifluoroacetic acid 0.1% (v/v).

MALDI-TOF-MS analysis

Prior to MALDI analysis, the sample was mixed with an equal volume of the MALDI matrix solution, 10 mg/ml

α–CHCA in trifluoroacetic acid 0.1% (v/v) and acetonitrile 50% (v/v). An aliquot of the sample/matrix solution

lix

(0.5 µL) was hand-spotted onto the MALDI sample plate and the sample was allowed to dry. The mass

spectrometric analyses were performed using the Applied Biosystems MALDI-TOF-MS system model Voyager-

DE PRO Biospectrometry Workstation equipped with a nitrogen laser radiating at 337 nm (Applied Biosystems,

Foster City, USA) and the laser intensity was set just above the threshold for ion production. MALDI mass

spectra were acquired in positive ion reflectron mode, with an accelerating voltage of 20 kV, a grid voltage of

15 kV, 0.4 V of guide wire and an ion extraction delay of 100 ns. The MS spectra for each sample were based

on the average of 700 laser shots per spot with an acquisition rate of 2 ns. MS acquisition data was calibrated

externally using the ProteoMass Peptide MALDI-MS Calibration Kit.

Data analysis and database searching

All data was processed using DataExplorer 4.5 software from Applied Biosystems. Peptide Mass Fingerprint

(PMF) data was used to search for candidate proteins using the MASCOT database search

(http://www.matrixscience.com) engine. SwissProt database was selected by default for all Mascot searches.

NCBInr database was used each time no significant identification was obtained with SwissProt. Database

searches were, by default, performed with no taxonomy restriction and allowing up to a maximum peptide mass

tolerance of 100 ppm. The number of allowed missed cleavages for trypsin was set to one.

Carbamidomethylation of cysteine and methionine oxidation were selected as fixed and variable modifications,

respectively. In order to provide accurate results, protein identification was considered positive for MASCOT

protein scores higher than 77 (p<0.01) that present a minimum of 4 peptides matching.

Proteome Profiler™ Array Assay

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Determination of relative levels of pre-selected mouse adipokines in conditioned and non-conditioned GL261

cell cultures was performed by using Mouse Adipokine Array Kit (R&D Systems Europe, UK). Cells were rinsed

with PBS before Lysis Buffer solubilization at, approximately, 1 x 107 cells/mL. Then, they were ressuspended

and rocked at 4°C for 30 minutes, following a microcentrifugation at 14,000 x g for 5 minutes. After supernatant

transfer into a clean test tube, proteins were quantified a total protein assay. Array Procedure was performed

according to manufacture instructions (Catalogue Number ARY024). Data analysis was based on pixel densities

on developed X-ray film collected and analysed using Gel-Doc XR (Bio-Rad© Laboratories), acquired by Image

Lab Software 5.2.1 (Bio-Rad© Laboratories) then quantified by ImageJ 1.49 (NIH, USA).


The differences in expression of proteins between glioma medium and CGl by Proteome Profile Microarray were

analysed by 2-way ANOVA for multiple comparisons with Prism Graphpad. P-values of <0.05 were considered

statistically significant. MALDI-TOF-MS was conducted by T-test at P-value cut off of 0.1 using default

parameters with Prism Graphpad.

Results and Discussion

In order to understand which adipocyte secreted proteins potentially influenced glioma tumor behavior, we

analysed the protein expression of glioma cells conditioned medium obtained from adipocytes cultures, by

means of bidimensional polyacrylamide gel electrophoresis under denatured conditions (2D-PAGE) followed by

mass spectrometry analysis of the selected spots. These spots were analysed by matrix assisted laser

lxi

desorption ionization time-of-flight/mass spectrometry (MALDI-TOF-MS). The non-conditioned glioma cells

(NCGl) were used as the control group for the conditioned glioma cells (CGl).

Differentially Expressed Proteins

Eleven differentially expressed spots were identified among the 1192 matched protein spots. Its detailed

information is presented in Table 1.

The 5 over-expressed proteins found on the CG1 cells. RFC1 corresponds to spot-1 with 9.38 isoelectric point,

126705 relative molecular mass and presenting a 2.32 ratio. KIF5C corresponds to spot-2, with 5.86 isoelectric

point, 109777 relative molecular mass and 2.91 ratio. ANXA2 corresponds to a spot-3 protein spot, with 7.55

isoelectric point, 38937 relative molecular mass and 2.23 ratio. NRAP, corresponds to spot-4, with 9.34

isoelectric point, 196716 relative molecular mass and 2.82 ratio. RACK 1, corresponds to spot-5, with 7.60

isoelectric point, 35511 relative molecular mass and 2.07 ratio (Table 1). The 3 spots significantly under-

expressed in CGl corresponding to 3 proteins identified as follows: STI1, corresponds to spot-6 and it presents

a 6.4 isoelectric point, 63170 relative molecular mass and 0.33 ratio; hnRNP L, corresponds to spot-7 spot, with

8.33 isoelectric point, 64550 relative molecular mass and 0.66 ratio; PGK1, corresponding to spot-8, with 8.02

isoelectric point, 44921 relative molecular mass and 0.4 ratio.

Only 2 spots were present in NCGl control group were aldose reductase, corresponds to spot-9, with 6.71

isoelectric point, 36052 relative molecular mass, and carbonic anhydrase, corresponds to spot-10, with 6.45

isoelectric point, 30124 relative molecular mass.

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The only spot present in the CGl was Citrate Synthase that corresponds to spot-11, with 8.72 isoelectric point

and 51988 of relative molecular mass.


Intending to further elucidate the involvement of adipose secretome in glioma, we evaluated the profile of GL261

under conditioned medium (CGL). The medium proteome is a desirable sample due to their accessibility and

representability due, in part, to the wide dynamic range of protein concentrations, which lead to the discovery

of new protein markers (Figure 1).

Figure 1 - Expression proteomic profile of GL261 with and without the effect of the secretome of 3T3-L1 mature

adipocyte cells.

As illustrated in Figure 1 different secretion patterns of numerous growth-factors, adipokines, cytokines and

angiogenesis-related molecules, were found between the different media. Endocan, IGFBPs, FGF acidic,

vascular endothelial growth factor (VEGF), among many others were found upregulated in GL-261 conditioned

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medium. Nevertheless, a significant reduction in adiponectin, serpin E1 and TIMP-1 levels were observed in

CGl (p≤ 0.01).

Conclusion

Adipose tissue is defined as a lipid store depot, inflammatory and endocrine organ 49. Thus, it provides a chronic

low grade inflammatory condition potentiated by the presence of multiple cytokines and hormones, sometimes

associated with disease. Most part of these factors are known to play a role in cell proliferation, apoptosis,

migration and invasiveness, as well as adhesion capacity, becoming expected to be overexpressed in

conditioned medium. Recent findings point that inflammatory, angiogenic or metabolic factors, significantly

influence tumor behavior 50,51. Taking into account in one hand the morbidity and the mortality associated to

gliomas and on the other hand the increased prevalence of obesity and its possible role in cancer, studies that

elucidate this relationship are of paramount importance in order to identify new glioma biomarkers, to grade

specific protein signatures to provide treatment efficacy, and to identify novel effective drug targets against

malignant glioma 52.

In the current study we analysed the proteome profile of a cellular model of brain cancer GL261 under the

presence of adipocyte secretome. Our general results suggest an increase of viability, metabolic fitness,

proliferation of tumor cell and reduced apoptosis. However, we are going to approach it by focusing on their

relationship within inflammation and angiogenesis.

lxiv

The proteomic analysis has allowed the identification of eleven well differentiated proteins that are secreted by

3T3-L1 adipocytes with a potential role in cancer biology. So we can see in table 1, it shows that we have over,

under and partially (only in one condition) expressed proteins.

65

Table 5 - Results retrieved for MALDI-TOF-MS/PMF queries in Mascot database search engine. Differential protein expression (> 2.0 fold up-regulated

and < 0.5-fold down-regulated in CGl/NCGl) of a total of 192 protein spots.

Protein

spot

Mascot

MOWSE

score 1

N matches

(n queries)

% sequence

coverage2

Ratio3 Protein ID Function

Spot-14 69 14 (43) 14

2.32

(↑)

Replication factor C subunit

(RFC1)

Clamp loader in eurcarioric replication. Role in

maintaining the stability and the integrity of the

genome 30.

Spot-2 80 25 (122) 25

2.91

(↑)

Kinesin heavy chain isoform

5C (KIF5C)

Kinesin motor protein present in motor neurons

and associated with trafficking of mitochondria and

vesicles 31,32.

66

Spot-3 102 20 (107) 52

2.23

(↑)

Annexin A2 (ANXA2)

Phospholipid-binding intracellular protein. Involved

in anti-inflammatory and anticoagulation, cell

proliferation and differentiation and apoptosis 33–35.

Spot-4 90 37 (109) 24

2.82

(↑)

Nebulin-related-anchoring

protein (NRAP)

Known for cytoskeletal stability, cell migration and

protein scaffolding 36.

Spot-5 125 14 (67) 53

2.07

(↑)

Guanine nucleotide-binding

protein subunit beta-2-like 1

(RACK1)

Intracellular adapter protein in signal-tranuction

pathways. Influences cell proliferation, migration,

adhesion and spreading and protein synthesis 37,38.

Spot-6 100 18 (68) 35

0.33

(↓)

Stress-induced

phosphoprotein 1 (STI1)

Co-chaperone in Hsp70 and Hsp90 folding and

protein transcription, signal transduction and cell

division. Associated with cell proliferation and

invasion 39.

67

Spot-7 93 17 (65) 35

0.66

(↓)

Heterogeneous nuclear

ribonucleoprotein L (hnRNP

L)

Central role in RNA metabolism. Also implicated in

cell proliferation and invasion 40,41.

Spot-8 93 21 (128) 61 0.4 (↓)

Phosphoglycerate Kinase 1

(PGK1)

Catayzes production one molecule of ATP during

glicose breackdown 42.

Spot-9 110 14 (72) 48

Only in

NCGl

Aldose reductase (AR)

Metabolic reduction of glucose to sorbitol and

catalyzes reduction of ROS and lipid peroxidation,

increasing mutagenesis risk 43,44.

Spot-

105

79 10 (54) 40

Only in

NCGl

Carbonic anhydrase (CA)

Metalloenzyme participating in respiration,

calcification and acid-base balance. Product of

HIF-1α, overexpressed in hypoxic solid tumors

45,46.

68

Spot-

114

64 8 (58) 17

Only in

CGl

Citrate synthase,

mitochondrial (CS)

Catalyzes mytochondiral reactions and it is

considered to play role in cancer cells metabolism

regarding metabolic reprogramming 47,48.

1 scores greater than 77 are significant for p<0.01

2 represents the percentage of the protein's sequence represented by the peptides identified in the MS run

3Ratio = CGl/NCGl (CGl – conditioned glioma cells, NCGl – non-conditioned glioma cells)

4 results obtained by limiting the research to entries from Mus musculus. In this case, scores greater than 62 are significant for p<0.01.

5 result obtained allowing up to 2 missed cleavages.

lxix

Our findings revealed lower expression of STI1, hnRNP L and PGK1 when glioma cells were exposed

to mature adipocytes conditioned medium. Regarding multiple effects of STI1, hnRNPs and PGK1

this under expression might reflect that the adipokines secreted by mature adipocytes might be able

to stop some important pathways in carcinogenesis, complemented with remodeling the oxidative

metabolism toward the utilization of fatty acids substrates instead of glucose. This interpretation may

be further supported by the increased expression of mitochondrial citrate synthase. Also, proteome

array profiling reveals under expression of TIMP-1, an inhibitor of ECM degradation, Serpin E1 and

adiponectin significant downregulation in conditioned medium, supporting adipokine results of

carcinogenesis development by adipocytes.

Additionally, data revealed that aldose reductase and carbonic anhydrase were present only in

glioma cells and that their expression was completely blocked when glioma cells were exposed to

mature adipocytes conditioned medium. Our findings suggested that this blockage may reflect a total

inhibition of aldose reductase and carbonic anhydrase, possibly associated to oxidative stress

response prevention, inhibition of inflammation and cancer proliferation, angiogenesis and

carcinogenesis, which may suggest some kind of protective mechanism related to obesity.

Considering overexpressed proteins in conditioned medium, RFC1, KIF5C, ANXA2, NRAP, RACK1

results were supported by overexpression of almost all the proteins profiled by arrays, comprising

angiogenesis, growth, metabolic and hormonal functions. Our analysis revealed that ANXA2

expression (2.23 fold) was more pronounced when glioma cells were exposed to CGl. This finding is

lxx

in agreement with previous studies that also revealed that ANXA2 is elevated in glioma, in which it

plays a central role in cell motility, migration, invasion and angiogenesis 34,33,35.

Moreover, and considering proteome array profiling, endocan, which is a proteoglycan secreted by

endothelial cells and upregulated by proangiogenic factors, appears to be overexpressed in tumour

vessels 53,54. Considering its overexpression in CGl, it may explain the involvement of adipose tissues

in tumor aggressiveness. In addition, adipose tissue contains diverse cells types including adipose

stromal cells, which produce a myriad of angiogenic factors, VEGF, HGF, FGF-2, and TGF,

contributing to the increment of released angiogenic and growth factor cytokines.

Thru microarray analysis we further identified the factors that are released by adipocyte cells, and

revealed that several pro-inflammatory factors (IL-6, IL-11, LIF, PAI-1, TNF-α), metabolic markers

(FGF-21, IGFBPs), angiogenic growth factors (endocan, HGF, VEGF IGF-I), and hormones (leptin,

resistin, RBP-4) were secreted to the medium into a high extent, whereas TIMP-1, an inhibitor of

ECM degradation, SerpinE1, wich encodes Plasminogen Activator Inhibitor 1 (PAI-1), were

underexpressed on CGl. Adiponectin, as it was expectable, was significantly downregulated in CGl

medium when compared with NCGl. Adiponectin modulates inflammatory responses, energy

expenditure in the CNS and periphery, glucose regulation and fatty acid catabolism. Its circulating

levels inversely correlate with insulin resistance, metabolic syndrome, obesity and cardiovascular

diseases. Curiously, different hormones related with insulin resistance and obesity, including TNF-α

and IL-6, downregulate adiponectin expression and secretion in fat cells in vitro 55,56. Our results on

lxxi

TNF-α (adipocytokine implicated in inflammation, apoptosis and cell survival), IL-6 (related to

proliferation in human glioblastoma cases, leading to poor prognosis) and IL-11, corroborate these

inverse relationship with adiponectin 57.

Endocan, as well as FGF-21, are extensively present along with pro-inflammatory and proangiogenic

molecules. It is associated with a vast number of pathological conditions where endothelium gets

highly vascularized cancers, making it a likely marker of the vascular growth and angiogenesis during

cancer evolution 58,59. These data support our findings in which endocan is overexpressed in CGl.

Moreover, and regarding other growth factors, FGF-21, HGF and IGFBPs, they all were

overexpressed in CGl. Hepatocyte Growth Factor (HGF), a multifunctional trophic factor activates a

tyrosine kinase signaling cascade, is elevated in obese adults and adolescents. In vitro, HGF

secretion from adipocytes of obese compared to lean individuals is greater 55,60.

lxxii

AgRP Leptin

Resistin RBP-4 IGF I IGF II

Adiponectin

Endocan HGF VEGF

RANTES RFC1 KIF5C

ANXA2 FGF acidic

FGF-21 IGFBPs

IGF I IGF II

TIMP-1

ST1

DPPIV Fetuin-A

FGF acidic FGF-21 IGFBPs PREF-1

Citrate Synthase

Serpin E1 Carbonic

anhydrase Aldose reductase

IL-6 IL-11 LIF

TNF-α Lipocalin

ANGPT-L3 RACK1 N-RAP

Serpin E1 hnRNPs

PGK1

Exp

ress

ion

un

der

Ad

ipo

cyte’s

Se

cre

tom

e

Exp

ress

ion

in t

he

abse

nce

of

A

dip

ocy

te’ s

Sec

reto

me

ENDOCRINE GROWTH METABOLIC ANGIOGENIC INFLAMMATORY

FGF acidic FGF-21

HGF IL-6

IL-11 LIF

TNF-α VEGF

Serpin E1 TIMP-1

lxxiii

Figure 2 - Proposed classification of molecules analysed by microarrays considering its cellular and

molecular functions. according to over and underexpression in different contexts.

Development and basic functions of organs and tissues relay on angiogenesis, and angiogenesis

role in adipose tissue development and obesity is crucial. Additionally, a great number of studies

point that an insufficiency in adipose tissue angiogenesis contributes to metabolic disease in obesity

61,62. These data from the literature reinforce our findings concerning the effect of conditioned medium

from adipose tissue in augmenting GL261 cell proliferation, migration, ability to invade and

decreasing apoptosis.

Concluding remarks

In conclusion, the present study presents a paradoxical relationship between obesity and cancer

(Figure 3). In a first view, conditioned medium (adipokines-enriched medium) may present a

protective effect against glioma, since STI1, hnRNPs and PGK1 that are generally overexpressed in

several types of cancer are under expressed. Similarly, both carbonic anhydrase and aldose

reductase that play an important role in cell physiology, inflammation and cancer metabolism are

even suppressed in glioma cells that grown under adipokines-enriched environment.

lxxiv

Contrariwise, RFC1, KIF5C, ANXA2, N-RAP and RACK1 that are generally involved in the matrix

remodeling, proliferation, migration, invasion of cancer cells is overexpressed in GL261 cell the in

the presence of the adipokines-enriched medium. Furthermore, along with these and CG1, and as

long as we can observe on protein secretome assay, our results unfolded the damaging effects of

adipocytes secretome towards glioma aggressiveness. Also the presence of citrate synthase only in

glioma cells cultured under conditioned medium and the decreased expression of PGK1 supports

the non-tested hypothesis that under an obesity environment, glioma cells may reshuffle their

metabolic patterns towards increased lipid consumption for aerobic ATP production over glucose

and other simple sugars.

Furthermore, our MALDI-TOF and proteomic array data suggests that, on a primary phase, obesity

contributes for decreasing of the tumor initiation. Even though, as time goes by, studied growth

factors positively influence proliferation and restricted tumorigenic growth. However, as we can

observe from diminished growth and hypoxic factors, and different metalloproteases, suggesting that

previously tumor proliferation due to obesity are restricted and confined, not metastizing. Thus, our

data reinforce the idea on obesity protecting against glioma initiation and metastization (Figure 3).

lxxv

Figure 2 - Influence of adipocyte secretome in glioma tumorigenesis. Under the influence of the

adipocyte secretome some proteomic alterations promote tumorigenesis (pink circles) and while

other alterations seem to be protective (green circles).

This study undoubtedly requires additional work to fulfill our understanding on the implication of

obesity in malignant brain tumors biology. Meanwhile, it discloses an interesting in vitro model for

the study of glioma biology under a “obesity” environment, that can be explored for the understanding

of cancer cells biology, for the search of biomarkers, prognostic markers and therapeutic

approaches.

Acknowledgments

Fernandes R. on the behalf of the authors deeply acknowledge Prof. Pedroso Lima, CNC, Coimbra,

Portugal by her kindness in providing the GL261 cells.

Costa J. was supported with an ERASMUS grant. Portuguese Government supported Coelho P. with

a PhD grant by FCT n. SFRH/BD/80434/2011. Sala C. was supported by AIRC grant n. 10807 and

lxxvi

PNR-CNR Aging Program 2012-2014. The project was partially supported by FCT (Strategic Project

Reference: UID/BIM/04293/2013).

Conflicts of interest

None to declare.

lxxvii

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