Tumor Microenvironment in Head and Neck Squamous Cell ... · Col15-binding integrins, PGE2 Glycolytic: (MCT4 ...

Tumor Microenvironment in Head and Neck SquamousCell Carcinoma

Joseph M. Curry,a John Sprandio,b David Cognetti,a Adam Luginbuhl,a Voichita Bar-ad,c

Edmund Pribitkin,a and Madalina Tulucd

The tumor microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) is

comprised of cancer-aGenetic changes in the

expression profiles, co

increased ROS, overprCAFs are among the m

metastasis. The adaptiv

cytokines, triggered aOverexpression of crit

to EMT, immune supp

forces in angiogenesmetabolism to fuel tu

cells of the TME. Incr

notion of “condemnedentire tissue comprise

Semin Oncol 41:217-2

0093-7754& 2014 Elshttp://dx.doi

Conflicts o

aDepartmenJefferson

bDepartmePhiladelp

cDepartmenPhiladelp

dDepartmePA.

Address coOtolarynversity,edu

Seminars

ssociated fibroblasts (CAFs), immune cells, and other supporting cells.carcinoma cells, such as alterations to TP53, NOTCH1, and specific gene

ntribute to derangements in cancer and microenvironment cells such as

oduction of cytokines, and epithelial to mesenchymal transition (EMT).ost critical elements of the TME contributing to proliferation, invasion, and

e immune response is suppressed in HNSCC through overexpression of

poptosis of T cells, and alterations in antigen processing machinery.ical cytokines, such as transforming growth factor-β (TGF-β), contributesression, and evolution of CAFs. Inflammation and hypoxia are driving

is and altered metabolism. HNSCC utilizes glycolytic and oxidativemorigenesis via coupled mechanisms between cancer cell regions and

eased understanding of the TME in HNSCC illustrates that the long-held

mucosa” reflects a process that extends beyond the epithelial cells to thed of each of these elements.

34 & 2014 Elsevier Inc. Open access under CC BY-NC-ND license.

Squamous cell carcinoma comprises more than90% of cancers of the head and neck and arises

from the squamous lining of the mucosal surfa-

ces of the upper aerodigestive tract, including the oralcavity, pharynx, larynx, and sinonasal tract. Head and

neck squamous cell carcinoma (HNSCC) is the sixth

most common cancer worldwide, and only 50%–60%of patients are alive at 5 years after diagnosis.1,2

Treatment can be quite morbid and result in significant

functional as well as aesthetic deficits, such as impair-ment of speech and swallowing and facial deformity.

evier Inc..org/10.1053/j.seminoncol.2014.03.003

f interest: none.

t of Otolaryngology Head and Neck Surgery, ThomasUniversity, Philadelphia, PA.

nt of Medical Oncology, Thomas Jefferson University,hia, PA.t of Radiation Oncology, Thomas Jefferson University,hia, PA.nt of Pathology, Thomas Jefferson University, Philadelphia,

rrespondence to Joseph M. Curry, MD, Department ofgology Head and Neck Surgery, Thomas Jefferson Uni-Philadelphia, PA 19107. E-mail: joseph.curry@jefferson.

in Oncology, Vol 41, No 2, April 2014, pp 217-234

Open access under CC BY-NC-ND license.

Treatment failure and locoregional recurrence arecommon and occur in up to 30% of patients and

account for the majority of deaths.3 The high rate of

local recurrence produced the long-held notion of“condemned mucosa” or “field cancerization” initially

described in the 1950s.4 This concept underscores not

only the difficulty in treating HNSCC but also denotesthe complexity of the molecular conditions under

which HNSCC develops and recurs. It is clear that

the notion of the condemned mucosa reflects a“condemned tissue” composed of the cancerous cells,

adjacent epithelial, stromal, and immune cells and their

surrounding matrix. Together these elements comprisethe tumor microenvironment (TME). In fact, this shift

in thought from the concept that cancer is derived

from a single cell type, to a disease occurring in acomplex tissue, has led some investigators to suggest

that the very definition of carcinoma be changed.5

Tumorigenesis requires multiple elements outlinedby Hanahan and Weinberg: (1) limitless replicative

potential, (2) self-sufficiency in growth signals, (3) insen-

sitivity to anti-growth signals, (4) ability to evadeapoptosis, (5) increased angiogenesis, and (6) invasion

and metastasis.6 Knowledge of the mechanisms

through which the cancer cells use the TME to executethese processes continues to evolve.7,8 There is great

interest in the downstream paracrine interactions with

217

J.M. Curry et al218

the stroma, immune interactions, and metabolic

changes and the role each plays in tumorigenesis.HNSCC is genetically heterogeneous, but a num-

ber of pathways have been found to be commonly

involved; the impact of several critical abnormalitieson the TME is highlighted below. The cellular

elements of the TME often coevolve with the tumor.

Stromal fibroblasts, T cells, macrophages, and othercell types develop abnormal phenotypes in a disor-

ganized response to the cancer (Figure 1). These non-

cancerous cells provide many of the paracrine signalsnecessary to turn on the pleotrophic abilities of

cancer cells.9 For example, fibroblasts become

cancer-associated fibroblasts and secrete factors suchas matrix metalloproteins (MMPs), contributing to

tumor invasiveness. Furthermore, as the chronic

inflammation of the TME remains unresolved, alter-ations in adaptive immune response such as apopto-

sis of cytotoxic T cells and activation of suppressor T

cells occurs.10 Additionally, tumors reprogram theirsurroundings creating a metabolically fertile environ-

ment to meet their high energy and anabolic require-

ments. This process was aptly described by Paget asthe “seed and soil” hypothesis.11 Fundamental tumor–non-tumor microenvironmental interactions such as

these represent potential points of intervention fortherapeutic strategies. Many critical targets, such as

nuclear factor-κB (NF-κB), hypoxia-inducible factor

(HIF)-1α, and vascular endothelial growth factor(VEGF), have been, and continue to be, explored as

therapeutic targets in the TME12–14 (Table 1).

IMPACT OF GENETIC AND EPIGENETICCHANGES OF THE EPITHELIUM ON THE TME

The initiating genetic alterations in the epithelial

cells of HNSCC are primarily the result of thecarcinogenic properties of tobacco and alcohol, and

in the oropharynx, oncogenic strains of the human

papilloma virus (HPV). Classically, HNSCC has beenthought of as a disease caused by tobacco and

alcohol, yet tobacco-related cancers are decreasing

Figure 1. Select elements and interaciotns of the TME. Theperivascular niche that commonly contain cancer stem cells ancompartment contains tumor cells that are glycolytic and less proDDR is shown between the leading edge tumor edge and normaadjacent to normal stroma and fibroblasts (yellow). CAFs expreprotein inducer (EMMPRIN) on cancer cells to activate MMP2activate TGF-β. CAFs and tumor cells produce elements like VEGinteractions between regulatory T cells (pink), cytotoxic T cells (r(blue/green) are shown. TGF-β and IL-10 produced by TAMs anMIF that recruits neutrophils. Regulatory T cells induce toleracytotoxic T cells in utlitzed by cancer cells to induce apoptosincrease angiogenesis and invasion by production of MMP-9, Vorange) are recruited to the TME by GM-CSF produced by cancTGF-β.

in incidence.15 Over the past several decades, onco-

genic strains of HPV have become apparent as anetiology for oropharyngeal squamous cell carcinoma

(OPSCC). HPV-related OPSCC accounts for up to 60%

of cases of oropharyngeal cancer in some regions;this has resulted in an increased incidence among

younger nonsmokers, and has been equated to an

epidemic by some investigators.16 Currently, this isthe second most common malignancy caused by

HPV.17 OPSCC is caused primarily by HPV16 (but

also HPV18, HPV31, and others), via the E6 and E7mechanisms established in cervical cancer.18

The most widely identified mutation in non–HPV-related HNSCC occurs in the tumor-suppressor geneTP53. This has been identified to occur in approx-

imately 50% of HNSCCs and is likely an early event, as

it is commonly found in premalignant lesions aswell.19–21 Mutations also have been shown to corre-

late with aggression and poor outcomes; for exam-

ple, p53 mutations have been found in 95% ofradioresistant tumors.22–24 Histologically negative

margins with p53 mutations have been shown to be

associated with a greater incidence of local recur-rence.25 Mutation of TP53 in tumor cells is associated

with increased migration of cancer-associated fibro-

blasts (CAFs) to the TME, while intact TP53 inhibitsmigration.26 Loss of functional p53 increases reactive

oxygen species (ROS) and reactive nitrogen species

(RNS) and may drive carcinogenesis via NF-κB andother inflammatory-mediated mechanisms. Altera-

tions in TP53 induce a DNA damage response

(DDR) in adjacent non-tumoral cells via productionof ROS. This effect was recently demonstrated in

esophageal SCC, and it increases with proximity to

and size of the primary tumor, with effects beingidentified several centimeters from the tumor.27–30

TP53 mutations also have been linked to abnormal

tumor metabolism, contributing to the Warburgeffect through increased activity of glucose trans-

porters and glycolytic enzymes furthering the pro-

duction of an acidic environment and high levels ofROS toxic to normal cells31 (Figure 1).

tumor is shown here with the leading tumor edge andd highly replicating tumor cells (blue). The more centralliferative (orange). Peritumoral epithelium demonstratingl epithelium. CAFs (purple) are shown in the tumor stromass MT-MMP that interacts with extrcellular metallomatrix. CSCs express CD144, which interacts with MMP9 toF, PGE2, and CXCL12 that trigger angiogenesis. Immuneed), M2 TAMs (green), and tumor-associated neurtrophilsd cancer cells suppress T-cell activity. TAMs also producence by cytotoxic T cells. The Fas receptor on activatedis. Tumor-associated neutrophils produce ROS, and alsoEGF, and HGF. CD34þ myeloid progenitor cells (yellow/er cells which in turn induce immunosuppression through

Tumor microenvironment in HNSCC 219

NOTCH1, the second most commonly mutated

gene in HNSCC occurring in approximately 15% ofcases, functions as a tumor-suppressor.21 It encodes a

transmembrane receptor that regulates cell differ-

entiation and embryonic development.21,32 InHNSCC, it is dependent on intercellular signaling in

the TME and contributes to proliferation and invasive-

ness through the pro-inflammatory cytokine, tumornecrosis factor-α (TNF-α). This TNF-α mechanism acts

on Slug and Twist, two other important transcription

factors that act as regulators of invasion and epithelialto mesenchymal transition (EMT).9,33 Evidence also

Table 1. Critical Cells of the TME in HNSCC

Cell Type Markers Secreted Factors Metabolism References

Squamous Cell CarcinomaKey Genetic Alterations: TP53,NOTCH1, EGFR, CDKN2a, STAT3,Cyclin D1, Rb

E-cadherin, cytokeratins,PD-L1, FasL

MMP 2, MMP 9, MMP 13,ROS,VEGF, CXCL1,CXCL8, PDGF, IL-8,FGF-2, TGF-β, TNF-α,IL-1, GMCSF

Tan et al,Koontongkaew S,Zhang Z et al,Smith A et al,Curry J et al, Feron O.

Central tumor compartment Glycolytic: (MCT4þ,MCT1�, TOMM20�,COX�)OXPHOS: (MCT1þ,MCT4-,TOMM20þ,COXþ)

Leading edge/invasive front,perivascular niche (proliferativecancer cells: high Ki-67)

Cancer stem cells CD33, CD144, ALDHEpithelial to mesenchymal

transitionN-cadherin, vimentin

Cancer-Associated Fibroblast α-SMA, integrin α6 HGF, CXCL12, TGF-β,MMP2, MMP9, PMF,PDGF, Type IV collagen,Col15-binding integrins,PGE2

Glycolytic: (MCT4þ,MCT1-LDH-Bþ)

Leef G, Curry J et al,Wheeler SE et al,Marsh D et al

Tumor-Infiltrating LymphocytesRegulatory T cells CD4þCD25þFoxP3þ IL-10, IL 12, TGF-β Young MR, Ferris RL

et al, Whiteside TLCytotoxic T cells CD8þ, TCR, Fas, PD-1 Perforin, granzymes,granulysin

Th2 suppressor cells CD4þ IL-4, IL-6, IL-10Myeloid progenitor cells CD34þ TGF-β

Tumor-Associated Macrophages(M2)

IL-10, TGF-β, MIF, EGF, CSF-1, MMP9, CXCL2,CXCL8,VEGF, ROS, RNS, PGEs

Lago Costa N et al,Dumitru C et al,Galdiero MR et al

Tumor-Associated Neutrophils MMP9, VEGF, HGF, elastase,ROS, PGEs

Galdiero MR et al,Dumtru C et al

Endothelial Cells Endothelins, CXCL1, CXCL8 Neiva KG et alAbbreviations: Rb, retinoblastoma gene; EGFR, epidermal growth factor receptor; CDKN2a, cyclin-dependent kinase inhibitor 2a; STAT 3, signal transducer and activator of transcription 3;

PD-L1, programmed death ligand-1; FasL, Fas ligand; MMP, matrix metalloprotein; ALDH, aldehyde dehydrogenase; ROS, reactive oxygen species; VEGF, vascular endothelial growthfactor; IL, inteleukin; FGF,fibroblast growth factor; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; PDGF, platelet-derived growth factor; GM-CSF, granulocye-macrophage colony-stimulating factor; EGF, epidermal growth factor; CSF-1, colony-stimulating factor-1; TCR, T-cell receptor; FoxP3, forked/winghead transcription factor; RNS, reactivenitrogen species; MCT, monocarboxylate transporter; PGE, protaglandin; PD-1, programmed death-1; TOMM20, translocase of outer mitochondrial membrane 20; COX, cytochrome Coxidase complex; LDH-B, lactate dehydrogenase B.

J.M.Curry

etal

220

Tumor microenvironment in HNSCC 221

has suggested that its activity is mediated through

MMPs and the inflammatory transcription factor, NF-κB, among other critical mechanisms34,35 (Figure 1).

EGFR is a membrane-bound tyrosine kinase recep-

tor that binds epidermal growth factor (EGF) and TGF-α. It is the target for the effective and widely used

monoclonal antibody, cetuximab. Mutation of the

EGFR gene is only present in about 10% of cases,but gene amplification is present in about 30% of cases

and overexpression has been identified in up to 90%

of cases. Increased expression and gene copy numbercorrelate with poor prognosis.36–39 After binding

one of its ligands, EGFR triggers multiple intracellular

signaling cascades that activate cell proliferation,survival, invasion, metastasis, and angiogenesis.21,40–43

It also allows for decreased response to radiotherapy

by enhancing proliferation, DNA-repair, and hypoxicresponses within the TME.44–46 Activation triggers

increased interleukin-8 (IL-8) and VEGF production,

promoting inflammation and angiogenesis.47,48

Cyclin-dependent kinase inhibitor 2a (CDKN2a) is

an important tumor-suppressor that is mutated in

9%–12% of HNSCC patients. The function of itsproduct, p16, is to block cell cycle progression from

the G1 to S phase, and is typically upregulated under

stress conditions in the cellular microenvironmentsuch as hypoxia.49 Mutations are common but alone

likely to be insufficient to result in tumorigenesis.21

Mutational loss of function correlates with a wors-ened prognosis, while overexpression of p16 is

common in HPV-related OPSCC and correlates with

an improved prognosis.45,48,50

Signal transducer and activation of transcription

(STAT) proteins are transcription factors that are

commonly overexpressed in cancer, and in HNSCC,STAT3 has been found to be commonly mutated and

overexpressed.5 STAT3 activation is linked to numer-

ous pathways, including TGF-β, IL-6, and EGFR, andit is involved in EMT, proliferation, apoptosis, and

inflammation.51 STAT3 is also central to mainte-

nance of self-renewal in cancer stem cells (CSCs).52

The STAT/JAK pathway is one of the critical targets

of cetuximab.53 Numerous other abnormalities have

been found to be prevalent in HNSCC; Tan et al hasaddressed many of these in an excellent review.21

Given the great variety of genetic abnormalities

that have been identified in HNSCC, gene expressionprofiles may offer greater accuracy for characteriza-

tion and diagnosis than analysis of single loci. Several

groups have established profiles that can differentiateHNSCC from surrounding normal tissues. For exam-

ple, in a early study Chung et al identified four

subtypes of SCC based on gene expression profiles,each with different survival and recurrence rates.54

They identified patterns that they classified as

(1) EGFR pathway subtype, (2) mesenchymal-enriched subtype, (3) normal-epithelium–like

subtype, and (4) high-antioxidant enzyme subtype.

The EGFR group had the worst outcome. The secondsubgroup had a high fibroblast component and

demonstrated evidence of EMT. The third group

demonstrated gene expression closest to normaltonsillar epithelium and had the best outcome. The

fourth group demonstrated patterns similar to that

induced by exposure to cigarette smoking with highlevels of antioxidant enzymes being expressed.54

Clatot et al recently published a series in which they

used high-throughput reverse transcriptase polymer-ase chain reaction to create a nine-gene model with

which they were able to classify patients with 90%

accuracy. Those in a cluster with higher expressionof the chemokine CXCL12 had significantly greater

disease-free survival compared to those in a low-

expression CXCL12 cluster. Among these nine genes,a high-fold change in survival was seen in the group

comprised of CXCL12, SCL16A4 (monocarboxylate

transporter 4, MCT4), and carbonic anhydrase IX(CA9). CXCL12 is an important cytokine in HNSCC

implicated in angiogenesis and other processes.55

SCL16A4/MCT4 is a lactate transporter that has beenshown to be overexpressed in response to hypoxia.

CA9 is also upregulated by hypoxia and functions to

regulate intracellular pH.56

A number of epigenetic changes have been found

to be common to HNSCC, including DNA methyla-

tion, histone modification, microRNA interference,and small interfering RNA. Epigenetic regulation such

as methylation of CDK2a and other genes has been

shown to occur.57 Methylation of death-associatedprotein kinase (DAPK) is associated with resistance

to anti-EGFR agents, like cetuximab.58 Jung et al

performed a combined analysis of the transcriptome,methylome, and miRNome of metastatic HNSCC and

non-metastatic HNSCC and identified a signature that

correlated with lower survival and metastatic pheno-type. The pathways involved in this group were

specifically related to cell–cell adhesion, EMT,

immune response, and apoptosis. For example, theyidentified decreased expression of desmoglein 3 (DSG

3), a component of desmosomes critical for cell–celladhesion. Desmosomes also have been shown to havetumor-suppressor function, and decreased expression

of DSG3 has been linked to a poor prognosis.59 They

also identified several elements significant in EMT,including upregulation of vimentin and downregula-

tion of cytokeratin intermediate fibers and activation

of TGF-β–related EMT pathways. Analysis of miRNAdemonstrated upregulation of pathways related to

DDR and immune response.60

CANCER-ASSOCIATED FIBROBLASTS

Normal squamous mucosal lining of the upperaerodigestive tract is organized into distinct

J.M. Curry et al222

compartments: the upper layer of differentiated

squamous or respiratory epithelial cells, a basalepithelial layer, the underlying basement membrane,

and stromal layer. Fibroblasts are abundant in the

stroma and are the primary element responsible forsecretion of the basement membrane proteins. They

secrete structural proteins such as type IV collagen

and laminin and also produce numerous cytokinesand paracrine signals. Accordingly, tumor- or cancer-

associated fibroblasts (CAFs) are among the most

critical cellular elements of the TME. CAFs arephenotypically altered fibroblasts, which are active

participants in the process of tumorigenesis, promot-

ing growth and metastasis.61

CAFs arise from the population of circulating

fibroblasts and co-evolve with the tumor developing

a distinct phenotype, and playing an active role incarcinogenesis.62–64 They produce a variety of con-

tractile proteins, giving them an “active” phenotype.Frequently, they demonstrate ultrastructural accu-mulation of α-smooth muscle actin (α-SMA), charac-

teristic of myofibroblastic (MF) differentiation.65–67

In HNSCC, CAFs frequently have this MF phenotypeand are associated with dense collagen deposition

and stromal desmoplasia.68,69 CAFs are also charac-

terized by expression of integrin α6, which is criticalto cell adhesion and surface signaling. It complexes

to bind laminins, components of the extrcellular

matrix, and interacts with CDKN1A, altering cellcycle progression. Lim et al demonstrated that

upregulation of α-SMA and integrin-α6 correlated

with worsened prognosis in oral cancer.70

CAFs express a variety of factors critical to

carcinogenesis, promoting cell motility by upregula-

tion of cytokines, such as paracrine motility factor,hepatocyte growth factor (HGF), CXCL12, and

TGF-β.71 HGF secreted by CAFs has been shown to

promote invasion and angiogenesis in HNSCC andesophageal SCC.65,72–74 CXCL12 binds to CXCR4;

this interaction plays a role in upregulation of MMP9,

EMT, and HIF-1α expression.75 TGF-β is a criticalelement in the TME that serves numerous functions,

including immunosuppression. Additionally, CAFs

directly contribute to extracellular matrix remodel-ing by secreting MMPs.76,77

Marsh et al demonstrated that the MF phenotype

seen in some oral carcinomas was strongly prognos-tic of a negative outcome.78 This study evaluated 282

oral HNSCC specimens and found that the presence

of MF stroma was the strongest prognostic variableassessed, as compared to surgical margins, extracap-

sular spread, and stage, among others. MF stroma

correlated with depth of invasion and with extrac-apsular spread in nodal metastasis. Interestingly,

tumor-containing lymph nodes with extranodal

spread were also surrounded with MF stroma. Inoral and lingual carcinoma cell lines, Lin et al were

able to demonstrate increased proliferation in asso-

ciation with CAFs.79,80 In a mouse model usingheterotopic injection of HNSCC cells with normal

fibroblasts or CAFs, Wheeler et al demonstrated that

HNSCC cells with CAFs resulted in increased growthof the primary tumor and nodal and distant meta-

stases compared to co-injection with normal

fibroblasts.61

CAFs are also critical to tumor metabolism. Recent

studies indicate that epithelial cancer cells may

derive nutrients from the CAFs via a coupled meta-bolic mechanism. Cancerous cells induce glycolysis

in adjacent stromal cells such as CAFs and then use

their high-energy byproducts, such as lactate andpyruvate.81 This is somewhat contrarian to the long

held belief of the Warburg effect, whereby tumors

are thought to rely on aerobic glycolysis to produceenergy for rapid growth. This has been labeled the

“reverse Warburg effect” and has been shown to be a

critical prognostic indicator in breast and otherhuman cancers. There is some evidence that sug-

gests this occurs in HNSCC as well.82

THE IMMUNE RESPONSE IN THE TME

The persistent unresolved inflammation associ-

ated with cancer results in a eventual decay and

malfunction of the normal immune processes, whichin turn contributes to tumorigenesis through

immune tolerance and suppression and also to

angiogenesis and production of ROS. Essentially,tumorigenesis is at least in part a byproduct of a

failure of the immune system.10,12,83,84 The adaptive

immune response contributes in a variety of ways totumorigenesis through the immune interactions in

the TME involving T lymphocytes, macrophages,

dendritic cells, and others.85

T Lymphocytes

T lymphocytes are the central component of theanti-tumor response. They serve to initiate and regulate

the adaptive immune response and to elicit the cyto-

toxic response to tumors.85 There is evidence thatdysfunction occurs at the local, regional, and systemic

levels in HNSCC. While a strong lymphocytic host

presence at the tumor interface is indicative of anadaptive immune response and correlates with an

improved survival,86–88 dysfunctional circulating T cells

and tumor-infiltrating T cells have been identified inHNSCC, suggesting that tumors can suppress a previ-

ously intact local and systemic immune response.84,89–93 Moreover on a regional level, metastatic lymph nodesof HNSCC show significantly decreased levels of CD8þ

lymphocytes.87,94 Common functional deficits of

tumor-infiltrating T cells include: (1) absent or lowexpression of a key molecule in the signaling receptor

Tumor microenvironment in HNSCC 223

receptor chain (CD3ζ), (2) decreased proliferation in

response to mitogens, (3) inability to kill tumor celltargets, (4) imbalance of their cytokine profile, and

(5) evidence of profound apoptotic features.84

Evasion of the adaptive response is executedthrough a variety of mechanisms such as decreased

expression of major histocompatibility complexes

(MHC I) or induction of apoptosis in T cells.Decreased expression of antigen-processing machi-

nery such as MHC glycoprotein allows escape of

subpopulations of tumor cells by avoiding activationof cell mediated immunity.95–97 This mechanism has

been demonstrated in HNSCC whereby tumor cells

produce gangliosides, which downregulate MHC I.98

Another means of evading detection is to induce

apoptosis in cytotoxic T cells. The FasL receptor

mechanism is expressed by activated cytotoxic T cells,which bind to FasL and typically result in triggering

the cytotoxic response. However, this also predis-

poses the T cell to apoptosis. Oral SCC cells have beenshown to contain membranous FasL-positive vesicles,

which trigger induction of T-cell apoptosis, circum-

venting the cytotoxic response.84,85,95

The cytotoxic response also can be dampened by

suppression. Intratumoral cytotoxic CD8þ T cells in

HNSCC show increased expression of programmeddeath-1 (PD-1), a marker of suppressed function.87,99

Its ligand, programmed death receptor ligand-1 (PD-

L1), is a surface protein that blocks function ofT lymphocytes and is expressed on malignant oral

SCC cells and also on CAFs.100 Cho et al demon-

strated that increased PD-L1 expression resulted inincreased apoptosis of intratumoral CD8þ TILs.101

Moreover, cytokines like, TGF-β, IL-10, and others

allow local naı̈ve T cells to be triggered to becomesuppressor T cells, while also exploiting the suppres-

sive functions of existing regulatory T cells.102PD-1 is

of particular interest in HPV-associated HNSCC, as alymphocytic infiltrate is one of the common features

of HPV-related OPSCC. Infiltration of the TME by PD-

1–positive T lymphocytes was correlated withimproved prognosis.103 While this is contrary to the

above findings, in the case of HPV-related OPSCC, the

PD-1–positive T lymphocytes, likely reflect an acti-vated chronic immune response due to long-standing

viral infection.103

ANTIGEN-PRESENTING CELLS AND TUMOR-ASSOCIATED MACROPHAGES

Dendritic cells are specialized antigen-presenting

cells (APCs) common in the TME of HNSCC.84,98,104

They have a high a capacity for antigen capture andalso stimulate T-cell maturation. In contrast, when

exposed to TGF-β and IL-10, they can promote

immune tolerance and differentiation of CD4þ Tcells into suppressive regulatory T cells.84,105–107

Langerhans cells are APCs located within the skin

and mucous membranes of the upper aerodigestivetract. They detect antigens in the mucosa and then

migrate to regional lymph nodes where they initiate

a primary immune response. Some evidence sug-gests that greater infiltration of HNSCC tumor sam-

ples with Langerhans cells correlates with improved

prognosis.84,108–110

Tumor-associated macrophages (TAMs) are

present with varying frequency in tumors, and are

common in HNSCC. TAMs are classified into twovarieties: proinflammatory (M1) and suppressive

(M2). Accordingly, studies in various cancers have

shown that TAMs can be associated with positive ornegative prognosis. M1 TAMs contribute to the anti-

tumor immune response via the production of

proinflammatory cytokines IL-12, IL-23, andinterferon-γ.84,111–113 While the M2 TAMs appear to

accumulate near blood vessels, promote angiogene-

sis,114,115 and produce a variety of suppressivecytokines such as IL-10 and TGF-β. They also serve

to promote tissue remodeling and inhibit anti-tumor

cytotoxic effects of M1 TAMs.84,111–113,116 Data inoral SCC suggest that TAMs are largely of the M2

type, as tumors with high levels of TAM infiltration

correlate with higher stage, lymph node metastasis,and extracapsular spread.114,117,118 Lago Costa et al

demonstrated that macrophages were increased in

the TME and the peripheral blood in HNSCC, andthat samples with increased TAMs showed increased

levels of TGF-β and its correlated immunosuppres-

sive effects.119 They produce ROS, RNS, and prosta-glandins (PGs), all of which can contribute to

inflammation and tumorigenesis. COX2 inhibitors

and nitric oxide synthase inhibitors (iNOS) havebeen used to antagonize these inflammatory agents

and their cytokines.120,121 TAMs in HNSCC also

produce significant levels of macrophage migrationinhibitory factor (MIF), which is an inflammatory

cytokine that stimulates neutrophils. MIF recruits

neutrophils to the tumor via a CXCR2 mechanismand then by feedback mechanisms increases inva-

siveness of the tumor cells.122 Neutrophils act on the

tumor in a variety of ways: inducing genetic insta-bility via ROS, increasing angiogenesis via MMP9 and

VEGF, and increasing invasion via HGF.123

THE BASEMENT MEMBRANE, INVASION, ANDMATRIX METALLOPROTIENASES

The basement membrane is barrier to tumor pro-

gression, and its degradation facilitates tumor invasion

and metastasis. For this to occur, cancer cells must(1) develop motility, (2) alter cell–cell adhesion, and(3) remodel the ECM.124 The basement membrane not

only serves as a structural framework for the overlyingepithelial cells but also provides paracrine signals that

J.M. Curry et al224

affect their behaviors such as differentiation and migra-

tion.125 Many of the key elements of the basementmembrane, including collagen type IV and fibronectin,

have been shown to be disregulated in HNSCC. MMPs

are most important group of proteolytic enzymes usedby cancer to degrade the ECM. MMPs in normal tissues

are expressed in balance with their inhibitors to main-

tain a well-organized system. MMPs are upregulated byNOTCH1 pathways, EGFR, TGF-β, HGF, and

granulocyte-macrophage colony-stimulating factor (GM-

CSF), which are commonly overexpressed inHNSCC.126–129 Among the most commonly identified

metalloproteinases in HNSCC are MMP-2, MMP-9 and

membrane-bound MMP (MT-MMP). MMP-2 and MMP-9are gelatinases and degrade collagen type IV, the most

critical step in degrading the BM.130 Increased levels of

MMP-2 and MMP-9 correlate with increased nodalmetastasis and poor prognosis.48 MMP-9 is the most

structurally complex and can degrade numerous ele-

ments of the TME, including elastin, fibrillin, laminin,gelatin, and types IV, V, XI, and XVI collagen.131,132

MMPs were initially thought to be produced solely by

the tumor cell, but further investigation has shownproduction also by the CAFs and surrounding inflam-

matory cells.48,133,134 CAFs are primarily responsible for

the increased production of MMP-2 in co-culture experi-ments.130 MT-MMP is critical in activating MMP-2.135,136

There are numerous other significant MMPs, such as

MMP-13, which participates in angiogenesis increasingthe level of VEGF at the invasive front.137

Importantly, the functions of MMPs extend

beyond protein degradation and invasion, as theytarget growth factors, growth factor receptors, and

cytokines.8 For example, MMP-9 also produces a

tolerogenic effect on dendritic APCs and also onregulatory T cells.43 Release of MMP-9 results in

endothelial cell invasion and vessel formation.12,55

MMPs impact differentiation and maturation of bonecells into osteoclasts, which is critical to the process

of bony invasion.138 HNSCC CSCs are characterized

by expression of CD44; CD44 is a surface proteinthat functions as a receptor for hyaluronic acid and

also is the docking receptor necessary for MMP-9

function.139 Given the broad significance of MMPsthey represent a possible target for therapy directed

at the TME. Interestingly, quercitin, a flavonoid

isolated from onions, inhibits MMP-2 and -9pathways.140

TGF-b AND EPITHELIAL–MESENCHYMALTRANSITION

A number of chemokines and cytokines providecritical paracrine signaling in the TME; here we focus

on TGF-β, as it broadly impacts many cellular behav-

iors in the TME. TGF-β has both growth-promotingand -suppressive effects on cells, and for some time

the role of TGF-β in malignancy had been controver-

sial. It typically inhibits epithelial cell proliferationand promotes secretion of matrix proteins and

proteases. Currently it is understood to act as a

tumor-suppressor early in tumorigenesis, then in laterphases it enhances the malignant phenotype.141 TGF-

β primarily acts through the SMAD family of tran-

scription factors and works in concert with mitogen-activated protein kinases (MAPKs), which regulate

diverse cellular activities such as mitosis, differentia-

tion, proliferation, cell survival, and apoptosis. Dys-regulation of TGF-β in malignancy occurs through

several mechanisms, including loss of response to its

ligand, defects in the transduction pathway, andothers.141 Oral SCC has been shown to be resistant

to the suppressive effects of TGF-β, secondary to

downregulation of TGF-β receptor II (TBRII).141,142

TGF-β is a primary factor triggering EMT in

HNSCC. EMT contributes to invasion allowing for

enhanced mobility via expression of a protein expres-sion patterns more characteristic of a mesenchymal

phenotype. Once established, nests of metastatic

tumor can transition back to a phenotype recapitulat-ing the original tumor in a distant site. EMT is

mediated through disruption of epithelial cell junc-

tions, remodeling of the actin cytoskeleton, andupregulation of mesenchymal markers like vimentin

and firbonectin.141 TGF-β pathways as well as those

triggered by the inflammatory cytokines TNF-α andIL-6 converge upon STAT3, upregulating it.52,143,144

STAT3 proteins are commonly overexpressed in

HNSCC. STAT3 interacts with Twist, Snail, and Slug(Snail2), transcription factors that contribute to EMT

in various cancers.52,144,145 Twist increases expres-

sion of N cadherin, a marker of a mesenchymalmotile phenotype, and decreases expression of E

cadherin, a marker of an epithelial phenotype. Slug

also decreases expression of E-cadherin. Loss ofE-cadherin and gained expression of N-cadherin is

critical to invasion and is referred to as cadherin

switching.146 Prime et al were able to demonstratemorphologic evidence of EMT and cadherin switch-

ing after several days exposure to TGF-β.138,147

Emerging evidence suggests that EMT is fundamentalto gaining “stemness” or the transition of cancer cells

to becoming CSCs. CSCs are thought to serve as a

fountainhead for tumors as they give rise to theremaining population of tumor cells, and contribute

to treatment resistance. CSCs accumulate at the

invasive front and perivascular spaces and are demar-cated by expression of markers such as CD133 and

CD44, and by aldehyde dehydrogenase activity.139

On the surface of CSCs, CD44 interacts with MMP-9and this allows for proteolytic activation of TGF-β.148

TGF-β extends beyond the epithelial cancer cells of

a tumor, and many of the effects have been describedabove. Lewis et al showed that TGF-β produced at the

Tumor microenvironment in HNSCC 225

invasive leading edge of the tumor induced a MF

phenotype in primary fibroblasts. They also showedthat this effect resulted in secretion of HGF by

myofibrblasts, which in turn promoted invasion

through the basement membrane. TGF-β serves toinhibit TH1 lymphocytes and cytotoxic T lympho-

cytes and the functions of natural killer cells.84

ANGIOGENESIS, INFLAMMATION, ANDHYPOXIA

Small tumor deposits of 1–3 mm can be supplied

by diffusion of nutrients from the surrounding tissue;

beyond this, the tumor is dependent on angiogenesisto supply its needs.149 A number of studies have

shown that angiogenesis is correlated with tumor

aggression.48,150–154 HNSCC often has large hypoxicareas of tumor necrosis where growth exceeds

angiogenesis.155–157 Hypoxic response and inflam-

mation are driving forces in angiogenesis.12,158 More-over, CSCs in HNSCC appear to be concentrated

along the invasive front of the tumor and in the

perivascular niche, an area within 100 μm of themicrovasculature. A variety of factors in the TME,

such as VEGF, NF-κB, and HIF-1α play central roles in

this process.VEGF enhances endothelial growth, migration of

endothelial precursors, and their differentiation. High

VEGF expression in oral SCC has been correlatedwith a poor prognosis, and a recent meta-analysis

suggested that VEGF overexpression could be a

useful prognostic marker.159 VEGF binds to its recep-tor, VEGFR1 in tumor cells, and induces expression

of Bcl-2, inducing chemokines like CXCL1 and

CXCL8. CXCL1 and CXCL8 promote endothelial cellproliferation and survival.160 Endothelial cells in turn

produce factors like EGF, which significantly increase

tumor cell survival and migration.161 VEGF and otherangiogenic factors such as IL-6 and IL-8 are increased

by a number of chemokines such as CXCL12, which

binds to chemokine receptors CXCR2 and CXCR4.High CXCR2 and CXCR4 levels have been shown to

be associated with increased microvessel density

within tumors.55,77,162

Chronic inflammation of the TME contributes to

tumor progression through a variety of mechanisms,

including production ROS and angiogenic factors.NF-κβ is an inflammatory signal transcription factor

playing a variety roles in invasion, proliferation, and

angiogenesis. Constitutive activation NF-κβ results inoverexpression of a variety of factors, including Il-6,

IL-8, and VEGF.43

There are many downstream inflammatorymarkers expressed as a result of NF-κβ and other

mechanisms, such as cyclooxygenases like COX-2.163

COX enzymes catalyze the production of PGs andlikely are the rate-limiting step in their synthesis.

COX-2 is usually overexpressed in inflammation and

preneoplastic lesions. PGs are increased in HNSCC,and PGE2 promotes invasion and angiogenesis and

inhibits apoptosis of cancer cells. COX-2 acts on

VEGF, fibroblast growth factor, and MMPs and is alsopro-angiogenic. COX-2 levels have been found to be

prognostic and selective COX-2 inhibitors have

been shown to increase the efficacy of radio-therapy in vitro.51,164 NF-κβ is the target of many

therapeutic interventions, such as curcumin, n-acetyl

cysteine (NAC), epigallocatechin gallate (EGCG), andothers.5

Intratumoral hypoxia is a key characteristic of

HNSCC, and is a negative prognostic factor, contri-buting to both chemotherapy and radiotherapy resist-

ance. Intratumoral hypoxia is generally accepted to

be a pO2 o10 mm Hg, and intratumoral pO2 levels≤2.5 mm Hg correlate with a worsened prognosis, as

does the overall volume of hypoxic tumor at the

primary site.165 HIF-1α is the most important factorinduced in adaptive response to hypoxia, and ele-

vated expression is also directly associated with a

poor prognosis.166 This transcription factor interactswith more than 100 genes to alter expression of

VEGF, CA9, lysyl oxidase, and many others.48,167 It

has been shown to alter cellular metabolism, and toincrease lymphatic vessel density and blood vessel

density in oral SCC.168,169 CA9 functions to regulate

pH homeostasis and alter the uptake of chemother-apeutic drugs, and also is purported to play a role in

proliferation and cell adhesion.165,170 Lysyl oxidase

catalyzes the crosslinking of collagens and elastins,and overexpression increases microvascular den-

sity.171,172 Agents such as reseveratrol, EGCG and

others may act by promoting degradation of HIF-1α.173–175 Resveratrol has been shown to decrease

expression of HIF-1α and VEGF in vitro.176

METABOLISM IN THE TME

Cancer cells have high bioenergetic requirementsneeded to maintain tumor growth. Tumor cells in

culture have long been demonstrated to rely heavily

on glycolysis with decreased, dysfunctional, or absentmitochondrial OXPHOS. Reliance on glycolysis in the

presence of oxygen is referred to as the Warburg

effect.177 This results in the generation of less ATPthan OXPHOS and yields high levels of pyruvate and

lactate. This is somewhat counterintuitive as there is

such a high bioenergetics requirement, yet OXPHOSis a more efficient means of energy generation than

glycolysis. Thus it is unclear why tumor cells would

thrive with a less efficient mechanism. It has beenhypothesized that glycolysis may confer a growth

advantage.178–180 Some normal, highly proliferative

cells, such as lymphocytes, favor aerobic glycolysisover oxidative metabolism, providing a rationale for

J.M. Curry et al226

the Warburg effect.181 Many cancer cells have defects

in critical components of the OXPHOS pathway, suchas the mitochondrial B-catalytic subunit of Hþ-ATPsynthase.182,183 Furthermore, when glycolytic flux is

high, the ATP yield can exceed that produced byOXPHOS.182,183 Additionally, the intermediates of

glycolytic metabolism can provide substrates for

amino acid, fatty acid, and nucleotide synthesis.167

The metabolic pressures induced by hypoxia in the

setting of rapid growth may then in turn select for

tumor cells which favor glycolytic metabolism evenin the presence of oxygen, as is suggested by the

frequent overexpression of HIF-1α in many cancers.

Hypoxic induction of HIF-1α favors this processspecifically inducing pyruvate dehydrogenase kinase

(PDK) and lactate dehydrogenase a (LDH-A). PDK

inactivates pyruvate dehydrogenase preventingimport of pyruvate to the mitochondria. LDH-A

restores NAD positivity and also uses pyruvate in

the cytosol, which together can reduce electron flowthough OXPHOS and also reduce oxidative stress.

Additionally, glycolytic metabolism results in the

acidotic efflux into the TME that assists in breakdownof the ECM and kills non-adapted normal cells.184

However, this is not likely the whole picture:

much recent evidence suggests that a metabolicsymbiosis exists within tumors cell between differ-

ent populations. Feron has likened this to the

coupling between fast and slow-twitch musclefibers. Fast-twitch glycolytic fibers release lactate

that is then taken up and utilized by slow twitch

fibers. MCT1 is a high-affinity transporter of lactate,which mediates influx into the cell; MCT4 is a low-

affinity transporter of lactate, which primarily medi-

ates efflux of lactate from cells. These transporterscouple cancer cells, so that hypoxic cells maintain

functioning glycolytic metabolism while aerobic

tumor cells recycle and utilize lactate and otherhigh-energy substrates produced by them. A similar

process in cancer would allow for an efficient intra-

tumoral metabolic coupling mechanism betweenoxygenated cells and hypoxic cells.179

Additional evidence favors multicompartmental

metabolism between the cancer cells and CAFs.Numerous co-culture experiments and in situ tumor

analyses have demonstrated this effect in breast and

other cancers.11,81,185 This work has brought to lighta “reverse Warburg effect”, where oxidative stresses

exerted by tumor cells induce aerobic glycolysis and

autophagy in CAFs. This, in turn, results in increasedlevels of intermediate catabolites such as lactate,

glutamine, and ketone bodies. These catabolites are

released into the TME and used for OXPHOS incarcinoma cells. This metabolically enriches the TME

and creates an environment that favors growth,

apoptosis resistance, invasion, and metastasis.81,185

This is Paget’s seed and soil hypothesis, a

phenomenon that may have been unnoticed in

previous homotypic culture experiments.11

Most studies on HNSCC cellular metabolism sug-

gest that the carcinoma cells are highly glycolytic

with high L-lactate generation, yet recent studiessuggest that metabolic heterogeneity and metabolic

coupling occur. Most HNSCC cells generate signifi-

cantly higher levels of lactate compared with normalhuman oral keratinocytes (NHOK), although several

cell lines generate significantly lower lactate levels

than NHOKs.186 It has been postulated that the cellswith decreased lactate production have increased

lactate uptake via MCTs, allowing them to utilize

OXPHOS.186 When some HNSCC cell lines that aretypically glycolytic are supplemented with excess

pyruvate, some of the effects were reversed, which

suggests that OXPHOS is important to supportHNSCC cell proliferation in the presence of a

catabolite-rich microenvironment.187 High tumor

lactate concentrations in HNSCC are associated withsubsequent nodal and distant metastatses.188,189

In our previously published work on oral SCC, we

demonstrated evidence of this multicompartmentmodel of metabolism. We have suggested that there

may be three metabolic compartments in HNSCC,

where the leading tumor edge relies on OXPHOSand the deeper layers of the tumor are more

glycolytic (aerobic or anaerobic) and tumor stroma

represents a third compartment undergoing aerobicglycolysis (Figure 2). This was demonstrated through

high expression of MCT4 in the stroma and deeper

tumor, while MCT1 was more highly expressed bythe leading tumor edge. We also confirmed OXPHOS

in the leading tumor edge with assays for TOMM20

and LDHb, both functional markers for mitochon-drial metabolism. This pattern of metabolic coupling

was demonstrated in a subset of our oral SCC

patients, and correlated with aggressive behaviorincluding a worsened disease-free survival and peri-

neural invasion. Interestingly, it also correlated with

increased specific uptake values (SUV) on positronemission tomography/computed tomography. We

further tested this metabolic coupling theory with

a squamous cell carcinoma line co-culture experi-ment. Using immortalized squamous cell lines we

were able to generate two divergent SCC popultions,

one RAS-dependent and another NF-κB–dependent.These cell lines were each able to induce metabolic

reprogramming of CAFs via oxidative stress. This

resulted in a lactate shuttling process that feeds thecancer cells fueling anabolic growth via and MCT1/

MCT4 metabolic couple between the tumor and the

stroma. Interestingly, this model also demonstratedthat the CAFs protected the cancer cells against

oxidative stress by reducing oxidative stresses within

the carcinoma cells. RAS-transformed cells were ableto reprogram adjacent epithelial cells, as well as

Figure 2. Metabolic coupling in HNSCC. The leading edge of the tumor relies on OXPHOS while the inner compartmentand CAFs rely on glycolytic metabolism. Higher expression of MCT1 is seen in the leading tumor edge, while higherexpression of MCT4 is seen in the central compartment and stromal CAFs.

Tumor microenvironment in HNSCC 227

fibroblasts, suggesting that cancer cells can subju-gate either group.

MCT4 may represent a possible target for meta-

bolic interruption and uncoupling of the tumor andstroma. In an animal model, we were able to

demonstrate that NAC was able to selectively inhibit

MCT4 induction in CAFs, halting mitochondrial bio-genesis in cancer cells but not in normal epithelial

cells. This may allow targeted therapy that selec-

tively starves cancer cells. MCT1 also has beenproposed as a possible target to prevent uptake of

lactate, forcing aerobic cells to use glucose and

depriving or decreasing availability to hypoxiccells.167 In fact, the MCT1 inhibitor, a-cyano-

hydroxycinnamate has been shown to slow tumorgrowth and potentiate the effect of radiotherapy in

MCT1-expressing tumors in mice.179,184

Hypoxia contributes to chemotherapy and radio-therapy resistance.155 Inhibition of HIF-1α can pre-

vent the induction of the hypoxic response blocking

angiogenesis.190 Zhang et al inhibited HIF-1α withsiRNA and oligonucleotides, which increased apop-

tosis in oral SCC.191 The EGFR inhibitor cetuximab

blocks downstream signaling activated by EGFR; thistriggers G1 phase arrest and can also trigger apop-

tosis.192 In addition, it has been shown to down-

regulate HIF-1α; this, in turn, downregulates theirLDH-a and glycolytic potential. This inhibition of

J.M. Curry et al228

glycolytic potential leads to inhibition of

proliferation.192

Metformin is a commonly used antihyperglycemic

drug in type 2 diabetics and has been proposed as a

potential anticancer therapy also that may impacttumor metabolism in the TME. Metformin has been

shown to inhibit cancer cell proliferation in several

human cancers, such as gastric, medullary thyroid,breast, and pancreatic cancers.193–196 Epidemiologic

studies also have shown significant effects from

metformin use in diabetics, lowering the risk ofcancer incidence and mortality.197 In oral SCC, Luo

et al demonstrated that metformin blocked cell cycle

progression at the G0/G1 phase and induced apop-tosis. Metformin triggered alterations in multiple

other pathways as well: increasing activation of the

adenosine monophosphate (AMP) kinase pathway,suppressing the mammalian target of rapamycin

(mTOR) pathway, decreasing cyclin D1 levels and

retinoblastoma (Rb) phosphorylation, and downregu-lating Bcl 2.198 They also were able to demonstrate

in vivo evidence of increased apoptosis in a xenograft

model. While this study demonstrated various effectson the cell cycle; the metabolic effects of metformin

on cancer have yet to be investigated.

CONCLUSION

Many elements of the TME beyond the cancerousepithelial cells impact progression of HNSCC.

Genetic alterations induced by tobacco and alcohol

or the HPV virus initiate the sequence of events thattrigger transformation of stromal cells, immune sup-

pression, and chronic inflammation. In turn,

unchecked growth, invasion, and metastasis prevail.The complexity of these processes reveals that the

long-held notion of “condemned mucosa” actually

reflects a “condemned tissue” comprised of many celltypes which have co-evolved during tumorigenesis.

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