Effects of non-cellular tumor microenvironment matrix on ... · Matrix molecules are produced and modifed by subclones of cancer cells & cells in tumor microenvironment (TME) Stroma-rich

3.7.2018

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Tuula Salo1, Susanna Teppo1, Sini Nurmenniemi1, Marilena Vered2, Dan Dayan2,

Carolina Bitu1 and Pia Nyberg1.

1Department of Diagnostics and Oral Medicine, Institute of Dentistry, University of Oulu, Oulu, Finland, and2 Institute of Pathology, Chaim Sheba Medical Center Center, Tel Hashomer, Ramat Gan, Israel. Tuula Salo

IAOP Vancouver25.6.2018

Universities of Helsinki & OuluFinland

Effects of non-cellular tumor microenvironment matrix on oral cancer prognosis and in vitro

experimentsLecture content

(I) In vivo - the importance of tumor extracellularmatrix (ECM) in oral cancer

(II) In vitro - ECM solid and mesh models for oral cancerstudies

(III) Preclinical cancer drug testing – role of TME matrices

Non-cellular matrix in primary oral cancers

Structural proteins

Enzymes

Active fragments

Extracellur vesicles

Cytokines

Growth Factors

Matrix molecules are produced and modifed bysubclones of cancer cells & cells in tumor microenvironment

(TME)

Stroma-rich group: In multivariate analyses Worse disease-free survival

HR 1.81 (95%CI 1.17-2.79, P= 0.008) Higher cancer-related mortality

HR 1.71 (95%CI 1.02-2.86, P= 0.03)

311 early-stage OTSCC cases

Stroma-poor (<50%) Stroma-rich (≥50%)

The prognostic value of tumor to stroma ratio (TSR) and budding & depth of invasion (BD)

in oral tongue cancer

Almangush et al. 2018

Combination of the highest-risk parameter scores for TSR and BD: Disease recurrence

HR 3.42 (95%CI 1.71-6.82 P= 0.004) Cancer-related mortality

HR 11.6 (95%CI 3.83-35.31 P< 0.001)

Budding & depth of invasion (BD) scores0: Tumor invasion depth (ID) < 4 mm, and <5 buds

at the invasive front (IF),1: ID ≥ 4 mm, but < 5 buds at IF, or

ID < 4 mm, but ≥ 5 buds at IF2: ID ≥ 4 mm and ≥ 5 buds (<5 cells) at the IF

Conclusion: Should we add the analyses of TSR and BD to pathology reports of OSCC?

Wu et al. TSR Meta-analyses. Oncotarget2016

Stiffness of TME:Type I collagen synthesis (PINP-ab) in OTSCC prognosis

Holle et al. 2016

LN metastasisSCC + TME

SCC cellsTME

Salo S. et al. 2013

Higher PINP expression in invasive vs superficial areas associated with worse prognosis of OTSCC patients; HR 1.924, 95% CI[1.127-3.285] p=0.004

Bagordakis et al. 2016

High PINP expression at the invasive front CAFsassociated with a poor prognosis of OSCC patients; HR 3.31, 95% CI [1.54-5.91] p=0.002

Ligands in TME: TN-C and FN in OTSCC prognosis

Negative Moderate Abundant

TNC expression

FN expression236 OTSCC cases

Conclusion: Expression of FN and TN-C in the TME, not in SCC cells, differentiate patients into low- and high-risk groups

5 year survival rate in early stage OTSCC:

TN-C: 88% if TN-C was not in TME42% if TN-C was abundant in TME

FN: 100% if FN was not in TME 24% if FN was abundant in TME

Sundquist et al. 2017

Tenascin-C (TN-C) & fibronectin (FN)

Both are present in most solid tumors

Both are related to cell adhesion & migration

Holle et al. 2016

100% 24%

88% 42%

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Cytokines in TME: IL-17F in OTSCC prognosis

• Il-17F belongs to IL-17 cytokine family; greatest homology with IL-17A

Extracellular - not intracellular -amount of IL-17F in IF front mast cells Disease specific survival In multivariate analyses: HR 4.18 (95% CI 1.01-17.26, p=0.04) for

early stage HR 3.51 (95%CI 1.48-8.34, P= 0.004)

for all stages

IL-17F is expressed mainly by mast cells

IL-17F MCT

mergedabi

OTSCC cells were in most cases negative (A), or sporadically (5-75%) positive (B,C) for IL-17F

Inflammatory cells showed strong expression

IL-17F

Mast cell tryptase

merge

early stage OTSCC all stages OTSCC

IL-17F inside mast cell IL-17F outside mast cell

Conclusion:Extracellular mast cell‐derived IL‐17F in the TME has anti-tumorigeniceffects in OTSCC

Almahmoudi et al. 201883 OTSCC cases

TME in cancer patients´ lymph nodes:structure & composition are different from the primary tumor

Structural changes in negative LNs architecture of thepatients with early stage OSCC

435 LN0 from pN0 neck dissections histopathological parameters:

Capsule thickness Sub-capsular and medullary sinus

ectasia Lobular architecture Percent of cortical reactive

follicles

-> prognosis is better

A thickened capsule & many reactive cortical follicles

-> prognosis is worse

A thin capsule and a few reactive cortical follicles

LN0 of close levels

“Even negative LNs in patients who are free of regional metastatic disease hold valuable prognostic information”

“A car wheel will go longer distance than a bicycle tire”

Vered et al. 2014

A) In vitro solid 3D matrix modelsfor cancer invasion

In vitro solid 3D matrix models for oralcancer invasion studies

Cloudberry

Human cancer cells

Rat tail type I collagen+

Mouse tumor(Matrigel)

+Human fibroblasts

Nylon membraneMedium

Steel grid

1) Organotypic ”rat-mouse-man”model, since 1980´s (Fusenig et al.)

3D TME invasion models to analyze the interaction between carcinoma cells and TME

Human has 78 less proteases than mouse

Overall & López-Otín 2002

2) Human uterus myoma disc model(Nurmenniemi et al. 2009)

Tongue carcinoma patient sample

AE1/AE3

HSC-3 tongue carcinoma cells in myoma disc

HSC-3 + fibroblasts in collagen disc

HSC-3 cells invade 7 times deeper in myoma than in thecollagen + fibroblasts discs

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HSC-3 cells do not invade within

pig tongue, human heart, or nose polyp tissues. Why?

Myoma+

HSC-3 14 days

Heart+

HSC-3 14 days

Tonguewounded

+ HSC-3

14 days

Polyp(nose)

+HSC-3

14 days

Sundquist et al. 2016 & 2017

Myoma is a mesenchymal tumor containing natural TME which is essential for carcinoma cell invasion

Around 120 discs by punch biopsy from an average size myoma

Myoma discs are simple to prepare and use

Myoma discs preparation:Human Tumor Tissue Based 3D in

vitro Invasion Assays: Åström et al. Methods Mol Biol,

2018

Every myoma is ”individual”- Invasion of the index cell line (HSC-3) varies in different myoma discs7A 28 16 34A

32C54

5026B49B47

32A 41C

39B 54 26A 53

35A235148

37

27

53

Nurmenniemi et al. 2009

Myoma discs composition

Collagen typesI, III, IV, lamininhyaluronic acidHA

Invasion +++ ++ +

VIM, fibroblasts;SMA, smooth musclecells; CD 45 and CD 68, inflammatory cells; FVIII, endothelial cells

ApopTag +

Most of the cellsare non-vitalafter storage in liquid nitrogen

TUNEL (green) +Tenascin-C +++ ++ +

SEM and TEM of HSC-3 cells in intact myoma

Protrusion of the membrane = Invadopodia(arrow head) of invading SCC in the deeper part of the myoma; breaks in BM

Basement membrane (arrow) surroundingthe ”resting” SCC cellin the upper part of the myoma

Sundquist et al. 2016

Rinsing of myoma tissue affects SCC cell invasion

”Anti-invasive” arr-HSC-3 cells(transfected with arresten)

3D Collagen gel+fbl Intact myoma

Myoma discs rinsed for 14 daysbefore the invasion assay withanti-invasive arr-HSC-3 cells

Media: SDS-PAGE + proteomic

Invaded similar to Ctrl

No invasion

Rinsed myoma

No invasion

2836

557295

130250

Gene Protein in rinsing myoma media Top enriched

IGFBP3 Insulin-like growth factor-binding protein 3 apoptotic process

LTBP3

Latent-transforming growth factor beta-binding

protein 3transforming growth factor beta receptor

signaling pathway

LTBP4

Latent-transforming growth factor beta-binding

protein 4transforming growth factor beta receptor

signaling pathway

FGF2 Fibroblast growth factor 2 apoptotic process

TGFB2 Transforming growth factor beta-2 axon guidance

EGF Pro-epidermal growth factor platelet activation

GDF9 Growth/differentiation factor 9 positive regulation of cell proliferation

TGFB1 Transforming growth factor beta-1positive regulation of transcription from

RNA polymerase II promoter

VEGFA Vascular endothelial growth factor Apositive regulation of transcription from

RNA polymerase II promoter

IGF1 Insulin-like growth factor Ipositive regulation of transcription from

RNA polymerase II promoter

HGF Hepatocyte growth factorpositive regulation of transcription from

RNA polymerase II promoter

TGFBR2 TGFbeta receptor type-2 apoptotic process

FGFR3 Fibroblast growth factor receptor 3 apoptotic process

EGFR Epidermal growth factor receptor negative regulation of apoptotic process

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Intact Myoma is hypoxic and contains severalinvasion inducing factors

Intact and rinsed myoma tissueWestern blot:

Intact Rinsed

MMP-11

LOX-1

• MMP-11 has pro-invasive & anti-apoptotic properties

• LOX is secreted by hypoxic tumors; facilitates invasion

Intact myoma disc with HSC-3 cells: invadingcells express CA-9

• CA-9 in HNSCC is associated with reduced survival

CA-9 (hypoxia factor carbonic anhydrase IX)

Intact Rinsed

Teppo et al. 2013

• The gene transfection - invasionÅström et al. 2017, 2018

• miRNA/mRNA/protein identificationsin invasive vs non-invasive cells- lazer capture Korvala et al. 2017

Solid myoma discs: used in studies related to various cancer cell lines or

co-culture experiments

• Myoma +/- cells irradiation +/- drugsVäyrynen et al. manuscript

• Co-cultures: cancer cells +/-

# M1 or M2 macrophages

Pirilä et al. 2015

# Activated mononuclear cells

Alsamadi et al. 2017

P=0.01 P=0.04

+MNC +Act-T-cell +Act-MNCHSC-3

HSC-3 HSC-3+M1 HSC-3+M2

Cancer

Myoma

Media +/

SCC cells

Myoma

Media +/- MNCs

A. Myoma +/- HSC-3

B. Collagen gel + fibroblasts +/- HSC-3

• precultured for 10 days

• Discs were transplanted onto the dorsal muscle fascia

C. Subcutaneous inj. HSC-3

• After 6 weeks B alb-c nude nu/nu mice) in group A and B were killed

• Mice in group C were sacrified when the tumor volume reached 1,000 mm3

• Implants and xenografts were collected in 4% formalin and embedded in paraffin

Myomaw/o HSC-3

Myomawith HSC-3

After 42 days

Effects of myoma, collagen+fbl, or no matrixon carcinoma cells growth pattern in mice

Same cancer cells in nude mice: different growth patterns - depends on the TME

• Stromal reaction

• EMT

• Invasion

• Dysplasia• Invasion

Myoma+HSC3 implants

Collagen gelfbl +HSC3 implants

• Encapsulation• NecrosisXenografts

Unpublished

For faster in vitro analysesgelatinous mesh matrices are needed

Bilberry

Matrigel®- the ”golden standard” for cancer in vitro studies

Interview 2013:

“Are you surprised that Matrigel was so successful?”Hynda Kleinman (NIH, USA):“I’m shocked that it’s this useful. I’m also shocked that no one has invented anything better. It’s still made the exact same way we made it 25 years ago. There must be thousands of tumors to make it…. Nobody’s done that.”

Engelbreth-Holm-Swarm(EHS) sarcomasubcutaneouslyin mice

Millions of micehave been killedto produceMatrigel!

Matrigel = basement membrane extract

• ECMatrix™ • Cultrex ® • BME ® • Geltrex ®

Composition:Laminins, Collagen IV, Heparin sulphateproteoglycan, Entactin/nidogen

Growth factors (TGF-b, EGF)

A mouse natural tumor: the composition varies from lot to lot!

Matrigel: 15-20 mil.€/yr

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We prepare Myogel similar to Matrigel

Salo et al. 2015; Naakka et al. unpublished

Proteomic analyses765 identified proteins:

• 34% were the same in both • 66% were different• Myogel: more small proteins than Matrigel

Breast cancer: MDA-MB-231 cells

Myogel

0.5mg/ml, 10 000 x

SEM• Myogel mesh is looser than Matrigel

Matrigel

0.5mg/ml, 10 000 x

We compared their properties in cancer invasion assays

Salo et al. 2015; Apu et al. unpublished; Tuomainen et al. Unpublished, Nees et al. unpublished

Matrigel

Myogel

MyogelMatrigel

24h 48h

Matrix conc. 2.4 mg/ml

Transwellverticalinvasion

HSC-3cells

Sandwichinvasion

Matrigel-collagen, HSC-3

Myogel-collagen, HSC-3

Matrigel-coated Myogel-coatedUT- SCC-42B cells

IncuCyte ZOOM -live Cell AnalysesScratch Wound Invasion Assay

HSC - 3 cells

Myogel-collagenMatrigel-collagen

Breast cancer: MDA-MB-231 cell line)

+Tanespimycin 100 nM

Carcinoma cellsinvade faster

with more streching spikeson top of and withinMyogel vs Matrigel

Potential use of Myogel for cancer patient´s tissue/ blood samples in pre-treatment tests

Media

Myogel

Spheroid

Spheroid wells are imaged and measured daily for:

• Spheroid size (minus original size)

• Invasion distance (spikes)

-> for drugs and irradiation analyses

Ilastik

Media

Media

Immune cells

Cancer tissue cells+ / - drug, embedded in Myogel

Cancer cells pass through theCancer cells = redImmune cells = blue

Al-Samadi, Tuomainen et al. unpublished

Microfluidicchipmodel

Lucariniet al.2017

Spheroidmodel Day 1

Day 3

Why still 95% of anti-cancer compounds

which have promising effects in preclinical studies

fail in Phase III clinical trials?

Designing activecompounds orantibodiesagainst cancer

High-throughput

screening (HTS)

in 2D cell culture

Rodent models

Efficacy and toxicity

& metabolism studies

Could we do something better in HTS?

ll line Sexa Ageb TNM Specimen site Typec Grade Passage

UT-SCC-8 M 42 T2N0M0 larynx pri G1 46

UT-SCC-14 M 25 T3N1M0 tongue pri(per) G2 31

UT-SCC-24A M 41 T2N0M0 tongue pri G2 43

UT-SCC-24B M 41 T2N1M0 neck met(per) G2 36

UT-SCC-28 F 48 T2N0M0 floor of mouth pri(per) G1 32

UT-SCC-42A M 43 T4N3M0 larynx pri G3 14

UT-SCC-42B M 43 T4N3M0 neck met G3 17

UT-SCC-40 M 65 T3N0M0 tongue pri G1 UT-SCC-44 F 71 T4N2BM0 gingiva of mandib. pri(per) G3 31

UT-SCC73 F 86 T1N0M0 tongue pri G2 16

UT-SCC81 M 48 T2N0M0 tongue pri G1 16

UT-SCC106A M 37 T1AN0M0 larynx pri G1 aM=male , F=female, b Age in years, C Pri=primary tumor, met=metastasis, per= persistent disease

We tested 12 HNSCC cell lines with HTS methodagainst 19 anti-cancer drugs

2D Myogel coating

2D Matrigel coating

3D Myogel (+ collagen)

3D Matrigel

2D Monolayer = ctrl19 drugs against:

Epidermal growth factor receptor(EGFR)

Mitogen-activated protein kinase (MEK) Phosphoinositide 3-kinases (PI3K) Mechanistic target of rapamycin

(mTOR)

Heat map of the drugs tested on cancer cell lines on top /withinMyogel, plastic, Matrigel

CONTROL

embedded coated coated embeddedErbitux 7 2 18 19 23

Gefitinib 6 3 16 17 21

Erlotinib 2 0 3 6 9

Afatinib 9 3 16 17 21

Canertinib 11 9 23 24 28

Erbitux 7 2 16 8 10

Gefitinib 10 6 17 12 13

Erlotinib 8 4 15 10 11

Afatinib 5 2 16 11 11

Canertinib 9 9 26 18 18

Erbitux 3 7 14 15 12

Gefitinib 0 1 2 5 5

Erlotinib 0 0 2 3 1

Afatinib 1 4 10 13 10

Canertinib 8 10 18 18 17

Erbitux 6 0 2 2 18

Gefitinib 0 0 1 3 12

Erlotinib 0 0 0 1 2

Afatinib 3 0 7 12 16

Canertinib 8 7 12 17 24

Erbitux 0 5 13 25 28

Gefitinib 0 2 6 8 14

Erlotinib 0 3 4 6 8

Afatinib 0 8 18 19 20

Canertinib 5 12 29 30 30

Erbitux 7 9 13 19 21

Gefitinib 2 3 3 4 6

Erlotinib 0 1 1 0 2

Afatinib 9 7 12 13 14

Canertinib 11 17 26 27 27

Erbitux 0 0 12 24 30

Gefitinib 2 1 4 11 23

Erlotinib 0 0 0 5 10

Afatinib 6 2 13 18 23

Canertinib 9 9 26 28 29

Erbitux 0 0 0 0 1

Gefitinib 0 0 0 0 1

Erlotinib 1 0 0 0 0

Afatinib 0 0 0 0 0

Canertinib 9 7 7 8 4

Erbitux 0 2 0 1 2

Gefitinib 1 1 0 1 0

Erlotinib 0 0 0 0 0

Afatinib 0 0 0 0 0

Canertinib 7 7 8 8 4

Erbitux 1 0 0 0 2

Gefitinib 2 0 0 1 0

Erlotinib 1 0 0 0 0

Afatinib 3 0 1 0 2

Canertinib 8 9 9 9 8

MYOGEL MATRIGEL

EGFR inhibitors 12 HNSCCcell lines

No effect -> highly effective

Tuomainen et al. unpublished

3.7.2018

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Low active

Inactive

Active

23 analyses show clear response in Matrigel and plastic; only 2 responded weakly in Myogel

0

10

20

30

40

50

60

70

80

90

100

Control Matrigel 2D Matrigel 3D Myogel 2D Myogel 3DRes

po

nse

rate

for

Erb

itu

x(%

)

Tuomainen et al. unpublishedSee poster #206

Monotherapy response objective patients rateclinical trials for HNSCC. Vermorken JB, et al. Cancer. 2008

*

Mean response rate of all the 12 HNSCC cell lines tested:Control & Matrigel 2D 68%, Myogel 2D 15%

Erbitux is a monoclonal ab, binds to EGFR and alters the TK-mediated signal transduction pathway - FDA approval for Cetuximab (Erbitux)

Phase III clinical trials for Erbitux- response rate was 13 %

Future pre-clinical drug screening & response rate testing?

Clinical trials

Should new active compounds be tested in cancer cell lines on top of or embedded in human tumor matrix (Myogel) wells?

Future pre-clinical drug screening & response rate testing?

Patient´s

Cancer tissue

LN metastases

Serum samples

Clinical trials

Should new active compounds be tested in cancer cell lines on top of or embedded in human tumor matrix (Myogel) wells?

Tests with human primary and metastase TME mimicking matrices

Immune checkpoint blockers

TowardsPersonalized Medicine

Microfluid chips? Spheroids or organotypic models

Carcinoma drugsIrradiation

HTS

Lymfogel?

Myogel?

Conclusion:The structure and composition of the non-cellular ECM plays a crucial role in cancer

growth both in vivo & in vitro

Rat type I collagen Human Myogel Mouse Matrigel

Human fibrin Human fibronectin

Bovine serum albumin

Tuula Salo1, Susanna Teppo1, Sini Nurmenniemi1, Marilena Vered2, Dan Dayan2,

Carolina Bitu1 and Pia Nyberg1.

1Department of Diagnostics and Oral Medicine, Institute of Dentistry, University of Oulu, Oulu, Finland, and2 Institute of Pathology, Chaim Sheba Medical Center Center, Tel Hashomer, Ramat Gan, Israel.

Researchers and collaborators behind these projectsOulu group: past and present membersMaija Risteli PhD, Johanna Korvala PhD, Mauricio Dourado PhD, Elias Sundquist PhD, Ilkka Alahuhta, PhD, Ehsanul Hoque Apu DDS, Sirpa Salo PhD, Sini Nurmenniemi PhD, Susanna Teppo MSc, Meeri Sutinen PhD, Pia Nyberg PhD, Emma Pirilä PhD, Pirjo Åström PhD, et.alOulu collaboratorsPetri Lehenkari, prof. group, Ilkka Miinalainen, PhDHelsinki groupAhmed Al-Samadi PhD, Alhadi Almangush PhD, Katja Tuomainen MSc, Rabeia Almahmoudi, DDSHelsinki Univ. collaboratorsPäivi Saavalainen, doc. groupAntti Mäkitie, prof. groupFIMM collaborators Krister Wennerberg, prof. group

Brazil:Coletta, Graner

Israel collaboratorsMarilena Vered, prof. groupBrazilian collaboratorsRicardo Coletta, prof. group , Adriana Leme, prof. groupSotiris Missailides, prof. group

Financial support

HUSLAB

Oulu group

Helsinki group