Effects of non-cellular tumor microenvironment matrix on ... · Matrix molecules are produced and...
Transcript of Effects of non-cellular tumor microenvironment matrix on ... · Matrix molecules are produced and...
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
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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