Cancer-associated fibroblasts and tumor cells: a diabolic cross-talk driving cancer progression

1. Cancer associatedfibroblastsand tumor cells: a diaboliccross-talkdrivingcancerprogression Prof. Dr. rer.nat. Gerhard UntereggerClinicofUrologySaarland UniversityMedical CenterHomburg/Saar - Germany

2. Crosstalk: Not restricted to malignant tissue! Tumor: A never healing wound ?! Invasiv growing carcinoma

3. The complex composition within the prostate tissue ...but the microevironment appears to be a little bit more complex! • epithelial cells • basal cells • luminal cells • transit amplifying cells • stem cells • neuroendocrine cells • stroma cells • fibroblats • myofibroblasts • endothelial (progenitor) cells • macrophages • epithelial – mesenchymal transitions • mesenchymal – epithelial transitions • mesenchymal – mesenchymal transitions Extracellular Matrix There is a strong communication within this network: Co-evolution!

4. Fibroblasts as Co-factors in Prostate Development and Cancer Fibroblasts are critical determinants in prostatic cancer growth and Dissemination. Chung LW Cancer Metastasis Rev. 1991 Oct; 10(3): 263-74 Stroma and Epithelial cells determine by paracrine interaction normal and malignant development!

5. Epithelial cells and Fibroblasts in Prostate Stroma Normal Prostate Gland Basal Cells Invasive Prostate Cancer Especially Basal Cells are in close contact to the stroma compartment

6. Receptors enable a permanent dynamic non-diabolic cross-talk

7. Cancer associatedfibroblastsand tumor cells: a diaboliccross-talkdrivingcancerprogression • We focus on CancerAssociatedFibroblasts (CAF) • aspivotal part of the tumor microenvironment • Are they a specificcelltype or only a phenotype? • Where do they come from? • Charcterization by phenotype? • Characterization by function?

8. Origin of CAF – from everywhere P. Cirri & P. Chiarugi: AmCancRes 2011; 1(4);482-497 = activatedMyofibroblast

9. Origin of CAF – modification of ECM Otranto et al. Cell Adesion & Migration 2012: 6:3; 203-219 Changes in the ECM stiffness

10. Feed-back-loop enables a co-evolution of CAF and Cancer cells P. Cirri & P. Chiarugi: AmCancRes 2011; 1(4);482-497 ECM-remodelling: Stiffness of ECM = Mechanical stress!

11. Fibroblasts as Co-factors in Prostate Development and Cancer Microenvironment as a pivotal co-factor was described a lot of years ago… Reconstituted basement membrane promotes morphological and functional differentiation of primary human prostatic epithelial cells. Fong CJ et al. Prostate 1991; 19(3):221-35 Acceleration of Human Prostate Cancer Growth in Vivo by Factors Produced by Prostate and Bone Fibroblasts. Gleave M et al. Cancer Research 1991; 51: 3733-3761 Growth of an androgen-sensitive human prostate cancer cell line, LNCaP, in nude mice. Lim et al. Prostate 1993; 22(2): 109-118 Regulation of prostate-specific antigen gene expression in LNCaP human prostatic carcinoma cells by growth, dihydrotestosterone, and extracellular matrix. Guo Y et al. Prostate 1994; 24(1):1-10 Prostate epithelial differentiation is dictated by its surrounding stroma. Chung LW and Davies R. Mol Biol Rep. 1996; 23(1):13-19 Co-evolution necessary for normal development!

12. Fibroblasts as Co-factors in Prostate Development and Cancer Strategies to disclose phenotype and function! • “Cancer –associated-Fibroblasts CAF”: Are they really defined? How they are defined? • How they are prepared? Distance between tumor-epithelial cells: 50-100-200-500μm ? • By isolation of “fibroblasts ” from TUR (normal) and Prostatectomy (CAF) using digestion • (SH Lang, M. Stower and NJ Maitland, Br.J. Cancer (2000) 82(4), 990-997 • Immortalization of NAF and CAF by hTERT • N. Paland et al. Mol Canc Res (2009) 7(8), 1212-1223 • Embryonic rat urogenital sinus mesenchyme (UGM) • K. Ishii et al. Endocrine-Related Cancer (2009) 16, 415-428 • NAF and CAF from BPH and Prostatectomy using digestion – activated by TGF-ß • E. Giannoni et al. CancRes (2010) 70(17), 6945-6956 • Isolation of CAF and NAF and outgrowth as explants using selective media and coatings • D.Peehl and R. Sellers Prostate (2000) 45, 115-123 • Isolation from explants and characterization by IC (Vimentin, SMC, CD90) • J.A. Tuxhorn ClinCancRes (2002) 8, 2912 -2923 • Isolation from histological examined regions*, digestion, centrifugation, MACS and FACS analysis! • L.E. Pascal et al. BMC Cancer (2009) 9, 317 * parallel sections were examined additionally! • Conclusions: Most NAF/CAF were not comprehensively characterized

13. Cancer associatedfibroblastsand tumor cells: a diaboliccross-talkdrivingcancerprogression • CancerAssociatedFibroblasts (CAF) : • Where do they come from? • Residentwithin the tumor/organ • PhenotypicswitchintoMyofibroblasts • EMT and MMT contribute to phenotypicalchanges • Circulatingcells of differentoriginwillcontribute, too • Disclosure of the nature and originremaina challenge Different cell types ore phenotypes? Remains to be disclosed!

14. Strategies to disclose the role of “Cancer associated fibroblasts” in tumor development and progression The most important question considering the experimental design: Different strategies for NAF/CAF preparation • Isolation of living cells* • Selection • Functional analysis • Changes in gene expression • Genetic modification • But: Really „CAF?“ • Isolation of stroma by LCM • Only descriptive • Alignment to Histology • Contamination • Heterogeneous • Really „CAF?“ * Stability of phenotype and function in vitro?

15. Strategies to disclose the role of “Cancer associated fibroblasts” in tumor development and progression: Fibroblasts as Modulators in Primary Cell Cultures of Prostatic Carcinoma Selection toward diploid cells in prostatic carcinoma derived cell cultures. Ketter R. et al. Prostate 1996; 28(6): 365-71. A new serial transfer explant cell culture system for human prostatic cancer tissues preventing selection toward diploid cells. Zwergel Th. et al. Cancer Genet. Cytogenet. 1998; 101 (1): 16-22 Origin of our „CAF“ used in these experiments: Selection of outgrowing cells from patients specimens after Prostatectomy: Examination by an experienced Pathologist and by parallel histological analysis –

16. Strategies to disclose the role of “Cancer associated fibroblasts” in tumor development and progression Recovery of epithelial cells and fibroblasts w/o digestion by selective outgrowth using coating and specific media Primary cell culture forming a monolayer Epithelial cells Primary cell culture forming a monolayer CAF/ NAF fibroblasts Particle

17. Cancer associatedfibroblastsand tumor cells: a diaboliccross-talkdrivingcancerprogression • We focus on CancerAssociatedFibroblasts (CAF) : • Are they a specificcelltype or only a phenotype? • Where do they come from? • Charcterization by phenotype • Characterization by function?

18. „Phenotypic Characterization of CAF´S“ D Wever O. et al. Int. J. Cancer 2008;129: 2229-2231 FAP SDF-1 PDGFR Proteom Secretom Transcriptom Genom Metabolom A Challenge!

19. Fibroblast derived Factors inducing EMT* • TGF-ß • oxidative stress • HIF-1α • ROS • Wnt//ß-Cat • KGF • HGF • * Acquisition of metastatic and aggressive phenotypes: • Mesenchymal morphology • Resistance to drugs , stress and anoikis • Enhanced migratory and invasive properties • Inhibition of senescence • Immunosuppression Metastasis Update: Human Prostate Carcinoma Invasion via Tubulogenesis. Nagle RB, Cress AE. Prostate Cancer. 2011;2011:249290. Epub 2011 Jun 21. Department of Pathology, The Arizona Cancer Center, The University of Arizona, Tucson, AZ 85724, USA. Abstract This paper proposes that human prostate carcinoma primarily invades as a cohesive cell collective through a mechanism similar to embryonic tubulogenesis, instead of the popular epithelial-mesenchymal transformation (EMT) model. Evidence supporting a tubulogenesis model is presented, along with suggestions for additional research. Additionally, observations documenting cell adhesion molecule changes in tissue and stromal components are reviewed, allowing for comparisons between the current branching morphogenesis models and the tubulogenesis model. Finally, the implications of this model on prevailing views of therapeutic and diagnostic strategies for aggressive prostatic disease are considered.

20. What is the best technology to characterize the environment completely? • Proteomic analysis of primary colon cancer-associated fibroblasts • using the SELDI-ProteinChip platform. • Z. Wang et al. Biomedicine & Biotechnology. 2012 13(3). • FAP only weak expressed • SMA overexpressed in 4/6 samples • 3 “new” proteins: FMRFamide related peptides, ILG-II, Thyß-4-like protein • Identification of stromally expressed molecules in the prostate by tag-profiling of • cancer-associated fibroblasts, normal fibroblasts and fetal prostate. • B Orr1 et al. Oncogene (2012) 31, • 671 transcripts enriched in CAF`s (organogenesis) • 356 transcripts decreased as compared to NAF´s (cell cycle associated) • Restriction to secreted/membrane bound proteins: • ASPN, CAV1, CFH, CTSK, DCN, FBLN1, FHL1, FN, NKTR, OGN, PARVA, S100A6, • SPARC, STC1 and ZEB1 • Specific and variable expression only in prostate cancer and fetal prostate fibroblasts

21. Changes in gene expression following/responsible for EMT • EMT Signature • Cancer Stem Cell Phenotype induced by CAF`s • E-Cadherin-Vimentin-Notch-1-PDFG-D-NF-kB-ZEB1 • Only Notch-1 overexpressed in bone metastasis • Variable expression and differences between ITF and center of the tumor • CAVE: EMT is a transient process and cells can re-differentiate by MET! • Sox2, Nanog, Oct4, Lin 28B, Notch1 increased • miR-200 decrased • Re-expression of miR-200 inhibits prostate spheres forming • Re-expression of miR-200 reduces Notch1 and Lin28B expression Molecular signature of epithelial-mesenchymal transitions (EMT) in human prostate cancer bone metastasis. S. Sethi et al. Am J Transl Res 2011, 3(1). Histological sections Epithelial to Mesenchymal Transition is Mechanistically linked with Stem Cell Signatures in Prostate Cancer Cells. D. Kong et al PLoS one 2010; 5(8. PC-3 cell line

22. miRNA-pattern as markers for CAF in bladder cancer* CAF exhibit a specific miRNA pattern *Enkelmann in prep. & J Cancer Res Clin Oncol (2011)

23. Proteom-pattern* as markers for CAF in bladder cancer * Enkelmann in prep.

24. mRNA Array analysis* of matches pairs of NAF/CAF • MAP3K1 (MEKK1) • Associated with focal adhesion • Overexpression in pancreatic cancer cells • Depletion by siRNA inhibits invasion and migration • Tranmitting signals for ECM homeostasis migration • Regulates detachment of migrating fibroblasts • Less data available from prostate cancer • SCARB1(SRB1) • Membrane bound protein receptor for HDL • Facilitates influx of Cholesterol into the cells • Increased expression in AR-independent cells • Enables the synthesis of DHT from Cholesterol • Expression involved in CRPC • S100A10 • Plasminogen receptor • Present on the cell surface as heterotetramer • Plasmin activates Marix-Metallo-Proteinases • Essential for Tumor-promoting macrophages into tumor sites • Increased expression in CRPC • Validation by qRT-PCR • Lack of genetic aberrations (CGH) • Gene expression variable during cultivation • Normal fibroblasts as control (skin) *Cooperation with M. Caraglia/G. Vitale

25. Cancer associatedfibroblastsand tumor cells: a diaboliccross-talkdrivingcancerprogression • We focus on CancerAssociatedFibroblasts (CAF) : • Are they a specificcelltype or only a phenotype? • Where do they come from? • Charcterization by phenotype • Characterization by function

26. Crosstalk's between Tumor cells and Microenvironment Considering the “Secretome” +ECM! O.E. Franco et al. (2010): Cancer associated fibroblasts in cancer pathogenesis. SemCell&DevelopBiol

27. Paracrine communication between CAF and tumor cells Co-cultivation systems (6-24 well systems) Cells are cultivated in the membrane insert (top) and/or bottom of a corresponding plate cell type 1 cell type 1 membrane Secretom cell type 2 cell type 2 Secretom • Important: Not easy to discriminate the origin of secretom! • Functional analysis is more convenient • Standard assays (e.g. WST) can be adapted • Microscopic examination difficult

28. Paracrine communication between CAF and tumor cells Changes from “normal” to “activated” fibroblasts by Genom Function Influence on Production of Genotyping - Epigenetic – Phenotype CGH Methylation SMA Proliferation Migration Invasion EMT AR-Activation Stemness Cytokines SDF Growth factors = Secretome

29. Paracrine communication between CAF and tumor cells Mechanisms of invasion: Movement and enyzm activities Prostate Stromal Cell-Derived Hepatocyte Growth Factor Induces invasion of Prostate Cancer Cell Line DU-145 through Tumor-Stromal Interaction. K. Nishimura et al. Prostate 1999; 41. Stromal-epithelial interactions influence prostate cancer cell invasion by altering the Balance of metallopeptidase expression. LA Dawson et al. Br J Cancer 2004; 90. Regulation of migration of primary prostate epithelial cells by secreted factors from prostate stromal cells. X. You et al. Experimental Cell Res. 2003; 288. Stromal myofibroblasts are drivers of invasive cancer growth. O. De Wever et al. Int J Cancer 2008; 123. Estrogens Promote Invasion of Prostate Cancer Cells in a Paracrine Manner through Up-Regulation of Matrix Metalloproteinase 2 in Prostatic Stromal Cells. L.Yu et al. Endocrinology 2011; 152. Function Influence on Proliferationn Migration Invasion EMT AR-Activation

30. Verification of CAF-mediated functions Co-culture as one tool to proof CAF-TC crosstalk Lang&Maitland Lang +/- conditioned Medium LNCaP PC-3 *Norman Maitland attending our annual AuF Meeting - Berlin 2012 Stroma CI-Gel bPS+/- tPS Stroma enhanced proliferationof Tumor cells (XTT)

31. Verification of CAF-mediated functions Changes from “normal” to “activated” fibroblasts Lang&Maitland (2000) Function +/- conditioned Medium PET- Membrane 0.4µm LNCaP Influence on Differential influence of normal and cancer-associated fibroblasts on the growth of human epithelial cells in an in vitro cocultivation model of prostate cancer. Paland N, et al. Mol Cancer Res. 2009 7(8) Stroma (fibroblasts) Proliferation Migration Invasion EMT AR-Activation Altered TGF-β signaling in a subpopulation of human stromal cells promotes prostatic carcinogenesis.Franco OE, Jiang M, Strand DW, Peacock J, Fernandez S, Jackson RS 2nd, Revelo MP, Bhowmick NA, Hayward SW. Cancer Res. 2011 71(4): The Gene Expression Program of Prostate Fibroblast Senescence Modulates Neoplastic Epithelial Cell Proliferation trough Paracrine Mechanisms. : Bavikl et al. Cancer Res. 2006 66(2)

32. CAF as attractant to select for invasive growing cancer cells Changes from “normal” to “activated” fibroblasts Tumor samples (Prostatectomy) PET-Insert Medium Function Membrane 8 µm Matrigel Tumor Spheroids Matrigel Compan.plate Collagen I Collagen I Fibroblasts (CAF) Replaced by SDF1 Influence on Proliferation Migration Invasion EMT AR-Activation CAF as attractant

33. CAF as attractant to select for invasive growing cancer cells CGH analysis of invasiv growing prostate cancer cells* In all cases, invasive sub-populations had either acquired additional chromosome abnormalities to those present in the tumor tissue or other abnormalities present in the tumor tissue were lost. Invasive cells were often characterized by gains on chromosomes 1p33-q36, 12q24-q25, 14q24q-qter, 15q24-qter, 16, 20q, 22 and losses on 4q, 6q11-q15 and 13q21-q23 . * Selected by CAF’s

34. Contribution of CAF to AR-mediated gene regulation: • CAF express AR at low levels • T/DHT mediated activation of these AR contributes to AR activities • AR activation occurs by synthesis of T/DHT from DHEA • T/DHT independent growth factor promote prostate cancer cells growth • *Prostate stromal cells and LNCaP cells coordinately activate the AR through synthesis • of testosterone and DHT from dehydroepiandrosterone DHEA. • A.Mizokami et al. Endocrine-related cancer 2009; 16. • Co-culture of CAF with LNCaP cells – Quantification of androgens by LC-ESI-MS/S • RT-PCR of 3ß-HSD and 17ß-HSD Enzymes (HSD = Hydroxysteroid-Dehydrogenase • Effects of Androgen Receptor and Androgen on Gene Expression in Prostate Stromal • Fibroblasts and Paracrine Signaling to Prostate Cancer Cells. • M.J. Tanner et al. PLoS one 2011, 6(1). • WPMY-1 transfected to express AR at levels like LNCaP • +/-DHT changes gene expression profile (like TGF-ß, Hedgehog, Wnt and MAP Kinase) • Conditioned medium from DHT/WPMY.1 increases growth of LNCaP cells

35. The long way from normal prostate development to CRPC Changes in the behavior, function and localisation of Prostate Epithelial (Cancer) Cells under the Control of T/DHT and AR (and several other factors) and triggered by the environment Differentiation Proliferation Invasion Survival* Extravasation 2nd Tumor+ Organ- restricted PrCa Attachment Proliferation Stem cells? AR+ Invasion MMP`s MET Association w. Macrophages Embolies Detachment Migration MMP`s EMT *as circulating tumor cells + Metastasis

36. Strategies to disclose the role of “Cancer associated fibroblasts” in tumor development and progression and for a comprehensive characterization! Changes from “normal” to “activated” fibroblasts by Genom Function Influence on Production of Genotyping Epigenetic Transcription Phenotype CGH Methylation Senescence Co-Evolution RNA SMA miRNA FAP PDFRß CD90 Proliferation Migration Invasion EMT AR-Activation Cytokines SDF Growth factors

37. Molecular genetic comparison of cancer and non-cancer-associated fibroblasts in prostate cancer* Epigenetic modifications in Fibroblasts Global hypomethylation of genomic DNA in cancer-associated myofibroblasts. Jiang L, Gonda TA, Gamble MV, Salas M, Seshan V, Tu S, Twaddell WS, Hegyi P, Lazar G, Steele I, Varro A, Wang TC, Tycko B Cancer Res. 2008 Dec 1;68(23):9900-8. Specific age-associated DNA methylation changes in human dermal fibroblasts. Koch CM, Suschek CV, Lin Q, Bork S, Goergens M, Joussen S, Pallua N, Ho AD, Zenke M, Wagner W;PLoS One. 2011 Feb 8;6(2):e16679. Comparison 23 vs 60 years old donors! Hypermethylation in the aged group! No significant changes during subcultivation!

38. Molecular genetic comparison of cancer and non-cancer-associated fibroblasts in prostate cancer* Conclusion and Future Trends Secretome by Proteomics • CAF/TC crosstalk highly complex and dynamic • Co-Evolution of CAF/TC • CAF characterisation difficult • Comprehensive characterisation of “Secretome” • New and better models required • Hormone metabolism in CAF unclear • Contribution of CAF to CRPC likely? Tumor cell ECM CAF/NAF Secretome by Hormones/Metabolites

39. Viabilityofprostatecancercelllines after ZOL-application * Cooperation with Prof. Dr. M. Caraglia & Dr. G. De Rosa Comparison between flat and 3D cell cultures*

40. Molecular genetic comparison of cancer and non-cancer-associated fibroblasts in prostate cancer* Gravity contributes to the Fibroblast-Tumor Cell Interaction in-vitro! Three-dimensional co-culture models to study prostate cancer growth, progression, and metastasis to bone Ruoxiang Wang, a, Jianchun Xua, Lisa Juliettec, Agapito Castillejac, John Loveb, Shian-Ying Sunga, Haiyen E. Zhaua, Thomas J. Goodwinb, Leland W.K. Chunga a Department of Urology, Molecular Urology and Therapeutics Program, Emory University School of Medicine, Atlanta, GA 30322, USA b NASA Johnson Space Center, Houston, TX 77058, USA c Wyle Laboratories, Houston, Columbia 1st Fev 2003 harbouring Co-Cultivation Experiments on board

41. Thanks to the audience! Thanks to all which contributes to our results: Helga Angeli Eva Schmidt Volker Jung Kerstin Junker Michele Caraglia Giovanni Vitale

42. 24 h w/o Flow 24 h + 48 h + 159p6 161p1 Aber auch die Bewegung spielt eine Rolle! HUVEC cultivated onto/into flow-chambers (IBIDI

43. The long way from normal prostate development to CRPC Changes in the behavior, function and localisation of Prostate Epithelial (Cancer) Cells are under control of T/DHT and AR (and several other factors) and triggered by the environment. Differentiation Proliferation Invasion Survival* Extravasation 2nd Tumor+ (vessel) (bone) Organ- restricted PrCa Attachment Proliferation Stem cells? AR+ Invasion MMP`s MET Association w. Macrophages Embolies *as circulating tumor cells + Metastasis Detachment Migration MMP`s EMT

44. EMT = Epithelial-Mesenchymal-Transition Temporary shift in differentiation allowing enhanced mobility

45. Niche-to-niche migration of BM and tumor cells. The transit of BM and tumor cells from their respective niches is a multidirectional pathway. Hematopoietic cells are mobilized from the BM niche in response to tumor-secreted chemokines and subsequently home to both the primary tumor microenvironment and peripheral niches. BMDCs homing to the primary tumor niche may remain in an undifferentiated state in the form of HPCs, EPCs, MSCs, or GR-1+ CD11b+ MDSCs; or may differentiate into more specialized cell types including TAMs. Early BMDCs in transit to premetastatic peripheral niches likely possess an undifferentiated status as HPCs or myeloid-precursor cells, and at later stages involve homing of EPCs. Metastasizing tumor cells subsequently travel to peripheral niches occupied by BMDCs. Wels et al.Migratoryneighbors and distantinvaders: tumor-associatednichecells. GENES DEVELOP. 2008

46. Medium Collagen I gel with fibroblasts Crosstalk: Not restricted to malignant tissue! • Bell et al., J. Invest. Dermatol. 1983: „The reconstitution of living skin.“ • Mackenzie & Fusenig, J. Invest. Dermatol.,1983: • “Regeneration of organized epithelial structure.” • Asselineau & Prunieras, Brit. J. Dermatol., 1984: • “Reconstruction of simplified skin: control of fabrication.” Keratinocytes Air-exposed +/- Ca2+ Collagen I