Cell and Tissue Research

, Volume 346, Issue 2, pp 223–236 | Cite as

Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion

  • Vivien J. Coulson-Thomas
  • Yvette M. Coulson-Thomas
  • Tarsis F. Gesteira
  • Cláudia A. A. de Paula
  • Ana M. Mader
  • Jaques Waisberg
  • Maria A. Pinhal
  • Andreas Friedl
  • Leny Toma
  • Helena B. Nader
Regular Article


During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.


Myofibroblasts Stromal reaction Proteoglycans Collagen 2D- and 3D-cultures 



Smooth muscle


Extracellular matrix


Small leucine-rich proteoglycans stromal reaction



This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (2007/59801-1), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We would like to thank Prof. Peter Reinach and Prof. Winston Kao for their kind support throughout this study. We also acknowledge Caroline Z. Romera and Elizabeth N. Kanashiro (INFAR/UNIFESP, Brazil) for their technical assistance.

Supplementary material

441_2011_1254_Fig6_ESM.jpg (50 kb)
Figure S1

Gene expression of SLRPs after heterotypic co-culture of colorectal cancer cells and fibroblasts. For bilayer co-culture the colorectal cancer cell lines Caco-2 and HCT 116 were seeded on top of previously seeded fibroblasts (CACO2/WPF5 and HCT116/WPF5, respectively) and fibroblasts were seeded on top of the previously seeded colorectal cancer cell lines (WPF5/CACO2 and WPF5/HCT116, respectively). After 24 hours, RNA was extracted for analysis by real-time PCR of decorin (a), biglycan (b), lumican (c) and fibromodulin (d). Controls were performed seeding the same cell type on top and underneath (WPF5/WPF5, CACO2/CACO2 and HCT116/HCT116). For the bi-compartmental (transwell) co-culture of colorectal cancer cells and fibroblasts the fibroblasts (WPF5) and colorectal cancer cells (CACO2 and HCT116) were grown both in transwell inserts (0.4-μm membrane pores) and in microplate wells to study the effect of soluble factors in the cross-talk between the cell lines. After 24 or 72 hours RNA was extracted from the cells in the bottom compartment and was analyzed through quantitative RT-PCR for the expression levels of decorin (e and i), biglycan (f and j), lumican (g and k) and fibromodulin (h and l). The experimental groups comprised seeding the colorectal tumor cell lines in the top compartment and the fibroblasts in the bottom compartment (tCACO2/WPF5 and tHCT116/WPF5) as well as the fibroblasts in the top compartment and the cancer cell lines in the bottom compartment (tWPF5/CACO2 and tWPF5/HCT116). The control groups entailed seeding the same cells in both the top and bottom compartments (tWPF5/WPF5; tCACO2/CACO2; tHCT116/HCT116). Gene expression was normalized against ribosomal protein S29 (RPS29). *p < 0.05. (JPEG 50 kb)

441_2011_1254_MOESM1_ESM.tif (6 mb)
High resolution image (TIFF 6106 kb)
441_2011_1254_MOESM2_ESM.pdf (204 kb)
Figure S2 3D co-culture of colorectal cancer cells and fibroblasts using a 3D stromatogenic model. Fibroblasts (CCD112) or colorectal cancer cells (Caco-2, HCT) were seeded on top of previously plated confluent fibroblasts (CCD112, and CCD112 + CACO2, CCD112 + HCT, respectively). The cultures were treated with ascorbic acid every other day to produce a 3D stromagenic system. Fibronectin, collagen I, biglycan, perlecan and versican were immunostained fluorescently labeled. Scale bar: 20 μm. (PDF 203 kb)


  1. Adany R, Heimer R, Caterson B, Sorrell JM, Iozzo RV (1990) Altered expression of chondroitin sulfate proteoglycan in the stroma of human colon carcinoma. Hypomethylation of PG-40 gene correlates with increased PG-40 content and mRNA levels. J Biol Chem 265:11389–11396PubMedGoogle Scholar
  2. Amatangelo MD, Bassi DE, Klein-Szanto AJ, Cukierman E (2005) Stroma-derived three-dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts. Am J Pathol 167:475–488PubMedCrossRefGoogle Scholar
  3. Angeli F, Koumakis G, Chen MC, Kumar S, Delinassios JG (2009) Role of stromal fibroblasts in cancer: promoting or impeding? Tumour Biol 30:109–120PubMedCrossRefGoogle Scholar
  4. Apte MV, Park S, Phillips PA, Santucci N, Goldstein D, Kumar RK, Ramm GA, Buchler M, Friess H, McCarroll JA, Keogh G, Merrett N, Pirola R, Wilson JS (2004) Desmoplastic reaction in pancreatic cancer: role of pancreatic stellate cells. Pancreas 29:179–187PubMedCrossRefGoogle Scholar
  5. Augoff K, Rabczynski J, Tabola R, Czapla L, Ratajczak K, Grabowski K (2008) Immunohistochemical study of decorin expression in polyps and carcinomas of the colon. Med Sci Monit 14:CR530–CR535PubMedGoogle Scholar
  6. Bosman FT, de Bruine A, Flohil C, van der Wurff A, ten Kate J, Dinjens WW (1993) Epithelial-stromal interactions in colon cancer. Int J Dev Biol 37:203–211PubMedGoogle Scholar
  7. Breuninger H, Schaumburg-Lever G, Holzschuh J, Horny HP (1997) Desmoplastic squamous cell carcinoma of skin and vermilion surface: a highly malignant subtype of skin cancer. Cancer 79:915–919PubMedCrossRefGoogle Scholar
  8. Caporale A, Vestri AR, Benvenuto E, Mariotti M, Cosenza UM, Scarpini M, Giuliani A, Mingazzini P, Angelico F (2005) Is desmoplasia a protective factor for survival in patients with colorectal carcinoma? Clin Gastroenterol Hepatol 3:370–375PubMedCrossRefGoogle Scholar
  9. Caporale A, Amore Bonapasta S, Scarpini M, Ciardi A, Vestri A, Ruperto M, Giuliani A (2010) Quantitative investigation of desmoplasia as a prognostic indicator in colorectal cancer. J Invest Surg 23:105–109PubMedCrossRefGoogle Scholar
  10. Compton CC, Fielding LP, Burgart LJ, Conley B, Cooper HS, Hamilton SR, Hammond MEH, Henson DE, Hutter RVP, Nagle RB, Nielsen ML, Sargent DJ, Taylor CR, Welton M, Willett C (2000) Prognostic factors in colorectal cancer - college of American pathologists consensus statement 1999. Arch Pathol Lab Med 124:979–994PubMedGoogle Scholar
  11. Conklin MW, Eickhoff JC, Riching KM, Pehlke CA, Eliceiri KW, Provenzano PP, Friedl A, Keely PJ (2011) Aligned collagen is a prognostic signature for survival in human breast carcinoma. Am J Pathol 178:1221–1232PubMedCrossRefGoogle Scholar
  12. Coulson-Thomas VJ, Gesteira TF, Coulson-Thomas YM, Vicente CM, Tersariol IL, Nader HB, Toma L (2010) Fibroblast and prostate tumor cell cross-talk: Fibroblast differentiation, TGF-beta, and extracellular matrix down-regulation. Exp Cell Res 316:3207–3226Google Scholar
  13. Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV (1997) Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J Cell Biol 136:729–743PubMedCrossRefGoogle Scholar
  14. Eyden B, Tzaphlidou M (2001) Structural variations of collagen in normal and pathological tissues: role of electron microscopy. Micron 32:287–300PubMedCrossRefGoogle Scholar
  15. Faouzi S, Le Bail B, Neaud V, Boussarie L, Saric J, Bioulac-Sage P, Balabaud C, Rosenbaum J (1999) Myofibroblasts are responsible for collagen synthesis in the stroma of human hepatocellular carcinoma: an in vivo and in vitro study. J Hepatol 30:275–284PubMedCrossRefGoogle Scholar
  16. Forster SJ, Talbot IC, Critchley DR (1984) Laminin and fibronectin in rectal adenocarcinoma: relationship to tumour grade, stage and metastasis. Br J Cancer 50:51–61PubMedCrossRefGoogle Scholar
  17. Goetz JG, Minguet S, Navarro-Lerida I, Lazcano JJ, Samaniego R, Calvo E, Tello M, Osteso-Ibanez T, Pellinen T, Echarri A, Cerezo A, Klein-Szanto AJ, Garcia R, Keely PJ, Sanchez-Mateos P, Cukierman E, Del Pozo MA (2011) Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis. Cell 146:148–163PubMedCrossRefGoogle Scholar
  18. Gress TM, Muller-Pillasch F, Lerch MM, Friess H, Buchler M, Adler G (1995) Expression and in-situ localization of genes coding for extracellular matrix proteins and extracellular matrix degrading proteases in pancreatic cancer. Int J Cancer 62:407–413PubMedCrossRefGoogle Scholar
  19. Gressner AM (1994) Activation of proteoglycan synthesis in injured liver—a brief review of molecular and cellular aspects. Eur J Clin Chem Clin Biochem 32:225–237PubMedGoogle Scholar
  20. Halvorsen TB, Seim E (1989) Association between invasiveness, inflammatory reaction, desmoplasia and survival in colorectal cancer. J Clin Pathol 42:162–166PubMedCrossRefGoogle Scholar
  21. Hanamura N, Yoshida T, Matsumoto E, Kawarada Y, Sakakura T (1997) Expression of fibronectin and tenascin-C mRNA by myofibroblasts, vascular cells and epithelial cells in human colon adenomas and carcinomas. Int J Cancer 73:10–15PubMedCrossRefGoogle Scholar
  22. Hasebe T, Mukai K, Tsuda H, Ochiai A (2000) New prognostic histological parameter of invasive ductal carcinoma of the breast: clinicopathological significance of fibrotic focus. Pathol Int 50:263–272PubMedCrossRefGoogle Scholar
  23. Hauptmann S, Zardi L, Siri A, Carnemolla B, Borsi L, Castellucci M, Klosterhalfen B, Hartung P, Weis J, Stocker G et al (1995) Extracellular matrix proteins in colorectal carcinomas. Expression of tenascin and fibronectin isoforms. Lab Invest 73:172–182PubMedGoogle Scholar
  24. Havenith MG, Arends JW, Simon R, Volovics A, Wiggers T, Bosman FT (1988) Type IV collagen immunoreactivity in colorectal cancer. Prognostic value of basement membrane deposition. Cancer 62:2207–2211PubMedCrossRefGoogle Scholar
  25. Hewitt RE, Powe DG, Carter GI, Turner DR (1993) Desmoplasia and its relevance to colorectal tumor invasion. Int J Cancer 53:62–69PubMedCrossRefGoogle Scholar
  26. Iozzo RV (1985) Neoplastic modulation of extracellular matrix. Colon carcinoma cells release polypeptides that alter proteoglycan metabolism in colon fibroblasts. J Biol Chem 260:7464–7473PubMedGoogle Scholar
  27. Iozzo RV, Bolender RP, Wight TN (1982) Proteoglycan changes in the intercellular matrix of human colon carcinoma: an integrated biochemical and stereologic analysis. Lab Invest 47:124–138PubMedGoogle Scholar
  28. Ishiwata T, Cho K, Kawahara K, Yamamoto T, Fujiwara Y, Uchida E, Tajiri T, Naito Z (2007) Role of lumican in cancer cells and adjacent stromal tissues in human pancreatic cancer. Oncol Rep 18:537–543PubMedGoogle Scholar
  29. Kalimo H, Lehto M, Nantosalonen K, Jalkanen M, Risteli L, Risteli J, Narva EV (1985) Characterization of the perivascular reticulin network in a case of primary brain lymphoma. Immunohistochemical demonstration of collagen types I, III, IV, and V; laminin; and fibronectin. Acta Neuropathol 66:299–305PubMedCrossRefGoogle Scholar
  30. Kunz-Schughart LA, Knuechel R (2002) Tumor-associated fibroblasts (part I): active stromal participants in tumor development and progression? Histol Histopathol 17:599–621PubMedGoogle Scholar
  31. Leppert PC, Baginski T, Prupas C, Catherino WH, Pletcher S, Segars JH (2004) Comparative ultrastructure of collagen fibrils in uterine leiomyomas and normal myometrium. Fertil Steril 82:1182–1187PubMedCrossRefGoogle Scholar
  32. Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SF, Csiszar K, Giaccia A, Weninger W, Yamauchi M, Gasser DL, Weaver VM (2009) Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139:891–906PubMedCrossRefGoogle Scholar
  33. Leygue E, Snell L, Dotzlaw H, Hole K, Hiller-Hitchcock T, Roughley PJ, Watson PH, Murphy LC (1998) Expression of lumican in human breast carcinoma. Cancer Res 58:1348–1352PubMedGoogle Scholar
  34. Leygue E, Snell L, Dotzlaw H, Troup S, Hiller-Hitchcock T, Murphy LC, Roughley PJ, Watson PH (2000) Lumican and decorin are differentially expressed in human breast carcinoma. J Pathol 192:313–320PubMedCrossRefGoogle Scholar
  35. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  36. Martin MS, Caignard A, Hammann A, Pelletier H, Martin F (1987) An immunohistological study of cells infiltrating progressive and regressive tumors induced by two variant subpopulations of a rat colon cancer cell line. Int J Cancer 40:87–93PubMedCrossRefGoogle Scholar
  37. Miura S, Kodaira S, Hosoda Y (1993) Immunohistologic analysis of the extracellular matrix components of the fibrous stroma of human colon cancer. J Surg Oncol 53:36–42PubMedCrossRefGoogle Scholar
  38. Nuernberger S, Cyran N, Albrecht C, Redl H, Vecsei V, Marlovits S (2011) The influence of scaffold architecture on chondrocyte distribution and behavior in matrix-associated chondrocyte transplantation grafts. Biomaterials 32:1032–1040PubMedCrossRefGoogle Scholar
  39. Painter KJ (2009) Modelling cell migration strategies in the extracellular matrix. J Math Biol 58:511–543PubMedCrossRefGoogle Scholar
  40. Palecek SP, Loftus JC, Ginsberg MH, Lauffenburger DA, Horwitz AF (1997) Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385:537–540PubMedCrossRefGoogle Scholar
  41. Persky B, Hendrix MJC (1990) Artificial matrix barriers—a diffusion study utilizing dextrans and microspheres. Anat Rec 228:15–22PubMedCrossRefGoogle Scholar
  42. Provenzano PP, Inman DR, Eliceiri KW, Knittel JG, Yan L, Rueden CT, White JG, Keely PJ (2008) Collagen density promotes mammary tumor initiation and progression. BMC Medicine 6:11Google Scholar
  43. Ramaswamy S, Ross KN, Lander ES, Golub TR (2003) A molecular signature of metastasis in primary solid tumors. Nat Genet 33:49–54PubMedCrossRefGoogle Scholar
  44. Sampaio Lde O, Bayliss MT, Hardingham TE, Muir H (1988) Dermatan sulphate proteoglycan from human articular cartilage. Variation in its content with age and its structural comparison with a small chondroitin sulphate proteoglycan from pig laryngeal cartilage. Biochem J 254:757–764PubMedGoogle Scholar
  45. Seya T, Tanaka N, Shinji S, Yokoi K, Koizumi M, Teranishi N, Yamashita K, Tajiri T, Ishiwata T, Naito Z (2006) Lumican expression in advanced colorectal cancer with nodal metastasis correlates with poor prognosis. Oncol Rep 16:1225–1230PubMedGoogle Scholar
  46. Shi Y, Niculescu R, Wang D, Ormont M, Magno M, San Antonio JD, Williams KJ, Zalewski A (2000) Myofibroblast involvement in glycosaminoglycan synthesis and lipid retention during coronary repair. J Vasc Res 37:399–407PubMedCrossRefGoogle Scholar
  47. Ueno H, Jones AM, Wilkinson KH, Jass JR, Talbot IC (2004) Histological categorisation of fibrotic cancer stroma in advanced rectal cancer. Gut 53:581–586PubMedCrossRefGoogle Scholar
  48. Wang J, Levenson AS, Satcher RL Jr (2006) Identification of a unique set of genes altered during cell-cell contact in an in vitro model of prostate cancer bone metastasis. Int J Mol Med 17:849–856PubMedGoogle Scholar
  49. Yang TY, Zaman MH (2010) Estimation of cellular adhesion forces using mean field theory. Cell Mol Bioeng 3:190–194CrossRefGoogle Scholar
  50. Yashiro M, Chung YS, Nishimura S, Inoue T, Sowa M (1996) Fibrosis in the peritoneum induced by scirrhous gastric cancer cells may act as "soil" for peritoneal dissemination. Cancer 77:1668–1675PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Vivien J. Coulson-Thomas
    • 1
    • 5
  • Yvette M. Coulson-Thomas
    • 1
  • Tarsis F. Gesteira
    • 1
  • Cláudia A. A. de Paula
    • 1
  • Ana M. Mader
    • 2
  • Jaques Waisberg
    • 3
  • Maria A. Pinhal
    • 1
  • Andreas Friedl
    • 4
  • Leny Toma
    • 1
  • Helena B. Nader
    • 1
  1. 1.Department of BiochemistryUniversidade Federal de São PauloSão PauloBrazil
  2. 2.Department of PathologyFaculdade de Medicina ABCSanto AndreBrazil
  3. 3.Department of GastrosurgeryFaculdade de Medicina ABCSanto AndreBrazil
  4. 4.Department of Pathology and Laboratory MedicineUniversity of Wisconsin-MadisonMadisonUSA
  5. 5.Departamento de Bioquímica, Escola Paulista de MedicinaUniversidade Federal de São PauloSão PauloBrazil

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