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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

Abstract

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.

Keywords

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

Abbreviations

SM

Smooth muscle

ECM

Extracellular matrix

SLRP

Small leucine-rich proteoglycans stromal reaction

Notes

Acknowledgements

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)

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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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