Cancer-associated fibroblasts (CAFs) are highly differentiated and heterogeneous cancer-stromal cells that promote tumour growth, angiogenesis and matrix remodelling.
We utilised an adapted version of a previously developed 3D in vitro model of colorectal cancer, composed of a cancer mass and the surrounding stromal compartment. We compared cancer invasion with an acellular stromal surround, a “healthy” or normal cellular stroma and a cancerous stroma. For the cancerous stroma, we incorporated six patient-derived CAF samples to study their differential effects on cancer growth, vascular network formation and remodelling.
CAFs enhanced the distance and surface area of the invasive cancer mass whilst inhibiting vascular-like network formation. These processes correlated with the upregulation of hepatocyte growth factor (HGF), metallopeptidase inhibitor 1 (TIMP1) and fibulin-5 (FBLN5). Vascular remodelling of previously formed endothelial structures occurred through the disruption of complex networks, and was associated with the upregulation of vascular endothelial growth factor (VEGFA) and downregulation in vascular endothelial cadherin (VE-Cadherin).
These results support, within a biomimetic 3D, in vitro framework, the direct role of CAFs in promoting cancer invasion, and their key function in driving vasculogenesis and angiogenesis.
Subscribe to Journal
Get full journal access for 1 year
only $16.63 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Chang, C. H., Qiu, J., O’Sullivan, D., Buck, M. D., Noguchi, T., Curtis, J. D. et al. Metabolic competition in the tumor microenvironment is a driver of cancer progression. Cell 162, 1229–1241 (2015).
Shiga, K., Hara, M., Nagasaki, T., Sato, T. & Takahashi, H. Cancer-associated fibroblasts: their characteristics and their roles in tumor growth. Cancers 7, 2443–2458 (2015).
Augsten, M. Cancer-associated fibroblasts as another polarized cell type of the tumor microenvironment. Front. Oncol. 4, 1–34 (2014).
Tommelein, J., Verset, L., Boterberg, T., Demetter, P., Bracke, M. & De Wever, O. Cancer-associated fibroblasts connect metastasis-promoting communication in colorectal cancer. Front. Oncol. 5, 1–11 (2015).
Attieh, Y. & Vignjevic, D. M. The hallmarks of CAFs in cancer invasion. Eur. J. Cell Biol. 95, 493–502 (2016).
Kalluri, R. The biology and function of fibroblasts in cancer. Nat. Publ. Gr. 16, 582–598 (2016).
Reid, S. E., Kay, E. J., Neilson, L. J., Henze, A., Serneels, J., McGhee, E. J. et al. Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium. EMBO J. 36, 2373–2389 (2017).
Mantovani, A., Marchesi, F., Malesci, A., Laghi, L. & Allavena, P. Tumour-associated macrophages as treatment targets in oncology. Nat. Rev. Clin. Oncol. 14, 399–416 (2017).
Cirri, P. & Chiarugi, P. Cancer-associated-fibroblasts and tumour cells: a diabolic liaison driving cancer progression. Cancer Metastasis Rev. 31, 195–208 (2012).
Darby, I. A., Laverdet, B., Bonté, F. & Desmoulière, A. Fibroblasts and myofibroblasts in wound healing. Clin. Cosmet. Investig. Dermatol. 7, 301–311 (2014).
Cirri, P. & Chiarugi, P. Cancer associated fibroblasts: the dark side of the coin. Am. J. 1 Cancer Res. 1, 482–497 (2011).
Madsen, C. D., Pedersen, J. T., Venning, F. A., Singh, L. B., Charras, G., Cox, T. R. et al. Hypoxia and loss of PHD 2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis. EMBO Rep. 16, 1394–1408 (2015).
Glentis, A., Oertle, P., Mariani, P., Chikina, A., Marjou, F. El, Attieh, Y. et al. Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane. Nat. Commun. 8, 1–13 (2017).
Maller, O., Dufort, C. C. & Weaver, V. M. YAP forces fibroblasts to feel the tension. Nat. Cell Biol. 15, 570–572 (2013).
Calvo, F., Ege, N., Grande-Garcia, A., Hooper, S., Jenkins, R. P., Chaudhry, S. I. et al. Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat. Cell Biol. 15, 637–646 (2013).
Cao, H., Xu, E., Liu, H., Wan, L. & Lai, M. Epithelial-mesenchymal transition in colorectal cancer metastasis: a system review. Pathol. Res. Pr. 211, 557–569 (2015).
Kakarla, S., Song, X.-T. & Gottschalk, S. Cancer-associated fibroblasts as targets for immunotherapy. Immunotherapy 4, 1129–1138 (2012).
Liu, T., Lin, B. & Qin, J. Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device. Lab. Chip 10, 1671–1677 (2010).
Pape, J., Magdeldin, T., Ali, M., Walsh, C., Lythgoe, M., Emberton, M. et al. Cancer invasion regulates vascular complexity in a three-dimensional biomimetic model. Eur. J. Cancer 119, 179–193 (2019).
Magdeldin, T., López-Dávila, V., Pape, J., Cameron, G. W. W., Emberton, M., Loizidou, M. et al. Engineering a vascularised 3D in vitro model of cancer progression. Sci. Rep. 7, 1–9 (2017).
Stamati, K., Priestley, J. V., Mudera, V. & Cheema, U. Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake. Exp. Cell Res. 327, 68–77 (2014).
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Rio, D. C., Ares, M., Hannon, G. J. & Nilsen, T. W. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb. Protoc. 5, 1–4 (2010).
Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CTmethod. Nat. Protoc. 3, 1101–1108 (2008).
Bolander, J., Chai, Y. C., Geris, L., Schrooten, J., Lambrechts, D., Roberts, S. J. et al. Early BMP, Wnt and Ca2+/PKC pathway activation predicts the bone forming capacity of periosteal cells in combination with calcium phosphates. Biomaterials 86, 106–118 (2016).
Choi, S. Y., Sung, R., Lee, S. J., Lee, T. G., Kim, N., Yoon, S. M. et al. Podoplanin, α-smooth muscle actin or S100A4 expressing cancer-associated fibroblasts are associated with different prognosis in colorectal cancers. J. Korean Med. Sci. 28, 1293–1301 (2013).
Madar, S., Goldstein, I. & Rotter, V. “Cancer associated fibroblasts”—more than meets the eye. Trends Mol. Med. 19, 447–453 (2013).
Knüpfer, H. & Preiss, R. Serum interleukin-6 levels in colorectal cancer patients-a summary of published results. Int J. Colorectal Dis. 25, 135–140 (2010).
Talele, N. P., Fradette, J., Davies, J. E., Kapus, A. & Hinz, B. Expression of α-smooth muscle actin determines the fate of mesenchymal stromal cells. Stem Cell Rep. 4, 1016–1030 (2015).
Hamada, K., Monnai, M., Kawai, K., Nishime, C., Kito, C., Miyazaki, N. et al. Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shi-scid IL2Rgammanull (NOG) mice. Int. J. Oncol. 32, 153–159 (2008).
Roudsari, L. C. & West, J. L. Studying the influence of angiogenesis in in vitro cancer model systems. Adv. Drug Deliv. Rev. 97, 250–259 (2016).
Vestweber, D. VE-cadherin: The major endothelial adhesion molecule controlling cellular junctions and blood vessel formation. Arterioscler Thromb. Vasc. Biol. 28, 223–232 (2008).
Liao, Y., Zhao, H., Liu, Q. & Peng, R. Fibulin-5 inhibits the cell proliferation, migration and angiogenesis in glioma. Int. J. Clin. Exp. Pathol. 9, 8943–8952 (2016).
Sullivan, K. M., Bissonnette, R., Yanagisawa, H., Hussain, S. N. & Davis, E. C. Fibulin-5 functions as an endogenous angiogenesis inhibitor. Lab. Investig. 87, 818–827 (2007).
Albig, A. R. & Schiemann, W. P. Fibulin-5 antagonizes vascular endothelial growth factor (VEGF) signaling and angiogenic sprouting by endothelial cells. DNA Cell Biol. 23, 367–379 (2004).
Jeanes, A., Gottardi, C. J. & Yap, A. S. Cadherins and cancer: how does cadherin dysfunction promote tumor progression? Oncogene 27, 6920–6929 (2008).
Grugan, K. D., Miller, C. G., Yao, Y., Michaylira, C. Z., Ohashi, S., Klein-Szanto, A. J. et al. Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion. Proc. Natl Acad. Sci. USA 107, 11026–11031 (2010).
Zhang, Y., Tang, H., Cai, J., Zhang, T., Guo, J., Feng, D. et al. Ovarian cancer-associated fibroblasts contribute to epithelial ovarian carcinoma metastasis by promoting angiogenesis, lymphangiogenesis and tumor cell invasion. Cancer Lett. 303, 47–55 (2011).
Hwang, R. F., Moore, T., Arumugam, T., Ramachandran, V., Amos, K. D., Rivera, A. et al. Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res. 68, 918–926 (2008).
Barcus, C. E., Keely, P. J., Eliceiri, K. W. & Schuler, L. A. Stiff collagen matrices increase tumorigenic prolactin signaling in breast cancer cells. J. Biol. Chem. 288, 12722–12732 (2013).
Wei, S. C. & Yang, J. Forcing through tumor metastasis: the interplay between tissue rigidity and epithelial-mesenchymal transition. Trends Cell Biol. 26, 111–120 (2016).
Tang, D., Gao, J., Wang, S., Ye, N., Chong, Y., Huang, Y. et al. Cancer-associated fibroblasts promote angiogenesis in gastric cancer through galectin-1 expression. Tumor Biol. 37, 1889–1899 (2016).
Hallaq, H. A null mutation of Hhex results in abnormal cardiac development, defective vasculogenesis and elevated Vegfa levels. Development 131, 5197–5209 (2004).
Kioi, M., Vogel, H., Schultz, G., Hoffman, R. M., Harsh, G. R. & Brown, J. M. Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J. Clin. Invest. 120, 694–705 (2010).
Weis, S., Cui, J., Barnes, L. & Cheresh, D. Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis. J. Cell Biol. 167, 223–229 (2004).
Li, W.-W., Wang, H., Nie, X., Liu, Y., Han, M. & Li, B.-H. Human colorectal cancer cells induce vascular smooth muscle cell apoptosis in an exocrine manner. Oncotarget 8, 62049–62056 (2017).
De Francesco, E. M., Lappano, R., Santolla, M. F., Marsico, S., Caruso, A. & Maggiolini, M. HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs). Breast Cancer Res. 15, 1–18 (2013).
Alarcón, T., Owen, M. R., Byrne, H. M. & Maini, P. K. Multiscale modelling of tumour growth and therapy: the influence of vessel normalisation on chemotherapy. Comput. Math. Methods Med. 7, 85–119 (2006).
Torres, S., Bartolomé, R. A., Mendes, M., Barderas, R., Fernandez-Aceñero, M. J., Peláez-García, A. et al. Proteome profiling of cancer-associated fibroblasts identifies novel proinflammatory signatures and prognostic markers for colorectal cancer. Clin. Cancer Res. 21, 6006–6019 (2013).
Ueno, H., Murphy, J., Jass, J. R., Mochizuki, H. & Talbot, I. C. Tumour “budding” as an index to estimate the potential of aggressiveness in rectal cancer. Histopathology 40, 127–132 (2002).
Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. K. The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8, 839–845 (2007).
You, W. K. & McDonald, D. M. The hepatocyte growth factor/c-met signaling pathway as a therapeutic target to inhibit angiogenesis. J. Biochem. Mol. Biol. 41, 833–839 (2008).
Ethics approval and consent to participate
Primary human colorectal cancer-associated fibroblasts were obtained in accordance with the Declaration of Helsinki. Samples were isolated from tumour tissues acquired from surgeries at the Royal Free Hospital. Patients provided informed consent for tissue donation for research under ethics reference 11/WA/0077 obtained through the North Wales Research Ethics Committee (Central and East) through TAPb biobank.
Consent to publish
The data that support the findings of this study are available from the corresponding author (U.C.) on reasonable request.
The authors declare no competing interests.
Judith Pape receives a stipend and EU fee funding from the EPSRC as part of the doctoral training programme (DTP). Mark Emberton receives research support from the United Kingdom’s National Institute of Health Research (NIHR) UCLH/UCL Biomedical Research Centre and became an NIHR Senior Investigator in 2015. This work was funded by the NIHR Invention for Innovation (i4i) programme. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.
Note This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Pape, J., Magdeldin, T., Stamati, K. et al. Cancer-associated fibroblasts mediate cancer progression and remodel the tumouroid stroma. Br J Cancer 123, 1178–1190 (2020). https://doi.org/10.1038/s41416-020-0973-9
Frontiers in Bioengineering and Biotechnology (2021)
Selective Targeting of Cancer-Associated Fibroblasts by Engineered H-Ferritin Nanocages Loaded with Navitoclax
Tissue-Engineering the Fibrous Pancreatic Tumour Stroma Capsule in 3D Tumouroids to Demonstrate Paclitaxel Response
International Journal of Molecular Sciences (2021)