Periodic Reporting for period 1 - CoCa (Collagen in Cancer: from the regulatory fibril forming function of collagen V in development to its implication in tumor progression)
Reporting period: 2016-06-01 to 2018-05-31
My research program builds on recent conceptual advances in how the physical and structural properties of the cell microenvironment contribute to tumor progression. While there is evidence that a slight increase in the hardness of the surrounding ECM perturbs tissue function, the influence of ECM topography on cancer progression remains largely unknown. Integrating molecular biology, transcription analysis, biochemistry, high-resolution imaging and mouse and cell models, the project investigated the role of COLV in tumor progression and the underlying mechanisms. This project is original and innovative for the following reasons: (1) it will comprehensively map COLV expression patterns in cancer. Central to this part of the project are two important findings: the ECM is a dynamic structure that influences tumour progression, and multiple cell types within the tumor can contribute to ECM production. It might provide the background for potential use of COLV as a biomarker and clues for understanding its function in cancer progression, helpful to clinicians. (2) Tumoral ECM topography related to the collagen V deposition. This burgeoning field of research focuses on how physical properties of ECM impact on cell behaviour during development. Investigating how disrupted ECM topography may contribute to cancer is even newer. We aimed to connect COLV overdeposition with a specific tumoral ECM topography to crucial aspects of the tumor phenotype and gene expression. (3) Upstream regulation of collagen V by hypoxia. Recent studies have established a direct link between hypoxia and the composition and the organization of the ECM. Blocking tumor hypoxia might provide a strategy to reduce COLV overdeposition and opens a possible avenue for anti-cancer therapy. I see this part of the project as a fundamental building block that will pave the way for future research to develop new therapeutic options offered to patients.
The main objective of this proposal was to decipher the mechanisms by which deregulation of COLV expression and subsequent extracellular deposition impacts cancer development. My aim was thus to provide a more complete picture of its role in tumor ECM topography, and how these changes impacts on cancer progression with a view to exploit this knowledge for cancer diagnosis and possibly in therapy. I had 3 more specific questions to address:
1. Establishment of a compilation data of COLV expression and localization in different tumor-type categories and its the cellular sources
2. Lay down the relationship beween Collagen V overdeposition and tumor ECM topography
3. What are the upstream mechanisms responsible for dysregulation of COLV expression in cancer? Does hypoxia regulate COLV overexpression in tumors in vivo?
For the second objective I developed human lung tumor model into immunodefficient mice inoculating A549 tumoral cells. First classical IHC for COLV, I, CA9 and HIF-1 was performed. The studied tumoral sections by SHG microscopy allowed us to see the topography of the matrix fibres, quantify and measured its dimensions. We also performed Raman spectrometry studies which gave the main biochemical composition of the tissue, being able to compare the matrix between the tumor and surrounding healthy tissue.
For the third objective in silico analysis of COLV proximal promoter revelled several putative regions susceptible of hypoxic regulation. This bioinformatic study found a very good candidate motive located 201 before the beginning of the COL5A1 gene. After, I established in vitro culture of different cell types that are present in the tumor (HUVEC, human Fibroblasts and A549 tumoral cells) to compare matrix production under hypoxic and normoxic conditions. We used Q-PCR and Immunofluorescence studies to quantify the production of matrix at transcriptional and protein level. In parallel, I performed a similar mouse experiment as in the objective 2, but in this case submitting the tumoral development to hypoxic conditions. Tumoral growth was followed by bioluminescence and samples were taken at different time points to establish the influence of hypoxia in the matrix structure and disease progression. At last, AFM microscopy was performed in hypoxic and normoxic samples to see the impact of hypoxia in the stiffness of the tumoral and healthy tissue.