Periodic Reporting for period 1 - FiBRO (Fibroblasts in the Biology of Rectal Cancer Progression)
Periodo di rendicontazione: 2016-05-01 al 2018-04-30
Although the therapeutic strategies adopted in the last years for the clinical management of patients with rectal cancer have improved patient’s quality of life, tumor relapse still constitutes to be a factor that determines patient survival. These patients, usually treated with chemo-radiotherapy (CRT), often develop a CRT-induced fibrosis, which is characterized by an overgrowth of fibroblasts at tumor-treated areas. The importance of tumor stroma for the development, progression and invasion of cancer is increasingly gaining weight, however, the biological basis of fibrosis, as well as the consequences that it has in terms of therapy resistance and tumor relapse remain unknown.
The main aim of the Marie Curie IF-FIBRO project was to elucidate the molecular changes that CRT could be exerting on the tumor stroma, particularly in the population of Cancer Associated Fibroblasts or CAFs. This, with the goal of understanding how stromal modifications can alter tumor development and aggressiveness and patient’s prognosis. Increasing knowledge in the process of tumor progression will have a clear impact in future’s medicine and society as it will determine the development of new diagnostic markers and targeted cancer therapies.
The main aim of the Marie Curie IF-FIBRO project was to elucidate the molecular changes that CRT could be exerting on the tumor stroma, particularly in the population of Cancer Associated Fibroblasts or CAFs. This, with the goal of understanding how stromal modifications can alter tumor development and aggressiveness and patient’s prognosis. Increasing knowledge in the process of tumor progression will have a clear impact in future’s medicine and society as it will determine the development of new diagnostic markers and targeted cancer therapies.
During the period 2016-2018, together with a team of surgeons and clinical oncologists at Institut Curie, Paris, we have collected samples from rectal cancer patients. To understand the effects of CRT both in the tumor and in the stroma, samples were taken before and after therapy from the same patient. This allows us to estimate the precise modifications exerted by chemoradiotherapy, avoiding variability due to patient’s heterogeneity. Using these samples I have carried out: i) the establishment of primary CAFs and normal fibroblasts from patient’s tissue samples (9 patients have been enrolled in the study so far), ii) fibroblasts characterization based on the expression of specific intracellular and membrane markers, iii) optimization development of high throughput proteomic techniques to analyze both the global proteomes and matrisomes of the obtained primary cultures, iv) development of 3D in vitro invasion assays to model tumor-stroma interaction and to evaluate how post-CRT CAFs influence the behavior of tumor cells. As well, we have established a series of mice models that help us to understand tumor cell invasion, and v) together with the pathology department of Institut Curie we started to perform a retrospective evaluation in tumor samples collected at the institute, in order to understand the relationship between fibrosis development and patient prognosis.
Description of main results so far
I have performed primary cultures from fibroblasts (CAFs and normal fibroblasts, NAFs) from 9 different rectal cancer patients, before and after radiotherapy. From those samples, I have obtained both intracellular protein extracts. Also, I have generated a system to purify extracellular matrix proteins from the primary cultures. In this system, cells are always plated on a soft substrate (30KPa), thus preventing the normal CAFs from getting activated. Together with the proteomics facility in Curie, we have performed proteomics for the samples available so far. Although this work is not finished yet, preliminary results point to a specific pattern of matrix protein expression in CAFs after radiotherapy.
Fibronectin (FN1) was among one of the most CAF expressed proteins. We have found that this protein plays an important role in the process of tumor metastasis, particularly in the process of circulating tumor cell (CTC) extravasation. Especially in tumor bearing mice, fibronectin was present in the form of aggregates attached to the walls of hepatic blood vessels. This specific location facilitates the attachment of CTCs and their extravasation to form new metastatic lesions. Indeed, cancer cells expressing Talin1, a protein that is involved in the formation of focal adhesions (structures used by the cells to attach Fibronectin), was key to allow CTC attachment to this fibronectin-based structures. Patients having higher levels of Talin1 in CTCs, showed a worse prognostic compared with those expressing less Talin1. In this work (Barbazan et al, Cancer Research, 2017), we concluded that both focal adhesion proteins in cancer cells, as well as hepatic fibronectin, could confer new targets against tumor dissemination.
Moreover, we have developed other methods to study tumor cell migration and invasion in 3D (Staneva R, Barbazan J, et al, Methods Mol Biol, 2018). Using some of these systems, we have evaluated how CAFs (pre and post-CRT) determine tumor invasion and stemness properties. Although these results have not been published yet, preliminary results point towards a cancer cell stemness promoting phenotype after radiotherapy.
References
- Barbazán J, Alonso-Alconada L, Elkhatib N, Geraldo S, Gurchenkov V, Glentis A, Van Niel G, Palmulli R, Fernández B, Viano P, Garcia-Caballero T, Lopez-Lopez R, Abal M & Vignjevic D. Liver metastasis is facilitated by the adherence of circulating tumor cells to vascular fibronectin deposits. Cancer Research, 2017. DOI: 10.1158/0008-5472.CAN-16-1917
- Staneva R, Barbazan J, Simon A, Vignjevic DM, Krndja D. Cell migration in tissues: Explant culture and live imaging. Methods Mol Biol, 2018.
DOI: 10.1007/978-1-4939-7701-7_13.
Description of main results so far
I have performed primary cultures from fibroblasts (CAFs and normal fibroblasts, NAFs) from 9 different rectal cancer patients, before and after radiotherapy. From those samples, I have obtained both intracellular protein extracts. Also, I have generated a system to purify extracellular matrix proteins from the primary cultures. In this system, cells are always plated on a soft substrate (30KPa), thus preventing the normal CAFs from getting activated. Together with the proteomics facility in Curie, we have performed proteomics for the samples available so far. Although this work is not finished yet, preliminary results point to a specific pattern of matrix protein expression in CAFs after radiotherapy.
Fibronectin (FN1) was among one of the most CAF expressed proteins. We have found that this protein plays an important role in the process of tumor metastasis, particularly in the process of circulating tumor cell (CTC) extravasation. Especially in tumor bearing mice, fibronectin was present in the form of aggregates attached to the walls of hepatic blood vessels. This specific location facilitates the attachment of CTCs and their extravasation to form new metastatic lesions. Indeed, cancer cells expressing Talin1, a protein that is involved in the formation of focal adhesions (structures used by the cells to attach Fibronectin), was key to allow CTC attachment to this fibronectin-based structures. Patients having higher levels of Talin1 in CTCs, showed a worse prognostic compared with those expressing less Talin1. In this work (Barbazan et al, Cancer Research, 2017), we concluded that both focal adhesion proteins in cancer cells, as well as hepatic fibronectin, could confer new targets against tumor dissemination.
Moreover, we have developed other methods to study tumor cell migration and invasion in 3D (Staneva R, Barbazan J, et al, Methods Mol Biol, 2018). Using some of these systems, we have evaluated how CAFs (pre and post-CRT) determine tumor invasion and stemness properties. Although these results have not been published yet, preliminary results point towards a cancer cell stemness promoting phenotype after radiotherapy.
References
- Barbazán J, Alonso-Alconada L, Elkhatib N, Geraldo S, Gurchenkov V, Glentis A, Van Niel G, Palmulli R, Fernández B, Viano P, Garcia-Caballero T, Lopez-Lopez R, Abal M & Vignjevic D. Liver metastasis is facilitated by the adherence of circulating tumor cells to vascular fibronectin deposits. Cancer Research, 2017. DOI: 10.1158/0008-5472.CAN-16-1917
- Staneva R, Barbazan J, Simon A, Vignjevic DM, Krndja D. Cell migration in tissues: Explant culture and live imaging. Methods Mol Biol, 2018.
DOI: 10.1007/978-1-4939-7701-7_13.
So far, there’s no studies comparing the effects of chemoradiotherapy in CAFs using primary cultures from tissue before and after therapy, from the same patient. The strength of out project resides in this, and we expect that the results obtained would be more reliable than what has been published so far.
The final results of FiBRO will determine the global role of CRT-derived CAFs in the behavior of cancer cells, and we expect the generation of new markers for therapy resistance evaluation, and possibly new therapeutic targets that could be tested.
The final results of FiBRO will determine the global role of CRT-derived CAFs in the behavior of cancer cells, and we expect the generation of new markers for therapy resistance evaluation, and possibly new therapeutic targets that could be tested.