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Cancer-associated fibroblasts are key modulators of the tumor microenvironment, facilitating tumor initiation, tumor growth, invasion and metastasis, in addition to function as sentinel cells of the immune system. Nevertheless, the impact of the abundant and heterogeneous CAF-community towards treatment resistance is only starting to emerge. With the ultimate goal of enhancing therapeutic outcome, our laboratory is exploring how CAFs are influencing responses to radiotherapy and immunomodulation.

Experiments were carried out with human CAFs freshly isolated from (NSCLC) lung tumor specimens. Primary CAF cultures were exposed to ionizing radiation delivered as single-high or fractionated medium-high doses. The secretory profiles of irradiated and control CAFs were compared by multiplex protein arrays and high-throughput proteomics. Immuno-regulatory properties of irradiated CAFs (iCAFs) were explored in functional in vitro assays, where different types of immune cells, such as macrophages, dendritic cells, natural killer cells or T-lymphocytes, were individually co-cultured with iCAFs or exposed to iCAF-conditioned medium or iCAF-derived exosomes. Radiation-induced events that could potentially interfere with anti-tumor immunity, including immunogenic cell death (ICD) and type-I interferon expression (IFN-I), was also explored in iCAFs. At the in vivo level, tumor development and inflammatory responses were studied upon transplantation of tumor cells admixed with irradiated or control CAFs.

Cultured CAFs display a noticeable radio-resistant phenotype. However, exposure to high radiation doses provoke induction of cellular senescence and important phenotypic changes including persistent DDR-responses, elevated surface levels of integrins and significant changes in the secretory profile. Importantly, radiation exposure does not elicit ICD or IFN-I release in iCAFs.  CAF-derived soluble factors, but not exosomes, show powerful immunosuppressive effects over activated lymphocytes, macrophages and dendritic cells, however, most of the observed effects seem to be unchanged upon radiation exposure. In vivo studies in xenografts propose that the net effect of IR on CAFs is advantageous, since the natural pro-tumorigenic effects of CAFs become abrogated.

Irradiated CAFs change substantially their phenotype upon exposure to single-high radiation doses, which could impact tumor responses to therapy. Of note, most of the studied CAF-mediated immunomodulatory functions are maintained following irradiation. However, the heterotypic nature of CAFs in true in vivo circumstances, and the existence of CAF subsets possibly exerting opposing effects in the context of radiotherapy and immunotherapy needs to be carefully considered for optimized treatment strategies.