“EFFECT OF IRRADIATION ON THE IMMUNOREGULATORY FUNCTIONS EXERTED BY TUMOUR FIBROBLASTS ON MACROPHAGES”

Abstract

Summary Background/aim of the study: Carcinoma-associated fibroblasts (CAFs) represents a heterogeneous population of cells and are considered one of the dominant stromal component of solid tumours, performing a crucial role in tumour proliferation and metastasis. The main objective of this study was to investigate the immunoregulatory features of CAFs isolated from non-small cell lung carcinomas on macrophages and the potential effects of ionizing radiation on observed effects. Methods: The study comprise primary cultures of CAFs isolated from freshly resected NSCLC (Non Small Lung Cancer) tumours (n=8) and monocyte-derived macrophages prepared from peripheral blood of healthy donors. The experimental settings included both co-cultures and incubations of macrophages with CAF-conditioned medium. Moreover, CAF-mediated effects were studied in both uncommitted and M1-polarized macrophages. Functional assays to study macrophage polarisation included expression of cell surface markers by flow cytometry, production of nitric oxide by flow cytometry and secretion of inflammatory cytokines by ELISA. Results: All functional assays illustrated that CAFs both in co-culture and by conditioned medium, promote changes on uncommitted macrophages (M0) that harmonize with both M1 and M2 phenotypes. CAFs, both in co-culture and by conditioned medium, could inhibit some of the pro-inflammatory features of M1 macrophages as demonstrated by strong inhibition of nitric oxide production, strong inhibition of proinflammatory cytokines secretion and a reduction of some M1 surface markers. Importantly, radiation given at high dose (1x18 Gy) or in fractioned regimens (3x6 Gy) is not able to modify substantially the immunoregulatory features exerted by CAFs over macrophages in vitro. Last, protein expression analyses in CAF supernatants show that both irradiated and non-irradiated CAFs produced approximately the same levels of cytokines. Conclusion: This study display that CAFs-derived soluble factors mediate measurable changes on uncomitted macrophages (M0) and down-regulate pro-inflammatory features of M1 polarized macrophages, even though the soluble factors accountable for this shift remains unknown. On the other hand, this study also illustrates that low dose fractioned radiotherapy and single high dose radiotherapy do not curtail the immunosuppressive effect of CAFs.