Abstract
The abilities of cancer-associated fibroblasts (CAFs) to regulate immune responses in the context of radiotherapy remain largely unknown. This study was undertaken to determine whether ionizing radiation alters the CAF-mediated immunoregulatory effects on macrophages. CAFs were isolated from freshly-resected non-small cell lung cancer tumors, while monocyte-derived macrophages were prepared from peripheral blood of healthy donors. Experimental settings included both (CAF-macrophage) co-cultures and incubations of M0 and M1-macrophages in the presence of CAF-conditioned medium (CAF-CM). Functional assays to study macrophage polarization/activation included the expression of cell surface markers, production of nitric oxide, secretion of inflammatory cytokines and migratory capacity. We show that CAFs promote changes in M0-macrophages that harmonize with both M1-and M2-phenotypes. Additionally, CAFs inhibit pro-inflammatory features of M1-macrophages by reducing nitric oxide production, pro-inflammatory cytokines, migration, and M1-surface markers expression. Radiation delivered as single-high dose or in fractioned regimens did not modify the immunoregulatory features exerted by CAFs over macrophages in vitro. Protein expression analyses of CAF supernatants showed that irradiated and non-irradiated CAFs produce approximately the same protein levels of immunoregulators. Thus, CAF-derived soluble factors mediate measurable changes on uncommitted macrophages and down-regulate pro-inflammatory features of M1-polarized macrophages. Notably, ionizing radiation does not curtail the CAF-mediated immunosuppressive effects.
Highlights
Radiotherapy (RT) is a highly efficient and widely used treatment modality for solid tumors and is largely understood as a localized treatment modality, aiming to induce tumor cell killing while keeping effects on the surrounding healthy tissue at a minimum
We explore if cancer-associated fibroblasts (CAFs)-mediated immunoregulatory effects on macrophages are changed after exposure of CAF cultures to different radiation regimens
CAFs were isolated from non-small cell lung cancer tissue by enzymatic digestion and outgrowth, as described by Hellevik et al [30]
Summary
Radiotherapy (RT) is a highly efficient and widely used treatment modality for solid tumors and is largely understood as a localized treatment modality, aiming to induce tumor cell killing while keeping effects on the surrounding healthy tissue at a minimum. The field of radiation oncology is embracing the notion that RT may trigger local and systemic immune-modulating effects [1,2,3,4]. Preclinical and clinical studies have shown that RT may profoundly alter cellular and acellular components of the tumor microenvironment (TME) [5] and transform it from immunosuppressive to immunoreactive [4,5,6]. A more thorough biological understanding of the effects elicited by ionizing radiation in TME elements may aid in the design of more efficient treatment strategies
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