Fibroblast-Mediated Immunoregulation of Macrophage Function Is Maintained after Irradiation

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.

Interaction between Fibroblasts and Immune Cells Following DNA Damage Induced by Ionizing Radiation

“…In contrast, M1 macrophages treated with CAF conditioned medium produce less CD40, CD206, IL-6, IL-10, IL-12, TNF-α, and nitric oxide. Moreover, molecules secreted by CAF abrogate migration of M1 macrophages [7,66] (Figure 3).…”

“…Macrophages are a part of the tumor microenvironment that interacts with CAFs and tumor cells. While CAFs influence both stimulated (M1) and unstimulated (M0) human macrophages in vitro, irradiation (18 Gy) does not affect these interactions [7]. Medium containing molecules secreted by CAFs, or conditioned medium, stimulates expression of CD40, CD80, CD163, CD206, IL-6 and IL-10 in M0 macrophages.…”

“…CAFs, as other fibroblasts, possess spindle-shaped morphology (Figure 1), although gained increased proliferation rates [1]. CAFs are extensively described in literature, including [1,[7][8][9][10][12][13][14][15][16][17]. In particular, CAFs can be defined as a heterogenous population of connective tissue cells that contribute to cancer progression by secreting specific molecules, including growth factors, proteases, chemokines and cytokines.…”

“…High levels of CAFs in tumors are associated with poor treatment outcome and prognosis [18,64,65]. Whether radiotherapy affects the interaction between CAFs and tumor cells was recently studied using several model systems, such as T cells [11] and macrophages [7]. It would be also important to investigate the relationship between radiated CAFs and other immune system cells, including but not limited to dendritic cells and natural killers.…”

Interaction between Fibroblasts and Immune Cells Following DNA Damage Induced by Ionizing Radiation

“…Radiotherapy is associated with increased radio-resistance of tumors, including NSCLC, likely due to the pro-tumorigenic activity of CAFs [6]. Pro-tumorigenic nature of irradiated CAFs is explained either by direct stimulation of tumor cell viability, or by inhibiting immune cells, such as macrophages, dendritic cells, T cells and natural killers [7][8][9][10][11]. Moreover, one can propose distinct mechanisms of tumor recovery following the therapy and role of CAFs in this scenario.…”