Abstract
Cancer-associated fibroblasts (CAFs) participate actively in tumor development and affect treatment responses, by among other mechanisms, promoting an immunosuppressive tumor microenvironment. In contrast to normal fibroblasts, reactive CAFs secrete a myriad of immunomodulatory soluble factors at high levels, i.e. growth factors, cytokines, and chemokines, which directly influence tumor immunity and inflammation. CAFs have been identified as important players in tumor radioresistance. However, knowledge on the immunomodulatory functions of CAFs during/after radiotherapy is still lacking. In this study, we investigated the effects of ionizing radiation on CAF-mediated regulation of dendritic cells (DCs). CAFs were obtained from freshly operated lung cancer tissues, while DCs were procured from peripheral blood of healthy donors. Experimental settings comprised both co-cultures and incubations with conditioned medium from control and irradiated CAFs. Functional assays to study DC differentiation/activation consisted on cytokine release, expression of cell-surface markers, antigen uptake, migration rates, T cell priming, and DC-signaling analysis. We demonstrate that CAFs induce a tolerogenic phenotype in DCs by promoting down-regulation of: i) signature DC markers (CD14, CD1a, CD209); ii) activation markers (CD80, CD86, CD40, and HLA-DR) and iii) functional properties (migration, antigen uptake, and CD4+ T cell priming). Notably, some of these effects were lost in conditioned medium from CAFs irradiated at fractionated medium-dose regimens (3x6 Gy). However, the expression of relevant CAF-derived regulatory agents like thymic stromal lymphopoietin (TSLP) or tryptophan 2,3-dioxygenase (TDO2) was unchanged upon irradiation. This study demonstrates that CAFs interfere with DC immune functions and unveil that certain radiation regimens may reverse CAF-mediated immunosuppressive effects.
Highlights
Recent studies in both pre-clinical and clinical settings have demonstrated that radiotherapy (RT) has the power to trigger immunological responses that can influence disease outcomes [1,2,3]
To investigate the effects of ionizing radiation (IR) on cancer-associated fibroblasts (CAFs)-mediated regulation of monocyte-to-dendritic cells (DCs) trans-differentiation, peripheral blood monocytes from health donors (CD14+ cells – 89% purity) were cultured in medium containing DC differentiation cytokines (IL-4 and Granulocyte-macrophage colony-stimulating factor (GM-CSF)) in the absence or presence of conditioned medium from irradiated or non-irradiated CAFs (CAF-Conditioned medium (CM)) or alternatively in (CAF-DC) co-cultures (CAF-CC)
We have investigated how CAFs from lung tumors influence monocyte-derived DC differentiation, maturation, and functions in vitro; and whether ionizing radiation is able to modify the CAF-mediated immunoregulatory features on DCs
Summary
Recent studies in both pre-clinical and clinical settings have demonstrated that radiotherapy (RT) has the power to trigger immunological responses that can influence disease outcomes [1,2,3]. Treatment outcomes will depend on the net effect of pro-immunogenic and antiimmunogenic signals. Recent studies have shown a correlation between cancer-associated fibroblasts (CAFs), a major component of the tumor stroma [9,10,11], and increased radiotherapy resistance in colorectal cancer [12] and non-small cell lung cancer (NSCLC) [13]. It is well established that CAFs play an important role in suppressing anti-tumor immune responses in the TME, with ability to negatively affect activation, trafficking, and state of differentiation of a vast population of immune cells [15,16,17]. Little is known about how RT is affecting the crosstalk between CAFs and immune cells
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