Immune-modulating properties of carcinoma-associated fibroblasts and their changes during radiotherapy. In vitro study of primary non-small cell lung carcinoma-associated fibroblasts

Abstract

Background/aim of the study: Carcinoma-associated Fibroblasts (CAFs) comprise a heterogeneous population of cells and often represent the major cellular component of solid malignancies playing critical roles in tumor progression and metastasis. The purpose of this study was to gain more knowledge on the immunosuppressive traits of CAFs isolated from non-small cell lung carcinomas (NSCLC), and to explore whether high-dose radiotherapy (HD-RT), which has been proven to have highly favorable outcomes in patients with early-stage inoperable peripheral lung cancer, modify CAFs-mediated immune-regulating functions in one or another direction.

Methods: The study included primary cultures of CAFs isolated from freshly resected NSCLC (n = 7) and freshly isolated Peripheral Blood Mononuclear Cells (PBMCs) isolated from randomly selected healthy volunteers. A potential crosstalk between irradiated or non-irradiated CAFs and T-lymphocytes was examined using in vitro co-cultures and PBMCs exposed to CAF-conditioned medium (CM). Relevant cell functions were analyzed by a series of assays including lymphocyte proliferation assays, lymphocyte migration assays, Treg assays and T-cell cytokine production. In the search for mechanisms behind the observed effects, a series of molecular assays including multiplex protein arrays, ELISAs, LC-MS/MS proteomics and cytokines specific blocking assays were performed. Finally the induction of immunogenic cell death (ICD) of CAFs in response to HD-RT (1 x 18 Gy) was studied by examining the release of high motility group box 1 (HMGB1) and ATP into the extracellular space.

Results: All functional assays showed that CAF-derived soluble factors exhibit strong immunosuppressive effects on Phytohaemagglutinin-L (PHA-L) stimulated T-cells, affecting both their function (P<0.001) and migration (P<0.05) rates, and this effect was sustained after a single radiation dose of 18 Gy. Moreover, the 18 Gy irradiated CAF-secretome contained the same levels of the immunosuppressive cytokines, IL-6, TGF-β, PGE2, IL-10 and IDO as the non-irradiated CA-secretome. In addition, HD-RT did not induce the release of HMGB1 and ATP by CAFs. Finally, by specific blockade of well-known factors it has been proven that IL-6, TGF-β, PGE2 and Galectin-3 do not play major roles in the immunosuppressive effects exerted by CAFs.

Conclusion: This study demonstrate that CAF-derived soluble factors mediate strong immunosuppressive effects over activated T-cells although the soluble factor responsible for these effects remains unknown. On the other hand, this study also demonstrates that HD-RT do not overcome the immunosuppressive effects exerted by non-irradiated CAFs and fails to induce substantial changes in the spectra of immune-regulatory molecules secreted by these cells. Moreover, CAFs do not switch on ICD responses after exposure to HD-RT.