Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth

Abstract

In the context of radiotherapy, collateral effects of ablative doses of ionizing radiation (AIR) on stromal components of tumors remains understudied. In this work, cancer-associated fibroblasts (CAFs) isolated from freshly resected human lung tumors were exposed to AIR (1x 18 Gy) and analyzed for their release of paracrine factors. Inflammatory mediators and regulators of angiogenesis and tumor growth were analyzed by multiplex protein assays in conditioned medium (CM) from irradiated and non-irradiated CAFs. Additionally, the profile of secreted proteins was examined by proteomics. In functional assays, effects of CAF-CM on proliferative and migratory capacity of lung tumor cells (H-520/H-522) and human umbilical vein endothelial cells (HUVECs) and their tube-forming capacity were assessed. Our data show that exposure of CAFs to AIR results in 1) downregulated release of angiogenic molecules such as stromal cell-derived factor-1, angiopoietin, and thrombospondin-2 (TSP-2); 2) upregulated release of basic fibroblast growth factor from most donors; and 3) unaffected expression levels of hepatocyte growth factor, interleukin-6 (IL-6), IL-8, IL-1β, and tumor necrosis factor-α. CM from irradiated and control CAFs did not affect differently the proliferative or migratory capacity of tumor cells (H-520/H-522), whereas migratory capacity of HUVECs was partially reduced in the presence of irradiated CAF-CM. Overall, we conclude that AIR mediates a transformation on the secretory profile of CAFs that could influence the behavior of other cells in the tumor tissue and hence guide therapeutic outcomes. Downstream consequences of the changes observed in this study merits further investigations.

Cancer-associated fibroblasts promote renal cell carcinoma progression

“…CAFs are a key component of the tumor microenvironment and orchestrate various aspects of cancer including tumor cell growth. Irradiated CAFs have been previously reported to promote tumor growth in various tumors, such as breast cancer and lung cancer [13]. It is thus logical that disruption of CAFs in the tumor microenvironment would influence clinical tumor behavior.…”

Proteomic overview and perspectives of the radiation-induced bystander effects

“…An increase in pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) was reported in the serum of irradiated mice using ELISA multiplex assays [36]. A decrease of angiogenic regulators (SDF-1alpha, ANG-1 and TSP-2) was observed following exposure of CAF to ablative doses of ionizing radiation, using multianalyte profiling assays [60].…”

“…The secretory profile of NSCLC-associated fibroblasts was investigated in vitro after ablative radiotherapy using a combination of shotgun proteomics and ELISA multiplex assays [60]. Cancer associative fibroblasts (CAF), isolated from freshly resected human lung tumors, were exposed to ablative doses of IR (18 Gy) and analyzed for the secretion of paracrine bystander factors.…”

“…Conditioned media from IR-induced senescent MCF-7 cells Several glycoproteins were modulated following high doses of radiation and serum cytokines (IL-1beta, IL-6 and TNF-alpha) were increased [60] 2013 In vitro Human CAF X-rays single 2, 6, 12, 18 Gy or 6 Â 3 Gy…”

“…8, left side). The use of cut-off membrane is an easy way to concentrate a protein solution and is widely used in biochemical studies of conditioned media [55,56,59,60,63].…”