Radiation treatment curtails pro-tumorigenic functions from cancer-associated fibroblasts in preclinical models

Background: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and active drivers of tumor progression, metastasis, and resistance to therapy. However, the effects of radiotherapy (RT) on CAFs and the consequent impact on tumor cell behavior remain controversial. Methods: Primary cultures of human CAFs isolated from non-small cell lung cancer (NSCLC) tumor samples were exposed to ionizing radiation by a single-high dose or a hypofractionated regimen. Different lung tumor cell lines were exposed to control or irradiated CAFs in direct co-cultures or to CAFs conditioned medium (CM), and changes in tumor cell proliferation, migration, epithelial-mesenchymal transition (EMT) and clonogenic survival were measured. The impact of RT on intratumoral CAF levels in different subcutaneous tumor models was measured by α-SMA expression in excised tumor specimens. In addition, pirfenidone, a CAF-reprogramming agent, was investigated for its capacity to influence tumor responses to radiation. Finally, correlations of CAF markers with treatment outcomes was studied in a cohort of RT-treated non-small cell lung cancer (NSCLC) patients. Results: Ionizing radiation induced premature senescence in CAFs; however, the expression of established CAF activation markers remained unchanged. Exposure of lung tumor cell lines to CM from irradiated CAFs did not alter their proliferation, migratory capacity, or clonogenic survival. Conversely, CAF-driven epithelial–mesenchymal transition (EMT) was attenuated in all tumor cell lines after incubations with CM from irradiated CAFs. Radiation delivered to subcutaneously transplanted tumors (1x12 Gy) did not change intra-tumoral CAF abundance in the LLC and CT26 murine models, whereas CAF numbers were significantly reduced in the stroma-rich 4662PDA pancreatic model. Pirfenidone-treated animals exhibited enhanced tumor radio-resistance, along with decreased collagen levels within tumors, and unchanged numbers of aSMA+CAFs. In clinical specimens, baseline tissue expression levels of the CAF markers FAP and aSMA were not associated with disease-specific survival. Conclusions: Our findings suggest that ionizing radiation may lessen certain pro-tumorigenic CAF functions—such as EMT induction—, whereas pharmacologic CAF reprogramming with pirfenidone paradoxically confers increased tumor radio-resistance, highlighting potential negative implications from CAF-targeted therapies.