Abstract
Accumulating evidence supports the notion that high-dose (>5 Gy) radiotherapy (RT) regimens are triggering stronger pro-immunogenic effects than standard low-dose (2 Gy) regimens. However, the effects of RT on certain immunoregulatory elements in tumors remain unexplored. In this study, we have investigated the effects of high-dose radiotherapy (HD-RT) on the immunomodulating functions of cancer-associated fibroblasts (CAFs). Primary CAF cultures were established from lung cancer specimens derived from patients diagnosed for non-small cell lung cancer. Irradiated and non-irradiated CAFs were examined for immunomodulation in experiments with peripheral blood mononuclear cells from random, healthy donors. Regulation of lymphocytes behavior was checked by lymphocyte proliferation assays, lymphocyte migration assays, and T-cell cytokine production. Additionally, CAF-secreted immunoregulatory factors were studied by multiplex protein arrays, ELISAs, and by LC-MS/MS proteomics. In all functional assays, we observed a powerful immunosuppressive effect exerted by CAF-conditioned medium on activated T-cells (p > 0.001), and this effect was sustained after a single radiation dose of 18 Gy. Relevant immunosuppressive molecules such as prostaglandin E2, interleukin-6, and -10, or transforming growth factor-β were found in CAF-conditioned medium, but their secretion was unchanged after irradiation. Finally, immunogenic cell death responses in CAFs were studied by exploring the release of high motility group box-1 and ATP. Both alarmins remained undetectable before and after irradiation. In conclusion, CAFs play a powerful immunosuppressive effect over activated T-cells, and this effect remains unchanged after HD-RT. Importantly, CAFs do not switch on immunogenic cell death responses after exposure to HD-RT.
Highlights
During the last decade, clinical implementation of high-dose radiotherapy (HD-RT) has revolutionized the therapeutic outcome for treatment-resistant cancers like non-small cell lung carcinoma (NSCLC)
High-dose radiotherapy is increasingly being used in the clinics for treatment of solid malignancies, and this approach is associated with higher rates of loco-regional tumor control and with the induction of secondary effects which influence therapy outcomes, such as changes in the vasculature and the immune status of tumors [4]
Three main observations can be highlighted from our study: (i) cancer-associated fibroblasts (CAFs) display a powerful immunosuppressive effect over Tcells, affecting both their function and migration rates; (ii) Neither HD-RT (1 × 18 Gy) nor LD-RT (4 × 2 Gy) is influencing the immunosuppressive functions of CAFs in vitro; and (iii) HD-RT do not ignite immunogenic cell death (ICD) responses in CAFs
Summary
Clinical implementation of high-dose radiotherapy (HD-RT) has revolutionized the therapeutic outcome for treatment-resistant cancers like non-small cell lung carcinoma (NSCLC). Oligo-fractionated HD-RT regimens are often associated with stereotactic. Minimal margins of normal tissue in the irradiated field combined with high-precision image-guided dose-delivery and steep dose-gradients constitute the fundament for increasing dose per fraction above the conventional 2 Gy while ensuring acceptable toxicity. On the ground that clinical RT is affecting a well-defined volume of heterotypic tumor tissue – and questioning the dogma that direct DNA damage to neoplastic cells is the sole mechanism for tumor control mediated by RT – researchers have instigated studies of radio-responses in different tumor compartments including both epithelial tumor cells and tumor-associated stromal cells. Radio-responses in the diverse population of stromal immune cells are probably the best explored of the non-malignant cells [6]
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