Hypofractionated radiotherapy followed by rhGM-CSF enhances immunogenic cell death in a murine model of pancreatic cancer.

PURPOSE: To investigate the optimal therapeutic sequence of rhGM-CSF combined with hypofractionated radiotherapy (HFRT) in treating mouse pancreatic cancer (PC) and explore the mechanisms.

METHODS

A PC-bearing model was established. The antitumor effects were observed under rhGM-CSF, HFRT, rhGM-CSF + HFRT, rhGM-CSF&HFRT, and HFRT + rhGM-CSF treatments. Tumor histopathological changes were examined using hematoxylin and eosin (H&E) staining. FCM was employed to detect calreticulin (CRT), mDCs, CD4+, and CD8+ T cells. Enzyme-linked immunosorbent assay (ELISA) was used to measure HMGB1, adenosine triphosphate (ATP), interleukin- (IL)-2, IL-4, IL-8, IL-10, IL-12, IFN-γ, tumor necrosis factor (TNF)-α, and iNOS levels. IF staining was performed to detect CD31 and α-smooth muscle actin, and immunohistochemistry was used to detect vascular endothelial growth factor (VEGF), soluble VEGF receptor-1 (sVEGFR-1), hypoxia-inducible factor- (HIF)-1α, and HIF-2α expression.

RESULTS

HFRT + rhGM-CSF inhibited tumor growth, promoted tumor necrosis, and increased inflammatory cell infiltration. This regimen also significantly enhanced immunogenic cell death by inducing CRT exposure and the release of HMGB1 and ATP. Furthermore, HFRT + rhGM-CSF markedly increased proportions of mDCs, CD4+ T cells, and CD8+ T cells, and upregulated expressions of IL-2, IL-8, IL-12, IFN-γ, TNF-α, and iNOS, but not IL-4 and IL-10. Additionally, rhGM-CSF synergized with HFRT to promote the normalization of blood vessels in the PC.

CONCLUSION

HFRT followed by rhGM-CSF had the best efficacy in PC, and the molecular mechanism may be related to immunogenic cell death.