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

## TL;DR

A treatment combining hypofractionated radiotherapy and rhGM-CSF was found to be most effective in reducing pancreatic cancer in mice by triggering immune responses.

## Contribution

The study identifies the optimal sequence of rhGM-CSF and hypofractionated radiotherapy for pancreatic cancer treatment.

## Key findings

- HFRT followed by rhGM-CSF inhibited tumor growth and promoted tumor necrosis.
- This treatment increased immunogenic cell death markers like CRT, HMGB1, and ATP.
- The regimen enhanced immune cell infiltration and upregulated pro-inflammatory cytokines.

## Abstract

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

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.

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.

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