Intraoperative radiation therapy induces immune response activity after pancreatic surgery

Abstract

Background: Pancreatic cancer has highly aggressive features, such as local recurrence that leads to significantly high morbidity and mortality and recurrence after successful tumour resection. Intraoperative radiation therapy (IORT), which delivers targeted radiation to a tumour bed, is known to reduce local recurrence by directly killing tumour cells and modifying the tumour microenvironment.

Methods: Among 30 patients diagnosed with pancreatic cancer, 17 patients received IORT immediately after surgical resection. We investigated changes in the immune response induced by IORT by analysing the peritoneal fluid (PF) and blood of patients with and without IORT treatment after pancreatic cancer surgery. Further, we treated three pancreatic cell lines with PF to observe proliferation and activity changes.

Results: Levels of cytokines involved in the PI3K/SMAD pathway were increased in the PF of IORT-treated patients. Moreover, IORT-treated PF inhibited the growth, migration, and invasiveness of pancreatic cancer cells. Changes in lymphocyte populations in the blood of IORT-treated patients indicated an increased immune response.

Conclusions: Based on the characterisation and quantification of immune cells in the blood and cytokine levels in the PF, we conclude that IORT induced an anti-tumour effect by activating the immune response, which may prevent pancreatic cancer recurrence.

Clinical trial registration: NCT03273374 .

Keywords: Cytokine; Immune response; Intraoperative radiation therapy; Pancreatic cancer.

Conflict of interest statement

The authors declare no conflict of interest.

© 2021. The Author(s).

Figures

Fig. 1

Intraoperative radiation therapy (IORT) leads to differential cytokine profile of the peritoneal fluid (PF) and affects PI3K/Smad signalling in pancreatic cancer cells. A Cytokine membrane array images. Each spot represents a cytokine hybridised in patient PF. B Heat map visualisation comparing cytokine array results. C. Cytokines significantly upregulated in the PF of IORT-treated patients (IORT n = 15, no IORT n = 11). D. Schematic overview of signalling pathways (PI3K-Akt, Smad2/3) in cancer cells associated with cytokines TGF-β, IFN-γ, and PDGF-BB. E. Western blot analysis of signalling-related protein xpression in Panc1 cells incubated with PF for 4 h. IORT: P3, P54, and P56; no IORT: P31, P33, and P42 (unpaired t-test; *p < 0.05 and **p ≤ 0.01)

Fig. 2

Peritoneal fluid (PF) stimulates cancer cell proliferation. A. Mia PaCa-2, Panc1, and Aspc1 cell lines were incubated for 4 days in the presence of PF at the indicated concentrations. As a positive control, foetal bovine serum (FBS) was used at the same concentration as PF. Incubated cells were treated with WST-1 solution, and absorbance was detected at 450 nm. B. Cells were incubated in the presence of PF until the colonies were formed. Colonies were stained with crystal violet, and the percentage of colony areas were calculated using ImageJ software. Columns correspond to the mean of three independent experiments; bars indicate SD. Statistical analyses were performed using the unpaired t-test

Fig. 3

Intraoperative radiation therapy (IORT)-treated peritoneal fluid (PF) decreased the invasiveness and wound healing ability of pancreatic cancer cells. A. Pancreatic cancer cell lines were tested for their invasiveness using Matrigel-coated Transwell chemotaxis assays. Random PF samples (7% in DMEM) were loaded in the bottom well of the transwell plate for pancreatic cancer cell treatment. Invaded cells were stained and analysed after 24 h. B. Pancreatic cancer cells cultured in 96-well plates were scratched using a wound maker and treated with PF for observing cell migration every 12 h

Fig. 4

Changes in EMT marker expression in cancer cells following treatment with peritoneal fluid (PF) collected after intraoperative radiation therapy (IORT). A. qPCR was used to measure the expression levels of EMT-related genes encoding mesenchymal markers (vimentin, snail, and N-cadherin) and epithelial markers (E-cadherin). B. Western blotting analysis of the expression of indicated EMT markers. RNA and protein were extracted from cells after 4 days of treatment. Used PF were from IORT (P1, P43, P56, and P64) and no IORT (P26, P42, P45, and P62). The results were statistically analysed using the unpaired t-test, and significance was marked at *: p < 0.05 and **: p < 0.01 using three independent biological replicates

Fig. 5

Changes in immune cell composition of PBMCs during the follow-up period of IORT-treated patients with pancreatic cancer. A. Representative flow cytometry plots illustrating the gating strategy used in the analysis of lymphocytes in the blood (left: CD3-CD56+ NK cell; middle: CD3 + CD4+ helper T cell and CD3 + CD8+ cytotoxic T cell; right: CD3-CD25 + FOXP3+ Treg cell). B. Rate of change in the indicated immune cells according to the blood collection time (baseline = post-operation day (POD) 1, normalised to each POD 1 value). C. Scheme of immune response by IORT treatment after pancreatic cancer surgery