Chemoradiation induces upregulation of immunogenic cell death-related molecules together with increased expression of PD-L1 and galectin-9 in gastric cancer

S. H. Petersen,S. Nakajima,L. F. Kua,K. Kono,W. P. Yong

doi:10.1038/s41598-021-91603-7

S. H. Petersen, S. Nakajima + Show 3 more

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https://doi.org/10.1038/s41598-021-91603-7

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Abstract

Surgery alone or combined with chemo- and/or radiation therapy remains the primary treatment for gastric cancer (GC) to date and immunotherapeutic tools such as monoclonal antibodies are only slowly being implemented. This is partly due to the fact that the immune microenvironment in GC during chemoradiation and other treatment modalities is still poorly understood. 7 gastric cancer (GC) cell lines were tested for their response to chemoradiation using 5-FU in combination with X-ray irradiation. We conducted flow cytometric analysis to determine the cells’ ability to undergo immunogenic cell death (ICD) and their expression of the two immunosuppressive proteins programmed death-ligand 1 (PD-L1) and galectin-9 (Gal-9). We evaluated the overall immunogenicity of two cell lines (MKN7, MKN74) in co-culture experiments with human monocyte-derived dendritic cells (Mo-DCs). Chemoradiation induces distinct responses in different GC cell lines. We observe ICD in vitro in all tested GC cell lines in the form of calreticulin (CRT) translocation to the plasma membrane. As a resistance mechanism, these cells also upregulated Gal-9 and PD-L1. Mo-DC maturation experiments showed that GCs provoked the maturation of Mo-DCs after chemoradiation in vitro. The addition of α-PD-L1 blocking antibody further enhanced the immunogenicity of these cells while improving DC viability. Blocking Tim-3, as the main receptor for Gal-9, had no such effect. Our findings suggest that the benefits of chemoradiation can substantially depend on tumor subtype and these benefits can be offset by induced immune evasion in GC. Combination treatment using checkpoint inhibitors could potentially lead to enhanced immune responses and yield better patient outcomes.

Highlights

Surgery alone or combined with chemo- and/or radiation therapy remains the primary treatment for gastric cancer (GC) to date and immunotherapeutic tools such as monoclonal antibodies are only slowly being implemented
We show for the first time that chemoradiation using X-ray radiation combined with a single dose of 5-FU caused immunogenic cell death (ICD) in GC cell lines, as evidenced by the translocation of CRT
By comparing median fluorescent intensities (MFI), we detected a significant increase of CRT surface expression after combination treatment of 5-FU plus radiation (P = 0.035), radiation treatment alone (P = 0.011) and doxorubicin alone (P = 0.031)

Summary

Introduction

Surgery alone or combined with chemo- and/or radiation therapy remains the primary treatment for gastric cancer (GC) to date and immunotherapeutic tools such as monoclonal antibodies are only slowly being implemented. Following clinical evidence of the beneficial effect of using PD-1 blocking antibody treatment[24,25,26], pembrolizumab and nivolumab received clinical approval for GC treatment Another checkpoint pathway is Gal-9 and its receptor T cell immunoglobulin and mucin-domain containing 3 (Tim-3), which is considered a co-inhibitory receptor on immune cells. The secretion of soluble or exosome-bound Gal-9 by cancer cells, and the subsequent reattachment to cancer cell’s surface, supports cancer immune evasion and tumor progression in different m alignancies[35,36,37,38,39,40,41] Both Tim[3] and PD1 receptor signaling are potential mechanisms of resistance against the potentiation of anticancer therapies by the immune system, such as through immunogenic cell death (ICD)

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