Radiation therapy (RT) is used in the management of several cancers; however, tumor radioresistance remains a challenge. Polymorphonuclear neutrophils (PMNs) are recruited to the tumor immune microenvironment (TIME) post-RT and can facilitate tumor progression by forming neutrophil extracellular traps (NETs). Here, we demonstrate a role for NETs as players in tumor radioresistance. Using a syngeneic bladder cancer model, increased NET deposition is observed in the TIME of mice treated with RT and inhibition of NETs improves overall radiation response. In vitro, the protein HMGB1 promotes NET formation through a TLR4-dependent manner and in vivo, inhibition of both HMGB1 and NETs significantly delays tumor growth. Finally, NETs are observed in bladder tumors of patients who did not respond to RT and had persistent disease post-RT, wherein a high tumoral PMN-to-CD8 ratio is associated with worse overall survival. Together, these findings identify NETs as a potential therapeutic target to increase radiation efficacy.

The combination of radiation with immune checkpoint inhibitors was reported in some cancers to have synergic effects both locally and distally. Our aim was to assess this combined therapy on both radiated and nonradiated bladder tumors and to characterize the immune landscape within the tumor microenvironment. Murine bladder cancer cells (MB49) were injected subcutaneously in both flanks of C57BL/6 mice. Mice were randomly assigned to the following treatments: placebo, anti-PD-L1 (four intraperitoneal injections over 2 weeks), radiation to right flank (10 Gy in two fractions), or radiationþanti-PD-L1. Tumor digestion, flow cytometry, and qPCR were performed. Logrank analysis was used for statistical significance. Radiationþanti-PD-L1 group demonstrated statistically significant slower tumor growth rate both in the radiated and nonirradiated tumors (P < 0.001). Survival curves demonstrated superior survival in the combination group compared with each treatment alone (P ¼ 0.02). Flow cytometry showed increased infiltration of immunosuppressive cells as well as CTL in the radiation and combination groups (P ¼ 0.04). Ratio of immunosuppressive cells to CTL shifted in favor of cytotoxic activity in the combination arm (P < 0.001). The qPCR analysis revealed downregulation of immunosuppressive genes (CCL22, IL22, and IL13), as well as upregulation of markers of CTL activation (CXCL9, GZMA, and GZMB) within both the radiated and distant tumors within the combination group. Combining radiation with immune checkpoint inhibitor provided better response in the radiated tumors and also the distant tumors along with a shift within the tumor microenvironment favoring cytotoxic activity. These findings demonstrate a possible abscopal effect in urothelial carcinoma with combination therapy.

Radical cystectomy (RC) together with bilateral pelvic lymph node dissection remains the standard treatment for muscle invasive bladder cancer (MIBC). However, radiation-based treatments such as tri-modal therapy (TMT) involving maximally performed transurethral resection of bladder tumor (TURBT), radiotherapy (XRT), and a chemosensitizer represent an attractive, less invasive alternative. Nevertheless, 25–30% of MIBC patients will experience local recurrence after TMT and half will develop metastasis. Radioresistance of tumor cells could potentially be one of the causes for local recurrence post treatment. High mobility group box-1 (HMGB1) was shown to play a role in bladder cancer radioresistance through its intracellular functions in promoting DNA damage repair and autophagy. Recently, HMGB1 was found to be passively released from irradiated tumor cells. However, less is known about the involvement of extracellular HMGB1 in impairing radiation response and its exact role in modulating the tumor immune microenvironment after XRT. We identified a novel mechanism of bladder cancer radioresistance mediated by the immunological functions of HMGB1. The combination of radiation plus extracellular HMGB1 inhibition markedly improved the radiation response of tumors and resulted in marked changes in the immune landscape. Moreover, combining radiation and HMGB1 inhibition significantly impaired tumor infiltrating MDSCs and TAMs -but not Tregs- and shifted the overall tumor immune balance towards anti-tumoral response. We conclude that extracellular HMGB1 is involved in bladder cancer radioresistance through promoting pro-tumor immune mechanisms.

No abstract