# Photothermal Therapy-Induced Immunogenic Cell Death Synergistically Enhances the Therapeutic Effect of Immune Checkpoint Inhibitors

## TL;DR

This study shows that photothermal therapy can induce immunogenic cell death and work better with immunotherapy to fight cancer.

## Contribution

PTT is shown to induce ICD more effectively than certain drugs and synergize with immunotherapy.

## Key findings

- PTT induced both HMGB1 release and CRT expression, unlike mitoxantrone and cisplatin.
- PTT combined with ICIs showed a synergistic anti-tumor effect in mice.
- PTT increased CD8+ T cell infiltration in tumor tissues.

## Abstract

Photothermal therapy (PTT), which kills cancer cells through thermal stress, can induce immunogenic cell death (ICD), thereby enhancing the therapeutic effect of immunotherapy. Although several ICD inducers exist, their relative ICD induction capabilities remain unclear. The purpose of this study is comparing the ICD-induction ability of anti-cancer drugs and PTT and explore the therapeutic potential of PTT combined with ICIs. In vitro assays demonstrated that cisplatin, a non-ICD inducer, failed to trigger high mobility group box protein 1 (HMGB1) release or upregulation of calreticulin (CRT) expression; whereas, mitoxantrone, an ICD inducer, promoted HMGB1 release but not CRT expression. In contrast, PTT induced both HMGB1 release and increased CRT expression. Furthermore, PTT promoted infiltration of CD8+ T cells in tumor tissues and exhibited a synergistic effect when combined with ICIs. These results suggest that PTT could be a drug-free, minimally invasive approach for ICD induction that can be combined with immunotherapy.

Background/Objectives: To improve the response rate of immune checkpoint inhibitors (ICIs), inducing immunogenic cell death (ICD) is a promising approach. Photothermal therapy (PTT) induces immunogenic cell death and activates anti-tumor immunity. While there are various ICD inducers, the difference in ICD induction by various modalities is poorly understood. In this study, we found previously unrecognized advantages of PTT compared to anti-cancer drugs and showed the usefulness of PTT as an anti-cancer drug-free approach to be combined with immunotherapy. Methods: Gold nanorods were synthesized as photothermal agents and added to culture medium or locally administered to tumor tissues. Mitoxantrone (MIT), an ICD inducer, and cisplatin (CDDP), a non-ICD inducer, were compared with PTT. To assess the induction of ICD, the subcellular localization and amounts of high mobility group box 1 (HMGB1) and calreticulin (CRT) were observed using immunofluorescent staining. FM3A tumor-bearing mice were treated with PTT or anti-cancer drugs, and cell death and DAMPs localization in tumor tissues were analyzed. Also, the supra-additive effect of PTT on ICI was observed. Tumor-infiltrating CD8+ T cells were examined to evaluate the immune status in tumor tissues. Results: In vivo assays showed that PTT induces HMGB1 release and increased expression of CRT on the cell membrane. Moreover, PTT showed a supra-additive effect in terms of therapeutic effect and anti-tumor activation when combined with an immune checkpoint inhibitor. Conclusions: In this study, we demonstrated that PTT induced ICD-related signaling and improved the response rate of ICI, which means PTT is a promising combination therapy with ICI.