Theranostics 2021; 11(2):540-554. doi:10.7150/thno.49517

Research Paper

Boiling histotripsy and in-situ CD40 stimulation improve the checkpoint blockade therapy of poorly immunogenic tumors

Mohit Pratap Singh1, Sri Nandhini Sethuraman1, Craig Miller2, Jerry Malayer1, Ashish Ranjan1✉

1. Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
2. Department of Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.

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Citation:
Singh MP, Sethuraman SN, Miller C, Malayer J, Ranjan A. Boiling histotripsy and in-situ CD40 stimulation improve the checkpoint blockade therapy of poorly immunogenic tumors. Theranostics 2021; 11(2):540-554. doi:10.7150/thno.49517. Available from https://www.thno.org/v11p0540.htm

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Abstract

Background: Advanced stage cancers with a suppressive tumor microenvironment (TME) are often refractory to immune checkpoint inhibitor (ICI) therapy. Recent studies have shown that focused ultrasound (FUS) TME-modulation can synergize ICI therapy, but enhancing survival outcomes in poorly immunogenic tumors remains challenging. Here, we investigated the role of focused ultrasound based boiling histotripsy (HT) and in-situ anti-CD40 agonist antibody (αCD40) combinatorial therapy in enhancing therapeutic efficacy against ICI refractory murine melanoma.

Methods: Unilateral and bilateral large (~330-400 mm3) poorly immunogenic B16F10 melanoma tumors were established in the flank regions of mice. Tumors were exposed to single local HT followed by an in-situ administration of αCD40 (HT+ αCD40: HT40). Inflammatory signatures post treatment were assessed using pan-cancer immune profiling and flow cytometry. The ability of HT40 ± ICI to enhance local and systemic effects was determined by immunological characterization of the harvested tissues, and by tumor growth delay of local and distant untreated tumors 4-6 weeks post treatment.

Results: Immune profiling revealed that HT40 upregulated a variety of inflammatory markers in the tumors. Immunologically, HT40 treated tumors showed an increased population of granzyme B+ expressing functional CD8+ T cells (~4-fold) as well as an increased M1 to M2 macrophage ratio (~2-3-fold) and CD8+ T: regulatory T cell ratio (~5-fold) compared to the untreated control. Systemically, the proliferation rates of the melanoma-specific memory T cell population were significantly enhanced by HT40 treatment. Finally, the combination of HT40 and ICI therapy (anti-CTLA-4 and anti-PD-L1) caused superior inhibition of distant untreated tumors, and prolonged survival rates compared to the control.

Conclusions: Data suggest that HT40 reprograms immunologically cold tumors and sensitizes them to ICI therapy. This approach may be clinically useful for treating advanced stage melanoma cancers.

Keywords: Boiling histotripsy, αCD40, Checkpoint blockade, Cold tumors, Anti-tumor immunity