(P 292) APPLICATION OF HIGH-INTENSITY FOCUSED ULTRASOUND THERMAL ABLATION AND BOILING HISTOTRIPSY WITH CHECKPOINT INHIBITORS IN A MURINE MODEL OF OSTEOSARCOMA: AN IN-DEPTH STUDY

Objective: Despite the significant advances of immune therapy in oncology, its’ efficacy has not been appreciated in pediatric solid tumors including osteosarcoma. This may be attributed to an immunosuppressive tumor microenvironment with low mutational burden, diminished immunogenicity, and reduced antigenicity. High intensity focused ultrasound (HIFU) has the potential to improve anti-tumor effects by triggering a localized inflammatory response, enhancing antigen presentation, and promoting T cell activation against tumor antigens. Previous studies demonstrated a survival advantage in treating refractory neuroblastoma using a combination of HIFU boiling histotripsy (BH) and checkpoint inhibitors (CPI), αCTLA-4 and αPD-L1. The objective of this study is to determine if the combination of BH or ablation combined with CPI can offer survival benefit in murine osteosarcoma.

Methods: Osteosarcoma tumors were established following subcutaneous injection of F420 cell line (0.100 x 106 cells) in the hind leg of C57BL/6J mice. Tumor size was monitored until it reached the goal 12 - 15mm across their largest dimension. Mice were randomly assigned to one of 6 experimental groups (n=15 each):1) control, 2 ) ablation alone, 3) BH alone, 4) CPI alone, 5) ablation + CPI, or 6) BH + CPI. CPI (100μg per dose) were injected intraperitoneally on days 1, 4 and 7 after HIFU or once tumors reached goal size in ‘CPI alone’ group. Mice were anesthetized and HIFU was performed with a 2000W RF amplifier driving a 1.5 MHz transducer and (BH: 450 mVpp, 13.33ms pulse, 1Hz pulse repetition frequency; ablation: 170mVpp continuous sonication). HIFU was guided by b-mode ultrasound imaging and performed at three adjacent foci (BH: 15 sec/focus; ablation: 5 sec/focus). ELISA for cytokine analysis was performed on serum and flow cytometry was performed on tumors, draining (DLN), contralateral lymph nodes (CLN) and spleen at 24, 48 and 72 h after treatment. Additional mice were used to assess survival (n=10 per group).

Results: No significant benefit was observed between the different treatment groups and control group, with a median survival of 10.5 days and 11 days respectively. Helper T-cells (CD4+ FoxP3-) were upregulated in the DLN following ablation + CPI at 48 and 72 h compared to the control group (40.1% P< 0.01, 95% CI [-22.4 to -1.3]). Cytotoxic T-cells (CD8+) were also upregulated in the DLN following ablation + CPI at 48 and 72 h compared to the control group (43.6% P< 0.001, 95% Cl [-22.1 to -1.81]) (Figure 1). Regulatory T cells (CD4+FoxP3+) were noted in the DLN and CLN following ablation and BH + CPI. BH alone or in combination with CPI led to significantly greater tumor immune cell infiltration (37.5%) compared to ablation alone or in combination with CPI. The CPI alone group had the highest increase in T-cell exhaustion (PD1+ TIM3- and PD1+ TIM3+).

Conclusion: The combination of ablation or HIFU BH with CPI did not improve survival rates compared to control. However, there was significant upregulation in CD4+ and CD8+T cells in the group receiving ablation with CPI. This immune response may highlight the potential of this combination therapy to mobilize the immune system against tumor cells. Furthermore, use of HIFU BH demonstrated improved tumor immune cells infiltration , which may be attributed to its ability to cause mechanical destruction of tumor tissue, potentially exposing a greater array of intact tumor antigens to the immune system. Reduced T cell exhaustion observed in the groups that received HIFU ablation and BH could be attributed to the mechanical and thermal effects of HIFU to cause tumor necrosis and subsequently stimulate a stronger immune response. Ongoing ELISA analysis of samples to quantify levels of relevant proteins and antibodies may further elucidate immunological alterations triggered by the different treatment modalities and lead to identification of new therapeutic targets for more effective combinatory treatment strategies.