Effect of HIFU on Murine Neuroblastoma Tumors

This study assess the role of HIFU on genetic change intratumorally post-treatment Immunotherapy promises unprecedented benefits to cancer patients. However, the majority of cancer types, including high-risk neuroblastoma remain immunologically unresponsive. High intensity focused ultrasound (HIFU) is a non-invasive technique that can mechanically fractionate tumors, transforming immunologically ‘‘cold’’ tumors into responsive ‘‘hot’’ tumors. We treated tumors with HIFU, and assessed systemic immune response using flow-cytometry, ELISA, and gene sequencing. In addition, we combined this treatment with αCTLA-4 and αPD-L1 to study its effect on the immune response and long-term survival. Combining HIFU with αCTLA-4 and αPD-L1 significantly enhances anti-tumor response, improving survival from 0 to 62.5%. HIFU alone causes upregulation of splenic and lymph node NK cells and circulating IL-2, IFN-Ɣ, and DAMPs, whereas immune regulators like CD4+Foxp3+, IL-10, and VEGF-A are significantly reduced. We also report significant abscopal effect following unilateral treatment of mice with large, established bilateral tumors using HIFU and checkpoint inhibitors compared to tumors treated with HIFU or checkpoint inhibitors alone (61.1% survival, p<0.0001). HIFU can effectively induce immune sensitization in a previously unresponsive murine neuroblastoma model, and promises a novel yet efficacious immuno-adjuvant modality to overcome therapeutic resistance. We used microarrays to help assess the role of HIFU-mechanical fracitonation on neuroblastoma tumors. mRNA was analyzed on Affymetrix Mouse Clariom S arrays using standard reagents and following the Affymetrix protocol (Thermofisher Scientific, Waltham, MA). Poly A controls were spiked into the reactions. Briefly, RNA was converted to cDNA, which was then in vitro transcribed into cRNA that was bead purified. Fifteen µg of purified cRNA was converted into ss-cDNA, which was then fragmented, cleaned, and hybridized (2.3 µg) for 16 hr (45°C) to the arrays. Hybridization solution was removed and arrays were washed and stained on an Affymetrix Fluidics 450 station and scanned with the Affymetrix GeneChip scanner.