High intensity focused ultrasound (HIFU) is a non-invasive ablative treatment that induces rapid temperature elevation in tissues in the focal zone, resulting in instantaneous coagulative necrosis. Previous studies by us demonstrated that repeated cycles of nonablative low energy focused ultrasound (LOFU), administered 1 day prior to ablative HIFU, induce antitumoral imunity. We hypothesized that LOFU modulates phenotypic changes in the tumor cell surface, resulting in enhanced antigen presentation and phagocytosis by dentritic cells (DC) and increased susceptibility to cytotoxic T cell (CTL) attack. In this study, we optimized the LOFU regimen based on immunomodulatory changes in tumor cells and cytotoxicity. Three murine tumor cell lines, Panc-2, RM-1, and E. G7 (OVA+) were exposed to LOFU using the Phillips therapy and imaging probe system, operated at 1 MHz, 100% duty cycle and 0-4 watts acoustic power for 1.5 seconds. Cells were harvested (6, 24, 48h and day 7) for detection of cell surface expression of MHC-I, Calreticulin, FAS and cytoplasmic HSP70 by flow cytometry and HSP70 ELISA of cell lysates. Phagocytosis assay was performed by coculturing marrow-derived DCs or JAWS-II cells with CellTracker-labeled LOFU-treated tumor cells, followed by flow cytometric detection of engulfed tumor cells. DC activation was analyzed by CD80, CD86, MHC-II and CD40 flow cytometry. T-cell activation was analyzed by coculturing OVA-specific splenocytes with LOFU-treated OVA+ E. G7 cells, followed by measurement of IFN-gamma secretion in culture supernatant by ELISA. Finally, C57BL/6 mice bearing palpable RM-1 tumors were treated with LOFU (weekly for 3wks) and sacrificed at 4th wk and tumor specific T cell response was assessed by IFN-gamma ELISPOT. Tumor cells treated with LOFU had significant upregulation of intracellular HSP70 (peak at 24hrs, 13.4±5.8 fold) and marginal cell killing (15.6±4.4%) at 3W acoustic power. Further studies were performed with LOFU at 3W. LOFU induced translocation of cytoplasmic calreticulin on the cell surface (50-79.5% at 24 hr). Immature DCs phagocytosed LOFU-treated tumor cells (10-16.3%), followed by induction of cell surface CD80, CD86 and MHC-II expression (2-3 fold induction) indicating increased activation. There was a 2-3-fold upregulation of surface expression of HSP70 and FAS in LOFU-treated E. G7 cells, resulting in enhanced OVA-specific CTL-mediated cytotoxicity. Finally, a significant increase in tumor specific T cells (0.1-0.3%) was observed in the splenocytes from mice with LOFU-treated RM-1 tumors. Control mice with untreated tumors had no detectable tumor-specific T cells.

These results demonstrate a potent tumor specific immune response induced by non-ablative LOFU. Further studies are warranted to augment LOFU/HIFU-induced antitumoral immunity for the treatment of recurrent and metastatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5630.