Low-Intensity Focused Ultrasound Produces Immune Response in Pancreatic Cancer

Abstract

Pancreatic adenocarcinoma is an aggressive malignancy with limited therapeutic treatments available and a 5-y survival less than 10%. Pancreatic cancers have been found to be immunogenically "cold" with a largely immunosuppressive tumor microenvironment. There is emerging evidence that focused ultrasound can induce changes in the tumor microenvironment and have a constructive impact on the effect of immunotherapy. However, the immune cells and timing involved in these effects remain unclear, which is essential to determining how to combine immunotherapy with ultrasound for treatment of pancreatic adenocarcinoma. We used low-intensity focused ultrasound and microbubbles (LoFU + MBs), which can mechanically disrupt cellular membranes and vascular endothelia, to treat subcutaneous pancreatic tumors in C57BL/6 mice. To evaluate the immune cell landscape and expression and/or localization of damage-associated molecular patterns (DAMPs) as a response to ultrasound, we performed flow cytometry and histology on tumors and draining lymph nodes 2 and 15 d post-treatment. We repeated this study on larger tumors and with multiple treatments to determine whether similar or greater effects could be achieved. Two days after treatment, draining lymph nodes exhibited a significant increase in activated antigen presenting cells, such as macrophages, as well as expansion of CD8⁺ T cells and CD4⁺ T cells. LoFU + MB treatment caused localized damage and facilitated the translocation of DAMP signals, as reflected by an increase in the cytoplasmic index for high-mobility-group box 1 (HMGB1) at 2 d. Tumors treated with LoFU + MBs exhibited a significant decrease in growth 15 d after treatment, indicating a tumor response that has the potential for additive effects. Our studies indicate that focused ultrasound treatments can cause tumoral damage and changes in macrophages and T cells 2 d post-treatment. The majority of these effects subsided after 15 d with only a single treatment, illustrating the need for additional treatment types and/or combination with immunotherapy. However, when larger tumors were treated, the effects seen at 2 d were diminished, even with an additional treatment. These results provide a working platform for further rational design of focused ultrasound and immunotherapy combinations in poorly immunogenic cancers.

Keywords: Immunotherapy; Pancreatic cancer; Tumor microenvironment; Ultrasound.