Abstract 2665: Immune cell modulation of pulsed focused ultrasound in murine melanoma and breast cancer models
Authors: Chandran Parwathy , Cohen Gadi , Burks Scott R. , Frank Joseph A. 
Published: September 2020

Abstract
Major therapeutic advances in the field of tumor immunotherapy are hindered by the ability to overcome the immunosuppressive tumor microenvironment (TME), which prevents the immune system from mounting an efficient anti-tumor response. Pulsed focused ultrasound (pFUS) studies has shown some promise as an immunomodulatory strategy for altering TME. This study evaluated the temporal changes following non-ablative pFUS on TME immune cell profiles in spleen (Sp), lymph nodes (LN), and tumors (B16 melanoma and 4T1 mammary carcinoma). Murine B16 and 4T1 cells were subcutaneously, bilaterally implanted into C57BL/6J and BALB/c mice (n=10 tumors/time-point), respectively. Ultrasound guided pFUS (VIFU 2000, Alpinion) was administered at 1 MHz, at 6 MPa PNP to 5 mm tumors. Entire tumor volume was sonicated with a 2 mm spacing between points using a 10 ms pulse length, 10% duty cycle and a pulse repetition frequency of 10 Hz. At days 1, 3, and 5 post-sonication, Sp, LN and tumors were harvested, fixed and processed for flow cytometry (FACS) analysis of immune cell populations [cytotoxic (Tcyt), helper (Th), and regulatory (Treg) T cells, natural killer (NK), dendritic cells (DC), F4/80+ macrophages (M1 and M2), myeloid-derived suppressor cells (MDSC)] and immune checkpoint markers (CTLA-4, PD-1 and PD-L1). The immune response to pFUS differed between the B16 and 4T1 tumor bearing mice. B16 mice at days 1, 3 and 5 post-sonication revealed a 3-4 fold increase in Tcyt, Th, NK and M1 macrophage activity in tumor, which peaked at day 3, and decreased by day 5, compared to control tumors. At day 5, a shift of cell populations toward LN, with increased Treg, Tcyt, NK, F4/80, M1 and M2 activity was observed. 4T1 mice showed high levels of Tcyt in Sp on day 1, and NK cells on day 3 in LN. Interestingly, these cell populations decreased in Sp and LN by day 5, with 3-4 fold increase in 4T1 tumors, along with increasing macrophage and DC activity. Despite the differences in both tumor types, results suggest that pFUS altered the TME by inducing anti-tumor responses by activating both innate (macrophages, NK cells) and adaptive (Tcyt, Th cells) arms of immunity. This study reveals the differences in immune cell profiles following sonication between the two tumor types and underscores the importance of not generalizing the effect of pFUS on the TME. This analysis also suggests that the global changes to the immune cell profile represent a shift from anti-inflammatory, immunosuppressive TME towards an anti-tumor TME. Optimizing pFUS parameters to enhance immune cell infiltration, would presumably lead to slowing tumor growth rates and when coupled with immune checkpoint inhibits could serve as neoadjuvant approach in treating malignancy.
Citation Format: Parwathy Chandran, Gadi Cohen, Scott R. Burks, Joseph A. Frank. Immune cell modulation of pulsed focused ultrasound in murine melanoma and breast cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2665.