Abstract 2763: Effect of murine vendor on anti-tumor immune responses to non-ablative pulsed focused ultrasound
Authors: Chandran Parwathy , Hutchison Ruby , Tomlinson Lauren , Cohen Gadi , Burks Scott R. , Frank Joseph A. 
Published: July 2021

Non-ablative image guided pulsed focused ultrasound (pFUS) has shown promise in murine tumor models as an immunomodulatory strategy for altering tumor microenvironment (TME). However, studies to date have demonstrated efficacy only in murine models procured from a single vendor. To determine whether differences in inbred mice obtained from 3 vendors would alter tumor growth and TME, we evaluated temporal expression of cytokine, chemokine and trophic factor (CCTF), and immune cell profiles following non-ablative pFUS to murine B16 melanoma flank tumors established in C57BL/6J mice. B16 cells (106 cells) were subcutaneously, bilaterally implanted into C57BL/6J mice (n=6/group/time-point) obtained from 3 vendors [Jackson Laboratories (JAX; n=36), Charles River (CR; n=36), Taconic Biosciences (Tac; n=36)]. Ultrasound guided pFUS (VIFU 2000, Alpinion) was administered at 1 MHz, at 6 MPa PNP to 5 mm tumors. Entire tumor volume was sonicated point-by-point along a 2×2 mm matrix with 20 msec pulse length, 10% duty cycle and a 5 Hz pulse repetition frequency. On days 1, 3, and 5 post-pFUS, spleens (Sp), lymph nodes (LN) and tumors were evaluated by proteomic (CCTF) and flow cytometry (FC) analyses. pFUS treatment demonstrated variable, vendor-dependent effects on tumor growth, CCTF and immune cell profiles. CR B16 volumes showed significant reduction (p&lt;0.05; 2-way ANOVA) starting at day 2 through day 5, but not in JAX or Tac mice. Proteomic analyses revealed a similar trend wherein CR TME exhibited upregulation of pro-inflammatory cytokines (TNFα, IL-1β, IL-12p70) on days 1 and 3, and downregulation of anti-inflammatory cytokines (VEGF, IL-10) over 5 days. Comparatively, less robust expression of anti-tumoral cytokines were observed in Tac and JAX TME over 5 days. FC corroborated tumor growth and molecular analyses, showing significant tumor infiltration of helper and cytotoxic T cells (Th, Tcyt), M1 macrophages and dendritic cells (DC) on days 3 and 5, compared to untreated controls. Day 5 CR Sp also exhibited a pro-inflammatory profile with T cells, macrophages and DC. Tac and JAX tumor profiles over days 1, 3 and 5 were relatively unchanged by pFUS with major, anti-tumoral immunological activity restricted to draining LN on days 5 and 3 in Tac and JAX, respectively. Moreover, molecular and immune cell differences were observed in B16 tumors from control mice on days 1, 3 and 5. Collectively, B16 bearing CR mice exhibited a more pronounced anti-tumor response to pFUS, resulting in reduced tumor growth and pro-inflammatory CCTF and immune cell profiles as compared to B16 tumors in Tac and JAX mice. Despite using the same mouse strain, tumor type, and pFUS parameters, profound vendor-dependent variability was observed, suggestive of the possible role of inherent genetic disparities among C57BL/6J mice from different vendors. These results suggest that vendor choice may be critical in evaluating anti-tumor effects of pFUS.

Citation Format: Parwathy Chandran, Ruby Hutchison, Lauren Tomlinson, Gadi Cohen, Scott R. Burks, Joseph A. Frank. Effect of murine vendor on anti-tumor immune responses to non-ablative pulsed focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2763.