Abstract A20: Enhancing checkpoint inhibitor therapy with ultrasound stimulated microbubbles
Authors: Bulner Sharshi , Prodeus Aaron , Gariepy Jean , Hynynen Kullervo , Goertz David E. 
Published: June 2021

Abstract
Checkpoint inhibitor (CI) therapies are playing an increasingly prominent role in the treatment of cancer but are only effective and durable in subsets of patients. This has prompted the investigation of combining multiple CIs or their use in conjunction with conventional therapies such as chemotherapy to achieve complementary effects. A significant consideration with combinatorial approaches is the associated increase in toxicity. This provides a compelling motivation to couple CI therapy with locally applied physical methods such as radiotherapy or therapeutic ultrasound. Therapeutic ultrasound is undergoing rapid development for oncological applications and can elicit therapeutically relevant effects through ablation or impacting vascular function. It is also known to evoke immune responses, though their use in combination with immunotherapy remains to be established. One therapeutic ultrasound approach is to acoustically stimulate systemically injected “microbubbles” to undergo violent oscillations in targeted tumor regions to shut down the vasculature. We have previously shown that this approach can potentiate the antitumor effects of a range of chemotherapeutic agents. In the present study, we investigate if this form of “antivascular” ultrasound can enhance the efficacy of anti-antagonistic PD-1 (aPD-1) therapy. To test this paradigm, we used a mouse colorectal cancer line (CT26.wt), which was initiated subcutaneously in the right hind limb of 8- to 12-week-old female Balb/c mice. Mice were split into four groups: MBs (control), aPD-1 (drug group), US (ultrasound plus microbubbles group) and US + aPD-1 (combo group). Experiments were initiated on mice when tumors were in the range of 50-100 mm3. The immunotherapy drug used in this study is an anti-mouse PD-1 (clone: RMP1-4, Bioxcell). The drug aPD-1 was administered intraperitoneally at a dosage of 200 μg to respective groups prior to treatment and subsequently administered every 3 days for a total of 5 doses. Longitudinal studies were done where tumor growth was monitored every 3 days until animals reached endpoint (tumor size&gt;1000 mm3). Acute experiments were done, which included flow cytometry and ELISPOT to assess how T-cell populations/activity changed with each respective treatment. Longitudinal experiments (n=5-6) showed that US + aPD-1 treatment significantly inhibited tumor growth relative to MB-only, US-only group and aPD-1 group at Day 6 (p&lt;0.0001, p&lt;0.01, p&lt;0.01, respectively) and at Day 9 (p&lt;0.0001, p&lt;0.01, p&lt;0.05, respectively). This inhibition of tumor growth with combinatorial treatment translated to longer survival times compared to MBs (p&lt;0.01), US (p&lt;0.01) and aPD-1 (p&lt;0.005). Flow cytometry (n=5-7) and ELISPOT (n=6) data did not clearly show a T cell-dependent mechanism for the inhibition of tumor growth. In conclusion, these results demonstrate the ability of “antivascular” US therapy to enhance CI therapy, while the specific mechanisms of enhancement remain to be elucidated.
Citation Format: Sharshi Bulner, Aaron Prodeus, Jean Gariepy, Kullervo Hynynen, David E. Goertz. Enhancing checkpoint inhibitor therapy with ultrasound stimulated microbubbles [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A20.