Thermally Ablative Focused Ultrasound Potentiates the Anti-Tumor Immunity of Neoadjuvant Checkpoint Inhibition and Vascular Normalization

Introduction: Many solid tumors (e.g. most breast, ovarian, and pancreatic cancers) exhibit an inherently immunosuppressive or “cold” tumor microenvironment (TME) that can hinder the tumor’s response to immune checkpoint inhibitor (ICI) therapy. One approach to reversing solid tumor immunosuppression entails the use of low-dose anti-angiogenic drugs to achieve “vascular normalization” (VN), an intervention that often improves recruitment and capture of beneficial immune cells by tumor endothelium. Yet another approach to addressing immunosuppression is non-invasive, sparse-scan, thermally ablative focused ultrasound (tFUS). tFUS is a modality that can noninvasively destroy tumor tissue while concurrently generating thermal transition zones that render the tumor more immunologically “visible” through damage-associated molecular pattern expression and antigen release. Here, we tested whether combining VN and tFUS with ICI (aPD1) could effectively inhibit primary breast tumor growth, as well as prevent secondary tumor growth via generation of systemic anti-tumor immunity.

Materials and

Methods: EMT6 or 4T1-bearing BALB/c mice were intraperitoneally injected with a VEGFR2-blocking antibody (DC101), an agent that is commonly used to induce VN. Cell adhesion molecule (P-selectin, E-selectin, and VCAM-1) expression on tumor endothelial cells, as well as immune cells (CD8+ T cells and Fox3+ Treg cells) in the TME, were assessed with flow cytometry on day 14. For one cohort of EMT6-bearing mice, aPD1 injections were also intraperitoneally performed. Following neoadjuvant DC101 and/or aPD1 (Fig. 1A), sparse-scan tFUS was applied to tumors on day 14 (Fig. 1B) in a grid pattern, with 1.5mm spacing between each sonication point. Each tFUS sonication was applied with 18W of power for 15 sec, yielding an estimated ablative temperature of 70°C. Primary (1°) tumor size was tracked for several weeks. For mice with either very small or completely eradicated 1° tumors on day 36, a secondary (2°) EMT6 tumor challenge was applied to the contralateral side. Mice were then monitored for several weeks.

Results, Conclusions, and Discussions:

Results: DC101 induced trends toward increased E- and P-selectin expression and significantly increased VCAM-1 expression on tumor endothelium in 4T1 tumors (Fig. 1C-E). DC101 also significantly improved CD8/Treg ratios in both 4T1 and EMT6 tumors (Fig. 1F,G). Combining DC101 with aPD1 ICI significantly controlled EMT6 tumor growth (Fig. 1H) and reduced integrated total tumor burden, as represented by the “area under the curve” metric (Fig. 1I). Individual tumor growth curves are depicted in Fig. 1J. Adding tFUS to DC101+aPD1 yielded greater growth control over time (Fig. 1K) and reduced total tumor burden (Fig. 1L), with individual growth curves shown in Fig. 1M. DC101+aPD1+tFUS eradicated most 1° tumors (Fig. 1N,O). When challenged with a 2° tumor on the contralateral side, the 2° tumor was rejected far more often when the 1° tumor had been treated with DC101+aPD1+tFUS, when compared to IgG control tumors (Fig. 1P).

Discussion: We developed and employed a novel combination of VN, tFUS, and checkpoint inhibition to control primary breast tumors and prevent secondary tumor growth, presumably through generation of systemic anti-tumor immunity. We first affirmed that DC101 modulates the capacity for intratumoral immune infiltration and improves CD8/Treg ratio, a common clinical prognostic indicator. Combining DC101 with aPD1 checkpoint blockade yielded synergistic 1° tumor growth control, reducing tumor burden by ~65% compared to DC101 and ~50% compared to aPD1. Even more strikingly, combining tFUS with DC101+aPD1 yielded greater 1° tumor control, reducing tumor burden by ~80% compared to control and completely eradicating 1° tumors in >80% of treated mice. Following contralateral rechallenge with a 2° EMT6 tumor, ~65% of DC101+aPD1+tFUS-treated mice rejected the 2° tumor, as compared to ~30% of DC101+aPD1-treated mice and 0% of IgG control mice, indicating that adaptive anti-tumor immunity was likely being effectively generated by the initial combination treatment.

Conclusion: Thermal ablation potentiates the anti-tumor control of neoadjuvant anti-angiogenic therapy and immune checkpoint blockade in primary EMT6 tumors. This response, in addition to enhanced secondary tumor rejection, suggests that DC101+aPD1+tFUS evokes a potent adaptive anti-tumor immune response.

Acknowledgements (Optional): MRS was supported by an NSF GRFP. Also supported by NIH R01EB030007 and R21CA230088 to RJP.