Image-guided Focused Ultrasound Ablation and CD40 Agonism for Immune-Mediated Protection in Breast Cancer

Introduction: Despite the marked promise of immunotherapies, breast cancer (BrCa) remains the most common cancer diagnosis in women, and the second leading cause of cancer-related deaths in women. Current treatment options are limited – with surgical resection being the first line of defense, followed by chemotherapy and radiation. Even as patients solicit these options, the risk of relapse and tumor metastasis to distal tissues (i.e., bone, lung, and brain) persists. As BrCa are largely recalcitrant to immunological rejection, there is a critically unmet need for better options. Focused ultrasound (FUS) is a non-ionizing, noninvasive, and tunable strategy for highly localized acoustic energy deposition into tumors. FUS offers a multitude of bioeffects at the targeted region of interest. When applied as a high intensity continuous wave, thermally ablative focused ultrasound (T-FUS) can cause coagulative necrosis within the focal zone. Between these necrotic and healthy regions exists a peri-ablative zone, where the sublethal damage induced by T-FUS can yield immunogenic sequelae, specifically in the form of damage-associated molecular patterns (DAMPs) (e.g. heat shock proteins) and tumor-associated antigen liberation. The immunomodulatory effects of T-FUS have also been shown to elicit greater infiltration of CD4 and CD8 T cells and combine favorably with various ITx in solid tumor settings. Given the clinical advancement of T-FUS and the unmet need in BrCa, we aim to investigate whether combining T-FUS with anti-CD40 offers protection in murine breast cancer.

Materials and

Methods: E0771 murine mammary carcinomas, consisting of 400,000 cells, were inoculated into the right flank of C57/Bl6 mice. After 28 days post-inoculation, mice were grouped using a non-biased automated MATLAB program to volume match into the following groups: IgG control, T-FUS, α-CD40, and T-FUS + α-CD40. Tumor measurements were carried out with daily precision caliper measurements and overall survival was monitored. As a secondary method, tumor volume was also determined with B-mode ultrasound imaging using a Vevo 2100 ultrasound imaging system. Following baseline measurements on day 28 post-inoculation, the α-CD40 priming regimen was initiated, with isotype IgG administered as a control. The antibodies were administered intraperitoneally at a dosage of 100 μg, and repeat administration occurred every 3 days. On day 35 post-inoculation, subtotal thermal ablation was performed with a custom ultrasound-guided FUS system (3.78MHz, 18W, 15s). The final administration of antibodies took place on day 42 post-inoculation, completing a total of 5 doses. After treatment, a selection of mice in the T-FUS + α-CD40 group experienced a complete response to the combination paradigm; this occurred exclusively in this group. Complete responder mice were then contralaterally rechallenged with a million E0771 cells following 100 days post-inoculation alongside age-matched, tumor/treatment-naïve wild-type controls. Multispectral flow cytometry was performed on serial blood samples from week 3-6 post-rechallenge.

Results, Conclusions, and Discussions: The combination of αCD40 and T-FUS offered superlative E0771 growth control relative to α-CD40, T-FUS monotherapy, and IgG controls (Fig 1A). Combination-recipient mice displayed marked improvement in overall survival, with 43% (3/7) of mice experiencing complete tumor regression (i.e. complete responders; CRs) (Fig 1B). The combination treatment group exclusively yielded complete response among the mice, whereas no instances of immunological rejection were observed in the α-CD40, T-FUS monotherapy, and IgG control groups (Fig 1B). On day 100 post-inoculation, mice that achieved complete response (CRs) were rechallenged, alongside age-matched tumor/treatment-naive wild-type control mice, to assess natural immune rejection in the absence of any therapeutic interventions. CRs rechallenged with contralateral E0771 inoculations did not display evidence of tumor outgrowth (Fig 1C). The observed outgrowth outcomes correlated with a 100% murine survival rate in the CRs, whereas all naïve mice succumbed to mortality based on tumor volume criteria (Fig 1D). To evaluate the memory T cell component of this response, blood was serially bled among all mice. Assessment of peripheral blood mononuclear cells in CRs revealed trends in the increased overall percentage of CD8+ and CD4+ T cells, as well as effector memory at week 3 post-rechallenge (Fig 1E). At week 4 post-rechallenge, there was a trend in the increased overall percentage of CD8+ and CD4+ central T cell memory relative to controls (Fig 1F).

The T-FUS in combination with CD40 agonism offers strong potential for conferring memory-driven responses against murine breast tumors. Current efforts aim to investigate how α-CD40 priming changes the tumor microenvironment prior to T-FUS and the immunological mechanisms for protection. Future studies will interrogate the role of therapeutic sequencing and mechanistic underpinnings. Moreover, we aim to evaluate the effect of T-FUS + α-CD40 treatment paradigm in a secondary BrCa model (4T1 triple negative murine model).

Numerous clinical trials are investigating thermal FUS approaches as a tool for potentiating immunotherapy in primary BrCa (NCT03237572, NCT04796220). We expect that findings from this study will be ripe for clinical translation.

Acknowledgements (Optional): This work was supported by NIH DP5OD031846 and T32CA009109, as well as the Wallace H.
Coulter Foundation for Translational Research.