Focused ultrasound thermal ablation and CD40 agonism reprograms breast tumor immunity to drive regression and memory

Zehra EF Demir; Aeryon Kim; Beyzanur G Ak; Micaiah S Lee; Thomas Sherlock; Stefanyda O Maslova; Andrew T Thede; Matthew R DeWitt; Melanie Rutkowski; Natasha D Sheybani

doi:10.1136/jitc-2026-015671

. 2026 Aug 4;14(8):e015671. doi: 10.1136/jitc-2026-015671

Focused ultrasound thermal ablation and CD40 agonism reprograms breast tumor immunity to drive regression and memory

Zehra EF Demir ¹, Aeryon Kim ^2,³, Beyzanur G Ak ¹, Micaiah S Lee ¹, Thomas Sherlock ¹, Stefanyda O Maslova ¹, Andrew T Thede ¹, Matthew R DeWitt ¹, Melanie Rutkowski ⁴, Natasha D Sheybani ^1,^✉

PMCID: PMC13448604 PMID: 42552061

Abstract

Background

Focused ultrasound thermal ablation (T-FUS) is a clinically accessible, non-invasive modality capable of inducing rapid tumor cytoreduction while mobilizing early immunologic danger signals. However, its capacity to synergize with potent co-stimulatory immunotherapies in breast cancer (BC) remains undefined. Here, we investigated whether subtotal T-FUS cooperates with CD40 agonism to elicit durable, T cell-dependent tumor control across immunologically and molecularly distinct murine BC models.

Methods

Subtotal ultrasound-guided T-FUS was applied to E0771, BRPKP110, EMT6, and 4T1 tumors in mice treated with systemic agonistic αCD40. Histology, ATP bioluminescence imaging, and multispectral flow cytometry were used to define acute tumor injury and immune remodeling. Therapeutic efficacy was evaluated by tumor growth, survival, T-cell depletion, and contralateral tumor rechallenge.

Results

Partial thermal ablation triggered canonical immunogenic cell-death signatures and acute remodeling of intratumoral myeloid populations, while expanding circulating CD4+ and CD8+ T cells. When layered onto this immunogenic milieu, αCD40 markedly constrained tumor outgrowth, yielding significant reductions in tumor burden across all models and complete tumor eradication in 30% of E0771 tumors, with additional complete responses in BRPKP110 and EMT6. Combination treatment also extended survival across three of four models. Efficacy required both CD4+ and CD8+ T cells, as depletion of either compartment abrogated tumor control. Complete responders mounted robust systemic immunity, rejecting contralateral tumor rechallenge with 100% protection and displaying persistent effector-memory T-cell activation.

Conclusions

Subtotal T-FUS cooperates with CD40 agonism to drive durable, T cell-dependent BC regression and immunologic memory. These findings establish T-FUS as an immune-potentiating partner for CD40 agonism and position this combination as a clinically scalable, in situ vaccination-like strategy with potential to broaden immunotherapy benefit across breast cancer subtypes, including luminal tumors that remain largely refractory to immune checkpoint blockade.

Keywords: Immunotherapy, Breast Cancer, Combination therapy, Immune modulatory, co-stimulatory molecules

WHAT IS ALREADY KNOWN ON THIS TOPIC

Thermally ablative focused ultrasound (T-FUS) can elicit immunogenic tumor injury and early antitumor immune signatures, but its ability to generate durable systemic immunity in breast cancer has remained incompletely defined. CD40 agonists can activate and license antigen-presenting cells, yet have shown limited efficacy as monotherapies in solid tumors, creating a need for rational partner interventions that kindle immunogenicity and productive T-cell priming.

WHAT THIS STUDY ADDS

To our knowledge, this is the first study to show that subtotal T-FUS can be paired with systemic CD40 agonism to drive durable, T cell-dependent tumor control across multiple murine breast cancer models, including luminal disease. The combination generated complete responses, required both CD4+ and CD8+ T cells, and induced robust systemic, tumor-specific immune memory marked by 100% rejection of contralateral rechallenge in complete responders.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

This study positions subtotal T-FUS plus CD40 agonism as a clinically accessible, incisionless, in situ vaccination-like strategy for breast cancer immunotherapy. These findings support further translational evaluation of T-FUS with next-generation CD40 agonists and provide rationale for clinical testing of this combination across breast cancer subtypes, including settings less responsive to checkpoint blockade.

Introduction

Breast cancer (BC) has long been among the most commonly diagnosed cancers worldwide and remains a leading cause of cancer-related deaths in women.^1–4 Across both sexes, it ranks among the top cancers by incidence and among the leading causes of cancer mortality.^5
6 Recently, BC screening guidance has shifted, with the US Preventive Services Task Force now recommending biennial mammography a decade earlier, starting at age 40.⁷ Against this backdrop of rising BC incidence in younger women, priorities are increasingly focused on treatment approaches that maintain efficacy while reducing cumulative toxicity and long-term morbidity. However, current standards of care for BC still impose substantial patient burden. Surgery is inherently invasive and can entail prolonged convalescence (particularly in older patients) as well as breast deformity and suboptimal cosmesis. Systemic therapies such as chemotherapy can be dose-limited by hematologic toxicity, and both chemotherapy and radiation can carry short- and long-term adverse effects, including potential impacts on fertility—an especially salient consideration for younger patients.^8
9

Less invasive, organ-sparing focal interventions have the potential to reshape BC treatment by reducing procedural morbidity and enabling unique opportunities for therapeutic synergy. Focused ultrasound (FUS) provides an incisionless modality for spatially precise thermal ablation (T-FUS)^10–12 with a clinical track record in the treatment of benign^13
14 and malignant^15
16 breast tumors, and is now being explored as an adjunct to immunotherapies (ITx) (NCT04796220, NCT03237572).^15–17 Preclinical and early clinical studies suggest that T-FUS can induce immunogenic tumor injury, including increased damage-associated molecular patterns (DAMPs; alarmins), upregulation of heat shock proteins, release of tumor-associated antigens, recruitment of antigen-presenting cells (APCs), and heightened local and systemic CD8+ and CD4+ T-cell responses against solid tumors.^12
15
18
19 Accordingly, a key next step is to define how T-FUS can be leveraged to broaden and deepen ITx responses—particularly in BC, where this potential has yet to be systematically evaluated across distinct subtypes and immunophenotypes.

Clinical interest in ITx for BC continues to expand,²⁰ with programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade improving outcomes in subsets of triple-negative disease.²¹ However, benefit remains subtype-restricted and inconsistent across BC more broadly,^22
23 motivating combination strategies designed to expand and improve responses.^20
24 Given the marked heterogeneity of immune contexture across BC subtypes—including differences in baseline tumor-infiltrating lymphocytes²⁵—there is increasing interest in approaches that enhance priming rather than relying solely on reinvigoration of pre-existing T-cell responses.²⁴ Among the most clinically advanced co-stimulatory strategies is CD40 agonism. CD40 (a tumor necrosis factor (TNF) receptor superfamily member) is expressed predominantly on APCs, including dendritic cells, B cells, and macrophages. CD40 engagement promotes APC activation and licensing to support effective T-cell priming.^26–28 Yet, despite encouraging activity in select settings, CD40 agonists have shown limited efficacy as monotherapies in solid tumors,^26
29 underscoring the need for rational partner interventions that increase immunogenic antigen availability and productive antigen presentation. In this context, T-FUS represents a mechanistically aligned modality to pair with CD40 agonism.^24
30

Building on timely recent clinical evidence that an Fc-engineered CD40 agonist antibody (2141-V11) can induce tumor regression, including a complete response in BC,³¹ we test the hypothesis that CD40 agonism cooperates with T-FUS to drive durable antitumor immunity in BC. Using a panel of murine models spanning diverse molecular subtypes and immunophenotypes, we show that combining αCD40 with a clinically aligned partial T-FUS regimen (NCT03237572) yields robust tumor control and survival benefit across multiple models. Mechanistically, therapeutic activity is T cell-dependent and can produce complete responses accompanied by evidence of immune memory and tumor specificity. Collectively, these findings position T-FUS plus CD40 agonism as a clinically accessible combination strategy with the potential to extend ITx benefit across BC subtypes, including luminal tumors that are typically less responsive to classic checkpoint blockade.^22
32

Results

Subtotal FUS thermal ablation results in clear zones of apoptotic and necrotic tissue destruction, concomitant with local upregulation of canonical thermal stress signatures

First, we developed experimental methods and procedures to achieve partial thermal ablation of BC tumors, consistent with partial T-FUS benchmarks established in ongoing FUS breast immuno-oncology clinical trials (NCT04796220, NCT03237572). Briefly, we used a custom ultrasound-guided FUS system equipped with four therapeutic transducers driven at 3.78 MHz in continuous wave mode to elicit thermal energy deposition; focal temperatures were three-dimensionally modeled in silico (figure 1A, online supplemental figure 1A–C). Tumors were acoustically coupled via a degassed water bath and positioned directly over an axially co-registered ultrasound imaging probe, enabling real-time B-mode ultrasound guidance for tumor localization and targeting (figure 1B). Widely used in clinical applications, the appearance of hyperechoic marks in sonography is often used as an indicator of ablation.^33–36 During or immediately following T-FUS treatment, hyperechoic signatures were evident on B-mode (figure 1C). Strikingly, this varied from model to model, presumably on the basis of tissue stiffness, vascularity, or other intertumoral heterogeneities.^37–39 Among the E0771, BRPKP110, EMT6, and 4T1 models, 78.6%, 88.9%, 31.3%, and 94.4% of the mice were reported as a grade 3, respectively (online supplemental table S1). Among these distinct BC models, we observed clear hyperechoic signals within all mice, with a majority (91.5%) at grade 2 or 3 (figure 1D, online supplemental table S1).

E0771 tumors were evaluated histologically 1-day post-treatment (figure 1E). H&E staining of T-FUS-treated tumors showed clear evidence of coagulative necrosis in the ablative zone, with patterning being distinct from tumor progression-associated necrosis in controls. In contrast with controls, ablative zones displayed higher levels and distinct patterning of apoptosis, as reflected by cleaved caspase 3 (Cl Casp 3) and poly(ADP-ribose) polymerase (PARP) staining (figure 1F). Furthermore, upregulation of heat shock protein 70 (HSP70) staining was evident in the so-called “periablative zone”, that is, regions neighboring those bearing signatures of coagulative necrosis and apoptosis—consistent with the notion of a transition zone from heat-mediated cell death to damage from ablative and periablative regions, respectively (figure 1F).

Release of ATP is considered a hallmark of immunogenic cell death, acting as a key DAMP.⁴⁰ T-FUS acutely conferred an ~146-fold increase in average luminescence relative to sham conditions in 4T1 cells in vitro (online supplemental figure 2A). Translated to orthotopic E0771 BCs in vivo, we observed that T-FUS conferred an ~10.3-fold increase in average maximum radiance, a proxy for ATP release, at 10-minute post-treatment (figure 1G). Longitudinal bioluminescence imaging revealed superlative intratumoral/peritumoral ATP levels in T-FUS-treated tumors compared with the volume-matched shams, and interestingly, this trend was sustained over the 40-minute imaging period (figure 1H).

3 days following T-FUS, tumors were excised for immunological characterization by multispectral flow cytometry. T-FUS, analogously deployed in E0771 and 4T1 tumors, did not confer changes to intratumoral B cells (CD19⁺, MHC-II⁺; figure 1I). However, the percentage of macrophages (MФs; F4/80⁺, Ly6C⁺), conventional dendritic cells (cDCs; CD11c⁺, MHC-II⁺), and monocytes (Ly6C⁺, F4/80⁻) was significantly decreased following ablation (figure 1J–L). Conversely, observed across both BC models, there was an acute enrichment in local granulocytes (Ly6G⁺, CD11b⁺), likely neutrophils and/or granulocytic myeloid-derived suppressor cells (G-MDSCs) (figure 1M). Thus, subtotal T-FUS produces a spatially patterned thermal injury—coagulative necrosis and apoptosis with a periablative heat-stress border—coupled to rapid ATP release and an acute remodeling of the intratumoral myeloid compartment toward granulocytic predominance.

Subtotal FUS thermal ablation promotes a systemic T-cell response

We also investigated shifts in the T lymphocyte population in E0771 mice 3 days post-treatment (figure 2A). As anticipated, there was a significant decrease in the absolute number per gram and percentage of CD8⁺ and CD4⁺ T cells (including regulatory T cell (Treg) subsets in the tumor (figure 2B–G). While we observed an overall decrease in intratumoral T-cell numbers, T-FUS-treated tumors exhibited a significantly higher CD8:CD4 T-cell ratio relative to control indicating a compositional shift toward CD8-skewed intratumoral T-cell balance 3 days post-treatment (figure 2H). At this time point, we did not anticipate T-FUS to augment intratumoral T cells, but existing literature suggesting that T-FUS can induce a systemic T-cell response^17
41 prompted us to postulate that T-FUS may elaborate systemic T-cell signatures. Indeed, in the tumor draining lymph node (TDLN), we observed strong trends toward enriched CD8⁺ and CD4⁺ T-cell numbers, while the percentage of these cells among total lymphocytes remained unchanged (figure 2I–L). In tandem, we sampled the circulating immune cell repertoire via terminal cardiac bleed. A dimensionality reduction analysis using t-distributed stochastic neighbor embedding (t-SNE) qualitatively relevaled a greater abundance of both CD8+ and CD4+ T cells in the blood following T-FUS treatment (figure 2M). Consistent with these qualitative shifts, quantification confirmed that T-FUS treatment significantly increased the number of CD8⁺ T cells, while driving a strong trend toward increased CD4⁺ T cells (~twofold) (figure 2N–O). Moreover, T-FUS drove a significant increase in the percentage of both CD8⁺ and CD4⁺ T cells (figure 2P,Q). Together, these results support that beyond its function as a local debulking modality, T-FUS functions as a systemic immunologic stimulus, yielding favorable shifts in local T-cell ratio and elevating T-cell representation in TDLN and blood.

Subtotal FUS thermal ablation in combination with a CD40 agonist constraints tumor outgrowth and promotes survival: conferring complete responses in multiple breast cancer models

Consistent with preclinical^11
18
19 and clinical^15
16
42
43 findings, our results support the role of partial thermal ablation as an immunomodulator. Accordingly, we sought to evaluate whether coupling of CD40 agonism would act in cooperation, enabling a promising combinatorial strategy for BC. To evaluate the efficacy of T-FUS and αCD40, across all BC models, we initiated treatment with intraperitoneal (i.p.) administrations of αCD40 (100 $\boldsymbol{\mu }$ g/mouse) every 3 days once tumors reached approximately 60 mm³ (figure 3A). We used multiple different murine models reflecting the diversity of human BC subtypes in their hormone receptor status, immune mosaic, and metastatic proclivity. These include basal-like triple-negative BC models including 4T1 and EMT6 (Balb/c background), as well as hormone receptor positive BC models including BRPKp110 and E0771 (C57BL/6 background)—which recapitulate Luminal A-like⁴⁴ and Luminal B-like⁴⁵ subtypes, respectively.

Across all BC models (E0771, BRPKP110, EMT6, 4T1), neither monotherapy offered significant benefit, whereas the combination treatment group offered the most superlative constraint of tumor outgrowth relative to controls (figure 3B–E). By a week following the conclusion of treatments, E0771, BRPKP110, EMT6, and 4T1 tumors exposed to T-FUS+αCD40 combination saw nearly 3.5X, 3.6X, 2.3X, and 3.1X reduction in the average tumor volume compared with IgG control, respectively. Area under the curve (AUC) analysis revealed significant reduction of cumulative tumor burden in E0771 and 4T1, with a striking 1.7-fold and 2-fold reduction in AUC compared with αCD40 monotherapy, respectively (online supplemental figure 2B,C). Serial three-dimensional (3D) B-mode ultrasound volumetry of 4T1 tumors independently confirmed a significant reduction in tumor burden in the combination group versus all other groups, corroborating digital caliper measurements (online supplemental figure 2D,E).

Combination of T-FUS and CD40 agonism also promoted significant survival benefit across 3 of 4 models (E0771, BRPKP110, and EMT6), underscoring the robustness of the combination across BC subtypes but also the importance of subtype-specific considerations (figure 3F–I). E0771 tumor-bearing mice receiving T-FUS+αCD40 additionally saw the most impressive extension in overall survival, exhibiting ~31% ~33%, and 22% increases in median survival time relative to IgG, T-FUS, and $\boldsymbol{\alpha }$ CD40 groups, respectively (figure 3F). Consistent with clinical response assessment frameworks, we assigned each mouse to a response category—progressive disease, stable disease, or complete response—to further probe response to T-FUS+αCD40 (figure 3F–I; wheel insets). Remarkably, E0771 mice treated with the combination displayed the highest proportion of stable and/or complete responses. In the combination treatment group exclusively, 30% of the mice demonstrated complete tumor regression (complete responders; CRs) (figure 3F). Furthermore, across two additional BC models—BRPKP110 and EMT6—CRs were observed only in the T-FUS+αCD40 group, although to a lesser degree (figure 3G,H). While 4T1 tumor-bearing mice did not receive explicit survival benefit, likely owing to onset of spontaneous pulmonary metastasis known to this model, ~58% of mice experienced stabilization of primary disease (figure 3I). In aggregate, T-FUS+αCD40 consistently outperformed controls across all models tested, delivering the most robust suppression of tumor outgrowth, extending survival across multiple models, and inducing stable/complete responses with BC subtype-specific magnitude. In light of the particularly robust response observed in E0771, subsequent experiments focused on this model to enable deeper mechanistic dissection.

αCD40 priming orchestrates potent local and systemic immune activation, enriching the abundance and maturity of APCs and T cells

Given the pronounced efficacy of the T-FUS+αCD40 regimen, we next sought to define how αCD40 priming reshapes the local and systemic immune landscape prior to ablation, thereby establishing the immune context in which T-FUS is delivered. We used multispectral flow cytometry to identify local and systemic changes in APCs in E0771 tumor-bearing mice (figure 4A). t-SNE visualization revealed qualitative shifts in the APC population in TDLN, guiding subsequent analysis by conventional gating (figure 4B). Consistent with findings in the literature,^26–28 αCD40 priming increased the absolute number and percentage of APCs including B cells, MФs, and cDCs (figure 4C–H). Additionally, the geometric mean fluorescent intensity (GMFI) of CD86, a canonical co-stimulatory marker of APC activation, on these cells was also significantly elevated with CD40 agonism (figure 4I–K). Moreover, the percentage of CD86 on these cells was also increased in the TDLN (figure 4L–N). On further disaggregation of the cDC population in the TDLN, we observed an increase in both the number and percent of cDC1s (CD11b⁺, XCR1⁺) and cDC2s (CD11b⁺, XCR1⁻, SIRPα⁺) (online supplemental figure 3A–D). Additionally, we observed an increase in the percentage of CD86⁺ cDC1s and GMFI of CD86 on cDC1s and cDC2s in the TLDN (online supplemental figure 3E–I). Beyond the TDLN, the global impacts of αCD40 priming were evident through shifts in the splenic and contralateral lymph node (cLN) B cell and MФ populations (online supplemental figure 4A,H), which displayed significantly elevated absolute numbers and maturity (online supplemental figure 4B–G, I–N).

We co-examined the local effects of tumor priming with CD40 agonism. While neither the absolute number per gram of tumor nor the percentage of cDCs changed, we observed an increase in the number and percentage of B cell and MФs (figure 4O–S, online supplemental figure 3J,K). Additionally, we observed a trend in greater CD86 GMFI on B cells, which was significant on MФs (figure 4T,U). In addition to augmenting intratumoral APCs, CD40 priming elicited shifts in the intratumoral T lymphocyte landscape (online supplemental figure 5A,B), specifically yielding significantly increased absolute number and percentage of intratumoral CD8⁺ T cells (online supplemental figure 5C,F); increased percentage of CD4⁺ T cells (online supplemental figure 5D,G); and significantly decreased percentage of Tregs (online supplemental figure 5E,H). Following αCD40 priming, both CD8⁺ and CD4⁺ T cells upregulated CD40L, exhibiting >twofold higher CD40L GMFI, consistent with an activated phenotype and enhanced capacity for CD40-dependent costimulatory signaling (online supplemental figure 5I,J). To begin distinguishing whether αCD40-associated shifts in T-cell abundance reflected local proliferation versus redistribution/trafficking, we also assessed Ki-67 on CD8⁺ and CD4⁺ T cells. T cells exhibited a significant increase in the percentage of Ki-67⁺ cells and in Ki-67 GMFI with CD40 agonism (online supplemental figure 5K–O). Taken together, these data indicate that αCD40 priming drives broad APC activation across compartments and markedly increases intratumoral T-cell abundance and proliferative state just prior to T-FUS.

Complete and non-complete responders to TFUS+αCD40 are distinguished by their T-cell signatures

A subset of mice (CRs) achieved complete tumor eradication in the combination arm (figure 5B–D). In an effort to identify immunologic correlates of durable tumor control, we leveraged the coexistence of these complete and “non-complete” responders (non-CRs) and profiled the T-cell landscape at a chronic endpoint (figure 5A). We dissected the immunological repertoire in tumors (when available) and secondary lymphoid organs via multispectral flow cytometry. Of note, no tumor data could be collected from CRs since their tumors had been entirely eliminated by the time of assessment. The tumors of combination-recipient non-CRs were compared with monotherapy and control counterparts. Despite appreciable tumor growth control in non-CRs, we observed that intratumoral T-cell representation was indistinguishable from mice receiving αCD40 alone (figure 5E–H). Both groups equivalently exhibited a trend towards increased CD8⁺ T-cell numbers and significant elevation in CD8⁺ T-cell percentage compared with T-FUS and IgG controls (figure 5E,G). Intratumoral CD4⁺ T-cell representation was largely unchanged at this time point (figure 5F,H). These data suggest that, in non-CRs, T-FUS may not have sufficiently augmented intratumoral T-cell signatures beyond those elicited by αCD40 alone, potentially contributing to incomplete tumor control.

We concomitantly evaluated spleens and TDLNs. While splenic T-cell numbers did not change appreciably (figure 5I,J), we observed a significant increase in their percentages across all combination recipients, irrespective of their response level (figure 5K,L). Contrastingly, T-

cell signatures within TDLNs were distinguishable by response level. Specifically, CRs exhibited reduced TDLN T-cell cellularity but increased relative representation of T cells (particularly CD4⁺), resulting in a lower CD8:CD4 ratio—consistent with a contracted or resolved TDLN state after tumor eradication; non-CRs retained relatively expanded, CD8⁺-skewed TDLNs consistent with persistent disease (figure 5M–R). These findings indicate that T-FUS+αCD40 elicits measurable systemic T-cell shifts while revealing response-associated differences in the draining lymph node T-cell landscape.

Protective effect of T-FUS+αCD40 is T cell-dependent

Given the robust efficacy of T-FUS+αCD40 and the systemic immune activation observed with αCD40 priming, we hypothesized that therapeutic benefit from the combinatorial regimen requires T cells. To test this, mice receiving T-FUS+αCD40 were depleted of CD4⁺ T cells, CD8⁺ T cells, or both during the post-treatment effector window (D41-59; figure 6A), with efficient depletion confirmed in peripheral blood by flow cytometry (figure 6B). Strikingly, loss of either CD4⁺ or CD8⁺ T cells abrogated tumor control. Compared with isotype-treated controls, mean tumor volumes were ~2.8–3-fold higher in CD4-depleted, CD8-depleted, and dual-depleted groups (figure 6C). Concordantly, T-cell depletion significantly reduced overall survival (figure 6D), eliminating the durable protection observed in isotype-treated controls—wherein a subset of mice saw complete response. Together, these findings demonstrate that the protective effect of T-FUS+αCD40 is T cell-dependent, with both CD4⁺ and CD8⁺ compartments contributing to therapeutic efficacy.

Complete responders reject contralateral re-challenge and exhibit systemic, functional tumor-specific T-cell memory

CRs in the T-FUS+αCD40 group remained tumor-free for >150 days following treatment, and on contralateral re-challenge with 1e6 E0771 cells (2× the primary inoculum; figure 7A), uniformly rejected tumor outgrowth compared with naïve, age-matched controls. This was reflected in complete protection from tumor establishment and 100% survival in the CR cohort, whereas controls developed tumor burden and succumbed to disease irrespective of age (figure 7B–D). To define immune correlates of this durable protection, we serially profiled peripheral blood by multispectral flow cytometry following re-challenge. CRs displayed a significant increase in the percentage of antigen-experienced/effector memory CD4⁺ T cells (CD44⁺, CD62L⁻) compared with treatment-naïve controls (figure 7E,F). Although there was no observable change in the number or percentage of peripheral CD8⁺ and CD4⁺ T cells following rechallenge (figure 7G–J), the absolute number of CD40L+ (figure 7K) and Ki-67+ (figure 7O) CD8+ T cells were significantly increased, with a strong trend in both CD40L+ and Ki-67+ CD4+ T cells (figure 7L,P). Moreover, the percentage of both CD40 and Ki-67 (figure 7M-N, Q-R) expression were significantly increased on both circulating T-cell subsets. These observations are consistent with an activated, proliferative recall state and enhanced CD40–CD40L costimulatory signaling capacity that could reinforce APC engagement during protective immunity in CR mice. Furthermore, the CD4 compartment in CRs exhibited an enhanced effector cytokine signature, with increased representation of interferon (IFN)-γ⁺ and IFN-γ⁺ TNF-α⁺ antigen-experienced CD4⁺ T cells (figure 7S–W).

Indeed, CRs rejected tumor rechallenge delivered at two times the original inoculum and exhibited distinct circulating T-cell signatures consistent with functional systemic antitumor immune memory. To determine tumor specificity of the T-cell response, CRs underwent a second rechallenge, and splenocytes were collected 7 days post-rechallenge for IFN-γ enzyme-linked immunospot assay (ELISpot) (figure 8A). Representative images illustrate that T cells stimulated with the appropriate tumor-associated antigens (ie, E0771, BRPKP110, or EMT6 cells) produced greater number of IFN-γ release spots compared with those stimulated with irrelevant antigen (B16F10) (figure 8B). On an individual basis, six of seven CRs showed a significant increase in IFN-γ spot counts compared with B16F10-stimulated controls, with one CR showing a strong trend toward significance (figure 8C). Furthermore, quantification of IFN-γ-secreting cells revealed a significant increase in tumor-specific responses compared with B16F10-stimulated controls (figure 8D). Collectively, these findings support that durable cures achieved with T-FUS+αCD40 are associated with a systemic recall-type T-cell response marked by expanded antigen-experienced T-cell memory, enhanced proliferation, and heightened tumor-specific effector function following re-challenge.

Discussion

In this study, we establish subtotal T-FUS as a potent partner for CD40 agonism in BC. To our knowledge, this is the first report to extend a T-FUS immuno-oncology paradigm across BC models that reflect the heterogeneity of molecular subtypes represented in human disease.^46
47 Across multiple genetically, hormonally, and immunologically distinct models,^45
48
49 T-FUS+αCD40 consistently outperformed either monotherapy, delivering the most robust constraint of tumor outgrowth and yielding complete responses in multiple settings. Immunotherapy responses in BC have largely been concentrated in triple-negative settings,²⁴ whereas hormone receptor-positive luminal disease is comparatively immunologically “silent”.^50
51 Thus, a critical contribution of our study stems from the inclusion of E0771, a luminal B-like HR⁺/ERα⁻/ERβ⁺,^45
48 and BRPKp110, a luminal A-like HR⁺/ERα⁺⁵² syngeneic model, which effectively expands exploration into a disease context that dominates human BC⁵⁰ but is to date understudied in FUS immuno-oncology. To our knowledge, BRPKp110 has not previously been evaluated in the setting of FUS. The breadth of efficacy that we observe with the combination of T-FUS and αCD40—extending into luminal-like contexts—underscores the potential of subtotal T-FUS to dramatically expand the immunotherapy-responsive landscape of BC going forward. With these findings, we conclude that T-FUS+αCD40 is a cooperative strategy capable of translating local tumor injury into adaptive, systemic antitumor immunity and unveil insights for future tuning of this combination in a BC subtype-specific manner.

Our results support a working model in which αCD40 priming establishes a permissive antigen presentation landscape that can be productively leveraged by a single session of intervening subtotal ablation. Prior to T-FUS, αCD40 increased the abundance and activation state of APCs—particularly within the TDLN—consistent with enhanced priming capacity and APC licensing effects corroborated within other studies.^48
53–55 Moreover, a recent publication corroborates our findings in E0771 and BRPKp110, demonstrating that the administration of a CD40 agonist effectively induces the recruitment of cDC1s and cDC2 within the TDLN, concurrently enriching trafficking of intratumoral T cells.⁴⁸ Indeed, this underscores the rationale behind alliance with T-FUS to induce a durable “vaccine-like” effect. T-FUS then triggered hallmark spatially patterned thermal denaturation, canonical thermal stress signatures (ie, heat shock protein), and rapid alarmin (ie, ATP) release that collectively drove appreciable acute shifts in the tumor-immune landscape.^11
18
19
43 Interestingly, across two BC models with fundamentally distinct vascular, stromal and immunological compositions,^11
56 T-FUS elicited convergent early immune remodeling—marked by significant reductions in intratumoral APCs (except B cells) and monocytes, as well as a pronounced granulocytic influx consistent with acute thermal injury.^57–59 Notably, this influx neither accelerated tumor progression nor was it deleterious to combinatorial benefit. While absolute intratumoral T-cell numbers contracted, their relative proportion was preserved acutely following T-FUS, coincident with early enrichment of CD4⁺ and CD8⁺ T-cell signatures in systemic compartments (TDLN and blood)—suggesting that T-FUS-driven cytoreduction is not inherently counterproductive to T-cell responses. Nevertheless, T-FUS monotherapy did not yield durable tumor control, suggesting that additional immune activation is required to yield sustained antitumor benefit.

It was only when T-FUS was combined with αCD40 that we observed significant primary growth control of BC tumors (four of four models) and survival benefit (three of four models). We observed the most durable control—and the highest proportion of CRs—in E0771 and more modest effects in BRPKP110, EMT6, and 4T1; this variability likely reflects differences in baseline immune contexture, metastatic propensity, host strain, and/or delivered thermal dose. Future studies should explore thermal dose, ablation volume, sonication pattern and density, and therapeutic sequencing to further define determinants of response. Critically, these data underscore that clinical translation requires careful consideration of the immunologic and hormonal diversity of BC subtypes.

We focused subsequent mechanistic studies on E0771 because it reproducibly yielded distinct response phenotypes (CRs vs non-CRs), enabling higher-resolution dissection of immune correlates associated with durable tumor control. We determined that therapeutic benefit and CRs conferred by the combination required adaptive immunity. Depletion of CD4⁺ and/or CD8⁺ T cells not only abrogated tumor control and survival benefit but also abolished complete responses—supporting a central role for T-cell effector function in the combination paradigm. The similar abrogation of tumor control after CD4⁺, CD8⁺, or dual depletion suggests that these subsets are not operating as independently additive effector arms, but rather as non-redundant components of a coordinated adaptive immune response in which loss of either CD4⁺ T-cell help/costimulatory support or CD8⁺ T-cell cytotoxic function is sufficient to disrupt therapeutic efficacy. Whether this T-cell dependence is conserved across BC models and subtypes remains an important question for future studies.

CRs rejected tumor re-challenge delivered at two times the original inoculum and exhibited distinct circulating T-cell signatures that were consistent with functional systemic antitumor immune memory. Specifically, they exhibited a clear recall phenotype after re-challenge, defined by CD4⁺ T cells enriched for markers of antigen experience, proliferation, CD40L-mediated costimulatory potential, and effector function. Consistent with these systemic signatures, IFN-γ ELISpot analysis confirmed that T cells from CRs mounted preferential responses to breast tumor cells relative to an unrelated B16F10 control, supporting a tumor-directed memory T-cell response as a mechanism of rechallenge rejection. While the specific antigenic targets remain to be defined, these data establish that T-FUS and CD40 agonism can generate durable, tumor-specific T-cell immunity.

T-cell depletion outcomes and CR versus non-CR-associated peripheral T-cell signatures support a central requirement for CD8⁺ and CD4⁺ T cells in durable protection, consistent with a model in which a sufficient magnitude of effector T-cell activity is germane for long-term protection. Furthermore, our longitudinal immunoprofiling findings rationalize CD40-licensed cDC subsets in the TDLN as potentially critical supporters of the observed T-cell-dependent tumor control. Future studies will directly test cDC subset requirements (eg, cDC1 dependence) and interrogate implicated pathways such as type I IFN signaling and the CD70–CD27 axis.^60
61 Another plausible alternative hypothesis is that combinatorial efficacy resulted from reprogramming of MDSCs into pro-inflammatory monocytes.^62–64 While we observed no changes in the number or percentage of intratumoral arginine-1⁺ monocytic MDSCs or G-MDSCs (online supplemental figure 6A–D), their frequency at a single time point may not fully capture myeloid functional state.

Our study aligns with others that have cemented the capacity of T-FUS to elicit immune-cooperative efficacy in BC.^11
18
19 However, no studies to date have combined a thermally ablative mode of FUS with CD40 agonism, nor have studies to date investigated FUS in any form with CD40 agonism in BC. Subablative FUS heating has been paired with CD40 agonism as an “in situ vaccination” strategy in melanoma. Indeed, in a bilateral B16F10 model, multiple sessions of mild FUS hyperthermia combined with repeat intratumoral αCD40 conferred moderate abscopal tumor control, elevated intratumoral T-cell responses, and macrophage repolarization.⁶⁵ A subsequent study using mechanical FUS ablation (boiling histotripsy; BH) in murine melanomas similarly supported that a single session of BH-induced tumor disruption can cooperate with intratumoral αCD40 to amplify inflammatory and effector programs yielding tumor control as well as sensitize tumors to dual immune checkpoint blockade⁶⁶ (ICB). Our study complements and expands this literature by showing that thermal ablation cooperates with CD40 agonism in the context of (1) a distinct solid tumor type and (2) a less invasive αCD40 administration route (systemic). Our study is also distinct in its implementation of an immunotherapy priming regimen as opposed to concomitant treatment. In this regard, our findings are consistent with other studies supporting the benefit of neoadjuvant staging of immunotherapy prior to T-FUS^18
19 or other interventions.^67
68

The degree to which FUS-mediated injury productively interfaces with CD40 agonism appears context-dependent and regimen-dependent. Indeed, recent work in a pancreatic cancer model demonstrated that a single BH treatment liberated tumor antigen locally but did not promote its appearance in the draining node or activate conventional DCs, nor did it enhance the activity of systemic αCD40⁶⁹—underscoring that factors such as ablation modality, drug dose, ablation coverage, and therapeutic sequencing may also dictate the coupling between innate immune priming and robust adaptive response driven by FUS and CD40 agonism. Where we observed intragroup heterogeneity within the same model and treatment arm, chronic immune profiling unveiled that TDLN T-cell landscapes diverged by response class. We postulate that this may reflect potential differences in residual antigen burden subsequent to T-FUS as well as the state of immune resolution versus ongoing disease. This heterogeneity is clinically relevant and motivates identification of actionable variables and early biomarkers that stratify non-responders such that they can be converted or rescued through rationally layered strategies such as ICB.^48
66

Several limitations should frame interpretation of these data. Absent a lack of combinatorial survival advantage in 4T1, we did not directly quantify spontaneous pulmonary metastatic burden in this model. Thus, whether this regimen can control disseminated disease remains unresolved and will require dedicated metastasis-focused studies across models. Immunophenotyping was performed at selected acute and chronic endpoints that capture informative but limited snapshots of a dynamic process. Longitudinal sampling across tumor, TDLN, blood, and metastatic sites will be necessary to further resolve response-associated compartmental immune shifts. Such studies should also help resolve why syngeneic tumors receiving the same nominal acoustic and drug regimen diverge in outcome, potentially reflecting baseline immune contexture, local tumor architecture, thermal dose deposition, antigen release, residual disease burden, and/or the biological “quality” of the T-FUS intervention.

Future studies should systematically tune acoustic exposure conditions, ablation coverage, sonication pattern/density, and thermal dose alongside αCD40 dose, route, and timing, including neoadjuvant versus adjuvant sequencing relative to T-FUS. In parallel, deeper mechanistic studies across models will be important to determine whether the cellular requirements observed in E0771 generalize across BC subtypes or reflect model-specific dependencies. In addition to defining CD4⁺ and CD8⁺ T-cell effector/help requirements, future work should interrogate cDC subsets, myeloid functional state, B cell-dependent antigen presentation, and T/B cell-interactive mechanisms that may be especially relevant to CD40 agonism. Finally, because PD-1/PD-L1 blockade remains clinically limited and subtype-restricted in BC, testing whether T-FUS+αCD40 can render poorly responsive tumors more permissive to checkpoint blockade represents a rational next step for amplifying or sustaining the antitumor immunity initiated by this combination.

Given the surge of clinical trials evaluating FUS in combination with immunotherapies (including those ongoing in BC: NCT04796220, NCT03237572), these findings bear near-term translational relevance for developing an incisionless, immunologically active strategy with robustness across BC subtypes. Multiple clinical FUS platforms provide feasible paths to implementation,¹² and the clinical landscape for CD40 agonism is rapidly evolving, including the surge in Fc-engineered agents designed to improve therapeutic index.^31
70
71 A timely next step will be to test whether subtotal T-FUS similarly potentiates next-generation CD40 agonists that offer improved safety profiles. Our findings position the clinically ripe combination of subtotal T-FUS and CD40 agonism as a non-invasive strategy for eliciting durable immunological protection and provide an early mechanistic framework to extend its relevance within and beyond BC.

Methods

Cell line and animal maintenance

E0771 and EMT6 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco #11965-092) with 10% fetal bovine serum (FBS, Gibco #11875-093). 4T1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (+ L glut, Gibco #11875-093) supplemented with 10% fetal bovine serum (FBS). BPRKP110 mouse mammary cancer cell line was obtained from Dr Melanie Rutkowski, University of Virginia. BRPKP110 cells were maintained in RPMI with 10% FBS, 1X L-glutamine (Gibco #25030-081), 1 mM sodium pyruvate (100 mM, Gibco #11360-070), 50 µM beta-mercaptoethanol (Gibco, #21985023). Cells were maintained at 37°C and 5% $CO_{2}$ . Cells were thawed up to a maximum of three passages and maintained in logarithmic growth phase for inoculations. Prior to expansion and freezing, all cell lines were authenticated and confirmed to be Mycoplasma-free.

All animal work was performed under a protocol approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in research. 8–10 weeks old female C57Bl/6 or BALB/c mice were obtained from The Jackson Laboratory (Jax #000664 and #000651, respectively). Mice were inoculated subcutaneously with E0771 or 4T1 cells (4×10⁵) or in the fourth mammary fat pad with BRPKP110 (5×10⁵) or EMT6 cells (4×10⁵). The administration volume was 100 µL per mouse. Mice were housed on a 12 hours/12 hours light/dark cycle and food was available to them at all times. Tumor growth was monitored daily with precision digital caliper measurements, and with B-mode ultrasound imaging at selected time points using the VEVO 2100 MS400. Digital caliper measurements were used to calculate tumor volume as follows: $volume=\left (length\times width^{2}\right)/2$ . When tumors reach approximately $60mm^{3}$ , 28 days (E0771), 16 days (BRPKP110), 10 days (EMT6), and 11 days (4T1) post-inoculation, mice were randomized with in-house Python code into experimental groups on the basis of pretreatment tumor volume.

Contralateral tumor rechallenge

E0771 tumor-bearing mice that experienced complete rejection of primary tumors following T-FUS+ $\alpha$ CD40 treatment were rechallenged on the contralateral flank. CRs were inoculated with a 2.5× dose of (1×10⁶ E0771 tumor cells) approximately 150 days after tumor eradication. Age-matched naïve control mice were simultaneously challenged alongside the CRs, serving as controls. Mice were housed and tumor growth was monitored as described above.

Ultrasound tumor measurements and analysis

While under inhaled isoflurane anesthesia, mice were placed on the Vevo Imaging Station (VisualSonics) equipped with a motor for automatic image acquisition. Tumors were covered with degassed ultrasound gel prior to 3D ultrasound imaging, acquired using a 30 MHz MicroScan linear ultrasound imaging array (MS400; Sonics). Tumors were segmented in 3D Slicer^72
73 by manually painting approximately every 10th slice followed by automatic filling between slices. Segment statistics were then calculated for each segmented tumor and volumes were obtained.

Anti-CD40 therapy

For anti-CD40 therapy, rat anti-mouse agonistic CD40 antibody ( $\alpha$ CD40; 500 $\mu$ g/kg in 100 $\mu$ L; clone FGK4.5; Ichorbio) was diluted in sterilized 1× phosphate-buffered saline (PBS) and administered i.p. every 3 days for a total of five doses. $\alpha$ CD40 treatment was initiated once tumors reached approximately $60mm^{3}$ . Mice that did not receive $\alpha$ CD40 received an i.p. injection of equivalently dosed rat IgG2a isotype control antibody (Ichorbio) at analogous time points. For all therapeutic efficacy studies, the control group received IgG2a isotype control and underwent “sham” treatment as described below.

In vivo ultrasound-guided FUS partial thermal ablation

Mice were treated with T-FUS 7 days after the initial dose of αCD40, on day 35 (E0771), day 23 (BRPKP110), day 17 (EMT6), or day 18 (4T1 and 4T1-ZSGreen) post-implantation. On treatment day, mice were anesthetized with an i.p. injection of ketamine (50 mg/kg; Zoetis) and dexmedetomidine hydrochloride (0.25 mg/kg; Dechra). Tumors and surrounding regions were depilated prior to T-FUS treatment, following which mice were positioned and treated. T-FUS was performed with a custom-built ultrasound-guided FUS system comprised of four 3.78 MHz single-element transducers (SU-102, Sonic Concepts), each with a diameter of 33 mm and a radius of curvature of 55 mm. The system was powered by a 200W amplifier (Electronics and Innovation 1020L) driven by an arbitrary function generator (Tektronix AFG3022C), and axially co-registered to a 15 MHz MicroScan linear ultrasound imaging array (MS200; VisualSonics). Degassed deionized water, maintained at 37°C, was used as the acoustic coupling medium. A motorized 3D motion stage was used to adjust the position of the mouse relative to fixed transducer positions for each sonication. B-mode ultrasound imaging (VEVO 2100 MS200, VisualSonics) was employed for tumor identification and sonication planning. Sonication points were arranged within the ultrasound-visible tumor margins across a single plane of the tumor, with each point separated by 1.5 mm. T-FUS was applied continuously for 15 s at 18 W acoustic power over 2–4 planes of treatment, each of which was separated by 1 mm. We deployed a grading system that assigned blinded scores of 1–3 to the visible degree of hyperechoic signal across treatment planes in the four preclinical models of BC that underwent T-FUS (E0771, BRPKP110, EMT6, 4T1). Scoring was based on the degree of hyperechoic signal across each treated plane. A score of 1 indicated minimal hyperechogenicity confined to a single plane and not present throughout the entire treated area of that plane. A score of 2 represented a moderate level of hyperechogenicity across 2–3 planes, while a score of 3 reflected a high degree of hyperechogenicity observed across all planes^3
4 and in the majority or entirety of the treated areas within those planes. Mice that did not receive T-FUS treatment underwent “sham” treatment, consisting of anesthesia, depilation, and analogous partial exposure to the 37°C degassed water bath for 3 min. At the conclusion of “sham” or T-FUS treatment, mice were transferred to a heating pad, given Antisedan for anesthesia reversal, and recovered.

T-cell depletion

E0771-bearing C57Bl/6 mice underwent depletion of CD4⁺ T cells, CD8⁺ T cells, or both 1 day following the conclusion of T-FUS+ $\alpha$ CD40 dosing paradigm (day 41). T-cell depletion was carried out with anti-CD8 ( $\alpha$ CD8; 2.43 clone; Bio X Cell) and anti-CD4 ( $\alpha$ CD4; GK1.5 clone; Bio X Cell). Depleting antibodies were administered intraperitoneally every 3 days for a total of seven doses (100 µg/mouse). To confirm depletions, tail vein bleeds were performed on day 46, and circulating CD4⁺ and CD8⁺ T cell levels were screened via flow cytometry.

Immunohistochemistry

On day 36 (E0771), IgG-control or T-FUS treated tumors underwent cardiac perfusion with a sequence of 1× PBS, 10% neutral buffered formalin, 1× PBS. 24 hours later, fixed tumors were paraffin embedded, sectioned, and stained for H&E, CL Casp 3 (Cell Signaling, Cat #9661), PARP1 (Abcam, ab32064), and HSP70 (Abcam, Clone: EPR16892, ab181606). Digital scans of stained slides were collected using ZEISS Axioscan 7. Demarcation of ablative and periablative zones was determined by evaluating margins of apoptosis signatures identified by H&E, CL Casp3, and PARP staining, as well as immunomodulatory changes indicated by HSP70 expression.

In vitro and in vivo ATP analysis

Luciferin-luciferase optical imaging was performed to quantify ATP production following T-FUS treatment. In vitro, 4T1 cells were washed with 1× PBS prior to being prepared in 60 µL serum-free, phenol-red-free RPMI and suspended in PCR tubes at 3×10⁶ cells per tube. Immediately following ablation of 4T1 cells in vitro, supernatants were collected for ATP quantification using the CellTiter-Glo Luminescent Cell Viability Assay (Promega G7571). Luminescence was measured with a TECAN Spark Multimode Microplate Reader.

In vivo ATP production was evaluated in E0771 tumors following T-FUS treatment. Mice were injected intravenously with 3 mg D-luciferin (Invitrogen, L2916) 1 min prior to T-FUS treatment. Firefly luciferase (Biotium #30020-1) was administered immediately after the conclusion of T-FUS treatment. Bioluminescence imaging (Lago X) was performed over 10–40-minute post-treatment. Tumor-localized luminescence was quantified using Aura Imaging Software (Spectral Instruments Imaging).

Flow cytometry

Tumor-bearing mice were euthanized at various time points for tissue and/or blood collection and subsequent immunoprofiling by multispectral flow cytometry. In E0771 tumor harvests, to distinguish between tissue-resident and vascular immune cell populations, mice were injected intravenously with rat anti-mouse CD45 PerCP (clone 30-F11; BioLegend) 3 min before euthanasia. Tumors, spleens, inguinal TDLNs, and non-draining inguinal cLN were mechanically homogenized with glass homogenizers and filtered through 100 $\mu$ m nylon filter mesh to obtain single-cell suspensions. Tumors went through an additional enzymatic digestion step prior to filtration with collagenase (Gibco #17018-029) and DNase 1 (Roche #10104159001). Following filtration, the lymphocyte layer was collected subsequent to the isolation procedure using Lympholyte-M Cell Separation Media (Cedarlane #CL5035). Spleen and blood samples were also subjected to hemolysis using BioLegend 1× red blood cell (RBC) lysis buffer (BioLegend #420301). Following RBC lysis, samples were quenched with complete media (RPMI or DMEM+10% FBS).

Following, cells were centrifuged at 1,500 revolutions per minute (RPM) for 5 min, washed with 1× PBS, and centrifuged again at the same parameters. Cells were stained with either Fixable Live/Dead Aqua or Nearinfrared (IR) for 30 min at 4°C. Following, cells were fixed with anti-mouse CD16/32 (Thermo Fisher #14-0161-86) to block Fc gamma receptors for 15 min at 4°C, then centrifuged and washed two times with 1× Flow Cytometry Staining Buffer (FACS; R&D Systems #FC001). Surface staining was performed in Brilliant Stain Buffer (BD Biosciences #566349) for 30 min at 4°C with fluorescent monoclonal antibodies for CD11b, MHC-II, CD317, F4/80, Ly6C, Ly6G, XCR1, CD19, CD40, CD11c, CD86, SIRP $\alpha$ , CD45, CD45.2, CD3, DX5, CD8, CD4, TIM3, LAG3, PD1, PD-L1, CD103, CD69, CD44, CD62L, CD40L, and CD25. Intracellular staining was carried out with Foxp3/Transcription Factor Staining Buffer Set (Tonbo Biosciences #TNB-0607-KIT). Samples were additionally stained for iNOS, Ki-67, Arg1, CD206, TNF-α, granzyme B, TOX, IFN-γ, FOXP3.

Online supplemental table S2 presents comprehensive details on all fluorescently labeled antibodies, including their clones and vendor. Samples were fixed with 1× FACS Lysis Solution (BD Biosciences #34902) for 10 min at 4°C, then centrifuged and resuspended with fluorescence-activated cell sorting (FACS) buffer. Flow cytometry was carried out using the Cytek Aurora Borealis five laser and SpectroFlo V.3.0.3 software. Gating and data analysis were performed using FlowJo V.10 or OMIQ software. Representative gating strategies are depicted in online supplemental figures 7 and 8.

Ex vivo T-cell stimulation

On day 7 post-E0771 tumor rechallenge, blood samples were prepared as described above. Cells were plated in two 96-well round-bottom plates: one stimulated and the other non-stimulated. Stimulated plates received the Cell Stimulation Cocktail (eBioscience #00497503) comprised of phorbol 12-myristate 13-acetate, ionomycin, brefeldin A (BFA) and monensin, while non-stimulated cells were plated in media. Plates were maintained at 37°C and 5% $CO_{2}$ for 2.5 hours. Cells were then stained for surface and intracellular markers (Ki-67, granzyme B, IFN-γ, and TNF-α).

Brefeldin A injection

To inhibit the secretion of cytokines from cells, E0771 tumor-bearing mice were treated with BFA (250 µg; Selleck Chemicals #S7046) diluted in sterilized 0.9% saline. BFA was administered i.p. to mice 4 hours prior to harvest.

Dimensionality reduction analysis of flow cytometry data

OMIQ software (Dotmatics; www.omiq.qi) was used to perform dimensionality reduction using t-SNE of the flow cytometry data. Briefly, gating strategies from FlowJo were imported into OMIQ to maintain consistent gating on CD45.2⁺ or CD45⁺ live cells by removing cell debris, doublets, and dead cells. All fluorescent parameters—excluding viability dye Blue or Aqua, CD45, and/or CD45.2—were included as dimensions. Subsampling was performed with 20,000 events. Single live CD45 cells for each file were concatenated for analysis by t-SNE dimensionality reduction followed by multicolor mapping of each group in the concatenated file to identify proportions of each population.

IFN-γ enzyme-linked immunospot assay

Approximately 150+ days after the first rechallenge, CR mice underwent a second rechallenge. Seven days later, spleens were harvested, and CD3+ T cells were isolated from CR spleens by negative selection using the MojoSort Mouse CD3 T Cell Isolation Kit (BioLegend). APCs were prepared from spleens of naïve C57BL/6 mice by depleting T cells using the MojoSort Mouse CD3 T Cell Selection Kit (BioLegend). One day before the assay, 96-well ELISpot plates were coated with anti-mouse IFN-γ capture mAb (BD ELISpot Mouse IFN-γ Set, BD Biosciences, San Diego, California, USA). For each well, 2 × 10⁵ CD3+ T cells from CR mice were co-cultured with 3 × 10⁴ live tumor cells (E0771, BRPKP110, or EMT6) and 1 × 10⁵ APCs. B16F10 cells were included as an irrelevant antigen negative control, whereas unstimulated T cells co-cultured with APCs served as the background control. Following 24-hour incubation at 37°C with 5% CO₂, cells were removed by washing. Biotinylated anti-mouse IFN-γ detection antibody was added, followed by streptavidin-horseradish peroxidase and 3-amino-9-ethylcarbazole substrate solution to develop the spots. Spots were quantified using an ImmunoSpot analyzer (Cellular Technology Limited, Shaker Heights, Ohio, USA). Antigen-specific IFN-γ responses were reported either as the mean spot counts from triplicate wells for each CR mouse or as background-subtracted spot counts normalized to the number of T cells.

Statistical analysis

Statistical analyses were performed using GraphPad Prism V.10.1.0. A comprehensive overview of the statistical techniques used in each experiment can be found in the accompanying figure legend. “n.s.” denotes that data are not significant. All data are represented as mean±SEM. Normality was assumed where no significant deviation was detected by Q–Q plots and the Shapiro-Wilk test. Equal variance was not assumed where homoscedasticity plot and Bartlett’s test indicated significant deviation. Therefore, Welch’s analysis of variance or Welch’s t-test was employed. For multiple comparisons, pairwise analyses were performed against the T-FUS+αCD40 group, and Dunnett’s test was used to adjust for multiple comparisons.

Supplementary material

online supplemental file 1

jitc-14-8-s001.pdf^{(4.3MB, pdf)}

DOI: 10.1136/jitc-2026-015671

Acknowledgements

We thank the Flow Cytometry Core Facility of the University of Virginia for their support and use of their cytometers, supported by the National Cancer Institute P30-CA044579 Center Grant. This work was supported by Biorepository and Tissue Research Facility which is supported by the University of Virginia School of Medicine, Research Resource Identifiers (RRID): SCR_022971. This work used FFPE embedded and sectioning in the Research Histology Core Facility which is supported by the University of Virginia School of Medicine, Research Resource Identifiers (RRID): SCR_025470. We thank Dr Drew Dudley (UVA) for providing the E0771 cell line. We thank Dr Frederic Padilla for his expertise and development of the ultrasound-guided FUS system used here. We thank Drs Patrick Dillon and David Brenin (UVA) for their invaluable clinical insights throughout the development of this work. This research was supported by state funding within the University of Virginia Comprehensive Cancer Center.

Study sponsors did not have any involvement in the collection, analysis and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication. Funders did not influence the results/outcomes of the study despite any author affiliations with the funder.

Footnotes

Funding: Supported by NIH DP5OD031846; US DOD Breast Cancer Research Program Era of Hope Scholar Award (HT9425-25-1-0411); the Focused Ultrasound Foundation; the University of Virginia’s Focused Ultrasound Immuno-Oncology (FUSION) Center; and the University of Virginia’s Wallace H. Coulter Foundation for Translational Research grants to NDS. ZED was supported by the UVA Cancer Center Training Fellowship. ZED and TS were supported by UVA Cancer Biology Training Grant (NIH T32CA009109). ZED and ATT were supported by UVA SEAS Endowed Graduate Fellowships: Victor Orphan Graduate Fellowship and John Bell McGaughy Graduate Fellowship, respectively. ATT was supported by the UVA Jefferson Scholars Foundation Fellowship.

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient consent for publication: Not applicable.

Ethics approval: The murine experiments were prospectively reviewed and approved by the University of Virginia Animal Care and Use Committee (#4393).

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

online supplemental file 1

jitc-14-8-s001.pdf^{(4.3MB, pdf)}

DOI: 10.1136/jitc-2026-015671

Data Availability Statement

All data relevant to the study are included in the article or uploaded as supplementary information.

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PERMALINK

Focused ultrasound thermal ablation and CD40 agonism reprograms breast tumor immunity to drive regression and memory

Zehra EF Demir

Aeryon Kim

Beyzanur G Ak

Micaiah S Lee

Thomas Sherlock

Stefanyda O Maslova

Andrew T Thede

Matthew R DeWitt

Melanie Rutkowski

Natasha D Sheybani

Abstract

Background

Methods

Results

Conclusions

WHAT IS ALREADY KNOWN ON THIS TOPIC

WHAT THIS STUDY ADDS

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

Introduction

Results

Subtotal FUS thermal ablation results in clear zones of apoptotic and necrotic tissue destruction, concomitant with local upregulation of canonical thermal stress signatures

Subtotal FUS thermal ablation promotes a systemic T-cell response

Subtotal FUS thermal ablation in combination with a CD40 agonist constraints tumor outgrowth and promotes survival: conferring complete responses in multiple breast cancer models

αCD40 priming orchestrates potent local and systemic immune activation, enriching the abundance and maturity of APCs and T cells

Complete and non-complete responders to TFUS+αCD40 are distinguished by their T-cell signatures

Protective effect of T-FUS+αCD40 is T cell-dependent

Complete responders reject contralateral re-challenge and exhibit systemic, functional tumor-specific T-cell memory

Discussion

Methods

Cell line and animal maintenance

Contralateral tumor rechallenge

Ultrasound tumor measurements and analysis

Anti-CD40 therapy

In vivo ultrasound-guided FUS partial thermal ablation

T-cell depletion

Immunohistochemistry

In vitro and in vivo ATP analysis

Flow cytometry

Ex vivo T-cell stimulation

Brefeldin A injection

Dimensionality reduction analysis of flow cytometry data

IFN-γ enzyme-linked immunospot assay

Statistical analysis

Supplementary material

Acknowledgements

Footnotes

Data availability statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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