Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity

Abstract

Background: Triple-negative breast cancer (TNBC) remains recalcitrant to most targeted therapy approaches. However, recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy. We therefore tested a strategy for immune sensitization of murine TNBC (4T1 tumors) through combination of focused ultrasound (FUS) thermal ablation and a chemotherapy, gemcitabine (GEM), known to attenuate myeloid-derived suppressor cells (MDSCs).

Methods: We applied a sparse-scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM. We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity. We also tested this combination in Rag1-/- mice or T cell-depleted wild-type mice to determine the essentiality of adaptive immunity. Further, we layered Programmed cell death protein 1 (PD-1) blockade onto this combination to evaluate its impact on tumor outgrowth and survival.

Results: The immune-modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1, consistent with the dominant MDSC-driven immunosuppression evident in this model. The combination of FUS+GEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice. Tumor control correlated with increased circulating antigen-experienced T cells and was entirely dependent on T cell-mediated immunity. The ability of FUS+GEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti-PD-1.

Conclusion: Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth, improves survival and enhances immunogenicity in a murine metastatic TNBC model. This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluation.

Trial registration numbers: NCT03237572 and NCT04116320.

Keywords: adaptive immunity; breast neoplasms; combined modality therapy; immunotherapy.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Partial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsets. (A) Design overview of a custom ultrasound-guided FUS system consisting of a 3.3 MHz single-element transducer orthogonally co-registered to an 8 MHz linear ultrasound imaging array. The tumor-bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at 37°C. ‘Sham’ mice were similarly positioned but did not undergo sonications. (B) Schematic illustration of FUS partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4T1 tumor-bearing mice. A grid of sonications was applied in a raster pattern onto the B-mode ultrasound-visible tumor. In total, two planes of sonication spaced 2 mm apart were applied to each tumor. Grid points were spaced 1 mm apart within a single plane. One week following thermal ablation, tumors and secondary lymphoid organs were excised for sham (n=6) or FUS-treated (n=5) mice and processed for flow cytometry. (C) Representative B-mode ultrasound images of ectopic 4T1 tumors either before (top) or during (bottom) FUS exposure. Sonication grid depicting targets (red points) is superimposed on B-mode image during treatment. Subsequent to thermal ablation, hyperechoic signatures (yellow arrow) are occasionally observed. (D) Representative H&E staining of either sham 4T1 tumors or those resected immediately following FUS partial thermal ablation. Zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone (scale bars=400 µm and 300 µm on left and right inset, respectively). (E) Absolute number of CD11c-hi DCs in the axillary tumor-draining lymph node (aDLN) of 4T1 tumor-bearing mice. *p=0.0136 vs sham. (F) Absolute number of CD86+ CD11c-hi DCs in the aDLN. **p=0.0063 vs sham. (G) Percentage of CD86+ subset out of total CD11c-hi DCs within aDLN. (H) Absolute number of intratumoral CD44+ CD8+ and CD44+ CD4+ T cells and regulatory T cells (Tregs) per gram tumor. (I) Percentage of CD11b+ myeloid cells out of total CD45+ immune cells across tumor, spleen, aDLN, inguinal DLN (iDLN), and nontumor draining axillary and inguinal LNs (nDLNs). *p<0.05 vs all other groups (irrespective of FUS exposure; specifically, tumor vs spleen: p=0.0226; tumor, spleen vs all other organs: p<0.0001). (J) Absolute number of intratumoral myeloid cells (CD11c-hi DCs, F4/80+ macrophages, Ly6C+ monocytic myeloid-derived suppressor cells (M-MDSCs), Ly6G+ granulocytic myeloid-derived suppressor cells (G-MDSCs)) per gram 4T1 tumor. ***p=0.0001 vs all other cell types (irrespective of FUS exposure). All data represented as mean±SEM. Significance assessed by unpaired t-test (F–H) or two-way analysis of variance followed by Tukey multiple comparison correction (I–K). ‘n.s.’=not significant. DCs, dendritic cells; FUS, focused ultrasound; HIFU, high-intensityfocused ultrasound.

Figure 2

Combination of focused ultrasound (FUS) partial thermal ablation with gemcitabine (GEM) constrains primary triple-negative breast cancer outgrowth and extends overall survival. (A) Overview of experimental design for evaluation combination of FUS with serial GEM treatment in murine mammary carcinoma. (B) Average 4T1 tumor outgrowth in sham (n=7), FUS monotherapy (n=5), GEM monotherapy (n=10), and combinatorial FUS+GEM therapy groups (n=10). Data are represented up to select time points corresponding with mouse dropout due to humane endpoints. All data represented as mean±SEM. Significance assessed on outgrowth up to day 40 by repeated measures mixed-effects model implementing restricted maximum likelihood method, followed by Tukey multiple comparison correction. *p

Figure 3

Combination of focused ultrasound (FUS) partial thermal ablation with gemcitabine (GEM) increases the levels of circulating T cells. (A) Overview of experimental design to understand the impact of FUS and/or GEM treatment on circulating immune cells. (B–C) Absolute number of circulating CD8+ T cells at day 21 (B) and day 28 (C). (D) Percentage of circulating CD8+ T cells expressing CD44 from days 21 to 28. (E–F) Absolute number of circulating CD4+ T cells at day 21 (E) and day 28 (F). (G) Percentage of circulating CD4+ T cells expressing CD44 from days 21 to 28. (H) Percentage of CD11b+ myeloid cells out of total CD45+ immune cell in circulation from days 21 to 31. (I–K) Percentage of myeloid cells (I), CD8+ T cells (J) and CD4+ T cells (K) out of total CD45.2+ immune cells. All data represented as mean±SEM. All data representative of sham (n=6–8), FUS monotherapy (n=4–6), GEM monotherapy (n=9), and combinatorial FUS+GEM therapy (n=6–7) groups. Significance assessed by analysis of variance followed by Tukey multiple comparison correction (for B, C, E, F) or Fisher’s least significant difference (LSD) without multiple comparisons correction (for I–K). Significance (for D, G and H) assessed by repeated measures mixed-effects model implementing restricted maximum likelihood method, followed by Fisher’s LSD without multiple comparisons correction. *p<0.05 vs all other groups unless otherwise indicated. **p<0.01, ***p<0.001 vs group(s) indicated.

Figure 4

Combinatorial FUS+GEM therapy does not promote robust local antitumor T cell responses. (A) Schematic of experimental design for analysis of immune milieu in tumors and secondary lymphoid organs following FUS and/or GEM treatment. (B, C) Absolute number per gram tumor of CD8+ (B) or CD4+ (C) T cells expressing CD44. (D, E) Percentage of intratumoral CD8+ CD44+ (D) or CD4+ CD44+ (E) T cells dually expressing granzyme B (GzB) and interferon-γ (IFNγ). (F) Percentage of intratumoral DCs expressing IL-12p40. (G) Percentage of intratumoral granulocytic myeloid-derived suppressor cells (G-MDSCs) expressing TNFα. Groups not significantly different in (B–E). All data represented as mean±SEM. All data representative of sham (n=6), FUS monotherapy (n=4), GEM monotherapy (n=9), and combinatorial FUS+GEM therapy (n=6) groups. Significance assessed by analysis of variance followed by Fisher’s leastsignificant difference without multiple comparisons correction for all panels. *p<0.05 vs indicated group(s). FUS, focusedultrasound; GEM, gemcitabine; IL, interleukin; TNFα, tumor necrosis factor-α.

Figure 5

Protection conferred by combination of focusedultrasound (FUS) with gemcitabine (GEM) is dependent on adaptive immunity. (A) Average 4T1 tumor outgrowth in wild-type (WT) or Rag1−/− mice receiving GEM monotherapy or combinatorial FUS+GEM therapy. Data are represented up to select time points corresponding with mouse dropout due to humane endpoints. n=5–7 mice per group. Significance assessed on outgrowth up to day 37 by repeated measures mixed-effects model implementing restricted maximum likelihood method, followed by Tukey multiple comparison correction. (B) Kaplan-Meier curve depicting overall survival of Rag1−/− 4T1 tumor-bearing mice receiving GEM monotherapy or combinatorial FUS+GEM therapy. n=7–9 per group. (C) Overview of experimental design for T cell depletions conducted on FUS+GEM background. αCD8 and αCD4 were administered on days 20, 23, 26, 29, 32, 35, and 39. On day 27, tail bleeds were performed to confirm CD8+ and CD4+ T cell depletion by flow cytometry. (D) Average 4T1 tumor outgrowth on FUS+GEM (WT) background with or without T cell depletion (‘αCD8/αCD4’). Data are represented up to select time points corresponding with mouse dropout due to humane endpoints. Significance assessed on outgrowth up to day 46 by repeated measures mixed-effects model implementing restricted maximum likelihood method. n=8–9 mice per group. (E) Kaplan-Meier curve depicting impact of T cell depletion on overall survival in FUS+GEM-recipient mice (WT) bearing 4T1 tumors. n=9–10 mice per group. All data represented as mean±SEM. Significance in (B, E) assessed by log-rank (Mantel-Cox) test. ‘n.s.’=not significant. *p<0.05 vs indicated group(s). **p=0.0022 vs FUS+GEM.

Figure 6

PD-1 blockade therapy moderately improves growth restriction conferred by FUS+GEM. (A) Representative histograms for intratumoral PD-1 expression on CD8+ CD44+ T cells across experimental groups. (B–C) Percentage of PD-1 expression (B) and PD-1 mean fluorescence intensity (C) on intratumoral CD8+ CD44+ T cells at 31 days postimplantation. (D–E) Percentage of PD-L1 expression on granulocytic myeloid-derived suppressor cells (G-MDSCs) (D) or CD45.2 (nonimmune) tumor/stromal cells (E) in 4T1 tumors at 31 days postimplantation with representative histograms. (F) Mice on a FUS+GEM background received αPD-1 every 3 days in either an ‘early’ (day 7–19) or ‘delayed’ (day 17–29) sequence. 4T1 tumor outgrowth in mice receiving FUS+GEM (n=8), early αPD-1 (n=5) or delayed αPD-1 (n=5). All data represented as mean±SEM. Data in (B–E) representative of sham (n=5–6), FUS monotherapy (n=3–4), GEM monotherapy (n=8–9), and combinatorial FUS+GEM therapy (n=4–6) groups. Significance in (B–E) assessed by analysis of variance followed by Fisher’s least significant difference without multiple comparisons correction for all panels. Significance in (F) assessed on outgrowth up to day 37 by repeated measures mixed-effects model implementing restricted maximum likelihood method, followed by Tukey multiple comparison correction. *p<0.05 vs indicated group. ***p=0.0003 vs FUS+GEM.