Focused ultrasound ablation of melanoma with boiling histotripsy yields abscopal tumor control and antigen-dependent dendritic cell activation

Abstract

Background: Boiling histotripsy (BH), a mechanical focused ultrasound ablation strategy, can elicit intriguing signatures of anti-tumor immunity. However, the influence of BH on dendritic cell function is unknown, compromising our ability to optimally combine BH with immunotherapies to control metastatic disease. Methods: BH was applied using a sparse scan (1 mm spacing between sonications) protocol to B16F10-ZsGreen melanoma in bilateral and unilateral settings. Ipsilateral and contralateral tumor growth was measured. Flow cytometry was used to track ZsGreen antigen and assess how BH drives dendritic cell behavior. Results: BH monotherapy elicited ipsilateral and abscopal tumor control in this highly aggressive model. Tumor antigen presence in immune cells in the tumor-draining lymph nodes (TDLNs) was ~3-fold greater at 24h after BH, but this abated by 96h. B cells, macrophages, monocytes, granulocytes, and both conventional dendritic cell subsets (i.e. cDC1s and cDC2s) acquired markedly more antigen with BH. BH drove activation of both cDC subsets, with activation being dependent upon tumor antigen acquisition. Our data also suggest that BH-liberated tumor antigen is complexed with damage-associated molecular patterns (DAMPs) and that cDCs do not traffic to the TDLN with antigen. Rather, they acquire antigen as it flows through afferent lymph vessels into the TDLN. Conclusion: When applied with a sparse scan protocol, BH monotherapy elicits abscopal melanoma control and shapes dendritic cell function through several previously unappreciated mechanisms. These results offer new insight into how to best combine BH with immunotherapies for the treatment of metastatic melanoma.

Keywords: boiling histotripsy; dendritic cells; focused ultrasound; immunotherapy; tumor antigen.

Figure 1

BH yields primary and abscopal control of B16F10-ZsG melanoma. 4x10⁵ B16F10-ZsG cells were inoculated in the left and right flanks of C57/Bl6 mice and tumors were exposed to BH or sham treatment 14 days post inoculation. A. Timeline for inoculation and treatment. B. Kaplan-Meier curve depicting overall survival (significance assessed by log-rank (Mantel-Cox) test: ∗ P<0.05). C-H. Ipsilateral and contralateral tumor growth. C & F. Individual tumor growth curves. D & G. Average logistic modeled tumor growth. n=8-11 per group. Full model, two-way repeated measures ANOVA from day 14 to 35, fixed effects: ∗∗ P<0.01. E & H. Area under the logistic average curve (AUC). Unpaired Welch's t-test: ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Means ± SEM.

Figure 2

BH transiently increases tumor antigen acquisition by immune cells in the tumor draining lymph nodes. 4x10⁵ B16F10-ZsG cells were inoculated in the right flanks of C57/Bl6 mice and tumors were exposed to BH or sham treatment 13 days post inoculation. A. Timeline for inoculation, treatment, and harvest for flow cytometry. Inguinal, axial, and brachial lymph nodes were harvested and pooled 24 h and 96 h post treatment. B. Scatter density plots indicating percent of LiveCD45⁺ cells that are ZsG⁺. C. Bar graph of flow cytometry analysis data. n=4-5 per group. Full model, two-way ANOVA followed by Tukey multiple comparison test: ∗∗ p < 0.01, ∗∗∗ p < 0.001. Means ± SEM.

Figure 3

BH enhances tumor antigen acquisition by antigen presenting and phagocytic cells. A. Diagram outlining immune cells interrogated for ZsG positivity. B-F. Bar graphs of ZsG⁺ cell counts. G-K. Bar graphs of proportions of ZsG⁺ cells. L-P. Bar graphs of geometric mean fluorescent intensity (GMF) of ZsG on ZsG⁺ cells. B, G, L. Total DCs (CD11c⁺MHCII⁺). C, H, M. B cells (CD19⁺CD3^-). D, I, N. Macrophages (CD11b⁺F4/80⁺). E, J, O. Monocytes (CD11b⁺F4/80^-Ly6C⁺Ly6G^-). F, K, P. Granulocytes (CD11b⁺F4/80^-Ly6C^midLy6G⁺). n = 10 per group. Unpaired Welch's t-test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Means ± SEM.

Figure 4

BH increases ZsG tumor antigen presence in cDCs in the tumor draining lymph nodes. A. Overview of CD8⁺ and CD4⁺ T cell activation by cDC1s and cDC2s. B. Density scatter plots of side scatter vs ZsG for cDC1s (top) and cDC2s (bottom). C & F. Bar graphs of numbers of cDCs that are ZsG⁺. D & G. Bar graphs of percentages of cDCs that are ZsG⁺. E & H. Bar graphs of ZsG GMF on cDCs that are ZsG⁺. n=10 per group. Unpaired Welch's t-test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Means ± SEM.

Figure 5

BH activates cDCs in tumor draining lymph nodes. A. Diagram illustrating the question of whether BH activates cDC1s and cDC2s. B. Frequency plots of side scatter vs CD86 for cDC1s (top) and cDC2s (bottom). C & F. Bar graphs of CD86 GMF of CD86⁺ cDC subsets. D & G. Bar graphs of number of activated cDCs (CD86^hiMHCII^hi). E & H. Bar graphs of percent of cDCs that are activated (CD86^hiMHCII^hi). n=10 per group. Unpaired Welch's t-test: ∗ p < 0.05, ∗∗ p < 0.01. Means ± SEM.

Figure 6

BH-induced cDC depends on ZsG acquisition. A. Diagram illustrating the overall question of whether ZsG acquisition is required for cDC1 and/or cDC2 activation. Data in this figure interrogate ZsG^- cDCs, so the ZsG⁺ portion is shaded. B. Frequency plots of side scatter vs CD86 for ZsG^- cDC1s (top) and ZsG^- cDC2s (bottom). C & F. Bar graphs of geometric mean fluorescence (GMF) intensity of CD86 on CD86⁺ZsG^- cDC subsets D & G. Bar graphs of number of specified ZsG^- cDC subset that are activated (CD86^hiMHCII^hi). E & H. Bar graphs of percent of specified cDC subset that are activated (CD86^hiMHCII^hi). n=10 per group. Unpaired Welch's t-test: not significant. Means ± SEM.

Figure 7

ZsG antigen promotes cDC maturation. A. Diagram illustrating the overall question of whether ZsG acquisition is required for cDC1 and/or cDC2 activation. Data in this figure interrogate ZsG⁺ cDCs, so the ZsG^- portion is shaded. B & C. Bar graphs of percent of ZsG⁺ cDC1s (B) and cDC2s (C) that are activated (CD86^hiMHCII^hi). n=10 per group. Unpaired Welch's t-test: ∗ p < 0.05. D-E & H-I. Geometric mean fluorescent (GMF) intensity of CD86 on ZsG^- and ZsG⁺ cDC1s (D-E) and cDC2s (H-I). n = 10. Paired t-test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. F & J. CD86 expression as a function of ZsG expression in ZsG⁺ cDC1s (F) and ZsG⁺ cDC2s (J) after sham treatment. G & K. CD86 expression as a function of ZsG expression in ZsG⁺ cDC1s (G) and ZsG⁺ cDC2s (K) after BH. Correlations are by linear regression with displayed R² values.

Figure 8

BH changes neither total DCs nor the proportions of ZsG⁺ cDC1s that are LN-resident (CD8α⁺) and migratory (CD103⁺). A. Diagram illustrating the question of whether cDC1s acquire BH-liberated ZsG in tumor and/or TDLN. B. Density scatter plots of CD11c vs MHCII with DCs (CD11c⁺MHCII⁺) in sham and BH treated TDLNs. C. Number of total DCs. D. Percentage of LiveCD45⁺ cells that are DCs. E. Density scatter plots of CD8α vs. CD103 to identify tissue-resident (CD8α⁺CD103^-) and migratory (CD103⁺) cDC1s in TDLNs of sham and BH treated mice. F & G. Number of tissue-resident (F) and migratory (G) cDC1s. H & I. Percentage of cDC1s that are tissue-resident (H) and migratory (I). J & K. Percentage of ZsG⁺ cDC1s that are tissue-resident (J) and migratory (K). L & M. Proportion of tissue-resident (L) and migratory (M) cDC1s that are ZsG⁺. n=10. Unpaired Welch's t-test: ∗ p < 0.05, ∗∗ p < 0.01. Means ± SEM.