Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation

Abstract

Introduction: Histotripsy is a novel focused ultrasound tumor ablation modality with potent immunostimulatory effects.

Methods: To measure the spatiotemporal kinetics of local andabscopal responses to histotripsy, C57BL/6 mice bearing bilateral flank B16 melanoma or Hepa1-6 hepatocellular carcinoma tumors were treated with unilateral sham or partial histotripsy. Treated and contralateral untreated (abscopal) tumors were analyzed using multicolor immunofluorescence, digital spatial profiling, RNA sequencing (RNASeq), and flow cytometry.

Results: Unilateral histotripsy triggered abscopal tumor growth inhibition. Within the ablation zone, early high mobility group box protein 1 (HMGB1) release and necroptosis were accompanied by immunogenic cell death transcriptional responses in tumor cells and innate immune activation transcriptional responses in infiltrating myeloid and natural killer (NK) cells. Delayed CD8+ T cell intratumoral infiltration was spatiotemporally aligned with cancer cell features of ferroptosis; this effect was enhanced by CTLA-4 blockade and recapitulated in vitro when tumor-draining lymph node CD8+ T cells were co-cultured with tumor cells. Inoculation with cell-free tumor fractions generated by histotripsy but not radiation or freeze/thaw conferred partial protection from tumor challenge.

Discussion: We propose that histotripsy may evoke local necroptotic immunogenic cell death, priming systemic adaptive immune responses and abscopal ferroptotic cancer cell death.

Keywords: ablation; ferroptosis; histotripsy; immunity; necroptosis; tumor; ultrasound.

Figure 1

Unilateral histotripsy tumor ablation induces antigen-specific abscopal inhibition of distant, untreated tumors and local immunogenic cell death. (A) C57BL/6 mice were inoculated with bilateral flank B16F10 tumors (“HT abscopal concordant”) or unilateral B16F10 flank tumors and contralateral Hepa1-6 flank tumors (“HT abscopal discordant”), and sham (“control”) or histotripsy ablation encompassing ~80-90% of unilateral B16F10 flank tumors (in control and abscopal concordant groups) or unilateral Hepa1-6 flank tumors (in the abscopal discordant group) was performed on day 10. In contrast to sham ablation controls, mice treated with unilateral partial histotripsy ablation demonstrated immediate local growth arrest of treated B16F10 tumors (“HT treated”) and immediate abscopal growth inhibition of distant untreated B16F10 tumors (“HT abscopal concordant”) but not distant untreated B16F10 tumors after contralateral Hepa1-6 tumor ablation (“HT abscopal discordant”). (B) In mice bearing bilateral flank Hepa1-6 tumors or unilateral Hepa1-6 flank tumors and contralateral B16F10 flank tumors, unilateral partial histotripsy ablation of Hepa1-6 tumors (in control and HT abscopal concordant groups) or B16F10 tumors (in the HT abscopal discordant group) demonstrated immediate growth arrest and regression of treated Hepa1-6 tumors (“HT treated”) and distant untreated Hepa1-6 tumors (“HT abscopal concordant”), but not of distant untreated Hepa1-6 tumors after contralateral B16F10 tumor ablation (“HT abscopal discordant”). (C) Multicolor immunofluorescence analysis of bilateral B16F10 tumors performed 2 days after unilateral sham or partial histotripsy ablation demonstrated homogeneous intranuclear staining of HMGB1 in control tumors. In contrast, significant loss of intranuclear HMGB1 staining was observed within the ablation zone of histotripsy-treated tumors; intranuclear HMGB1 was retained outside of the ablation zone. (D) RNASeq of CD45- tumor cells performed on day 13 revealed marked differences in transcriptional activity between control tumors (blue) and histotripsy-treated (“HT-Tx”) tumors (red) as evidenced by principal component analysis. (E) qRT-PCR of tumors performed on day 13 revealed an approximately 20-fold increase in TNFα mRNA in histotripsy-treated tumors (red) compared with control tumors (blue). (F) Multicolor immunohistochemistry 1 day after sham or histotripsy ablation revealed no measurable expression of the necroptosis markers pRIPK3 and pMLKL in control tumors; in contrast, profound co-localized expression of pRIPK3 and pMLKL was seen along the periphery of ablated zones in histotripsy-treated tumors. (G) Serial quantitation of pRIPK3 fluorescence intensity in control (“C”) and histotripsy-treated (“HT-Tx”) tumors over various time points demonstrated rapid and transient upregulation of necroptosis-associated phosphorylated protein levels following histotripsy ablation. [(A, B): n=7-9 mice per group; *=p<0.05 compared with control tumors; †=p<0.05 compared with HT abscopal concordant tumors. (C-G): n=3-4 mice per group; *=p<0.05 compared with control day 1 tumors; **=p < 0.01 compared with control day 1 tumors; ***=p < 0.0001 compared with control day 1 tumors].

Figure 2

Early cellular stress and immunogenic cell death is observed in treated tumors but not distant untreated tumors following histotripsy. Mice bearing bilateral B16F10 tumors were treated with unilateral sham (control) or partial histotripsy ablation on day 10. (A) Multicolor immunofluorescence microscopy analysis of control, histotripsy-treated, and histotripsy-abscopal tumors on days 1, 3 or 10-12 after unilateral sham or partial histotripsy ablation revealed no significant release of intranuclear HMGB1 in control tumors at any time point. Histotripsy-treated tumors exhibited significant and immediate loss of nuclear HMGB1 staining that was highest within the ablation zone and the junction of ablated and peripheral non-ablated zones at early time points. At later time points, progressive loss of nuclear HMGB1 was observed within peripheral non-ablated zones of histotripsy-treated tumors. Similarly, whereas contralateral histotripsy-abscopal tumors demonstrated no HMGB1 translocation at early time points, progressive loss of intranuclear HMGB1 staining was observed at later time points. (B) RNASeq of CD45- tumor cells from sham-treated control tumors (blue) and histotripsy-abscopal (“HT-Abs”) tumors (red) performed 3 days after unilateral partial histotripsy revealed no substantial differences in overall mRNA transcriptional activity as evidenced by principal component analysis. (C) RNASeq of CD45- tumor cells 3 days after sham or unilateral histotripsy ablation demonstrated upregulated transcription of genes associated with necroptosis, ER stress, cellular response to LPS, TNFα signaling and inflammatory response in histotripsy-treated tumors (“HT-Tx”) as compared to control and histotripsy-abscopal (“HT-Abs”) tumors. (n=3-5 mice per experimental group).

Figure 3

Histotripsy ablation is followed by infiltration of innate and adaptive immune cell populations. Mice bearing bilateral B16F10 tumors were treated with unilateral sham (control) or partial histotripsy ablation on day 10. Multicolor immunofluorescence performed 1, 3 and 10 days after sham or histotripsy partial histotripsy ablation revealed (A) early infiltration of NK1.1+ cells initially localized within the histotripsy ablated zone that gradually migrated outward toward peripheral, non-ablated zones on days 3 and 10, and (B) early and transient infiltration of CD11b+ cells within the histotripsy ablated zone on day 1 and delayed infiltration of CD8+ T cells within the non-ablated zone on day 10. (C) Quantitation of NK1.1+ cells within ablated zones (red) and non-ablated zones (blue) of histotripsy-treated tumors at various time points revealed gradual migration of NK cells away from ablated zones toward non-ablated zones. (D) Quantitation of Ly6GC+ cells at various time points after histotripsy confirmed immediate but short-lived infiltration strictly localized to the ablated zone. (E) RNASeq of CD45+ immune cells performed 3 days after unilateral partial histotripsy ablation revealed marked differences in transcriptional activity between control tumors (blue) and histotripsy-treated (“HT-Tx”) tumors (red) as evidenced by principal component analysis. (F) Upregulated transcription of genes associated with neutrophil chemotaxis was observed in histotripsy-treated but not histotripsy-abscopal (“HT-Abs”) tumors. (G) Mice bearing bilateral Hepa1-6 tumors were treated with unilateral partial histotripsy ablation on day 10. Flow cytometric analysis of tumor-draining lymph nodes of control (blue), histotripsy-treated (green) and histotripsy-abscopal (red) tumors performed on day 13 revealed significant increases in cDC1 and cDC2 populations within lymph nodes draining histotripsy-treated tumors. (H) Similarly, flow cytometric analysis revealed significant increases in activated CD8+ T cells within lymph nodes draining histotripsy-treated tumors. (n=3-6 mice per group; *=p<0.05 between groups; **=p < 0.01 between groups; ****=p < 0.0001 between groups).

Figure 4

The temporal kinetics of intratumoral cell infiltration are dissimilar at early time points and similar at later time points between histotripsy-treated and histotripsy-abscopal tumors. Mice bearing bilateral B16F10 tumors were treated with unilateral sham (control) or partial histotripsy ablation on day 10. (A) Multicolor immunofluorescence 10 days after unilateral B16F10 histotripsy ablation showed an influx of NK1.1+ and CD8+ cell populations within the non-ablated zones of histotripsy-treated (“HT-Tx”) tumors and diffusely in histotripsy-abscopal (“HT-Abs”) tumors. (B) Quantitation of CD8+ staining at various time points revealed delayed infiltration that was strictly localized to non-ablated zones (blue) and not ablated zones (red). (C) Multicolor immunofluorescence at early time points revealed infiltration of NK1.1+ cells in histotripsy-abscopal (“HT-Abs”) tumors. (D-F) Multicolor immunohistochemistry performed 1, 3 and 10 days after partial histotripsy ablation revealed minimal increases in NK1.1+ and CD8+ cells in control tumors over time (D). In contrast, histotripsy-treated tumors exhibited rapid influx of CD11b+ and Ly6GC+ and NK1.1+ cell populations immediately after ablation; whereas CD11b+ and Ly6CG+ cell infiltration was short-lived, NK1.1+ cell populations followed a biphasic pattern of early and delayed intratumoral infiltration; CD8+ and CD4+ cell infiltration followed a delayed pattern of delayed intratumoral infiltration (E). Whereas histotripsy-abscopal tumors did not exhibit the early pattern of CD11b+ and Ly6GC+ cell infiltration seen in histotripsy-treated tumors, patterns of NK1.1+ and CD8+ and CD4+ cell populations were similar to those observed in histotripsy-treated tumors (F). (G-I) Digital spatial profiling of immune-relevant protein expression within regions of lymphocytic infiltration was performed on control, histotripsy-treated (“HT-Tx”) and histotripsy-abscopal (“HT-Abs”) B16F10 tumors 10 days after unilateral sham or histotripsy ablation. Principal component analyses demonstrated differences in overall expression of immune-relevant proteins between control (purple) and histotripsy-treated (gold) tumors (G), and between control (purple) and histotripsy-abscopal (bronze) tumors (H); however, patterns of immune-relevant protein expression were largely superimposable between histotripsy-treated (brown) and histotripsy-abscopal (green) tumors (I). (J-L) RNASeq analyses of intratumoral CD45+ cells were performed on control, histotripsy-treated (“HT-Tx”) and histotripsy-abscopal (“HT-Abs”) B16F10 tumors 10 days after unilateral sham or histotripsy ablation. Principal component analyses demonstrated differences in overall transcriptional activity between control (blue) and histotripsy-treated (red) tumors (J) and between control (red) and histotripsy-abscopal tumors (blue) (K), but not between histotripsy-treated (blue) tumors and histotripsy-abscopal (red) tumors (L). (n=3-10 mice per experimental group; ****=p < 0.0001 between groups).

Figure 5

Late intratumoral CD8+ cell infiltration co-localizes with immunogenic and ferroptotic cancer cell death following histotripsy. Mice bearing bilateral B16F10 tumors were treated with unilateral sham (control) or partial histotripsy ablation on day 10. (A) Multicolor immunofluorescence performed on day 7 after sham or histotripsy ablation revealed minimal CD8+ cell infiltration and minimal extranuclear translocation of HMGB1 among cancer cells in control tumors. CD8+ cell infiltration was co-localized with loss of intranuclear HMGB1 staining in histotripsy-treated tumors (B) and in histotripsy-abscopal tumors (C). (D) Significantly higher percentages of cancer cells located within 50 μm of a CD8+ cell exhibited loss of nuclear HMGB1 in histotripsy-treated and histotripsy-abscopal tumors as compared with controls. (E) Multicolor immunofluorescence performed on days 3, 5 and 7 after sham or histotripsy ablation revealed spatial co-localization of CD8+ cell infiltration with cancer cell accumulation of 4-HNE, a byproduct of ferroptosis, in histotripsy-treated tumors but not in control tumors. (F) Strong co-localization was observed between CD8+ cell infiltration and 4-HNE on day 7 in histotripsy-treated and histotripsy-abscopal tumors, but not in control tumors. (G) Gradual accumulation of 4-HNE staining intensity was observed in histotripsy-treated (“HT Tx”) and histotripsy-abscopal (“HT Abs”) tumor cells. (H) A linear relationship was observed between number of CD8+ cells present (x axis) and intensity of 4-HNE expression within 50 μm (y-axis) in histotripsy-treated tumors but not in control tumors. (I, J) Mice bearing B16F10 flank tumors were treated with sham or histotripsy tumor ablation on day 10, and tumor-draining lymph nodes were harvested on day 15. CD8+ T cells derived from tumor-draining lymph nodes were co-cultured with B16F10 melanoma cells in vitro. Significant accumulation of 4-HNE was observed within B16F10 melanoma cells co-cultured with CD8+ T cells derived from lymph nodes draining histotripsy-treated but not sham-treated tumors. (n=3-6 mice per group; *=p < 0.05 compared with control day 1 tumors; ***=p < 0.001 compared with control day 1 tumors).

Figure 6

The combination of histotripsy with checkpoint inhibition results in additive intratumoral infiltration of CD8+ cells and synergistic induction of cancer cell ferroptosis. Mice bearing bilateral B16F10 tumors were treated with no therapy (control), checkpoint inhibition (CI) with anti-CTLA-4 mAb on days 6, 9 and 12 (“CI”), unilateral partial histotripsy ablation (“HT”) on day 7, or both (“HT+CI”). (A) Non-ablated abscopal tumor growth on day 18 was suppressed in mice treated with contralateral HT and CI, but maximal in mice treated with both. (B) Multicolor immunofluorescence of non-ablated abscopal tumors revealed increases in intratumoral CD8+ cell infiltration, loss of nuclear HMGB1, and 4-HNE accumulation after both CI and HT, with maximal effects seen after combinatorial HT+CI. (C) Quantitation of CD8+ cell density demonstrated an additive effect between CI and the abscopal effect of HT. (D) A similar additive effect was observed between CI and the abscopal effect of HT in extranuclear HMGB1 translocation. (E) The combination of HT+CI appeared to be a greater than additive effect on abscopal 4-HNE expression. The additive effects of CI and HT on abscopal CD8+ cell infiltration, HMGB1 release, and 4-HNE expression are shown in line graph form (F) and dot plot form (G). (n=4-7 mice per group; *=p<0.05 compared with controls; **=p<0.01 compared with controls; ***=p<0.001 compared with controls).

Figure 7

Tumor lysates generated by histotripsy confer partial protection when administered as vaccines. Mice bearing B16F10 tumors were treated with no therapy, 15 Gy radiation therapy (XRT), or histotripsy (“HT”) ablation on day 9. Tumors were explanted on day 10 and untreated tumors were dissociated by 3 cycles of alternative freezing and thawing. The resulting lysates were centrifuged to generate cell-free fractions, and fractions were delivered via intraperitoneal injection into naïve mice one day prior to B16F10 flank injection. (A, B) Whereas vaccination with tumor lysates generated by XRT or freeze-thaw conferred no protection against B16F10 challenge tumor growth as compared with unvaccinated controls, mice receiving tumor lysates generated by histotripsy exhibited slower challenge tumor growth kinetics. (C) Multicolor immunohistochemistry of challenge tumors growing in unvaccinated control and HT lysate-vaccinated mice showed marked increases in both CD8+ cell infiltration and 4-HNE accumulation. (n=3-6 mice per group; *=p<0.05 compared with controls).