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. 2023 Jan 23;14:1012799. doi: 10.3389/fimmu.2023.1012799

Figure 1.

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].