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

Histotripsy is a novel focused ultrasound tumor ablation modality with potent immunostimulatory effects. To measure the spatiotemporal kinetics of local and abscopal 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 Controltralateral untreated (abscopal) tumors were analyzed using multicolor immunofluorescence, digital spatial profiling, RNA sequencing (RNASeq), and flow cytometry. Flow-sorted CD45+ and CD45- cells were lysed and RNA was extracted per manufacturer instructions using the Qiagen RNeasy Mini Kit. RNA was submitted to the University of Michigan Advanced Genomics Core for library preparation and QuantSeq analysis. Pooled libraries were subjected to 101 bp single-end sequencing with Illumina NovaSeq following manufacturer protocols . Bcl2fastq2 Controlversion Software (Illumina) was used to generate de-multiplexed Fastq files. Reads were trimmed using Trim Galore (v0.5.0) using default settings. Reads were aligned using STAR (v 2.6.0) using default settings . Unique molecular identifiers were collapsed using Lexogen Quantseq (v2.3.6). Reads were mapped to the mouse genome GRCm38 from the Ensembl genome database. Reads were counted using BioControlductor R package Rsubread and featureCounts function . RNASeq data analysis and visualizations were performed using BioJupies.