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Background Despite advances in therapy, such as immune checkpoint inhibitors (ICI), outcomes of head and neck squamous cell carcinoma (HNSCC) remain suboptimal, with ICI response rates at ~17% in the recurrent and metastatic setting. The oscillation and subsequent cavitation of ultrasound-sensitive microbubbles (clinically approved as diagnostic ultrasound contrast agents) have been shown to alter the tumor microenvironment (TME) by temporarily increasing vascular permeability and initiating cancer cell apoptosis. We hypothesize that stable cavitation of ultrasound contrast agents using noninvasive ultrasound can alter the TME and augment ICI therapy.Methods In this IACUC approved study, C57BJ/L mice (8-10 weeks old) subcutaneously injected with a syngeneic MOC1 HNSCC cells were treated two weeks post inoculation with mouse aPD-1 (treatment-only control; 200µg InVivoMAb anti-mouse PD-1 (Bio X Cell) administered IP), ultrasound alone (control), or aPD-1 and Definity microbubbles (treatment arm) (n=3 each, N=9 total). Ultrasound was performed using a Siemens Sequia with a 10L4 probe operating in cadence pulse sequencing mode at a mechanical index of 0.4. Animals were euthanized 24 hours post-treatment, and tumors were harvested and embedded in paraffin. As an initial screen, CD45 staining was performed and quantified using a customized script. Subsequently, Visium spatial transcriptomics was performed on a subset of mice using CytAssist (10xGenomics) and libraries sequenced on Illumina NovaSeq 6000. Raw .fastq files were processed with 10x Space Ranger v3.0.0, and and spatial gene expression were visualized in Loupe Browser v8.0. Pairwise comparisons were performed in Seurat v5.3.0 with the hypothesis that microbubbles may augment aPD-1 treatment. ClusterProfiler v4.16.0 was used to identify GO terms enriched in the significantly differentially (log2FC >1 with FDR<0.05) expressed genes.Results The combination of ultrasound-triggered microbubble cavitation and aPD1 demonstrated no toxicity in any animal. Additionally, perfusion of the ultrasound contrast was well visualized in all tumors, demonstrating the ability of contrast-enhanced ultrasound to noninvasively evaluate HNSCC vascularity. Immunohistochemistry showed increased CD45 counts in the treatment + bubble condition compared to the control and treatment only groups (p=0.08;). Unsupervised gene set expression analysis revealed spatially related changes in critical hallmark genesets, angiogenesis, glucose transporters, and immune pathways ( figure 1). Of note, adaptive immune response was markedly upregulated in the aPD-1 + microbubble condition compared to aPD-1 treatment alone (figure 1).Conclusions Our preclinical results demonstrated that ultrasound-triggered microbubble cavitation can potentially augment aPD-1 therapy in HNSCC, and this effect is exerted via an increased immunogenicity of the tumor microenvironment.Ethics Approval IACUC approval included.Abstract 15 Figure 1Microbubble cavitation induced changes in immunogenomic signatures measured with Visium spatial analysis. A) Visium clustering and annotations with aPD-1 treatment alone. From left to right: tissue with Visium cluster overlay, angiogenesis vs glycolysis (predominantly the glucose and lactate transporter gene expression, which is essential for Warburg effect/tumor preferential glucose metabolism), angiogenesis vs adaptive immune response. On the right panel, adaptive immune response is generally low (blue areas). B) Comparative data from a tumor treated with both aPD-1 and microbubble cavitation. The relative expression of angiogenesis and glycolysis gene markers are mostly homogenous (mostly green patches)(left panels). However, the adaptive immunity gene signature was significantly increased in the aPD-1+ microbubble group compared to aPD-1 only (right panel). C) Volcano plot of significant genes shown in red (-log10p plotted vs log 2 fold change) with the addition of microbubble cavitation. D) ORA enrichment analysis of these genes shows enrichment in adaptive immune response, chemotaxis, and cytokine production with the addition of microbubble cavitation