Abstract B099: Deletion of <i>Piezo1</i> in CD11b+ Dendritic Cells Amplifies the Local and Systemic Rhabdomyosarcoma Anti-Tumor Response

Abstract Rhabdomyosarcoma (RMS) is a rare, yet aggressive skeletal muscle malignancy predominantly affecting children under ten years old. RMS tumors are canonically enriched with several myeloid populations, many of which can sense and respond to soluble and biophysical cues in the tumor microenvironment (TME). Store-operated Ca2+ Entry (SOCE) was the presumed calcium signaling mechanism that myeloid cells, specifically dendritic cells (DCs) and macrophages, utilize to perform their critical functions. Shockingly, in 2015, Vaeth et. al determined that SOCE was not necessary for their functionality begging the question: what calcium channel(s) underly myeloid cells’ phenotypic and functional plasticity in the highly dynamic TME? We propose expression of the mechanosensitive cation channel, PIEZO1, supports a pro-tumor phenotype in tumor-associated myeloid cells (TAMCs) while its deletion skews this population to anti-tumor functioning thereby propagating the RMS anti—tumor response. The impact of myeloid cells’ expression of PIEZO1 on tumor progression was assessed by orthotopically inoculating a novel murine CD11b-conditional Piezo1 knockout (CD11b cKO) strain with syngeneic RMS cells and performing downstream analyses. Compared to wild-type (WT) mice, we found CD11b cKO mice developed tumors at slower rates and had smaller primary tumors and metastases at the pre-clinical endpoint. Analysis of the bulk tumors’ supernatants revealed enhanced anti-tumor changes in the cKO mice including increased CCL21 and CXCL10 and decreased EGF. Flow cytometry revealed anti-tumor immunophenotype changes in the cKO mice including increased numbers of intra-tumoral monocytes, CD11b+ dendritic cells, CD8+ T-cells, and FOXP3- CD4+ T-cells. To home in on the population(s) driving the CD11b cKO phenotype, we performed parallel tumor growth experiments in our newly generated CX3CR1-conditional homozygous Piezo1 knockout (CX3CR1 cKO) mouse strain. We found although the CX3CR1 cKO tumors were smaller than WT, their size was not significantly different from either WT or CD11b cKO. Combined, these in vivo data suggest CD11b+ DCs, specifically monocyte-derived dendritic cells, are a main driver of my CD11b cKO phenotype. To further characterize these DCs, we performed phenotypic and functional analyses on isolated splenic CD11b+ DCs and bone marrow-derived dendritic cells (BMDCs). Flow cytometric analysis demonstrated that WT and CD11b cKO DCs had similar activation marker expression. Surprisingly, co-culture of either CD11b cKO DC population with naïve OT-I CD8+ T-cells after soluble OVA pulse invoked enhanced CD69 and Interferon-g expression in the T-cells. Together, our findings suggest Piezo1 deletion in CD11b+ DCs drives a robust RMS anti-tumor response. Without PIEZO1, the ability to endocytose and cross-present soluble antigen to CD8+ T-cells was unleashed- functions never described as being PIEZO1-related. We will further characterize these mechanisms through scRNA-Seq of bulk tumors with the ultimate hope of developing better DC-related cancer therapeutics. Citation Format: Melissa Romain Bonner, David Askew, Saada Eid, Suzanne L Tomchuck, muta Abiff, Jay Myers, Justin Garyu, Alex Huang. Deletion of Piezo1 in CD11b+ Dendritic Cells Amplifies the Local and Systemic Rhabdomyosarcoma Anti-Tumor Response [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B099.