Abstract 637: CyTOF analysis of heterogeneity between Ewing sarcoma cell lines in vitro and in vivo.

Abstract Introduction: Ewing sarcoma (ES) is the second most common bone cancer in children and is known to be driven by an oncogenic fusion, most commonly EWS:FLI1. Recent work has identified that ES tumors are heterogenous and there are populations of cells within tumors with varying levels of the EWS:FLI1 fusion. These populations have differences in morphology, proliferation, drug sensitivities, and invasiveness. Further, it is known that cancer stem cell populations are also found within ES tumors and these cells also have differences in morphology and drug sensitivity. The purpose of this study was to use CyTOF to assess differences in EWS:FLI1 fusion and cancer stem cell heterogeneity and immune infiltration between various ES cell lines in vitro and in vivo. Methods: In vitro, A673, TC71, RDES, and SL00015 cells were seeded separately at equal numbers and stained for CyTOF the following day. In vivo, A673, RDES, or SL00015 cells were injected subcutaneously into Nude mice. Once tumors reached 1500-2000mm3, they were harvested and dissociated for CyTOF analysis. The CyTOF panel used was designed to assess EWS:FLI1 fusion heterogeneity, the cancer stem cell population, and immune cell infiltration. OMIQ was used for data analysis which included phenograph clustering with UMAP visualization, heatmap of expression patterns, and changes in tumor and immune cell populations. Results: In vitro, each of the 4 cell lines have varying morphologies and their growth ranges from adherent to clusters of cells in suspension. Phenograph clustering revealed different expression patterns among the cell lines. Interestingly, the expression patterns of different cell lines grown in vivo clustered together more than the in vivo grown cells did with their in vitro counterpart. Overall, the cells grown in vivo had a higher percentage of the EWS:FLI1low population compared to the respective cell line grown in vitro. The amount of cancer stem cells varied among cell lines and sample types, though the percentage was generally higher in the in vitro samples compared to in vivo. Further, immune infiltration also varied across tumors formed from different cell lines with the RDES tumors having the most overall infiltration. Conclusions: The CyTOF panel used in this study can identify distinct expression patterns among cell lines and sample types. The differences in expression patterns identified between a cell line grown in vitro or in vivo could be an important consideration in drug efficacy or mechanism studies in the context of Ewing sarcoma. Citation Format: Piper Harrell, Lily Golzar, Kenzie Wells, Poornima Gourabathini, Kimberly Q. McKinney, Erin M. Trovillion, Javier Oesterheld, Kaitlyn H. Smith. CyTOF analysis of heterogeneity between Ewing sarcoma cell lines in vitro and in vivo abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 637.