Objectives/Goals: KSHV is well documented to infect and establish latency in endothelial cells; however, infected cell types in the Kaposi Sarcoma (KS) tumor microenvironment are not well defined. Here, we present the first application of the Lunaphore COMETTM multiplexed immunofluorescence platform for spatial proteomic analyses of KS lesions. Methods/Study Population: Custom antibody panels were validated and optimized for characterization of the TME components, as compared to marginal structures. The Ab panels supported the detection, quantification, and comparison of >15 cell types in regions of interest in the KS tissues in a single automated run. For data analysis, we developed a rule-based decision tree for accurate cell classification and used the resulting cell populations to perform a nearest-neighbor analysis. We assessed spatial proximity among cell types located within, adjacent to, or distal from the areas with a large number of cells expressing KSHV LANA. Results/Anticipated Results: The highest KSHV load was detected in CD34+ cells with or without CD31, consistent with an endothelial progenitor phenotype (EPCs). We also defined 12 cell types with KSHV-LANA detected in <1% of each cell type. Among them, tumor-infiltrating CTLs were proximal to or overlapping with infected EPCs, suggesting they have cytolytic activities. In addition, we observed areas with T-cells detected at high frequency near normal vascular endothelial cells (VECs), indicating immune cell trafficking toward the lesion in these marginal areas. While supporting previous findings that T-cells are not infected by KSHV, our data also revealed areas having high viral Ag load and abnormal vasculature, versus surrounding areas with much lower Ag load, normal vasculature, and robust immune infiltration. Discussion/Significance of Impact: Our approach not only demonstrated its ability to characterize the KS TME at a previously unapproachable level of complexity but has also revealed gaps in lineage assignments in substantial proportions of the tumor, which will be subjected to further characterization.
Yalcin et al. (Wed,) studied this question.