Identification of immune evasion mechanisms in the osteosarcoma microenvironment

Division Of Basic Sciences - Nci

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Abstract

We have successfully completed spatial transcriptomic profiling of a cohort of pediatric metastatic osteosarcoma specimens to thoroughly characterize the molecular landscape of this disease. Our analysis revealed that these tumors exhibit conserved regional gene expression programs and are characterized by (1) minimal intratumoral inflammatory cytokine expression; (2) heterogeneous and widespread signaling of TGFb family members throughout the tissues; (3) lymphocyte exclusion; and (4) a peritumoral fibrocollagenous border resemblant of foreign body encapsulation. These data have been published and made publicly available (PMID: 40173049). Our current focus is to further investigate the cellularly dense peritumoral microenvironment of osteosarcoma pulmonary metastases. To accomplish this, we have generated high-resolution spatial transcriptomic data and are actively analyzing these data to reveal the identity, gene expression profiles, cellular/biological processes, regulatory networks, intracellular communication patterns, and inferred function of each cell within this region. We will also use these data identify cell types of interest for subsequent cell depletion studies using appropriate models of metastatic osteosarcoma to credential these cellular targets and determine how the cell types of interest contribute to the overall phenotype and function of the microenvironment of osteosarcoma pulmonary metastases. Our current analysis of the spatial profiling data, together with a detailed histopathological analysis of patient tissue specimens, and in vitro cellular and molecular biology experiments supports our previously published work suggesting that endothelial cells in metastatic osteosarcoma specimens are dysfunctional (PMID: 31428529). Given these data, we hypothesize that the tumor vasculature in osteosarcoma pulmonary metastases functions to selectively prevent lymphocytes from extravasating out of the blood supply to infiltrate the core of these lesions and that this serves as a major impediment for successful immunotherapeutic intervention in this disease setting. To investigate this further, we have successfully performed single nuclei multiome sequencing of 24 metastatic osteosarcoma specimens in which both RNA and ATAC sequencing are simultaneously performed on individual nuclei extracted from frozen tissues. Our analysis has revealed that the endothelial cells in these specimens have adopted an altered cellular state as evidenced by distinct transcriptional profiles, pathways, processes, and gene regulatory networks. We have also detected the expression of various inhibitory immune checkpoints within these cells. We have validated these findings using immunohistochemistry and in vitro co-culture assays. We are actively pursuing this line of research and are currently investigating the functional significance of these candidate pathways using a combination of genetic (CRISPRa/CRISPRi) and pharmacological approaches both in vitro and in vivo using relevant models of metastatic osteosarcoma.