Abstract 6211: Targeting the tumor microenvironment of metastasis to treat metastatic Ewing sarcoma
Authors: Offenbacher Rachel , Lin Yu , Karagiannis George , Oktay Maja , Loeb David 
Published: June 2022

Abstract
Introduction: Ewing sarcoma (EWS) is the second most common bone cancer in adolescents and young adults. Intensive multimodal treatment cures almost 75% of patients who present with localized disease. However, only 25% of patients who present with metastases become long term survivors, and those who suffer a metastatic relapse are almost never cured. Recently, a 3-cell assembly consisting of an invasive cancer cell, a Tie2-high expressing macrophage, and an endothelial cell (termed the Tumor Microenvironment of Metastasis, or TMEM), has been described as the doorway through which breast cancer cells enter the vasculature and disseminate to distant sites. We hypothesized that EWS cells use a similar mechanism for dissemination, and that targeting TMEM offers a novel therapeutic opportunity to prevent metastatic recurrence.
Methods: Formalin-fixed, paraffin embedded tissue samples were obtained from 16 patients with EWS treated at our institution, and these were evaluated for the presence of TMEM structures by immunohistochemistry. We also evaluated samples obtained from our novel, clinically relevant murine model of spontaneous distant EWS metastasis. Fragments of EWS either from cell line-derived tumors or patient-derived xenografts (PDX) orthotopically or subcutaneously. After tumors grew, mice were injected with fluorescent dextran in the tail vein and tumors harvested 60 minutes later. Intravascular dextran remains inside blood vessels because the molecular weight is too large to allow diffusion between endothelial cells. As TMEM disrupts the endothelial barrier, dextran leaks out of blood vessels and can be detected and quantified using fluorescence microscopy. Tumors were evaluated for the presence of TMEM structures by immunohistochemistry (IHC), and for the presence of localized vascular leakiness, (the hallmark of functional TMEM), by fluorescence microscopy.
Results: We found TMEM assemblies in all patient samples, at numbers well in excess of those seen in samples of metastatic breast cancer. Interestingly, although there was no difference in TMEM counts from patients who were survivors compared with those who died from metastatic disease, there was an increase in TMEM counts in samples from tumors that were exposed to chemotherapy compared to diagnostic biopsy samples, something also reported in breast cancer patients. We also identified TMEM assemblies and localized vascular leakiness in EWS PDX tumors implanted orthotopically.
Discussion: TMEM assemblies are present in both EWS patient samples and PDX models, suggesting that this important mechanism of tumor intravasation into the vasculature may be contributing to distant metastasis. Future work will focus on identifying drugs that inhibit the function of TMEM, with the goal of translating this work into clinical trials aimed at diminishing the burden of metastasis in EWS patients.
Citation Format: Rachel Offenbacher, Yu Lin, George Karagiannis, Maja Oktay, David Loeb. Targeting the tumor microenvironment of metastasis to treat metastatic Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6211.