Abstract INTRODUCTION: Tumor-released exosomes have pleiotropic functions in promoting autocrine signaling to distant cells. Elucidating the mechanistic modulation of the immune system by these exosomes provides insights into potential biomarkers for detection, recurrence and response and identifies potential new therapeutic targets. METHODS: Exosomes were isolated from human glioblastoma stem cells (GSCs) and fibroblasts (control) using differential centrifugation and characterized by nanosight technology, electron microscopy, and western blotting. Fluorescent-labeled exosomes were co-cultured with human immune cells. Confocal microscopy was used to determine the preferential uptake in various immune populations and to evaluate the intracellular trafficking. The cell-secreted exosome content was characterized by mass spectrometry and nanostring technology. The phenotypic and functional skewing of the monocyte lineage was analyzed given its propensity to take up exosomes. RESULTS: GSC-secreted exosomes were homogenous in morphology, ranged from 50-120 nm in size, and expressed CD63 and CD9 surface molecules. The GSCs-produced exosomes were preferentially absorbed by CD14+ monocytes (precursors to macrophages) and Gr-1+ derived myeloid cells isolated from healthy volunteers and/or glioblastoma patients. When activated, CD4+ and CD8+ T cells, but not NK cells could also uptake exosomes. Longitudinal kinetic studies established that the highest uptake of PKH67-labeled GCS-secreted exosomes occurred at 48 hours after exposure. Confocal microscopy revealed that monocytes could only internalize GSC-released exosomes but not fibroblast-secreted exosomes. The exposure to GSC-secreted exosomes induced a phenotypic change in monocytes and prevented them from undergoing apoptosis. Studies of M1/M2 macrophage markers by flow cytometry revealed that GSCs-secreted exosomes, but not the fibroblast-secreted exosomes, increased expression of CD80, CD163, CD206 and decreased expression of MHC class II. This profile was similar to myeloid suppressor cells and macrophages that were obtained directly ex vivo from glioblastomas (n = 17). The GSC-secreted exosomes were preferentially enriched relative to fibroblast-secreted exosomes in transcriptional regulators that induced the M2 phenotype. CONCLUSIONS: Monocytes demonstrate preferential uptake of GSC-secreted exosomes which then induces a glioma-supportive M2 phenotype - similar to the phenotype observed in myeloid cells and macrophages isolated from human glioblastomas. This data indicates that the GSC-secreted exosomes can be a contributing factor in the M2 skewing within the tumor microenvironment. Citation Format: Konrad Gabrusiewicz, Yuuri Hashimoto, Jun Wei, Maiti Sourindra, John Yu, Shinji Yamashita, Anna Zal, Tomasz Zal, Laurence Cooper, Amy B. Heimberger. Glioblastoma stem cell-secreted exosomes can induce a tumor supportive M2 response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5088. doi:10.1158/1538-7445.AM2015-5088