**1. Introduction**

Glioblastoma multiforme (GBM) is the most aggressive brain tumor of glial origin and has a poor median survival of 14 months [1]. One of the reasons for its malignancy and challenging therapeutics development lies in its heterogeneous nature at the cellular and molecular levels. It is now generally recognized that GBM is composed of a subpopulation of glioma stem cells (GSCs), capable of tumor initiation and progressive self-renewal upon treatments, and other cells within the tumor microenvironment (TME). The TME contains cancerous cells surrounded by parenchymal cells, including endothelial/vascular cells, microglia, and immune cells [2]. One of the major cell types from the GBM TME is glioblastoma-associated macrophages (GAMs). GAMs have been shown to contribute to the progression of GBM. For instance, the presence of M2 GAMs has been shown to promote the growth and metastasis of GBM cells [2,3].

More importantly, emerging evidence indicates the dynamic intercellular communications within the GBM TME via secretions of cytokines, chemicals, and signaling molecules. Among these, secreted exosomes represent a class of small bilayered particles (ranging from 50 to 150 nm in diameter) which have been extensively explored for their roles in GBM tumorigenesis over the past few years [4]. Recent studies have shed light on the diverse functions of exosomes involved in GBM tumorigenesis. For instance, exosomes released from human GBM cell lines contain various types of heat shock proteins and transforming growth factor beta 1(TGF-β1) which are proposed to exert immune suppressive roles in GBM [5]. In addition, serum-derived exosomes from GBM patients and Cerebrospinal fluid (CSF) derived exosomes were shown to contain a high level of miR-221, serving as a potential GBM biomarker [6]. A recent study demonstrated that microglia also communicate and affect the function of glioma via the release of exosomes [7]. These findings suggest that there is a potential area for therapeutics development via interrupting the intracellular communications between GBM cells and their TME by means of exosomes. However, the role of exosomes derived from M2 GAMs has not been fully appreciated.

In this study, we first demonstrated that when human GBM cell lines were cocultured with clinically isolated glioblastoma-associated macrophages, this significantly enhanced colony formation ability and tumor sphere generation in association with an increased expression of Sox2, STAT3, interleukin 6 (IL-6), and Nestin and a decrease in glial fibrillary acidic protein (GFAP). Subsequently, exosomes released into the culture medium of GAMs were isolated and cocultured with GBM cell lines. A similarly increased tumorigenic property was observed in addition to the increased resistance against temozolomide (TMZ). More importantly, miR-21, a oncomiR, was identified as the most abundant microRNA species in the exosomes released from the GAMs. We then provided evidence for the positive association between miR-21 level and GBM malignancy. Exogenously increased miR-21 in GBM cells increased their ability to polarize GAMs towards the M2 phenotype, and the reduction of miR-21 reversed these properties. In addition, we showed that miR-21-mediated oncogenic properties were associated with their targeting/inhibitory function on PDCD4 (a tumor suppressor). An increased

miR-21 level in the GBM cells led to their increased ability to polarize GAMs towards the M2 phenotype by the increased secretion of the M2 cytokines IL-6 and TGF-β1.

Subsequently, we examined the feasibility of applying pacritinib, an inhibitor of the STAT3-associated pathway, as an anti-GBM agent. We showed that pacritinib treatment significantly reduced cell viability and colony/tumor sphere formation in association with reduced levels of STAT3, Sox2, PDCD4, and miR-21; it also reduced the ability to generate M2 GAMs. Notably, pacritinib-treated GAMs released fewer miR-21-enriched exosomes. Finally, we demonstrated preclinical support for using pacritinib to overcome TMZ-resistance using a TMZ-resistant LN18-bearing mouse model.
