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Glioblastoma multiforme is the most common, highly aggressive malignant brain tumor which is marked by highest inter- and intra-tumoral heterogeneity. Despite, immunotherapy, and combination therapies developed; the clinical trials often result into large number of failures. Often cancer cells are known to communicate with surrounding cells in tumor microenvironment (TME). Extracellular vesicles (EVs) consisting of diverse cargo mediates this intercellular communication and is believed to modulate the immune function against GBM. Tumor-associated microglia (TAM), though being the resident innate immune cell of CNS, is known to attain pro-tumorigenic M 2 phenotype, and this immunomodulation is aided by extracellular vesicle-mediated transfer of oncogenic, immunomodulatory molecules. Besides, oncogenic proteins, long non-coding RNAs (lncRNAs), are believed to carry oncogenic potential, and therefore, understanding the mechanism leading to microglial dysregulation mediated by GBM-derived extracellular vesicle (GDEV) lncRNAs becomes crucial. This review focuses on current understanding of role of GDEV and lncRNA in microglial dysfunction and its potential as a therapeutic target.

The brain is often reported as the first site of recurrence among breast cancer patients overexpressing human epidermal growth factor receptor 2 (HER2). Although most HER2+tumors metastasize to the subcortical region of the brain, a subset develops in the cortical region. We hypothesize that factors in cerebrospinal fluid (CSF) play a critical role in the adaptation, proliferation, and establishment of cortical metastases. We established novel cell lines using patient biopsies to model breast cancer cortical and subcortical metastases. We assessed the localization and growth of these cells in vivo and proliferation and apoptosis in vitro under various conditions. Proteomic analysis of human CSF identified astrocyte-derived factors that support the proliferation of cortical metastases, and we used neutralizing antibodies to test the effects of inhibiting these factors both in vivo and in vitro. The cortical breast cancer brain metastatic cells exhibited greater proliferation than subcortical breast cancer brain metastatic cells in CSF containing several growth factors that nourish both the CNS and tumor cells. Specifically, the astrocytic paracrine factors IGFBP2 and CHI3LI promoted the proliferation of cortical metastatic cells and the formation of metastatic lesions. Disruption of these factors suppressed astrocyte-tumor cell interactions in vitro and the growth of cortical tumors in vivo. Our findings suggest that inhibition of IGFBP2 and CHI3LI signaling, in addition to existing treatment modalities, may be an effective therapeutic strategy targeting breast cancer cortical metastasis.

Prognosis of patients with HER2+ breast-to-brain-metastasis (BBM) is dismal even after current standard-of-care treatments, including surgical resection, whole-brain radiation, and systemic chemotherapy. Radiation and systemic chemotherapies can also induce cytotoxicity, leading to significant side effects. Studies indicate that donor-derived platelets can serve as immune-compatible drug carriers that interact with and deliver drugs to cancer cells with fewer side effects, making them a promising therapeutic option with enhanced antitumor activity. Moreover, human induced pluripotent stem cells (hiPSCs) provide a potentially renewable source of clinical-grade transfusable platelets that can be drug-loaded to complement the supply of donor-derived platelets. Here, we describe methods for ex vivo generation of megakaryocytes (MKs) and functional platelets from hiPSCs (hiPSC-platelets) in a scalable fashion. We then loaded hiPSC-platelets with lapatinib and infused them into BBM tumor-bearing NOD/SCID mouse models. Such treatment significantly increased intracellular lapatinib accumulation in BBMs in vivo, potentially via tumor cell-induced activation/aggregation. Lapatinib-loaded hiPSC-platelets exhibited normal morphology and function and released lapatinib pH-dependently. Importantly, lapatinib delivery to BBM cells via hiPSC-platelets inhibited tumor growth and prolonged survival of tumor-bearing mice. Overall, use of lapatinib-loaded hiPSC-platelets effectively reduced adverse effects of free lapatinib and enhanced its therapeutic efficacy, suggesting that they represent a novel means to deliver chemotherapeutic drugs as treatment for BBM.

Prognosis of patients with HER2+ breast-to-brain-metastasis (BBM) is dismal even after current standard-of-care treatments, including surgical resection, whole-brain radiation, and systemic chemotherapy. Radiation and systemic chemotherapies can also induce cytotoxicity, leading to significant side effects. Studies indicate that donor-derived platelets can serve as immune-compatible drug carriers that interact with and deliver drugs to cancer cells with fewer side effects, making them a promising therapeutic option with enhanced antitumor activity. Moreover, human induced pluripotent stem cells (hiPSCs) provide a potentially renewable source of clinical-grade transfusable platelets that can be drug-loaded to complement the supply of donor-derived platelets. Here, we describe methods for ex vivo generation of megakaryocytes (MKs) and functional platelets from hiPSCs (hiPSC-platelets) in a scalable fashion. We then loaded hiPSC-platelets with lapatinib and infused them into BBM tumor-bearing NOD/SCID mouse models. Such treatment significantly increased intracellular lapatinib accumulation in BBMs in vivo, potentially via tumor cell-induced activation/aggregation. Lapatinib-loaded hiPSC-platelets exhibited normal morphology and function and released lapatinib pH-dependently. Importantly, lapatinib delivery to BBM cells via hiPSC-platelets inhibited tumor growth and prolonged survival of tumor-bearing mice. Overall, use of lapatinib-loaded hiPSC-platelets effectively reduced adverse effects of free lapatinib and enhanced its therapeutic efficacy, suggesting that they represent a novel means to deliver chemotherapeutic drugs as treatment for BBM.

Long non-coding RNAs (lncRNAs) are increasingly being recognized as major players in various cellular processes such as gene regulation, chromatin packaging, genomic imprinting, dosage compensation, cell differentiation, and development. HOX antisense intergenic RNA (HOTAIR) is a 2.2 kb long lncRNA, localized in chromosome 12 within the homeobox C (HOXC) gene cluster and is transcribed by RNA polymerase II (RNAPII) from the antisense strand. HOTAIR interacts with polycomb repressive complex-2 (PRC2), which is a multiprotein complex containing a histone H3 lysine27 (H3K27)-specific methyltransferase and histone H3K4-specific demethylases LSD1 and recruits them to the target gene loci leading to gene repression Studies have demonstrated that HOTAIR misregulation is associated with severe human diseases including cancers. We demonstrated that HOTAIR is transcriptionally upregulated by estrogen (E2), by endocrine disrupting chemicals that are estrogenic in nature (e.g. diethylstilbestrol (DES) and bisphenol-A (BPA)) in vitro, in estrogen receptor positive (ER +ive) breast cancer cells such as MCF7 and T47D cells as well as in vivo in the mammary glands of ovariectomized female rats. The HOTAIR promoter analyses and luciferase based reported assay revealed that it contains multiple functional EREs. Estrogen receptors α and β (ERα and ERβ) bind to the functional EREs in an E2, BPA and DES dependent manner. We also observed that MLL1 and MLL3 are recruited to the HOTAIR EREs in an E2/BPA/DES dependent manner. In contrast, MLL2 and N-CoR (Nuclear receptor co-repressor) are bound to the HOTAIR promoter in the absence of E2/BPA/DES but get delocalized upon exposure to E2/BPA/DES, indicating the critical role played by N-CoR and MLL2 in maintaining the HOTAIR transcription at a basal level (under non-activated conditions; in the absence of E2/BPA/DES). Histone acetyltransferases such as CBP/p300 are also recruited to the HOTAIR EREs in an E2/BPA/DES dependent manner. MLL1 and MLL3 mediated increase in histone H3K4-trimethylation levels and CBP/p300 mediated increase in the histone acetylation levels at the HOTAIR promoter loci, in presence of E2, BPA or DES leads to recruitment of general transcription factors (e.g. TAF250) and RNAPII in the HOTAIR promoter loci. We also showed that enhancer of zeste homolog 2 (EZH2), a H327 methyltransferase that is overexpressed in breast cancer and a functionally interacts with HOTAIR is transcriptionally induced by E2, BPA and DES in vitro and in vivo. EZH2 promoter contains multiple functional EREs that are bound by ERs and other ER coregulators such as MLL2, MLL3 and CBP/P300 in the presence of E2/BPA/DES and aids in the transcriptional induction of EZH2. Various studies have shown that HOTAIR promotes tumorigenesis and metastases and the HOTAIR levels are significantly higher in metastatic tumor cells. Hypoxic microenvironments also have been known to play a critical role in the tumor development and augmentation of metastatic potential of various solid tumors. Therefore, we hypothesized that HOTAIR is transcriptionally regulated in the tumor microenvironment where hypoxia predominates. Herein, we have demonstrated that HOTAIR is upregulated by hypoxia in a variety of cancer cells including colon cancer cells. We also demonstrate that HIF-1α binds to the hypoxia response elements (HRE) present in the HOTAIR promoter under hypoxic condition. Along with HIF-1α, p300, MLL1, and RNAPII were enriched at the HOTAIR promoter HRE. The levels of H3K4 trimethylation and histone acetylation were significantly increased at the HOTAIR HRE. Thus, upregulation of HOTAIR might contribute to development of metastasis and malignancy in the hypoxic microenvironment.

Rationale: Mean platelet volume (MPV) and platelet count (PLT) are platelet measures that have been linked to cardiovascular disease (CVD) and mortality risk. Identifying protein biomarkers for these measures may yield insights into CVD mechanisms. Objective: We aimed to identify causal protein biomarkers for MPV and PLT among 71 CVD-related plasma proteins measured in Framingham Heart Study (FHS) participants. Methods and Results: We conducted integrative analyses of genetic variants associated with PLT and MPV with protein quantitative trait locus (pQTL) variants associated with plasma proteins followed by Mendelian randomization (MR) to infer causal relations of proteins for PLT/MPV, and tested protein-PLT/MPV association in FHS participants. Utilizing induced pluripotent stem cell (iPSC)-derived megakaryocyte (MK) clones that produce functional platelets, we conducted RNA-sequencing and analyzed transcriptome-wide differences between low- and high-platelet producing clones. We then performed small interfering RNA (siRNA) gene knockdown experiments targeting genes encoding proteins with putatively causal platelet effects in MK clones to examine effects on platelet production. Protein-trait association analyses were conducted for MPV (n = 4,348) and PLT (n = 4,272). Eleven proteins were associated with MPV and 31 with PLT. MR identified four putatively causal proteins for MPV and four for PLT. Glycoprotein V (GP5), granulin (GRN), and melanoma cell adhesion molecule (MCAM) were associated with PLT in both protein-trait and MR analyses. Myeloperoxidase (MPO) showed significant association with MPV in both analyses. MK RNA-sequencing analysis results were directionally concordant with observed and MR-inferred associations for GP5, GRN, and MCAM. In siRNA gene knockdown experiments, silencing GP5, GRN, and MPO decreased platelet counts. Conclusions: By integrating population genomics data, epidemiological data, and iPSC-derived MK experiments, we identified four proteins that are causally linked to platelet counts. These proteins and genes may be further explored for their utility in increasing platelet production in bioreactors for transfusion medicine purposes as well as their roles in the pathogenesis of CVD via a platelet/blood coagulation-based mechanism.