PTEN plays a crucial role in preventing the development of glioblastoma (GBM), a severe and untreatable brain cancer. In GBM, most PTEN deficiencies are missense mutations that have not been thoroughly examined. Here, we leveraged genetically modified mice and isogenic astrocyte cell cultures to investigate the role of clinically relevant mutations (G36E, L42R, C105F, and R173H) in the development of EGFR-driven GBM. We report that the loss of tumor suppression from these mutants is unrelated to their lipid phosphatase activity and rather relate to elevated localization at the cell membrane. Moreover, expression of these PTEN mutations heightened EGFR activity by sequestering EGFR within endomembranes longer and affected its signaling behavior. Through comprehensive studies on global protein phosphorylation and kinase library analyses in cells with the G36E and L42R PTEN mutations, we identified distinct cancer-promoting pathways activated by EGFR, offering targets for treating GBM with these PTEN alterations.

High‐sensitivity flow cytometers have been developed for multi‐parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high‐sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single‐molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di‐8‐ANEPPS and with PE‐conjugated anti‐EGFR or anti‐tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross‐calibrated, hard‐dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di‐8‐ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin‐labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super‐resolution/single‐molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter‐platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter‐instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi‐quantitative LOD values for other flow cytometers.

Supplementary Figure 2 related to Figure 2. Presence of EVM supports pro-oncogenic traits of P GSCs in vitro.

Supplementary material and methods

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Supplementary Figure 4 related to Figure 4. GSC EV proteome predicts patients' outcome

Immunotherapy clinical trials have not shown efficacy in glioblastoma (GBM), arguably the deadliest of all cancers. A microenvironment, characterized by a paucity of T cells recognizing tumor peptides displayed on major histocompatibility complexes of tumor cells is one of the main reasons for GBM’s evasion. Virotherapy provides the opportunity to reshape the GBM TME towards pro-inflammatory phenotype. Data from a recent clinical trial with herpes simplex-1 based oncolytic virus (oHSV) have shown that the administration of these agents leads to a rapid influx of CD8 T cells, and activation of myeloid cells, suggesting that oHSV may offer an in-situ vaccination approach to increase availability of actionable antigens. C57BL/6-derived GBM models do not mimic the outcome observed with patient derived tumors in terms of virus replication. To decipher the impact of oHSV on tumor antigen specific responses, better immunocompetent models are needed. To assess whether oHSV treatment modulates the immunopeptidome profile (IP) of tumor, we used two transplantable GEMM-derived GBM models, 1620 and 1694. Both have wild type human EGFR, loss of Pten and Cdkn2a in BALB/c background. These models were more receptive to infection than CT2A. In vivo, virus persisted in tumors for up to 6 days, but not for 4 days in CT2A. IP analyses revealed that oHSV promotes epitope presentation on tumor cells and in TME. Moreover, low dose of virus efficiently induced antigen availability in both models though it differentially modulated the MHC ligandome. Using two new GBM models, we showed that an oncolytic virus used in clinical trial with recurrent GBM (NCT03152318) modulates IP of tumors, and low dose of virus is sufficient to induce the presentation of tumor epitopes. This project is supported by Bridge Fund given to Drs. Chiocca and White and P01 CA236749 to Dr. Chiocca.

Autophagy and telomere maintenance are two cellular survival processes that show a strong correlation during human ageing and cancer growth, however, their causal relationship remains unclear. In this study, using an unbiased transcriptomics approach, we uncover a novel role of autophagy genes in regulating telomere extension and maintenance pathways. Concomitantly, the pharmacological inhibition of ULK1 (Unc-51 like autophagy activating kinase 1) attenuated human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in HepG2 cells. Furthermore, the suppression of telomerase activity upon ULK1 inhibition was associated with telomere shortening and onset of cellular senescence in HepG2 cells. These results, thus, demonstrate a direct role of autophagy in maintaining cellular longevity via regulation of telomerase activity, which may have implications in the pathophysiology of ageing and cancers.

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Genes coding for protein sets that vary coherently between GSC subtypes within 89- protein signature, were retrieved from TCGA GBM dataset and identified by subtype prediction.

Supplementary Figure 3 related to Figure 3. Presence of P GSC supports pro-oncogenic traits of M GSCs in vivo

M and P GSCs display distinct intra-spherical organization in vitro. Representative time lapse movie

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Supplementary Figure 1 related to Figure 1. Analysis of GSC EV proteome heterogeneity reveals coherent molecular modules.

Abstract The composition of the tumor immune microenvironment (TIME) is considered a key determinant of patients’ response to immunotherapy. The mechanisms underlying TIME formation and development over time are poorly understood. Glioblastoma (GBM) is a lethal primary brain cancer for which there are no curative treatments. GBMs are immunologically heterogeneous and impervious to checkpoint blockade immunotherapies. Utilizing clinically relevant genetic mouse models of GBM, we identified distinct immune landscapes associated with expression of EGFR wild-type and mutant EGFRvIII cancer driver mutations. Over time, accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) was more pronounced in EGFRvIII-driven GBMs and was correlated with resistance to PD-1 and CTLA-4 combination checkpoint blockade immunotherapy. We determined that GBM-secreted CXCL1/2/3 and PMN-MDSC–expressed CXCR2 formed an axis regulating output of PMN-MDSCs from the bone marrow leading to systemic increase in these cells in the spleen and GBM tumor-draining lymph nodes. Pharmacologic targeting of this axis induced a systemic decrease in the numbers of PMN-MDSC, facilitated responses to PD-1 and CTLA-4 combination checkpoint blocking immunotherapy, and prolonged survival in mice bearing EGFRvIII-driven GBM. Our results uncover a relationship between cancer driver mutations, TIME composition, and sensitivity to checkpoint blockade in GBM and support the stratification of patients with GBM for checkpoint blockade therapy based on integrated genotypic and immunologic profiles.

Figure Legend to all supplementary data

Supplementary Figures 1-7 from ROS Fusion Tyrosine Kinase Activates a SH2 Domain–Containing Phosphatase-2/Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Signaling Axis to Form Glioblastoma in Mice

List of the 89 proteins from GSC EV proteome profile distinguishing P from M GSC subtypes; signal intensity

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.