Abstract
Clinical trials of systemic T cell checkpoint blockade in GBM patients showed only disappointing results. This may be attributed in part to the immunosuppressive components of the GBM immune ...tumor microenvironment (iTME). Therefore, major efforts have been undertaken to describe the GBM iTME on a single cell level. However, human data on the composition of the iTME in different tumor regions (contrast enhancing tumor center versus peripheral infiltration zone) remain scarce. Here, we performed high-depth single-cell RNA sequencing (scRNAseq) on patient-matched biopsies from the tumor center and the peripheral infiltration zone of five primary GBM patients. Additionally, peripheral blood mononuclear cells (PBMC) of the same patients were included in the scRNAseq analysis to explore the transcriptional changes occurring during tumor infiltration of circulating immune cells. Through analysis of > 45’000 cells, we revealed a distinct regional transcriptional profile of microglia (MG) and monocyte-derived macrophages (MdM), a non-reactive/exhausted MG subcluster in the GBM iTME and an impaired interferon-response signature in the peripheral cytotoxic cell compartment. Comparing CD8+ T-cells from the tumor periphery to PBMC-derived CD8+ T-cells of the same patient revealed a CX3CR1+ CD8+ T-cell population with effector memory phenotype which is enriched in the PBMC but lacking in the tumor periphery. Peripheral CD8+ T-cells were mainly composed of tissue-resident memory CD8+ T-cells with exhausted effector functions. Our analysis provides a large-scale dissection of GBM-associated cell types complemented by patient-matched PBMCs, serving as a high dimensional reference map of the human GBM iTME.
Abstract
Glioblastoma (GBM) is the most common primary tumor of the central nervous system in adults. GBM patients have a particularly poor prognosis and extremely short survival time due to lack of ...effective therapies and rapid tumor relapse. Neoplastic cells should be effectively recognized and destroyed by the immune system, but its antitumor activity is often inhibited by tumor-secreted factors that contribute to the tumor immunosuppressive microenvironment (TME). As TME plays a key role in cancer progression and immune evasion, understanding the interplay between GBM cells and myeloid and lymphoid populations is pivotal in creating new therapeutic strategies for GBM patients. To identify subtypes and functional diversity of immune cells in glioma TME we employed single-cell RNA and protein sequencing (CITEseq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing) and Visium (10XGenomics) spatial transcriptomics. We characterized the populations of myelo- and lymphoid cells, and examined their unique transcription profiles, functional diversity and localization in TME. By combining analysis of CITE-seq with spatial transcriptomics we characterized and described 35 phenotypes of immune cells, which then we localized spatially within TME in GL261 mouse gliomas. Our results indicated that the peripheral monocytes/macrophages (Cd49d) localized in the tumor core, while microglia (Tmem119) accumulated at the invasive edge. Moreover, we observed a ring of activated astrocytes and rare T lymphocytes dispersed around the tumor. Finally, Ligand-Receptor and CellChat analysis of our CITE-seq results revealed the interplay between GBM, myeloid cells and lymphocytes, indicated on potential factors responsible for accumulation and tumor-evoked reprograming of immune cells. Single-cell technologies provide high-resolution insights into cellular and functional heterogeneity of gliomas, the analysis of which, in the future, will provide us with new therapeutic strategies for GBM patients. Studies were supported by NSC grant 2020/39/B/NZ4/02683 (BK) and PACIFIC Call 1 PAS (MG).
Abstract
Tissue organization plays an important role in tumor development and evolution. Systematic investigation of the differences in the local composition of the tumor microenvironment (TME) in ...glioblastoma (GBM) in distinct regions of the tumor is needed for identification of traits driving tumor recurrence. Thus, we performed an analysis of three tumors, six spatially distant biopsies each, representing tumor surface, core, and deep margin. GBM cell fluorescence generated by the 5-aminolevulinic acid (5-ALA) metabolic labeling prior to surgery allowed us to enrich for the tumor cell fractions and the cells of the microenvironment, respectively, from each biopsy. Using RNA-seq and scRNA-seq we compared the transcriptomic profiles of GBM and tumor microenvironment (TME) at distinct locations within the tumor. We observed a gradient of GBM subtypes across the tumor regions, with a molecular identity progressively less defined from the tumor core towards the margins. Interestingly, the single-cell based copy number analysis revealed a subset of GBM cells that appeared to evade the 5-ALA labelling. The high prevalence of these 5-ALA-negative GBM cells within the deep tumor margins points to a metabolically distinct subpopulation that could be responsible for local tumor recurrence. The phenotypic gradient of cancer cells from tumor core towards the margins was also mirrored by tumor microenvironment-enriched fractions. Deep margin samples from the profiled tumors were transcriptionally more divergent than biopsies from other regions. Moreover, they were depleted from major populations of TME cells. In situ analysis revealed that TERT promoter mutant cells are not equally distributed across the tumor indicating the coexistence of GBM sub-clones with distinct evolutionary potential. Altogether, our results provide a novel insight into co-evolution of tumor microenvironment and GBM clonal heterogeneity within the context of the tumor tissue.
Abstract
Patients with glioblastoma (GBM) have an overall survival of 15 months. These catastrophic survival rates are correlated with systematic relapses that might arise from remaining glioblastoma ...stem cells (GSCs) left behind after treatments. Our goal was identifying whether radioresistance of GBM is caused by GSCs and how hypoxia and TAMs affect this process. First, we established a glioblastoma-brain cortical organoid (GBM-BCOs) to model human GBM. We co-cultured GFP-labeled GBM neurosphere with BCOs for 14 days and we observed that tumor take rate was 100% for all GBM neurosphere tested. Next, we evaluated whether hypoxia enhanced TAMs migration into GBM-BCOs. We incorporated THP-1 cells into GFP-GBM-BCOS for 14 days and incubated for 24 h under normoxic or hypoxic conditions. Hypoxia increased THP-1 cells influx into GBM-BCOs. Further, we investigated whether TAMs enhanced the enrichment of GSCs in GBM neurosphere and in GBM-BCOs. We found that, co-culture of THP-1 cells with GBM neurosphere or with GBM-BCOs system, enhanced CD133+ population after 72 h hypoxic condition. In addition, IF of GBM human tissue revealed increased of CD133+ and IBA1+ cells in peri-necrotic area compared to non-necrotic area and non-necrotic tumor. To examine whether TAMs enhanced the radioresistant phenotype and the quiescent population, we irradiated GBM neurosphere grown under hypoxic and normoxic conditions with different doses of γ-rays. After irradiation, these cells were co-cultured with TAMs for 72 h under normoxic and hypoxic conditions. MTT assay showed that TAMs enhanced the radioresistant phenotype promoted by hypoxia in GBM neurosphere and an increase in quiescent markers (γ-H2AX and IL-6) were found in our co-culture model after hypoxia exposure. These results indicate, not only hypoxic condition play a key role in the GSCs enrichment and radioresistance, but also crosstalk between TAMs and GSCs promote tumor oncogenesis and contribute to an ineffective anti-tumor response.
Abstract
Myeloid cells comprising up to 50% of total tumor mass in glioblastoma (GBM) contribute to tumor progression and immunosuppression. Restraining glioma-induced myeloid cell infiltration ...increases functional T cells within the tumors, improves the efficacy of checkpoint blockade, and significantly extends mouse survival. Here, we focus on an ion channel, voltage-gated proton channel Hv1, which is mainly expressed in myeloid cells and shapes the physiological functions of myeloid cells. Our bioinformatics analysis demonstrated that elevated Hv1 expression in the tumor mass correlates with poor disease prognosis in patients. Using the leading immunocompetent model of GBM in mice, we showed that Hv1 knockout mice (Hv1-/-) exhibit slower glioma progression and prolonged survival. Notably, in vivo two-photon imaging, immunofluorescence, and full-spectrum flow cytometry revealed that Hv1-/- mice have reduced monocyte/macrophage infiltration, higher frequency of MHCII+ among all infiltrating myeloid cells, and increased PD-1+CD4+ T cells in the tumor-burdened hemisphere. As a result, we combined anti-PD-1 treatment with Hv1 knockout in the middle stage of glioma, and found that approximately 30% of Hv1-/- mice were cured. Together, our results demonstrated that Hv1 regulates myeloid infiltration and could be a novel therapeutic target for the treatment of GBM.
Abstract
OBJECTIVE
The median age of onset for glioblastoma (GBM; IDHwt) is 68-70 years. Age is a strong prognostic factor for GBM patient outcomes such that overall survival in older adults is less ...than their younger counterparts – even after adjustment for MGMT promoter methylation status. Aging is associated with increased levels of senescence in the brain. Several age-related neurological disorders have been shown to improve with senolytic treatments. Here, we explored the effects and therapeutic neutralization of syngeneic brain tumors on increasing senescence levels in the extratumoral brain (ie. outside of the brain tumor) of young and old mice.
METHODS
General RNA-sequencing, as well as single-cell (sc) RNA-sequencing was performed on extratumoral tissue from young (8-12 weeks) and older adult (80-90 weeks) C57BL/6 mice with or without GL261 and key markers were validated with RT-PCR. The combined effects of the senolytics, dasatinib and quercetin, with radiation, anti-PD-1 mAb, and IDO enzyme inhibitor treatment, was also investigated.
RESULTS
General- and sc-RNA sequencing revealed a distinct gene expression profile in the extratumoral brain of older mice with syngeneic GL261 as compared to all other groups. RT-PCR results confirmed that the brain tumor increased gene expression for senescence levels, p53, and NFkB signaling in the older adult extratumoral brain. Expression of the senescence marker p16INK4A was primarily localized to oligodendrocyte progenitor cells in the older adult brain. The combinatorial treatment of senolytics with RT, anti-PD-1 mAb, and IDO enzyme inhibitor led to a synergistic survival benefit in older adult mice with GL261 as compared to the treatment with senolytics, or immunotherapy, alone.
CONCLUSIONS
The data suggest that the extratumoral brain may be responsible in-part for the poorer outcomes of older adults with GBM and that treatment approaches that target senescent cells may provide clinical benefit.
Abstract
Glioblastoma (GBM), a high-grade glial tumor, is highly aggressive and is characterized by intra-tumoral heterogeneity and widespread infiltration, impairing therapeutic success. Our ...laboratory discovered dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal features called “oncostreams” inside GBM tumors that facilitate invasion into the normal brain. We found that collagen 1α1 (Col1A1) is essential for oncostream structure and function. Using in vivo intravital imaging and ex vivo explant glioma models, we established that Col1A1 suppression abolishes oncostreams, reprograms the malignant histopathological phenotype, and extends median survival in mice. However, the signaling through which collagen communicates to maintain an invasion-permissive glioma tumor microenvironment remains unclear. We propose to analyze a poorly understood collagen receptor family -the discoidin domain receptors (DDRs) - which is expressed in glioma and perivascular stromal cells. The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) data suggest DDR1/DDR2 overexpression correlates with tumor progression and poor prognosis in glioma patients. Our RNA-seq data further confirm overexpression of DDRs in NPA and NPD glioma compared to the normal brain. Our preliminary data suggests that inhibiting collagen receptors DDR1 or DDR2 sensitizes glioma cells to radiotherapy (IR). DDR1 inhibition completely disrupts oncostreams` structure in the ex vivo explant glioma model. Moreover, pharmacological inhibition of DDR1 combined with irradiation significantly enhances the median survival of NPA (Nras, shP53, shATRX) tumors in an orthotopic mouse glioma model. Furthermore, we are currently testing whether inhibition of DDR1 using a genetically engineered mouse model (GEMM) of glioma called NPAD1 (Nras, shP53, shATRX, shDDR1) will show an increase in overall survival. We hypothesize that blocking collagen receptors DDR1 or DDR2 will control tumor growth, invasion and mediate anti-glioma immunity. This study will eventually uncover a novel therapeutic treatment for GBM, targeting DDR1 and DDR2 in human patients.
Abstract
Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median survival of only 15 months and a 5-year survival of less than 5% despite the best available treatments. ...Tumor recurrence/progression and therapy resistance are major obstacles for GBM treatment. GBM is characterized by intensive vascular proliferation that is associated with tumor cell growth, invasion, resistance to chemo/radiotherapy, and decreased disease-free survival. Increasing data have shown the existence of cells with endothelial characteristics called tumor derived endothelial cells, which come from the transdifferentiation of GBM cells and, more specifically, from GBM stem cells (GSCs). However, the molecular mechanisms underlying this process remain largely unknown. GSCs are tightly regulated by distinct transcriptional programs that are driven by nuclear transcription factors. Brachyury is a transcription factor expressed in normal, undifferentiated embryonic notochord in the axial skeleton and plays an important role in stem cell development and differentiation during normal embryonic development. Here, we show that brachyury is highly expressed in patient-derived GBM cells. Functional studies demonstrate that brachyury is regulated by fibroblast growth factor receptor 1 (FGFR1)/mitogen-activated protein kinase (MAPK). Our studies further disclose that FGFR1/MAPK-directed brachyury activation promotes GSC survival and endothelial formation via vascular endothelial growth factor receptor 2 (VEGFR2), whereas pharmacological inhibition of FGFR1 and MAPK or shRNA-mediated downregulation of brachyury decreases GSC transdifferentiation into endothelial cells and suppresses GBM cell growth and stemness via VEGFR2. Our findings highlight the importance of FGFR1/MAPK-regulated brachyury activation in GSC transdifferentiation into endothelial cells via VEGFR2, and targeting the FGFR1-MAPK-brachyury-VEGFR2 signal pathway may represent a novel therapeutic strategy by reprogramming GSCs driving vascular transdifferentiation and tumor progression.
Abstract
INTRODUCTION
Patients with glioblastoma have a very poor prognosis. Tumor-associated microglia and macrophages (TAMs) constitute up to 30 % of the cells in glioblastoma, and they secrete ...cytokines, chemokines and growth factors that influence the microenvironment. The existence of different TAM subtypes appears to be more complex than the established M1 and M2 phenotypes, but their role in glioblastoma is not fully understood and rarely considered therapeutically. This could explain why many clinical trials fail despite of promising preclinical results. This project aims to interrogate the existence and characteristics of different TAM subtypes in human glioblastoma in order to identify novel subpopulations and therapeutic targets. MATERIALS AND
METHODS
Live CD11b+ TAMs were isolated from patient glioblastoma tissue, and single-cell RNA sequencing (scRNA-seq) was performed using the 10X Genomics Chromium platform. The data was processed and analyzed with the R-package Seurat.
RESULTS
We have sequenced 74,000 TAMs/microglia from three glioblastomas and two control brain biopsies. In the controls, we detected mostly microglia, while the primary glioblastomas showed a predominance of monocyte-derived TAMs. We identified 11 TAM subtypes, such as hypoxic, proliferating, interferon-induced, chemokine-producing and TNF-producing TAMs, as well as a novel subtype potentially involved in tumor progression.
CONCLUSION AND PERSPECTIVES
We have detected a spectrum of TAM subtypes, which is more complex than the established M1 and M2 phenotypes. Our findings confirm a recent TAM scRNA-seq study, and in addition, we identify a novel subpopulation, which express known tumor-promoting genes, normally expressed by cancer cells. We are currently validating our findings. This study contributes to the elucidation of the glioblastoma microenvironment. It will potentially lead to identification of novel clinically relevant targets affecting the TAM-glioblastoma interactions.
Abstract
Medulloblastoma (MB) is the most common pediatric brain malignancy. MB comprises 5 major subgroups known as WNT, SHH p53wt, SHH p53mut, Group 3 and Group 4. Among the four MB subgroups SHH ...group is the most dominant molecular subgroup in infants and adults. These tumors are proposed to arise from cerebellar granule neuron precursors (CGNPs), whose developmental expansion requires SHH signaling from the neighboring Purkinje neurons. Previous reports suggest that SHH group features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB cells interact with Tumor Microenvironment, we performed cytokine array analysis of culture media from SHH group Patient Tumor cells, spontaneous SHH MB mouse tumor cells and SHH MB cell lines. Further, confirmed these results using ELISA, Western blot, and immunofluorescence from human SHH MB cell lines, Smo/A1 mouse tumor primary cells and PZp53Med cell lines. In continuation to the observation of IGFBP2 expression in various cell types in single cell analysis, we analyzed the presence of IGFBP2 in astrocytes using Smo/A1 mouse tumor Immunohistochemistry. Our data showed increased levels of IGFBP2 produced by SHH MB cell lines compared to others. We analyzed the role of IGFBP2 in SHH MB tumor growth and metastasis. IGFBP2 knock-down stable cell lines showed phenotypic changes including reduced cell proliferation, cell migration and EMT. Further western blot analysis of IGFBP2 KD cells showed reduced expression of EMT markers also reduced the activation of STAT3. Our preliminary in vitro data suggest IGFBP2 exerts it metastasis-promoting role in SHH MB by regulating the expression of EMT marker proteins and matrix remodeling proteins. Further functional studies suggest that in SHH MB, IGFBP2 may regulate a STAT3-mediated EMT program to metastasize. These findings provide a strong rationale for further pursuing how IGFBP2 promotes medulloblastoma tumor cell growth and migration in vivo.
