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
Mitochondrial transfer occurs both in stroke (central nervous system) and inflammatory pain (peripheral nerves). However, its role in glioblastoma (GBM) remains poorly understood. We ...hypothesized that mitochondrial transfer from non-malignant to GBM cells supports tumor metabolism and growth. Using transgenic mice expressing fluorophore-tagged mitochondria, we found that ~50% of orthotopically-implanted mouse GBM cells acquire mitochondria. Brain-resident cells, especially astrocytes, were the primary mitochondrial donors in vitro and in vivo. Mitochondrial transfer also occurred from immortalized human astrocytes to patient-derived xenograft (PDX) models in vitro at rates of 15-35%. GBM cells that acquired mitochondria expressed higher levels of the ATP-synthase subunit ATP5A and produced more ATP, while metabolomics revealed multiple upregulated pathways in recipient cells. These data point to increased metabolic activity in recipient cells. In vivo, mouse GBM cells that acquired mitochondria were more likely to be in S/G2/M cell cycle phases. We observed a similar effect in PDX that acquired astrocyte mitochondria in vitro, suggesting that transfer drives GBM proliferation. Using sorted mouse and human GBM cells with/without in vitro astrocyte mitochondrial acquisition, we found that mitochondrial transfer promoted in vitro self-renewal and in vivo tumorigenicity, leading to significant reduction in survival and increased penetrance in orthotopic GBM models. Transfer in mouse and human systems was contact-dependent and was abrogated by physical separation of donor and recipient cells by transwell inserts. Pharmacologic inhibition of cytoskeleton and gap junctions did not affect transfer rate, while blocking growth-associated protein 43 (GAP43) function by c-Jun N-terminus kinase inhibition decreased transfer rate by 15-30%, suggesting a potential role of GAP43. Taken together, mitochondrial transfer comprises a fundamental, protumorigenic mechanism of GBM, enhancing metabolic activity and driving tumor cell proliferation. Elucidating the molecular machinery regulating astrocyte mitochondrial transfer and its downstream protumorigenic effects will lead to therapeutic opportunities targeting this understudied tumor microenvironment interaction.
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
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.
Abstract
All GBM molecular subsets share the common trait of accelerated progression following necrosis which cannot be adequately explained by cellular proliferation arising from accumulated genetic ...alterations. We suggest that development of necrosis is much more than a passive phenomenon related to rapid growth but rather is a driving force behind TME restructuring responsible for sustaining accelerated expansion. Current tumor models fail to adequately mimic the magnitude of post-necrotic restructuring within the microenvironment and remain overly reliant on post-mortem analyses, obligating researchers to extrapolate causal relationships between necrosis and progression phenomena during tumor evolution. In GBM (WHO grade 4), the most malignant primary brain tumor, vascular pathology and central necrosis precede rapid, radial expansion. Mechanisms enabling selective fitness within a hypoxic/anoxic GBM setting remain poorly understood. Nanostring GeoMX spatial profiling demonstrates M1-like TAM enrichment in non-necrotic human samples and M2-like TAMs in perinecrotic regions. Our immunocompetent RCAS/tv-a model aptly captures events seen in human gliomas, exposing dynamic temporal and spatial changes that facilitate GBM progression, incorporating unique microenvironmental stressors typically absent from GBM animal models, specifically emerging central necrosis. Simultaneously, our in vitro models scrutinize how hypoxia-dependent signaling between GBM cells, microglia and monocytes alter TAM accumulation and function in the TME. TAMs increase dramatically with the onset of necrosis, with a preferential localization to the hypoxic zone in the perinecrotic niche, which supports their survival. Flow cytometry on digested pre- and post-necrotic GBMs, showed increased TAM accumulation at 6 weeks vs. 2 weeks (necrosis emerges in week 4-5). Our in vivo data along with in silico analysis of Ivy GAP and TCGA datasets suggests GBM cells within the peri-necrotic niche attract and polarize TAMs through MIF secretion, necrotic DAMP (adenosine, S100B) release, and arginase secretion to suppress T cells.
Abstract
INTRODUCTION
Glioblastoma (GBM) typically recurs after standard of care therapies. A major obstacle is that GBMs employ various mechanisms to suppress the host immune system, preventing ...destruction and removal of cancer cells. A better understanding of the crosstalk mechanisms between tumor and immune cell types is needed to advance immunotherapeutic approaches against GBM. GBMs contain primary cilia, organelles likened to both cellular ‘antennae’ and transmitters, and their presence is associated with more aggressive and treatment resistant tumors.
OBJECTIVE
To explore whether ciliated GBM cells associate with infiltrating immune cells and to determine if these interactions are specific for certain immune cell populations.
METHODS
We immunostained GBM patient specimens and sections from syngeneic mouse models of GBM for markers of cilia (ARL13B and gTub) together with various immune cell markers (CD45, CD11b, Ly-6B.1, CD3). Cilia were also examined in brain tumors generated in CCR2+/rfp/CX3CR1+/gfp mice, to evaluate the proximity of glioma cell cilia to CCR2- and CX3CR1-expressing myeloid cell subpopulations (brain resident microglia as well as peripheral-derived macrophages and monocyctic- MDSCs (M-MDSCs). Proximity of tumor cell cilia to different markers or cell types was quantified using nearest neighbor analyses.
RESULTS
In patient samples, CD45+ cells extended processes that contacted the tumor cilia and cilia tip. Similar observations were made in intracranial GBM-bearing mice where we detected juxtaposition of cilia with recruited immune cells (i.e. CD45, CD11b and Ly-6B.1). Notably, cilia predominately associated with immune-suppressive M-MDSCs. Further, CD3+ T cells rarely juxtaposed tumor cilia and maintained greater distances away from ciliated tumor cells compared to M-MDSCs.
CONCLUSION
Our data suggest specific interactions between ciliated tumor cells and select immune-suppressive cell types, raising the possibility immunologically cold tumors may contain more ciliated cells. Ongoing studies are examining how the tumor immune cell landscape develops depending on the presence of tumor cilia.
Abstract
The symbiotic interactions between cancer stem cells and the tumor microenvironment (TME) are critical for tumor progression. However, the molecular mechanism underlying this symbiosis in ...glioblastoma (GBM) remains enigmatic. Here, we show that circadian locomotor output cycles kaput (CLOCK) and its heterodimeric partner brain and muscle ARNT-like 1 (BMAL1) in glioma stem cells (GSCs) drive immunosuppression in GBM. Integrated analyses of the data from transcriptome profiling, single-cell RNA sequencing, and TCGA datasets, coupled with functional studies, identified legumain (LGMN) as a direct transcriptional target of the CLOCK–BMAL1 complex in GSCs. Moreover, CLOCK-directed olfactomedin-like 3 (OLFML3) upregulates LGMN in GSCs via the hypoxia-inducible factor 1-alpha (HIF1A) signaling. Consequently, LGMN promotes microglial infiltration into the GBM TME via upregulating CD162, and polarizes infiltrating microglia towards an immune-suppressive phenotype. In GBM mouse models, inhibition of the CLOCK–OLFML3–HIF1A–LGMN–CD162 axis reduces intratumoral immune-suppressive microglia, increases CD8+ T cell infiltration, activation and cytotoxicity, and synergizes with anti-PD1 therapy. In human GBM, the CLOCK-regulated LGMN signaling correlates positively with microglial level and poor prognosis. Together, these findings uncover the CLOCK–OLFML3–HIF1A–LGMN axis as a molecular switch that controls microglial biology and immunosuppression, thus revealing potential new therapeutic targets for GBM patients.
Abstract
We show the therapeutic potential of myeloid-directed immunomodulatory treatment for medulloblastoma. Patients with medulloblastoma, the most common malignant pediatric brain tumor, need new ...treatments, as standard therapy produces disabling neurotoxicities and fails 20% of patients. About 30% of medulloblastomas show hyperactivation of the SHH (Sonic Hedgehog) signaling pathway, and these tumors have large myeloid populations in the tumor microenvironment (TME). We analyzed whether activating toll-like receptors on these myeloid cells would slow tumor growth. Our prior single-cell RNA sequencing studies in both patient samples and mouse models showed that 10% of the cells in SHH medulloblastoma are myeloid cells and that these cells uniquely express TLR7 and TLR8. We treated mice genetically engineered to develop SHH medulloblastomas with the TLR7/8 agonist resiquimod, administered systemically either as free drug or in polyoxazoline nanoparticles (POx-resiquimod), and compared POx-resiquimod+radiotherapy to radiotherapy alone. We found that POx-resiquimod extended the survival time of mice with medulloblastoma, while free drug failed to show benefit. PK studies showed that POx-resiquimod increased tumor drug exposure, consistent with increased efficacy. Mechanistically, POx-resiquimod increases tumor myeloid populations and decreased the fraction of myeloid cells that expressed IGF1. POx-resiquimod plus radiation therapy, moreover, was superior to either POx-resiquimod alone, or radiotherapy alone. Together our data show that the TLR7/8 agonist resiquimod, delivered in nanoparticle formulation, produces a significant anti-tumor effect in SHH medulloblastoma, and increased the efficacy of radiotherapy. As radiotherapy is the mainstay of current medulloblastoma treatment, we propose that TLR7/8-agonist therapy such as resiquimod may be added to current regimens to reduce the radiation dose needed for efficacy, and to increase the fraction of successfully treated patients.
Abstract
Characterizing intra- and inter-tumoral heterogeneity of glioblastoma has historically relied on discrete classifications of malignant cell populations leaving immune and other cell ...populations, known to exist admixed with the malignant tumor cells, relatively neglected. Manifold learning algorithms can manage deconvolving multiple cell populations and are often used to track cell state transitions in single cell transcriptomics. We applied a manifold learning approach to TCGA microarray data (Nf525) and bulk transcriptomics of 134 image localized biopsies across 30 patients with primary and 9 with recurrent glioblastoma to further elucidate how to organize biopsies across a spectrum of possible tissue states. The algorithm revealed a low-dimensional manifold graph for which each biopsy lives across 3 polarizing tissue states - one that is associated with diffusely invaded brain, one that is enriched in mesenchymal genes, and one that is enriched in classical proliferative tumor signatures. We deconvolved the bulk transcriptomics of the image-localized biopsies to reveal the relative abundance of 18 malignant, immune, and other cell subpopulations in each biopsy. Overlaying the cellular decomposition onto the manifold graph visualizing the tissue state distributions revealed that transitions between states correlate with changes in cellular cohabitation composition. The tumor cellular cohabitation ecologies have the lowest diversity, as inferred by ecological measures such as Shannon entropy and evenness, at the distal poles of the graph when compared to the transitional arms. Further, we found that the relationship between imaging appearance of contrast enhancement on T1-weighted MRI and the biopsy cellular composition varies with sex and primary vs recurrent biopsy status. The limited spectrum of possible tissue states revealed by the manifold learning is suggestive of a limited continuum along which tumor and non-tumoral cell populations can cohabitate. Such a limited low-dimensional biological space may constrain the dynamics of tumor biology in a predictable manner.
Abstract
Glioblastoma (GBM) is the most common primary malignant brain tumor and shows poor outcomes as a median survival is 12-18 months with current standard-of-care of therapy. GBM exhibits sex ...differences in incidence and overall survival, with males experiencing a higher incidence and a worse prognosis compared to females. However, the role of immune cells in GBM sex differences outside of myeloid cells remains poorly understood. Using a syngeneic mouse GBM model, we recapitulated the sex differences observed in patients, with shortened survival in male hosts compared to female hosts. These findings were not recapitulated in immuno-deficient mouse strains such as NSG and RAG1KO mice, suggesting a role for immune system, specifically T cells, in GBM sex differences. Flow cytometry analysis of tumor-infiltrating leukocytes revealed that more T cells were found in female tumors, whereas male tumors were enriched in macrophages. Additionally, more T cells in male tumors exhibited high levels of inhibitory receptors, whereas most female T cells were more functional as measured by expression of anti-tumor cytokines such as TNF, IFN-gamma, and granzyme B. A bone marrow chimera model (BMC) revealed that male T cells retained their phenotype in female hosts, whereas female T cell behavior was affected by the male environment. Yet survival analysis on BMC model and adoptive transfer model suggests strong immune cell-intrinsic effect on controlling tumor progression, further supported by in vitro T cell exhaustion model. Lastly, we found that males have more progenitor exhausted T cells, which led to better response to anti-PD1 treatment. Collectively, these results suggest that both cell-intrinsic and cell-extrinsic factors regulate T cell activity in a sex-specific manner, providing insights to develop sex-specific therapeutic approaches.
