Chitinase-3-like protein 1 (CHI3L1) is a secreted, non-enzymatic glycoprotein that interacts with cell-surface and extracellular-matrix proteins, proteoglycans, and polysaccharides. Many studies reported the overexpression of CHI3L1 in various cancers, but its exact role in tumorigenesis/cancer progression remains elusive. We performed a comprehensive analysis of CHI3L1 expression in public repositories including single-cell RNAseq datasets to determine the cellular source of CHI3L1 expression in gliomas. The highest CHI3L1 expression was detected in glioblastoma (GBM), a high-grade diffusive brain tumor with dismal survival prognosis. CHI3L1 knockout (KO) in human U87-MG glioma cells grossly affected transcriptional profiles and in vitro invasiveness of these cells, and strongly reduced the growth of intracranial U87-MG tumors in athymic mice. CHI3L1 KO in glioma cells resulted in normalization of tumor vasculature. Co-culture of CHI3L1 KO glioma cells with astrocytes upregulated aquaporin 4 (AQP4) in p38/MAPK and IL-1β/NFκB-dependent manner. Diminished infiltration of glioma-associated myeloid cells in CHI3L1 KO tumors was associated with reduction in SPP1 expression in CHI3L1 KO cells. Altogether, we demonstrate that CHI3L1 depletion affects several mechanisms crucial for GBM progression, therefore its targeting represents a novel strategy to treat GBM patients.

Glioblastoma (GBM) is a highly diffusive tumor which restricts the efficacy of surgical resection and facilitates tumor recurrence. Matrix metalloproteinase 2 (MMP2) is the extracellular matrix protease that is activated by MMP14 expressed by microglia and plays a central role in tissue remodeling and tumor progression. The R132H mutation in isocitrate dehydrogenase 1 (IDH1R132H) is commonly observed in gliomas, results in hypermethylator phenotype and in GBMs is associated with better survival. The impacts of IDH1R132H on the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. We employed unique mouse glioma models which mimic human GBM pathology, coupled with high-dimensional CITEseq and spatial transcriptomics to analyze the immune landscape in different gliomas. Additionally, we performed gelatin zymography, qPCR, wound healing, and DNA methylation qPCR techniques to validate the obtained results. Mmp2 expression and activity varied with IDH1 status in experimental gliomas. Spatial transcriptomics demonstrated that Mmp2 expression is localized within the IDH1 wild-type tumors and absent in IDH1 mutated ones. Gelatin zymography showed the presence of Mmp2 activity in extracts from IDH1 wild-type tumors. Methylation of Mmp2 gene promoter was increased and resulted in decreased Mmp2 expression in IDH1 mutated gliomas. The IDH1 wild-type gliomas showed more diffuse growth patterns (tumors were stained with Nestin antibody) whereas IDH1 mutant gliomas showed more constrained growth patterns. In the TCGA glioma dataset, methylation of the MMP2 gene promoter was increased which was associated with decreased MMP2 expression in IDH1 mutated gliomas. We found that the IDH1 mutation results in hypermethylation of the Mmp2 gene promoter, leading to decreased Mmp2 expression and Mmp2 activity in experimental gliomas and human GBMs. Reduced Mmp2 activity and decreased tumor invasion result in more restricted tumor growth, which may facilitate resection, and radiotherapy, thereby improving patient survival. Studies were supported by National Science Center Poland grant 2020/39/B/NZ4/02683.

Astrocytes comprise ~50% of all brain cells and present distinct morphological, molecular and functional properties in different brain regions. In glioblastoma (GBM), an aggressive primary brain tumor, astrocytes become activated and tumor-associated astrocytes (TAAs) exhibit different transcriptomic profiles, morphology, and functions supporting disease progression. Heterogeneity and specific roles of TAAs within various regions of tumors are poorly known. Advancements of single-cell and spatial transcriptomics allow to profile tumors at unprecedented resolution revealing cell phenotypes, hidden functionalities, and spatial architecture in disease-specific context. We combined spatial transcriptomics and multiple immunofluorescent staining to visualize TAAs heterogeneity and location of various subpopulations in three intracranial murine glioma models. Using distinct gene expression profiles, we identified subtypes of TAAs with distinct localization and inferred their specialized functionalities. Gene signatures associated with TAAs reflected their reprograming in the tumor microenvironment (TME), revealed their multiple roles and potential contributing factors shaping the local milieu. Using spatial correlation analysis of the spots, we inferred the interactome of Slc1a2 (encoding a glutamate transporter) with the other markers of TAAs based on segregated areas of the tumor. The designer RGD peptide that blocked tumor-microglia communications, altered the spatial distribution of TAAs in GL261 gliomas providing new insights into cell-to-cell communication. Spatial transcriptomics combined with multiple staining unveiled multiple functional phenotypes of TAAs and interactions within TME. Altogether, we demonstrate distinct morphology of TAAs and different roles in various regions of the tumor. Glioma-induced heterogeneity of TAAs allows adaptation to the pharmacologically induced modification of the immunosuppressive TME.

Immune checkpoint inhibitors (ICIs) present clinical benefits in many cancer patients but invariably fail in glioblastoma (GBM), the most common and deadly primary brain tumor. The lack of ICIs efficacy in GBM is attributed to the accumulation of tumor-reprogrammed glioma-associated myeloid cells (GAMs) that create a “cold” immunosuppressive tumor microenvironment (TME), impeding the infiltration and activation of effector T cells. GBM-derived αvβ3/αvβ5-integrin ligands, including SPP1, were shown to mediate the emergence of GAMs. We hypothesized that a combination strategy aiming to block the reprogramming of GAMs using a synthetic 7aaRGD peptide that targets SPP1/integrin signaling might overcome resistance to ICIs and reinvigorate anti-tumor immunity. Matrigel invasion assay was used to test the efficacy of 7aaRGD in glioma-microglia co-cultures. We determined the impact of 7aaRGD, administered as a monotherapy or combined with PD-1 blockade, on tumor growth, GAMs accumulation and phenotypes, arginase-1 levels and neovasculature in experimental gliomas. The effects of treatments on the tumor immune landscape were dissected using multiparameter flow cytometry, immunocytochemistry, cytokine profiling and RNA-seq analysis of sorted GAMs followed by CITE-seq based data deconvolution. 7aaRGD efficiently blocked microglia-dependent invasion of human and mouse glioma cells in vitro. Intratumorally delivered 7aaRGD alone did not reduce tumor growth in orthotopic gliomas but prevented the emergence of immunosuppressive GAMs and led to normalization of peritumoral blood vessels. Combining 7aaRGD with anti-PD-1 antibody resulted in reduced tumor growth, with an increase in the number of proliferating, interferon-ɣ producing CD8+T cells and depletion of regulatory T cells. Transcriptomic profiles of myeloid cells were altered by the combined treatment, reflecting the restored “hot” inflammatory TME and boosted immunotherapy responses. Intratumoral administration of 7aaRGD similarly modified the phenotypes of GAMs in human U87-MG gliomas in immunocompromised mice. Exploration of transcriptomic datasets revealed that high expression of integrin receptor coding genes in pre-treatment biopsies was associated with a poorer response to immune check-point blockade in patients with several types of cancers. We demonstrate that combining the blockade of SPP1/integrin signaling with ICIs modifies innate immunity and reinvigorates adaptive antitumor responses, which paves the way to improve immunotherapy outcomes in GBM.

Immune checkpoint inhibitors (ICI) presented clinical benefits in many cancer patients but invariably fail in glioblastoma (GBM), the most common and deadly primary brain tumor. Lack of ICI efficacy in GBM is attributed to the accumulation of immunosuppressive myeloid cells that create the “cold” tumor microenvironment (TME) impeding infiltration and activation of effector T cells. We developed a designer RGD peptide that hindered glioma-instigated, integrin-mediated pro-tumoral reprogramming of myeloid cells and blocked microglia-dependent invasion of human and mouse glioma cells in co-cultures in vitro . Intratumorally-delivered RGD alone did not reduce glioma growth in syngeneic mice but prevented the emergence of immunosuppressive myeloid cells and led to peritumoral blood vessels normalization. Furthermore, combining RGD with immunotherapy using PD-1 blockade reduced tumor growth, led to upsurge of proliferating, interferon-ɣ producing CD8+T cells and depleted regulatory T cells. Transcriptomic profiles of myeloid cells were altered by the combined treatment, consistently with the restored “hot” inflammatory TME and boosted immunotherapy responses. RGD modified the phenotypes of myeloid cells in human gliomas in nude mice. Thus, combining the integrin blockade with ICI reinvigorates antitumor immunity and paves the way to improve immunotherapy outcomes in GBM. ### Competing Interest Statement The authors have declared no competing interest.

Chitinase-3-like protein 1 (CHI3L1) is a secreted, non-enzymatic glycoprotein that interacts with cell-surface and extracellular-matrix proteins, proteoglycans, and polysaccharides. Many studies reported the overexpression of CHI3L1 in various cancers, but its exact role in tumorigenesis/cancer progression remains elusive. We performed a comprehensive analysis of CHI3L1 expression in public repositories including single-cell RNAseq datasets to determine the cellular source of CHI3L1 expression in gliomas. The highest CHI3L1 expression was detected in glioblastoma (GBM), a high-grade diffusive brain tumor with dismal survival prognosis. CHI3L1 knockout (KO) in human U87-MG glioma cells grossly affected transcriptional profile and in vitro invasiveness of these cells and strongly reduced the growth of intracranial U87-MG tumors in athymic mice. CHI3L1 KO in glioma cells resulted in normalization of tumor vasculature. Co-culture of CHI3L1 KO glioma cells with astrocytes upregulated Aquaporin 4 in p38 MAPK-dependent manner. Diminished infiltration of glioma-associated myeloid cells in CHI3L1 KO tumors was associated with reduction in SPP1 expression in CHI3L1KO cells. Altogether, we demonstrate that CHI3L1 depletion affects several mechanisms crucial for GBM progression, therefore its targeting represents a novel strategy to treat GBM patients.

Abstract Clinical trials with immune checkpoint inhibitors (ICI) have benefited many cancer patients but failed in a number of tumors, including glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. The main obstacle for ICI efficacy in GBM is the continuously evolving tumor microenvironment (TME), in which antitumor immunity is inhibited or eluded by tumor-secreted factors. Accumulation and reprogramming of myeloid cells (GAMs) creates a “cold” immunosuppressive TME with a poor infiltration and exhaustion of effector T cells. We employ Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) with 40 protein markers along with spatial transcriptomics and immunophenotyping to dissect identities and functionalities of immune cells (CD45+) in experimental GL261 gliomas. The results revealed identities of immune cells instrumental for creating the immunosuppressive milieu and cell-cell communication networks. We have developed a designer RGD peptide that blocks the reprogramming of GAMs by targeting tumor-GAMs interactions. We demonstrate that RGD efficiently blocks microglia-dependent invasion of murine and human glioma cells in vitro. We explored if the intratumorally delivered RGD can revert tumor-induced changes in the TME of experimental gliomas and improve anti-PD-1 immunotherapy. While RGD alone did not reduce tumor growth in vivo, it prevented reprogramming of myeloid cells into protumoral GAMs and led to the normalization of peritumoral blood vessels. Combining RGD with anti-PD-1 antibody resulted in reduced tumor growth, increase in percentages of proliferating, interferon-ɣ producing CD8+T cells and depletion of regulatory T cells. Transcriptomic profiles of GAMs were altered by the combined treatment, consistently with the restored “hot” inflammatory TME which resulted in boosting immunotherapy responses. Intratumorally delivered RGD modified in the same way functionalities of GAMs in the TME of human U87MG gliomas in nude mice. Our results demonstrate that the integrin blockade combined with immune checkpoint inhibitors reinvigorates both innate and adaptive antitumor immunity. The results pave the way to improve responses to immunotherapy in GBM and other cancers.

Astrocytes comprise ~50% of all brain cells and present distinct morphological, molecular and functional properties in different brain regions. In glioblastoma (GBM), an aggressive primary brain tumour, tumour-associated astrocytes (TAAs) become activated and exhibit different transcriptomic profiles, morphology and functions supporting disease progression. Heterogeneity and specific roles of TAAs within various regions of tumours are poorly known. Advancements of single-cell and spatial transcriptomics allow to profile tumours at unprecedented resolution revealing cell phenotypes, hidden functionalities and spatial architecture in disease-specific context. We combined spatial transcriptomics and multiple immunofluorescent staining to visualize TAAs heterogeneity and location of various subpopulations in intracranial murine gliomas. Using distinct gene expression profiles, we identified subtypes of TAAs with distinct localization and inferred their specialized functionalities. Gene signatures associated with TAAs reflected their reprograming in the tumour microenvironment (TME), revealed their multiple roles and potential contributing factors shaping the local milieu. Using spatial correlation analysis of the spots, we inferred the interactome of Slc1a2 (encoding a glutamate transporter) with the other markers of TAAs based on segregated areas of the tumour. The designer RGD peptide blocking tumour-microglia communications, alters the spatial distribution of TAAs in experimental gliomas providing insights into potential mechanisms. Spatial transcriptomics combined with multiple staining unveils multiple functional phenotypes of TAAs and interactions within TME. It shows their distinct morphology and unveils different roles in various regions of the tumour. We demonstrate the glioma-induced heterogeneity of TAAs and their adaption to the pharmacologically-induced modification of the immunosuppressive TME. ### Competing Interest Statement The authors have declared no competing interest.

Immune checkpoint inhibitors (ICI) presented clinical benefits in many cancer patients but invariably fail in glioblastoma (GBM), the most common and deadly primary brain tumor. Lack of ICI efficacy in GBM is attributed to the accumulation of immunosuppressive myeloid cells that create the 'cold' tumor microenvironment (TME) impeding infiltration and activation of effector T cells. We developed a designer RGD peptide that hindered glioma-instigated, integrin-mediated pro-tumoral reprogramming of myeloid cells and blocked microglia-dependent invasion of human and mouse glioma cells in co-cultures in vitro. Intratumorally-delivered RGD alone did not reduce glioma growth in syngeneic mice but prevented the emergence of immunosuppressive myeloid cells and led to peritumoral blood vessels normalization. Furthermore, combining RGD with immunotherapy using PD-1 blockade reduced tumor growth, led to upsurge of proliferating, interferon-ɣ producing CD8+T cells and depleted regulatory T cells. Transcriptomic profiles of myeloid cells were altered by the combined treatment, consistently with the restored 'hot' inflammatory TME and boosted immunotherapy responses. RGD modified the phenotypes of myeloid cells in human gliomas in nude mice. Thus, combining the integrin blockade with ICI reinvigorates antitumor immunity and paves the way to improve immunotherapy outcomes in GBM.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glio-blastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.

Abstract Chitinase-3-like protein 1 (CHI3L1) is a secreted, non-enzymatic glycoprotein that binds proteins and carbohydrates and interacts with cell-surface and extracellular-matrix proteins, proteoglycans, and polysaccharides. Multiple interacting partners of CHI3L1 make dissection of its functions challenging. While many studies reported an upregulation of CHI3L1 mRNA/protein in various tumors, its exact roles in tumorigenesis remain elusive. We performed a comprehensive analysis of CHI3L1 expression in multiple public datasets including TCGA and single-cell RNAseq datasets to determine the cellular source of CHI3L1 expression in gliomas. The highest CHI3L1 mRNA/protein levels were detected in glioblastoma (GBM), a high-grade diffusive brain tumor. CHI3L1 knockout in human U87-MG glioma cells grossly affected transcriptional profile and in vitro invasiveness of these cells and strongly reduced the growth of intracranial U87-MG tumors in athymic mice. Remarkably, CHI3L1 knockout in glioma cells resulted in normalization of tumor vasculature and diminished infiltration of glioma-associated myeloid cells. Mechanistically, CHI3L1 depleted cells had reduced MMP2 expression/activity, which was associated with reduced invasion; and downregulated SPP1 (osteopontin), a crucial factor driving myeloid cell accumulation in GBM. Altogether, we demonstrate that CHI3L1 is a key player in GBM progression, and its targeting represents a novel strategy to treat GBM patients.

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).

Immunotherapies with immune checkpoint inhibitors or adoptive cell transfer have become powerful tools to treat cancer. These treatments act via overcoming or alleviating tumor-induced immunosuppression, thereby enabling effective tumor clearance. Glioblastoma (GBM) represents the most aggressive, primary brain tumor that remains refractory to the benefits of immunotherapy. The immunosuppressive immune tumor microenvironment (TME), genetic and cellular heterogeneity, and disorganized vasculature hinder drug delivery and block effector immune cell trafficking and activation, consequently rendering immunotherapy ineffective. Within the TME, the mutual interactions between tumor, immune and endothelial cells result in the generation of positive feedback loops, which intensify immunosuppression and support tumor progression. We focus here on the role of aberrant tumor vasculature and how it can mediate hypoxia and immunosuppression. We discuss how immune cells use immunosuppressive signaling for tumor progression and contribute to the development of resistance to immunotherapy. Finally, we assess how a positive feedback loop between vascular normalization and immune cells, including myeloid cells, could be targeted by combinatorial therapies with immune checkpoint blockers and sensitize the tumor to immunotherapy.

Immune checkpoint blockers (ICBs) have revolutionized the treatment of some solid cancers but have failed to benefit the majority of glioblastoma (GBM) patients. Two reasons underlying limited ICB benefit are: 1) immune-related adverse events, and 2) resistance conferred by the tumor microenvironment. Here, we show that ICBs induce cerebral edema in patients and GBM mouse models. This edema results from an inflammatory response to ICB treatment that disrupts the blood-tumor-barrier, as confirmed by intravital imaging, mechanistic blocking studies, and single-cell RNA sequencing. Losartan – a commonly prescribed antihypertensive agent – controls ICB-induced edema, reprograms the immunosuppressive tumor microenvironment, and improves survival under ICB therapy. In combination with a standard of care regimen in mice mimicking clinical treatment (surgical resection, chemoradiation), losartan increases the percent of long-term surviving (cured) mice under ICB therapy from 16% to 43%. Finally, a bihemispheric “resect-and-response” model to establish predictive biomarkers from the tumor microenvironment reveals that cured mice have an immunostimulatory (“hot”) immune tumor compartment prior to therapy. These results provide the basis for clinical testing of adding to losartan to ICB treatment for GBM patients.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.

Abstract The compression of brain tissue by a tumor mass is believed to be a major cause of the clinical symptoms seen in patients. However, the biological consequences of these physical stresses on the brain tissue are unknown. Using clinical imaging and preclinical studies, we discovered that a subgroup of primary and metastatic brain tumors, classified as nodular based on the growth pattern, exert compressive solid stress on the surrounding brain tissue, leading to a decrease in local vascular perfusion, as well as neuronal death and impaired function. We demonstrated a causal link between solid stress and neurological dysfunction, by applying and removing cerebral compression, mimicking the mechanics of tumor growth and surgical resection respectively. Finally, we showed that treatment with lithium reduced solid stress-induced neuronal death and improved motor coordination in mice. Our results indicate that brain tumor-generated solid stress impairs neurological function in patients and show lithium as a potential therapeutic intervention to counter these effects.

The compression of brain tissue by a tumour mass is believed to be a major cause of the clinical symptoms seen in patients with brain cancer. However, the biological consequences of these physical stresses on brain tissue are unknown. Here, via imaging studies in patients and by using mouse models of human brain tumours, we show that a subgroup of primary and metastatic brain tumours, classified as nodular on the basis of their growth pattern, exert solid stress on the surrounding brain tissue, causing a decrease in local vascular perfusion as well as neuronal death and impaired function. We demonstrate a causal link between solid stress and neurological dysfunction by applying and removing cerebral compression, which respectively mimic the mechanics of tumour growth and of surgical resection. We also show that, in mice, treatment with lithium reduces solid-stress-induced neuronal death and improves motor coordination. Our findings indicate that brain-tumour-generated solid stress impairs neurological function in patients, and that lithium as a therapeutic intervention could counter these effects.

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

Water soluble fluorescent carbon nano onions (wsCNO) cross the blood brain barrier (BBB) in the CADASIL murine model as well as in GBM induced mice.