Glioblastoma (GBM) is the most aggressive form of glioma with a high rate relapse despite intensive treatment. Tumor recurrence tightly linked to radio-resistance, which in turn associated hypoxia. Here, we discovered strong link between hypoxia and local complement signaling using publicly available bulk, single-cell, spatially resolved transcriptomic data from patients GBM. Complement component 3 (C3) receptor C3AR1 were both disease shorter survival human glioma. In genetically engineered...

<p>FH is expressed in human glioma and correlates with disease severity. FH produced lower-grade (<b>A</b>) GBM (<b>B</b>). <b>C,</b> expression decreased overall survival (<b>C</b>) disease-free (<b>D</b>) of patients glioma. Expression primary therapy outcome (<b>E</b>), cancer type (<b>F</b>), neoplasm histological grade (<b>G</b>), new event post-initial (<b>H</b>),...

<p>Supplementary figure 2. FH in Ntv-a mouse model (A) Mouse binds to derived primary Tregs. Tregs were incubated for 2 h at 4oC with fluorescently labeled 25 or 100 μg/mL FH. The binding was detected using flow cytometry. (B) Western blot detecting supernatants of tumor cells isolated from mice. partially depleted antibody against bound Dynabeads. (C) FH-rendered increase survival cell supernatant and FH-depleted supernatant. After 7 days viability assessed by Annexin V Via-Probe...

<p>FH increases the viability of Tregs. FH-rendered increase in survival CD4<sup>+</sup> (<b>A</b>) but not CD8<sup>+</sup> (<b>B</b>) or naïve (<b>C</b>) T cells. FH increased Tregs (<b>D</b>) and did Treg-depleted cells (<b>E</b>). Cells were incubated with medium, 150 μg/mL FH, α1-AT for 7 days, assessed by Annexin V Via-Probe staining. <b>F,</b> Gating according to ICOS expression....

<p>FH binds to T cells via ICOS. <b>A,</b> Biotinylated FH CD4<sup>+</sup> upon incubation for 2 hours with TexMACS medium alone and supplemented 50 100 μg/mL FH. <b>B,</b> Fractionation detecting binding but not internalization into cells, incubated 150 at 4°C or 37°C. <b>C,</b> HuProt microarray showing top-specific proteins CCP19-20. <b>D</b> <b>E,</b> PLA activated were 37°C, the interaction between ICOS was...

<div>Abstract<p>The survival rate of patients with glioma has not significantly increased in recent years despite aggressive treatment and advances immunotherapy. The limited response to treatments is partially attributed the immunosuppressive tumor microenvironment, which regulatory T cells (Treg) play a pivotal role immunologic tolerance. In this study, we investigated impact complement factor H (FH) on Tregs within microenvironment found that FH an ICOS ligand. binding immune...

<p>Supplementary figure 3. Direct effect of FH on glioma cells Proliferation PIGPC treated with medium only, 25- or 100-μg/mL (A) and H4 mock FH-transfected (B) was analyzed after 24, 48, 72 96 hours using CyQUANT assay. Data obtained at 24 used for the normalization. (C) Correlation between Ki67 gene expression in tumor from patients, GEO database. Survival pretreated 100 μg/mL (D-F) (G-I) rendered apoptotic by treatment 0.75 uM staurosporine h. Viability assessed Annexin V Via-Probe...

<p>Supplementary figure 4. Staining control for human samples and additional information about patients (A) Histology (B) clinical parameters of glioma patients. MGMT-methylation status; TMZ-temozolomide; Adj-adjusted. WT – wild type.</p>

<p>FH knockdown is associated with a lower number of ICOS<sup>+</sup> Tregs in murine glioma model. FH expression was investigated gliomas generated using the RCAS/tv-a vectors to induce <i>PDGFB</i> (<b>A</b> and <b>B</b>) <i>shp53</i> (<b>B</b>). <b>C,</b> Schematic illustration mouse Increased survival (<b>D</b>), decrease Olig2<sup>+</sup> tumor cells (<b>E</b>),...

<p>ICOS is associated with poor prognosis of patients glioma. <b>A,</b> ICOS expression correlates decreased survival Expression neoplasm histological grade (<b>B</b>), cancer type (<b>C</b>), and <i>EGFR</i> mutations (<b>D</b>). positively infiltration Tregs (<b>E</b>) negatively CD8<sup>+</sup> T cells (<b>F</b>) CD4<sup>+</sup> (<b>G</b>) in lower-grade...

<p>FH enhances the immunosuppressive function of Tregs. <b>A,</b> FH increases effect Tregs were incubated in medium only or supplemented with 150 μg/mL FH. CD4<sup>+</sup> T cells isolated from same donor and cocultured presence CD2/CD3/CD28 MACSiBeads <sup>3</sup>H-thymidine. Secretion IL10 (<b>B</b> <b>D</b>) TGFβ (<b>C</b>) by only, α1-AT, FH, dFH. Cells stimulated anti-CD3 anti-CD28 microbeads IL2, secreted...

<p>Supplementary figure 6. FH expression in glioma correlates with pro-tumorigenic markers</p>

<p>FH in glioma correlates with Treg occurrence and increased viability. FH expression positively infiltration of Tregs lower-grade (<b>A</b>) GBM (<b>B</b>). <b>C,</b> Western blot detecting supernatants FH-transfected cell lines depletion MRC-OX24-bound Dynabeads. <b>D,</b> incubated H4 line exhibited survival, the effect is weaker when partially depleted. Correlation analysis immune was performed using TIMER2.0, based on...

<p>Supplementary figure 5. FH, ICOSL and ICOS dependence on glioma patient survival. The survival data of <i>n</i> = 509 patients from TCGA provisional dataset brain lower-grade glioma, analyzed with cBioPortal. Statistical tests: Logrank Test.</p>

<p>Supplementary figure 1. FH binds to T-cells via ICOS</p>

Glioblastoma is characterized by extensive necrotic areas with surrounding hypoxia. The cancer cell response to hypoxia in these well-described; it involves a metabolic shift and an increase stem cell-like characteristics. Less known about the hypoxic of tumor-associated astrocytes, major component glioma tumor microenvironment. Here, we used primary human astrocytes genetically engineered mouse model investigate this stromal type We found that became reactive intermediate severe hypoxia,...

CD59 is a glycosylphosphatidylinositol (GPI)‐anchored cell surface inhibitor of the complement membrane attack complex (MAC). We showed previously that highly expressed in pancreatic islets but down‐regulated rodent models diabetes. knockdown not enzymatic removal led to loss glucose‐stimulated insulin secretion (GSIS), suggesting an intracellular pool required. In this current paper, we now report non‐GPI‐anchored present cytoplasm, colocalizes with exocytotic protein vesicle‐associated 2,...

The complement inhibitor CSMD1 acts as a tumor suppressor in various types of solid cancers. Despite its high level expression the brain, function gliomas, malignant brain tumors originating from glial cells, has not been investigated. Three cohorts glioma patients comprising 1500 were analyzed our study along with their clinical data. H4, U-118 and U-87 cell lines used to investigate gliomas. PDGFB-induced model was utilized for validation vitro downregulation correlated reduced overall...

Abstract Glioblastoma (GBM) is characterized by fast progression, an infiltrative growth pattern, and a high rate of relapse. A defining feature GBM the existence spatially functionally distinct cellular niches, i.e. hypoxic niche, leading-edge perivascular in which malignant cells engage paracrine crosstalk with cell types comprising tumor microenvironment. Here, analysis single-cell transcriptomic data human transgenic mouse models GBM, we unexpectedly identified pericytes, mural...

Abstract The survival rate of glioma patients has not significantly increased in recent years despite aggressive treatment and advances immunotherapy. limited response to treatments is partially attributed the immunosuppressive tumor microenvironment, where regulatory T cells (Tregs) play a pivotal role immunological tolerance. In this study, we investigated impact complement factor H (FH) on Tregs within microenvironment found that FH an ICOS ligand. binding immune checkpoint molecule...

Abstract Glioblastoma (GBM) is the most aggressive form of glioma with a high rate relapse despite intensive treatment. Tumor recurrence tightly linked to radio-resistance, which in turn associated hypoxia. Here, we discovered strong link between hypoxia and local complement signaling using publicly available bulk, single cell, spatially resolved transcriptomic data from human GBM patients. Complement component 3 ( C3 ) receptor C3AR1 were both disease shorter survival glioma. In genetically...

Abstract BACKGROUND Despite early response to radiotherapy, glioblastomas invariably recur, frequently within the field receiving high-dose irradiation. Stromal radiation responses remain poorly characterized, but recent evidence suggest that cells of tumor microenvironment generate tumor-supportive conditions in post-radiotherapy brain. Here, we explored character astrocyte reactivity states induced during progression and treatment, identified compounds target such states, evaluated their...