RAGE ablation attenuates glioma progression and enhances tumor immune responses by suppressing galectin-3 expression.

BACKGROUND: Malignant gliomas consist of heterogeneous cellular components that have adopted multiple overlapping escape mechanisms that overcome both targeted and immune-based therapies. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that is activated by diverse proinflammatory ligands present in the tumor microenvironment. Activation of RAGE by its ligands stimulates multiple signaling pathways that are important in tumor growth and invasion. However, treatment strategies that only target the interaction of RAGE with its ligands are ineffective as cancer therapies due to the abundance and diversity of exogenous RAGE ligands in gliomas. METHODS: As an alternative approach to RAGE ligand inhibition, we evaluated the genetic ablation of RAGE on the tumorigenicity of 2 syngeneic murine glioma models. RAGE expression was inhibited in the GL261 and K-Luc gliomas by shRNA and CRSPR/Cas9 techniques prior to intracranial implantation. Tumor growth, invasion, and inflammatory responses were examined by histology, survival, Nanostring, and flow cytometry. RESULTS: Intracellular RAGE ablation abrogated glioma growth and invasion by suppressing AKT and ERK1/2 activities and by downregulating MMP9 expression. Interestingly, RAGE inhibition in both glioma models enhanced tumor inflammatory responses by downregulating the expression of galectin-3 and potentiated immunotherapy responses to immune checkpoint blockade. CONCLUSIONS: We demonstrated that intracellular RAGE ablation suppresses multiple cellular pathways that are important in glioma progression, invasion, and immune escape. These findings strongly support the development of RAGE ablation as a treatment strategy for malignant gliomas.

RAGE Inhibitors as Alternatives to Dexamethasone for Managing Cerebral Edema Following Brain Tumor Surgery.

Resection of brain tumors frequently causes injury to the surrounding brain tissue that exacerbates cerebral edema by activating an inflammatory cascade. Although corticosteroids are often utilized peri-operatively to alleviate the symptoms associated with brain edema, they increase operative morbidities and suppress the efficacy of immunotherapy. Thus, novel approaches to minimize cerebral edema caused by neurosurgical procedures will have significant utility in the management of patients with brain tumors. We have studied the role of the receptor for advanced glycation end products (RAGE) and its ligands on inflammatory responses to neurosurgical injury in mice and humans. Blood-brain barrier (BBB) integrity and neuroinflammation were characterized by Nanostring, flow cytometry, qPCR, and immunoblotting of WT and RAGE knockout mice brains subjected to surgical brain injury (SBI). Human tumor tissue and fluid collected from the resection cavity of patients undergoing craniotomy were also analyzed by single-cell RNA sequencing and ELISA. Genetic ablation of RAGE significantly abrogated neuroinflammation and BBB disruption in the murine SBI model. The inflammatory responses to SBI were associated with infiltration of S100A9-expressing myeloid-derived cells into the brain. Local release of pro-inflammatory S100A9 was confirmed in patients following tumor resection. RAGE and S100A9 inhibitors were as effective as dexamethasone in attenuating neuroinflammation. However, unlike dexamethasone and S100A9 inhibitor, RAGE inhibition did not diminish the efficacy of anti-PD-1 immunotherapy in glioma-bearing mice. These observations confirm the role of the RAGE axis in surgically induced neuroinflammation and provide an alternative therapeutic option to dexamethasone in managing post-operative cerebral edema.

Local and Systemic Immune Dysregulation Alters Glioma Growth in Hyperglycemic Mice.

PURPOSE: Unlike most cancers, no clear epidemiological correlation between diabetes (Db) and malignant glioma progression exists. Because hyperglycemia activates proinflammatory pathways through the receptor for advanced glycation endproducts (RAGE), we hypothesized that Db can also promote malignant glioma progression. EXPERIMENTAL DESIGN: We compared the growth of two phenotypically diverse syngeneic glioma models in control and diabetic mice. Tumor growth and antitumor immune responses were evaluated in orthotopic and heterotopic models and correlated to RAGE and RAGE ligand expression. RESULTS: Irrespective of tumor implantation site, growth of a "classical" glioma model, GL261, increased in hyperglycemic mice and was mediated by upregulation of RAGE and its ligand, HMGB1. However, growth of a "mesenchymal" glioma subtype, K-Luc, depended on tumor implantation site. Whereas heterotopic K-Luc tumors progressed rapidly in Db mice, intracranial K-Luc tumors grew slower. We further showed that hyperglycemia inhibited the innate antitumor inflammatory responses in both models. Although this contributed to the accelerated growth of heterotopic tumors, suppression of tumor inflammatory responses dampened the growth of orthotopic K-Luc gliomas. CONCLUSIONS: Hyperglycemia may enhance glioma growth through promotion of RAGE expression and suppression of antitumor immune responses. However, abrogation of the proinflammatory milieu in tumors may also dampen the growth of inflammatory glioma subtypes in the brains of diabetic mice. This dichotomy in glioma growth response to hyperglycemia may partly explain why conflicting epidemiological studies show both an increased risk and a protective effect of Db in patients with malignant gliomas.

S100B suppression alters polarization of infiltrating myeloid-derived cells in gliomas and inhibits tumor growth.

S100B, a member of the multigene family of Ca2+-binding proteins, is overexpressed by most malignant gliomas but its biological role in gliomagenesis is unclear. Recently, we demonstrated that low concentrations of S100B attenuated microglia activation through the induction of STAT3. Furthermore, S100B downregulation in a murine glioma model inhibited macrophage trafficking and tumor growth. Based on these observations, we hypothesized that S100B inhibitors may have antiglioma properties through modulation of tumor microenvironment. To discover novel S100B inhibitors, we developed a high-throughput screening cell-based S100B promoter-driven luciferase reporter assay. Initial screening of 768 compounds in the NIH library identified 36 hits with >85% S100B inhibitory activity. Duloxetine (Dul, an SNRI) was selected for the initial proof-of-concept studies. At low concentrations (1-5 µM) Dul inhibited S100B and CCL2 production in mouse GL261 glioma cells, but had minimal cytotoxic activity in vitro. In vivo, however, Dul (30 mg/kg/14 days) inhibited S100B production, altered the polarization and trafficking of tumor-associated myeloid-derived cells, and inhibited the growth of intracranial GL261 gliomas. Dul therapeutic efficacy, however, was not observed in the K-Luc glioma model that expresses low levels of S100B. These findings affirm the role of S100B in gliomagenesis and justify the development of more potent S100B inhibitors for glioma therapy.

RAGE expression in tumor-associated macrophages promotes angiogenesis in glioma.

Interaction of RAGE (the receptor for advanced glycation endproducts) with its ligands can promote tumor progression, invasion, and angiogenesis. Although blocking RAGE signaling has been proposed as a potential anticancer strategy, functional contributions of RAGE expression in the tumor microenvironment (TME) have not been investigated in detail. Here, we evaluated the effect of genetic depletion of RAGE in TME on the growth of gliomas. In both invasive and noninvasive glioma models, animal survival was prolonged in RAGE knockout (Ager(-/-)) mice. However, the improvement in survival in Ager(-/-) mice was not due to changes in tumor growth rate but rather to a reduction in tumor-associated inflammation. Furthermore, RAGE ablation in the TME abrogated angiogenesis by downregulating the expression of proangiogenic factors, which prevented normal vessel formation, thereby generating a leaky vasculature. These alterations were most prominent in noninvasive gliomas, in which the expression of VEGF and proinflammatory cytokines were also lower in tumor-associated macrophages (TAM) in Ager(-/-) mice. Interestingly, reconstitution of Ager(-/-) TAM with wild-type microglia or macrophages normalized tumor vascularity. Our results establish that RAGE signaling in glioma-associated microglia and TAM drives angiogenesis, underscoring the complex role of RAGE and its ligands in gliomagenesis.

S100B promotes glioma growth through chemoattraction of myeloid-derived macrophages.

PURPOSE: S100B is member of a multigenic family of Ca(2+)-binding proteins, which is overexpressed by gliomas. Recently, we showed that low concentrations of S100B attenuated microglia activation through the induction of Stat3. We hypothesized that overexpression of S100B in gliomas could promote tumor growth by modulating the activity of tumor-associated macrophages (TAM). EXPERIMENTAL DESIGN: We stably transfected GL261 glioma cell lines with constructs that overexpressed (S100B(high)) or underexpressed (S100B(low)) S100B and compared their growth characteristics to intracranial wild-type (S100B(wt)) tumors. RESULTS: Downregulation of S100B in gliomas had no impact on cell division in vitro but abrogated tumor growth in vivo. Interestingly, compared to S100B(low) tumors, S100B(wt) and S100B(high) intracranial gliomas exhibited higher infiltration of TAMs, stronger inflammatory cytokine expression, and increased vascularity. To identify the potential mechanisms involved, the expression of the S100B receptor, receptor for advanced glycation end products (RAGE), was evaluated in gliomas. Although S100B expression induced RAGE in vivo, RAGE ablation in mice did not significantly inhibit TAM infiltration into gliomas, suggesting that other pathways were involved in this process. To evaluate other mechanisms responsible for TAM chemoattraction, we then examined chemokine pathways and found that C-C motif ligand 2 (CCL2) was upregulated in S100B(high) tumors. Furthermore, analysis of The Cancer Genome Atlas's glioma data bank showed a positive correlation between S100B and CCL2 expression in human proneural and neural glioma subtypes, supporting our finding. CONCLUSIONS: These observations suggest that S100B promotes glioma growth by TAM chemoattraction through upregulation of CCL2 and introduces the potential utility of S100B inhibitors for glioma therapy.

HMGA1 expression in human gliomas and its correlation with tumor proliferation, invasion and angiogenesis.

High-mobility group A1 (HMGA1) protein is an architectural transcription factor widely expressed during embryonic development and tumor progression. The purpose of this research was to investigate the expression of HMGA1 in malignant gliomas with different WHO classification and to study the correlation of HMGA1 expression with tumor proliferation, invasion, and angiogenesis. Expression of HMGA1, Ki-67, MMP-9, VEGF-A, and MVD in malignant gliomas and their correlation were studied in 60 samples of different WHO classification by use of immunohistochemistry, and in 27 randomly selected samples by use of real-time quantitative PCR. Immunohistochemistry results showed that nuclear immunostaining of HMGA1 protein was not observed in normal brain tissues but was observed in 96.7% (58 of 60) of malignant gliomas including high (+++) in 15 (25.0%), moderate (++) in 28 (46.7%), and negligible to low (0-+) in 17 (28.3%) samples. Expression of HMGA1 protein was significantly higher in glioblastoma multiforme than in WHO grade II (P = 0.002) and WHO grade III gliomas (P = 0.024). HMGA1 protein expression correlated significantly with expression of Ki-67 (r = 0.530, P = 0.000), MMP-9 (r = 0.508, P = 0.000), VEGF-A (r = 0.316, P = 0.014), and MVD (r = 0.321, P = 0.012), but not with sex (r = 0.087, P = 0.510) and age (r = -0.121, P = 0.358). Real-time quantitative PCR results, also, were indicative of HMGA1 overexpression in glioblastoma multiforme compared with WHO grade II (P = 0.043) and WHO grade III (P = 0.031) gliomas. HMGA1 gene expression correlated significantly with gene expression of Ki-67 (r = 0.429, P = 0.025), MMP-9 (r = 0.443, P = 0.024), and VEGF-A (r = 0.409, P = 0.034). These results indicated that expression of HMGA1 correlates significantly with malignancy, proliferation, invasion, and angiogenesis of gliomas. We conclude that HMGA1 may be a potential biomarker and rational therapeutic target for human tumors.

The different HMGA1 expression of total population of glioblastoma cell line U251 and glioma stem cells isolated from U251.

The high-mobility group A1 (HMGA1) protein is a non-histone architectural nuclear factor and participates in diverse biological processes, including gene transcription, embryogenesis, cell cycle regulation, apoptosis, and even neoplastic transformation. In our study, glioma stem cells (GSCs) expressing the surface marker CD133 from human glioblastoma cell line U251 were isolated using MACS column and were analyzed using immunofluorescence and flow cytometry (FCM). The different expression of HMGA1 was detected using real-time RT-PCR and Western blot at transcriptional and translational levels between U251 and isolated GSCs. The results show that GSCs were successfully isolated from U251 and cultured in serum-free medium (SMF). The percentage of GSCs in U251 was 0.32%±0.07%. HMGA1 expression was significantly higher in GSCs than in glioblastoma cells (P<0.05), up to 6.13±0.25-fold and 2.75±0.99-fold at transcriptional and translational levels, respectively. These results indicated HMGA1 is overexpressed in GSCs as compared to glioblastoma cell line U251, which points to the expression of HMGA1 being closely related to malignant proliferation, invasion, and differentiation of tumors from the prospective of tumor stem cells (TSCs). We conclude that HMGA1 may be a potential biomarker and rational therapeutic target for glioblastoma and GSC.