GL261 Murine Model Research Articles

Combination of αPD-1 and extended half-life IL-2 is effective against the GL261 glioma and uniquely reverses GBM-associated immunosuppression

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12–18 months post diagnosis. The unique immune environment of the brain and the immunosuppressive features of GBM complicate the use of immunotherapies against these tumors. We aimed to promote the efficacy of αPD-1 against the GL261 murine model of GBM by supplementing this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. Neither αPD-1 nor MSA-IL-2 are effective monotherapies for GL261 tumors, but the combination provides durable tumor clearance and prevents subsequent rechallenge. Most strikingly, this combination therapy cleared established GL261 tumors even in mice incapable of MHC class I restricted antigen presentation. Instead, therapeutic efficacy was abrogated by the depletion of CD4 T cells. Our combination therapy also reversed multiple key features of GBM-associated peripheral immune suppression, including depressed CD4 T cell counts and dampened MHC class II expression. Our combination therapy’s ability to overcome these immune derangements associated with glioma growth and to function independent of MHC class I restricted antigen presentation enhance the appeal of this therapy for future translational adaptation. This work was supported by the National Institutes of Health (R01 NS103212) and the Mayo Clinic-Koch Institute Cancer Solutions Team Grant

αPD-1 and extended half-life IL-2 combination therapy clears established GL261 gliomas in an MHC class I independent fashion

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12 months post diagnosis despite aggressive treatment. Immunotherapy approaches for GBM have been attempted but the unique immune environment of the brain and the immunosuppressive features of GBM complicate these efforts. Even αPD-1 checkpoint blockade, highly useful in the context of melanoma, has proved ineffective in the treatment of GBM. In order to promote the efficacy of αPD-1 against the GL261 murine model of GBM, we supplemented this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated dangerously high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. While neither αPD-1 nor MSA-IL-2 are effective monotherapies against the GL261 model tumor, we have found this combination immunotherapy to provide durable tumor clearance and to create immunologic memory capable of resisting GL261 rechallenge. Most strikingly, this combination therapy cleared established GL261 tumors even in mice incapable of MHC class I restricted antigen presentation. Instead, therapeutic efficacy was abrogated by the depletion of CD4 T cells. Independence of MHC class I restricted antigen presentation makes this combination immunotherapy using αPD-1 checkpoint blockade both highly novel and translatable to clinical use.

Combination immunotherapy of αPD-1 and extended half-life IL-2 clears established GL261 gliomas in an MHC class I independent fashion

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12 months post diagnosis despite aggressive treatment. Immunotherapy approaches to treat GBM are being developed, but the unique immune environment of the brain and the immunosuppressive features of GBM have complicated early efforts. Even αPD-1 checkpoint blockade, highly useful in the context of melanoma, has proved ineffective in the treatment of GBM. In order to promote the efficacy of αPD-1 against the GL261 murine model of GBM, we supplemented this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated dangerously high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. While neither αPD-1 nor MSA-IL-2 are effective monotherapies against the GL261 model tumor, we have found this combination immunotherapy to provide durable tumor clearance and to create immunologic memory capable of resisting GL261 rechallenge. Most strikingly, this combination therapy is independent of the CD8 T cell function, clearing established GL261 tumors in mice incapable of MHC class I restricted antigen presentation. Independence of MHC class I restricted antigen presentation makes this combination immunotherapy using αPD-1 checkpoint blockade both highly novel and translatable to clinical use.

Abstract 1303: Targeted inhibition of galectin-1 suppresses glioblastoma growth

Abstract Background: Galectin-1 (Gal1), a carbohydrate-binding protein is implicated in cancer cell proliferation, invasion and tumor angiogenesis. An earlier report from our laboratory demonstrated that glioblastoma (GB) cells express high levels of Gal-1, which promotes tumor progression through upregulation of CXCR4 viaNF-κB in in vitro GB models. Recently, several Gal1-targeting compounds have emerged. OTX008 is a calixarene derivative designed to bind the Gal1 amphipathic β-sheet conformation. Our study aimed to validate the effect of the non-peptidic galectin-1 inhibitor OTX008 in human GB in relevant pre-clinical models. Methods: In vitro cell culture and in vivo GL261 murine models of GB were utilized to assess efficacy of treatment with OTX008 via Alzet osmotic pumps. To investigate the mechanistic effect of OTX008 on GB cells, we used RT-PCR and western blots techniques. Magnetic resonance imaging (MRI), and immunohistochemistry (IHC)-staining methods were used to detect tumor growth in in vivo GL261 murine model following OTX008 treatments. Results: In cultured human and mouse GB cells, OTX008 inhibited proliferation and invasion at micromolar concentrations. We also found that the anti-proliferative effects correlated with Gal1 expression in human and mouse GB cell lines. Furthermore, OTX008 inhibited Gal1 expression and AKT-dependent survival pathways, and decreased CXCR4 expression. In vivo, continued 100ng/kg OTX008 treatment via Alzet osmotic pumps over 14 days reduced tumor growth of GL261 murine model after 3 weeks of implantation. OTX008 treatment was associated with downregulation of Gal1 and Ki67 in treated tumors, as well as decreased micro-vessel density and VEGFR2 expression. Conclusion: These findings suggest that targeting galectin-1 may be an effective approach in the treatment of GB patients. Citation Format: David Cachia, Arindam Rano Chatterjee, William Alex Vandergrift, Sunil J. Patel, Gabriel A. Rabinovich, Arabinda Das. Targeted inhibition of galectin-1 suppresses glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1303.

Up-Regulation of the Alpha Prime Subunit of Protein Kinase CK2 as a Marker of Fast Proliferation in GL261 Cultured Cells.

Glioblastoma (GB) is the most prevalent malignant primary brain tumor in adults. The preclinical glioblastoma model GL261 is widely used for investigating new therapeutic strategies. GL261 cultured cells are used for assessing preliminary in vitro data for this model although very little is known about the molecular characteristics of this cell line. Protein Kinase CK2 is a pleiotropic serine-threonine kinase and its inhibition may be a promising therapeutic strategy for GB treatment. In our group we follow treatment response with CK2 inhibitors in vivo using the GL261 murine model. For that, it is of our interest to assess the differential expression of α, α', β CK2 subunits as well as CK2 activity in the GL261 GB model. CK2α' expression changed along the growth curve of GL261 cells, undergoing downregulation in postconfluent phase cells, whereas CK2α and CK2β expression remained essentially unchanged. Furthermore, a marked decrease in CK2 activity in slowly proliferating postconfluent phase GL261 cells was observed. Finally, CK2α' expression in orthotopic GL261 tumors was intermediate between CK2α' expression found in cultured cells in exponentially growing or postconfluent phase, reflecting the heterogeneous nature of GL261 tumours growing in vivo. The results obtained suggest that, in the GL261 cell line, CK2α' could play a specific role in highly proliferative cells. Also, the decrease in CK2 activity in slowly proliferating GL261 cells could imply a differential susceptibility to subunit-specific CK2 inhibitors in this cell line, although further studies are needed to confirm this hypothesis.

Convective forces increase CXCR4-dependent glioblastoma cell invasion in GL261 murine model

Glioblastoma is the most common and malignant form of brain cancer. Its invasive nature limits treatment efficacy and promotes inevitable recurrence. Previous in vitro studies showed that interstitial fluid flow, a factor characteristically increased in cancer, increases glioma cell invasion through CXCR4-CXCL12 signaling. It is currently unknown if these effects translate in vivo. We used the therapeutic technique of convection enhanced delivery (CED) to test if convective flow alters glioma invasion in a syngeneic GL261 mouse model of glioblastoma. The GL261 cell line was flow responsive in vitro, dependent upon CXCR4 and CXCL12. Additionally, transplanting GL261 intracranially increased the populations of CXCR4+ and double positive cells versus 3D culture. We showed that inducing convective flow within implanted tumors indeed increased invasion over untreated controls, and administering the CXCR4 antagonist AMD3100 (5 mg/kg) effectively eliminated this response. These data confirm that glioma invasion is stimulated by convective flow in vivo and depends on CXCR4 signaling. We also showed that expression of CXCR4 and CXCL12 is increased in patients having received standard therapy, when CED might be elected. Hence, targeting flow-stimulated invasion may prove beneficial as a second line of therapy, particularly in patients chosen to receive treatment by convection enhanced delivery.

Aquaporin‐9 expression in malignant glioma

Recent studies described enhanced expression of the water channel aquaporin 9 (AQP9) in biopsies of glioblastoma patients. This may indicate a function for AQP9 in glioma progression as water channels are known to contribute to cell migration. Furthermore, AQP9 is a glycerol and lactate channel and could play a role in the energy metabolism of malignant glioma cells.Glial tumors usually consist of several cell types, including malignant glial cells, endothelial cells, microglia and other cells of the immune system. Currently, the specific cellular localization of AQP9 has not been determined. The aim of our studies was to determine the cellular expression of AQP9 in glioblastoma biopsies. Furthermore, we are currently investigating the cellular expression of AQP9 in various murine models of glioblastoma.Human tissue revealed positive AQP9 labeling in a subpopulation of tumor cells. Moreover, we discovered strong immunolabeling of AQP9 co‐localized with the neutrophil marker CD15. Similar to the situation in human tumors, AQP9 was expressed in neutrophils invading murine Gl261 derived tumors. However, Gl261 derived cells did not express AQP9.We conclude, that AQP9 is expressed in human glioma cells and tumor associated neutrophils. Furthermore, the Gl261 murine model of glioblastoma may be suitable to study a function of AQP9 in tumor associated neutrophils.Study supported by Lundbeck Foundation

Chemokine receptor CXCR3 promotes growth of glioma

Human glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of GBM indicates a need to identify new therapeutic targets. In this study, we examined the role of CXCR3 in glioma progression using the GL261 murine model of malignant glioma. Intracranial GL261 tumors express CXCL9 and CXCL10 in vivo. Glioma-bearing CXCR3-deficient mice had significantly shorter median survival time and reduced numbers of tumor-infiltrated natural killer and natural killer T cells as compared with tumor-bearing wild-type (WT) mice. In contrast, pharmacological antagonism of CXCR3 with NBI-74330 prolonged median survival times of both tumor-bearing WT and CXCR3-deficient mice when compared with vehicle-treated groups. NBI-74330 treatment did not impact tumor infiltration of lymphocytes and microglia. A small percentage of GL261 cells were identified as CXCR3(+), which was similar to the expression of CXCR3 in several grade IV human glioma cell lines (A172, T98G, U87, U118 and U138). When cultured as gliomaspheres (GS), the human and murine lines increased CXCR3 expression; CXCR3 expression was also found in a primary human GBM-derived GS. Additionally, CXCR3 isoform A was expressed by all lines, whereas CXCR3-B was detected in T98G-, U118- and U138-GS cells. CXCL9 or CXCL10 induced in vitro glioma cell growth in GL261- and U87-GS as well as inhibited cell loss in U138-GS cells and this effect was antagonized by NBI-74330. The results suggest that CXCR3 antagonism exerts a direct anti-glioma effect and this receptor may be a potential therapeutic target for treating human GBM.

CX3CL1 and CX3CR1 in the GL261 murine model of glioma: CX3CR1 deficiency does not impact tumor growth or infiltration of microglia and lymphocytes

Human glioblastoma multiforme (GBM) is the most malignant form of human brain tumors. A characteristic of GBM is the marked presence of tumor infiltrated microglia/macrophages and lymphocytes. The goal of this study was directed toward understanding the role of the chemokine system CX3CL1 and its receptor CX3CR1 in the GL261 murine model of malignant glioma. In situ hybridization analysis identified CX3CL1 and CX3CR1 expression in GL261 tumors. The impact of CX3CR1 deletion on the growth of intracranial GL261 gliomas and associated immune cell infiltration was evaluated in CX3CR1 gene-disrupted C57BL/6 mice. A slight increase in the tumor growth rate in CX3CR1−/− mice was evident with similar numbers of microglia and CD4 +, CD8 +, FoxP3 +, or Ly49G2 + lymphocytes within tumors established in CX3CR1 +/− and −/− mice. These data indicate that CX3CR1 has little or no effects on either gliomagenesis or the migration of microglia and lymphocytes into GL261 tumors.