The expression of BST2 in human and experimental mouse brain tumors.
ABSTRACT: Glioblastoma multiforme (grade IV astrocytoma) is a highly malignant brain tumor with poor treatment options and an average lifespan of 15 months after diagnosis. Previous work has demonstrated that BST2 (bone marrow stromal cell antigen 2; also known as PDCA-1, CD137 and HM1.24) is expressed by multiple myeloma, endometrial cancer and primary lung cancer cells. BST2 is expressed on the plasma membrane, which makes it an ideal target for immunotherapy. Accordingly, several groups have shown BST2 mAb to be effective for targeting tumor cells. In this report, we hypothesized that BST2 is expressed in human and mouse brain tumors and plays a critical role in brain tumor progression. We show that BST2 expression is upregulated at both the mRNA and protein level in high grade when compared to low grade human astrocytoma (p<0.05). To test the relevance of BST2, we utilized the intracranially (IC)-injected GL261 cell-based malignant brain tumor mouse model. We show that BST2 mRNA expression is increased in mouse brain IC-injected with GL261 cells, when compared to mouse brain IC-injected with saline at 3 weeks post-operative (p<0.05). Furthermore, BST2 immunofluorescence predominantly localized to mouse brain tumor cells. Finally, mice IC-injected with GL261 cells transduced with shRNA for BST2±preincubated with BST2 mAb show no difference in overall lifespan when compared to mice IC-injected with GL261 cells transduced with a scrambled shRNA±preincubated with BST2 mAb. Collectively, these data show that while BST2 expression increases during brain tumor progression in both human and mouse brain tumors, it has no apparent consequences to overall lifespan in an orthotopic mouse brain tumor model.
Project description:Numerous monoclonal antibodies (mAb) targeting tumor antigens have recently been developed. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) via effector cells such as tumor-infiltrating natural killer (NK) cells and macrophages are often involved in mediating the antitumor activity of mAb. CpG oligodeoxynucleotides (ODN) have a potent antitumor activity and are considered to increase tumor infiltration of NK cells and macrophages. Our group previously reported significant antitumor activity of anti-bone marrow stromal antigen 2 (BST2) mAb against BST2-positive endometrial cancer cells through ADCC. In this study, we evaluated the synergistic antitumor activity of combination therapy with anti-BST-2 mAb and CpG ODN using SCID mice and elucidated the mechanisms underlying this activity. Anti-BST2 mAb and CpG ODN monotherapy had a significant dose-dependent antitumor activity (P = 0.0135 and P = 0.0196, respectively). Combination therapy with anti-BST2 mAb and CpG ODN had a significant antitumor activity in SCID mice (P < 0.01), but not in NOG mice. FACS analysis revealed significantly increased numbers of NK cells and macrophages in tumors treated with a combination of anti-BST2 mAb and CpG ODN and with CpG ODN alone in SCID mice (P < 0.05 and P < 0.01, respectively). These results suggested that the combination therapy with anti-BST2 mAb and CpG ODN has a significant antitumor activity and induces tumor infiltration of NK cells and macrophages. Combination therapy with CpG ODN and anti-BST2 mAb or other antitumor mAb depending on ADCC may represent a new treatment option for cancer.
Project description:Mebendazole (MBZ), first used as an antiparasitic drug, shows preclinical efficacy in models of glioblastoma and medulloblastoma. Three different mebendazole polymorphs (A, B, and C) exist, and a detailed assessment of the brain penetration, pharmacokinetics, and antitumor properties of each individual mebendazole polymorph is necessary to improve mebendazole-based brain cancer therapy.In this study, various marketed and custom-formulated mebendazole tablets were analyzed for their polymorph content by IR spectroscopy and subsequently tested in an orthotopic GL261 mouse glioma model for efficacy and tolerability. The pharmacokinetics and brain concentration of mebendazole polymorphs and two main metabolites were analyzed by LC/MS. We found that polymorph B and C both increased survival in a GL261 glioma model, as B exhibited greater toxicity. Polymorph A showed no benefit. Polymorph B and C both reached concentrations in the brain that exceeded the IC?? in GL261 cells 29-fold. In addition, polymorph C demonstrated an AUC????h brain-to-plasma (B/P) ratio of 0.82, whereas B showed higher plasma AUC and lower B/P ratio. In contrast, polymorph A presented markedly lower levels in the plasma and brain. Furthermore, the combination with elacridar was able to significantly improve the efficacy of polymorph C in GL261 glioma and D425 medulloblastoma models in mice.Among mebendazole polymorphs, C reaches therapeutically effective concentrations in the brain tissue and tumor with fewer side effects, and is the better choice for brain cancer therapy. Its efficacy can be further enhanced by combination with elacridar.
Project description:Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. GL261 murine glioma cells are widely used as a syngeneic animal model of glioma, however, it has become common practice to transfect these cells with luciferase for fluorescent tumor tracking. The aim of this study was to compare the survival of mice injected with fluorescent or non-fluorescent GL261 cells and characterize the differences in their tumor microenvironment. Mice were intracranially implanted with GL261, GL261 Red-FLuc or GL261-Luc2 cells at varying doses. Cytokine profiles were evaluated by proteome microarray and Kaplan-Meier survival analysis was used to determine survival differences. Median survival for mice implanted with 5?×?104 GL261 cells was 18 to 21 days. The GL261 Red-FLuc implanted mice cells did not reach median survival at any tumor dose. Mice injected with 3?×?105 GL261-Luc2 cells reached median survival at 23 days. However, median survival was significantly prolonged to 37 days in mice implanted with 5?×?104 GL261-Luc2 cells. Additionally, proteomic analyses revealed significantly elevated inflammatory cytokines in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Our data suggest that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators.
Project description:Although blood-based liquid biopsies have emerged as a promising non-invasive method to detect biomarkers in various cancers, limited progress has been made for brain tumors. One major obstacle is the blood-brain barrier (BBB), which hinders efficient passage of tumor biomarkers into the peripheral circulation. The objective of this study was to determine whether FUS in combination with microbubbles can enhance the release of biomarkers from the brain tumor to the blood circulation. Two glioblastoma tumor models (U87 and GL261), developed by intracranial injection of respective enhanced green fluorescent protein (eGFP)-transduced glioblastoma cells, were treated by FUS in the presence of systemically injected microbubbles. Effect of FUS on plasma eGFP mRNA levels was determined using quantitative polymerase chain reaction. eGFP mRNA were only detectable in the FUS-treated U87 mice and undetectable in the untreated U87 mice (maximum cycle number set to 40). This finding was replicated in GL261 mice across three different acoustic pressures. The circulating levels of eGFP mRNA were 1,500-4,800 fold higher in the FUS-treated GL261 mice than that of the untreated mice for the three acoustic pressures. This study demonstrated the feasibility of FUS-enabled brain tumor liquid biopsies in two different murine glioma models across different acoustic pressures.
Project description:Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an average survival time of 15 months. Previously, we and others demonstrated that CD4(+)FoxP3(+) regulatory T cells (Tregs) infiltrate human GBM as well as mouse models that recapitulate malignant brain tumors. However, whether brain tumor-resident Tregs are thymus-derived natural Tregs (nTregs) or induced Tregs (iTregs), by the conversion of conventional CD4(+) T cells, has not been established. To investigate this question, we utilized the i.c. implanted GL261 cell-based orthotopic mouse model, the RasB8 transgenic astrocytoma mouse model, and a human GBM tissue microarray. We demonstrate that Tregs in brain tumors are predominantly thymus derived, since thymectomy, prior to i.c. GL261 cell implantation, significantly decreased the level of Tregs in mice with brain tumors. Accordingly, most Tregs in human GBM and mouse brain tumors expressed the nTreg transcription factor, Helios. Interestingly, a significant effect of the brain tumor microenvironment on Treg lineage programming was observed, based on higher levels of brain tumor-resident Tregs expressing glucocorticoid-induced tumor necrosis factor receptor and CD103 and lower levels of Tregs expressing CD62L and CD45RB compared with peripheral Tregs. Furthermore, there was a higher level of nTregs in brain tumors that expressed the proliferative marker Ki67 compared with iTregs and conventional CD4(+) T cells. Our study demonstrates that future Treg-depleting therapies should aim to selectively target systemic rather than intratumoral nTregs in brain tumor-specific immunotherapeutic strategies.
Project description:Eltd1 is widely expressed in endothelial cells. We have reported that the expression of Eltd1 is highly up-regulated in glioma vessels. Other studies have shown that Eltd1 is up-regulated in vessels in other types of tumors, and has been suggested as a vascular target for anti-angiogenesis therapy. However, the role of Eltd1 in vessels is largely unknown. We isolated endothelial cells from healthy brain tissue and GL261 tumor tissue, and analyzed the role of Eltd1 in tumor angiogenesis by analyzing the endothelial gene profile from wild type and Eltd1-/- mice. Overall design: We injected GL261 intracranially to C57BL/6 wild type and Eltd1-/- mice. Tumors were dissected under a stereological microscope. To isolate endothelial cell mRNA from heathy brain tissue and tumor tissue, we took advantage of the mouse that expressing a fusion eGFP-L10a ribosomal protein under control of the VE-cadherin promoter, and crossed it with the Eltd1-/- mouse. Using translating ribosome affinity purification (TRAP) strategy, endothelial specific mRNA was isolated and were further prepared for RNAseq analysis.
Project description:Peripheral macrophages and resident microglia constitute the dominant glioma-infiltrating cells. The tumor induces an immunosuppressive and tumor-supportive phenotype in these glioma-associated microglia/brain macrophages (GAMs). A subpopulation of glioma cells acts as glioma stem cells (GSCs). We explored the interaction between GSCs and GAMs. Using CD133 as a marker of stemness, we enriched for or deprived the mouse glioma cell line GL261 of GSCs by fluorescence-activated cell sorting (FACS). Over the same period of time, 100 CD133(+?)GSCs had the capacity to form a tumor of comparable size to the ones formed by 10,000 CD133(-) GL261 cells. In IL-6(-/-) mice, only tumors formed by CD133(+?)cells were smaller compared with wild type. After stimulation of primary cultured microglia with medium from CD133-enriched GL261 glioma cells, we observed an selective upregulation in microglial IL-6 secretion dependent on Toll-like receptor (TLR) 4. Our results show that GSCs, but not the bulk glioma cells, initiate microglial IL-6 secretion via TLR4 signaling and that IL-6 regulates glioma growth by supporting GSCs. Using human glioma tissue, we could confirm the finding that GAMs are the major source of IL-6 in the tumor context.
Project description:Glioblastoma (GBM) is the most aggressive type of brain tumor. In this context, animal models represent excellent tools for the early detection and longitudinal mapping of neuronal dysfunction, that are critical in the preclinical validation of new therapeutic strategies. In a mouse glioma model, we developed sensitive behavioral readouts that allow early recognizing and following neurological symptoms. We injected GL261 cells into the primary motor cortex of syngenic mice and we used a battery of behavioral tests to longitudinally monitor the dysfunction induced by tumor growth. Grip strength test revealed an early onset of functional deficit associated to the glioma growth, with a significant forelimb weakness appearing 9 days after tumor inoculation. A later deficit was observed in the rotarod and in the grid walk tasks. Using this model, we found reduced tumor growth and maintenance of behavioral functions following treatment with Cytotoxic Necrotizing Factor 1 (CNF1) at a symptomatic stage. Our data provide a detailed and precise examination of how tumor growth reverberates on the behavioral functions of glioma-bearing mice, providing normative data for the study of therapeutic strategies for glioma treatment. The reduced tumor volume and robust functional sparing observed in CNF1-treated, glioma-bearing mice strengthen the notion that CNF1 delivery is a promising strategy for glioma therapy.
Project description:While immunomodulatory monoclonal antibodies (mAbs) have therapeutic efficacy against many tumors, few patients are cured. Attempting to improve their therapeutic efficacy we have applied the TC1 mouse lung carcinoma model and injected established subcutaneous tumors intratumorally with 3 weekly doses of various combinations of mAbs. Combinations of mAbs to CTLA4/PD1/CD137 (the 3 mAb combination) and to CTLA4/PD1/CD137/CD19 (the 4 mAb combination) were most efficacious to induce complete regression of both the injected tumor and an untreated tumor in the same mouse. Tumor cure was consistently associated with shifting a Th2 to a Th1 response in tumor-draining lymph nodes and spleen and it involved epitope specific and long-lived memory T cells as well as M1 macrophages. This shift and accompanying tumor rejection was harder to achieve as the treated tumors increased in size. Relapse of tumors which had initially regressed following treatment with immunomodulatory mAbs was associated with return of a Th2 microenvironment in tumors, tumor-draining lymph nodes and spleens rather than the emergence of immune-resistant tumor cells. While mAbs to CTLA4 plus PD-1 were therapeutically ineffective, combining the 2 of them with intraperitoneal cisplatin, 10?mg/kg, induced long-term complete tumor regression in most mice with small TC1 tumors and the therapeutic efficacy against larger tumors improved by administrating cisplatin together with the 3 or 4 mAb combination.
Project description:<b>Aims: </b>Glioblastoma multiforme (GBM) is the most devastating malignant primary brain tumor known. Life expectance is around 15 months after diagnosis. Several events contribute to the GBM progression such as uncontrolled genetic cancer cells proliferation, angiogenesis (mostly vascular endothelial growth factor (VEGF)-mediated), tissue invasion, glioma stem cell activity, immune system failure, and a hypoxic and inflammatory tumor microenvironment. Tumor cells antiproliferative effect of 20 nm citrate-covered gold nanoparticles (cit-AuNP) has been reported, along with anti-inflammatory and anti-oxidative effects. We aimed to test whether either chronic treatment with 20 nm cit-AuNP or anti-VEGF antibody (Ig)-covered AuNP could reduce GBM progression in mice.<br><br><b>Main methods: </b>Effect of the gold nanoparticles on the GL261 glioblastoma cells proliferation <i>in vitro</i>, and on the GL261-induced glioblastoma cell growth in C57BL/6 mice <i>in vivo</i> were tested. Besides, fluorophore-conjugated gold nanoparticles penetration through the GL261 plasma cell membrane, gold labelling in brain parenchyma of glioblastoma-carrying mice, and VEGF expression into the tumor were evaluated.<br><br><b>Key findings: </b>We observed cit-AuNP did no change the GL261 cells proliferation. Similarly, we demonstrated chronic treatment with either cit-AuNP or anti-VEGF Ig-covered AuNP did not modify the GL261 cells-induced GBM progression in mice. By the end, we showed AuNPs did not trespass in appreciable amount both the GL261 plasma cell membrane and the tumoral blood brain barrier (BBB), and did not change the VEGF expression into the tumor.<br><br><b>Significance: </b>20 nm cit-AuNP or anti-VEGF Ig covered-AuNP are not good tools to reduce GBM in mice, probably because they do not penetrate both tumor cells and BBB in enough amount to reduce tumor growing.