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Background: Glioblastoma (GB) recurrence is associated with a highly immunosuppressive tumor microenvironment. Previous research in our lab has illustrated that the carbohydrate-binding protein Galectin-1 (Gal-1) has high expression in GB patient samples and plays vital pro-tumorigenic roles within the tumor microenvironment, regulated by hypoxia-inducible factor-1 (HIF-1). However, since Gal-1 displays many different activities by binding to a number of diverse N- or O-glycan modified target proteins, it has been difficult to fully understand how Gal–1 supports tumor growth and promotes resistance to anti-VEGF mAb. Thus, our aim is to elucidate the role of tumor hypoxia which may in turn increase tumor aggressiveness by up-regulating the expression of Gal-1 in relevant GB in vitro and in vivo model. Methods and Results: GB cells were cultured in a hypoxic environment simulated by cobalt chloride (CoCl2), which is a chemical inducer of hypoxia-inducible factor-1α (HIF-1α). HIF-1α is a transcription factor that has an important role in tumor cell adaptation to hypoxia and controls the expression of several genes. Various CoCl2 concentrations were used to simulate degrees of hypoxia in GB cells. Following treatment with 250 µM CoCl2, an MTT assay was conducted to determine the growth and anti-VEGF mAb (+/- Radiation + Temozolomide) sensitivity of GB cells. We observed that CoCl2(chemical activator of HIF-1α)-treatment simulated hypoxia and induced resistance to anti-VEGF mAb treatment via upregulation of HIF-1α, MDR1/P-gp and MRP protein levels. In addition, proapoptotic members of the Bcl-2 protein family, Bax and Bad, were downregulated. The anti-apoptotic member Bcl-2 exhibited no significant change in expression, whereas the ratio of Bcl-2/Bax was increased. We observed that hypoxia induced Gal-1 through mechanisms that are independent of HIF 1α and HIF-2α but involved reactive oxygen species-dependent activation of the transcription factor nuclear factor κB. Since upregulation of hypoxia inducible transcription factors (HIFs) via Gal-1 is the primary adaptive cellular response to a hypoxic environment, we analyzed the effects of Gal-1 inhibition treatment in vivo. We observed significant alteration of hypoxic tumor microenvironment (TME) in recurrent GB by alteration of tumor associate microglia phenotypes from M2 to. M1, suppression of CD4+CD25+Foxp3+ regulatory T cells and activation of CD 8+ T cells in in vivo GB models. Conclusion: The present study revealed that a CoCl2-simulated hypoxic microenvironment was able to effectively induce resistance to anti-VEGF mAb treatment in GB cells. The results demonstrate the inhibitory effect of Gal-1 in the tumor hypoxia microenvironment and inhibition may be a viable therapeutic target for recurrent GB treatment. Citation Format: Wayne Glore, Arabinda Das, Scott M. Lindhorst, Abhay K. Varma, William A. Vandergrift, Sunil J. Patel, David Cachia. Tumor hypoxia drives immunosuppression via upregulation of galectin-1 in recurrent Glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2685.

Background: Despite aggressive treatment, including surgical resection, radiation and chemotherapy, over 90% of glioblastoma (GB) patients experience tumor recurrence. This may be due to high migratory potential, abnormal angiogenesis, molecular heterogeneity and a strong immunosuppressive environment. An earlier report from our laboratory demonstrated that GB cells express high levels of carbohydrate-binding galectin protein: galectin-1 (Gal-1). However, the role of galectin-1 in GB remains elusive. The aim of this study was to evaluate whether galectin-1 promotes tumor progression through upregulation of CXCR4 via NF-κB in in vitro GB models. Methods: To investigate the molecular mechanisms of Gal-1 on tumor progression, we used RT-PCR and western blots techniques. Gal-1 was stably over-expressed in the U118 and U87MG cell line. Gal-1 (+/-) sub-clones were assayed for attachment, proliferation, migration, and tube formation. Results: Gal-1 protein expression was assessed in human GB cells. We found increased Gal-1 protein expression in human GB cells and tissue. To investigate the effects of Gal-1 silencing on angiogenesis in vitro, we collected conditioned media from Gal-1-knockdown U118 and U87MG cells then applied this to human umbilical vein endothelial cells (HUVEC) culture to analyze endothelial cell proliferation, migration, and tube formation. Proliferation rate, migrating capability, and tube formation decreased significantly when HUVECs were cultured with concentrated conditioned media from Gal-1-knockdown cells. This data suggests that Gal-1 in GB cells may promote tumor angiogenesis both in vitro and co-culture. Results showed that silencing of Gal-1 in U118 and U87MG cells significantly repressed CXCR4 expression at the mRNA level and protein levels. To further explore the role of CXCR4 in GB cells, CXCR4 knockdown resulted in significant inhibition of cell migration and clonogenic ability in vitro. Thus, Gal-1 might upregulate CXCR4 expression to promote tumor progression. A previous laboratory study indicated that NF-κB promotes cancer cell motility by upregulating CXCR4 expression. Luciferase reporter assay was used to determine the involvement of NF-κB in Gal-1-induced CXCR4 expression in U118 and U87MG cells. NF-κB luciferase activity was significantly decreased in Gal-1-knockdown cells. Moreover, decreased CXCR4 promoter activity and protein expression in Gal-1-knockdown cells were observed. This data indicates that Gal-1 might modulate CXCR4 expression via NF-κB activation. Conclusion: Our in vitro cell culture results suggest that upregulation of galectin-1 and CXCR4 might at least in part explain the aggressive infiltrative biology of GB and the Gal-1-CXCR4 axis may serve as a therapeutic target. Further studies in different animal models are warranted. Citation Format: David Cachia, Rachel Malley, William A. Vandergrift, Sunil Patel, Gabriel A. Rabinovich, Arabinda Das. Galectin-1 upregulates CXCR4 in glioblastoma through upregulation of CXCR4 via NF-κB activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3559.

Glioblastoma is the most common and highly aggressive human brain cancer. Most of the glioblastoma patients do not survive more than a few months after the diagnosis. The prognosis for glioblastoma patients remains very poor even after aggressive treatments. Traditional therapeutic strategies are not yet satisfactory for controlling the growth of this deadly malignancy in humans. Heterogeneity of cells in glioblastoma is one of biggest hurdles in its management. Novel and innovative therapeutic agents need to be explored for targeting the key cell signaling pathways that promote proliferation and survival of different human glioblastoma cells. Receptor tyrosine kinases have been implicated in the progression of this malignancy. Anaplastic lymphoma kinase (ALK) and c-Met are two such proteins. ALK/c-Met are associated with the development and progression of many human cancers, including glioblastoma. Also, overexpression or mutation of c-ros proto-oncogene encodes a receptor tyrosine kinase, which also leads to oncogenic development of glioblastoma. In the present study, we examined the activity of the Pfizer drug crizotinib (PF-02341066, a dual ALK/c-Met inhibitor) for suppression of both ALK/c-Met pathways and induction of apoptotic death in different human glioblastoma cell lines (U87MG, T98G, U118MG, and U138MG) and fresh tumor biopsies surgically extracted from 6 glioblastoma patients. Our dose-response studies indicated that treatment with PF-02341066 for 24 h could induce morphological features (as detected by Wright staining) and also biochemical features (as detected by DNA fragmentation assay and Western blotting) of apoptosis. We used Western blotting for investigation into the molecular mechanisms of apoptotic death and found that induction of apoptosis was associated with decreases in expression of ALK, c-Met, c-Ros, Survivin, and Bcl-2 proteins after the treatment. Western blotting also demonstrated that treatment with PF-02341066 caused apoptosis due to an increase in Bax:Bcl-2 ratio and activation of caspase-3 in glionlastoma cells and tissues. Increase in activity of caspase-3 was confirmed by colorimetric assay. Most importantly, PF-02341066 significantly enhanced the anti-cancer effect of temozolomide (TMZ), a widely used anti-glioblastoma drug, in human glioblastoma cell lines (U87MG, T98G, U118MG, and U138MG) as well as in fresh tumor biopsies from 6 glioblastoma patients. Our studies also suggested that induction of apoptosis by PF-02341066 does not depend on the functional status of p53, PTEN, ALK, c-Met, or c-Ros in the glioblastoma cell lines or in the glioblastoma tissues. Further studies in different animal models of glioblastoma are warranted in the future to determine whether PF-02341066 may be useful as an anti-cancer agent alone or in combination with TMZ for the management of human glioblastomas in vivo. This investigation was supported in by Pfizer Oncology. Citation Format: Arabinda Das, Gerald C. Wallace, Catherine Haar, Michele L. DeCandio, W Alex Vandergrift, Sunil J. Patel, Swapan K. Ray, Naren L. Banik, Pierre Giglio. PF-02341066 (crizotinib), a dual ALK/c-Met inhibitor, for inhibiting growth of glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2183. doi:10.1158/1538-7445.AM2013-2183

BACKGROUND: Glioblastoma, the most common and malignant brain tumor in humans, still remains incurable because of inefficacy of the currently available chemotherapeutic agents. Conventional chemotherapeutic agents have not been able to show promising results in glioblastoma patients for the last several decades. Therefore, new and non-conventional therapeutic agents also must be explored for controlling the growth of glioblastoma in vivo. Organosulfur compounds from garlic are well characterized and used as alternative medicine in many Asian countries. As a new treatment strategy in this investigation, we examined the anti-tumor properties of the most potent garlic compound diallyl trisulfide (DATS) in human glioblastoma U87MG xenografts in SCID mice. METHODS: We developed human glioblastoma U87MG xenografts in SCID mice for treatment with DATS for 7 days consecutively. The efficacy of DATS treatments in controlling tumor growth was assessed by histopathological examinations and also molecular analyses such as in situ immunofluorescent labeling and Western blotting. RESULTS: Our findings indicated that a range of DATS doses (10 μg/kg - 10 mg/kg) was effective in reducing tumor volume and weight in SCID mice ectopically xenografted with exponentially growing human glioblastoma U87MG cells. Histopathological examinations of the tumor sections showed significant amounts of inhibition of cell proliferation and induction of cell death. In addition, these findings were associated with a significant reduction in the number of mitotic bodies. Increase in TUNEL-positive staining was also detected in the tumor following treatment, suggesting that DATS induced apoptotic death in glioblastoma xenografts in SCID mice. Further, immunofluorescent staining of the tumor sections confirmed increase in expression of glial fibrillary acidic protein (GFAP), an acclaimed astrocyte-specific marker, in the tumors after the treatments. Our Western blotting demonstrated increases in the apoptosis promoting factors, reduction in the anti-apoptotic proteins (e.g., Bcl-2, BIRC proteins), and activation of the cysteine proteases such as calpain and caspase-3 to favor induction of apoptotic death in the tumors. It should also be mentioned that light microscopic examinations tissue sections revealed no damage in the livers and kidneys from the SCID mice following DATS treatments. CONCLUSIONS: Taken together, our results clearly demonstrated that DATS could be an effective therapeutic agent in preventing not only tumor progression but also induction of apoptosis in human glioblastoma in vivo. Success of our in vivo treatment of human glioblastoma xenografts with DATS strongly suggests that DATS should also be explored as a potential therapeutic agent for treatment glioblastoma in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2350. doi:1538-7445.AM2012-2350

Neuroblastoma is a pediatric tumor that has the highest rate of spontaneous regression. But the metastatic malignant neuroblastomas keep growing and eventually kill the young children. Because immature neuroblasts of the adrenal glands in most cases lead to malignant neuroblastomas, there are ongoing endeavors for finding suitable combination of therapeutic agents for induction of differentiation and apoptosis in malignant neuroblastomas. Retinoids (retinoic acid derivatives) have been shown to inhibit growth and induce neurite outgrowth in human malignant neuroblastoma cell lines. The mechanism by which retinoids inhibit cancer cell growth has not been completely elucidated; however, retinoids have been shown to affect multiple signal transduction pathways. In the present investigation, we compared the capabilities of all-trans retinoic acid (ATRA), 13-cis retinoic acid (13-CRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) at low doses in causing growth inhibition due to induction of differentiation with alterations in expression of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in human malignant neuroblastoma SK-N-BE2 and SK-N-DZ cells. Expression of RARβ was upregulated in SK-N-BE2 and SK-N-DZ cells following treatment with 1 μM of ATRA or 13-CRA for 7 days. Treatment of neuroblastoma cells with 1 μM ATRA, 1 μM 13-CRA, or 0.5 μM 4-HPR for 7 days induced neuronal differentiation with down regulation of telomerase activity. Thereafter, we examined the effects of various combinations of retinoid and flavonoid in SK-N-BE2 and SK-N-DZ cells. Treatment with a flavonoid such as 25 μM (-)-epigallocatechin-3-gallate (EGCG) or 25 μM genistein (GST) for 24 h induced more apoptosis in differentiated cells than undifferentiated cells. Western blotting showed alterations in the levels of Bax and Bcl-2 proteins resulting in an increase in Bax: Bcl-2 ratio in apoptotic cells. Treatment with the combination of retinoid and flavonoid dramatically down regulated the expression of BIRC proteins so as to promote proteolytic activities of the caspases for increasing apoptosis in neuroblastoma cells. Colorimetric assays using chromogenic synthetic peptide substrates confirmed the contribution of different proteolytic mechanisms to apoptosis. The results of this investigation suggested that retinoids induced neuronal differentiation, downregulated telomerase activity, and potentiated the action of flavonoids for increasing apoptosis in neuroblastoma cells. Therefore, combination of retinoid and flavonoid appears to be an effective therapeutic strategy for controlling the growth of human malignant neuroblastoma cells. This investigation was supported in part by the R01 grants (NS-57811 and CA-91460) from the National Institutes of Health (Bethesda, MD). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 678.