Author:

Abstract:

Despite major advances in the management of malignant gliomas GBM remains incurable. The aggressive nature of GBM is believed to be due to the extensive spread of tumor cells into surrounding regions of the brain and to the development of treatment resistance. A large amount of experimental evidence has highlighted a role for galectin-1, a hypoxia-sensitive, glycan-binding protein, in the aggressiveness of astrocytic tumors. Within astrocytic tumors, a positive correlation has been described between galectin-1 expression levels and the grade of malignancy. Moreover, high-grade astrocytic tumors with high levels of galectin-1 expression are associated with dismal prognosis as compared to high-grade astrocytic tumors with low expression levels of galectin-1. Galectin-1 regulation of glioma aggressiveness is multimodal. Galectin-1 has been shown to modulate tumor astrocyte migration. In addition, intratumoral galectin-1 expression was demonstrated to protect glioma cells against cytotoxic insults such as chemotherapy and radiotherapy. Moreover, a pro-angiogenic role for galectin-1 has been described in several other malignancies as well as in gliomas. In a variety of cancer models, the presence of galectin-1 within the tumor microenvironment has been shown to contribute significantly to the establishment of local immune resistance. Although the abundance of galectin-1 in malignant glioma is well-known, it remains to be determined whether this lectin also plays an important role in glioma-mediated immune evasion. Hence, we investigated whether glioma-derived galectin-1 is capable of modulating innate or adaptive anti-glioma immune responses in an experimental murine glioma model. This syngeneic mouse model comprises the intracranial challenge of immune competent mice with GL261 HGG cells. To investigate whether glioma-derived galectin-1 exerts an immune modulating function, we developed a GL261 tumor cell line in which the galectin-1 expression was permanently and stably silenced. Among the different galectin-1 knockdown clones that were generated we selected one monoclonal cell line (GL261-KD) in which a strong decrease in galectin-1 protein and gene expression level was observed. In a first set of experiments, we analyzed the impact of intratumoral galectin-1 downregulation on in vivo glioma progression. These experiments revealed that the silencing of glioma-derived galectin-1 significantly improves the outcome of glioma-bearing mice. This observation could not be explained by a direct role of galectin-1 on tumor cell proliferation, as wild-type GL261-wt tumor cells as well as GL261-KD tumor cells exhibited a similar in vitro proliferation rate. Interestingly, absence of host-derived galectin-1 had no effect on in vivo glioma progression. In a next step, we analyzed whether the silencing of glioma-derived galectin-1 induced proportional or phenotypic changes in brain-infiltrating myeloid cells. Both flow cytometric and pathological analysis revealed a significant decrease in the percentage of brain-infiltrating macrophages and MDSC amongst the total population of infiltrating CD45high immune cells upon intratumoral silencing of the galectin-1 expression. To further explore the link between tumor-derived galectin-1 and myeloid cell recruitment towards the tumor microenvironment, we analyzed CCL2 and VEGF mRNA expression levels in in vitro cultured GL261-WT and GL261-KD tumor cells. This experiment revealed that the downregulation of intratumoral galectin-1 reduced the mRNA expression levels of both CCL2 and VEGF in in vitro cultivated GL261 tumor cells. Moreover, CCL2 expression was also significantly downregulated in brain-infiltrating myeloid cells that were isolated from GL261-KD tumor-bearing mice, suggesting the presence of a negative, autocrine feedback loop. Interestingly, we observed a similar negative correlation between intratumoral galectin-1 expression levels and the magnitude of macrophage / microglia influx in biopsies of patients with newly diagnosed GBM. Whether glioma-derived galectin-1 also regulates macrophage function in the GL261 glioma model remains elusive. From our experiments, it is clear that myeloid cells that are recruited to a galectin-1-depleted glioma microenvironment have a phenotype that is considerably different from the phenotype of myeloid cells that were recruited to a galectin-1 enriched glioma microenvironment. However, further ex vivo functional studies are required to address the role of glioma-derived galectin-1 in the education of TAM. To investigate the involvement of the adaptive immune response in the prolonged survival of GL261-KD tumor-bearing mice, tumor cells were injected into immune compromised NSG mice which lack mature T cells, B cells and functional NK cells. These experiments demonstrated that the prolonged survival upon an intracranial challenge with GL261-KD tumor cells requires the presence of an intact adaptive immune system. Whereas compelling evidence has accumulated regarding the modulating effects of tumor-derived galectin-1 on T cell viability, we found no correlation between the amounts of tumor-derived galectin-1 and the apoptosis of brain-infiltrating lymphocytes. However, we did observe a modulating effect of galectin-1 on T cell function, as in the absence of galectin-1 the brain-infiltrating T cells produced significantly higher amounts of IFN-ɣ. Interestingly, quantification of the number of blood vessels in GL261-WT or GL261-KD tumor-bearing mice revealed that the silencing of glioma-derived galectin-1 also impaired angiogenesis, thereby underscoring the multimodal role of this lectin in the glioma microenvironment. Finally, we explored whether the silencing of glioma-derived galectin-1 could improve the survival of vaccinated tumor-bearing mice. These experiments revealed that prophylactic DC vaccination of either GL261-WT or GL261-KD tumor-bearing mice significantly prolongs the survival of tumor-bearing mice. In addition, prophylactic DC vaccination protected a small subset of mice against primary intracranial tumor challenge. Moreover, comparison of the outcome of vaccinated GL261-WT tumor-challenged mice and vaccinated GL261-KD tumor-challenged mice revealed the presence of a significant survival benefit in vaccinated GL261-KD tumor-challenged mice. Moreover, we demonstrated that this survival benefit was only observed upon the silencing of tumor-derived but not of host-derived galectin-1. Collectively, these data demonstrate that tumor-derived galectin-1 is a modulator of anti-glioma immune responses and that the silencing of intratumoral galectin-1 expression improves the outcome of tumor-bearing mice both in the presence or absence of DC vaccination. Hence, the targeting of galectin-1 might offer an adjuvant modality in the treatment of patients with malignant gliomas.Thus far, it is unknown whether increased galectin-1 expression levels are exclusively found at the site of the tumor or whether altered galectin-1 levels can also be found in the serum of patients with HGG as a consequence of a leaky brain tumor vasculature. Hence, we evaluated the galectin-1 serum levels in a prospective dataset of 43 healthy controls and 125 patients with newly diagnosed or recurrent HGG. Patient blood samples were collected at the moment of neurosurgical intervention and/or at the moment of leukapheresis (approximately 2-3 weeks after surgery) in case of postoperative tumor vaccination therapy. Galectin-1 serum levels were measured by means of ELISA. The comparison of the galectin-1 serum levels in healthy controls revealed that the galectin-1 serum levels depended significantly on age and sex. Therefore, a multivariable model for galectin-1 with age and sex as predictors has been fitted in the control patients. Age- and sex-adjusted galectin-1 serum levels were significantly higher in all patient subgroups compared to healthy controls with a high discriminative ability that increased with age. Collectively, the data presented in this study may represent a first step to establish galectin-1 as a biomarker in glioma disease monitoring. Further longitudinal evaluation is required and ongoing to investigate the value of galectin-1 serum levels in HGG patients as an additional diagnostic marker and as a predictor of treatment response and prognosis. Furthermore, galectin-1 serum levels could also provide an important tool for the identification of HGG patients that could benefit from galectin-1 directed therapies that are currently under development.