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- Material and Methods
- Supporting Information
Galectin-1 is a glycan-binding protein, which is involved in the aggressiveness of glioblastoma (GBM) in part by stimulating angiogenesis. In different cancer models, galectin-1 has also been demonstrated to play a pivotal role in tumor-mediated immune evasion especially by modulating cells of the adaptive immune system. It is yet unknown whether the absence or presence of galectin-1 within the glioma microenvironment also causes qualitative or quantitative differences in innate and/or adaptive antitumor immune responses. All experiments were performed in the orthotopic GL261 mouse high-grade glioma model. Stable galectin-1 knockdown was achieved via transduction of parental GL261 tumor cells with a lentiviral vector encoding a galectin-1-targeting miRNA. We demonstrated that the absence of tumor-derived but not of host-derived galectin-1 significantly prolonged the survival of glioma-bearing mice as such and in combination with dendritic cell (DC)-based immunotherapy. Both flow cytometric and pathological analysis revealed that the silencing of glioma-derived galectin-1 significantly decreased the amount of brain-infiltrating macrophages and myeloid-derived suppressor cells (MDSC) in tumor-bearing mice. Additionally, we revealed a pro-angiogenic role for galectin-1 within the glioma microenvironment. The data provided in this study reveal a pivotal role for glioma-derived galectin-1 in the regulation of myeloid cell accumulation within the glioma microenvironment, the most abundant immune cell population in high-grade gliomas. Furthermore, the prolonged survival observed in untreated and DC-vaccinated glioma-bearing mice upon the silencing of tumor-derived galectin-1 strongly suggest that the in vivo targeting of tumor-derived galectin-1 might offer a promising and realistic adjuvant treatment modality in patients diagnosed with GBM.
Glioblastoma (GBM) is the most frequent and malignant human brain tumor, accounting for ∼50% of all primary brain tumor cases in adults. Despite the availability of multimodal treatments, including maximal, safe neurosurgical resection and chemoradiotherapy, the prognosis of GBM remains dismal with a median survival expectancy of ∼15 months and a mortality rate of 90% within 3 years. Hence, these patients are in high need of new, efficient treatment strategies that improve their quality of life and clinical outcome. During the last decade, significant advances have been made in the development of immunotherapeutic strategies to combat primary intracranial tumors. Our research group[3, 4] and others have demonstrated that DC-based immunotherapy is a promising new treatment strategy in the fight against GBM. However, the extent of regression of GBM that is induced by tumor vaccination remains limited. The absence of robust tumor regression can be partially explained by the abundance of immune modulating mechanisms used by tumor cells to dampen antitumor immune responses. These findings suggest that additional interference with these local immunosuppressive mechanisms will be required to facilitate the development of more potent antiglioma immune responses.
In this respect, galectin-1 could potentially offer an interesting target. Galectin-1 is a natural immunosuppressive glycan-binding protein whose expression is upregulated in several types of cancer, including GBM. 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. Through interactions with β-galactoside-expressing glycoproteins on the T cell surface, galectin-1 can negatively regulate T cell survival,[8, 9] antagonize effector T cell signaling, block pro-inflammatory cytokine secretion and suppress transendothelial migration. Furthermore, galectin-1 can blunt T cell responses by promoting the accumulation and expansion of regulatory T cells (Tregs). The first link between galectin-1 expression at tumor sites and T cell-mediated tumor rejection was reported by Rubinstein et al., who demonstrated that the targeted inhibition of galectin-1 gene expression in mouse melanoma cells resulted in increased T cell-mediated tumor rejection. In addition, animal studies have demonstrated that the metabolic inhibition of galectin-1-binding carbohydrates can delay tumor growth by enhancing tumor lymphocyte infiltration, increasing IFN-ɣ production while lowering IL-10 production. Moreover, in several human malignancies, an inverse correlation was demonstrated between lymphocyte infiltration and galectin-1 expression. Thus far, most studies that have reported an immunosuppressive role for galectin-1 in the tumor microenvironment have revealed a modulating effect of galectin-1 on cells of the adaptive immune system. It is yet unknown whether tumor-derived galectin-1 can also induce quantitative or qualitative differences in tumor-infiltrating myeloid cells.
A number of different studies have already demonstrated a pivotal role for tumor-derived galectin-1 in the aggressiveness of GBM. Galectin-1 has been shown to enhance glioma cell migration, stimulate angiogenesis and to promote the development of chemotherapy and radiotherapy resistance. The importance of galectin-1 in glioma growth and progression is further underscored by the observation that intratumoral galectin-1 expression levels are positively correlated with the grade of malignancy and with worse prognosis. Although the abundance of galectin-1 in malignant glioma is well-known and several mechanisms of galectin-1-mediated immune modulation have been described in different types of cancer, the role of galectin-1 in glioma-mediated immune evasion is still elusive. The primary goal of this study was to examine whether the absence or presence of galectin-1 in the glioma microenvironment results in quantitative or qualitative differences in innate and/or adaptive antitumor immune responses. Moreover, we explored the potency of prophylactic DC-based immunotherapy to protect mice against primary tumor inoculation in the presence or absence of tumor- or host-derived galectin-1. To address these questions, we used the orthotopic GL261 mouse glioma model, which is the most abundantly used immune-competent model to study the potency of immunotherapy approaches against intracranial high-grade glioma.
- Top of page
- Material and Methods
- Supporting Information
In this study, we explored whether the presence of galectin-1 in the glioma microenvironment exerts a modulating role on antiglioma immune responses. Accumulating research in different cancer models has recognized an essential role for galectin-1 in tumor-mediated immune evasion, suggesting that specific interference with the local galectin-1 expression might contribute to overcome tumor immune resistance and boost immunotherapeutic strategies.[7, 14, 15]
In this study, we have provided evidence that the local immune responses in glioma-bearing mice are different in the presence or absence of tumor-derived galectin-1. The most remarkable difference was observed in the accumulation of brain-infiltrating macrophages of tumor-bearing mice. Both flow cytometric and pathological analysis revealed that the silencing of glioma-derived galectin-1 significantly decreased the percentage of brain-infiltrating macrophages in tumor-inoculated mice. To our knowledge, this is the first report that provides in vivo evidence for a role of tumor-derived galectin-1 in myeloid cell recruitment towards the tumor microenvironment. Although myeloid cells, such as macrophages, can exert cytotoxicity towards tumor cells and stimulate adaptive antitumor immune responses, the accumulation of these cells within the tumor microenvironment is associated with poor prognosis in many types of cancer. These TAMs have been shown to promote the aggressiveness of tumors by stimulating tumor cell survival, growth and migration. Especially in high-grade gliomas, it has been shown that myeloid-derived cells in the tumor microenvironment far outweigh the presence of T cells, already revealing the underestimated importance of this innate arm of immunity in this disease. Within GBM, the number of tumor-infiltrating macrophages is increased compared to low-grade glioma. Conflicting data have been reported regarding the role of tumor-infiltrating macrophages in the murine GL261 high-grade glioma model. Galarneau et al. reported enhanced tumor progression and faster mortality in GL261 tumor-inoculated mice that were depleted of macrophages. In contrast, Zhu et al. noted that the systemic neutralization of CCL2 production prolonged the survival of C57BL/6 mice bearing intracranial GL261 gliomas. This observation was concomitant with a strong decrease in TAM and MDSC within the tumor microenvironment, suggesting a protumoral role exerted by these cells. Of note, unpublished data from our research group revealed that prophylactic vaccination of immune-competent mice with lysate-loaded DC improved the median survival rate, which was concomitant with a reduction of MDSC within the tumor microenvironment.
To further examine the link between galectin-1 and myeloid cell recruitment, we evaluated the mRNA expression levels of CCL2 in in vitro cultured GL261-WT and GL261-KD tumor cells. This experiment revealed that the downregulation of the intratumoral galectin-1 expression significantly reduced the mRNA expression levels of CCL2 in cultivated GL261-KD tumor cells. CCL2, a chemokine that recruits both macrophages and MDSCs, was originally identified in and purified from human gliomas. Increased CCL2 expression levels have been identified in biopsies of high-grade gliomas compared with low-grade gliomas, suggesting a role in glioma aggressiveness. Although GL261 cells have been shown to produce low levels of CCL2 in vitro, we were not able to detect CCL2 in nonconcentrated supernatants of in vitro cultured GL261-WT or GL261-KD tumor cells. It is, however, probable that the hypoxic in vivo tumor microenvironment upregulates CCL2 in GL261 tumor cells. In addition to CCL2, we also observed a downregulation of VEGF mRNA and protein expression levels in glioma tumor cells upon silencing of intratumoral galectin-1. VEGF has been shown to be an important chemoattractant for myeloid cells. Moreover, VEGF also stimulates the accumulation of MDSCs within the tumor environment. To present, extensive research has been dedicated to the development of drugs capable of depleting myeloid cells from the tumor microenvironment. In this regard, galectin-1 might represent an important therapeutic target. Likewise, VEGF blockers could theoretically result in therapeutic advantage in combination with immunotherapeutic strategies.
Whether glioma-derived galectin-1 also regulates macrophage function remains an open question. Additional ex vivo functional studies are required to address this issue. Glioma-derived galectin-1 might modulate macrophage phenotype and activity, as several studies have reported a regulatory effect of galectin-1 on myeloid cells.[34-36] Interestingly, in none of these reports galectin-1 binding had an effect on macrophage viability, suggesting that the differences in macrophage numbers observed in this study cannot be explained by galectin-1-mediated modulation of macrophage survival.[37, 38] Moreover, all of the data reported on galectin-1 and macrophage function have been reported in the context of the exposure of macrophages or monocytes to the monomeric form of galectin-1 and not to dimeric galectin-1, which is implicated in the regulation of T cell viability and function.
Whereas compelling evidence has accumulated regarding the modulatory effects of tumor-derived galectin-1 on T cell viability, we found no correlation between the amounts of tumor-secreted galectin-1 and the apoptosis of brain-infiltrating lymphocytes of tumor-inoculated mice. Accumulating data have demonstrated that the outcome of galectin-1-glycan interactions is complex and depends on the biochemical structure of galectin-1 and the nature of the target cell. Galectin-1-triggered T cell death has been shown to require its dimerization. The dimeric form of galectin-1 exhibits a rapid dissociation constant, which limits wide spread T cell-modulatory effects. In addition, the binding of galectin-1 to extracellular ligands preserves its dimeric conformation. The rapid dissociation of homodimeric galectin-1 within the glioma microenvironment might represent a possible explanation for the absence of galectin-1-mediated T cell apoptosis. We did, however, observe a difference in the extent of IFN-ɣ production amongst brain-infiltrating CD8+ T cells isolated from tumor-inoculated mice. In the absence of glioma-derived galectin-1, brain-infiltrating CD8+ T cells produced significant higher levels of IFN-ɣ suggesting that also the adaptive immune system is different in the presence or absence of tumor-derived galectin-1.
Finally, we explored the potency of prophylactic DC-mediated immunotherapy to protect mice against primary tumor inoculation in the presence or absence of galectin-1. Absence of host-derived galectin-1 did not change glioma-bearing mice survival neither in the presence nor in the absence of prophylactic DC vaccination. Interestingly, absence of tumor-derived galectin-1 prolonged the survival of glioma-bearing mice even in the absence of prophylactic DC vaccination. By analyzing the extent of angiogenesis in tumor-inoculated mice, we observed a remarkable and significant reduction in the amount of blood vessels in mice inoculated with galectin-1-depleted glioma cells. These findings further underscore the multimodal role of galectin-1 in the glioma microenvironment. Furthermore, we observed that the silencing of tumor-derived galectin-1 significantly improved the survival of DC-vaccinated tumor-bearing mice. These data are consistent with a recently published study by Stannard et al., who provided the first and thus far, the only evidence showing that combining galectin-blocking carbohydrates with immunotherapy can decrease tumor progression and improve the outcome of tumor-bearing mice. However, in contrast to that study in which thiodigalactoside, a nonmetabolizable dissacharide, was used to none-specifically target the in vivo interaction between galectin-1 and β-galactoside-expressing macromolecules, we developed a galectin-1-targeting miRNA construct to permanently suppress the expression of glioma-derived galectin-1. In this way, we have avoided the non-specific silencing of other possible expressed galectins. Moreover, the use of sequence-specific, post-transcriptional gene silencing has allowed for the interference of both intracellular and extracellular functions of galectin-1. This is interesting because an intracellular role for galectin-1 has been implicated in Ras oncogene activation, in temozolomide resistance (42;42;42) and also in angiogenesis.[25, 26] However, the in vitro transduction of glioma cells with a galectin-1-targeting miRNA construct remains an artificial system that is not easily translatable into a clinical setting. We therefore are currently assessing in vivo delivery systems for galectin-1-targeting siRNAs, the feasibility of which has already been demonstrated in a preliminary study. Note that the presented experiment did not include a group of mice that was treated with mock-loaded DCm (DCm-mock). Previous studies from our research group have demonstrated that prophylactic vaccination with DCm-mock can induce a small, significant improvement in glioma-bearing mice survival, but was not capable of inducing long-term survival.
Overall, these findings suggest that the targeting of glioma-derived galectin-1 could be a promising and realistic new treatment modality to improve the clinical outcome of high-grade patients both in the absence or presence of DC-based immunotherapy.