A single-step, rapid, and versatile method for simultaneous detection of cell surface glycan profiles using fluorochrome-conjugated lectins.

Cell surface glycans play essential roles in diverse physiological and pathological processes and their assessment has important implications in biomedicine and biotechnology. Here we present a rapid, versatile, and single-step multicolor flow cytometry method for evaluation of cell surface glycan signatures using a panel of selected fluorochrome-conjugated lectins. This procedure allows simultaneous detection of cell surface glycans with a 10-fold reduction in the number of cells required compared with traditional multistep lectin staining methods. Interestingly, we used this one-step lectin array coupled with dimension reduction algorithms in a proof-of-concept application for discrimination among different tumor and immune cell populations. Moreover, this procedure was also able to unveil T-, B-, and myeloid cell subclusters exhibiting differential glycophenotypes. Thus, we report a rapid and versatile lectin cytometry method to simultaneously detect a particular repertoire of surface glycans on living cells that can be easily implemented in different laboratories and core facilities.

Untangling Galectin-Mediated Circuits that Control Hypoxia-Driven Angiogenesis.

Development of an aberrant vascular network is a hallmark of the multistep pathological process of tumor growth and metastasis. In response to hypoxia, several pro-angiogenic factors are synthesized to support vascularization programs required for cancer progression. Emerging data indicate the involvement of glycans and glycan-binding proteins as critical regulators of vascular circuits in health and disease. Galectins may be regulated by hypoxic conditions and control angiogenesis in different physiopathological settings. These ß-galactoside-binding proteins may promote sprouting angiogenesis by interacting with different glycosylated receptors and triggering distinct signaling pathways. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current anti-cancer modalities and overcoming resistance to these treatments. Here we describe selected strategies and methods used to study the role of hypoxia-regulated galectins in the regulation of blood vessel formation.

Galectin-1 fosters an immunosuppressive microenvironment in colorectal cancer by reprogramming CD8+ regulatory T cells.

Colorectal cancer (CRC) represents the third most common malignancy and the second leading cause of cancer-related deaths worldwide. Although immunotherapy has taken center stage in mainstream oncology, it has shown limited clinical efficacy in CRC, generating an urgent need for discovery of new biomarkers and potential therapeutic targets. Galectin-1 (Gal-1), an endogenous glycan-binding protein, induces tolerogenic programs and contributes to tumor cell evasion of immune responses. Here, we investigated the relevance of Gal-1 in CRC and explored its modulatory activity within the CD8+ regulatory T cell (Treg) compartment. Mice lacking Gal-1 (Lgals1-/- ) developed a lower number of tumors and showed a decreased frequency of a particular population of CD8+CD122+PD-1+ Tregs in the azoxymethane-dextran sodium sulfate model of colitis-associated CRC. Moreover, silencing of tumor-derived Gal-1 in the syngeneic CT26 CRC model resulted in reduced number and attenuated immunosuppressive capacity of CD8+CD122+PD-1+ Tregs, leading to slower tumor growth. Moreover, stromal Gal-1 also influenced the fitness of CD8+ Tregs, highlighting the contribution of both tumor and stromal-derived Gal-1 to this immunoregulatory effect. Finally, bioinformatic analysis of a colorectal adenocarcinoma from The Cancer Genome Atlas dataset revealed a particular signature characterized by high CD8+ Treg score and elevated Gal-1 expression, which delineates poor prognosis in human CRC. Our findings identify CD8+CD122+PD-1+ Tregs as a target of the immunoregulatory activity of Gal-1, suggesting a potential immunotherapeutic strategy for the treatment of CRC.

Reprogramming the tumor metastasis cascade by targeting galectin-driven networks.

A sequence of interconnected events known as the metastatic cascade promotes tumor progression by regulating cellular and molecular interactions between tumor, stromal, endothelial, and immune cells both locally and systemically. Recently, a new concept has emerged to better describe this process by defining four attributes that metastatic cells should undergo. Every individual hallmark represents a unique trait of a metastatic cell that impacts directly in the outcome of the metastasis process. These critical features, known as the hallmarks of metastasis, include motility and invasion, modulation of the microenvironment, cell plasticity and colonization. They are hierarchically regulated at different levels by several factors, including galectins, a highly conserved family of ß-galactoside-binding proteins abundantly expressed in tumor microenvironments and sites of metastasis. In this review, we discuss the role of galectins in modulating each hallmark of metastasis, highlighting novel therapeutic opportunities for treating the metastatic disease.

Enhanced Antitumor Immunity via Endocrine Therapy Prevents Mammary Tumor Relapse and Increases Immune Checkpoint Blockade Sensitivity.

The role of active antitumor immunity in hormone receptor-positive (HR+) breast cancer has been historically underlooked. The aim of this study was to determine the contribution of the immune system to antiprogestin-induced tumor growth inhibition using a hormone-dependent breast cancer model. BALB/c-GFP+ bone marrow (BM) cells were transplanted into immunodeficient NSG mice to generate an immunocompetent NSG/BM-GFP+ (NSG-R) mouse model. Treatment with the antiprogestin mifepristone (MFP) inhibited growth of 59-2-HI tumors with similar kinetics in both animal models. Interestingly, MFP treatment reshaped the tumor microenvironment, enhancing the production of proinflammatory cytokines and chemokines. Tumors in MFP-treated immunocompetent mice showed increased infiltration of F4/80+ macrophages, natural killer, and CD8 T cells, displaying a central memory phenotype. Mechanistically, MFP induced immunogenic cell death (ICD) in vivo and in vitro, as depicted by the expression and subcellular localization of the alarmins calreticulin and HMGB-1 and the induction of an ICD gene program. Moreover, MFP-treated tumor cells efficiently activated immature dendritic cells, evidenced by enhanced expression of MHC-II and CD86, and induced a memory T-cell response, attenuating tumor onset and growth after re-challenge. Finally, MFP treatment increased the sensitivity of HR+ 59-2-HI tumor to PD-L1 blockade, suggesting that antiprogestins may improve immunotherapy response rates. These results contribute to a better understanding of the mechanisms underlying the antitumor effect of hormonal treatment and the rational design of therapeutic combinations based on endocrine and immunomodulatory agents in HR+ breast cancer. SIGNIFICANCE: Antiprogestin therapy induces immunogenic tumor cell death in PRA-overexpressing tumors, eliciting an adaptive immune memory response that protects mice from future tumor recurrence and increases sensitivity to PD-L1 blockade. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/5/1375/F1.large.jpg.

Sweetening the hallmarks of cancer: Galectins as multifunctional mediators of tumor progression.

Hanahan and Weinberg have proposed 10 organizing principles that enable growth and metastatic dissemination of cancer cells. These distinctive and complementary capabilities, defined as the "hallmarks of cancer," include the ability of tumor cells and their microenvironment to sustain proliferative signaling, evade growth suppressors, resist cell death, promote replicative immortality, induce angiogenesis, support invasion and metastasis, reprogram energy metabolism, induce genomic instability and inflammation, and trigger evasion of immune responses. These common features are hierarchically regulated through different mechanisms, including those involving glycosylation-dependent programs that influence the biological and clinical impact of each hallmark. Galectins, an evolutionarily conserved family of glycan-binding proteins, have broad influence in tumor progression by rewiring intracellular and extracellular circuits either in cancer or stromal cells, including immune cells, endothelial cells, and fibroblasts. In this review, we dissect the role of galectins in shaping cellular circuitries governing each hallmark of tumors, illustrating relevant examples and highlighting novel opportunities for treating human cancer.

Glycans Pave the Way for Immunotherapy in Triple-Negative Breast Cancer.

The clinical efficacy of therapies targeting the PD-1/PD-L1 pathway is still limited. In this issue of Cancer Cell, Li and colleagues identify a PD-L1 glycosylation-based mechanism in triple-negative breast cancer that fosters immunosuppression by enhancing interactions with PD-1. Targeting glycosylated PD-L1 with a drug-conjugated antibody opens new avenues for treatment.

Differential response of myeloid-derived suppressor cells to the nonsteroidal anti-inflammatory agent indomethacin in tumor-associated and tumor-free microenvironments.

Myeloid-derived suppressor cells (MDSCs) are key regulatory cells that control inflammation and promote tumor-immune escape. To date, no specific immunomodulatory drug has proven efficacy in targeting the expansion and/or function of these cells in different pathophysiologic settings. In this study, we identified a context-dependent effect of the nonsteroidal anti-inflammatory drug indomethacin (IND) on MDSCs, depending on whether they were derived from tumor microenvironments (TME) or from tumor-free microenvironments (TFME). Treatment of mice bearing the LP07 lung adenocarcinoma with IND inhibited the suppressive activity of splenic MDSCs, which restrained tumor growth through mechanisms involving CD8(+) T cells. The same effect was observed when MDSCs were treated with IND and conditioned media from LP07 tumor cells in vitro. However, in the absence of a tumor context, IND enhanced the intrinsic suppressive function of MDSCs and amplified their protumoral activity. In a model of autoimmune neuroinflammation, IND-treated MDSCs differentiated in TFME attenuated inflammation, whereas IND-treated MDSCs differentiated in TME aggravated clinical symptoms and delayed resolution of the disease. Mechanistically, IND reduced arginase activity as well as NO and reactive oxygen species production in MDSCs differentiated in TME but not in TFME. Moreover, expression of the C/EBP-ß transcription factor isoforms correlated with the suppressive activity of IND-treated MDSCs. Our study unveils the dual and context-dependent action of IND, a drug that serves both as an anti-inflammatory and anticancer agent, which differentially affects MDSC activity whether these cells are derived from TME or TFME. These results have broad clinical implication in cancer, chronic inflammation and autoimmunity.

Study of galectins in tumor immunity: strategies and methods.

During the past decade, a better understanding of the cellular and molecular mechanisms underlying tumor immunity has provided the appropriate framework for the development of therapeutic strategies for cancer immunotherapy. Under this complex scenario, galectins have emerged as promising molecular targets for cancer therapy responsible of creating immunosuppressive microenvironments at sites of tumor growth and metastasis. Galectins, expressed in tumor, stromal, and endothelial cells, contribute to thwart the development of immune responses by favoring the expansion of T regulatory cells and contributing to their immunosuppressive activity, driving the differentiation of tolerogenic dendritic cells, limiting T cell viability, and maintaining T cell anergy. The emerging data promise a future scenario in which the selective blockade of individual members of the galectin family, either alone or in combination with other therapeutic regimens, will contribute to halt tumor progression by counteracting tumor-immune escape. Here we describe a selection of methods used to investigate the role of galectin-1 in tumor-immune escape.

Regulation of galectins by hypoxia and their relevance in angiogenesis: strategies and methods.

Formation of an aberrant and heterogeneous vascular network is a key pathological event in the multistep process of tumor growth and metastasis. Pro-angiogenic factors are synthesized and released from tumor, stromal, endothelial, and myeloid cells in response to hypoxic and immunosuppressive microenvironments which are commonly found during cancer progression. Emerging data indicate key roles for galectins, particularly galectin-1, -3, -8, and -9 in the regulation of angiogenesis in different pathophysiologic settings. Each galectin interacts with a preferred set of glycosylated receptors, triggers different signaling pathway, and promotes sprouting angiogenesis through different mechanisms. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current clinical approaches. Here we describe selected strategies and methods used to study the galectin-1 regulation by hypoxia and its role in blood vessel formation.

Microbially driven TLR5-dependent signaling governs distal malignant progression through tumor-promoting inflammation.

The dominant TLR5(R392X) polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

Glycosylation-dependent lectin-receptor interactions preserve angiogenesis in anti-VEGF refractory tumors.

The clinical benefit conferred by vascular endothelial growth factors (VEGF)-targeted therapies is variable, and tumors from treated patients eventually reinitiate growth. Here, we identify a glycosylation-dependent pathway that compensates for the absence of cognate ligand and preserves angiogenesis in response to VEGF blockade. Remodeling of the endothelial cell (EC) surface glycome selectively regulated binding of galectin-1 (Gal1), which upon recognition of complex N-glycans on VEGFR2, activated VEGF-like signaling. Vessels within anti-VEGF-sensitive tumors exhibited high levels of α2-6-linked sialic acid, which prevented Gal1 binding. In contrast, anti-VEGF refractory tumors secreted increased Gal1 and their associated vasculature displayed glycosylation patterns that facilitated Gal1-EC interactions. Interruption of ß1-6GlcNAc branching in ECs or silencing of tumor-derived Gal1 converted refractory into anti-VEGF-sensitive tumors, whereas elimination of α2-6-linked sialic acid conferred resistance to anti-VEGF. Disruption of the Gal1-N-glycan axis promoted vascular remodeling, immune cell influx and tumor growth inhibition. Thus, targeting glycosylation-dependent lectin-receptor interactions may increase the efficacy of anti-VEGF treatment.

Clinical relevance of galectin-1 expression in non-small cell lung cancer patients.

BACKGROUND: Identification of biomarkers in lung cancer, a leading cause of cancer-related mortality, has a meaningful clinical relevance in the quest of novel prognostic factors and therapeutic targets. The glycan-binding protein galectin-1 (Gal-1) modulates tumor progression by mediating cell-cell and cell-extracellular matrix interactions, as well as angiogenesis and tumor immune-escape. Previous works reported the expression of Gal-1 in lung cancer, although its clinical significance remains uncertain. OBJECTIVE: To assess the clinicopathologic relevance and prognostic value of Gal-1 expression in a cohort of 103 Stage I-III non-small cell lung cancer (NSCLC) patients. METHODS: Gal-1 expression was determined by immunohistochemistry in tumor tissue samples. The percentage of immunoreactive tumor cells and stroma, as well as the presence of blood vessels with positively stained endothelium in the tumor and surrounding normal tissue, were recorded. Results were correlated with the clinicopathologic factors of the patients (Spearman's rank correlation coefficient, chi-square test) and overall survival by univariate (Kaplan Meier) and multivariate analyses (Cox regression hazard model). RESULTS: We did not observe significant associations between Gal-1 expression and relevant clinicopathologic features at diagnosis of NSCLC. However, Kaplan Meier analysis revealed a significant association between Gal-1 expression and overall survival, when Gal-1 expression was analyzed on tumor cells alone ("tumor cell percentage") or when an integrated score accounting for tumor cell as well as stromal expression of Gal-1 ("total score") was assessed. Patients showing high Gal-1 expression evidenced a poorer clinical outcome. Furthermore, "total score" remained significantly associated with survival by multivariate Cox regression analysis in the whole cohort of patients, even when controlling for the classical predictors and prognostic factors of NSCLC. CONCLUSION: We conclude that Gal-1 expression may be a useful biomarker for better prediction of the clinical outcome and management of NSCLC patients.

Expansion of CD11b(+)Ly6G (+)Ly6C (int) cells driven by medroxyprogesterone acetate in mice bearing breast tumors restrains NK cell effector functions.

The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b(+)Gr-1(+), mostly CD11b(+)Ly6G(+)Ly6C(int) and CD11b(+)Ly6G(-)Ly6C(high) cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b(+)Ly6G(+)Ly6C(int) cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b(+)Ly6G(+)Ly6C(int) cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b(+)Gr-1(+) cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b(+)Gr-1(+) cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b(+)Ly6G(+)Ly6C(int) cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.

Thwarting galectin-induced immunosuppression in breast cancer.

Novel therapeutic strategies are needed to counteract breast cancer-associated immunosuppression. Silencing the expression of galectin-1 in a breast carcinoma model inhibited tumor growth and prevented lung metastasis by reducing the frequency and immunosuppressive activity of CD4+CD25+ FOXP3+ regulatory T cells.

Targeting galectin-1 overcomes breast cancer-associated immunosuppression and prevents metastatic disease.

Galectin-1 (Gal1), an evolutionarily conserved glycan-binding protein, contributes to the creation of an immunosuppressed microenvironment at sites of tumor growth. In spite of considerable progress in elucidating its role in tumor-immune escape, the mechanisms underlying the inhibitory functions of Gal1 remain obscure. Here, we investigated the contribution of tumor Gal1 to tumor growth, metastasis, and immunosuppression in breast cancer. We found that the frequency of Gal1(+) cells in human breast cancer biopsies correlated positively with tumor grade, while specimens from patients with benign hyperplasia showed negative or limited Gal1 staining. To examine the pathophysiologic relevance of Gal1 in breast cancer, we used the metastatic mouse mammary tumor 4T1, which expresses and secretes substantial amounts of Gal1. Silencing Gal1 expression in this model induced a marked reduction in both tumor growth and the number of lung metastases. This effect was abrogated when mice were inoculated with wild-type 4T1 tumor cells in their contralateral flank, suggesting involvement of a systemic modulation of the immune response. Gal1 attenuation in 4T1 cells also reduced the frequency of CD4(+)CD25(+) Foxp3(+) regulatory T (T(reg)) cells within the tumor, draining lymph nodes, spleen, and lung metastases. Further, it abrogated the immunosuppressive function of T(reg) cells and selectively lowered the expression of the T-cell regulatory molecule LAT (linker for activation of T cells) on these cells, disarming their suppressive activity. Taken together, our results offer a preclinical proof of concept that therapeutic targeting of Gal1 can overcome breast cancer-associated immunosuppression and can prevent metastatic disease.

Disrupting galectin-1 interactions with N-glycans suppresses hypoxia-driven angiogenesis and tumorigenesis in Kaposi's sarcoma.

Kaposi's sarcoma (KS), a multifocal vascular neoplasm linked to human herpesvirus-8 (HHV-8/KS-associated herpesvirus [KSHV]) infection, is the most common AIDS-associated malignancy. Clinical management of KS has proven to be challenging because of its prevalence in immunosuppressed patients and its unique vascular and inflammatory nature that is sustained by viral and host-derived paracrine-acting factors primarily released under hypoxic conditions. We show that interactions between the regulatory lectin galectin-1 (Gal-1) and specific target N-glycans link tumor hypoxia to neovascularization as part of the pathogenesis of KS. Expression of Gal-1 is found to be a hallmark of human KS but not other vascular pathologies and is directly induced by both KSHV and hypoxia. Interestingly, hypoxia induced Gal-1 through mechanisms that are independent of hypoxia-inducible factor (HIF) 1α and HIF-2α but involved reactive oxygen species-dependent activation of the transcription factor nuclear factor κB. Targeted disruption of Gal-1-N-glycan interactions eliminated hypoxia-driven angiogenesis and suppressed tumorigenesis in vivo. Therapeutic administration of a Gal-1-specific neutralizing mAb attenuated abnormal angiogenesis and promoted tumor regression in mice bearing established KS tumors. Given the active search for HIF-independent mechanisms that serve to couple tumor hypoxia to pathological angiogenesis, our findings provide novel opportunities not only for treating KS patients but also for understanding and managing a variety of solid tumors.

The aggressiveness of murine lymphomas selected in vivo by growth rate correlates with galectin-1 expression and response to cyclophosphamide.

Although lymphomas account for almost half of blood-derived cancers that are diagnosed each year, the causes of new cases are poorly understood, as reflected by the relatively few risk factors established. Galectin-1, an immunoregulatory ß-galactoside-binding protein, has been widely associated with tumor-immune escape. The aim of the present work was to study the relationship between tumor growth rate, aggressiveness, and response to cyclophosphamide (Cy) therapy with regard to Gal-1 expression in murine T-cell lymphoma models. By means of a disruptive selection process for tumor growth rate, we generated two lymphoma variants from a parental T-cell lymphoma, which have unique characteristics in terms of tumor growth rate, spontaneous regression, metastatic capacity, Gal-1 expression and sensitivity to Cy therapy. Here, we show that Gal-1 expression strongly correlates with tumor growth rate, metastatic capacity and response to single-dose Cy therapy in T-cell lymphoma models; this association might have important consequences for evaluating prognosis and treatments of this type of tumors.

Neuroblastoma triggers an immunoevasive program involving galectin-1-dependent modulation of T cell and dendritic cell compartments.

The immunosuppressive strategies devised by neuroblastoma (NB), the most common solid extracranial childhood cancer, are poorly understood. Here, we identified an immunoevasive program triggered by NB through secretion of galectin-1 (Gal-1), a multifunctional glycan-binding protein. Human and mouse NB cells express and secrete Gal-1, which negatively regulates T cell and dendritic cell function. When injected subcutaneously in syngeneic A/J mice, knockdown transfectants expressing low amounts of Gal-1 (NXS2/L) showed reduction of primary tumor growth by 83-90% and prevented spontaneous liver metastases in contrast to NXS2 cell variants (NXS2/H, NXS2 wildtype) expressing high amounts of Gal-1. Splenocytes from mice receiving Gal-1 knockdown NXS2/L cells secreted higher amounts of IFN-γ and displayed enhanced cytotoxic T-cell function compared to NXS2/H or NXS2 controls. Immunohistochemical analysis revealed a six- to tenfold increase in the frequency of CD4+ and CD8+ T cells infiltrating tumors from mice receiving knockdown transfectants. This effect was confirmed by in vitro migration assays. Finally, supernatants of NXS2/H or NXS2 cells suppressed dendritic cell (DC) maturation and induce T cell apoptosis, whereas these effects were only marginal on DCs and T cells exposed to supernatants from NXS2/L cells. These results demonstrate a novel immunoinhibitory role of the Gal-1-glycan axis in NB, highlighting an alternative target for novel immunotherapeutic modalities.