Low-dose cyclophosphamide modulates galectin-1 expression and function in an experimental rat lymphoma model

In recent years, one of the most important insights into tumor immunity was provided by the identification of negative regulatory pathways and immune escape strategies that greatly influence the magnitude of antitumor responses. Galectin-1 (Gal-1), a member of a family of highly conserved β-galactoside-binding proteins, has been recently shown to contribute to tumor cell evasion of immune responses by modulating survival and differentiation of effector T cells. However, there is still scarce information about the regulation of Gal-1 expression and function in vivo. Here we show that administration of a single low-dose cyclophosphamide (Cy), which is capable of restraining metastasis in the rat lymphoma model L-TACB, can also influence Gal-1 expression in primary tumor, metastasis, and spleen cells and modulate the effects of this protein on T cell survival. A time-course study revealed a positive correlation between Gal-1 expression and tumor volume in primary tumor cells. Conversely, Gal-1 expression was significantly reduced in spleen cells and lymph node metastasis throughout the period studied. Interestingly, cyclophosphamide treatment was capable of restoring the basal levels of Gal-1 expression in primary tumors and spleens. In addition, this antimetastatic agent rendered spleen T cells from tumor-bearing animals resistant to Gal-1-induced cell death. Our results suggest that, in addition to other well-known functions of cyclophosphamide, this immunomodulatory agent may also modulate Gal-1 expression and function during tumor growth and metastasis with critical implications for tumor-immune escape and immunotherapy.Mariano F. Zacarías Fluck and María J. Rico contributed equally to this work.     

Th2/Th1 switch induced by a single low dose of cyclophosphamide in a rat metastatic lymphoma model

Cyclophosphamide (Cy) is an alkylating agent widely used in cancer chemotherapy. It has a bimodal effect on the immune system, depending on the dose and schedule of administration. We have previously demonstrated that a single low dose of Cy has an antimetastatic effect, achieved through immunomodulation, in lymphoma bearing rats. Such a treatment reduced the splenic production of IL-10, TGF-beta, and NO, restoring the lymphoproliferative capacity. A shift from immunosuppression to immunopotentiation induced by low-dose Cy treatment was mainly mediated by a decrease in IL-10 production. The present study focused on the analysis of the modulation of type-1 cytokine levels by treatment with a single low dose of Cy and the effect these cytokines (IL-2 and IFN-gamma) and IL-10 have on primary tumor and metastatic cell growth. Our results suggest that a single low dose of Cy induces a Th2/Th1 shift in the cytokine profile of lymphoma-bearing rats, which may be responsible for its antimetastatic effect. A direct action of IL-10 as a growth factor and IFN-gamma as a cytotoxic factor on metastatic cells is also shown.     

Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.     

Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinases-1 in the liver: efficient protection against T-cell lymphoma and colon carcinoma metastasis

BACKGROUND: Matrix metalloproteinases (MMPs) are critical for metastasis of tumor cells. Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural MMP inhibitor, was shown to reduce metastasis in different models. Here, we investigated whether increased TIMP-1 levels in the liver achieved by adenoviral gene transfer will effectively inhibit liver metastasis of two independent tumor cell lines. METHOD: TIMP-1 was transferred with adenoviral vectors into the livers of DBA/2 and Balb/c mice, which were subsequently challenged by hematogenous experimental metastases of the T-cell lymphoma cell line L-CI.5s or the colorectal carcinoma cell line CT-26, respectively. RESULTS: MMP-9 expression in the liver was induced upon metastasis in both tumor types. Adenoviral gene transfer led to high transduction efficacy as indicated by lacZ expression in 60% of hepatocytes. TIMP-1, a key inhibitor of MMP-9, was expressed at 10(5)-fold higher levels by adenoviral gene transfer as compared with levels achieved in TIMP-1 transgenic mice, previously shown to be inefficient to reduce T-cell lymphoma metastasis. High local and systemic (serum) levels of TIMP-1 led to substantial (94%) reduction of T-cell lymphoma and colorectal carcinoma (73%) experimental liver metastasis. CONCLUSIONS: Adenoviral gene transfer led to systemic and local TIMP-1 levels sufficient to inhibit metastasis of a highly aggressive T-cell lymphoma, pointing at the requirement of threshold levels for effective anti-metastatic efficacy. This approach was also efficient in a colon carcinoma solid tumor model. We propose that viral gene transfer of TIMP-1 can provide a suitable defense strategy to prevent metastatic spread to the liver.     

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.     

Development and characterization of anti-galectin-9 antibodies that protect T cells from galectin-9-induced cell death

Antibodies that target immune checkpoint proteins such as programmed cell death protein 1, programmed death ligand 1, and cytotoxic T-lymphocyte–associated antigen 4 in human cancers have achieved impressive clinical success; however, a significant proportion of patients fail to respond to these treatments. Galectin-9 (Gal-9), a β-galactoside-binding protein, has been shown to induce T-cell death and facilitate immunosuppression in the tumor microenvironment by binding to immunomodulatory receptors such as T-cell immunoglobulin and mucin domain–containing molecule 3 and the innate immune receptor dectin-1, suggesting that it may have potential as a target for cancer immunotherapy. Here, we report the development of two novel Gal-9-neutralizing antibodies that specifically react with the N-carbohydrate-recognition domain of human Gal-9 with high affinity. We also show using cell-based functional assays that these antibodies efficiently protected human T cells from Gal-9-induced cell death. Notably, in a T-cell/tumor cell coculture assay of cytotoxicity, these antibodies significantly promoted T cell-mediated killing of tumor cells. Taken together, our findings demonstrate potent inhibition of human Gal-9 by neutralizing antibodies, which may open new avenues for cancer immunotherapy.     

Identification of galectin-1 as a critical factor in function of mouse mesenchymal stromal cell-mediated tumor promotion

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development.These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression.     

Galectin-1: a small protein with major functions

Galectins are a family of carbohydrate-binding proteins with an affinity for beta-galactosides. Galectin-1 (Gal-1) is differentially expressed by various normal and pathological tissues and appears to be functionally polyvalent, with a wide range of biological activity. The intracellular and extracellular activity of Gal-1 has been described. Evidence points to Gal-1 and its ligands as one of the master regulators of such immune responses as T-cell homeostasis and survival, T-cell immune disorders, inflammation and allergies as well as host-pathogen interactions. Gal-1 expression or overexpression in tumors and/or the tissue surrounding them must be considered as a sign of the malignant tumor progression that is often related to the long-range dissemination of tumoral cells (metastasis), to their dissemination into the surrounding normal tissue, and to tumor immune-escape. Gal-1 in its oxidized form plays a number of important roles in the regeneration of the central nervous system after injury. The targeted overexpression (or delivery) of Gal-1 should be considered as a method of choice for the treatment of some kinds of inflammation-related diseases, neurodegenerative pathologies and muscular dystrophies. In contrast, the targeted inhibition of Gal-1 expression is what should be developed for therapeutic applications against cancer progression. Gal-1 is thus a promising molecular target for the development of new and original therapeutic tools.     

Galectin-1 activates carbonic anhydrase IX and modulates glioma metabolism

Galectins are a family of β-galactose-specific binding proteins residing within the cytosol or nucleus, with a highly conserved carbohydrate recognition domain across many species. Accumulating evidence shows that Galectin 1 (Gal-1) plays an essential role in cancer, and its expression correlates with tumor aggressiveness and progression. Our preliminary data showed Gal-1 promotes glioma stem cell (GSC) growth via increased Warburg effect. mRNA expression and clinical data were obtained from The Cancer Genome Atlas database. The immunoblot analysis conducted using our cohort of human glioblastoma patient specimens (hGBM), confirmed Gal-1 upregulation in GBM. GC/MS analysis to evaluate the effects of Gal-1 depletion showed elevated levels of α-ketoglutaric acid, and citric acid with a concomitant reduction in lactic acid levels. Using Biolog microplate-1 mitochondrial functional assay, we confirmed that the depletion of Gal-1 increases the expression levels of the enzymes from the TCA cycle, suggesting a reversal of the Warburg phenotype. Manipulation of Gal-1 using RNA interference showed reduced ATP, lactate levels, cell viability, colony-forming abilities, and increased expression levels of genes implicated in the induction of apoptosis. Gal-1 exerts its metabolic role via regulating the expression of carbonic anhydrase IX (CA-IX), a surrogate marker for hypoxia. CA-IX functions downstream to Gal-1, and co-immunoprecipitation experiments along with proximity ligation assays confirm that Gal-1 physically associates with CA-IX to regulate its expression. Further, silencing of Gal-1 in mice models showed reduced tumor burden and increased survival compared to the mice implanted with GSC controls. Further investigation of Gal-1 in GSC progression and metabolic reprogramming is warranted.Subject terms: Cancer metabolism, Cell signalling     

Apoptotic cell death and related gene expression in metastatic tumors of AKR lymphomas of varying malignancy

Resistance to apoptosis may be related to tumor progression, due to the implications it might have on both tumor mass and genetic instability. We compared the tendency to spontaneous apoptosis and the proliferative capacity of metastatic growths of several AKR lymphoma variants (TAU-45, TAU-47, TAU-44, TAU-33, TAU-42 and TAU-46, in the order of increasing metastatic potential). We further compared the expression of several apoptosis-related genes. Cell proliferative capacity did not appear to determine malignant behavior since, on the whole, a decrease in S + G2M fraction was observed with increasing malignancy. Sensitivity to apoptotic cell death decreased with increasing malignancy when comparing the TAU-45, TAU-47, TAU-44 and TAU-33 variants, suggesting a role of reduced apoptosis in this T-cell lymphoma. An increase in Bcl-2 content with increasing aggressiveness among these variants, implicates this protein in this tumor progression-related resistance to apoptosis. However, the two variants of highest malignancy, TAU-42 and TAU-46, did not follow the same trend, since they displayed a relatively high content in apoptotic cells and a low Bcl-2 content. Fas receptor expression did not correlate with tendency to apoptosis, indicating that malignant behavior in the AKR lymphoma does not depend on CD95/Fas/APO1 downregulation. Overexpression of p53 was observed only in one of the variants of lowest malignancy.     

Galectins and their ligands: negative regulators of anti-tumor immunity

Cytotoxic CD8⁺ T cells are major players of anti-tumor immune responses, as their functional activity can limit tumor growth and progression. Data show that cytotoxic T cells efficiently control the proliferation of tumor cells through major histocompatibility complex class I-mediated mechanisms; nevertheless, the presence of tumor-infiltrating CD8⁺ T cells in lesional tissue does not always correlate with better prognosis and increased survival of cancer patients. Similarly, adoptive transfer of tumor-specific cytotoxic T cells has only shown marginal improvement in life spans of patients with metastatic disease. In this report, we discuss experimental evidence showing that expression of tumor-derived galectins, galectin (Gal)-1, Gal-3 and Gal-9, and concomitant presence of their ligands on the surface of anti-tumor immunocytes directly compromise anti-tumor CD8⁺ T cell immune responses and, perhaps, undermine the promise of adoptive CD8⁺ T cell immunotherapy. Furthermore, we describe novel strategies designed to counteract Gal-1-, Gal-3- and Gal-9-mediated effects and highlight their targeting potential for creating more effective anti-tumor immune responses. We believe that Gal and their ligands represent an efficacious targeted molecular paradigm that warrants clinical evaluation.     

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19⁺ tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19⁺ human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2^−/−γc^−/− immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.     

Immune rejection of a large sarcoma following cyclophosphamide and IL-12 treatment requires both NK and NK T cells and is associated with the induction of a novel NK T cell population

Combined immunotherapy with cyclophosphamide (Cy) and IL-12, but not IL-12 alone, stimulates eradication of a large established solid tumor (20 mm), MCA207, a methylcholanthrene-induced murine sarcoma. In these studies we demonstrate that NK1.1(+) cells and CD1d-dependent NK T cells each play important yet distinct roles in regression of a large tumor in response to Cy and IL-12, and we define a novel NK T cell subset, selectively increased by this treatment. Mice depleted of NK1.1(+) cells demonstrated more rapid initial tumor growth and prolonged tumor regression following treatment, but tumors were eventually eradicated. In contrast, initial tumor regression following therapy was unimpaired in CD1d(-/-) mice, which are deficient in most NK T cells, but tumors recurred. No tumor regression occurred following Cy and IL-12 therapy in CD1d(-/-) mice that were depleted of NK1.1(+) cells. We found that Cy and IL-12 induced the selective increase in liver and spleen lymphocytes of a unique NK T subpopulation (DX5(+)NK1.1(-)CD3(+)). These cells were not induced by treatment in CD1d(-/-) mice. Our studies demonstrate a contribution of both NK and NK T cells to the Cy- and IL-12-stimulated anti-tumor response. We describe the selective induction of a distinct NK T cell subset by Cy and IL-12 therapy, not seen following IL-12 therapy alone, which we suggest may contribute to the successful anti-tumor response induced by this immunotherapeutic regimen.     

A new multi-parameter flow cytometric assay for monitoring lymphoma growth and spread in a pre-clinical murine model for human lymphoma.

BACKGROUND: Mouse survival is commonly used as an indirect measure of lymphoma tumor response to anti-idiotype vaccine; however, this gives no information regarding residual lymphoma cells at primary or metastatic sites. We aimed to develop a method with which to monitor lymphoma tumor kinetics in the mouse as an independent measure of vaccine efficacy. METHODS: We developed a multi-parameter flow cytometric (MPFC) assay for 38C13 mouse lymphoma cells using sequential gating to detect aberrant antigen expression and binding by an anti-idiotype antibody (S1C5). Subsequently, we tested the utility of the MPFC assay in a 38C13 tumor modeling study in the C3H/HeN mouse. RESULTS: The MPFC assay was demonstrated in vitro to have both high specificity and sensitivity for 38C13 lymphoma cells. In tumor kinetic studies in the C3H/HeN mouse, as the tumor enlarged, the MPFC assay showed increasing prevalence of 38C13 cells at the inoculation site in addition to metastases to lymphoid organs and bone marrow. The latter findings were confirmed by both histology and immunohistochemistry. CONCLUSIONS: The MPFC assay is an independent parameter for monitoring 38C13 lymphoma kinetics and could be used to monitor tumor response to individual vaccines or other lymphoma therapy prior to clinical trials.     

NM23 and metastasis suppressor genes: update

Metastatic dissemination represents a leading cause of death in cancer patients. Elucidating the mechanisms of the metastatic process is therefore essential to control it. Since 1988, when the NME (NM23) gene was discovered, several genes specifically suppressing the metastatic potential of tumor cells, have been identified. These metastasis suppressor genes, which exhibit a reduced expression in metastatic tumor cells, are defined by their capacity to suppress metastatic dissemination in vivo without inhibiting primary tumor growth when transfected into metastatic cell lines and injected into experimental animals. Their decreased expression in a subset of human tumor cohorts is associated with a high metastatic potential, thus confirming the data obtained in experimental models. Most of these genes affect key signal transduction pathways, including mitogen-activated protein kinases, Rho-GTPases and G-protein-coupled receptors. These signaling categories control cell-cell and cell-matrix interactions, which are important in monitoring adhesion, invasion and migration properties of metastatic tumor cells. Reduced expression of metastasis suppressor genes is most often due to epigenetic mechanisms, suggesting that their re-expression could constitute a new anti-metastatic therapy. In this paper, we review the literature on metastasis suppressor genes, with a particular focus on NM23.     

Galectin-1: biphasic growth regulation of Leydig tumor cells

Galectin-1 (Gal-1) is a widely expressed beta-galactoside-binding protein that exerts pleiotropic biological functions. To gain insight into the potential role of Gal-1 as a novel modulator of Leydig cells, we investigated its effect on the growth and death of MA-10 tumor Leydig cells. In this study, we identified cytoplasmic Gal-1 expression in these tumor cells by cytofluorometry. DNA fragmentation, caspase-3, -8, and -9 activation, loss of mitochondrial membrane potential (DeltaPsim), cytochrome c (Cyt c) release, and FasL expression suggested that relatively high concentrations of exogenously added recombinant Gal-1 (rGal-1) induced apoptosis by the mitochondrial and death receptor pathways. These pathways were independently activated, as the presence of the inhibitor of caspase-8 or -9 only partially prevented Gal-1-effect. On the contrary, low concentrations of Gal-1 significantly promoted cell proliferation, without inducing cell death. Importantly, the presence of the disaccharide lactose prevented Gal-1 effects, suggesting the involvement of the carbohydrate recognition domain (CRD). This study provides strong evidence that Gal-1 is a novel biphasic regulator of Leydig tumor cell number, suggesting a novel role for Gal-1 in the reproductive physiopathology.     

Numerical Simulation of the Inhibitory Effect of Angiostatin on Metastatic Tumor Angiogenesis and Microenvironment

The present work formulates and analyzes the inhibitory effect of anti-angiogenic factor angiostatin excreted by the primary tumor on metastatic tumor angiogenesis, blood perfusion, and interstitial fluid flow in the tumor microenvironment by means of a numerical experiment. The simulation results demonstrate that angiostatin has an obvious impact on the morphology, growth rate, and the number of branches of microvascular network inside and outside the metastatic tumor, and angiostatin has the capacity to regulate and inhibit the formation of new blood vessels. Heterogeneous blood perfusion, widespread interstitial hypertension, and low convection within the metastatic tumor have obviously improved under the inhibitory effect of angiostatin, which are consistent with physiological observed facts. The simulation results may provide beneficial information and theoretical models for clinical research of anti-angiogenic therapy strategies.