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

Abstract

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-c 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 1 and CD8 1 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.Neuroblastoma (NB) is the most common extracranial solid malignancy in childhood. 1 This neuroendocrine tumor arising from neural crest cells is responsible for over 10-15% of pediatric cancer deaths. 1 The disease exhibits extreme heterogeneity, resulting in most recent stratification into very low, low, intermediate or high risk. 2 NB of lower risk groups occur during the first 18 months of life, and good outcomes are typical after surgery. 2 In contrast, high-risk NB, notably characterized by the amplification of NB-derived myelocytomatosis viral related oncogene (N-MYC), is resistant even to the most intensive treatment protocols available. 3,4 This suggests the need of more effective therapeutic strategies targeting nontraditional targets and lacking toxicity for treating high-risk aggressive NB.The introduction of monoclonal antibodies or chimeric T cells engineered to express chimeric antigen receptors targeting the GD2 antigen together with the implementation of dendritic cell (DC)-based vaccination have validated the critical function of the immune system in promoting NB regression. 5 Treatment with IL-2, GM-CSF, CD40 agonists or CTLA-4 blockade, in combination with other traditional approaches, has successfully improved immune effector functions in high-risk NB patient. 5,6 However, the success of these adjuvant immunotherapeutic approaches is hindered by a number of strategies used by tumors or tumor-associated stroma cells to elude immune recognition or thwart immune

Targeting of heme oxygenase‐1 as a novel immune regulator of neuroblastoma

“…46 Recently, we could demonstrate that NB promotes iDC to promote its own survival. 20 Here, we extended this finding and showed that targeted HO-1 inhibition in NB cells could abrogate HO-1-mediated blockade of DC maturation. As iDCs produce IL-10 and, additionally, express HO-1 at higher levels, 21 a further characterization of these cells and their immunosuppressive function in relation to HO-1 in NB is urgently needed.…”

“…Passages between 5 and 12 were used for the in vitro and in vivo experiments. 20 Zinc (II) protoporphyrin IX (ZnPPIX) (Frontier Scientific Europe, Carnforth, UK) and cobalt protoporphyrin (CoPPIX) (Sigma Aldrich) were both dissolved in 0.2 M NaOH for the in vitro experiments, distilled water. was added up to 1 mg/ml (pH 7.4 adjusted with 1 M HCl) (Millipore, Schwalbach, Germany).…”

“…20 On Day 7, BMDCs were resuspended in serumfree condition medium (SFCM) (0.3 3 10 6 /ml) generated from 24-hr supernatants of confluent NXS2 cultures simultaneously with LPS (100 ng/ml; Sigma Aldrich) to induce DC maturation. 26 Three median cutoffs were calculated by the online analysis.…”

“…19 We recently described a novel program of immune tolerance triggered by NB-derived galectin-1 to negatively regulate DCs and T-cell function. 20 In this context, HO-1 deserves further consideration as it was found to be an important molecule in defining the activation state of DCs via the activation of the IFN regulatory factor 3 pathway. 21,22 In experimental models for transplantation, inflammation and pregnancy, HO-1 expression facilitates the generation of tolerogenic DCs that in turn promote the formation of T-regulatory cells (Treg).…”

Adaptive Immune Responses During Pregnancy

“…Gal-1 is known to define autoimmunity, 70 inflammatory neurodegeneration, 71 cardiac inflammation, 72 and tumor escape. 73 74 and recent data confirm that it is not only expressed by the placenta itself 75 but also in uterine NK cells, 76 tolerogenic DCs, 55 and recent data show its importance when expressed in uterine MCs. 58 Pregnancies of Gal-1-deficient mice were first described as normal as the number of born embryos was apparently unaffected by this mutation.…”