Melanoma Pulmonary Metastasis Research Articles

Chitinase 3-like-1 Inhibits Innate Antitumor and Tissue Remodeling Immune Responses by Regulating CD47-SIRPα- and CD24-Siglec10-Mediated Phagocytosis.

Innate immune responses such as phagocytosis are critically linked to the generation of adaptive immune responses against the neoantigens in cancer and the efferocytosis that is essential for homeostasis in diseases characterized by lung injury, inflammation, and remodeling as in chronic obstructive pulmonary disease (COPD). Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it inhibits adaptive immune responses by stimulating immune checkpoint molecules (ICPs) and portends a poor prognosis. CHI3L1 is also induced in COPD where it regulates epithelial cell death. In this study, we demonstrate that pulmonary melanoma metastasis inhibits macrophage phagocytosis by stimulating the CD47-SIRPα and CD24-Siglec10 phagocytosis checkpoint pathways while inhibiting macrophage "eat me" signals from calreticulin and HMGB1. We also demonstrate that these effects on macrophage phagocytosis are associated with CHI3L1 stimulation of the SHP-1 and SHP-2 phosphatases and inhibition of the accumulation and phosphorylation of cytoskeleton-regulating nonmuscle myosin IIa. This inhibition of innate immune responses such as phagocytosis provides a mechanistic explanation for the ability of CHI3L1 to stimulate ICPs and inhibit adaptive immune responses in cancer and diseases such as COPD. The ability of CHI3L1 to simultaneously inhibit innate immune responses, stimulate ICPs, inhibit T cell costimulation, and regulate a number of other oncogenic and inflammation pathways suggests that CHI3L1-targeted therapeutics are promising interventions in cancer, COPD, and other disorders.

Immunomodulatory Function of Pien Tze Huang in T Cell-Mediated Anti-tumor Activity against B16-F10, MC38 and Hep1-6 Tumor Models.

To investigate the anti-tumor effects of Pien Tze Huang (PZH) in mouse models of B16-F10 melanoma, MC38 colorectal cancer, Hep1-6 hepatocellular carcinoma and chemically induced hepatocellular carcinoma model. Various tumor models, including B16-F10, MC38 and Hep1-6 tumor hypodermic inoculation models, B16-F10 and Hep1-6 pulmonary metastasis models, Hep1-6 orthotopic implantation model, and chemically induced hepatocellular carcinoma model, were utilized to evaluate the anti-tumor function of PZH. Tumor growth was assessed by measuring tumor size and weight of solid tumors isolated from C57BL/6 mice. For cell proliferation and death of tumor cells in vitro, as well as T cell activation markers, cytokine production and immune checkpoints analysis, single-cell suspensions were prepared from mouse spleen, lymph nodes, and tumors after PZH treatment. PZH demonstrated significant therapeutic efficacy in inhibiting tumor growth (P<0.01). Treatment with PZH resulted in a reduction in tumor size in subcutaneous MC38 colon adenocarcinoma and B16-F10 melanoma models, and decreased pulmonary metastasis of B16-F10 melanoma and Hep1-6 hepatoma (P<0.01). However, in vitro experiments showed that PZH only had slight impact on the cell proliferation and survival of tumor cells (P>0.05). Nevertheless, PZH exhibited a remarkable ability to enhance T cell activation and the production of interferon gamma, tumor necrosis factor alpha, and interleukin 2 in CD4+ T cells in vitro (P<0.01 or P<0.05). Importantly, PZH substantially inhibited T cell exhaustion and boosted cytokine production by tumor-infiltrating CD8+ T cells (P<0.01 or P<0.05). This study has confirmed a novel immunomodulatory function of PZH in T cell-mediated anti-tumor immunity, indicating that PZH holds promise as a potential therapeutic agent for cancer treatment.

A New Synthetic Curcuminoid Displays Antitumor Activities in Metastasized Melanoma.

The semisynthetic derivatives MePip-SF5 and isogarcinol, which are aligned with the natural products curcumin and garcinol, were tested for their antitumor effects in a preclinical model of pulmonary melanoma metastasis. MePip-SF5 was almost five times more effective in inhibiting B16F10 melanoma cell proliferation than its original substance of curcumin (IC50 MePip-SF5 2.8 vs. 13.8 µM). Similarly, the melanoma cytotoxicity of isogarcinol was increased by 40% compared to garcinol (IC50 3.1 vs. 2.1 µM). The in vivo toxicity of both drugs was assessed in healthy C57BL/6 mice challenged with escalating doses. Isogarcinol induced toxicity above a dose of 15 mg/kg, while MePip-SF5 showed no in vivo toxicity up to 60 mg/kg. Both drugs were tested in murine pulmonary metastatic melanoma. C57BL/6 mice (n = 10) received 500,000 B16F10 melanoma cells intravenously. After intraperitoneal injection of MePip-SF5 (60 mg/kg) or isorgarcinol (15 mg/kg) at days 8, 11 and 14 and sacrifice at day 16, the MePip-SF5-treated mice showed a significantly (p < 0.05) lower pulmonary macroscopic and microscopic tumor load than the vehicle-treated controls, whereas isogarcinol was ineffective. The pulmonary RNA levels of the mitosis marker Bub1 and the inflammatory markers TNFα and Ccl3 were significantly (p < 0.05) reduced in the MePip-SF5-treated mice. Both drugs were well tolerated, as shown by an organ inspection and normal liver- and kidney-related serum parameters. The novel curcuminoid MePip-SF5 showed a convincing antimetastatic effect and a lack of systemic toxicity in a relevant preclinical model of metastasized melanoma.

The role of tumor-associated macrophages and soluble mediators in pulmonary metastatic melanoma.

Skin malignant melanoma is a highly aggressive skin tumor, which is also a major cause of skin cancer-related mortality. It can spread from a relatively small primary tumor and metastasize to multiple locations, including lymph nodes, lungs, liver, bone, and brain. What’s more metastatic melanoma is the main cause of its high mortality. Among all organs, the lung is one of the most common distant metastatic sites of melanoma, and the mortality rate of melanoma lung metastasis is also very high. Elucidating the mechanisms involved in the pulmonary metastasis of cutaneous melanoma will not only help to provide possible explanations for its etiology and progression but may also help to provide potential new therapeutic targets for its treatment. Increasing evidence suggests that tumor-associated macrophages (TAMs) play an important regulatory role in the migration and metastasis of various malignant tumors. Tumor-targeted therapy, targeting tumor-associated macrophages is thus attracting attention, particularly for advanced tumors and metastatic tumors. However, the relevant role of tumor-associated macrophages in cutaneous melanoma lung metastasis is still unclear. This review will present an overview of the origin, classification, polarization, recruitment, regulation and targeting treatment of tumor-associated macrophages, as well as the soluble mediators involved in these processes and a summary of their possible role in lung metastasis from cutaneous malignant melanoma. This review particularly aims to provide insight into mechanisms and potential therapeutic targets to readers, interested in pulmonary metastasis melanoma.

The Role of Surgery in the Treatment of Melanoma Pulmonary Metastases in the Modern Era.

The lung represents a frequent site of spread for metastatic melanoma, which has historically been managed with surgical resection achieving promising outcomes. We hypothesized that the role of surgery in the management of melanoma pulmonary metastases (MPM) is evolving among the development of less invasive diagnostic and novel systemic therapeutic strategies. A single-center thoracic surgery database was reviewed and patients who underwent surgical resection of MPM between 1998 and 2019 were identified. Demographic, clinicopathologic, and surgical data were collected and analyzed, as were the annual volumes and indications for surgical resection. A Cochran-Armitage test was used to assess the trend in surgical indication. Three hundred and seventy seven surgical procedures for MPM were performed during the years of study in the care of 347 patients. Patients were predominantly male, with a mean age of 59.3y. The mean number of annual resections was 17 and while this number initially increased from six in 1998 to a peak of 39 cases in 2008, a decline was subsequently observed. Diagnostic resection decreased from 22% in 1998-1999 to 5% at the peak of procedures in 2008-2009 and to 0 in 2018-2019 (P=0.02). Curative resection increased from 44% in 1998-1999 to 73% in 2008-2009 (P<0.001) and remained the dominant reason for surgery in later years. Surgical indications in the management of MPM have transformed in conjunction with systemic modalities, and the volume of resections has decreased in the modern era. Despite innovations in systemic management and shifting goals of operative interventions, surgeons continue to play a vital role in caring for these patients with an advanced disease.

EASINESS: E. coli Assisted Speedy affINity-maturation Evolution SyStem.

Antibodies are one of the most important groups of biomolecules for both clinical and basic research and have been developed as potential therapeutics. Affinity is the key feature for biological activity and clinical efficacy of an antibody, especially of therapeutic antibodies, and thus antibody affinity improvement is indispensable and still remains challenging. To address this issue, we developed the E. coli Assisted Speed affINity-maturation Evolution SyStem (EASINESS) for continuous directed evolution of Ag–Ab interactions. Two key components of EASINESS include a mutation system modified from error-prone DNA polymerase I (Pol I) that selectively mutates ColE1 plasmids in E. coli and a protein–protein interaction selection system from mDHFR split fragments. We designed a GCN4 variant which barely forms a homodimer, and during a single round of evolution, we reversed the homodimer formation activity from the GCN4 variant to verify the feasibility of EASINESS. We then selected a potential therapeutic antibody 18A4Hu and improved the affinity of the antibody (18A4Hu) to its target (ARG2) 12-fold in 7 days while requiring very limited hands-on time. Remarkably, these variants of 18A4Hu revealed a significant improved ability to inhibit melanoma pulmonary metastasis in a mouse model. These results indicate EASINESS could be as an attractive choice for antibody affinity maturation.

A Theranostic Cellulose Nanocrystal-Based Drug Delivery System with Enhanced Retention in Pulmonary Metastasis of Melanoma.

Metastatic melanoma can be difficult to detect until at the advanced state that decreases the survival rate of patients. Several FDA-approved BRAF inhibitors have been used for treatment of metastatic melanoma, but overall therapeutic efficacy has been limited. Lutetium-177 (177 Lu) enables simultaneous tracking of tracer accumulation with single-photon emission computed tomography and radiotherapy. Therefore, the codelivery of 177 Lu alongside chemotherapeutic agents using nanoparticles (NPs) might improve the therapeutic outcome in metastatic melanoma. Cellulose nanocrystals (CNC NPs) can particularly deliver payloads to lung capillaries in vivo. Herein, 177 Lu-labeled CNC NPs loaded with vemurafenib ([177 Lu]Lu-CNC-V NPs) is developed and the therapeutic effect in BRAF V600E mutation-harboring YUMM1.G1 murine model of lung metastatic melanoma is investigated. The [177 Lu]Lu-CNC-V NPs demonstrate favorable radiolabel stability, drug release profile, cellular uptake, and cell growth inhibition in vitro. In vivo biodistribution reveals significant retention of the [177 Lu]Lu-CNC-V NPs in the lung, liver, and spleen. Ultimately, the median survival time of animals is doubly increased after treatment with [177 Lu]Lu-CNC-V NPs compared to control groups. The enhanced therapeutic efficacy of [177 Lu]Lu-CNC-V NPs in the lung metastatic melanoma animal model provides convincing evidence for the potential of clinical translation for theranostic CNC NP-based drug delivery systems after intravenous administration.

Gelated Vorinostat with inner-lysosome triggered release for tumor-targeting chemotherapy

Histonedeacetylase inhibitor (HDACi) has great potential in targeted antitumor therapy by inhibiting tumor migration, invasion, and metastasis. As one of the typical HDACis, vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) was approved as a therapeutic agent for cancer therapy, however, challenges remain due to their poor solubility, short half-life and low efficiency in cellular penetration. Considering the disadvantages of usual drug carriers, folate and vorinostat bound BSA nanogel (FVBN)was fabricated to implement higher solubility, stability, cellular uptake, and lipase-responsive release. With good dispersion and stability, FVBN significantly increased the cellular uptake of vorinostat through folate-mediated endocytosis. FVBN exhibited comparable cytotoxicity with free SAHA, and the growth of tumor cells was blocked in G1/G0 phase just like SAHA performed in cell cycle arrest tests. Moreover, FVBN not only effectively inhibited the growth of melanoma but also observably prevented pulmonary metastasis of melanoma. In the experiment against nude mice bearing solid ovarian cancer, FVBN showed excellent antitumor effect without liver damage, demonstrating the superiority of gelated and inner-lysosome triggered release strategies to the free SAHA, and it is promising to expand the scope of application of HDACi in clinical cancer therapy.

Abstract A59: Inhibition of Glycogen Synthase Kinase 3 (GSK-3) synergizes with anti-PD-1 by potentiating tumor-infiltrated CD8+ T-cells

Abstract Anti-programmed cell death-1 (anti-PD-1) blockade in tumor therapy results in an expansion of CD8+ T cells and can restore the function of exhausted CD8+ T cells. We identified GSK-3 as a key upstream kinase of regulating PD-1 expression. Inhibition of GSK-3 in CD8+ T cells increased Tbx21 (Tbet), which suppressed Pdcd1 (PD-1) transcription (Taylor et al., Immunity 2016). In controlling spontaneous pulmonary metastasis of B16 melanoma and EL-4 lymphoma cells, GSK-3 blockade is as effective as anti-PD-1 blocking antibody (Taylor et al., Cancer Research 2018). Given the clinical importance in devising strategies to overcome tumor resistance to immune checkpoint blockade, we assessed whether the combined use of GSK-3 small-molecule inhibitor (SMI) and anti-PD-1 can overcome resistance to anti-PD-1 monotherapy. Indeed, GSK-3 SMI and anti-PD-1 synergized in therapy against solid B16 tumors that are resistant to monotherapy, leading to delayed tumor growth. Further, the synergy of GSK-3 SMI and anti-PD-1 therapy has been successfully confirmed by using adoptive transfer of OT1 cells into B16-OVA tumor-bearing mice. High-dimensional profiling of tumor-infiltrated lymphocytes (TILs) using mass cytometry (CyTOF) showed that the combination therapy gave an increase in cellularity of CD8+ effector memory T cells, which correlated to better tumor control. Transcriptome analysis of these CD8+ TILs highlighted the increased cytotoxicity as shown by significant changes of granzymes. Our findings outline a next-generation approach of potentiating PD-1 antibody therapy with SMIs of GSK-3 that synergizes as therapy by boosting the cellularity and cytotoxicity of tumor infiltrated CD8+ T cells. Citation Format: Janna Krueger, Alison Taylor, Alexandra Kazanova, Saloua Jeidane, Vinicius Motta, Ian Watson, Christopher E. Rudd. Inhibition of Glycogen Synthase Kinase 3 (GSK-3) synergizes with anti-PD-1 by potentiating tumor-infiltrated CD8+ T-cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A59.

Cancer Targeted Gene Therapy for Inhibition of Melanoma Lung Metastasis with eIF3i shRNA Loaded Liposomes.

Eukaryotic translation initiation factors 3i (eIF3i) is a proto-oncogene that is overexpressed in various tumors, reducing its expression by eIF3i shRNA is a promising strategy to inhibit tumor growth or metastasis. Tumor cell is the target of eIF3i shRNA so that tumor-site accumulation could be important for fulfilling its therapeutic effect. Thus, the iRGD modified liposome (R-LP) was rationally synthesized to enhance the antitumor effect by active targeted delivery of eIF3i shRNA to B16F10 melanoma cells. R-LP encapsulating eIF3i shRNA gene (R-LP/sheIF3i) were prepared by a film dispersion method. The transfection experiment proves that R-LP could effectively transfect B16F10 cells. R-LP/sheIF3i notably restrained the migration, invasion, and adhesion of melanoma cells in vitro. In a mouse model of lung metastasis, R-LP/sheIF3i administered by intravenous injection suppressed pulmonary metastasis of melanoma by dramatically downregulated eIF3i expression and subsequently inhibiting tumor neovascularization and tumor cells proliferation in vivo. Our results provide a basis for tumor cells targeting strategies to reduce the expression of eIF3i by RNAi in the treatment of tumor metastasis.

Folate-mediated and pH-responsive chidamide-bound micelles encapsulating photosensitizers for tumor-targeting photodynamic therapy.

Background: Nonspecific tumor targeting, potential relapse and metastasis of tumor after treatment are the main barriers in clinical photodynamic therapy (PDT) for cancer, hence, inhibiting relapse and metastasis of tumor is significant issues in clinic.Purpose: In this work, chidamide as a histone deacetylases inhibitor (HADCi) was bound onto a pH-responsive block polymer folate polyethylene glycol-b-poly(aspartic acid) (PEG-b-PAsp) grafted folate (FA-PEG-b-PAsp) to obtain the block polymer folate polyethylene glycol-b-poly(asparaginyl-chidamide) (FA-PEG-b-PAsp-chidamide, FPPC) as multimodal tumor-targeting drug-delivery carrier to inhibiting tumor cell proliferation and tumor metastasis in mice.Methods: Model photosensitizer pyropheophorbide-a (Pha) was encapsulated by FPPC in PBS to form the polymer micelles Pha@FPPC [folate polyethylene glycol-b-poly(asparaginyl-chidamide) micelles encapsulating Pha]. Pha@FPPC was characterized by transmission electron microscope and dynamic light scattering; also, antitumor activity in vivo and in vitro were investigated by determination of cellular ROS level, detection of cell apoptosis and cell cycle arrest, PDT antitumor activity in vivo and histological analysis.Results: With favorable and stable sphere morphology under transmission electron microscope (TEM) (~93.0 nm), Pha@FPPC greatly enhanced the cellular uptake due to its folate-mediated effective endocytosis by mouse melanoma B16-F10 cells and the yield of ROS in tumor cells induced by PDT, and mainly caused necrocytosis and blocked cell growth cycle not only in G2 phase but also in G1/G0 phase after PDT. Pha@FPPC exhibited lower dark cytotoxicity in vitro and a better therapeutic index because of its higher dark cytotoxicity/photocytotoxicity ratio. Moreover, Pha@FPPC not only significantly inhibited the growth of implanted tumor and prolonged the survival time of melanoma-bearing mice due to both its folate-mediated tumor-targeting and selectively accumulation at tumor site by EPR (enhanced permeability and retention)effect as micelle nanoparticles but also remarkably prevented pulmonary metastasis of mice melanoma after PDT compared to free Pha, demonstrating its dual antitumor characteristics of PDT and HDACi.Conclusion: As a folate-mediated and acid-activated chidamide-grafted drug-delivery carrier, FPPC may have great potential to inhibit tumor metastasis in clinical photodynamic treatment for cancer because of its effective and multimodal tumor-targeting performance as photosensitizer vehicle.

Blocking podoplanin suppresses growth and pulmonary metastasis of human malignant melanoma

BackgroundPodoplanin (PDPN), a transmembrane O-glycoprotein, is up-regulated in many tumors and is involved in tumor metastasis and malignant progression. In previous studies, we generated a functional blocking monoclonal antibody (mAb, SZ168) against the extracellular domain of human PDPN. This study is aimed to investigate whether blocking PDPN by SZ168 inhibits tumor growth and metastasis.MethodsMalignant melanoma xenograft model by inoculating subcutaneously human malignant melanoma cell line C8161 into the back of BALB/c nude mice was used. Endogenous PDPN expression in C8161 cells and nasopharyngeal cancer cell line CNE-2 was detected using western blot and flow cytometry.ResultsSZ168 significantly inhibited C8161 or CNE-2 cell-induced platelet aggregation in a dose-dependent manner with a maximal inhibition of 73.9 ± 3.0% in C8161 cells or 77.1 ± 2.7% in CNE-2 cells. Moreover, SZ168 inhibited the growth and pulmonary metastasis of C8161cells in vivo. The number of lung metastatic foci in the SZ168-treated group was significantly decreased compared with that in the control mouse IgG group (1.61 ± 0.44 vs.3.83 ± 0.60, P < 0.01). Subcutaneous tumor volume, weight, and incidence were also significantly reduced in the SZ168-treated group compared to the control group (P < 0.05). Additionally, SZ168 recognized PDPN in immunohistochemical analyses of tumor tissue sections.ConclusionsSZ168 blocks growth and pulmonary metastasis of human malignant melanoma by inhibiting the interaction between tumor PDPN and platelet CLEC-2 and therefore is a promising antibody for therapeutic development against malignant melanoma.

Mesenchymal stem cells-curcumin loaded chitosan nanoparticles hybrid vectors for tumor-tropic therapy

The combination of controlled release technology and targeted drug delivery has become a promising strategy for cancer therapy. In this study, cell-nanoparticle hybrid vector was constructed using mesenchymal stem cells as the targeting cellular carrier and biotinylated chitosan polymer nanoparticles as the drug depot. Drug-loaded nanoparticles (hydrodynamic size =377.0 ± 14.6 nm and zeta potential = 9.6 ± 1.9 mV) were prepared by encapsulating hydrophobic model drug curcumin into biotinylated chitosan polymer. The biotin-modified nanoparticles were anchored on biotinylated mesenchymal stem cells surface by biotin-avidin binding, achieving an upload of 54.73 ± 3.95 pg/cell. The anchorage of nanoparticles on mesenchymal stem cells had no effect on their viability and homing property. Biotin-avidin binding lasted over 48 h, which could be sufficient for cell-directed tumor-tropic delivery. The in vitro and in vivo anti-tumor results advocate that cell-nanoparticle hybrid vector could prove beneficial in pulmonary melanoma metastasis therapy.

Glycogen synthase kinase 3 (GSK-3) synergizes with immune-checkpoint blockade (ICB) to overcome tumor resistance due to unique tumor infiltrating T-cell subsets as revealed by mass cytometry (CyTOF)

Abstract We have shown that the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) is a major regulator of programmed cell death-1 (PD-1) transcription and expression in T-cells (Taylor et al., 2016, Immunity). Further, small molecule inhibitors (SMIs) increased Tbx21 transcription for enhanced Tbet expression which suppressed Pdcd1 transcription in CD8+cytolytic T-cells. SMIs against GSK-3 are as effective as anti-PD-1 blocking antibody in the control of spontaneous pulmonary metastasis of B16 melanoma and EL-4 lymphoma cells (Taylor et al., 2018, Cancer Research). This introduced a new chemical approach to down-regulate PD-1 for the treatment of cancer. In addition, GSK-3 effects in Tbet expression can induce the expression of interferon-γ1 (IFNγ1) and granzyme B (GZMB) for enhanced cytolytic T-cell (CTL) killing of tumors suggesting that GSK-3 SMIs might synergize with check-point blockade. We now show that GSK-3α/β inhibition can cooperate with anti-PD-1 blockade to overcome tumor resistance. Using high dimensional profiling of tumor infiltrated lymphocytes (TILs) with mass cytometry (CyTOF), we show that the synergy of GSK-3 SMI and anti-PD-1 is accompanied by 2 specific findings: (i) an increase in the presence of a unique subset of CD8+ TIL effector-memory T-cells beyond that observed with anti-PD-1 alone, and (ii) the significant decrease of a subset of CD4+ suppressor regulatory T-cells (Tregs) not observed with anti-PD-1 alone. Our findings offer a small molecule approach for synergy with anti-PD-1 that leads to an increase in CD8+ T-cells and the marked decrease of Tregs in tumors.

GTPase-activating protein (RASAL1) associates with ZAP-70 of the TCR and negatively regulates T-cell proliferation and anti-tumor immunity

Abstract T-cell activation is needed for responses to antigen and controls responses to foreign antigen and cancer neo-antigens in immunotherapy. The full range of signaling events that inhibit T-cell activation and limit anti-tumor reactivity is unclear. Previous studies have identified the importance of phosphatases and E-3 ligases as inhibitors of activation. In this study, we show by a combination of tandem affinity chromatography, co-precipitation and proximity ligation analysis (PLA) that RASAL1, a novel GTPase-activating protein (GAP), binds to ZAP-70 of the TCR complex and inhibits anti-CD3 activation of ERKs and proliferation in T-cells. RASAL1 inhibited via two pathways where it binds and inhibits ZAP-70 activity, and acts as a GAP to inhibit the p21ras-ERK pathway. As a negative regulator, its expression is induced as a consequence of T-cell activation, where it reduced in vitro responses to anti-CD3 and to antigenic peptides presented by dendritic cells (DCs), while having no effect on T-cell dwell times. Further, we show that siRNA knock-down of RASAL1 expression increased in vivo CD4+ T-cell responses to peptide antigen while reducing the pulmonary metastasis of B16 melanoma and the growth of solid EL-4 lymphoma tumors. This anti-tumor effect was accompanied by a marked increase in CD8+ tumor-infiltrating T-cells (TILs) expressing effector molecules granzyme B (GZMB) and interferon γ-1 (IFNγ1). These findings identify RASAL1 as a new negative regulator of T-cell activation and tumor immunity.

Abstract A091: IL-33 activates antitumoral toxicity in eosinophils through stimulation of contact-dependent degranulation

Abstract The alarmin IL-33 plays pleiotropic roles in allergy, autoimmunity and inflammation through binding to its specific receptor ST2 expressed by most hematopoietic cells. Emerging evidences suggest an involvement of this cytokine also in cancer immunity, although its function remains ill-defined. Eosinophils (EOS) are a rare blood population playing critical roles in allergic inflammation and parasitic responses. We recently showed that EOS play an essential role in anti-tumor responses against melanoma growth and pulmonary metastasis mediated by IL-33 in vivo.In the present study we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumoral activities of EOS. We show that IL-33 indirectly stimulates the recruitment of EOS inducing tumor-derived chemokines CCL24 and CCL5. Furthermore, IL-33 directly activates EOS inducing the expression of adhesion molecules, such as the integrin CD11b, resulting in efficient contact-dependent tumor cell killing. In co-culture experiments, IL-33 activated EOS tightly bond to tumor cells, forming increased numbers of conjugates, with respect to resting eosinophils. Confocal laser-scanning microscopy (CLSM) of eosinophil-tumor cell conjugates revealed polarization of the pore-forming eosinophilic cationic protein (ECP) and of CD11b on the cell synapses exclusively in IL-33-activated, but not resting, EOS. Furthermore, we show that IL-33 activated EOS release larger amounts of extracellular vesicles (EV) with respect to resting EOS. Transmission electron microscopy (TEM) revealed increased degranulation and EV release of IL-33-activated EOS following cell contact with target tumor cells. Our results advocate for an eosinophil-mediated tumoricidal function promoted by IL-33, thus opening perspectives for novel cancer immunotherapy strategies. Citation Format: Fabrizio Mattei, Carla Buccione, Sara Andreone, Francesca Spadaro, Adele De Ninno, Jacopo Mancini, Cristiana Zanetti, Isabella Parolini, Francesca Iosi, Antonella Tinari, Valeria Lucarini, Annamaria Gerardino, Giovanna Ziccheddu, Luca Businaro, Claudia Afferni, Giovanna Schiavoni. IL-33 activates antitumoral toxicity in eosinophils through stimulation of contact-dependent degranulation [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A091.

Glucocorticoid receptor signaling during prolonged psychological stress compromises the ability of invariant NKT cells to participate in antitumor immune surveillance

Abstract The nervous system serves critical roles in the regulation of immune responses. Consequently, the neuroimmune functional interface can be disrupted by psychological stress, potentially impeding our ability to combat malignancies. Invariant natural killer T (iNKT) cells are innate-like T cells that, upon activation by glycolipid antigens and/or select environmental cues such as inflammatory cytokines, participate in antitumor immune responses. However, how mediators of stress may impact iNKT cell functions in this context remains unexplored. We subjected BALB/c and/or C57BL/6 mice to prolonged psychological stress, in a well-established model of physical restraint, before administering α-GalCer (a glycolipid agonist of iNKT cells) or a combination of IL-12 and IL-18. We report that stress abrogates IFN-γ production by activated iNKT cells in vivo, an effect that is reversible by blockade of glucocorticoid receptors. Accordingly, α-GalCer-stimulated iNKT cells in stressed mice failed to protect against pulmonary metastasis of B16-F10 melanoma. We also observed that otherwise α-GalCer-transactivated NK cells from stressed mice had a diminished capacity to lyse YAC-1 lymphoma cells. Finally, the suppressive effect of stress on iNKT cells was not due to their apoptotic death. In fact, unlike conventional T cells, iNKT cells were resistant to glucocorticoid-induced apoptosis. Taken together, our findings define a novel mechanism underlying stress-induced immunosuppression, which involves innate-like but not conventional T lymphocytes.

Angiotensin II promotes pulmonary metastasis of melanoma through the activation of adhesion molecules in vascular endothelial cells

Hypertension is considered as one of the cancer progressive factors, and often found comorbidity in cancer patients. Renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, and angiotensin II (Ang II) is well known pressor peptide associated with RAS. Ang II has been reported to accelerate progression and metastasis of cancer cells. However, its precise mechanisms have not been fully understood. In this study, we sought to elucidate the mechanisms by which Ang II exacerbates hematogenous metastasis in mouse melanoma cells, focusing the adhesion pathway in vascular endothelial cells. For this purpose, B16/F10 mouse melanoma cells, which do not express the Ang II type 1 receptor (AT1R), were intravenously injected into C57BL/6 mice. Two weeks after cell injection, the number of lung metastatic colonies was significantly higher in the Ang II-treated group (1 μg/kg/min) than in the vehicle-treated group. The AT1R blocker valsartan (40 mg/kg/day), but not the calcium channel blocker amlodipine (5 or 10 mg/kg/day), significantly suppressed the effect of Ang II. In endothelium-specific Agtr1a knockout mice, Ang II-mediated acceleration of lung metastases of melanoma cells was significantly diminished. Ang II treatment significantly increased E-selectin mRNA expression in vascular endothelial cells collected from lung tissues, and thus promoted adherence of melanoma cells to the vascular endothelium. Ang II-accelerated lung metastases of melanoma cells were also suppressed by treatment with anti-E-selectin antibody (20 mg/kg). Taken together, Ang II-treatment exacerbates hematogenous cancer metastasis by promoting E-selectin-mediated adhesion of cancer cells to vascular endothelial cells.

Ginkgo biloba Exocarp Extract Inhibits the Metastasis of B16-F10 Melanoma Involving PI3K/Akt/NF-κB/MMP-9 Signaling Pathway.

In recent years, interest in natural plant extracts for cancer treatment is growing in the drug development field. Ginkgo biloba exocarp extract (GBEE) is known for possessing inhibitory effects on various mouse and human cancer cells. And no adverse reactions were observed during its clinical application to cancer patients. The aim of this study is to investigate the inhibitory effect of GBEE on the metastasis of B16-F10 melanoma and its related mechanisms. The B16-F10 melanoma lung metastasis model was established in C57BL/6J mice. It was found that GBEE inhibited the growth and pulmonary metastasis of B16-F10 melanoma transplanted tumor and downregulated the level of MMP-9 protein. Meanwhile, the B16-F10 cells were used to study in vitro. The results showed that GBEE inhibited the proliferation and migration of B16-F10 cells. Simultaneously, it suppressed the heterogeneous adhesion of B16-F10 cells to human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. In addition, the levels of p-PI3K, p-Akt, NF-κB, and MMP-9 were decreased, while the PI3K and Akt were not significantly changed. These results indicate that GBEE can inhibit the metastasis of B16-F10 melanoma via multiple links and the molecular mechanism involved the regulation of PI3K/Akt/NF-κB/MMP-9 signaling pathway.

ABCB5 promotes melanoma metastasis through enhancing NF-κB p65 protein stability

Melanoma is the most aggressive type of skin cancer. Melanoma has an extremely poor prognosis because of its high potential for vascular invasion, metastasis and recurrence. The mechanism of melanoma metastasis is not well understood. ATP-binding cassette sub-family B member 5 (ABCB5) plays a key role in melanoma growth. However, it is uncertain what function ABCB5 may exert in melanoma metastasis. In this report, we for the first time demonstrate ABCB5 as a crucial factor that promotes melanoma metastasis. ABCB5 positive (ABCB5+) malignant melanoma initiating cells (MMICs) display a higher metastatic potential compared with ABCB5 negative (ABCB5-) melanoma subpopulation. Knockdown of ABCB5 expression reduces melanoma cell migration and invasion in vitro and melanoma pulmonary metastasis in tumor xenograft mice. ABCB5 and NF-κB p65 expression levels are positively correlated in both melanoma tissues and cell lines. Consequently, ABCB5 activates the NF-κB pathway by inhibiting p65 ubiquitination to enhance p65 protein stability. Our finding highlights ABCB5 as a novel pro-metastasis factor and provides a potential therapeutic target for melanoma.