Fusogenic vesicular stomatitis virus combined with natural killer T cell immunotherapy controls metastatic breast cancer.

BACKGROUND: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. METHODS: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. RESULTS: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. CONCLUSION: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.

Pilot study of jadomycin B pharmacokinetics and anti-tumoral effects in zebrafish larvae and mouse breast cancer xenograft models.

Despite numerous therapeutic options, multidrug resistance (MDR) remains an obstacle to successful breast cancer therapy. Jadomycin B, a natural product derived from Streptomyces venezuelae ISP5230, maintains cytotoxicity in MDR human breast cancer cells. Our objectives were to evaluate the pharmacokinetics, toxicity, anti-tumoral, and anti-metastatic effects of jadomycin B in zebrafish larvae and mice. In a zebrafish larval xenograft model, jadomycin B significantly reduced the proliferation of human MDA-MB-231 cells at or below its maximum tolerated dose (40 µm). In female Balb/C mice, a single intraperitoneal dose (6 mg/kg) was rapidly absorbed with a maximum serum concentration of 3.4 ± 0.27 µm. Jadomycin B concentrations declined biphasically with an elimination half-life of 1.7 ± 0.058 h. In the 4T1 mouse mammary carcinoma model, jadomycin B (12 mg/kg every 12 h from day 6 to 15 after tumor cell injection) decreased primary tumor volume compared to vehicle control. Jadomycin B-treated mice did not exhibit weight loss, nor significant increases in biomarkers of impaired hepatic (alanine aminotransferase) and renal (creatinine) function. In conclusion, jadomycin B demonstrated a good safety profile and provided partial anti-tumoral effects, warranting further dose-escalation safety and efficacy studies in MDR breast cancer models.

Selective Cannabinoid 2 Receptor Agonists as Potential Therapeutic Drugs for the Treatment of Endotoxin-Induced Uveitis.

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.

Reconstitution models to evaluate natural killer T cell function in tumor control.

Natural killer T (NKT) cells are glycolipid-reactive T lymphocytes that function in immunosurveillance and immune regulation. However, reduced tumor control in NKT cell-deficient Jα18(-/-) mice may be confounded by an overall reduction in T-cell receptor (TCR) repertoire diversity in these animals. Mechanistic studies are also hindered by a lack of tools to target molecules specifically in NKT cells. To address these issues, we developed protocols to expand functional NKT cells and stably reconstitute them in Jα18(-/-) mice. In vivo delivery of α-galactosylceramide (α-GalCer)-loaded dendritic cells expanded NKT cells in wild-type mice without skewing CD4 or TCR Vß expression profiles. Expanded NKT cells exhibited enhanced cytokine responses upon re-stimulation with glycolipid or CD3 ligation. Adoptive transfer of recently expanded wild-type or interferon (IFN)-γ(-/-) NKT cells protected recipient Jα18(-/-) mice from B16 melanoma metastasis without the need for additional glycolipid stimulation. However, NKT cell reconstitution in recipient Jα18(-/-) mice was short lived. Long-term reconstitution was only achieved when expanded NKT cells were transferred into sublethally irradiated recipients. Thirty days after transfer, NKT cell numbers, phenotype and α-GalCer-induced cytokine responses were equivalent to naive wild-type mice. Jα18(-/-) recipients reconstituted with wild-type or IFN-γ(-/-) NKT cells were both protected from B16 melanoma metastasis following α-GalCer treatment, and NK cell transactivation was intact in mice reconstituted with IFN-γ(-/-) NKT cells. These studies validate the use of reconstitution protocols to investigate the mechanisms of NKT cell immune function, demonstrating that NKT cell-derived IFN-γ and the altered TCR repertoire in Jα18(-/-) mice do not impact NKT cell-mediated antitumor responses.

The reversible P2Y12 inhibitor ticagrelor inhibits metastasis and improves survival in mouse models of cancer.

Tumor cells use activated platelets to promote their proliferation and metastatic potential. Because platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets, we investigated the potential of the reversible P2Y12 inhibitor ticagrelor, a clinical agent used in the prevention of cardiovascular and cerebrovascular events, to inhibit tumor adhesion and metastasis. In B16-F10 melanoma intravenous and intrasplenic metastasis models, mice treated with a clinical dose of ticagrelor (10 mg/kg) exhibited marked reductions in lung (84%) and liver (86%) metastases. Furthermore, ticagrelor treatment improved survival compared to saline-treated animals. A similar effect was observed in a 4T1 breast cancer model, with reductions in lung (55%) and bone marrow (87%) metastases following ticagrelor treatment. In vitro, B16-F10 cells exhibited decreased interaction with platelets from ticagrelor-treated mice compared to saline-treated mice, an effect similar to that observed with blockade of glycoprotein IIbIIIa. Similarly, B16-F10 cells co-incubated with platelets from ticagrelor-treated mice exhibited reduced adhesion to endothelial monolayers compared to those co-incubated with platelets from saline-treated animals, an effect also observed in vivo. Interestingly, pretreatment of endothelial monolayers with ticagrelor did not result in reduced tumor cell adhesion. These findings support a role for P2Y12-mediated platelet activation in promoting metastases, and provide proof-of-concept for the clinical use of ticagrelor in the prevention of tumor metastasis.

Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2.

The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated ß-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.

Thymic retention of CD4+CD25+FoxP3+ T regulatory cells is associated with their peripheral deficiency and thrombocytopenia in a murine model of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a bleeding disorder in which antibodies and/or T cells lead to enhanced peripheral platelet destruction and reduced bone marrow platelet production. Several reports have observed that ITP is associated with a peripheral deficiency of tolerance-inducing CD4+CD25+FoxP3+ T regulatory cells (Tregs). Using a murine model of ITP, we analyzed Tregs in the spleen and thymus. CD61 knockout mice were immunized against wild-type (CD61+) platelets, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice. Compared with SCID mice receiving naive splenocytes, within 2 weeks after transfer, the ITP SCID mice became thrombocytopenic (< 200 × 10(9) platelets/L) and had increased serum anti-CD61 antibodies. The quantity of thymic Tregs by 2 weeks after transfer was significantly elevated, whereas Tregs in the spleens were significantly reduced. Treatment of the ITP mice with 2 g/kg intravenous immunoglobulin raised the platelet counts, reduced antibody production, and normalized the thymic and splenic Treg populations. Compared with thymocytes from ITP mice treated with intravenous immunoglobulin, thymocytes from untreated ITP mice delayed the onset of ITP when administered before engraftment with immune splenocytes. These results suggest that ITP in mice is associated with a peripheral Treg deficiency because of thymic retention and therapy normalizes the Tregs.

Natural killer T cell immunotherapy combined with IL-15-expressing oncolytic virotherapy and PD-1 blockade mediates pancreatic tumor regression.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death, with a 5-year -year survival rate of less than 10%. This results from late detection, high rates of metastasis, and resistance to standard chemotherapies. Furthermore, chemotherapy and radiation are associated with significant morbidity, underscoring the need for novel therapies. Recent clinical studies have shown that immunotherapies can provide durable outcomes in cancer patients, but successes in pancreatic cancer have been limited. It is likely that novel and combined therapies will be needed to achieve clinical benefits. METHODS: Using experimental mouse models of pancreatic ductal adenocarcinoma, we examined natural killer T (NKT) cell activation therapy in combination with a recombinant oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express the cytokine IL-15 (VSV-IL-15). Panc02 pancreatic ductal carcinoma cells were implanted subcutaneously or orthotopically into syngeneic C57BL/6 mice. Mice were then treated with VSV expressing green fluorescent protein (VSV-GFP) or VSV-IL-15 and/or NKT cell activation therapy via delivery of α-GalCer-loaded DCs. We further assessed whether the addition of PD-1 blockade could increase the therapeutic benefit of our combination treatment. Three days after NKT cell activation, some groups of mice were treated with anti-PD-1 antibodies weekly for 3 weeks. RESULTS: VSV-GFP and VSV-IL-15 mediated equal killing of human and mouse pancreatic cancer lines in vitro. In vivo, VSV-IL-15 combined with NKT cell activation therapy to enhance tumor regression and increase survival time over individual treatments, and was also superior to NKT cell therapy combined with VSV-GFP. Enhanced tumor control was associated with increased immune cell infiltration and anti-tumor effector functions (cytotoxicity and cytokine production). While ineffective as a monotherapy, the addition of blocking PD-1 antibodies to the combined protocol sustained immune cell activation and effector functions, resulting in prolonged tumor regression and complete tumor clearance in 20% of mice. Mice who cleared the initial tumor challenge exhibited reduced tumor growth uponon rechallenge, consistent with the formation of immune memory. CONCLUSION: TThese results demonstrate that NKT cell immunotherapy combined with oncolytic VSV-IL-15 virotherapy and PD-1 blockade enhances tumor control and presents a promising treatment strategy for targeting pancreatic cancer.

The Inhibitory Receptor Siglec-8 Interacts With FcεRI and Globally Inhibits Intracellular Signaling in Primary Mast Cells Upon Activation.

Immunomodulation of mast cell (MC) activity is warranted in allergic and inflammatory diseases where MCs have a central role in pathogenesis. Targeting Siglec-8, an inhibitory receptor on MCs and eosinophils, has shown promising activity in preclinical and clinical studies. While the intracellular pathways that regulate Siglec-8 activity in eosinophils have been well studied, the signaling mechanisms that lead to MC inhibition have not been fully elucidated. Here, we evaluate the intracellular signaling pathways of Siglec-8-mediated inhibition in primary MCs using an anti-Siglec-8 monoclonal antibody (mAb). Phospho-proteomic profiling of FcεRI-activated MCs revealed Siglec-8 mAb-treatment globally inhibited proximal and downstream kinases, leading to attenuated MC activation and degranulation. In fact, Siglec-8 was found to directly interact with FcεRI signaling molecules. Siglec-8 inhibition was dependent on both cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that interact with the SH2 containing protein phosphatase Shp-2 upon Siglec-8 phosphorylation. Taken together, these data support a model in which Siglec-8 regulates proximal FcεRI-induced phosphorylation events through phosphatase recruitment and interaction with FcεRIγ, resulting in global inhibition of MCs upon Siglec-8 mAb engagement.

Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis.

BACKGROUND: Oncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches. METHODS: Using experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro. RESULTS: VSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin. CONCLUSION: Taken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.

Mast Cell and Eosinophil Activation Are Associated With COVID-19 and TLR-Mediated Viral Inflammation: Implications for an Anti-Siglec-8 Antibody.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection represents a global health crisis. Immune cell activation via pattern recognition receptors has been implicated as a driver of the hyperinflammatory response seen in COVID-19. However, our understanding of the specific immune responses to SARS-CoV-2 remains limited. Mast cells (MCs) and eosinophils are innate immune cells that play pathogenic roles in many inflammatory responses. Here we report MC-derived proteases and eosinophil-associated mediators are elevated in COVID-19 patient sera and lung tissues. Stimulation of viral-sensing toll-like receptors in vitro and administration of synthetic viral RNA in vivo induced features of hyperinflammation, including cytokine elevation, immune cell airway infiltration, and MC-protease production-effects suppressed by an anti-Siglec-8 monoclonal antibody which selectively inhibits MCs and depletes eosinophils. Similarly, anti-Siglec-8 treatment reduced disease severity and airway inflammation in a respiratory viral infection model. These results suggest that MC and eosinophil activation are associated with COVID-19 inflammation and anti-Siglec-8 antibodies are a potential therapeutic approach for attenuating excessive inflammation during viral infections.

A monoclonal antibody to Siglec-8 suppresses non-allergic airway inflammation and inhibits IgE-independent mast cell activation.

In addition to their well characterized role in mediating IgE-dependent allergic diseases, aberrant accumulation and activation of mast cells (MCs) is associated with many non-allergic inflammatory diseases, whereby their activation is likely triggered by non-IgE stimuli (e.g., IL-33). Siglec-8 is an inhibitory receptor expressed on MCs and eosinophils that has been shown to inhibit IgE-mediated MC responses and reduce allergic inflammation upon ligation with a monoclonal antibody (mAb). Herein, we evaluated the effects of an anti-Siglec-8 mAb (anti-S8) in non-allergic disease models of experimental cigarette-smoke-induced chronic obstructive pulmonary disease and bleomycin-induced lung injury in Siglec-8 transgenic mice. Therapeutic treatment with anti-S8 inhibited MC activation and reduced recruitment of immune cells, airway inflammation, and lung fibrosis. Similarly, using a model of MC-dependent, IL-33-induced inflammation, anti-S8 treatment suppressed neutrophil influx, and cytokine production through MC inhibition. Transcriptomic profiling of MCs further demonstrated anti-S8-mediated downregulation of MC signaling pathways induced by IL-33, including TNF signaling via NF-κB. Collectively, these findings demonstrate that ligating Siglec-8 with an antibody reduces non-allergic inflammation and inhibits IgE-independent MC activation, supporting the evaluation of an anti-Siglec-8 mAb as a therapeutic approach in both allergic and non-allergic inflammatory diseases in which MCs play a role.

Myeloid-derived suppressor cell depletion therapy targets IL-17A-expressing mammary carcinomas.

Triple-negative breast cancer (TNBC) is an invasive subtype of breast cancer but paradoxically associated with increased tumor-infiltrating leukocytes. The molecular and cellular mechanisms underlying TNBC immunobiology are incompletely understood. Interleukin (IL)-17A is a pro-inflammatory cytokine that has both pro- and anti-tumor effects and found in 40-80% of TNBC samples. We report here that IL-17A mRNA and protein are detectable in some human TNBC cell lines and further upregulated by IL-23 and LPS stimulation. Furthermore, the impact of tumor-derived IL-17A in host immune response and tumor growth was examined using murine TNBC 4T1 mammary carcinoma cells transduced with an adenoviral vector expressing IL-17A (AdIL-17A) or control vector (Addl). Compared to Addl-transduction, AdIL-17A-transduction enhanced 4T1 tumor growth and lung metastasis in vivo, which was associated with a marked expansion of myeloid-derived suppressor cells (MDSCs). However, AdIL-17A-transduction also induced strong organ-specific and time-dependent immune activation indicated by dynamic changes of NK cells, B cells, CD4, and CD8 T cells in peripheral blood, lung, and tumor site, as well as the plasma levels of IFNγ. Such findings highlight that tumor-associated IL-17A induces concurrent immune activation and immune suppression. Administration of anti-Gr1 or anti-G-CSF antibody effectively depleted MDSCs in vivo, markedly reducing the growth of AdIL-17A-transduced 4T1 tumors, and eliminating lung metastasis. Collectively, our study demonstrates that MDSC depletion is an effective and practical approach for treating IL-17A-enriched mammary carcinomas.

Promotion of Primary Murine Breast Cancer Growth and Metastasis by Adipose-Derived Stem Cells Is Reduced in the Presence of Autologous Fat Graft.

BACKGROUND: Cell-assisted lipotransfer involves enrichment of autologous fat with supraphysiologic numbers of adipose-derived stem cells to improve graft take. Adipose-derived stem cells have been shown to promote cancer progression, raising concerns over the safety of adipose-derived stem cells and cell-assisted lipotransfer in postoncologic breast reconstruction. The authors compared the effect of adipose-derived stem cells alone, cell-assisted lipotransfer, and conventional fat grafting on breast cancer growth and metastasis. METHODS: Proliferation and migration of murine 4T1 breast cancer cells cultured in control medium or mouse adipose-derived stem cell- or fat graft-conditioned medium were assessed by flow cytometry and scratch assay, respectively. Transcription levels of arginase-1, transforming growth factor-ß, and vascular endothelial growth factor were assessed in adipose-derived stem cells and fat graft by quantitative reverse transcription polymerase chain reaction. An orthotopic mouse tumor model was used to evaluate breast cancer progression and metastasis. 4T1 cells were injected into the mammary pad of female BALB/c mice. Six days later, tumors were injected with saline, adipose-derived stem cells, fat graft, or cell-assisted lipotransfer (n = 7 per group). Two weeks later, primary tumors were examined by immunohistochemistry and lung metastasis was quantified. RESULTS: Adipose-derived stem cell-conditioned medium increased cancer cell proliferation (p = 0.03); migration (p < 0.01); and transcription of arginase-1, transforming growth factor-ß, and vascular endothelial growth factor compared to fat graft-conditioned or control medium (p < 0.02). Tumor-site injection with adipose-derived stem cells alone led to increased primary tumor growth and lung metastasis compared to control, fat graft, or cell-assisted lipotransfer groups (p < 0.05). Adipose-derived stem cell injection increased CD31 vascular density in tumors (p < 0.01). CONCLUSION: Adipose-derived stem cells alone, but not conventional fat graft or cell-assisted lipotransfer, promote breast cancer cell proliferation and invasiveness in vitro and in vivo.

Ticagrelor inhibits platelet-tumor cell interactions and metastasis in human and murine breast cancer.

Activated platelets promote the proliferation and metastatic potential of cancer cells. Platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets. We examined the potential of the reversible P2Y12 inhibitor ticagrelor, an agent used clinically to prevent cardiovascular and cerebrovascular events, to reduce tumor growth and metastasis. In vitro, MCF-7, MDA-MB-468, and MDA-MB-231 human mammary carcinoma cells exhibited decreased interaction with platelets treated with ticagrelor compared to untreated platelets. Prevention of tumor cell-platelet interactions through pretreatment of platelets with ticagrelor did not improve natural killer cell-mediated tumor cell killing of K562 myelogenous leukemia target cells. Additionally, ticagrelor had no effect on proliferation of 4T1 mouse mammary carcinoma cells co-cultured with platelets, or on primary 4T1 tumor growth. In an orthotopic 4T1 breast cancer model, ticagrelor (10 mg/kg), but not clopidogrel (10 mg/kg) or saline, resulted in reduced metastasis and improved survival. Ticagrelor treatment was associated with a marked reduction in tumor cell-platelet aggregates in the lungs at 10, 30 and 60 min post-intravenous inoculation. These findings suggest a role for P2Y12-mediated platelet activation in promoting metastasis, and provide support for the use of ticagrelor in the prevention of breast cancer spread.

Natural Killer T-cell Immunotherapy in Combination with Chemotherapy-Induced Immunogenic Cell Death Targets Metastatic Breast Cancer.

Natural killer T (NKT) cells are glycolipid-reactive lymphocytes that promote cancer control. In previous studies, NKT-cell activation improved survival and antitumor immunity in a postsurgical mouse model of metastatic breast cancer. Herein, we investigated whether NKT-cell activation could be combined with chemotherapeutic agents to augment therapeutic outcomes. Gemcitabine and cyclophosphamide analogues enhanced the potential immunogenicity of 4T1 mammary carcinoma cells by increasing the expression of antigen-presenting molecules (MHC-I, MHC-II, and CD1d) and promoting exposure or release of immunogenic cell death markers (calreticulin, HMGB1, and ATP). In 4T1 primary tumor and postsurgical metastasis models, BALB/c mice were treated with cyclophosphamide or gemcitabine. NKT cells were then activated by transfer of dendritic cells loaded with the glycolipid antigen α-galactosylceramide (α-GalCer). Chemotherapeutic treatments did not impact NKT-cell activation but enhanced recruitment into primary tumors. Cyclophosphamide, gemcitabine, or α-GalCer-loaded dendritic cell monotherapies decreased tumor growth in the primary tumor model and reduced metastatic burden and prolonged survival in the metastasis model. Combining chemotherapeutics with NKT-cell activation therapy significantly enhanced survival, with surviving mice exhibiting attenuated tumor growth following a second tumor challenge. The frequency of myeloid-derived suppressor cells was reduced by gemcitabine, cyclophosphamide, or α-GalCer-loaded dendritic cell treatments; cyclophosphamide also reduced the frequency of regulatory T cells. Individual treatments increased immune cell activation, cytokine polarization, and cytotoxic responses, although these readouts were not enhanced further by combining therapies. These findings demonstrate that NKT-cell activation therapy can be combined with gemcitabine or cyclophosphamide to target tumor burden and enhance protection against tumor recurrence. Cancer Immunol Res; 5(12); 1086-97. ©2017 AACR.

Reovirus FAST Protein Enhances Vesicular Stomatitis Virus Oncolytic Virotherapy in Primary and Metastatic Tumor Models.

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral fusogens (∼100-150 amino acids) and efficiently induce cell-cell fusion and syncytium formation in multiple cell types. Syncytium formation enhances cell-cell virus transmission and may also induce immunogenic cell death, a form of apoptosis that stimulates immune recognition of tumor cells. These properties suggest that FAST proteins might serve to enhance oncolytic virotherapy. The oncolytic activity of recombinant VSVΔM51 (an interferon-sensitive vesicular stomatitis virus [VSV] mutant) encoding the p14 FAST protein (VSV-p14) was compared with a similar construct encoding GFP (VSV-GFP) in cell culture and syngeneic BALB/c tumor models. Compared with VSV-GFP, VSV-p14 exhibited increased oncolytic activity against MCF-7 and 4T1 breast cancer spheroids in culture and reduced primary 4T1 breast tumor growth in vivo. VSV-p14 prolonged survival in both primary and metastatic 4T1 breast cancer models, and in a CT26 metastatic colon cancer model. As with VSV-GFP, VSV-p14 preferentially replicated in vivo in tumors and was cleared rapidly from other sites. Furthermore, VSV-p14 increased the numbers of activated splenic CD4, CD8, natural killer (NK), and natural killer T (NKT) cells, and increased the number of activated CD4 and CD8 cells in tumors. FAST proteins may therefore provide a multi-pronged approach to improving oncolytic virotherapy via syncytium formation and enhanced immune stimulation.

Cannabinoid 2 receptor is a novel anti-inflammatory target in experimental proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) can develop after ocular trauma or inflammation and is a common complication of surgery to correct retinal detachment. Currently, there are no pharmacological treatments for PVR. Cannabinoids acting at cannabinoid 2 receptor (CB2R) can decrease inflammation and fibrosis. The objective of this study was to examine the anti-inflammatory actions of CB2R as a candidate novel therapeutic target in experimental PVR. PVR was induced by intravitreal injection of dispase in wild-type (WT) and CB2R genetic knockout (CB2R-/-) mice. Ocular pathology was studied at 24 h or one week after dispase injection. CB2R modulation was examined in WT mice, using the CB2R agonist, HU308, and the CB2R antagonist, AM630. Histopathological scoring and quantification of microglia was used to evaluate tissue pathology. Quantitative PCR and multiplex assays were used to assess changes in proinflammatory cytokines. Intravital microscopy (IVM) was used to visualize and quantify leukocyte-endothelial adhesion to the iridial microcirculation. Activation of CB2R with HU308 in WT mice with PVR decreased mean histopathological scores, the number of microglia, and leukocyte adhesion compared to vehicle-treated animals. Conversely, an increase in histopathological scores and activated microglia was observed in PVR animals after treatment with AM630. CB2R-/- mice with PVR exhibited exacerbated ocular histopathology, increased microglia numbers, and elevated protein levels of cytokines as compared to WT mice. In conclusion, our results indicate that intervention at early stage PVR with CB2R agonists reduces ocular inflammation and disease severity. CB2R may represent a therapeutic target to prevent PVR progression and vision loss. This article is part of the Special Issue entitled 'Lipid Sensing G Protein-Coupled Receptors in the CNS'.

CXCL16-positive dendritic cells enhance invariant natural killer T cell-dependent IFNγ production and tumor control.

Crosstalk interactions between dendritic cells (DCs) and invariant natural killer T (iNKT) cells are important in regulating antitumor responses elicited by glycolipid antigens. iNKT cells constitutively express the chemokine receptor CXCR6, while cytokine-activated DCs upregulate the transmembrane chemokine ligand, CXCL16. This study examined the co-stimulatory role of CXCR6/CXCL16 interactions in glycolipid-dependent iNKT cell activation and tumor control. Spleen and liver DCs in wild-type mice, but not iNKT cell deficient (Jα18(-/-)) mice, transiently upregulated surface CXCL16 following in vivo administration of the glycolipid antigen α-galactosylceramide. Recombinant CXCL16 did not directly induce iNKT cell activation in vitro but enhanced interferon (IFN)-γ production when mouse or human iNKT cells were stimulated with plate-bound anti-CD3. Compared with glycolipid-loaded CXCL16(neg) DCs, CXCL16(hi) DCs induced higher levels of IFNγ production in iNKT cell cultures and following adoptive transfer in vivo. The number of IFNγ(+) iNKT cells and expansion of T-bet(+) iNKT cells were reduced in vivo when CXCL16(-/-) DCs were used to activate iNKT cells. Enhanced IFNγ production in vivo was not dependent on CXCR6 expression on natural killer (NK) cells. Adoptive transfer of glycolipid-loaded CXCL16(hi) DCs provided superior protection against tumor metastasis compared to CXCL16(neg) DC transfers. Similarly, wild-type DCs provided superior protection against metastasis compared with CXCL16(-/-) DCs. These experiments implicate an important role for CXCR6/CXCL16 interactions in regulating iNKT cell IFNγ production and tumor control. The selective use of CXCL16(hi) DCs in adoptive transfer immunotherapies may prove useful for enhancing T helper (Th) type 1 responses and clinical outcomes in cancer patients.