BackgroundThe receptors of Notch family play an important role in controlling the development, differentiation, and function of multiple cell types. The aim of this study is to investigate the role of Notch1 signaling upon immune suppression induced by melanoma cells.MethodsMelanoma cell line B16 cells were transfected by lentivirus containing mouse Notch1 gene or Notch1 shRNA to generate B16 cell line that highly or lowly expressed Notch1. Notch1 in anti-tumor immune response was comprehensively appraised in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice. The ratios of CD3+CD8+ cytotoxic T cells, CD49b+NK cells, CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+ MDSCs in tumor-DLN or spleen were examined by flow cytometry. After the co-culture of B16 cells and CD8+ T cells, the effects of Notch1 on the proliferation and activation of T cells were assessed by CCK8 assay, CFSE dilution and Chromium-release test. The mRNA expression and supernatant secretion of immunosuppressive cytokines, TGF-β1, VEGF, IL-10 and IFN-γ were measured by RT-PCR and ELISA, respectively.ResultsDownregulation or overexpression of Notch1 in B16 melanoma cells inhibited or promoted tumor growth in immunocompetent mice, respectively. Notch1 expression in B16 melanoma cells inhibited the infiltration of CD8+ cytotoxic T lymphocytes and NK cells and reduced IFN-γ release in tumor tissue. It could also enhance B16 cell-mediated inhibition of T cell proliferation and activation, and upregulate PD-1 expression on CD4+ and CD8+ T cells. The percentage of CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+MDSCs were significantly increased in tumor microenvironment, and all these were attributed to the upregulation of TGF-β1.ConclusionThese findings suggested that Notch1 signaling in B16 melanoma cells might inhibit antitumor immunity by upregulation of TGF-β1.

BackgroundThe high incidence of recurrence and metastasis of hepatocellular carcinoma (HCC) necessitate the discovery of new predictive biomarkers of invasion and prognosis. Minichromosome maintenance complex component 6 (MCM6), which has been reported to up-regulate in multiple malignancies, was considered to be a novel diagnoses biomarker in HCC. However, its functional contributions and prognostic value remain unclear.MethodsThe expression of MCM6 was analyzed in 70 HCC tissues and 5 HCC cell lines by immunohistochemistry and real-time RT-PCR. The roles of MCM6 in HCC cell proliferation, migration and invasion were explored by CCK8, Wound healing and Transwell assays, respectively. Western blotting and Immunofluorescence staining were conducted to detect the protein expressions of ERK signaling pathway and EMT-related markers. To verify the above findings in vivo, we established subcutaneous xenograft tumor and orthotopic xenograft tumor models in nude mice. Finally, Enzyme-linked immunosorbent assay was used to evaluate the serum MCM6 level.ResultsMCM6 was significantly up-regulated in HCC tissues. Increased MCM6 expression was associated with aggressive clinicopathological features and worse prognosis in HCC patients. These results were consistent with our analyses of The Cancer Genome Atlas database (TCGA). Furthermore, knockdown of MCM6 significantly decreased proliferative and migratory/invasive capability of HCC cells in vitro, as well as decreased tumor volume, weight and the number of pulmonary metastases in vivo. Mechanistic analyses indicated that MCM6 promoted EMT and activated MEK/ERK signaling. More importantly, serum MCM6 levels in HCC patients were significantly higher than those in cirrhosis and healthy controls (P < 0.0001), and allowed distinguishing early recurrence with high accuracy (AUC = 0.773).ConclusionsOur findings indicate that MCM6 predicts poor prognosis and promotes metastasis in HCC. Postoperative serum MCM6 level could be valuable to detect preclinical early recurrence, indicative of a need for more careful surveillance and aggressive therapeutic intervention.Electronic supplementary materialThe online version of this article (10.1186/s13046-017-0669-z) contains supplementary material, which is available to authorized users.

Immune checkpoint therapies for cancer, like the anti-programmed cell death 1 (PD-1) agent pembrolizumab, have gained considerable attention. However, the use of immune checkpoint inhibitors in the context of adoptive immunotherapy is poorly characterized. We investigated the therapeutic efficacy of dendritic cell-stimulated CIK (DC-CIK) cells pretreated with pembrolizumab against hepatocellular carcinoma (HCC) in cytotoxicity assay in vitro and in a nude mouse xenograft model. We used time-lapse imaging to investigate tumor killing. We also performed a survival analysis based on lymphocyte subpopulation-specific mRNA signatures using The Cancer Genome Atlas (TCGA) HCC cohort (n=371 patients). The results indicated that PD-1 inhibition increased the anti-tumor effects of DC-CIK cells over those of DC-CIK cells alone, resulting in a survival benefit importantly. Time-lapse imaging revealed that DC-CIK cells appeared to be more effective and aggressive after anti-PD-1 treatment than after culture in control conditions. The PD-1 inhibitor also induced more effective immune cell infiltration of the tumor. Our analysis of the TCGA HCC cohort confirmed that a genetic signature consistent with a high degree of intratumoral CD8+ T cell infiltration is associated with good prognosis. These results suggest that blockade of the PD-1/PD-L1 axis in DC-CIK cells with a PD-1 inhibitor prior to infusion is a promising therapeutic strategy against HCC.

Various types of vaccines have been proposed as approaches for prevention or delay of the onset of cancer by boosting the endogenous immune system. We previously developed a senescent‐cell‐based vaccine, induced by radiation and veliparib, as a preventive and therapeutic tool against triple‐negative breast cancer. However, the programmed death receptor‐1/programmed death ligand‐1 (PD‐1/PD‐L1) pathway was found to play an important role in vaccine failure. Hence, we further developed soluble programmed death receptor‐1 (sPD1)‐expressing senescent cells to overcome PD‐L1/PD‐1‐mediated immune suppression while vaccinating to promote dendritic cell (DC) maturity, thereby amplifying T‐cell activation. In the present study, sPD1‐expressing senescent cells showed a particularly active status characterized by growth arrest and modified immunostimulatory cytokine secretion in vitro. As expected, sPD1‐expressing senescent tumor cell vaccine (STCV/sPD‐1) treatment attracted more mature DC and fewer exhausted‐PD1+ T cells in vivo. During the course of the vaccine studies, we observed greater safety and efficacy for STCV/sPD‐1 than for control treatments. STCV/sPD‐1 pre‐injections provided complete protection from 4T1 tumor challenge in mice. Additionally, the in vivo therapeutic study of mice with s.c. 4T1 tumor showed that STCV/sPD‐1 vaccination delayed tumorigenesis and suppressed tumor progression at early stages. These results showed that STCV/sPD‐1 effectively induced a strong antitumor immune response against cancer and suggested that it might be a potential strategy for TNBC prevention.