| Part I:Notch1activation promotes renal cell carcinoma growth via PI3K/Akt signalingObjective:Both Notchl and PI3K/Akt pathways are aberrantly activated in clear-cell renal cell carcinoma (CCRCC) and involved in tumorigenesis. The aim of this study was to test our hypothesis that elevated Notchl signaling activity exerts its growth-promoting effects via the PI3K/Akt pathway in CCRCC.Experimental Design:First, the protein levels of Notchl pathway components were assessed in matched fresh tumor and normal tissues from16CCRCC patients by Western blot. Then, three CCRCC cell lines and one normal counterpart were used in the functional experiments. Finally, immunohistochemistry analysis was performed on tissue microarrays containing CCRCC from125patients. To investigate the correlation between Notchl and PI3K/Akt pathways, we enhanced and suppressed Notchl activity respectively in a CCRCC cell line through diverse means, and then evaluated ensuing phosphorylated Akt (pAkt) levels. To further study their collaboration in promoting tumor growth, cell proliferation assay, colony formation assay and cell cycle analysis were conducted under several different conditions. Immunostaining of the tissue microarrays was utilized to determine whether the phenomena we observed also existed in vivo.Results:Notchl signaling was activated in CCRCC tissue samples and cell lines. Notchl activation increased CCRCC cell proliferation, enhanced anchorage-independent growth, and accelerated G1/S cell cycle progression. Such effects of Notchl signaling was mediated by the PI3K/Akt pathway. Correlations between Notchl, pAkt and Ki-67in tissue microarrays reinforced our in vitro findings.Conclusions:The current study established a functional link between Notchl and PI3K/Akt pathways in CCRCC. Part II:Enhancing whole-tumor cell vaccination by engaging innate immune system through NY-ESO-1/dendritic cell interactionsObjective:NY-ESO-1is a cytoplasmic cancer/germline antigen with distinctively strong immunogenicity. We have previously demonstrated that NY-ESO-1serves as an endogenous adjuvant by engaging dendritic cell (DC)-surface receptors of calreticulin and to11-like receptor4. In the present study, NY-ESO-1was investigated for its immunomodulatory roles as a molecular adjuvant in whole-tumor cell vaccines.Experimental Design:Renca cells were genetically engineered to express NY-ESO-1on the cell surface. We investigated the impact of ectopic cell-surface expression of NY-ESO-1on tumor immunogenicity, DC activation, cytotoxic T lymphocytes against model tumor-associated antigens and the effectiveness of the modified tumor cells as a therapeutic whole-cell vaccine.Results:Cell-surface expression of NY-ESO-1reduced the tumor growth of Renca cells in BALB/c mice, although the modification did not alter cell proliferation rate in vitro. Directly engaging the innate immune system through NY-ESO-1facilitated the interaction of tumor cells with DC, leading to enhanced DC activation and subsequent tumor-specific T cell priming. When used as a therapeutic whole-cell vaccine, Renca cells with NY-ESO-1on the surface mediated stronger inhibitory effects on tumor growth and metastasis compared with parental Renca or Renca cells expressing a control protein GFP on the surface. Augmented antitumor efficacy correlated with increased CD8+T cell infiltration into tumors and decreased myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) in the spleen.Conclusion:As a cancer/germline antigen and as an immunomodulatory adjuvant through engaging innate immune receptors, NY-ESO-1offers a unique opportunity for cancer immunotherapy.
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