The Mechanism Of Inhibiting Tumor Stem Cells And Enhancing Anti-tumor Immunity By Blocking STAT3 In Head Neck Squamous Cell Carcinoma (HNSCC)

Head and neck squamous cell carcinomas (HNSCC) account for 6% of all malignancies. There are 600,000 new cases worldwide every year, ranking 6th among all cancer types, and an increase in the incidence has been noted. The complex anatomic features of head and neck regions and secluded location of a large proportion of head and neck tumor has been an obstacle in the diagnosis of HNSCC. Therefore, a remarkable proportion of patients were diagnosed at an advanced stage which lowered the 5-year survival rate of HNSCC. Recent articles reported an overall 5-year survival rate of 53% despite great progress in the treatment procedures such as reconstructive surgery, minimally invasive surgery, precise radio-chemotherapy and immunotherapy, etc. This problem warrants extensive research into practicable therapeutic targets and corresponding antineoplastic agents in the context of rapidly evolving fundamental biomedical and interdisciplinary research aiming at profound understanding of the mechanisms of tumorigenesis, development, recurrence and metastasis of HNSCC. It is believed such efforts would deepen our understanding of HNSCC and allow doctors to achieve a better prognosis of this lethal disease.HNSCC is a group of histologically heterogeneous tumors with complex tumor microenvironments, which is one of the major characteristics of this disease. Cancer stem cells (CSCs) and suppressive tumor-infiltrating immune cells (myeloid-derived suppressive cells, MDSCs; tumor associated macrophages, TAMs) are among the important components of tumor and its microenvironment. CSC, which is responsible for tumorigenesis, development, recurrence and chemo-resistance of HNSCC, are a small part of cancer cells residing in the tumor capable of self-renew, infinite proliferation and differentiation into tumors cells. In light of this, drugs targeting CSCs would be critical to the overall treatment. However, the medical community still lacks sufficient research on immunotherapies in the field of HNSCC, though immunotherapy for cancer has been listed as one of top ten scientific breakthroughs in 2013. HNSCC can be regarded as a kind of immunosuppressive cancer as MDSCs and TAMs play a pivotal part in the formation and maintenance of immunosuppressive microenvironment.Signal transducers and activators of transcription 3 (STAT3) exists in various tissues and cells, and this DNA-binding protein functions as a transducer and transcriptional regulator. Presiding in the cytoplasm, when STAT3 is phosphorylated and activated, it dimerizes and translocases to the cell nuclei, binding to specific DNA sequences and modulating downstream signal pathways which lead to the regulation of cell physiology. As a pleiotropic transcriptional regulator, STAT3 is constantly activated in the tumor microenvironment and in the immune cells, which will promote tumorigenesis and tumor development.Nanotechnology, which includes nanoparticles, nanostructures, nanomaterials and nanodevices, is the strategic commanding height in the 21st century. Characteristics of nanoparticles like long half-life, invisible to the naked eyes and sterically stability give those advantages in in vivo researches. In comparison with traditional chemotherapies and target therapies, nano drug-delivery system has greater drug-loading capacity, higher bioavailability, more effective targeting effects and less toxicity. There has been much fundamental cancer biological research and relatively less translational research due to the excessive focus on fundamental scientific mechanisms. It has been a traditional scientific routine to discover problems in the clinical practices and then dig into its reasons. Even with much effort, this routine has encountered major setback in cancer research, whose objects is continuing evolving and in some extent can be taken as collectively intelligent. Taken together, it is the future to concocting multi-target effects and nanotechnologies to develop cancer drugs that can hit multiple cancer targets. PartⅠ Discovering the function of STAT3 in HNSCC cancer stem cell regulation and chemoresistance Objective:HNSCC cancer stem cell is an important part in tumorigenesis, cancer development, recurrence and metastasis. Current part aims at studying multi-functional of signal transducer STAT3 and its expression in HNSCC; correlation between STAT3 and cancer stem cell biomarkers (SOX2, OCT4 ALDH1, and CD44); STAT3’s role in HNSCC chemoresistance and the effects of targeting STAT3 in cancer stem cell regulation. Methods:Firstly, we analyzed public database Oncomine (cancer microarray database) to study STAT3 signaling in available cancer research; then we used tissue microarrays to study STAT3 expression pattern and its relationship with cancer stem cell biomarkers SOX2, OCT4 ALDH1, CD44 by immunohistochemistry; expression data were quantified and analyzed by Aperio Scanscope, and differential expression pattern were analyzed by Pearson correlation and hierarchical cluster; effects of STAT3 inhibitor on HNSCC cancer stem cells were analyzed by sphere formation assay and flow cytometry (side population assay); effects of STAT3 inhibitor in combination with traditional chemotherapies (cisplatin,5-Fu and docetaxel) on chemoresistance were analyzed by HNSCC nude mice model.Results:Activated STAT3 was highly expressed in HNSCC. Positive signal was concentrated in nucleus because STAT3 must translocate to nucleus before it begin to function. Correlation analysis revealed STAT3’s close relationship with cancer stem cell biomarkers. In vitro and in vivo experiments indicated that targeting STAT3 signaling could effectively reducing HNSCC stem cell proliferation and targeting STAT3 signaling is useful to overcome chemoresistance originated from treatment by traditional chemotherapies.Conclusions:Highly activation of STAT3 and its close relationship with HNSCC cancer stem cell were detected in most HNSCC tissue samples and HNSCC tissue samples underwent inductive chemotherapy. It was also found that targeting STAT3 effectively reduced HNSCC cancer stem cells, indicating an important role of STAT3 in HNSCC cancer stem cell self-renewal.Part ⅡInvestigating the function of STAT3 in HNSCC anti-tumor immunity via regulating immature myeloid-derived cellsObjective:As a multi-functional regulator, STAT3 not only play important role in CSC, it also play privital role in anti-tumor immunity. This chapter aims at detecting STAT3 signal expression in immature myeloid-derived immunocytes and its functions in anti-tumor immunity.Methods:Detecting expression levels of STAT3 and immature myeloid-derived immunocytes biomarkers (CD11b, Gr1, MDSC; CD68, CD163, TAM) in HNSCC tissue microarray by immunohistochemistry. Results were quantified and analyzed by Aperio Scanscope. Pearson correlation and Cluster analysis were used to analyze differential expression. Double-labeled immunohistofluorescence were used to detect STAT3 signals in immature myeloid-derived immunocytes. Using STAT3 specified small molecular inhibitor in immunocompetent Tgfbrl/Pten conditional double knockout mice and detecting immunosuppressive cells MDSC, TAM; effector cells CD4, CD 8 and immune check point (PD-1, CTLA4) in transgenic mice by flow cytometry, it is possible to assess the function of STAT3 in disrupting HNSCC immunosuppressive microenvironment.Results:STAT3 was highly correlated with immature myeloid-derived markers. Double labeled immunofluorescence has shown STAT3 positive signals in these immunosuppressive cells. Targeting STAT3 signaling could effectively lower the number of immunosuppressive cells MDSC and TAM in transgenic mice and enhance immunoreaction in HNSCC.Conclusion:Targeting STAT3 could both remarkably impair HNSCC cancer stem cells and immunosuppressive cells which is a pivotal part in complex tumor microenvironment, indicating the benefits of inhibiting STAT3 signaling on both reducing tumor size and promoting tumor immune status.Part ⅢStudying the function of gelatin nanoparticle capsuled STAT3 specific inhibitor and Docetaxel in HNSCCObjective:Above-mentioned studies demonstrated the important function of STAT3 signaling in HNSCC tumorigenesis and tumor development with respects of cancer stem cells and tumor-associated immunocytes. The potential therapeutic role of targeting STAT3 in HNSCC treatment was promising. This part of research is focused on encapsulating STAT3 small molecular inhibitor and traditional chemotherapies like docetaxel into biocompatible gelatin nanoparticles to investigate the possibility of translational research in HNSCC on nanotechnology.Methods:Detecting expression level of gelatin enzymes (MMP-2, MMP-9) in HNSCC by immunohistochemistry and in TCGA data by bioinformatics; using nanotechnology to produce nanoparticles from biocompatible gelatin and inspecting its characteristics by transmission electron microscope; using CCK-8 kit to detect bio-toxicity of gelatin nanoparticles; loading STAT3 inhibitor and docetaxel on gelatin nanoparticles and measure its loading efficacy; using HNSCC cell line to compare the therapeutic effects of STAT3 inhibitor, docetaxel used alone and loaded on gelatin.Results:Gelatin enzymes MMP-2, MMP-9 were highly expressed in HNSCC. Custom-made gelatin nanoparticles has significant nanoparticle features; gelatin nanoparticles had no toxic effects on normal oral epithelial cells or HNSCC cells; STAT3 inhibitor and docetaxel could be loaded on nanoparticles; when STAT3 inhibitor were loaded on gelatin nanoparticles, it exhibited less toxic effects without losing its therapeutic efficacy.Conclusion:When STAT3 inhibitor or docetaxel were loaded on gelatin nanoparticles, they exhibited less toxic effects without losing its therapeutic efficacy,...