1 The Molecular Mechanism Of Reversing T Cell Exclusion In Triple-negative Breast Cancer By Blocking FGFR Signaling Pathway 2 The Molecular Mechanism Of Circ-1073 To Inhibit The Malignant Biological Behaviors In Cancer Cells

PART1 THE MOLECULAR MECHANISM OF REVERSING T CELL EXCLUSION IN TRIPLE-NEGATIVE BREAST CANCER BY BLOCKING FGFR SIGNALING PATHWAYObjective:To investigate the molecular mechanism of cancer-associated fibroblasts(CAFs)in promoting T cell exclusion and the effect of fibroblast growth factor receptor inhibitor(FGFRi)combined with anti-PD-1 monoclonal antibody treatment on tumor growth in mouse triple-negative breast cancer(TNBC)model.Methods:The bioinformatics methods were used to compare the differential gene expressions between excluded-type and inflamed-type of TNBC.In vivo experiments explored the effect of FGFRi monotherapy on tumor growth and tumor immune microenvironment(TIME).In vitro experiments including CFSE staining,migration assay,and IFN-γsecretion assay were conducted to investigate the direct effects of FGFRi on T cells.CCK8,real-time quantitative PCR(RT-q PCR),induced differentiation assay and migration assay were performed in vitro to detect the direct effects of FGFRi on CAFs.A co-culture system was used to explore the effect of FGFRi on CAFs-inhibited T cell migration.Western blot was analyzed to clear the influences of FGFRi on downstream signaling pathways in a mouse embryo fibroblast cell line NIH/3T3.FGFRi combined with anti-programmed death receptor-1 monoclonal antibody(anti-PD-1 m Ab)was used to treat mouse TNBC.Flow cytometry and high-throughput sequencing were performed to detect the changes of TIME.Results:Bioinformatics analysis revealed that 6 FGFR-related signaling pathways were enriched in excluded-type of TNBC.The hindered tumor growth and the increased number of tumor infiltrating lymphocytes(TILs)were manifested simultaneously in the FGFR-treated group.However,in vitro assays showed that there were no significant difference in proliferation,migration and IFN-γsecretion of T cells between the control group and the FGFRi-treated group.Interestingly,impeded proliferation,migration,and secretion of CAFs were found in the FGFRi-treated group.Notably,the migratory T cells were significantly increased by FGFRi treatment during in vitro co-culture system consisted of CAFs and mouse spleen lymphocytes.Moreover,the expression level of p-MEK 1/2,but not p-AKT in NIH/3T3 cells was evidently downregulated in the FGFRi-treated group.Importantly,the significant inhibition of tumor growth accomplished by more CD8~+T cells and activated/memory CD4~+T cells infiltration with more IFN-γproduction in CD8~+T cells,but less myeloid-derived suppressor cells(MDSCs)and tumour-associated macrophages(TAMs)was observed in the FGFRi combined anti-PD-1m Ab group,compared with those in the other groups.However,there was no significant difference in Tregs infiltration in these groups.Conclusion:FGFRi promoted T cells infiltration by inhibiting the bio-functions of CAFs.FGFRi combined with anti-PD-1 m Ab plays a significant tumor suppressive role in mouse TNBC,providing a new strategy for immune-based therapy in TNBC.PART2 THE MOLECULAR MECHANISM OF CIRC-1073 TO INHIBIT THE MALIGNANT BIOLOGICAL BEHAVIORS IN CANCER CELLSObjective: To investigate the correlations between the expression level of circ RNAs 0001073(circ-1073)and the clinical characteristics of breast cancer,and research the suppressive effect of circ-1073 on tumor progression in breast cancer for exploring a new therapy of breast cancer.Methods: The expression levels of circ RNAs 0001073(circ-1073)in breast cancer cell lines(BCCs),breast cancer tissues and breast cancer peritumoral tissues were detected by RT-q PCR.The Kaplan-Meier method and a receiver operating characteristic curve were used to evaluate relapse-free survival(RFS)and the diagnostic value of circ-1073 in breast cancer,respectively.Biological functions of circ-1073 were determined by Cell Counting Kit,colony formation assay,flow cytometry,wound-healing assay,migration assay and xenograft models.Results: Low circ-1073 expression was discovered in BCCs and breast cancer tissues compared with normal mammary epithelial cells and peritumoral tissue,respectively.Larger tumor volumes,a higher axillary lymph node metastasis positive rate,higher TNM stages and a shorter RFS were found in the low circ-1073 expression group.In vitro,the significant reduction of the proliferation,the acceleration of apoptosis,and the evidently elevation of the protein expression levels of Cleaved Caspase-3and Cleaved Caspase-9 were detected in BCCs of the circ-1073 overexpression group.In vivo,the significantly inhibition of tumor growth was observed after injection of circ-1073 overexpression plasmid or a mixture of circ-1073 plasmid and nanoparticles into the tumors of the xenograft model.Conclusion: The downregulation of circ-1073 in breast cancer is significantly associated with poor breast cancer prognosis.Circ-1073 functions as a tumor suppressor in BCCs and xenograft model,indicating circ-1073 may provide a new treatment strategy for breast cancer.