白血病抑制因子(LIF) 調控多種類型細胞的分化，然而其如何調控口腔鱗狀上皮癌尚待進一步研究。透過分析TCGA資料庫，我們發現口腔癌患者中較高的LIF表現量與較差的總體生存率相關。GSEA結果顯示，LIF的表現與T細胞與干擾素信息傳導，及上皮間質轉化的功能相關。IHC結果顯示約30％的口腔癌檢體存有中等至強度LIF表現的免疫細胞群。LIF也調控癌細胞的免疫反應，我們發現高LIF表現會抑制抗原呈現基因的表現。由t-SNE及heap maps分析單細胞定序結果顯示腫瘤組織中免疫細胞種類傾向免疫抑制clonal types。本計畫結合細胞定序轉錄體及寡核甘酸標記抗體深入探討口腔癌的微環境變化，將利用單細胞RNA定序方法分析20對口腔癌檢體各類細胞群，並以獨立檢體群及實驗進一步驗證。具體目標包括：(1）評估LIF與口腔癌惡性化的相關性，(2）瞭解免疫細胞群的進行性變化及特異性新抗原受體的表現圖譜，(3)分析LIF與免疫反應基因的共表達圖譜與對免疫細胞極化反應的調控，（4）建立免疫標誌分子組預測免疫反應及預後，（5）評估抑制LIF的潛在治療效益，(6)分離辨識LIF或腫瘤新抗原之TCR T細胞。 ;The progression of OSCC is profoundly affected by a delicate interacting network consisting of cancer cells, infiltrating immune cells, and microenvironmental factors such as the infection of human papillomavirus. Emerging evidences indicate that leukemia inhibitory factor (LIF) plays a critical role in facilitating cancer progression by promoting tumor growth and resistance to anti-cancer therapy. Nevertheless, few studies have been conducted to elucidate the functional role of LIF in OSCC progression or the LIF-mediated immune modulations in OSCC tumor microenvironment. We analyzed the TCGA NGS database and found that higher LIF expression was correlated with a poorer overall survival in OSCC patients. Expression of LIF was associated with functions of the T cell signaling, interferon signaling, and epithelial-mesenchymal transition (EMT) based on results of gene-set enrichment analysis (GSEA). IHC data showed that approximately 30 % (11/35) of OSCC cases we investigated exhibited medium to high infiltration of LIF+-immune cells. LIF might also play a role in the immune modulation of cancer cells. Our data demonstrated that higher LIF expression in cancer cells would suppress expression of key antigen presentation molecules. Using t-SNE and heap map analyses on single cell RNA sequencing data of paired-OSCC samples, we found distinct clonal types of immune cells distributed within OSCC microenvironment. Overall, an immunosuppressive immune phenotype was observed compared to cells derived from adjacent normal tissue. In this project, we combine the oligonucleotide-barcoded surface antibodies, the 3’-single-cell RNA sequencing, and 5’-V(D)J expression technology to investigate the dynamic changes of immune repertoires with more focus on how LIF modulates immune responses within the microenvironment of OSCC. Systemic analyses of single cell RNA sequencing on 20-paired OSCC samples are utilized to obtain insights of OSCC microenvironment and the roles of LIF in immune regulation, followed by strict validations and functional/mechanistic studies. Specific aims include: (1) determine the clinical relevance of LIF expression in oral cancer cells and tumor infiltrating immune cells; (2) determine the profiles of 3’-single cell transcriptome and 5’V(D)J expression in cells derived from OSCC tissues; (3) investigate whether LIF regulates TILs/macrophage polarization in OSCC microenvironment; (4) establish a set of immune marker panel for predicting therapeutic response and evaluate the predictive power on OSCC diagnosis/prognosis; (5) evaluate the potential benefits by targeting to LIF or combining LIF inhibitors in both in vitro and in vivo models; (6) isolate clonal T cells that recognize LIF or selected neoantigens. As we gain a greater understanding of factors in modulating OSCC tumor microenvironment, we should be able to tailor therapeutic strategies by converting the immunosuppressive microenvironment for a selected OSCC patients.
