초록/요약

Cancer tissue heterogeneity, as well as the communications between cancer cellular components, may exert critical roles in cancer origination and development, finally influencing the effects of anti-cancer therapeutics. In this study, I established a manual single-cell seperation system, MDA (multiple displacement amplification) based single-cell exom sequencing (scWES) method, and SMART-seq2 (switching mechanism at 5' end of the RNA transcript sequencing version 2) based single-cell transcriptome sequencing (scRNA-seq) method. And I conducted these approaches on cancer cell lines and clinical cancer tissues to charecterize the cancer heterogeneity and its consequence on various therapies. In clinical colorectal and esophageal neoplasma/cancer tissues, with scWES, I found that all the neoplasma/cancer tissues were of monoclonal origin, and each showed a specific spectrum of heterogeneous somatic mutations. Novel driver mutations were also identified that related with the development of colorectal adenoma and cancer, particularly OR1B1 (GPCR signaling pathway) for colorectal adenoma evolution, LAMA1 (PI3K-Akt signaling pathway) and ADCY3 (FGFR signaling pathway) for colorectal cancer evolution. By comparing the cancer tissues before and after irradiation in esophageal squamous cell carcinoma (ESCC) at single-cell level, I identified 74 radiosensitive and radioresistant candidates, which decreased or increased their proportion of cells after irradiation, respectively. Further experimental validations showed that knock-down of the recurrent candidate genes, AHNAK2, EVPL and LAMA5 significantly sensitize the ESCC cells to radiotherapy. In scRNA-seq analyses of esophageal cancer cell lines, I found the dynamics and heterogeneities, which formed the intrinsic and acquired resistance during the chemotherapy with paclitaxel. The expression level of KRT19 was identified that is responsible for the intrinsic paclitaxel resistance, while high expressed proteasomes and low expressed HIF-1 signaling genes showed significant corelations with acquired paclitaxel resistance. Finally, by using scRNA-seq, I revealed the characteristics of various cell types and identified unique gene signatures and crucial cancer-related signaling pathways from both carcinoma and non-carcinoma cells in the esophageal carcinoma, including the ESCC and esophageal adenocarcinoma (EAC) tissues. In high stemness level of cancer cells, signaling pathway of DNA damage repair was significantly enriched in ESCC, while cell cycle signaling pathway was enriched in EAC. Furthermore, NOTCH and WNT signaling pathways were also investigated at single-cell level in ESCC and EAC. Different WNT activation axis were identified in ESCC (WNT4/WNT2b – FZD3/FZD6 – DVLs) other than in EAC (WNT10A – FZD5 – DVLs), while NOTCH signaling was specifically activated in ESCC, but not in EAC. Collectively, single-cell sequencing technologies have been the promising systematic and comprehensive approaches for analyzing the intratumoral heterogeneity, and reconstructing cancer clonality or subpopulations with different treatments, which provides more therapeutic targets for the future drug deveolpment in cancer presicion medicine.