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Immunoselection Enhances Tumor Initiating Cell-like Properties by Up-regulating NANOG Expression

  • 노경희 (대학원 의학과 종양생물학전공)

초록/요약

Tumor immunotherapy, despite its initial efficacy, produces later resistant tumor recurrences via shaping tumor phenotype. Tumor initiating cells (TICs) are thought to be responsible for tumor recurrence after various cancer therapies including tumor immunotherapy although a direct confirmation of the causality remains to be elucidated. Therefore, in this study, I investigated whether tumor immunotherapy could enrich and maintain TICs, and whether the high tumorigenic and resistant abilities of TICs were based on their marked ability of cognate stem cell factors to stimulate tumor cell proliferation and resistance against anti-tumor immunity. TC-1 P3 tumor cells which were previously developed a highly immune-resistant murine cancer cell line using an in vivo immunoselection strategy had increased TICs properties in terms of in vitro proliferation and tumor sphere formation and in vivo tumor formation compared to their original TC-1 P0 tumor cells. Through western blotting and immune fluorescent image analysis, one of key embryonic stem cell factors, NANOG was found to be up-regulated in TC-1 P3 as a consequence of immunoselection. Specific knock-down of NANOG in TC-1 P3 cells using its siRNA dramatically decreased tumor sphere-forming capacity while over-expression of NANOG in TC-1 P0 parental cells increased in the number of in vitro tumor sphere-forming colony and in vivo tumorigenicity. In addition, NANOG took a key role in immune-resistance of TC-1 P3 cells. Importantly, the siRNA-mediated knock-down of NANOG in TC-1 P3 cells restored E7-specific CD8+ immune sensitivity while over-expression of NANOG rendered TC-1 P0 parental cells resistant to E7-specific CD8+ CTL killing. More importantly, the tumor-initiating and immune-resistant properties of NANOG were also reproduced in various human cervical cancer cells. Taken together, these findings suggest that immunotherapy could lead to enrichment of TICs-like cells. Thus, the high tumorigenic and immune-resistant potentials of TICs-like cells are associated with NANOG expression that may represent a new mechanism of immune-resistance and a potential target to increase the efficacy of cancer immunotherapy.

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목차

ABSTRACT i
CONTENTS iii
I. INDRODUCTION1
II. MATRIALS AND METHODS 5
1. Mice 5
2. DNA constructs 5
3. In vitro and in vivo delivery of siRNAs 6
4. Cells 7
5. In vitro tumor sphere assays 8
6. In vivo tumorigenicity assay 8
7. Real-time quantitative RT-PCR 9
8. Immunocytochemical Analyses 9
9. Western blot 10
10. Flow cytometry analysis 10
11. Intracellular delivery of Granzyme B and detection of apoptosis 12
12. Cytotoxicity T Lymphocyte assays for measurement of Nanog-mediated immunoselection and immunoresistance 12
13. In vivo tumor treatment experiments 13
14. Statistical analysis 14
III. RESULTS 15
1. Vaccination enhances the growth and tumor initiating cell-like properties of
tumor cells 15
2. Immunoselection drives induction of Nanog in tumor cells 21
3. Vaccination induces Nanog expression in tumor cells 23
4. Enhancement of the tumor stem-like phenotype by vaccination requires Nanog 27
5. Nanog mediated immune resistance phenotype in vitro and in vivo 30
6. Nanog mediates the immune-resistant phenotype of tumor cells selected by vaccination 33
7. Gene silencing of Nanog using siNANOG-loaded chitosan nanoparticles reduces tumor volume in mice bearing TC-1 P3 36
8. Nanog confers tumor-initiating cells features and immune resistance properties to human cancer cells 40
IV. DISSCUSSION 44
V. REFFERENCES 50

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