The role of NK/LAK cells in modulation of migratory/ invasive behavior of putative cancer stem cells in the floating but not adherent subset of the lymph node metastatic UP-LN1 carcinoma cell line

• Main Authors: Hung Chang Chen, 陳宏昌
• Other Authors: S. K. Liao
• Format: Others
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/97409983768186248732

碩士 === 長庚大學 === 生物醫學研究所 === 98 === UP-LN1 is a poorly differentiated carcinoma cell line established from a lymph node (LN) metastatic lesion of a patient with unknown primary. The phenotype of CK7-/CK20+/CEA+/SCCA- detected in the original lesion and cultured cells led us to believe this cell line to be originated from a primary cancer of the gastrointestinal tract. UP-LN1 cells exhibited unique in vitro morphological characteristics with naturally occurring floating (F) and adherent (A) cells. The aim of this study was to determine if there were differences between F and A cells in terms of the phenotype, distribution of cancer stem cells (CSCs), susceptibility to NK/LAK cytolysis, and IFN-γ-mediated induction/enhancement of surface CXCR4. Comparative tumorigenicities (i.e., CSC or tumor initiating cell (TIC) properties) between F and A cells were carried out by xenotransplantation in NOD/SCID mice. For differential expression of selected tumor markers and genes associated with TICs/CSCs between F and A cells, DNA-microarray, RT-PCR and cytofluorometric analyses were used. Immunofluorescence (IF)-confocol microscopy was performed to examine the CD44/CD24 expression profile in the original lymph node metastatic lesion, from which the UP-LN1 cell line was established. Relative sensitivities of the two cell types to cytolysis of non-MHC-restricted effector cells (activated NK and LAK) were also determined using a 51Cr release cytotoxicity assay. The cytofluorimetric migration and invasion assays were used to evaluate the mobility and invasiveness of P, A and F cells with and without IFN-γ treatment. Injection of F cells at as low as 103 cells could initiate a tumor formation in NOD/SCID mice, while an equivalent value for A cells was 105. F and A cells were tested for a panel of drug resistant gene mRNAs, of which ABCG2, ABCB1, ABCC1 and ABCC4 were expressed in greater amounts in F cells. Interestingly, F and A cells exhibited distinctively different phenotypes, namely CD44bright/CD24dim and CD44dim/CD24bright, respectively. Consistently, results of IF-confocol microscopy on sections of the patient’s LN metastasis revealed that the CD44bright/CD24dim phenotype was mostly observed in the region containing loose cells, representing F cells, while the region of compact cells showed the CD44dim/CD24bright phenotype, consistent with that of A cells. The surface HLA class I expression was down-regulated in a greater extent in F cells, which could however be restored as that expressed by A cells after exposure to IFN-γ. F cells but not A cells exhibited extreme resistance to activated allogeneic NK cells (CD56dim/CD16+ cytotoxic subset) sorted out from peripheral blood mononuclear cells followed by IL-2 activation, as well as to LAK cells. In contrast to A cells in which the basal surface expression of CXCR4 was essentially not affected by IFN-γ, F cells turned out to be highly sensitive to IFN-γ to induce/enhance surface CXCR4 expression. Further, IFN-γ treatment increased the mobility and invasiveness of both A and F cells toward CXCL12 chemoattraction, with F cells displaying a greater ability to do so, suggesting that the induction of CXCR4+ metastatic CSCs (mCSCs) in F cells could functionally contribute to cancer metastasis in a greater efficiency. We herein have identified the resistance to activated NK/LAK killing and drugs, depressed HLA class I expression, and CD44bright/CD24dim collectively to be the phenotype of F cells, suggesting F cells of the UP-LN1 cell line to be enriched for the predominant CSCs/TICs. A different class or classes of CSCs/TICs could also be harbored in A cells because of demonstrated xeno-tumorigenicity. Finally, IFN-γ-mediated induction of CXCR4 expression by F but not by A cells appeared to be essential for UP-LN1 cells to exhibit metastatic behavior, and hence F cells are the niche for mCSCs.