Side-Population and Hedgehog Signaling Analyses on Floating vs Attachment Subpopulations of the CEA-Producing UP-LN1 Cell Line as a Study Model---Identification and Eradication of Cancer Stem Cells

We have previously documented the establishment and preliminary characterization of a CEA-producing carcinoma cell line from a lymph node metastatic lesion (poorly differentiated ca) of the neck of a patient with unknown primary [Chang et al., 2005; see Appendix]. This tumor was likely of adenocarcinoma of GI-tract origin, based on its particular phenotype, CK7-/CK20+/CEA+/SCCA- in both the original clinical lymph node tumor specimen and cultured cells. The persistent appearance of adherent (A cells) and floating (F cells) under normal monolayer culture conditions prompted us to investigate differential properties of these two cell types further. Unexpectedly, A cells were found to be highly susceptible to lymphokine-activated killer (LAK)-mediated cytolysis, whereas F cells were essentially resistant to LAK cell killing. Using DNA microarrays, we identified expression of 17 genes in F cells which were upregulated by ≧2-fold than in A cells. We also identified expression of other 38 genes to be upregulated by ≧2-fold in A cells than in F cells. At least 3 cancer stem cell (CSC) markers were identified in the 17 genes upregulated in F cells, suggesting that CSC and their progenitors are more abundant in F subpopulation. In the past 5 months after the initiation of the new grant supported by the NSC, we found that surface CXCR4+ cells can be enhanced in F cells but not A cells by IFN-γ, and that the expression of basal surface HLA-ABC, which can be enhanced by IFN-γ, are much less than on A cells. With these as the background information, we hypothesized that “greater numbers of cancer stem-like cells and their progenitors are in F cells than in A cells, and thus it is important to find ways to eradicate these cells for effective cancer therapeutic strategies for this malignancy of the GI tract.”We here therefore propose to extend this study with the following specific objectives: 1) To elucidate the mechanism(s) underlying the high sensitivity of A cells but not F cells to LAK-mediated cytolysis; 2) To sort out and analyze the cancer stem cells (CSC) and their immediate progenitors from F cells and A cells using a side-population (SP) assay or by a FACS machine using a panel of appropriate mAbs. The role of depressed expression of HLA-ABC and IFN-γ-enhanced CXCR4 in relation to CSCs and migrating/metastatic CSCs, respectively will be investigated. 3) Side-populations (SPs) and migrating CSCs in F and A cells independently isolated will be compared with respect to the expression of CSC genes, including the Hedgehog (Hh) pathway related genes, Ihh, Gli and Ptch.. 4) To determine if any of the expression listed in #3) is associated with clinical lymph node metastasis, tumor progression in GI tumor specimens, and/or colorectal and gastric tumor cell lines (i.e., to correlate Gli or Ptch or CXCR4 immunochemical positivity with patient prognosis). 5) To determine whether Gli or CXCR4, for instance, is necessary for maintaining the proliferation potential of cells of the UP-LN1 cell line, after transient transfection of siRNA targeting Gli-mRNA or CXCR4- mRNA into F cells of the UP-LN1 cell line. 6) To search for agents (drugs, biological, peptides including CXCR4 antagonists) are able to selectively eradicate most, if not all, SP cells or CXCR4+CD133+ cells, or CXCR4+CD133- cells of the UP-LN1 cell line. Xenografts in the NOD-SCID mouse model will be used to verify for the effectiveness of the selected drugs or molecules proved to be indeed useful as shown in anti-CSCs in vitro results. We feel strongly that CSC may have direct translational importance for cancer therapy.

Project ID:PC9808-1028
External Project ID:NSC98-2314-B182-018