Figure 1. Inducible KLF4 overexpression suppresses stem-like properties of human cancer cells.

(A) Top 10% of CD44-positive or -negative subpopulations of L3.7 PDA cells isolated by flow cytometry sorting (Ai) were subcutaneously injected into nude mice (50,000 cells/mouse; n=5 mice/group) for tumorigenesis analysis. The representative tumor size and the average tumor weight in each group are shown (Aii). RT-PCR analysis of KLF4 expression in CD44-positive or -negative subpopulations of L3.7 and SW620 cells (Aiii).

(B) Addition of doxycycline (Dox) into tumorsphere culture containing single-cell suspension of primary spheroid-forming RF4-L3.7 cells (Bi) or RF4-SW620 cells (Bii) on KLF4 expression (left panel, WB assay) and their secondary spheroid formation, the representative images of spheroids formed and the quantitative data are shown, respectively.

(C) Addition of a low dose of Dox into the single-cell suspension of tumorsphere culture drastically sensitized the primary spheroid-forming RF4-SW620 and RF4-L3.7 cells to 5-fluorouracil (5-FU) and gemcitabine (GEM) treatment, as determined by spheroid formation assay. Sensitivity is expressed as the 50% inhibitory concentrations (IC₅₀) of 5-FU and GEM.

(D) Administration of Dox in drinking water inhibited tumorigenesis and liver metastasis after subcutaneous or ileocolic vein injection of a single-cell suspension of primary RF4-L3.7SF cells into mice (n=8/group). Representative images of tumor growth in mice (Di) and average tumor weight (Dii) and number of liver metastases (Diii) are shown. (*P<0.05; **P<0.01).