Tumor xenografts resemble the original primary tumor. (A) IHC of low-molecular weight cytokeratins (CK-LMW), cytokeratin 7 (CK7), surfactant protein C (SP-C), transcription thyroid factor 1 (TTF-1), CD133, and MIB-1 performed in LT45 parental tumor, corresponding xenograft, and xenograft tumor derived from injection of CD133⁺ cells. (B) FACS analysis of CD133⁺ESA⁺cells in xenografts compared with parental tumors.

CD133⁺ cells survive cisplatin treatment. (A) FACS analysis of CD133 expression in A549 parental cell line and A549 treated for 1 h with cisplatin (IC₈₀). CD133 expression increased in the A549 cell line 72 h after treatment. (B) FACS analysis of CD133 expression in stable cisplatin-resistant A549/Pt cells compared with the A549 parental cell line. (C) Enrichment of CD133⁺ABCG2⁺ (Upper) and CD133⁺CXCR4⁺ (Lower) populations in xenografts LT45 and LT56 after in vivo cisplatin treatment. (D) CD133 expression in advanced NSCLC patients treated with platinum-containing regimens. Progression-free survival curve was calculated with the Kaplan–Meier method and compared by using the log-rank test.

IHC analysis of CD133 expression in two representative positive (LT74 and LT60) and one negative (LT66) adenocarcinoma lung tumor samples at low and high magnifications. As controls (CTR) for staining specificity, a normal lung sample negative for CD133 (Bottom Left) and a control antibody-stained tumor sample (Bottom Right) are also shown.

In vitro characterization of CD133⁺ cells. (A) FACS analysis for CD133 expression in freshly dissociated primary LT73 and corresponding adherent cell culture after four passages in vitro. (B) PKH26 staining of LT73 and A549 spheres. (Top) Single cells from dissociated spheres were separately labeled with PKH26 and PKH67 red and green fluorescent dyes, and cultures were harvested by mixing red and green labeled cells. (Middle and Bottom) A549 (Middle) and LT73 (Bottom) spheres, derived from mixed cultures, showed a separate green or red fluorescent staining, with a decreasing gradient of fluorescence intensity within the spheres. Microscopic images of spheres derived from labeled cells were acquired at 20× magnification in bright field, with rhodamine filter for PKH26 fluorescence (Left) and with fluorescein filter for PKH67 fluorescence (Right). (C) Time-course analysis of CD133⁺ cells sorted from A549 spheres. FACS analysis of CD133 after sorting and at the first and second population doubling (PD). (D) Real-time PCR analysis of stemness gene expression in CD133⁺ and CD133⁻ fractions, sorted from LT28 xenograft (Left) and A549 spheres (Right). Unsorted cells were used as calibrator for the relative quantification of gene expression. (E) Real-time PCR analysis of transporters of ABCC-B-G families in CD133⁺ and CD133⁻ cells sorted from A549/s, and ABCC family in CD133⁺ and CD133⁻ cells sorted from LT73/s and LT45 xenograft. Unsorted spheres or LT45 unsorted cells were used as calibrators for the relative quantification of gene expression.