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. 2009 Sep 22;106(38):16281-6.
doi: 10.1073/pnas.0905653106. Epub 2009 Sep 10.

Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment

Giulia Bertolini  1 , Luca Roz, Paola Perego, Monica Tortoreto, Enrico Fontanella, Laura Gatti, Graziella Pratesi, Alessandra Fabbri, Francesca Andriani, Stella Tinelli, Elena Roz, Roberto Caserini, Salvatore Lo Vullo, Tiziana Camerini, Luigi Mariani, Domenico Delia, Elisa Calabrò, Ugo Pastorino, Gabriella Sozzi
Affiliations

Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment

Giulia Bertolini et al. Proc Natl Acad Sci U S A. .

Abstract

The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133+, epithelial-specific antigen-positive (CD133+ESA+) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133(-) counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133+ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133+ABCG2+ and CD133+CXCR4+ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133+ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
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.
Fig. 2.
Fig. 2.
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.
Fig. 3.
Fig. 3.
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.
Fig. 4.
Fig. 4.
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.
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