The specific cellular subset of a tumour mass responsible for initiating and maintaining the disease is widely accepted as coincident with a niche of so-called cancer stem cells (CSCc), or tumor-initiating cells (TICs), endowed with properties such as aberrant differentiation, self-renewal and drug resistance. We recently showed that the bronchoalveolar duct junctions of normal lung harbour epithelial-specific stem cells (Tesei A., et al. Cell Proliferation 2009; 42: 298-398). Here we aimed at isolating and characterizing CSCc/TICs from 1) a human adenocarcinoma cell line (RAL) that we originally established at the Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) at Meldola (Gasperi-Campani A., et al., Cancer Genetics and Cytogenetics 1998; 107:11–20) and from 2) surgical human tissue samples. Cellular suspensions derived from the RAL cell line were cultured in ultralow attachment plates in a serum-free medium. Neoplastic lung tissue samples, derived from patients affected by adenocarcinoma at different stages, were mechanically and enzymatically dissociated before expansion in culture. Expression of stem-like phenotype-related genes in cell clusters was evaluated with RT-PCR and Real Time RT-PCR. Floating spherical colonies developed within about ten days in culture. The spheroids obtained by our originally established lung adenocarcinoma cell line RAL, resulted enriched in CD133 and OCT-3/4 transcripts. RT-PCR analysis of spheres obtained from surgical tissue samples showed a higher expression of BCRP-1, CD133, BMI-1, OCT-3/4, Lef-1, CD44 and Slug, when compared to original tissue. Floating spherical RAL cell colonies and the original adherent cell culture counterpart was also evaluated for their level of CpG methylation in OCT-3/4 and CD133 gene promoters, by means of bisulfite sequencing. They both showed a coincident hypermethylated profile suggesting that this level of transcriptional control is not involved in modulating their expression in human lung adenocarcinoma stem-like cells in our floating spheroid colonies.
Human lung adenocarcinoma cells in floating spheroid colonies display stem-like phenotypic hallmarks / TESEI A.; ARIETI C.; PAGANELLI G.; PASINI A.; BRIGLIADORI G.; CALISTRI D.; GIORDANO E.; ZOLI W.. - STAMPA. - (2011), pp. 83-83. (Intervento presentato al convegno Recapturing Pluripotency: Links between Cellular Reprogramming and Cancer tenutosi a CNIO, Madrid, Spagna. nel 7-9 Novembre 2011).
Human lung adenocarcinoma cells in floating spheroid colonies display stem-like phenotypic hallmarks
PASINI, ALICE;GIORDANO, EMANUELE DOMENICO;
2011
Abstract
The specific cellular subset of a tumour mass responsible for initiating and maintaining the disease is widely accepted as coincident with a niche of so-called cancer stem cells (CSCc), or tumor-initiating cells (TICs), endowed with properties such as aberrant differentiation, self-renewal and drug resistance. We recently showed that the bronchoalveolar duct junctions of normal lung harbour epithelial-specific stem cells (Tesei A., et al. Cell Proliferation 2009; 42: 298-398). Here we aimed at isolating and characterizing CSCc/TICs from 1) a human adenocarcinoma cell line (RAL) that we originally established at the Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) at Meldola (Gasperi-Campani A., et al., Cancer Genetics and Cytogenetics 1998; 107:11–20) and from 2) surgical human tissue samples. Cellular suspensions derived from the RAL cell line were cultured in ultralow attachment plates in a serum-free medium. Neoplastic lung tissue samples, derived from patients affected by adenocarcinoma at different stages, were mechanically and enzymatically dissociated before expansion in culture. Expression of stem-like phenotype-related genes in cell clusters was evaluated with RT-PCR and Real Time RT-PCR. Floating spherical colonies developed within about ten days in culture. The spheroids obtained by our originally established lung adenocarcinoma cell line RAL, resulted enriched in CD133 and OCT-3/4 transcripts. RT-PCR analysis of spheres obtained from surgical tissue samples showed a higher expression of BCRP-1, CD133, BMI-1, OCT-3/4, Lef-1, CD44 and Slug, when compared to original tissue. Floating spherical RAL cell colonies and the original adherent cell culture counterpart was also evaluated for their level of CpG methylation in OCT-3/4 and CD133 gene promoters, by means of bisulfite sequencing. They both showed a coincident hypermethylated profile suggesting that this level of transcriptional control is not involved in modulating their expression in human lung adenocarcinoma stem-like cells in our floating spheroid colonies.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
