Nair, Rohini M and Balla, Murali MS and Khan, Imran and Kalathur, Ravi Kiran Reddy and Kondaiah, Paturu and Vemuganti, Geeta K (2017) In vitro characterization of CD133lo cancer stem cells in Retinoblastoma Y79 cell line. In: BMC Cancer, 17 (1). ISSN 1471-2407
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Abstract
Background: Retinoblastoma (Rb), the most common childhood intraocular malignant tumor, is reported to have cancer stem cells (CSCs) similar to other tumors. Our previous investigation in primary tumors identified the small sized cells with low CD133 (Prominin-1) and high CD44 (Hyaluronic acid receptor) expression to be putative Rb CSCs using flow cytometry (FSClo/SSClo/CD133lo/CD44hi). With this preliminary data, we have now utilized a comprehensive approach of in vitro characterization of Y79 Rb cell line following CSC enrichment using CD133 surface marker and subsequent validation to confirm the functional properties of CSCs. Methods: The cultured Rb Y79 cells were evaluated for surface markers by flow cytometry and CD133 sorted cells (CD133lo/CD133hi) were compared for CSC characteristics by size/percentage, cell cycle assay, colony formation assay, differentiation, Matrigel transwell invasion assay, cytotoxicity assay, gene expression using microarray and validation by semi-quantitative PCR. Results: Rb Y79 cell line shared the profile (CD133, CD90, CXCR4 and ABCB1) of primary tumors except for CD44 expression. The CD133lo cells (16.1 ± 0.2%) were FSClo/SSClo, predominantly within the G0/G1 phase, formed larger and higher number of colonies with ability to differentiate to CD133hi cells, exhibited increased invasive potential in a matrigel transwell assay (p < 0.05) and were resistant to Carboplatin treatment (p < 0.001) as compared to CD133hi cells. The CD133lo cells showed higher expression of several embryonic stem cell genes (HOXB2, HOXA9, SALL1, NANOG, OCT4, LEFTY), stem cells/progenitor genes (MSI2, BMI1, PROX1, ABCB1, ABCB5, ABCG2), and metastasis related gene- MACC1, when compared to the CD133hi cells. Conclusions: This study validates the observation from our earlier primary tumor study that CSC properties in Rb Y79 cell line are endowed within the CD133lo population, evident by their characteristics- i.e. small sized, dormant in nature, increased colony forming ability, differentiation to CD133hi cells, higher invasiveness potential, drug resistance and primitive gene expression pattern. These findings provide a proof of concept for methodological characterization of the retinoblastoma CSCs with future implications for improved diagnostic and treatment strategies.
| Item Type: | Journal Article |
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| Publication: | BMC Cancer |
| Publisher: | BioMed Central Ltd. |
| Additional Information: | The Copyright of this article belongs to the Authors. |
| Keywords: | Cancer stem cell markers; CD133 (Prominin); Flow cytometry; Retinoblastoma; Stem-like cancer cells; AC133 Antigen; Biomarkers; Cell Cycle; Cell Line, Tumor; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunophenotyping; Neoplastic Stem Cells; Phenotype; Retinoblastoma; Tumor Stem Cell Assay; ABC transporter B5; BMI1 protein; breast cancer resistance protein; carboplatin; CD133 antigen; chemokine receptor CXCR4; Hermes antigen; left right determination factor; multidrug resistance protein 1; octamer transcription factor 4; Thy 1 antigen; transcription factor HoxA9; transcription factor NANOG; biological marker; CD133 antigen; ABCB1 gene; ABCB5 gene; ABCG2 gene; Article; BMI1 gene; cancer cell culture; cancer resistance; cancer stem cell; cell cycle assay; cell cycle G0 phase; cell cycle G1 phase; cell differentiation assay; cell invasion assay; cell selection; cell size; controlled study; cytotoxicity assay; flow cytometry; gene expression; HOXA9 gene; HOXB2 gene; human; human cell; in vitro study; LEFTY gene; MACC1 gene; microarray analysis; MSI2 gene; NANOG gene; OCT4 gene; oncogene; polymerase chain reaction; PROX1 gene; retinoblastoma cell line; SALL1 gene; upregulation; cancer stem cell; cell cycle; gene expression profiling; gene expression regulation; genetics; immunophenotyping; metabolism; pathology; phenotype; retinoblastoma; tumor cell line; tumor stem cell assay |
| Department/Centre: | Division of Biological Sciences > Molecular Reproduction, Development & Genetics |
| Date Deposited: | 17 Jun 2022 07:16 |
| Last Modified: | 17 Jun 2022 07:16 |
| URI: | https://eprints.iisc.ac.in/id/eprint/73501 |
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