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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 27, 2012 |
| Title |
CD20 positive cells are undetectable in the majority of multiple myeloma cell lines and are not associated with a cancer stem cell phenotype |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Although new therapies have doubled the survival of multiple myeloma (MM) patients, this remains an incurable disease. It has been postulated that the so-called MM Cancer Stem Cells (MM-CSC) would be responsible for tumor initiation and relapse but their unequivocal identification remains unclear. Here, we investigated in a panel of MM cell lines the presence of CD20+ cells harboring a MM-CSC phenotype. Among the multiple cell lines investigated, only a small population of CD20dim+ cells (0.3%) in the RPMI-8226 cell line was found. CD20dim+ RPMI-8226 cells expressed the plasma cell markers CD38 and CD138 and were CD19-CD27-. Additionally, CD20dim+ RPMI-8226 cells did not exhibit stem-cell markers as shown by gene expression profiling and the aldehyde dehydrogenase (ALDH) assay. Moreover, we demonstrated that CD20dim+ RPMI-8226 cells are not essential for CB17-SCID mice engraftment and show lower self-renewal potential than the CD20- RPMI-8226 cells. These results do not support CD20+ expression for the identification of MM-CSC.
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| Overall design |
For detailed description of Material and Methods, see supplementary Methods.
The human MM cell lines RPMI-8226, U266, MM1S, MM1R, NCI-H929, RPMI-LR5, U266-LR7 and U266-Dox4 were studied. Cells were cultured as previously described [Ocio, 2009, 3781].
MM cell lines were immunophenotyped using a 7-color immunofluorescence technique [Paiva, 2011, 697], with the following combination of monoclonal antibodies (Pacific Blue (PB)/ anemonia majano cyan (AmCyan)/ fluorescein isothiocyanate (FITC)/ peridinin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5)/ PE-cyanin 7 (PE-Cy7)/ allophycocyanin (APC)/ alexafluor 700 (AF700)): CD19/CD45/CD20/CD138/CD27/CD56/CD38. Data were stored for a minimum of 3x105 events. CD20dim+ and CD20- RPMI-8226 cells were sorted after incubation with CD20-APC/7AAD and acquisition on a FACSAria cytometer (Becton Dickison Biosciences). Sorting was performed only for viable cells (7AAD-) and debris were excluded by scatter properties. The CD20- and CD20dim+ RPMI-8266 sorted cells had a final purity of 98% and 88%, respectively.
CD20dim+ and CD20- RPMI-8226 cells were extensively characterized. Morphological characterization was done with May-Grünwald-Giemsa staining. Characterization of VDJH and IGKappa rearrangements was performed as described elsewhere [BIOMED-2, BMH4-CT98-3936]. The expression of aldehyde dehydrogenase (ALDH) was assessed using the Aldefluor Kit (StemCell Technologies) following manufacturer’s instructions with further staining with a CD20-APC antibody. For gene expression profile studies, RNA was isolated, labeled and hybridized to Human Gene 1.0 ST array (Affymetrix) according to Affymetrix protocols [Gutiérrez, 2005, 402]. The arrays were analyzed using the DNA-Chip Analyzer software (DChip). Fold change ≥2 was considered significant.
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| Contributor(s) |
Paíno T, Ocio EM, Paiva B, San-Segundo L, Garayoa M, Gutiérrez NC, Sarasquete ME, Pandiella A, Orfao A, San Miguel JF |
| Citation(s) |
22315496 |
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| Submission date |
Oct 17, 2011 |
| Last update date |
Jul 26, 2018 |
| Contact name |
Teresa Paíno |
| E-mail(s) |
[email protected]
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| Phone |
+34 923294812
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| Organization name |
Centro de investigación del cáncer
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| Street address |
Campus Miguel de Unamuno
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| City |
Salamanca |
| ZIP/Postal code |
37007 |
| Country |
Spain |
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| Platforms (1) |
| GPL6244 |
[HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version] |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA148017 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE33020_RAW.tar |
26.7 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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