CD20 positive cells are undetectable in the majority of multiple myeloma cell lines and are not associated with a cancer stem cell phenotype

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nHIB19) and CD27-PE-Cy7 (clone O323) antibodies were purchased from eBioscience (San Diego CA, USA) and CD38-AlexaFluor700 antibody (clone HIT2) was obtained from Exbio (Vestec, Czech Republic). CD20 dim+ and CD20 -RPMI-8226 cells were extensively characterized. For real time quantitative PCR (qRT-PCR), total RNA was extracted from CD20 dim+ and CD20 -RPMI-8226 cells using an RNeasy Mini Kit (Qiagen, Valencia, USA) following the manufacturer's protocol. RNA quality and quantity were assessed with the RNA Nano LabChip (Agilent Tech. Inc., Palo Alto, CA, USA). The retrotranscription reaction was performed with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Foster City, CA, USA) according to the manufacturer's recommendations. Finally, real time quantitative PCR was performed using TaqMan gene expression assay kits (Applied Biosystems Foster City, CA, USA): Hs_00544819 for MS4A1 (CD20) and Hs99999905_m1 GAPDH as a control gene. Relative gene expression was calculated by the 2 -ΔCt method, ΔCt=Ct (gene) -Ct (GAPDH). Morphological characterization was performed with May-Grünwald-Giemsa staining. May-Grünwald and Giemsa stains were obtained from Merck (Darmstadt, Germany). Characterization of VDJH and IGκ rearrangements was performed in genomic cDNA as described elsewhere. 13 The expression of aldehyde dehydrogenase (ALDH) was assessed using the Aldefluor Kit (StemCell Technologies, Grenoble, France) following the manufacturer's instructions with further staining with a CD20-APC antibody. For microarray studies, RNA from 3 independent CD20 dim+ or CD20 -RPMI-8226 samples was isolated, labeled and hybridized to Human Gene 1.0 ST array (Affymetrix) according to Affymetrix protocols.14 The arrays were analyzed using the DNA-Chip Analyzer software (DChip). Fold change of 2 or more was considered significant. All microarray data have been deposited with the Gene Expression Omnibus under accession number GSE33020.For serial colony assays, 1000-1500 CD20 dim+ or CD20 -RPMI-8226 cells/mL were plated in Methocult ® (StemCell Technologies, catalog n. H4230) and incubated at 37°C and 5% CO2. After 14 days, colonies (≥40 cells) were scored and subsequently collected, rinsed with PBS and plated again in fresh Methocult ® . A sample was used to evaluate CD20 expression in the colonies. For engrafting assays, unfractionated or sorted CD20 dim+ or CD20 -RPMI-8226 cells were subcutaneously inoculated into 6-7 week old CB17-SCID mice (Charles River, Spain).11 In selected mice, tumors were isolated, mechanically minced and filtered through 40 µm cellstrainers to perform phenotypic studies and/or reinjection into secondary recipients. All animal experiments were performed according to the protocol previously approved by the ethical committee of the University of Salamanca, Spain.Statistical analyses were performed using the SPSS-15.0 software (SPSS, Chicago, IL, USA); significant differences between groups were assessed by the Student's t and Mann-Whitney U tests....

Reply to "Response to "CD20 positive cells are undetectable in the majority of multiple myeloma cell lines and are not associated with a cancer stem cell phenotype". Haematologica 2012;97(7):1110-1114

“…We read with interest the letter by Van Hoef 1 about the article by Paino et al 2 In that study, we were unable to identify a CD20 + population among 8 myeloma cell lines leading us to conclude that CD20 may not be a suitable antigen for the identification of myeloma cancer stem cells.…”

Differential Protein Expression Profile Between CD20 Positive and Negative Cells of the NCI-H929 Cell Line

“…In patients, it was believed that CD20-CD138+ was the origin of clonogenic myeloma cells (Chiron et al, 2012). Moreover, some researchers could not detect CD20 positive cells in NCI-H929 cells and believed that CD20 positive cells were not associated with a cancer stem cell phenotype (Paíno et al, 2012). At present, the nature of clonogenic myeloma cells remains unclear.…”

“…It was showed that CD20+ and CD138-cells were not the mark of clonogenic cells (Jakubikova et al, 2011;Chiron et al, 2012;Hosen et al, 2012). Moreover, some study found that NCI-H929 cells did not contain CD20 positive cells and suggested that CD20 positive cells were not associated with a cancer stem cell phenotype (Paíno et al, 2012).…”

The myeloma stem cell concept, revisited: from phenomenology to operational terms

“…During the last decade, MSCNET has performed detailed studies to confirm or refute previous studies [47][48][49][50][51][52][53][54][55][56][57][58][59][60] and established protocols 17,[61][62][63][64][65][66][67][68][69][70] to delineate the phenotypes of subpopulations of cells in randomly selected primary tumor samples and in preclinical disease models. While our investigations did not confirm that pre-PC B cells are myeloma initiating, 50 low/-memory B cells engrafted into human bone grafts, resulting in the repopulation of polyclonal B cells, which supports the hypothesis that memory B cells have the ability to self-renew.…”