Cancer Stem Cells of Diffuse Large B Cell Lymphoma Are Not Enriched in the CD45+CD19- cells but in the ALDHhigh Cells

Abstract

Although the existence of cancer stem cells (CSCs) has been suggested in diffuse large B cell lymphoma (DLBCL), there is still no definitive marker. CD45⁺CD19^- has been regarded as a potential marker of CSCs in mantle cell lymphoma (MCL). So, we explored the role of CD45⁺CD19^- in DLBCL. However, both CD45⁺CD19^- cells and CD45⁺CD19⁺ cells did not generate tumors until more than 100,000 cells were inoculated in NOD/SCID mice, even CD45⁺CD19⁺ cells generated more and larger tumors, as well as the soft agar colony formation in vitro; The aldehyde dehydrogenase (ALDH) activity was also identified in this study. Only 1,500 ALDH^high cells were enough to generate tumors in mice while the same number of ALDH^- cells were not. Moreover, both groups formed tumors when more cells were inoculated, but ALDH^high cells formed more and larger tumors. The similar result was obtained in vitro clonogenicity experiments. OCT4, SOX2, Nanog, and ABCG2 genes did not show any difference in CD45⁺CD19⁺, CD45⁺CD19^-, ALDH^high and ALDH^- cells. Taken together, CSCs are not enriched in the CD45⁺CD19^- cells but in the ALDH^high cells in DLBCL cell lines.

Keywords: ALDH activity; CD45+CD19-; CSCs markers; diffuse large B cell lymphoma.

Figure 1

The surface markers of LCL by flow cytometry analysis. CD45, CD19, CD20 and CD23 are positive.

Figure 2

CD45⁺CD19⁺ and CD45+CD19^- cells were analyzed and sorted by flow cytometer. (A) The proportion of CD45⁺CD19^- cells and the post-sort purity in LCL cell line. (B) The proportion of CD45⁺CD19^- cells and the post-sort purity in Farage cell line.

Figure 3

Characterization of CD45⁺CD19⁺ and CD45⁺CD19^- cells in LCL cell line. (A) the weight of tumors generated by CD45⁺CD19⁺ cells were higher than CD45⁺CD19^- cells, *p≤0.05. (B) Ki-67 analysis of CD45+CD19+ and CD45+CD19- cells derived tumor xenografts by IHC (×400). (C) cells were plated at clone density (2500 cells and 1250 cells per well) and cultured for 14 days, colony formation was quantified using the fluorescent CyQUANT GR Dye. Three independent experiments were performed, ***p≤0.001. (D) the expression levels of ABCG2, Nanog, OCT4 and SOX2 were evaluate. β-actin was used as a loading control. There was no difference between the two groups.

Figure 4

Characterization of CD45⁺CD19⁺ and CD45⁺CD19^- cells in Farage cell line. (A) the weight of tumors generated by CD45⁺CD19⁺ cells were higher than CD45⁺CD19^- cells, **p≤0.01. (B) Ki-67 analysis of CD45⁺CD19⁺ and CD45⁺CD19^- cells derived tumor xenografts by IHC (×400). (C) cells were plated at clone density (2500 cells and 1250 cells per well) and cultured for 14 days, colony formation was quantified using the fluorescent CyQUANT GR Dye. Three independent experiments were performed. ***p≤0.001. (D) the expression levels of ABCG2, Nanog, Oct4 and SOX2 were evaluated. β-actin was used as a loading control. There was no difference between the two groups.

Figure 5

Characterization of ALDHhigh and ALDH- cells in Farage cell line. (A) The proportion of ALDH^high cells and the post-sort purity in Farage cell line. (B) the weight of tumors generated by ALDH^high cells were higher than ALDH- cells. *p≤0.05. (C) cells were plated at clone density (2500 cells and 1250 cells per well) and cultured for 14 days, colony formation was quantified using the fluorescent CyQUANT GR Dye. Three independent experiments were performed. ***p≤0.001. (D) and (E) the expression levels of ABCG2, Nanog, Oct4 and SOX2 were evaluated by RT-PCR and WB analysis. GAPDH and β-actin were used as loading controls respectively. There was no difference between the two groups.