Side Population Cells of Human B-Cell Non-Hodgkin Lymphoma Cell Lines May Contain Lymphoma-Stem Cells Responsible for Lymphoma Initiation
Authors: Lee Ha Yeon , Lee Mi Ran , Kim Won Seog , Kim Seok Jin 
Published: October 2019

Abstract
Abstract 3105

Background:
Cancer stem cells are considered to initiate cancer development, and such populations might be related with the resistance to treatment and relapse. There are accumulated data supporting the presence of cancer stem cells in several human cancers such as breast and colon cancers. In hematologic malignancy, the presence of cancer stem cells was supported by the previous studies from leukemia. However, there are few reports regarding the possibility of lymphoma stem cells. Side population cells are defined as cells that efficiently extrude the Hoechst 33342 dye, and hence remain negative for this fluorescent marker in flow cytometric analysis. The isolation of SP cells from total tumor cells has been used as a tool for cancer stem cell research.

Methods:
We performed the side-population analysis to isolate a stem-like cell population. The side population analysis was based on the modified Hoechst 33342 dye efflux assay. Thus, cells expressing ATP binding cassette (ABC) transporter subfamily G number 2 (ABCG2) on their surface efflux Hoechst 33342 dye. On the other hand, cells without ABCG2 expression cannot efflux dye. According to this different nature, we discriminated side population from non-side population. We isolated side population from three lymphoma cell lines, 1A2 (B-cell lymphoblastic lymphoma cell line), Raji (Burkitt lymphoma cell line), and Toledo (Diffuse large B-cell lymphoma cell line). We compared the SP cells with NSP cells from each cell line via in vitro propagation and colony forming assay. We also evaluated the ability of tumorigenesis in NOD/SCID mice and compared the gene expression via performing microarray.

Results:
We isolated a subset of cells (side population) from three cell lines. The number of side population was extremely small, so the ratio of SP to NSP was from 0.01% to 0.25% regardless of the type of lymphoma cell lines. The in vitro propagation for 3 weeks showed that the growth rate of the side population was significantly higher than non-side population in three cell lines. The life span of non-side population was limited while side population could continuously proliferate. Thus, non-side population could not be cultured over four weeks, and the proportion of viable cells was higher in the side population (≥95%) than non-side population (≤80%). The RT-PCR and confocal microscopy demonstrated the higher expression of ABCG2 in the side population compared to the non-side population. The inoculation of lymphoma cells (200 – 1000) into NOD/SCID mice showed the tumor formation in mice inoculated with side population cells while there was no tumor in non-side population. The hierarchical clustering of differentially expressed genes showed the different pattern of gene expression between side and non-side population in each cell line.

Conclusion:
This study suggests that the side population may contain a cell population highly capable of proliferation, and this population may have the characteristics of lymphoma stem-like cells.

Disclosures:
No relevant conflicts of interest to declare.