Identification and gene expression analysis of the side population subclone in mantle cell lymphoma

Abstract

Introduction: It has been increasingly understood that seemingly effective treatments merely remove the bulk of tumor cells, while more rare cancer stem cells reside. The treatment resistant cells soon repopulate and form new tumor mass. These are often selected by the exposure to therapy and thus increasingly resistant. One way of identifying such cells is through analysis of the side population (SP) in tumor masses. In this study, we investigated the presence of SP in twenty-five lymphoma cell lines, representing five different types of B-cell lymphomas. Methods: Cells were labelled with fluorescent DNA-binding dye Hoechst 33342, and SP cells were identified as a subpopulation of cells that have an elevated rate of Hoechst efflux. All samples were analyzed on a FACS Aria II (BD Bioscience, San Jose, CA, USA). Using cell sorting, both SP and non-SP cells could be isolated. The presence of SP was assessed in MCL patient samples and cell lines derived from MCL, FL, DLBCL, Burkitt's lymphoma (BL), and CLL. Subsequent gene expression analysis was performed using Affymetrix Human Transcriptome Array 2.0 (HTA 2.0; Affymetrix Inc, Santa Clara, CA, USA). Results: The presence of SP was limited to one mantle cell lymphoma (MCL) derived primary sample (P3) and cell line (REC-1). Downstream analysis involving in vitro cultivation of sorted SP shows that SP cells are able to grow and differentiate into daughter cells and non-SP cells. Of interest, the frequency of SP cells was enriched when wild type REC-1 cells were exposed and became resistant to chemotherapy. Moreover, a differential gene expression profile between SP cells and non-SP cells was revealed by comprehensive gene expression analysis showing deregulation of group of genes involved in cell proliferation, cell cycle regulation, cell migration, BCR signaling, cancer progression, and tumorigenesis. Of major importance, validation of gene expression results reveal that SP, compared to non-SP, exhibits significantly higher expression levels of CD44 and CD69, known to be involved in cancer stem cells and lymphocyte proliferation, respectively. Conclusion: We show that SP is found among MCL patients and that genes involved in several cell signaling pathways are deregulated in SP derived from both REC-1 and patient material. This indicates that REC-1 can serve as a tool for future screening efforts. Importantly, we show evidence that CD69 and CD44 are commonly upregulated in SP cells from MCL patient material and could have important functional roles in relapse and treatment resistance. Keywords: flow cytometry; mantle cell lymphoma (MCL)