PF573 SINGLE CELL RNA-SEQ REVEALED A RARE ITGA2 EXPRESSING CELL SUBPOPULATION WITH DISTINCT CANCER STEM CELL MARKER SIGNATURE IN MULTIPLE MYELOMA

Abstract

Background: Multiple myeloma (MM) stem cells (MMSC) are a rare cell population capable of self-renewal and differentiation into the predominating mature myeloma cells. Specific targeting this cell population could effectively eradicate the tumors in the patients. Previous studies reported CD138 non-expressing myeloma cells as putative MMSC. However, conflicting results by subsequent studies demonstrated the tumor-initiating and clonogenic cells expressed CD138 and apoptotic features in CD138 non-expressing cells. Aims: In this study, we aimed at identification of MMSC by examination of the transcriptome of myeloma cells at single cell level. Methods: Single cell RNA-Seq with flow-based SMART-Seq2 protocol was employed to determine the transcriptome of 768 myeloma cells from MM1 s and NCIH929 cell lines (384 cells each line) and 68 cells from a clinical MM bone marrow sample. The percentages of myeloma cells expressing the candidate biomarker(s) protein were determined by flow cytometry. Results: Results of quality control analyses indicated ∼400K read counts per cell with 15–20% of multiple mapping representing ribosomal RNAs and 12–15% mitochondrial RNAs and with 6700–8600 variable genes for transcriptome clustering analyses. By unsupervised analysis using t-distributed stochastic neighbor embedding (t-SNE), myeloma cells were clustered according to their cell cycle statuses. After controlling the cell cycle effects by regression, two clusters with similar cell numbers were observed in each cell line. Interestingly, the results of supervised analysis with t-SNE on 24 cancer stem cell markers expression revealed a rare subpopulation of myeloma cells (MM1 s: 2.6%; NCIH929: 3.6%) that were signified by Integrin alpha 2 (ITGA2) expression but no expression of OCT4 and NANOG stem cell markers. Flow cytometry using anti-ITGA2 antibody confirmed the presence of this rare cell population (∼0.3%) in MM cells. For the cells from the clinical sample, 3 clusters were observed by unsupervised analysis after controlling cell cycle effects. Studies of the signature genes for each cluster indicated two clusters were derived from erythroid cells and lymphocytes; while the 3rd cluster (n = 49) from myeloma cells, within which one ITGA2-expressing cell was idenitifed. ITGA2 encodes a cell surface protein CD49b integrin receptor as a marker for primitive human hematopoietic stem cells. In leiomyoma, CD49b marked the side population cells and the stemness of CD49b-expressing cells was also demonstrated. Recently, increased NOTCH1 and its downstream targets expression implying high activity of NOTCH signaling, which are involved in stem cell self-renewal and differentiation, were reported in CD49b-expressing trophoblast progenitor cells in human placenta. All these findings suggested the link of ITGA2 and stem cell properties. Functional studies of the ITGA2 positive myeloma cells are currently in progress regarding their clonogenic potentials and self-renewal capacity. Summary/Conclusion: Our study revealed a rare subpopulation of ITGA2-expressing cells with distinct cancer stem cell markers signature in MM cell lines and a clinical sample, potentially representing MMSC.