There is increasing evidence that the Cancer Stem Cell (CSC), a small percentage of cancer cells that have the characteristic to self-renew, proliferate, differentiate and replenish the bulk of the tumor, may be the leading cause of cancer relapse after chemotherapy. A distinct subpopulation within cancer cells known as the side population (SP), distinguished via Hoechst 33342 staining, has been repeatedly reported to be enriched with cells possessing CSC characteristics. In this study, we first examine if SP can be identified in four established myeloma cell lines. Hoechst-33342 stained cells were analyzed through FACS LSRII using a blue and red dual-wavelength analysis after UV excitation to detect the important red-blue differential dye emission feature of the SP cells. An average of 26.5% of SP cells was found in RPMI8226, 21.4% in KMS-11, 5.4% in U266 and 10.1% in OPM-2 within the viable cells compartment in four separate experiments. In addition, 4.9% of SP cells were also identified in the plasma cell leukemia cell line ARH77.

SP and non-SP cells of RPMI8226 and KMS-11 were then sorted and respective RNAs were extracted, amplified and labeled for subsequent Affymetrix microarray scanning. The gene-expression profiles of SP and non-SP cells from RPMI8226 and KMS-11 were compared before selecting statistically significant genes of interest. Two genes were found to be highly upregulated in SP cells. The first one was the Receptor for Hyaluronan-Mediated Motility (RHAMM) gene of 29.0 and 3.9 fold increase in RPMI8226 and KMS-11 respectively. Increased expression of RHAMM has been previously reported to correlate with human B cell malignancies. The second upregulated gene was the human inhibitor of apoptosis protein-family gene, Apollon, of 4.4 and 3.2 fold increase in RPMI8226 and KMS-11 respectively. Another gene - interferon gamma receptor 1 (IFNGR1) gene was found to be down-regulated in SP cells, of 4.3 and 1.2 fold decrease in RPMI8226 and KMS-11 that may provide an explanation of their escape from immune response via T cells.

We then examine if SP cells that represent putative CSC exist in primary myeloma cells. Seven myeloma patients’ bone marrow samples were investigated and 0.1 to 5.2 %, of an average of 1.4% of SP cells were identified in the CD38++ plasma cells compartments in 6 out of 7 bone marrow samples. This study reports the existence of SP in both myeloma cell lines and primary myeloma cases. Further studies will be required to confirm the protein expression of selected genes selected from the gene-expression profiles of SP versus non-SP from myeloma cells for the insights into the characteristic and tumorigenicity of CSC in myeloma.

Disclosures: Cancer Institute NSW Fellowship.

Dr. Daniel Sze is supported by the Cancer Institute NSW Career Development and Support Fellowship.

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