Abstract C8: Putative tumor‐initiating cell populations in mantle cell lymphoma exhibit microenvironmentally influenced differences in stem cell‐like and tumorigenic properties

The presence of subpopulations of cells possessing enhanced tumor formation capabilities has been described in several tissues. These cells have been termed tumor‐initiating cells (TICs) or cancer stem cells and have been isolated using various techniques, including the side population (SP), the use of cell surface markers including CD133 and CD44 and aldehyde dehydrogenase activity. We have elected to evaluate the stem cell‐like and tumor forming capabilities of SP and CD133 + /CD44 + cells in Mantle Cell Lymphoma (MCL), an aggressive and relatively therapy unresponsive form of Non‐Hodgkin9s Lymphoma. All analyses were conducted in the presence of micro‐environmental niche cells (murine stromal 5 cells) or extracellular matrix components (Matrigel) as these factors are known to play an integral role in processes associated with stem cell self‐renewal and differentiation. SP cells and CD133 + /CD44 + cells were present in Granta 519, an aggressive MCL cell line. The in‐vitro technique of the Cobblestone Area Forming Cell (CAFC) assay was utilized to assess the self‐renewal capabilities of SP and CD133 + /CD44 + cells when the two cell types were cultured in the presence and absence of Matrigel, a mixture of extracellular matrix proteins derived from the Englebreth‐Holm murine sarcoma. CAFC assays revealed enhanced self‐renewal capabilities for Granta SP cells over non‐SP cells, with the addition of Matrigel increasing the self‐renewal frequency of SP and non‐SP cells by 5.8 and 8.6 fold respectively. Conversely, Granta CD133 + /CD44 + and CD133 − /CD44 − cells possessed similar self‐renewal capabilities, with the addition of Matrigel having a negligible effect. When evaluated for chemotherapy resistance, the murine stromal 5 (MS5)‐adherent CD133 + /CD44 + cells and the non‐adherent SP cells were more resistant to hydroxyurea while all MS5‐adherent Granta cells were resistant to this agent. In‐vivo analyses demonstrated tumor formation of Matrigel mixed SP and non‐SP populations but not for Matrigel mixed CD133 + /CD44 + or CD133 − /CD44 − cells. Although micro‐metastatic tumors were not detected for SP and non‐SP injected mice, flow cytometric analyses identified Granta cells, characterized by CD20 expression, in all murine tissues examined. IL‐8, a proangiogenic cytokine implicated in tumor metastasis, may be involved in the metastatic spread of Granta cells as it was significantly present in the supernatants of Granta SP MS5 co‐cultures, but was negligible or undetected when non‐SP, CD133 + /CD44 + and CD133 − /CD44 − cells were co‐cultured with MS5 cells. These studies suggest that the SP phenotype may be a better marker for the isolation of stem‐like cells in MCL. Moreover, the micro‐environmental components found in Matrigel, rather than cell phenotype differences, appear to play a vital role in the initiation of tumor development by SP and non‐SP cells. The tumor micro‐environmental niche may therefore be an important target for development of novel therapeutic agents. Citation Information: Cancer Res 2009;69(23 Suppl):C8.