escapees with progenitor c ell properties in breast, lung and ovarian cancer.

A123

The cancer stem cell (CSC) hypothesis states that the cancer-initiating cell is a transformed tissue stem cell, which retains the essential property of self-protection through the activity of multiple drug resistance. When tumor cells are exposed to chemotherapeutic agents or radiation, they undergo the premature or accelerated cellular senescence (ACS): terminal growtharrest and adopt morphologic and marker features suggestiveof cellular senescence. It was shown that few tumor cells can escape the senescence. Here, we test the hypothesis thatsenescence escapees have stem cell properties. Three cell lines, H460 (lung cancer), OVCAR3 (ovarian cancer) and MCF7 (breast cancer) were treated with a clinically relevant dose of adriamycin or cisplatin.Two weeks following induction of senescence , clonal outgrowthswere expanded and characterized in terms of senescence-associatedß-galactosidase activity. Propogated senescence escapee and untreated cultured cells were analyzed by flow cytometry using Hoechst 33342 for identification of a sub-population termed "side population: SP" that is thought to correspond to CSC. Concentration of SP cells in untreated cells did not exceed 0.01%, whereas in escapees SP increased up to 60%. Next, senescence escapee cells were analyzed for stem cell surface markers using immunohistochemistry and flow cytometry methods. Cultured senescence escapee cells expressed ABCG2, CD117, CD90 markers and the putative stem cell marker Oct-4. Moreover, propagated senescence H460 escapees gave rise to new tumors when as few as 10⁴ cells were injected into the SCID mice thus confirming their cancer CSC phenotype. Inflammation is a well known risk factor for tumor progression. To assess possible correlation between CSC population and inflammation during tumor formation we analyzed the multiple cytokines and growth factors secreted into the culture medium using xMAP technology from Luminex, Inc. We observed secretion of multiple cytokines by CSC cells. These cytokines may be responsible for pro-inflammatory effects during tumor progression. In addition, cytokines, such as VEGF, IL-6, IL-8 and others may induce neoangiogenesis and protect surrounding cells by inhibiting apoptosis and inducing cell proliferation. In this study, we retrospectively confirm that senescence escapee cells represent a tumorogenic stem cell like subpopulation within lung, breast and ovarian cancers.