Differential expression study in heterogeneous tumoral cell clones

Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic alterations. This results in phenotypic and molecular heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed at dissecting the molecular mechanisms underlying the cooperation between different clones. We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allows tracking of multiple clones by colour: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones in vitro revealed clear differences at genetic levels, that induce differences in growth rate, morphology and cytokine expression among them. In vivo, all clonal cell lines were able to form tumors, however an injection of an equal mix of clones, led to the formation of tumors where mKO E10 was almost depleted. Additionally, the mKO E10 clonal cell line showed a significant deficiency in the formation of lung metastasis. These results confirm that even in stable cell lines heterogeneity is present. In vitro the complementation of growth medium with medium or exosomes from either parental or clonal cell lines, increased the growth rate of the other clones. Co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration, respectively. These findings support a model where the interplay of clones confers aggressiveness and may allow to identify factors involved in cellular communication which can be involved in clonal cooperation and be new targets preventing tumor progression. Tumoral cell line has been labeled with a plasmid harboring a fluorescent protein gene (transposon). Cells has been sorted to single cell and cultured again (clones). We have observed different properties in each clone when they are injected in mice. We are interested in study if different gene expression among the clones, is the responsible of the heterogeneity in the properties. We used microarray to study different gene expression among clones. 1.5 million cells were seeded in 100x17mm-dish in triplicate. After 72 hours cells were trypsinized and centrifuged. RNA was extracted using the PureLink™ RNA Mini Kit (ThermoFisher).