The quality of each library was evaluated with the Agilent bioanalyzer high sensitivity assay, and quantified by qPCR. The libraries were pooled together at 10 nM based on the qPCR results, and then the pool was quantified again by qPCR. The pooled library was sequenced in one lane of a HiSeq2000 paired end 100bp flow cell. This allowed us to detect which transcripts are differentially expressed among these types of CR4 cells, thus C. We have found that small CR4 cells grown either as separate holoclones adherent to type I collagen or as 3D floating spheroids possess a large number of differentially expressed genes in comparison to the bulk tumor cells grown under standard culture conditions. Thus, in adherent small CR4 cells and 3D spheroids, 357 and 365 genes, respectively, were overexpressed compared to the CR4 long tumor cells. Among these genes, 287 were commonly upregulated, which means that both culture conditions allow for maintenance of the CR4 small cells at stemness state. It is largely accepted now that effective anticancer therapies should be focused not only on the bulk mass of the tumor, but most importantly, on functionally significant cells possessing high tumor-initiating Ferrostatin-1 capacity and high resistance to treatment, i.e. CICs. It is also well established that human Chloroquine Phosphate tumors are highly heterogeneous both intra-and interindividually. Given the fact that the majority of established long-term passaged cancer cell lines do not reflect either complexity, or individual features of the parental tumors, it is conceivable that modern cancer research and anti-cancer drug development should be based on novel in vitro and in vivo systems allowing for physiologically and clinically more relevant modeling and more objective evaluation of drug efficacy. Molecular, biochemical, genomic and proteomic characterization of human tumors aims to discover novel targets that potentially can cure cancer. But in reality, clinically and physiologically relevant targets are covered by noise from the dominating cell populations in unselected, bulk tumor masses, often contaminated with different levels of non-tumor cells, including normal fibroblasts and CAFs, endothelial cells, pericytes, inflammatory and other cells. Therefore, the selection of clinically and physiologically appropriate molecular targets should be fundamentally based on the selection of appropriate, clinically and physiologically significant cells, even if they represent a very minor population within the individual tumor.