In addition, when Il6 was knocked-down in the niche cells (Supplementary Figure S5A), and such modified niche cells were co-cultured with TICs, a decrease mammosphere forming efficiency was observed (Supplementary Figure S5B). CD29HCD24L niche cells enhanced TICs tumor initiation potential shown by limiting dilution co-transplantation assay These in vitro assays were suggestive of a functional conversation between the TICs and niche cells. cells resulted in reduced tumorigenicity and increased tumor latency. These studies illustrate the non-cell autonomous properties and importance of cooperativity between tumor subpopulations. mammosphere assays using a syngeneic p53 null mouse mammary tumor model (20). Using FACS and microarray analysis, these studies also Evodiamine (Isoevodiamine) identified a unique group of cells in these tumors expressing mesenchymal-like cell markers. Factors such as cytokines, chemokines, growth factors and secretory Wnt proteins that have been reported to function as niche components in various tissues, were significantly increased within the mesenchymal-like tumor cell subpopulation. The stem cell niches characterized to date in the mouse use Wnt signaling, Notch signaling, IL6, or CXCL12 to regulate stem Rabbit polyclonal to Vang-like protein 1 cell function (21). All these factors are important autocrine or paracrine cues that affect diverse processes in normal tissue development and tumorigenesis. The functional conversation between niche cells and TICs, therefore, were investigated by comparing the properties of the combined mesenchymal-like and TIC subpopulations to the individual isolated subpopulations alone. Co- and transwell-cultures of putative niche cells with TICs in serum-free suspension mammosphere assays revealed that both the self-renewal ability and the proliferation potential of the TICs Evodiamine (Isoevodiamine) were enhanced in the presence of the niche cells or factors secreted from the niche cells. co-transplantation assays indicated that this niche cells enhanced the TIC tumor initiation potential when a limited number of TICs was present. Transduction of niche cells with lentiviral expressed short hairpin RNAs (shRNAs) directed against Wingless-type MMTV integration site family, member 2 (Wnt2) and Cxcl12 ligands differentially expressed within the niche population, resulted in reduced mammosphere frequency and decreased in vivo tumorigenic potential with increased latency. Knockdown of the receptors for these ligands in the TIC subpopulation also provided additional evidence of the importance of functional interactions between these tumor subpopulations. Results A Lin?CD29HCD24Low(L) subpopulation from p53 null mammary tumors displays a mesenchymal-like gene expression profile Cell surface markers CD29 and CD24 separated dissociated p53 null tumor cells into four subpopulations: CD29HCD24H, CD29HCD4L, CD29LCD24H, and CD29LCD24L. The lineage (Lin)?CD29HCD24H subpopulation displayed a significantly increased tumorigenic potential as compared to the other subpopulations (20). PCR genotyping performed using p53 primers (X7/X6.5 defining p53 wild-type, and X7/NEO19 defining p53 null) confirmed the p53 null status of all the individual subpopulations suggesting their non-host cell of origin when 30-cycle of PCR was performed (Supplementary Determine S1A, left). A small trace of p53 wild type product was detected when a 35-cycle of PCR was performed most Evodiamine (Isoevodiamine) likely due to infiltrating immune cells within the tumors (Supplementary Physique S1A, right). To determine whether there exist genomic copy-number differences among the four subpopulations, we performed high resolution mouse whole-genome bacterial artificial chromosome (BAC)-based comparative genomic hybridization (CGH) array which covers the entire mouse genome (22, 23). The syngeneic Balb/c mouse tail DNA was used as control. The chromosomal copy-number profiles performed around the four subpopulations of the p53 null tumor did not show significant variations (Supplementary Physique S1B). We have previously shown that this Lin?CD29HCD24L subpopulation identified in most of the heterogeneous p53 null tumors studied (including estrogen receptor positive (ER)+ and unfavorable (ER?) tumors, tumors expressing basal/myoepithelial markers K5/K14, as well as those only expressing luminal marker K8), was usually <5% of the total cell populace. The TIC subpopulation (i.e. Lin?CD29HCD24H) was able to generate tumors with as few as 10 Evodiamine (Isoevodiamine) cells. The Lin?CD29HCD24L subpopulation was also able to generate tumors, but only when more cells were transplanted indicating a reduced tumorigenic potential as compared to.