Whereas TIC have been identified in a wide variety of human and mouse malignancies 
, little is known about their underlying biology, and few compounds have been identified that selectively target these cells 
. Targeting TICs is an important cancer therapeutic objective as these cells are resistant to current cancer therapies, including chemo- and radiation-therapy 
. Hence whereas standard therapies result in tumor shrinkage, they may fail to provide long lasting cures because rare TIC survive and seed tumor relapse.
The use of genomic and drug discovery technologies, such as global gene expression profiling and high-throughput screening would greatly aid the search for anti TIC therapies. However, implementation of these methodologies has been confounded by a lack of suitable human BTIC-enriched populations for study. Typically BTIC represent an exceedingly small percentage (~0.01%) of the total tumor cell population 
, and even in the most highly enriched populations, BTIC rarely achieve more than 1–2% purity 
. Moreover, means of propagating BTIC-enriched tumor cell populations in vitro
have not been described. To overcome these obstacles, we have studied BTICs from mouse mammary tumors of breast cancer prone transgenic models because they comprise a high BTIC frequency, averaging ~30% in most tumors and companion tumorspheres 
To determine whether the Wnt/Beta-catenin pathway is required for the survival and/or self-renewal of BTIC, we employed three small-molecular weight tool compounds, PKF118–310, PKF115–584 and CGP049090, which were originally identified in a high throughput screen to identify those that abrogate the binding of β-catenin to Tcf4 in vitro
. Follow up analyses of these compounds revealed their capacity to: block β-catenin binding to GST-Tcf4 in vivo
; reduce expression of a Wnt/β-catenin luciferase reporter; restore the β-catenin induced axis duplication of Xenopus
embryos when co-injected with β-catenin; inhibit expression of the Wnt target genes Myc
; and retard the proliferation of colon cancer cell lines known to display hyperactive Wnt signaling in vitro
. Collectively, the latter findings suggest that PKF118–310, PKF115–584 and CGP049090 reduce Wnt/β-catenin signaling leading to the inhibition of cancer cell line proliferation in vitro
. To the best of our knowledge the effect of these compounds on breast tumorigenesis has not previously been assessed.
Several studies have implicated Wnt/β-catenin signaling in both the pathogenesis of breast cancer and the regulation of normal mammary epithelial stem cell processes 
. Our data suggests that the Wnt/β-catenin pathway is hyperactive in BTIC compared to normal mammary epithelial stem/progenitor cells or to their more differentiated descendants. We made use of the small molecule inhibitors to investigate the consequences of inhibiting Wnt/β-catenin signaling in both breast tumor cells and normal mammary epithelial stem/progenitor cell populations. Due to the limited availability of the natural compounds PKF115–584 and CGP049090, we focused primarily on the use of PKF118–310, which can be chemically synthesized. Our initial experiments showed that each of the 3 compounds inhibited sphere and colony formation by primary tumor cells and primary mammary epithelial cells, as well as by established tumorsphere- and mammosphere-derived cells without any apparent selectivity. However, both PKF115–584 and CGP049090 displayed somewhat increased selectivity of between 6–7 fold (IC50MMS
, ) for primary tumor cells over primary mammary epithelial cells in sphere forming assays compared to PKF118–310 (2–3 fold selectivity), indicating that further investigation of the potential selectivity these compounds is warranted.
We did not observe any significant selectivity of PKF118–310 for either the survival and/or self-renewal of tumorsphere-initiating cells compared to mammosphere-initiating cells in primary sphere-forming assays (). However, a single exposure of primary tumor cells to PKF118–310 in a primary sphere-forming assay was sufficient to block subsequent secondary sphere formation in the absence of the compound. By contrast, mammary epithelial cells exposed to PKF118–310 were not impaired in their capacity to form secondary spheres, suggesting that the effect of PKF118–310 on secondary sphere formation is specific to BTICs. Taken at face value these observations suggest that PKF118–310 inhibited tumorsphere formation by an irreversible mechanism, whereas the compound acted reversibly to affect mammosphere formation. Inhibition of Wnt/Beta-catenin signaling by PKF118–310 may be cytotoxic for tumorsphere-initiating cells, perhaps because they are addicted this pathway, whereas pathway inhibition may be cytostatic for mammosphere-initiating cells.
Whereas sphere formation is a convenient and relatively rapid surrogate in vitro
assay for stem/progenitor and TIC activity, the nature of sphere-forming cells is controversial and consequently we employed additional means to identity the tumor cells that might be targeted by PKF118–310 
. To this end we transplanted primary tumor cell populations that had been incubated with PKF118–310 under the same conditions as had been used in primary sphere-forming assays and thereafter measured the capacity of the remaining viable tumor cells to seed tumor growth after transplantation into syngeneic FVB/N female mice. These transplantation assays directly assess BTIC frequency and demonstrated that PKF118–310 targeted these cells as manifested by a concentration-dependent reduction in tumor incidence in recipient mice resulting from transplant of the compound-treated tumor cells.
Administration of PKF118–310 to tumor-bearing mice blocked tumor growth during the 10-day treatment period, an interval during which the tumors expanded by 2–3 fold in tumor-bearing mice that were administered the vehicle. Histological analyses of tumor sections from mice administered PKF118–310 revealed loss of tumor architecture manifested as reduced cellularity and phenotypic features associated with reduced tumor grade. Whereas no evidence of apoptotic cell death or altered expression of differentiation markers was evident in tumor sections from mice administered the compound (data not shown), the frequency of Ki67-positive cells, a biomarker of cell proliferation, was markedly reduced. Importantly, tumor cells harvested from mice exposed to PKF118–300 formed tumor grafts in only 3 of 20 mice transplanted with these cells some 2 weeks after tumors had already formed in 20/20 mice transplanted with vehicle-treated tumor cells. Our transplantation assay can detect single tumor cells in the bulk tumor cell population, which on average comprise ~30% functional BTIC as established by limiting dilution cell transplantation assays 
. Hence, our findings suggest that the frequency of BTIC comprising the tumors was dramatically reduced by inhibiting Wnt/Beta-catenin signaling in tumors 
. Taken together these multiple lines of investigation suggest that antagonists of Wnt/Beta-catenin signaling target BTIC and provide proof-of-principle that eradicating these cells leads to durable breast cancer remission.