J Natl Cancer Inst. BMC-2147 tumorgrafts by inhibition of the Notch pathway. GSI enhanced the efficacy of docetaxel in preclinical studies. In the clinical trial, 30 patients with advanced breast malignancy were Betamethasone valerate (Betnovate, Celestone) treated with escalating doses of MK-0752 plus docetaxel. Clinically meaningful doses of both drugs were possible, with manageable toxicity and preliminary evidence of efficacy. A decrease in CD44+/CD24?, ALDH+, and MSFE were observed in tumors of patients undergoing serial biopsies. Conclusions These preclinical data demonstrate that pharmacological inhibition of the Notch pathway can reduce BCSCs in breast tumorgraft models. The clinical trial demonstrates feasibility of combination GSI and chemotherapy, and together these results encourage further Betamethasone valerate (Betnovate, Celestone) study of Notch pathway inhibitors in combination with chemotherapy in breast malignancy. by determining the ability of a single cell to generate a differentiated tissue . In breast cancer models, BCSCs can be identified by their ability to form non-adherent mammospheres in serum-free media (MSFE), and to initiate tumors upon re-transplantation. Although the MSFE and re-transplantation approaches to stem cell identification are possible to execute in a preclinical setting, these are impractical to carry out in most clinical situations. Breast malignancy stem cells have been characterized by the cell surface marker phenotype (CD44+/CD24?/low)  and aldehyde dehydrogenase activity (ALDH+) [23, 24], which identifies these assays as potential surrogate biomarkers for BCSC directed therapies in clinical applications. In summary, although previous studies have examined the effect of GSI on BCSCs and in cell lines, none have translated this work to clinical studies. In the studies reported herein, we performed a Phase I clinical trial to establish a safe and potentially efficacious combination of a GSI in combination with docetaxel chemotherapy in patients with advanced breast cancer. We tested GSI, docetaxel, and combination therapy modeled after the clinical trial regimen on mice bearing human tumorgrafts in order to evaluate the effects of treatment on tumor volume and/or BCSCs as determined by MSFE, re-transplantation, and the surrogate markers ALDH+ and CD44+/CD24?. In addition, we obtained serial biopsies on a subset of clinical trial participants in order to preliminarily evaluate the effect of GSI plus docetaxel therapy on BCSCs using ALDH+ Betamethasone valerate (Betnovate, Celestone) and CD44+/CD24? in clinical samples. MATERIALS AND METHODS Preclinical Studies For preclinical evaluation of BCSC inhibitors, the Chang laboratory in collaboration with Michael T. Lewis has developed stable breast cancer-in-mice xenograft models by transplanting human breast malignancy tumor biopsies into the mammary gland excess fat pad of immune deficient mice (herein referred Nkx1-2 to as tumorgrafts). To evaluate the effectiveness of stem cell targeted brokers in altering the tumorigenic BCSC populace, the mice Betamethasone valerate (Betnovate, Celestone) are treated with the brokers and the tumor subsequently excised for rigorous evaluation in BCSC assays. These BCSC assays include: 1) flow cytometric analysis of BCSC cell-surface markers and aldehyde dehydrogenase activity (CD44+/CD24? and ALDH+, respectively); 2) mammosphere forming efficiency (MSFE); and 3) re-transplantation to measure the presence of tumor-initiating cells (TICs). Preparation of Tumorgrafts All animal protocols were reviewed and approved by the Animal Protocol Review Committee at Baylor College of Medicine or The Methodist Hospital Research Institute. Tumorgrafts were initially generated by transplantation of patient breast malignancy tumor biopsies or a fragment of surgical specimens into the cleared fat-pad of SCID/Beige mice (Harlan Laboratories, Indiana, IN, USA) as previously described [20, 21, 25]. As previously described, MC1 human tumors were originally derived from a pleural effusion and are estrogen and progesterone receptor unfavorable and HER-2 unfavorable [22, 24]. BCM-2147 breast tumorgrafts were generated by transplantation of estrogen and progesterone receptor unfavorable and HER-2 unfavorable human breast tumor biopsy tissue into the cleared fat-pad of SCID/Beige mice. Xenografted tumors were maintained as tumor lines by serial passage of tumor tissue into the cleared fat-pad of SCID/Beige mice, without intervening culture. Currently thirty-eight stable tumor lines representing twenty-eight impartial patients have been generated and rigorously characterized for quality control, including STR analysis to document.
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