Cell therapy with TRIL-armed, genetically engineered or phenotypically redirected, effectors
Progetto Aim of this Unit is to provide data of tumoricidal activity of mTRAIL useful for the design of clinical studies. It has been reported that breast carcinoma cells belonging to different molecular subtypes presented differential sensitivity to sTRAIL-mediated apoptosis (Keane MM Cancer Res 1999, Rahman et al., Breast Cancer Res. Treat, 2009), therefore a differential tumoricidal activity of mTRAIL on different breast cancer cells is expected. Specifically we will:
- analyze lines (BT20, BT474, BT549, HBL100, HCC1937, HCC70, MCF10A, MCF7, MDAMB157, MDAMB175VII, MDAMB231, MDAMB361, MDAMB435, MDAMB436, MDAMB453, MDAMB468, SKBR3, T47D, ZR751) representing the three major subgroups of breast cancer: ER positive, HER2 amplified and triple-negative breast carcinomas, for sensitivity to mTRAIL in vitro by co-culturing tumor cells with CD34 positive cells infected with mTRAIL. Tumor cell death will be evaluated at different effector: target ratio by annexin V-propidium iodide double staining as previously described (Carlo-Stella Hum Gene Ther, 2006). (month 1-9)
- analyze in animal models the capability of CD34 positive cells infected with mTRAIL to inhibit the tumor growth. Based on in vitro data concerning the sensitivity to CD34-TRAIL + cells, sensitive and resistant cell lines will be selected, xenotransplanted in immunocompromised mice (NOD/SCID) and treated with CD34-TRAIL + cells or CD34 mock starting at early (3 days after injection of tumor cells) or advanced (when tumors reach a volume of about 0.5 mm3) stage. mTRAIL anti-tumor activity will be monitored by measuring tumor diameters. (month 9-18)
- since vimentin expression levels have been found predictors of sTRAIL sensitivity (Raham Breast Cancer Res Treat, 2009) the correlation between mTRAIL sensitivity in vitro and in vivo, and the expression of vimentin mRNA and/or protein level will be also investigated. (month 9-18). If vimentin results associated with efficacy of CD34-TRAIL + cells, this marker will be useful to select patients for anti-mTRAIL treatment. Furthermore, since vimentin-positive breast cancer cells have features consistent with putative breast cancer stem cells, breast carcinoma cell lines treated in vitro with mTRAIL will be tested for their capability to form mammospheres in vitro as well as to grow when xenotransplanted in immunodeficient mice. Once more, the percentage of cells expressing stemness markers, i.e. CD44 and ALDH, will be investigated in breast carcinoma cell lines of different molecular subtypes before and after mTRAIL treatment. (month 18-30)
- on the contrary, if no association between vimentin and anti- mTRAIL activity is found, the genes most highly correlated with mTRAIL sensitivity will be defined using the published microarray data of cell lines (Neve RM et al, Cancer cell 2006). At the same time, xenografted tumors, defined as sensitive or resistant by above in vivo experiments, will be profiled on an Illumina platform and correlation of gene expression and mTRAL sensitivity evaluated. All analyses will be performed using BRB array tools software. (month 12-15) Proteins encoded by genes significantly correlated with mTRAIL sensitivity will be analyzed by immunohistochemistry on cell lines and tumor xenografts, to select biomarkers with predictive value for mTRAIL response in patients with breast cancer. (month 24-36)