Characterization of breast cancers and therapy response by MRS and quantitative gene expression profiling in the choline pathway.

Tumor choline metabolites have potential for use as diagnostic indicators of breast cancer phenotype and can be non-invasively monitored in vivo by MRS. Extract studies have determined that the principle diagnostic component of these peaks is phosphocholine (PCho), the biosynthetic precursor to the membrane phospholipid, phosphatidylcholine (PtdCho). The ability to resolve and quantify PCho in vivo would improve the accuracy of this putative diagnostic tool. In addition, determining the biochemical mechanisms underlying these metabolic perturbations will improve the understanding of breast cancer and may suggest potential molecular targets for drug development. Reported herein is the in vivo resolution and quantification of PCho and glycerophosphocholine (GPC) in breast cancer xenografts in SCID mice via image-guided 31P MRS, localized to a single voxel. Tumor metabolites are also detected using ex vivo extracts and high-resolution NMR spectroscopy and are quantified in the metastatic tumor line, MDA-mb-231. Also reported is the quantification of cytosolic and lipid metabolites in breast cells of differing cancer phenotype, and the identification of metabolites that differ among these cell lines. In cell extracts, PCho and the PtdCho breakdown products, lysophosphatidylcholine, GPC and glycerol 3-phosphate, are all raised in breast cancer lines relative to an immortalized non-malignant line. These metabolic differences are in direct agreement with differences in expression of genes encoding enzymes in the choline metabolic pathway. Results of this study are consistent with previous studies, which have concluded that increased choline uptake, increased choline kinase activity, and increased phosholipase-mediated turnover of PtdCho contribute to the observed increase in PCho in breast cancer. In addition, this study presents evidence suggesting a specific role for phospholipase A2-mediated PtdCho catabolism. Gene expression changes following taxane therapy are also reported and are consistent with previously reported changes in choline metabolites after the same therapy in the same tumor model.

Response of choline metabolites to docetaxel therapy is quantified in vivo by localized (31)P MRS of human breast cancer xenografts and in vitro by high-resolution (31)P NMR spectroscopy of cell extracts.

Choline-containing compounds (CCCs) are elevated in breast cancer, and detected in vivo by the (1)H MRS total choline (tCho) resonance (3.25 ppm) and the (31)P MRS phosphomonoester (PME) resonance (3.8 ppm). Both the tCho and PME resonances decrease early after initiation of successful therapy. The single major component of these composite resonances, phosphocholine (PCho), also responds to therapy by decreasing. The ability to resolve and quantify PCho in vivo would thus increase the sensitivity of this biomarker for early detection of therapeutic response. Herein, the in vivo resolution and quantification of PCho is reported in human mouse xenograft tumors of the human breast cancer cell lines MCF-7 and MDA-mb-231. Significant decreases in tumor PCho are observed within 2 to 4 d posttreatment with the antimicrotubule drug, docetaxel. To determine whether these decreases are a general tumor response or an intracellular metabolic response, high-resolution NMR spectroscopy was performed on extracts of cells treated with docetaxel. Significant decreases in intracellular PCho and increases in glycerophosphocholine (GPC) were observed. These decreases are coincident with other tumor and cellular responses such as tumor growth delay (TGD), cell-cycle arrest, and modes of cell death such as mitotic catastrophe, necrosis, and apoptosis, with mitotic catastrophe predominating.