Increased levels of choline metabolites are an early marker of docetaxel treatment response in BRCA1-mutated mouse mammary tumors: an assessment by ex vivo proton magnetic resonance spectroscopy

Abstract

Background: Docetaxel is one of the most frequently used drugs to treat breast cancer. However, resistance or incomplete response to docetaxel is a major challenge. The aim of this study was to utilize MR metabolomics to identify potential biomarkers of docetaxel resistance in a mouse model for BRCA1-mutated breast cancer.

Methodology: High resolution magic angle spinning (HRMAS) (1)H MR spectroscopy was performed on tissue samples obtained from docetaxel-sensitive or -resistant BRCA1-mutated mammary tumors in mice. Measurements were performed on samples obtained before treatment and at 1-2, 3-5 and 6-7 days after a 25 mg/kg dose of docetaxel. The MR spectra were analyzed by multivariate analysis, followed by analysis of the signals of individual compounds by peak fitting and integration with normalization to the integral of the creatine signal and of all signals between 2.9 and 3.6 ppm.

Results: The HRMAS spectra revealed significant metabolic differences between sensitive and resistant tissue samples. In particular choline metabolites were higher in resistant tumors by more than 50% with respect to creatine and by more than 30% with respect to all signals between 2.9 and 3.6 ppm. Shortly after treatment (1-2 days) the normalized choline metabolite levels were significantly increased by more than 30% in the sensitive group coinciding with the time of highest apoptotic activity induced by docetaxel. Thereafter, choline metabolites in these tumors returned towards pre-treatment levels. No change in choline compounds was observed in the resistant tumors over the whole time of investigation.

Conclusions: Relative tissue concentrations of choline compounds are higher in docetaxel resistant than in sensitive BRCA1-mutated mouse mammary tumors, but in the first days after docetaxel treatment only in the sensitive tumors an increase of these compounds is observed. Thus both pre- and post-treatment tissue levels of choline compounds have potential to predict response to docetaxel treatment.

Figure 1

¹ H HRMAS spectra and Principal Component Analysis (PCA) of control tumor samples. A: The mean spectra of resistant tumor (red) show increased choline compounds compared to those of sensitive tumors (blue). B: Biplot [42] showing PCA analysis of HRMAS spectra. Docetaxel sensitive samples are shown with black triangle and resistant samples with squares. Arrows are drawn based on the loading plots to show the important metabolites responsible for the demarcation between the groups. Resistant samples have higher levels of GPC, PCho and Choline.

Figure 2

Choline compound over creatine signal ratios (A-C) and normalized choline compound integrals (D-F) of docetaxel resistant and sensitive tumor tissue samples pre- and post-treatment. Docetaxel resistant and sensitive tumor samples were monitored pretreatment at day 0 and after treatment at time points 1-2, 3-5 and 6-7 days. Statistical significant differences are indicated with an asterisk (*p < 0.05, **p < 0.01). The number of samples of resistant and sensitive tumor tissue for the different time points were: [7,5], [7,5], [5,5] and [4,4].

Figure 3

Principal Component Analysis (PCA) biplots [ 42 ] of the sensitive (A) and resistant (B) strains of mouse models. Plots show the differences in the metabolic profiles depending on the number of days after the administration of docetaxel. The sensitive strain shows distinct clusters based on the number of days after treatment (A) while the resistant strain does not (B) show such clusters.