Introduction
Basal-like breast cancer (BLBC) accounts for approximately 15-20% of breast cancers, and has the least favorable prognosis of all breast cancer subtypes. BLBC often occurs in women younger than 40 years and is associated with short time to metastasis and short overall survival compared with other subtypes of breast cancer [
1,
2]. Introduction of drugs targeting oncogenic signaling pathways may represent a new paradigm in the treatment of BLBC [
1,
3]. Basal-like breast cancer frequently exhibits the triple negative phenotype. In contrast to other breast cancer subtypes, these patients currently lack targeted treatment alternatives and would therefore benefit from the introduction of new, molecularly targeted drugs. However, introduction of targeted therapy will also depend on the development of diagnostic approaches to evaluate whether the relevant target is driving tumor progression.
For breast cancer, the presence of human epidermal growth factor receptor 2 (HER2) amplification predicts possible positive effects of injected neutralizing antibodies [
4]. Predicting efficacies of a targeted drug from DNA sequence variations have proven useful for treatment of lung cancers with epidermal growth factor receptor inhibitors [
5,
6]. However, predicting the activity in the phosphatidylinositol 3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) pathway based on DNA sequence alterations is complex. The activity in the pathway seems to depend on a number of alternative mechanisms, including amplification or activating mutations in
PIK3CA, loss of phosphatase and tensin homolog (PTEN) protein at a DNA, mRNA or protein level, or activating mutations/amplification in
AKT1/AKT2 [
7‐
10]. Owing to the number of different mechanisms that, directly or indirectly and at different levels, can lead to elevated PI3K pathway activity, development of methods that quantitatively report on signaling activity in the tumor tissue is tempting. Conventional immunohistochemistry using antibodies for active, phosphorylated Akt has been suggested, but this approach is limited by its low linear range and by the difficulty in introducing a second stain for normalizing purposes.
To accelerate the introduction of targeted drugs into clinical practice, identification of molecular biomarkers for early monitoring of response to therapy and development of resistance is required [
11,
12]. Assessment of tumor metabolism using magnetic resonance spectroscopy (MRS) is a promising approach for biomarker discovery, since the metabolic characteristics of cancer are inherently different from normal tissue and since oncogenic signaling regulates energy metabolism in cancer cells [
13,
14]. Identification of metabolic biomarkers is therefore an important step in the introduction of rational, personalized treatment of BLBC patients with drugs targeting oncogenic signaling.
Inhibitors targeting components of the PI3K pathway are a promising new class of drugs currently evaluated in various cancers. They are of particular interest in BLBC, because abnormal activity in the PI3K/Akt/mTOR signaling axis has been described both in preclinical models and in clinical cohorts in this breast cancer subtype [
8,
15‐
17]. Metabolic effects of PI3K inhibition in cancer have been studied
in vitro and
in vivo [
12]. However, data on metabolic effects in basal-like breast cancer are lacking, and the effect of PI3K inhibition on choline metabolism in breast cancer has not yet been studied in
in vivo models. Different subtypes of cancer have distinct metabolic profiles and the flux through metabolic pathways is in part governed by the oncogenic signaling.
We have therefore studied PI3K/mTOR/Akt pathway activity in basal-like and luminal-like breast cancer xenografts, and the effect of the pan-Akt inhibitor MK-2206 and the dual PI3K/mTOR inhibitor BEZ235 in these models in vivo. The response to treatment was evaluated both with respect to tumor volume, cellular proliferation and blockade of PI3K signaling. Metabolic changes in the tumor tissue were examined by ex vivo high-resolution magic angle spinning (HR MAS) MRS. The objectives of the study were to use a novel immunofluorescence imaging method to quantify the level of pAktser473 in tumor tissue sections, to determine whether inhibition of the PI3K signaling pathway caused anti-tumor effects in the basal-like xenograft model, and to identify metabolic biomarkers associated with response to treatment.
Materials and methods
Animal models
The MAS98.12 (basal-like) and MAS98.06 (luminal-like) breast cancer xenograft models have previously been established by orthotopic implantation of biopsy tissues from primary mammary carcinomas in severe combined immunodeficiency mice [
18].
For both xenograft models, animals were randomized into the following treatment groups (
n = 8 per group): vehicle control (0.2 ml/day), MK-2206 (120 mg/kg/day) and BEZ235 (50 mg/kg/day). MK-2206 (Selleck Chemicals, Houston, TX, USA) was dissolved in dimethyl sulfoxide and diluted in 30% Captisol
® (CyDex Pharmaceuticals, Inc., Lenexa, KS, USA) to a final concentration of 15 mg/ml. BEZ235 (Selleck Chemicals) was dissolved in
N-methyl-2-pyrrolidone and diluted in 30% Captisol
® to a final concentration of 6.5 mg/ml. Vehicle control solution consisted of dimethyl sulfoxide,
N-methyl-2-pyrrolidone and 30% Captisol
® (1:1:2). These dose levels have previously shown efficacy in murine xenograft models [
19,
20].
The drugs were administered by gavage for 3 consecutive days. Tumor volume was measured before and after treatment using external calipers (volume = πab2 / 6, where a and b represent the longest diameter and shortest diameter, respectively). After treatment, tumor tissue was harvested and preserved for histopathology (4% neutral buffered formalin) or snap frozen in N2(l) for metabolic profiling.
An additional batch of mice (n = 5 or 6 per group) was randomly assigned to treatment as described above when the tumor diameter reached approximately 5 mm and was treated with MK-2206 or BEZ235 for up to 26 days. The tumor volumes were measured regularly with calipers during the treatment period.
All procedures and experiments involving animals were approved by the National Animal Research Authority, and were carried out according to the European Convention for the Protection of Vertebrates used for Scientific Purposes.
Histopathology
Tumor tissue was fixed in 4% formaldehyde immediately after isolation from the animal and embedded in paraffin. Sections were cut at 4 µm and mounted on glass slides. Immunohistochemical staining for the mitosis marker phosphohistone H3 (PHH3) was carried out as previously described [
21]. Mitotic activity was counted in PHH3-stained sections according to Skaland and colleagues [
22], and was reported as the number of positive counts per 10 fields of view. Necrotic areas were avoided.
For analysis of the activity in the PI3K/Akt pathway, sections were co-stained with mouse anti-pan-Akt antibody (#2920; Cell Signaling Technology, Beverly, MA, USA) and rabbit anti-pAktser473 antibody (#4060; Cell Signaling Technology). Four different secondary antibodies were used to image binding of the primary antibodies. For confocal microscopy, anti-mouse conjugated with Alexa 488 and anti-rabbit conjugated with Alexa 555 (Invitrogen, Paisley, UK) were used. For near-infrared (NIR) immunofluorescence imaging, anti-rabbit conjugated with IR-680 dye and anti-mouse conjugated with IR-800 dye (Li-Cor Biosciences, Lincoln, NE, USA) were used. All secondary antibodies were added simultaneously in a 1:1 ratio to allow combined low-resolution quantifications using NIR fluorescent scanning and high resolution of regions of interest using confocal microscopy in the visible area of the light spectra. Negative control sections were prepared by staining with secondary antibodies only.
Near-infrared immunofluorescence imaging
Stained tissue sections (n = 4 in all treatment groups) were scanned on an Odyssey Infrared Imaging System (Li-Cor Biosciences) with a spatial resolution of 21 µm. The samples were scanned simultaneously to enable quantitative image analysis. The signals were recorded in separate channels for concurrent imaging of pAktser473 (700 nm) and total Akt (800 nm) levels. The images were exported as colorized 32-bit .tiff files and the signal intensity was quantified using ImageJ (National Institute of Health, Bethesda, MD, USA). Regions of interest enveloping the entire tumor area were defined and the mean signal intensity for each region of interest was determined. Compensation for autofluorescence and unspecific antibody binding was performed by subtraction of the mean signal from adjacent negative control sections. The signal intensity was compared across treatment groups using the Student's t test with the threshold for statistical significance defined as P ≤0.05. Confocal microscopy was carried out using an Axiovert microscope (Carl Zeiss MicroImaging Inc., Jena, Germany) with 20× and 63× magnification, and images were captured and analyzed using Zeiss LSM Meta and Zeiss LSM Image Examiner (Carl Zeiss MicroImaging Inc.).
Human breast cancer biopsies
To evaluate the feasibility of the NIR immunofluorescence imaging method in human tumor tissue, five paraffin-embedded specimens from patients with BLBC were retrieved from the Breast Cancer Subtypes research biobank, NTNU, which has been approved by the Regional Research Ethics Committee. The tumors were classified as BLBC using immunohistochemical and in situ hybridization methods as surrogates for gene expression profiling. On immunohistochemical stained tissue microarrays, the tumors were estrogen receptor negative (249R-16/SP1; Cell Marque, Rocklin, CA, USA) and progesterone receptor-negative (NCL-PGR 312-CE; Leica Biosystems, North Ryde, Australia) but were positive for cytokeratin 5 (Ncl-CK5-L-CE; Leica Biosystems) and/or epidermal growth factor receptor (K1494/2-18C9; Dako Denmark/Glostrup, Denmark) developed using pharmDx™ (Dako, Denmark). The tumors were also negative for HER2 using chromogenic in situ hybridization for the HER2 gene and the chromosome 17 centromere (HER2 CISH pharmDx™; Dako, Denmark) (gene:chromosome ratio <2.0).
For NIR fluorescence staining, the clinical samples were stained and imaged according to the protocol described above. The primary antibodies were omitted as a negative control of the immunostaining. The sections were stained, imaged and processed simultaneously and quantifications were performed using the Li-Cor software. After subtracting the signal intensity for the negative control in each channel, the mean intensity for the anti-pAktser473 labeling was divided by the signal intensity for the total Akt labeling. One of the biopsies contained both normal and cancerous tissue and allowed comparison of the pAktser473 signal in the different parts of the section.
Western blotting
Snap-frozen tumor samples were thawed and immediately lysed in a lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1% NP-40, 0.25% Triton X-100) with phosphatase inhibitor (Complete Lysis-M; Roche Diagnostics, Indianapolis, IN, USA) and a combination of phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St Louis, MO, USA). The protein concentration was determined in clear cell lysates and equal amounts of total protein (50 μg) were separated by SDS-PAGE. After immunoblotting, the membranes were developed using a mixture of the anti-pAktser473 and pan-Akt antibodies and were imaged after labeling with NIR fluorescent secondary antibodies. PTEN levels in the tumor lysates were detected using a C-terminal PTEN antibody (#18-0256; Invitrogen) and pAktthr308 detected by a monoclonal rabbit antibody (#2965; Cell Signaling Technology). The amount of β-actin (#ab6276; Abcam, Cambridge, UK) in the lysates was used as control of equal protein loading. Binding of the respective primary antibodies was detected using secondary antibodies labeled with NIR fluorescent dyes. The images from the Odyssey Infrared Imaging System were processed using the Li-Cor software and mounted using Canvas (ACD Systems International Inc, Seattle, WA, USA).
Frozen xenograft tissue was cut to fit into 30 µl disposable inserts (Bruker BioSpin, Ettlingen, Germany) filled with 3 µl PBS/D2O buffer containing trimethylsilylpropanoic acid as a chemical shift reference. The average weight of the tissue samples was 12 ± 3 mg. Samples were analyzed using a Bruker AVANCE DRX-600 spectrometer equipped with a 1H/13C HR MAS probe (Bruker BioSpin). Samples were spun at 5 kHz and the instrument temperature was maintained at 4°C for all experiments. A single-pulse experiment (zgpr; Bruker) was performed for all samples. The water resonance was suppressed using a presaturation delay of 3 seconds and an acquisition time of 3.40 seconds. A sweep width of 16 ppm was used for signal collection. Thirty-two free induction decays were acquired into 64k points.
A creatine reference solution (9.05 μmol/g) was analyzed under identical conditions for use as an external calibration standard. Post-processing of spectra included 0.3 Hz exponential line broadening and baseline correction using a fifth-order function.
Chemical shifts were calibrated to the trimethylsilylpropanoic acid at 0.0 ppm. Assignment of metabolite peaks was performed with reference to previously published data [
23]. The peak area of each metabolite was calculated by polynomial regression (PeakFit v 4.12; Systat Software Inc, Chicago, IL, USA). The correlation coefficient of the fit (
r2) for all spectra was ≥0.95. Concentration of each metabolite was calculated with reference to the recorded sample weight and the peak area of the creatine reference solution. Metabolite concentrations were compared across treatment groups using Student's
t test with the threshold for statistical significance defined as
P ≤0.05.
Discussion
In this study, the response to two PI3K inhibitors with different molecular targets was evaluated in two different breast cancer xenograft models. Combined NIR and confocal immunofluorescence imaging was used to evaluate the baseline level of PI3K signaling in the tumors and to determine the pharmacodynamic effects of drugs targeting the PI3K pathway. Ex vivo HR MAS MRS was used to identify metabolic biomarkers for response to therapy. Basal-like xenografts had significantly higher pAktser473 levels at baseline, but the phosphorylation was significantly reduced after treatment with BEZ235 and MK-2206. This response was accompanied by early changes in phospholipid and glucose metabolism, reflecting the long-term tumor growth delay caused by PI3K inhibition in this model.
The basal-like and luminal-like xenograft models are established from human primary breast carcinomas directly transplanted to immunodeficient mice. They represent breast cancer with poor (basal-like) and good (luminal-like) prognosis, and have retained the gene expression profile and morphology from the primary tumors [
18]. Since patient-derived xenografts represent the cellular heterogeneity of human breast cancer, they are considered to be of high clinical relevance [
27]. Previous studies have shown that the basal-like xenograft has a triple-negative phenotype, active angiogenesis and a rapid growth rate compared with the hormone-sensitive luminal-like xenograft model [
21]. Gene set enrichment analysis has suggested overactivity in the PI3K signaling pathway [
18].
Using a flat-bed NIR fluorescence imager, the levels of pAkt
ser473 could be assayed with minimal autofluorescence interference. Subtraction of the signal intensity from tissue sections representing the background levels has been shown to allow quantitative measurement of fluorescent probes with high accuracy [
28]. This method allowed semiquantitative analysis of the signal intensity originating from the specifically bound antibodies. This was confirmed by western blotting of the same tissue specimens. The NIR imaging method opens for automated, quantitative imaging of PI3K pathway activity in tumor samples. As for immunostaining in general, this NIR imaging approach is highly dependent on the quality of the antibodies and we have not yet identified an anti-pAkt
thr308 antibody that can be used for immunostaining.
The resolution of the images (21 μm) was sufficient to determine relatively fine spatial differences in signaling activity and the scan area is sufficiently large to scan a high number of tumor samples at the same time. However, the method depends on the presence of the phosphorous group at serine 473 in Akt that is responsible for kinase activity. This modification has previously been found labile and is lost over time from isolation of the tumor tissue until fixation or freezing [
29]. In the present study, the tumor samples were immediately divided into two parts: one-half was immediately snap-frozen in liquid nitrogen, and the other was immediately fixed. In addition, the fixative was injected into the tumors to avoid dephosphorylation of Akt deeper inside the tumor tissue. In the xenograft tissue, the use of anti-mouse secondary antibodies gave rise to a significant signal in tissue with a high content of murine stromal components. However, the feasibility study performed in human BLBC specimens demonstrated that both total Akt and pAkt
ser473 levels could be quantified with high specificity without contribution from unspecific binding of the secondary antibodies. In the clinical setting, the method could be useful for determining activity of Akt for stratification of patients to treatment with PI3K inhibitors. The finding that pAkt
ser473 is clearly elevated in only one in five cases of BLBC underscores the importance of subgrouping these patients. Using conventional immunohistochemistry, Lopez-Knowles and coworkers found an elevated level of pAkt
ser473 in 24% of 258 invasive breast cancer cases [
8]. Interestingly, there is a clear correlation between increased pAkt and loss of PTEN (but not with mutations in PIK3CA) in human tumors and breast cancer cell lines [
8,
24].
In vitro sensitivity for the small-molecule inhibitor LY294002 has been shown to correlate with loss of PTEN [
24]. Our finding that the pAkt
ser473-positive and PTEN-negative basal-like xenograft is sensitive towards both MK-2206 and BEZ235 is thus in line with previous
in vitro observations.
In this study, two different inhibitors of PI3K signaling were evaluated. MK-2206 is an allosteric pan-Akt inhibitor with broad preclinical anti-tumor activity [
19]. BEZ235 is a dual PI3K/mTOR inhibitor, which also has broad antiproliferative effects in a wide range of
in vitro and
in vivo cancer models [
20]. Both drugs are currently in phase I/phase II clinical trials [
30]. PIK3CA mutations, loss of PTEN and increased pAkt levels occur frequently in BLBC. PI3K inhibitors have therefore been suggested as a potentially suitable class of drugs for treatment of this patient group [
8,
17]. BLBC is strongly associated with the triple-negative phenotype, and because no molecularly targeted treatment options exist for this patient group, PI3K inhibitors have been suggested to be of particular benefit [
17]. However, several studies have failed to identify a correlation between PIK3CA mutations and response to PI3K inhibition. The importance of PTEN loss as a single predictive biomarker for response is also debatable [
31]. Owing to the complex relationships that determine response to treatment, identification of predictive biomarkers is difficult. Functional biomarkers such as pAkt
ser473, which more directly is linked to signal transduction activity, may therefore have higher predictive specificity. The current lack of predictive biomarkers for response to PI3K inhibitors calls for alternative stratification strategies. One approach is to identify biomarkers that are associated with changes in the cancer cells after initiation of therapy. Since oncogenic signaling directly regulates key metabolic pathways in cancer, identification of metabolic biomarkers for response to therapy could represent a promising alternative [
32].
In this study, the effect of PI3K inhibitors was markedly different in basal-like and luminal-like xenografts. In the luminal-like xenografts, no treatment-related effects on tumor volume, cellular proliferation or pAkt
ser473 levels were observed. This indicates that PI3K signaling is not the driving force of tumor growth in this model, which is in accordance with its estradiol addiction [
21] and the low baseline level of pAkt
ser473. The lack of pharmacodynamic response was reflected in the absence of metabolic changes seen in the HR MAS MRS data. In contrast, the basal-like xenograft had a high baseline activity in the PI3K pathway and responded strongly to treatment with both MK-2206 and BEZ235. A long-term delay in tumor growth was observed compared with vehicle-treated controls, concurrent with a reduction in mitotic activity. Furthermore, the levels of pAkt
ser473 were reduced to very low levels after 3 days of treatment with the PI3K inhibitors. This observation confirms that the drug indeed hits the target in this model, with concurrent effects on cellular proliferation and tumor metabolism. Both the PHH3 assay and the immunofluorescence imaging analysis suggested that BEZ235 had a stronger inhibitory effect than MK-2206 in basal-like xenografts, with a significant correlation between Akt
ser473 phosphorylation and mitotic activity. This differential pharmacodynamic effect between the drugs was also reflected in the metabolic profiles. MK-2206 caused increased PCho concentration and reduced lactate concentration. The magnitude of change in these metabolite concentrations was larger in BEZ235-treated xenografts. In addition, GPC and glucose concentrations were significantly increased. The HR MAS MRS data indicated that PCho, GPC, lactate and glucose are potential metabolic biomarkers for response to PI3K inhibitors. These findings are in accordance with previous studies demonstrating that phospholipid and glucose metabolism pathways contain potential metabolic biomarkers for response to molecularly targeted drugs [
11,
12].
An abnormally high rate of glucose uptake and utilization is seen in most cancers. In contrast to normal cells, cancer cells extract energy from glucose through glycolysis rather than oxidative phosphorylation, even under normoxic conditions [
33]. The low ATP yield is compensated by a high metabolic flux [
34]. This way, cancer cells can produce energy while conserving carbon for production of proteins and nucleotides. The glycolytic activity is governed by the cellular microenvironment, but is also regulated by oncogenic signaling [
35‐
37]. The regulatory effect of PI3K signaling on glucose metabolism is complex and multilayered, and includes both Akt-mediated induction of glucose transport and hexokinase activity as well as stimulation of glycolytic rate and lactate production mediated by HIF-1 and Myc [
14,
38]. Blockade of the PI3K/Akt/mTOR signaling axis has been shown to reduce glycolytic rate and lactate production in cancer
in vitro [
39,
40]. The high sensitivity and spectral resolution achieved in our study allowed determination of both glucose and lactate concentration
ex vivo, demonstrating that inhibition of PI3K signaling both increased glucose concentration and reduced lactate concentration. As the lactate concentration can be measured using
in vivo MRS, this biomarker is interesting with respect to preclinical therapy monitoring [
41]. In the clinical setting, it is difficult to measure the lactate concentration in breast cancer due to the interference from lipids in this tissue. Hyperpolarized
13C pyruvate may therefore be the best approach for clinical assessment of glucose metabolism using MRS [
42].
The oncogenic signaling pathways that regulate glucose metabolism have also been shown to regulate choline metabolism [
13,
43]. In breast cancer, abnormally high concentrations of choline metabolites are observed both
in vitro and
in vivo [
44]. High levels of PCho, GPC and choline were initially associated with a high turnover of cell membrane components in rapidly proliferating cells. Later studies indicated that the abnormal choline metabolism in fact is directly linked to malignant transformation [
45]. Although the mechanisms are not fully elucidated, accumulating evidence indicates that synthesis and hydrolysis of PtdCho generates mitogenic messenger molecules such as diacylglycerol, phosphatidic acid, arachidonic acid metabolites and PCho itself [
46‐
49]. Abnormal expression of both choline kinase and phospholipases has been associated with development of cancer [
44,
50]. It is therefore not surprising that interfering with this metabolic system is considered a valuable therapeutic approach. As an example, drugs inhibiting choline kinase have shown promising anti-tumoral effects in preclinical models and have now entered clinical trials [
44,
51,
52]. However, changes in choline metabolites in response to targeted therapy are poorly understood [
53].
From
in vitro studies, cancer aggressiveness has generally been assumed to be associated with high PCho concentration, and response to therapy assumed to be reflected in decreased concentrations of this metabolite [
54,
55]. However, it is increasingly recognized that GPC may be a relevant biomarker both in breast cancer and other cancers [
26,
56,
57]. Response to targeted therapy may also be associated with increased concentration of PCho and/or GPC [
40,
58,
59]. The use of choline metabolites as metabolic biomarkers for therapy monitoring therefore requires knowledge about both subtype-specific metabolic profiles and the changes associated with various targeted treatments in each distinct subtype. Choline metabolism may respond differentially to targeted treatment
in vitro and
in vivo, and this aspect must also be taken into account [
60,
61]. In this study, both PCho and GPC increased in basal-like xenografts after blockade of the PI3K signaling. Previous
in vitro studies of PI3K inhibitors in prostate cancer, colon cancer and breast cancer cell lines have suggested a reduced PCho concentration and an increased GPC concentration, whereas
in vivo studies in glioblastoma xenografts have suggested a decrease in tCho [
40,
62,
63]. However, we anticipate that the metabolic changes depend on the oncogenic signaling abnormalities seen in different cancer subtypes. The basal-like xenograft model has previously been shown to have a distinct metabolic phenotype, which also was found in a corresponding cohort of human breast cancer biopsies [
26]. Our data demonstrate a relationship between PI3K/Akt/mTOR signaling and choline metabolism. As the basal-like xenograft is driven by PI3K signaling, and as its distinct metabolic profile may be associated with this signaling activity, the increased PCho and GPC concentrations observed in this study might possibly be unique features of the MAS98.12 basal-like xenograft. Further studies in a larger panel of basal-like xenografts, representing various genetic backgrounds and metabolic profiles, are needed to elucidate these mechanisms and determine whether the metabolic effects are representative for basal-like breast cancer in general. From a clinical perspective, increased PCho and GPC concentration translates into an increase in tCho, which can be assessed
in vivo using
1H MRS. Alternatively,
in vivo 31P spectroscopy could be a possible approach for clinical applications, because this method allows spectral resolution of the phosphomonoesters and diesters PCho, phosphoethanolamine, GPC and glycerophosphoethanolamine in clinical magnetic resonance systems [
64].
This study indicates that PI3K inhibitors may be of value in treatment of basal-like breast cancer with high pAkt levels and/or PTEN loss. Early metabolic changes reflected the long-term inhibitory effect on tumor growth. Several studies have suggested that PI3K inhibitors must be combined with other targeted drugs or classical chemotherapy in order to induce apoptosis or kill the cancer cells, and this may also be the case in basal-like breast cancer [
65]. As choline metabolism generally is more complex and variable than glucose metabolism in terms of response to therapy, one could assume that assessment of the glycolytic rate and downstream metabolites of glucose may be the most universally applicable approach for identifying relevant metabolic biomarkers. On the contrary, choline metabolism is richer in information and could potentially provide prognostic value in addition to use in therapy monitoring. Finally, it is plausible that lack of a metabolic response, or return to the pretreatment metabolic profile, is associated with primary or acquired drug resistance.
Authors' contributions
SAM conceived and designed the study, conducted the in vivo experiments, contributed to data collection and interpretation, and led the writing of the manuscript. CGD developed the methodology for immunofluorescence imaging, and performed the immunofluorescence analysis and the immunoblotting. SSG performed the HR MAS MRS analysis and analyzed the data. AK conducted in vivo experiments, and collected and analyzed data. AB supervised the histopathology analysis. GMM and OE established the xenograft models, and participated in the study design and preparation of the manuscript. ISG contributed to study design and supervised data analysis. GB supervised the studies, performed confocal microscopy, interpreted the data and contributed to the preparation of the manuscript. All authors participated in drafting and critically revising the manuscript. All authors read and approved the final manuscript.