4.8.2. Breast

In the TNBC xenograft mouse models previously described (see 4.6 Diffusion-Weighted Imaging, [146]), MRS was associated with DWI to detect early tumor response after combination therapy with TRA-8 and carboplatin. The therapeutic efficacy was assessed by monitoring tumor volume, ADC changes, and lipid concentration through the fat–water ratio (FWR) MRS. The MRI acquisitions with DWI and MRS were performed before, during, and at the end of the therapeutic protocol. Significant changes in FWR required seven days for detection. Both ADC and FWR changes were confirmed as useful imaging biomarkers to evaluate the therapeutic efficacy, but ADC changes were detected earlier than FWR. This imaging protocol should be translated into clinical trials to improve outcomes by stopping ineffective therapies [146].

In the brain metastasis model previously described (see 4.6 Diffusion-Weighted Imaging, [147]), MRS was again added to DWI. A single-voxel MRS was used to analyze the metabolic patterns of the lesions in ten subjects. Only eight out of ten mice had brain metastases, and just two of them showed a field homogeneity good enough and a correct voxel position inside the lesion. However, when the lesions increased in size and infiltrated the brain parenchyma, the spectral changes indicated the replacement of the healthy tissue pattern with the tumor tissue pattern. The data proved a decrease in N-acetyl aspartate (NAA) as the earliest sign of metastasis growth, followed by a decrease in Cho and Cr levels. Those results suggest that this approach, in addition to DWI, may help in discriminating brain metastasis growth and the classification of distinct progression stages, and it might open the way to its use in the diagnosis and therapy monitoring [147].

Phosphorus (31P) MRS was used to quantify the levels of phosphorylated metabolites in breast cancer tissue. In particular, phosphocholine (PCho), glycerophosphocholine (GPC), phosphoethanolamine (PEtn), and glycerophosphoethanolamine (GPE) may be considered valuable biomarkers for the diagnosis and noninvasive monitoring of therapies both in preclinical studies and in the clinical phase. To this end, the metabolic response to the oral treatment with phosphatidylinositol-3-kinase/mammalian target of the rapamycin (PI3K/mTOR) inhibitor BEZ235 was evaluated in basal-like (MAS98.12) and luminal-like (MAS98.06) breast xenograft models. These models were established by direct implantation of primary human breast cancer tissues in the mammary fat pad of immunodeficient mice, and then serially transplanted in BalbC nu/nu mice. As a result, a significant increase in GPC and PCho was found in basal-like xenografts, whereas PEtn decreased. No significant changes were observed in phosphorylated metabolites in luminal-like xenografts, which did not respond to treatment with BEZ235. These data demonstrate the usefulness of 31P MRS in the metabolic profiling of breast cancer subtypes and the evaluation of the metabolic response to targeted anticancer drugs [156].

In a pilot study, the use of MRS was tested for monitoring breast cancer therapy. Indeed, such neoplastic tissue contains high levels of choline-containing compounds (tCho). The changes in tCho concentration were determined to predict the early clinical response to neoadjuvant chemotherapy in the first 24 h after initial treatment in patients with locally advanced breast cancer. Sixteen women, aged 18 to 80 years, with biopsy-confirmed, locally advanced breast cancer, were enrolled. Thirteen patients received combined doxorubicin hydrochloride and cyclophosphamide treatment that was administered on day one, with additional doses at 21-day intervals for a total of 64 days. Patients underwent MRS before treatment, within 24 h after the first dose, and then after the fourth dose. As a result, eight out of thirteen patients showed a significant correlation between changes in tCho concentration and lesion-size reduction. These patients had a lower tCho level within 24 h after the first dose compared to baseline and a further decrease in the tCho concentration after the fourth dose of the

combined therapy. The other five patients out of the thirteen showed no changes and had a baseline tCho concentration less than or equal to that measured within 24 h after the first dose. Therefore, MRS may be used to assess early response to neoadjuvant chemotherapy and to customize an effective regimen for individual patients [157].

#### 4.8.3. Prostate

In the prostate cancer model described (see 4.6 Diffusion-Weighted Imaging, [148]), MRS was applied to obtain metabolic maps of total choline and lactate/lipid ratio, and extracellular pH (pHe). Total choline and lactate/lipid levels were significantly higher in orthotopic compared with xenograft tumors, whereas the pHe maps showed a significantly lower pH in orthotopic tumors compared with subcutaneous tumors. As already stated, such an imaging approach may prove to be extremely helpful in studying the tumor microenvironment, demonstrating changes that might be linked the metastatization process, like in this case, total choline and a more acidic extracellular pH. Once validated, these techniques may also support the development of novel strategies to reduce metastatization [148].

#### *4.9. Chemical Exchange Saturation Transfer*

Chemical exchange saturation transfer (CEST) is an MRI method to detect low concentrations of metabolites for probing specific molecular and physiological events. The sensitivity of this approach is enhanced by the use of a set of new specific contrast agents, and endogenous as well as exogenous molecules can be used. Indeed, a variety of molecules have been demonstrated as potential contrast agents in this technique, including small diamagnetic molecules, paramagnetic ions complexes, liposomes, nanoparticles, and hyperpolarized gases [158–160]. The CEST applications in the clinic aim to monitor different metabolites, such as glycogen concentration (glycoCEST), glycosaminoglycans levels (gagCEST), or glutamate (gluCEST). Moreover, this is a valid imaging modality for detecting and monitoring the progression of tumors and assessing their responses to therapy by avoiding exposing the patient to radiation [159,161,162]. In preclinical studies, CEST has been used to measure the rate of metabolites' uptakes, such as glycogen and glucose, which are hallmarks of the tumor microenvironment. The evaluation of extracellular pH (acidoCEST), which is linked to the increase in lactic acid production after an increase in glycolysis, may be used for assessing tumor aggressiveness and early responses to treatments that inhibit glycolytic metabolism [161,163,164]. The CEST measurement of glucose metabolism (glucoCEST) in tumor mouse models allows studying the tumor microenvironment, and it may represent a potential replacement of the PET approach [159,164].

#### 4.9.1. Breast

The CEST has been proposed as a new molecular imaging approach to detect glucose or its analogs in the diagnosis of tumors. D-glucose and deoxy-D-glucose (2DG) were commonly employed to that end, but their toxicity at high concentrations precludes their clinical use and limits preclinical applications. A preliminary experiment was conducted to examine the validity of 3-O-methyl-D-glucose (3OMG) as a nontoxic alternative, which has been demonstrated to be able to detect tumors in several models of murine and human breast cancer. Moreover, this method was compared with glucoCEST and [18F]-FDG PET on the same animals. Orthotropic tumors were induced in mice by injecting human MDA-MB-231 or MCF7 cells. The CEST MRI sequences were performed on mice before and after the administration (intravenous, intraperitoneal, or oral) of 3OM. The same animals were then injected with [18F]-FDG for PET imaging, and D-glucose for glucoCEST after a specific time. The results showed that the CEST MRI following the administration of 3OMG produced patterns that reflected the metabolic activity of tumors and clearly distinguished them from other body districts. A marked 3OMG-CEST MRI contrast was obtained, and the most aggressive breast cancer models produced the highest CEST contrast. The contrast reached its maximum at 20 min post administration and lasted for more than one hour, without any difference in effect levels or timing between the three routes of administration. The 3OMG CEST method compared to the glucoCEST showed a higher CEST contrast than D-glucose. Moreover, a good correlation was found between 3OMG CEST contrast and FDG uptake, providing clear validation of this technique. Therefore, the validation of the 3OMG-CEST MRI method in the clinic would offer significant advantages for evaluation, detection, and monitoring of tumors' progressions, and assessing their responses to therapy, avoiding radiation exposure [161].

#### 4.9.2. Prostate

The CEST approach, using amide proton transfer (APT) MR imaging, was used in a preliminary study to localize prostate cancer better and to detect the difference in cancer aggressiveness, discriminating between cancerous and non-cancerous tissues. The APT MRI does not require the injection of a contrast agent since it uses endogenous amide protons in tissue, which allows detecting micromolar concentrations of mobile proteins with high sensitivity. Therefore, the applicability of this imaging modality to the detection of prostate cancer was based on the high rate of tumor cell proliferation and on the cellular density of this tumor, which leads to high levels of mobile proteins. In this study, twelve patients with biopsy-proven prostate cancer scheduled for prostatectomy were enrolled and underwent T2 and APT MRI acquisitions. The APT ratio in the tumor zone was significantly higher than that in the benign regions of the peripheral zone; hence, distinguishing them. Such results were confirmed by both the T2-weighted imaging and histopathological findings. The CEST APT imaging technique may, thus, represent a potential approach to detecting and discriminating between low and high-grade prostate cancer, and it might be more specific compared with DCE or DWI sequences [164].
