*3.4. High-Frequency Ultrasonography (HFUS)*

Compared to other molecular imaging modalities, HFUS represents a noninvasive, more cost-effective modality, with excellent temporal and spatial resolutions (up to 30 μm axial resolution and 70 μm lateral resolution). It shows the ability to obtain real-time anatomo-functional data rapidly. Moreover, this modality can be implemented by using microbubbles as a molecular-target contrast agent to enhance image quality and specificity. Ultrasound imaging is clinically used for routine screening examinations on breast, abdomen, neck, and other body districts, as well as for therapy monitoring. In this setting, over the last few years, the sensitivities and specificities of ultrasound devices to detect microbubbles have progressively improved. Indeed, beyond the frequent use of color or power dopplers, non-targeted microbubbles are used as intravascular contrast agents improving the detection and characterization of cancerous lesions, inflammatory processes, and cardiovascular pathologies [83,84,88]. In preclinical investigations, HFUS can be applied to monitor tumor growth and vasculature development, and in combination with contrast-enhanced microbubbles agents (CEMAs), is used to assess tumor angiogenesis, inflammation, and therapeutic effects. For example, the vascular endothelial growth factor receptor 2 (VEGFR2), which is a molecular marker of angiogenesis and is overexpressed on tumor vascular endothelial cells, is widely used in preclinical cancer research as a marker of therapy responsiveness [88–92]. Therefore, this modality could help enhance the translation of antiangiogenic agents and contribute positively to human patients' treatments [93].

#### *3.5. Magnetic Resonance Imaging (MRI)*

TheMRI is a noninvasive imagingmodality without ionizing radiation, which providesmorphological images with excellent soft-tissue contrast and high spatial resolution. The MRI is one technique with multiple possible outcomes; indeed, depending on the sequences, it allows studying, other than the merely displaying the anatomy, of various aspects of the same lesion. In particular, diffusion of water molecules can be studied with diffusion-weighted sequences (DWI); spectroscopy allows detecting and quantifying small amounts of known molecules; angiographic sequences (like time-of-flight or phase contrast angiography) allow evaluating large blood vessels, even without the need of contrast agents, and so on [17,82–84]. The MRI has also proven to be useful in preclinical research, allowing the evaluation of biological processes at the molecular level in in vivo rodent models of diseases. The MRI needs complete immobility of the subject since even the respiratory and cardiac motions can induce substantial artifacts, reducing the quality and the information available from images; hence, requiring the use of respiratory gating to minimize the former effect [93].

The use of anesthesia may minimize animal motion, but some anesthetic agents may alter animal physiology, and thereby, potentially affect the biological processes under investigation [94]. Pre-anesthesia fasting times, the anesthetics, and their dose, may all impact biological processes. Pre-anesthetic fasting in mice is generally considered unnecessary, and prolonged fasting can cause hypoglycemia. However, in imaging studies, a full stomach may interfere with adjacent structures. From the metabolic point of view, fasting may display profound effects on glucose metabolism, and hence, for example, it may significantly influence the results of FDG-PET studies. Body temperature should be monitored and kept within thermoneutrality, since anesthesia induces more or less marked hypothermia, which can also negatively affect the quality of some molecular imaging procedures. The choice of the anesthetic protocol is paramount for particular MRI sequences as well. Indeed, anesthetics may display a significant impact on vascular tone and hemodynamics, masking or altering critical experimentally-related results. Nowadays, great drug selection is available, and the choice, as mentioned, should be in accordance to the specific experimental aims. Nonetheless, an ideal anesthetic agent should be easy to administer, produce a rapid and adequate immobilization, have limited side effects, and be reversible and safe for the animals [20,94].

On the other hand, significant technical challenges exist in the transfer of preclinical MRI sequences to the clinic, due to the higher strength of the magnetic fields used in preclinical settings. However, preclinical systems often lack the wide selection of coils available for medical use, and hence, they require the development of specific coils. Therefore, preclinical MRI is essential for the development and validation of novel techniques, but these technical and biological challenges can partly hinder translation [93,95].

#### *3.6. Multimodality Imaging*

The combination of different imaging methods, the so-called multimodal imaging approach, offers the opportunity to correlate the most advantageous capabilities of each method and provide complementary information while compensating for their limitations. In preclinical research, all possible combinations of molecular imaging techniques are usually employed. In human medicine, the first example is the PET/CT combination, which offers highly specific functional information, with an excellent anatomic co-registration. Such a system is used in preclinical imaging as well. Another fascinating combination is the PET/MRI, which has been used in the last few years, in both clinical and preclinical settings, even if with some critical technical drawbacks. In any case, the multimodal approach should offer an excellent anatomical resolution with functional information, allowing for a multiparametric evaluation within a single study [8,9,83,96–100].

#### **4. Magnetic Resonance Imaging Sequences in the Translational Context**

The MRI is one of the primary preclinical and clinical imaging modalities, ideal for non-invasive longitudinal studies and useful for monitoring multiple parameters. In the oncological field, MRI is suitable for the definition of lesions with a high spatial resolution and anatomical detail, allowing the identification of primary tumors and metastatic disease. Moreover, it is also useful for the evaluation of quantitative characteristics that provide physiological, biochemical, and molecular details able to predict the biological behavior of cancer [9,32,101]. Various MRI sequences have been successfully developed by preclinical research and translated into clinical applications, as described below. Indeed, the preclinical MRI scanners can be easily considered a translational platform, since both preclinical and clinical scanners work with the same hardware settings and with almost identical sequences [9,102]. However, it is always necessary to establish the reproducibility of the technique chosen before being introduced in the clinic.

As mentioned, the MRI offers the ability to evaluate numerous tumors' biological properties, such as angiogenesis, perfusion, pH, hypoxia, metabolism, and macromolecular content. Such biological features are used as indicators in preclinical research to monitor a cancer's response to therapy [93,95]. Moreover, the complementary nature of the information accessible, applying different MRI sequences in the same subject, can undoubtedly help cancer research; clinical management as well [101].

#### *4.1. T1 and T2 Weighted Sequences*

Image contrast in MRI acquisition is determined by tissue properties, sequence type, and sequence parameters. Several relaxation constants are used to describe the decay and recovery of the MR signal; in particular, the two most commonly used parameters are the T1 and T2, the so-called spin-lattice relaxation time (T1) and spin-spin relaxation time (T2), which are both weighted. Based on the T1 and T2 weighting, many sequences have been developed [103–105]. In clinical applications, the T1 and T2-weighted sequences are the standard for anatomical imaging, and they are useful in the oncology field, for the detection and evaluation of macroscopic changes in the lesion, and staging. In addition, exogenous contrast agents, such as gadolinium, manganese and iron-based contrast agents, can be applied to enhance the contrast between tissues or organs [104,106]. In preclinical studies, T1 and T2-weighted sequences are used for anatomical acquisition, with the employment of contrast agents as well [107].

#### 4.1.1. Thyroid

In thyroid cancer, MRI texture analysis derived from conventional sequences reflects different histopathologic features and represents a possible association that can be used as a prognostic biomarker. Thirteen patients with histopathologically-confirmed thyroid carcinoma were enrolled and subjected to MRI acquisitions. The T1-precontrast and T2-weighted images were analyzed; overall, 279 texture features for each sequence were examined and correlated to the histopathological parameters Ki67 and p53, which are considered prognostic biomarkers. Several significant correlations were identified; indeed, sum square average features derived from T1-weighted images and entropy-based features derived from T2-weighted images are associated with p53 count in thyroid cancer. Similarly, different texture features derived from T1- and T2-weighted images showed associations with the Ki67 index. Hence, this relatively simple MRI technique, combined with texture analysis, does not replace histopathological examinations, but may be a novel, noninvasive modality for further characterizing thyroid cancer in clinical oncology [108].

The effects of the tyrosine kinase inhibitor gefitinib on MRI parameters, and the ability of such parameters to help schedule chemotherapy, were tested in a murine model of breast cancer. In this study, a xenograft model with BT474 cells (ductal carcinoma) was induced, and mice were treated with gefitinib either daily for ten days or "pulsed" for two consecutive days (higher dose for two administrations). The MRI acquisitions were performed at 2-day intervals over two weeks, and T1 and T2 weighted sequences were used to follow-up tumor volume. Moreover, diffusion and DCE MRI were performed, and relative results were reported in the respective paragraphs. Treatment with gefitinib resulted in significant tumor growth inhibition, both with pulsed and daily treatment. Hence, T1 and T2 proved to help evaluate growth inhibition but did not give any further information on which therapeutic protocol gave the best results [109].

#### 4.1.2. Breast

In a preliminary study on breast cancer, the MRI texture analysis was used to analyze the correlation between textural features and tumor volume, and to distinguish the underlying molecular subtypes luminal A and B. Patients with histopathologically-proven, invasive, ductal breast cancer were selected. The first frame of T1 and T2-weighted sequences pre-contrast was acquired and followed by the administration of a gadolinium-based contrast agent. The data analyzed mainly in the pre-contrast images showed that luminal A and B types had different textural features. Luminal B types of cancer have, in fact, a more heterogeneous appearance in MR images compared to luminal A types. These subtypes also showed a difference in tumor volume, with luminal B types showing a larger volume than luminal A types. The MRI texture analysis, combining information from both T1 and T2-weighted images, may provide further information on the biological aggressiveness of breast tumors that may improve therapeutic efficacy and management [110].

#### 4.1.3. Prostate

Anatomical MRI sequences may improve the accuracy of evaluating prostate volume, especially in subjects under hormonal treatment. The T2-weighted MRI was performed to evaluate the normal prostate volume in male C57/BL6 mice. The mice underwent castration, and they were repeatedly imaged to follow the castration-induced regression of the prostate. In addition to the T2-weighted sequence, a chemical shift-selective sequence (CHESS) was performed to suppress abdominal fat signal, and hence, to improve prostate distinction. Forty days after castration, each mouse was treated daily with the androgen dihydrotestosterone (DHT) subcutaneously (s.c.) to induced prostate re-growth. These sequences, in particular, the CHESS, allowed good discrimination of both the prostate margins and the ventral lobe from the dorsal and lateral lobes. Hence, this approach may improve the ability to differentiate the prostate from surrounding tissues and to better visualize the boundaries of the organ, even in human patients [111].

#### *4.2. Dynamic Contrast-Enhanced*

The most common MRI methods available to quantify perfusion, hemodynamic, and vascular properties of a tissue, are the dynamic contrast-enhanced (DCE) MRI and the arterial spin labeling (ASL) methods [93]. The DCE MRI represents an indirect measure of angiogenesis that evaluates the dynamic passage of a contrast agent through the tumor vessels to derive pharmacokinetic properties, measuring the contrast agent extravasation rate (Ktrans) and volume fraction (ve) of the extracellular extravascular space (EES), as thoroughly described elsewhere [93,112–114]. Such methods have been used to monitor perfusion, as a marker of responsiveness to antiangiogenic treatments, both in preclinical and clinical trials, in terms of either efficacy or early identification of treatment failure [14,99,115–120]. Therefore, the clinical ability of this method is remarkable not only for the functional information on the tumor vascularization pattern but also for the chemotherapy planning, monitoring response, and implementing a new line of therapies [121].

#### 4.2.1. Breast

In the preclinical model of ductal breast cancer with BT474 cells previously described (see 4.1 T1 and T2 Weighted Sequences, [109]), the DCE was also performed at 2-day intervals over two weeks. Transendothelial permeability (Kps), and fractional plasma volume (fPV) were measured. Tumor Kps decreased with pulsed treatment but then rebounded and increased with daily treatment. Tumor fPV increased in both treated groups, subsequently decreasing with pulsed treatment. Therefore, such quantitative MRI parameters may provide a sensitive measure to distinguish treatment regimens, and they might be useful for determining correct treatment scheduling, and hence, enhance chemotherapeutic efficacy [109].

In a preliminary study of breast cancer in human patients, the predictive ability of MRI was demonstrated using the DCE or DWI methods to predict tumor response following neoadjuvant therapy (NAT). This model integrated the heterogeneity of MRI-derived parameters (i.e., efflux rate constant—*Kep*, and ADC, as further described in the respective paragraph) with the hormone receptor status (i.e., estrogen receptor—ER, progesterone receptor—PR, and human epidermal growth factor receptor 2—HER2) and clinical data which were obtained before and after the first cycle of NAT. Thirty-three breast cancer patients underwent neoadjuvant chemotherapy, and therefore, were scanned for DW and DCE-MRI in the following three time-points: before NAT, after the first cycle of NAT, and after all NAT cycles. The median number of treatment cycles was 14 cycles. After completion of NAT, the pathological complete response (pCR) and nonresponse (non-pCR) were determined at the time of surgery. Twelve patients exhibited a pCR, and twenty-one patients were non-pCR. For patients with pCRs, the mean Kep had decreased between the pre and post-first NAT cycles, while for a non-pCR patient, Kep increased. Measurements after all NAT cycles were not obtainable in pCR patients since there was no residual tumor identifiable in the MRI scan. The immunohistochemical evaluation of hormone receptor status has defined the molecular subtypes of breast cancer for all patients; indeed, among the thirty-three patients, nine, six, eight, and ten patients are classified into luminal A, B, HER2, and basal subtypes, respectively. The MRI approach in question seems to accurately predict a treatment response right after a single cycle of therapy. Moreover, this method may improve the accuracy of evaluating a tumor's response to NAT, showing a higher predictive power than models based on tumor size changes, and it may be used as a short-term surrogate marker of outcome in breast cancer patients [122].

#### 4.2.2. Prostate

Male BALB/c nude mice were implanted subcutaneously with the human-derived, androgen-sensitive CWR22 cell line, to evaluate the predictive capacity of DCE in a prostate cancer xenograft mouse model. These mice received combinations of androgen-deprivation therapy, obtained by surgical castration and/or radiotherapy. The MRI sequences applied were DCE and DWI, which will be discussed in the dedicated paragraph, and they were performed pre-radiation, and on day one and nine; the ADC value, the *Ktrans,* tumor volume, and PSA were used to measure the therapeutic response. The *Ktrans* and PSA showed a high level of correlation with treatment response, and thus, this parameter might be included in the evaluation of treatment responses in prostate cancer patients [123].

#### *4.3. Arterial Spin Labeling*

As mentioned, the ASL is a noninvasive and quantitative technique that allows perfusion measurement without requiring the administration of a contrast agent. This technique uses arterial blood water as an endogenous diffusible tracer by "labeling" it; i.e., by inverting the magnetization of the blood with radiofrequency pulses. As a result, studies can be repeated in the same subject over time [93,101,124–127]. Measurement of tumor blood flow with this technique is strongly helpful for tumor grading and evaluation of anticancer treatment [124,126]. The ASL is widely used in the preclinical and clinical fields, but in the latter case, it is still an emerging technique and has not yet replaced more invasive procedures, such as contrast-enhanced MRI, probably due to the complexity of the method and the relatively high sensitivity to motion artifacts [124].

#### Breast

ASL might gain a significant impact on the diagnosis and therapy management of breast cancer, thanks to its ability to quantify perfusion without the use of contrast agents, as examined in a pilot study. Quantification of perfusion of normal fibroglandular tissue and breast cancer using a flow-sensitive, alternating-inversion, recovery-balanced, steady-state free precession (FAIR TrueFISP) ASL sequence was performed in twenty-two individuals, including eighteen patients with suspected breast tumors and four healthy controls, in addition to the routine clinical imaging protocol. The definitive diagnosis was obtained by histology after biopsy or surgery. The results showed that ASL perfusion was successfully acquired in thirteen of eighteen tumor patients and all healthy controls. The mean ASL perfusion of invasive ductal carcinoma tissue was significantly higher than the perfusion of the normal breast parenchyma and invasive lobular carcinoma. No significant difference was found between the mean ASL perfusion of the normal breast parenchyma and invasive lobular carcinoma tissue. Hence, these results indicate that ASL perfusion can differentiate malignant lesions from normal breast parenchyma as well as breast tumor types. This MRI modality may be useful to detect early changes in response to neoadjuvant chemotherapy, and the signal changes proportional to the blood flow may represent a property that allows identifying potential biological markers, and consequently, developing targeted therapies. Furthermore, image acquisition can be repeated without the concern of cumulative doses of paramagnetic contrast agent, and in patients with renal insufficiency, who may not be safely injected with contrast agents [128].

#### *4.4. Blood Oxygen Level-Dependent Functional Magnetic Resonance Imaging*

The blood oxygen level-dependent (BOLD) functional MRI (fMRI) provides information on changes in oxygenation in tissue to measure the hemodynamic response. The BOLD "contrast" reflects a variation in the transverse relaxation rate of tissue, and the paramagnetic effects influence it by the concentration of deoxyhemoglobin [93,129]. Generally speaking, this technique is heavily used to study cerebral activity, but it has the potential to evaluate metabolism, angiogenesis, and variations of oxygenation in tumors, as well. Indeed, in preclinical models and human tumors, it has been applied as a noninvasive method to monitor antiangiogenic therapies [129,130]. However, BOLD provides an indirect estimate of oxygen delivery and has a variable and scarce relationship with tumor tissue hypoxia, which is a significant negative prognostic factor. The ability to "map" hypoxia might help therapy planning and predicts treatment failure; thus, driving early changes of therapeutic strategy [131–133]. Furthermore, BOLD measurements are influenced by variations in vessel caliber, by the presence of hemorrhage and movement artifacts, which hinders its implementation as a clinical biomarker of hypoxia and its readiness to translate into clinical use [131,132].

#### Breast

The BOLD MRI method has been employed as a simple, noninvasive method to assess tumor oxygenation during a preliminary observational study. Eleven patients with locally advanced breast cancer were enrolled for preoperative neoadjuvant chemotherapy with doxorubicin and cyclophosphamide for four cycles. Of these, seven patients completed all chemotherapy treatments and underwent MRI before, during, and after chemotherapy. Breast tumor response was divided into complete response, partial, and stable response based on clinical palpation. The BOLD study applied a 6-min oxygen-breathing challenge; BOLD contrast enhancement was observed in all tumors, but patients with complete responses showed a significantly higher BOLD before the start of chemotherapy compared with both partial or stable response; furthermore, there was no significant difference between latter groups. The correlation between high BOLD response and better treatment outcome suggests that this may be an excellent, noninvasive prognostic tool for cancer management, providing early predictive information on the response to chemotherapy [134].
