Preclinical Imaging Evaluation of miRNAs’ Delivery and Effects in Breast Cancer Mouse Models: A Systematic Review

Simple Summary The purpose of this systematic review was to assess the advancements in preclinical molecular imaging protocols used to study the delivery, tracking and therapeutic efficacy of miRNAs in mouse models of breast cancer. For this aim we have interrogated several browsers (PubMed, EMBASE, BIOSIS™ and Scopus) using the following terms: breast cancer, mouse, mice, microRNA(s) and miRNA(s). From 114 articles selected according to a PRISMA protocol, we focused on mouse models, routes of miRNA administration, therapy efficacy and molecular imaging. Importantly, we highlight here the advancements made in all imaging techniques’ applications used, providing a useful tool, on the basis of the current evidence, with which to suggest the best preclinical imaging protocol. Abstract Background: We have conducted a systematic review focusing on the advancements in preclinical molecular imaging to study the delivery and therapeutic efficacy of miRNAs in mouse models of breast cancer. Methods: A systematic review of English articles published in peer-reviewed journals using PubMed, EMBASE, BIOSIS™ and Scopus was performed. Search terms included breast cancer, mouse, mice, microRNA(s) and miRNA(s). Results: From a total of 2073 records, our final data extraction was from 114 manuscripts. The most frequently used murine genetic background was Balb/C (46.7%). The most frequently used model was the IV metastatic model (46.8%), which was obtained via intravenous injection (68.9%) in the tail vein. Bioluminescence was the most used frequently used tool (64%), and was used as a surrogate for tumor growth for efficacy treatment or for the evaluation of tumorigenicity in miRNA-transfected cells (29.9%); for tracking, evaluation of engraftment and for response to therapy in metastatic models (50.6%). Conclusions: This review provides a systematic and focused analysis of all the information available and related to the imaging protocols with which to test miRNA therapy in an in vivo mice model of breast cancer, and has the purpose of providing an important tool to suggest the best preclinical imaging protocol based on available evidence.


Introduction
As has been recently estimated, breast cancer (BC) alone accounts for~30% of all new diagnoses in women [1]. Although improvements in BC's early diagnostic strategies and therapy have increased survival rates, this malignant tumor remains one of the most frequent causes of cancer-related mortality among females worldwide [1]. To date, it is All of the studies published in the last six years (2015-2021) which reported precli ical molecular and diagnostic imaging results in vivo or ex vivo in murine models of B were included in this systematic review. Reference lists from relevant reviews identifie in the database searches were manually searched to identify other eventual studie Searches were concluded on 1 May 2021.

Study Selection and Eligibility Criteria
An electronic spreadsheet was prepared to report, for each study, the title, list of a thors list, date of publication and language; moreover, whenever indicated, it was r ported if the study was a review, an extensive research paper, an abstract or a letter/ed torial.
All non-English language papers were excluded for the authors' and readers' co venience. After removing all duplicates, congress abstracts and posters, letters to Edito or editorials, each author was asked to screen the studies based on their titles and a

Data Extraction
A.G., L.A. and A.Z. independently extracted from the selected studies (i) the mouse strain, (ii) the cell line used and all its peculiar characteristics, i.e., all eventual genetic modification of the original cell line, (iii) the model generated with the cell line, i.e., orthotopic, subcutaneous xenograft or metastatic, (iv) the miRNAs studied and their administration route, (v) the imaging modality or the multimodal approach used and (vi) the outcome measure, i.e., tumor volume reduction or changes in pathophysiologic aspects. Two other authors (G.S. and F.M.O.) independently extracted from the selected studies (i) the cell line used and all its peculiar characteristics, i.e., all eventual genetic modification of the original cell line, (ii) the model generated with the cell line, i.e., orthotopic, subcutaneous xenograft or metastatic, and (iii) the miRNAs studied, their administration route and the presumed effect. L.A. and F.M.O. reviewed and summarized all the information retrieved and discussed with all authors whenever discrepancies were detected and a consensus was needed.

Literature Search
As reported in Figure 1, the search strategy using the eight research strings identified 6860 scientific manuscripts on PubMed ® (MEDLINE ® ), 4294 on EMBASE, 2631 on BIOSIS™ and 5120 on Scopus. The eight lists were compared within each search engine, deleting all duplicates, hence the final number of manuscripts was 875 for PubMed ® (MEDLINE ® ), 1441 for EMBASE, 844 for BIOSIS™ and 1447 for Scopus. At this point the four lists were merged together, erasing duplicates again, with a definitive list of 2073 records. Such a list was individually screened by each author, relying on the title and abstract, excluding all non-English papers, congress abstracts and posters, letter to editors, clear reference to cancers other than BC or to other pathologies at all in addition to reviews. Reviews were, however, searched for other relevant references, but no other eligible papers were detected. In this phase 852 records were excluded and 1221 papers were assessed for full text eligibility, excluding all those in which there was no use of mouse models of BC, there was not in vivo imaging or ex vivo on whole organs, i.e., were excluded imaging techniques applied to histological samples. At this point, 170 papers were studied to prepare the qualitative-quantitative synthesis, further excluding all manuscripts in which the animal models were used to study long non-coding, small interfering or other RNAs, as well as genes or other signaling molecules, without a clear link to a miRNAs. The final data extraction was from a total of 114 manuscripts.

Mouse Models of Breast Cancer
Various factors play a role in the study of preclinical models, in particular the mouse strain, the cell line and the engraftment route. A summary of the murine strain used in the articles analyzed and the relative references are shown in Table 1, while Figure 2 shows the absolute number of experiments for each strain. Table 1. Murine strains used in miRNA experiments.
Regarding cell lines, in most of the experiments human-derived cell lines were used, and only few used syngeneic, i.e., mouse-derived, cell lines of BC. Figure 3 shows the absolute number of experiments for each cell line, and Table 2 shows the different specific modification to each cell line and the relative references. Figure 2. Number of experiments regarding the murine strain used in the articles analyzed. NOD: non-obese diabetic; SCID: severe combined immunodeficient mice; and NSG: NOD SCID gamma mouse. In detail, MDA-MB-231 was used in most experiments (62.5%), followed by MCF-(16.7%). SKBR3 and SUM-derived cells were used in two experiments each (1.4% each), whereas R2N1d-labeled with green fluorescent protein (GFP) and transfected with miR-, BT549-transfected with miR-and T47D-TR (tamoxifen-resistant) cell lines were used in one experiment each (0.7% each). Two experiments (1.4%) used breast cancer stem cells (BrCSCs): in one experiment, BrCSCs were obtained after the induction of the differentiation of BC cells purified from fresh tissues from patients' mastectomies and then transduced with GFP via lentivirus infection, prior to being used in an orthotopic model [84]. In the second experiment BrCSCs were obtained from both patients' tissues and from MDA-MB-231 and MCF-7 cell lines [59]. One experiment (0.7%) used a patient-derived xenograft (PDX) labeled with luciferin and modulated for miR precursor expression [95]. The only syngeneic cell line used was 4T1 (13.8%).  In detail, MDA-MB-231 was used in most experiments (62.5%), followed by MCF-(16.7%). SKBR3 and SUM-derived cells were used in two experiments each (1.4% each), whereas R2N1d-labeled with green fluorescent protein (GFP) and transfected with miR-, BT549-transfected with miR-and T47D-TR (tamoxifen-resistant) cell lines were used in one experiment each (0.7% each). Two experiments (1.4%) used breast cancer stem cells (BrCSCs): in one experiment, BrCSCs were obtained after the induction of the differentiation of BC cells purified from fresh tissues from patients' mastectomies and then transduced with GFP via lentivirus infection, prior to being used in an orthotopic model [84]. In the second experiment BrCSCs were obtained from both patients' tissues and from MDA-MB-231 and MCF-7 cell lines [59]. One experiment (0.7%) used a patient-derived xenograft (PDX) labeled with luciferin and modulated for miR precursor expression [95]. The only syngeneic cell line used was 4T1 (13.8%).
Regarding the murine model, the three models for cellular engrafting, i.e., subcutaneous and orthotopic xenografts as well as a metastatic model obtained by the intravenous injection (IV)of cancer cells, were all represented ( Table 3). The most frequently used model was the IV metastatic model (46.8%), which was obtained either via intravenous injection (68.9%)-in one paper it was indicated as intraarterial [58]-in the tail vein or in the left ventricle (9.8%). Direct intratibial injection to study osseous metastasis was applied in a few experiments (6.6%), as was direct intrapulmonary injection (1.6%). Finally, the development of spontaneous metastasis after orthotopic injection was obtained either after surgical resection of the primary nodule (4.9%)-with lymph node [112] or pulmonary metastasization [23,34]-or with the primary orthotopic implant on site (8.2%). In two papers, it was not specified how the metastatic model was obtained [70,106].
The orthotopic model, with injection in the second or forth mammary gland or fat pad, transcutaneously, after surgical exposure or intra nipple, was the second most frequently used model (29.2%). A subcutaneous xenograft, implanted in various sites, i.e., on the shoulder, the armpit, the flank and thigh, was used in 24% of the experiments.
Nanoparticle (NP)-based delivery represents a promising strategy for BC treatment, preventing miRNA degradation in the bloodstream and improving miRNA delivery in tissue-specific targeting. Indeed, we found that 29 experiments (25.4%) were conducted using different formulation of NPs, including natural lipid-based NPs (LNPs) and synthetic NPs composed of inorganic materials such as silica (SiO 2 ), gold (Au) or polymer (e,i. polyamidoamine-PAMAM-dendrimers) [137] (Table 4).
In a few studies, multiple miRNAs were simultaneously co-delivered using polymeric NPs triggered in BC tissue by the urokinase plasminogen activator peptide (uPA) [108], by ultrasound [33] or by RNA-triple-helix hydrogel scaffolds [82].
Recently, the anticancer activity of miRNAs derived from marine invertebrate marsupenaeus japonicus shrimp was analyzed in two experiments in which tumor-bearing mice were fed shrimp that were fed mja-miR-35-expressing bacteria [64] or treated with synthesized shrimp miR-34 [93].

Therapy Effect and Efficacy
The potential role of miRNAs could be categorized based on their mode of action and therapeutic efficacy established in preclinical BC mouse models. The number of experiments and references regarding the effects of therapy and its efficacy are summarized in Figure 5 and in Table 5. The combined delivery of miRNA and a chemotherapeutic drug into tumor sites was obtained using polymeric hybrid NPs (Dox + miR-34a) [28], polydopamine (PDA)-based NPs (Dox + antisense-miR-21) [68], magnetic NPs (Dox + miR-10b) [112], calcium/phosphate lipid NPs (paclitaxel + miR-124) [66] and a lipid nanocarrier coated by cationic albumin (docetaxel + miRNA-34a) [135]. Interestingly, specific NPs were developed to codeliver the photosensitizer indocyanine green (ICG) and the inhibitor of miR-21 [41].
Exosomes are small extracellular vesicles (EVs) of 30-150 nm in diameter, which are released by cancer cells in the tumor microenvironment for intercellular communication.
Recently, the anticancer activity of miRNAs derived from marine invertebrate marsupenaeus japonicus shrimp was analyzed in two experiments in which tumor-bearing mice were fed shrimp that were fed mja-miR-35-expressing bacteria [64] or treated with synthesized shrimp miR-34 [93].

Therapy Effect and Efficacy
The potential role of miRNAs could be categorized based on their mode of action and therapeutic efficacy established in preclinical BC mouse models. The number of experiments and references regarding the effects of therapy and its efficacy are summarized in Figure 5 and in Table 5.  Among the biological effects reported in mice, tumor growth alone (30.7%) or in combination with tumor metastasis (34.2%) are determined to be the effects most frequently studied.
Twenty animal models (17.5%) were conducted to study the effects of miRNA delivery on lung metastasis, and only a few experiments were performed analyzing bone (7.9%), brain (1.7%) and liver metastasis (0.87%).
The co-delivery of miRNA and small-molecule chemotherapy drugs in tumor sites represents a promising strategy to fight the progression of cancer in mice. In this context, the co-delivery of Dox with miR-34a [28] or with miR-159 [46] to cancer sites suppressed tumor growth. A regression of lung metastasis disease was established by the cotreatment of miR-10b and Dox [112]. The combination treatment of taxol and AC1MMYR2 (a small molecule that reduces miR-21 expression) [63] or of miRNA-34a and docetaxel [135] impaired tumor growth and metastasis. Paclitaxel and miR-124 coloaded in a lipid nanosystem impaired lung metastasis formation in orthotopic mice [66]. Co-delivery of miR-96/-182 with cisplatin, using NPs, reduced primary tumor formation and prevented lung metastasis formation [35].
Currently, the detection of miRNAs in cancer tissues could help to monitor the progression of cancer. From our research, biodistribution studies were found in six articles (5.3%). miR-155 expression was monitored in two different studies by intravenously injecting a PA nanoprobe [27] and by the synthesized peptide nucleic acid (PNA) mimic loaded with [ 99m Tc] [45]. A molecular beacon (MB) circuit was developed to monitor the expression of miR-34a in BC tissue with high sensitivity [125]. A nanosensor conjugated with MN-NPs allowed for the discrimination of BC cells from non-tumoral cells based on miR-10b expression [111]. Monitoring the expression of miR-200c [103] and of miR-14/-21/-9 [39] in tumor-bearing mice was useful to determine the therapeutic approach. Finally, tumor angiogenesis was evaluated in three studies reporting that miR-497 exhibited anti-angiogenesis and antitumor effects targeting VEGFR2 [126] and miR-210 promoted angiogenesis [69], while miR-125a-5p affected tumorigenesis, metastasis and angiogenesis in vivo [73].

Molecular Imaging
Most of the known preclinical imaging techniques have been applied in studying miRNAs' delivery and/or efficacy. Figure 6 shows the absolute number of experiments for each imaging modality, and Table 6 shows the number of experiments for each specific modality and aim, in addition to the relative references.  [71], -125b [106] and -30 family members [58], the inhibition of miR-218-5p [130] or by intratumorally injecting synthetic miR-135/-203 mimics [76] or osteoclast-targeting An-tagomiR-214-3p using (D-Asp)8-liposome [133]. Currently, the detection of miRNAs in cancer tissues could help to monitor the progression of cancer. From our research, biodistribution studies were found in six articles (5.3%). miR-155 expression was monitored in two different studies by intravenously injecting a PA nanoprobe [27] and by the synthesized peptide nucleic acid (PNA) mimic loaded with [ 99m Tc] [45]. A molecular beacon (MB) circuit was developed to monitor the expression of miR-34a in BC tissue with high sensitivity [125]. A nanosensor conjugated with MN-NPs allowed for the discrimination of BC cells from non-tumoral cells based on miR-10b expression [111]. Monitoring the expression of miR-200c [103] and of miR-14/-21/-9 [39] in tumor-bearing mice was useful to determine the therapeutic approach. Finally, tumor angiogenesis was evaluated in three studies reporting that miR-497 exhibited anti-angiogenesis and antitumor effects targeting VEGFR2 [126] and miR-210 promoted angiogenesis [69], while miR-125a-5p affected tumorigenesis, metastasis and angiogenesis in vivo [73].

Molecular Imaging
Most of the known preclinical imaging techniques have been applied in studying miRNAs' delivery and/or efficacy. Figure 6 shows the absolute number of experiments for each imaging modality, and Table 6 shows the number of experiments for each specific modality and aim, in addition to the relative references. Bioluminescence was determined to be the most frequently used tool (64%); this technique was used as a surrogate for tumor growth for efficacy treatment or for the evaluation of tumorigenicity in miRNA-transfected cells (29.9%); for tracking, evaluation of engraftment and response to therapy in metastatic models (50.6%); and for both the aforementioned aims in the same experiment, evaluating metastasis either in vivo or ex vivo on whole organs (16.1%). As already reported, in one experiment (1.1%) a transgenic VEGFR2-luc mouse was used to evaluate the expression of VEGFR by non-invasive  Table 6. Number of experiments for each specific modality and aim.
Bioluminescence was determined to be the most frequently used tool (64%); this technique was used as a surrogate for tumor growth for efficacy treatment or for the evaluation of tumorigenicity in miRNA-transfected cells (29.9%); for tracking, evaluation of engraftment and response to therapy in metastatic models (50.6%); and for both the aforementioned aims in the same experiment, evaluating metastasis either in vivo or ex vivo on whole organs (16.1%). As already reported, in one experiment (1.1%) a transgenic VEGFR2luc mouse was used to evaluate the expression of VEGFR by non-invasive bioluminescence, and to evaluate the effect of miRNA mimic treatment as anti-angiogenetic therapy [126]. Bioluminescence was also used for vector uptake and intercellular target repression (2.3%), although most of these experiments were performed by fluorescence imaging.
Fluorescence imaging was the second most frequently used technique (21.3%), and was used primarily to trace vector biodistribution (73.2%) by using different strategies, e.g., by directly conjugating the miR to the fluorophore, or simply uploading the fluorophore within the vector. In one interesting report, the vector was neither an NP nor an extracellular vesicle nor a liposome, but a folate, directly linked to the miR as well as to a near-infrared (NIR) fluorophore for fluorescent detection [115]. Fluorescence was rarely used for tumor growth evaluation (11.5%), analysis of tumor persistence after direct intratumoral injection of the miR labeled with fluorophore [41] or within fluorescent SiO 2 NPs [99] (7.7%), and for cell tracking (3.8%). One interesting experiment (3.8%) showed the ability of a molecular beacon to detect and image endogenous miRNAs with a high level of specificity in vivo [125]. Besides these two mostly used imaging techniques, other tools were used to study the biodistribution or different aspects of miRNAs' treatment efficacy. Microcomputed tomography (µCT) was used to analyze in vivo or ex vivo osteolytic lesions in metastatic bone models or identify pulmonary metastases (5.2%). The former evaluation was performed with standard radiography (1.5%) in two other experiments [58,131]. Magnetic resonance imaging (MRI) was used in 2.9% of the experiments, mainly for the detection of magnetic NPs' biodistribution, and only in one experiment for the evaluation of invasiveness of adjacent tissue [74]. Positron emission tomography (PET)/CT was applied with [18F]-FDG administration to evaluate tumor growth, in terms of tumor glucose metabolism, or for the detection of pulmonary metastases (2.2%). High-frequency ultrasonography (HFUS) was performed to evaluate tumor growth or microbubble-mediated nanoparticles delivery as a therapeutic intervention (1.5%). Photoacoustic (PA) (0.7%) imaging was used to determinate the ability of self-assembling nanoprobes to identify specific miRNAs. In brief, in the presence of a specific miRNA, aurum aggregation from the nanoprobes, via a hybridization chain reaction, allowed for the identification of the PA signal [27]. Finally, single-photon emission computed tomography (SPECT) (0.7%) was used to label and track a molecular probe and to evaluate both the specificity in detecting the selected miR and both for biodistribution purposes [45].

Discussion
We systematically analyzed the most recent studies using preclinical imaging technologies to investigate the potential of specific miRNAs as therapeutic and diagnostic tools in BC. Although several systematic reviews focused on the crucial role played by miRNA in BC biology and as therapeutics [140], to our knowledge this review is the first systematic review that specifically focused on the use of preclinical molecular imaging for the evaluation of miRNAs' delivery and effects in BC. Numerous are the advantages offered by the application of different imaging techniques to study animal models of cancer [16]. First, the possibility to perform in real time non-invasive longitudinal studies of the same mice. This allows the number of animals to be analyzed to be reduced, in accordance with Directive 2010/63/EU, the principle of the 3Rs (Replacement, Reduction and Refinement), and animal welfare considerations. It is noteworthy that it is the translational aspect of preclinical imaging that may be considered as a bridge from basic to clinical research. In this context, this systematic review documented all investigations that used different imaging technologies dedicated to small animals, such as optical imaging (OI), HFUS, MRI, CT and PET/CT, to evaluate and validate miRNAs as anti-cancer agents as well as shed light on molecular mechanisms.
Most of the studies included in this systematic review were performed on miR-10b (five studies), miR-21 (eleven studies), the miR-34 family (eight studies) and the miR-200 family (ten studies). A high level of miR-10b indicates a poor prognosis in BC, correlating with angiogenesis and metastatic behaviors (increased tumor size, lymph node positivity and a high Ki-67 score) [141][142][143]. Importantly, in NOD-SCID mice, a high miR-10b level led to distant metastasis, while in the 4T1 mouse mammary tumor metastasis model the delivery of AntagomiR specific for the silencing of miR-10b suppressed the distant metastasis [144,145]. Overexpression of miR-21, one of the most studied oncomiRs in BC, is associated with lymph node metastasis, resistance to anticancer agents and a poor prognosis [146][147][148]. The up-regulation of miR-21 in this cancer induces the silencing of several tumor suppressor genes, such as programmed cell death 4 (PDCD4) [149] and leucine zipper transcription factor-like 1 (LZTFL1) [150]. A tumorigenicity assay was recently performed in Balb/c-nude mice inoculated with BC cells silenced for miR-21 using specific peptide nucleic acids (PNA). In vivo, functional studies showed that PNA-antimiR-21 inhibits tumor growth in vivo [151]. Another important family correlated to cancer is the miR-34 family: it is comprised of miR-34a, miR-34b and miR-34c. They exert a tumor suppressor role in various cancers and are regulated by p53 [152]. In BC, miR-34a plays a crucial role in proliferation, motility and stemness [153]. Identified targets of miR-34a are SIRT1 and BCL2 [154]. Another example of oncomiR is miR-200a, which promotes epithelial-mesenchymal transition (EMT), drug resistance and metastasis by targeting tumor protein P53-inducible nuclear protein 1 (TP53INP1) and yes-associated protein 1 (YAP1) in human BC [155,156]. Importantly, miR-200a belongs to the miR-200 family that appears to be crucial for BC progression. In particular, the miR-200 family is composed of five members (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) that are reported to be involved in EMT and angiogenesis of BC cells [157][158][159]. Besides, it is reported that deregulated levels of miR-200a and miR-200c occurred in the tamoxifen-resistant BC model, where they induced a reduction in the mRNA of c-MYB [159].
A major problem in the clinical use of miRNAs is the delivery method. This is due to several reasons, such as the destabilization of the RNA in circulation due to serum ribonucleases, ineffective targeting to the tumor cells because of the tumor microenvironment and a poor uptake of the miRNA. Several delivery methods have been tested, such as the lentiviral-and liposomal-mediated delivery of the tumor-suppressive miRNA miRNA-34a (miR-34a), which reduces the tumor burden in non-small-cell lung cancer (NSCLC) mouse models [160]. In addition to vehicle-and viral-mediated miRNA delivery, systemic injection of vehicle-free oligonucleotides has also been tested. However, this approach has proven problematic because of the pharmacokinetic and stability limitations associated with intravenous delivery, and thus either relies on local delivery or necessitates achieving a high oligonucleotide concentration that is often only seen in the kidneys and liver. Although local delivery is an option, achieving delivery beyond sites that are accessible to local delivery, such as to micrometastatic lesions, is not achievable.
Most of the imaging studies reported in this review used optical imaging (OI) to analyze miRNAs' effects on cancer murine models, in particular bioluminescence (BLI) (64%) and fluorescence (21.3%). One of the most common applications of OI is to monitor tumor growth and metastasis formation in orthotopic xenograft models and transgenic animal models [161]. Furthermore, it is a highly sensitive technique and allows non-invasive monitoring of disease-relevant processes and permits the tracking of cells [162]. The main advantages of OI compared to other imaging platforms are the low cost and the absence of ionizing radiation, as well as the possibility to more easily translate the observations obtained in vitro on the corresponding cell line injected into animals. Fluorescence shows some disadvantages due to background signals and autofluorescence, which are absent in the BLI, which in turn has brightness and low spatial-temporal resolution [161]. Among the numerous studies analyzed in this review regarding BLI with luciferase to monitor tumor growth and/or metastatic spread, it is worthy to note the investigation that used VEGFR2-luc transgenic mice to monitor the effect of miR-497 mimic not only on tumor growth but also on tumor angiogenesis [126]. The results demonstrated that overexpression of miR-497 showed inhibitory effects on VEGFR2 activation [126]. The limits of fluorescence can be overcome by using near-infrared fluorophores that penetrate deeper into tissues and exhibit very low autofluorescence. An interesting study conjugated microRNAs to folate (FolamiR) for delivering them into cells that overexpress the folate receptor. In particular, the tumor-suppressive FolamiR, FolamiR-34a, was labeled with NIR fluorophore, and its delivery to TNBC xenografts was evaluated by OI [115]. Furthermore, Tu et al.
reported a novel strategy for miRNA detection through enzyme-free signal amplification by self-circulation of the hybridization between the miRNAs and molecular beacon (MB) circuits. This approach allowed miRNA to be detected in the BC xenografts by amplifying the fluorescence signal and contributing improving detection sensitivity [125].
HFUS is the most suitable technique to monitor tumor growth due to the capability of this technique to perform accurate morphologic imaging. Most interesting, HFUS has also been used to deliver directly therapeutic microRNAs (AmiR-21 and miR-100) and TK-p53-NTR triple therapeutic gene, co-loaded in PLGA-PEG-PEI polymer NPs to tumor models of TNBC [33]. As our research group has demonstrated in past experiments, ultrasound mediated therapy, enhanced vascular permeability and microbubbles cavitation improve drug delivery directly into tumor sites [163]. PET has also been used to evaluate the response to miRNA therapy in a tumor model of TNBC by targeting tumor glycolysis [36], as well as to assess metastasis in an in vivo mice model of TNBC. The limitation of this technique is related to the high-cost relative to the radiotracer and/or to the necessity of having a cyclotron close to the animal facility, plus the necessity of using radiations. Thus, specific and long training is necessary to have a person able to perform experiments of nuclear medicine. An emergent methodology is high-field MRI, which combines the possibility of performing a morphologic analysis of the primary tumor to follow the spread of metastasis, plus the possibility of therapeutic delivery of miRNAs or miRNAs combined with chemotherapeutic agents with magnetic NPs. MRI has the advantage of avoiding the use of ionizing radiation, but has the disadvantage of requiring a long time for the acquisition of high-quality diagnostic images. A good anesthetic protocol and continuous monitoring of the mice model could compensate for the last issue. Finally, what mainly emerges from the cited papers, is the advantage that we could obtain by having a multimodal imaging approach to diagnose both the mice model of BC and to perform an efficient therapy. The last issue could also be better addressed through a targeted nanosystem with directed delivery against molecular markers of breast cancer.

Conclusions
The studies reported and discussed in this systemic review highlight the utility of preclinical molecular imaging focused on the development of novel therapeutic strategies for miRNAs based in breast cancer management. To date, despite the multiple advances in imaging technology, this extensive and focused literature review shows that optical imaging remains the most widely used method in preclinical investigations, probably due to its low cost and ease of use. In fact, only few of the papers we cited demonstrated the advantages that we could obtain by having a multimodal imaging approach to diagnose mice models of BC and perform an efficient therapy. Therefore, given the large amount of information that can be extrapolated from multimodal imaging and its strong translational power to the clinic, future studies using multiple imaging modalities are desirable. Finally, the development of NPs engineered to encapsulate miRNAs alone or in combination with other drugs and their delivery to specific targets will provide deeper knowledge in this research field, and will be certainly be one of the fields that will be improved in the future.
We aimed to highlight the role of preclinical imaging and its potentiality to test new experimental therapies for breast cancer patients, aiding the translation from in vitro studies to the clinic. Preclinical imaging is a continuously evolving field, and new nanoprobes could represent novel systems for personalized therapy in the future.