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Background: Breast cancer is the most common malignancy in women, with high morbidity and mortality. Molecular alterations in breast cancer involve the expression or upregulation of various molecular targets that can be used for diagnostic nuclear medicine imaging and radiopharmaceutical treatment. Theragnostics is based on the binding of radionuclides to molecular targets. These radionuclides can induce a cytotoxic effect on the specific tumor cell (target) or its vicinity, thus allowing a personalized approach to patients with effective treatment and comparably small side effects. Aim: This review aims to describe the most promising molecular targets currently under investigation for theragnostics and precision oncology in breast cancer. Methods: A comprehensive literature search of studies on theragnostics in breast cancer was performed in the PubMed, PMC, Scopus, Google Scholar, Embase, Web of Science, and Cochrane library databases, between 2010 and 2022, using the following terms: breast neoplasm*, breast, breast cancer*, theragnostic*, theranostic*, radioligand therap*, RLT, MET, FLT, FMISO, FES, estradiol, trastuzumab, PD-L1, PSMA, FAPI, FACBC, fluciclovine, FAZA, GRPR, DOTATOC, DOTATATE, CXC4, endoglin, gastrin, mucin1, and syndecan1. Results: Fifty-three studies were included in the systematic review and summarized in six clinical sections: 1) human epidermal growth factor receptor 2 (HER2); 2) somatostatin receptors (SSTRS); 3) prostate-specific membrane antigen radiotracers (PSMA); 4) fibroblast activation protein-α targeted radiotracers; 5) gastrin-releasing peptide receptor-targeted radiotracers; 6) other radiotracers for theragnostics. Conclusion: The theragnostic approach will progressively allow better patient selection, and improve the prediction of response and toxicity, avoiding unnecessary and costly treatment.

The file contains three supplementary figures with legends. Figure S1: Characterization of cell lines expressing CITK-specific shRNAs. Figure S2: The expression of CITK correlates with MB patients'' prognosis and CITK is highly expressed in mouse SmoA1 MB. Figure S3: Engineering and characterization of conditional CITK inactivation in mice.

Medulloblastoma is the most common malignant brain tumor in children. Current treatment for medulloblastoma consists of surgery followed by irradiation of the whole neuraxis and high-dose multiagent chemotherapy, a partially effective strategy associated with highly invalidating side effects. Therefore, identification and validation of novel target molecules capable of contrasting medulloblastoma growth without disturbing brain development is needed. Citron kinase protein (CITK), encoded by primary microcephaly gene MCPH17, is required for normal proliferation and survival of neural progenitors. Constitutive loss of CITK leads to cytokinesis failure, chromosome instability, and apoptosis in the developing brain, but has limited effects on other tissues. On this basis, we hypothesized that CITK could be an effective target for medulloblastoma treatment. In medulloblastoma cell lines DAOY and ONS-76, CITK knockdown increased both cytokinesis failure and DNA damage, impairing proliferation and inducing cell senescence and apoptosis via TP53 or TP73. Similar effects were obtained in the NeuroD-SmoA1 transgenic mouse model, in which CITK deletion increased apoptotic cells and senescence markers such as P21CIP1, P27KIP1, and P16INK4A. Most importantly, CITK deletion decreased tumor growth and increased overall survival in these mice, with no apparent side effects. These results suggest that CITK can be a useful molecular target for medulloblastoma treatment.Significance: In vitro and in vivo proof of concept identifies citron kinase protein as a suitable target for medulloblastoma treatment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4599/F1.large.jpg. Cancer Res; 78(16); 4599–612. ©2018 AACR.

A total of 20% to 50% of prostate cancer (PCa) patients leave the surgery room with positive tumour margins. The intraoperative combination of fluorescence guided surgery (FGS) and photodynamic therapy (PDT) may be very helpful for improving tumour margin delineation and cancer therapy. PSMA is a transmembrane protein overexpressed in 90–100% of PCa cells. The goal of this work is the development of a PSMA-targeted Near InfraRed Fluorescent probe to offer the surgeon a valuable intraoperative tool for allowing a complete tumour removal, implemented with the possibility of using PDT to kill the eventual not resected cancer cells. PSMA-617 binding motif was conjugated to IRDye700DX-NHS and the conjugation did not affect the photophysical characteristics of the fluorophore. The affinity of IRDye700DX-PSMA-617 towards PCa cells followed the order of their PSMA expression, i.e., PC3-PIP > LNCaP > PC3, PC3-FLU. NIRF imaging showed a significant PC3-PIP tumour uptake after the injection of 1 or 5 nmol with a maximum tumour-to-muscle ratio (ca. 60) observed for both doses 24 h post-injection. Importantly, urine, healthy prostate, and the bladder were not fluorescent at 24 h post-injection. Flow cytometry and confocal images highlighted a co-localization of PSMA+ cells with IRDye700DX-PSMA uptake. Very interestingly, ex vivo analysis on a tumour specimen highlighted a significant PSMA expression by tumour-associated macrophages, likely attributable to extracellular vesicles secreted by the PSMA(+) tumour cells. FGS proved that IRDye700DX-PSMA was able to easily delineate tumour margins. PDT experiments showed a concentration-dependent decrease in cell viability (from 75% at 10 nM to 12% at 500 nM), whereas controls did not show any cytotoxicity. PC3-PIP tumour-bearing mice subjected to photodynamic therapy showed a delayed tumour growth. In conclusion, a novel PSMA-targeted NIRF dye with dual imaging-PDT capabilities was synthesized and displayed superior specificity compared to other small PSMA targeted molecules.

Since the very beginnings of the chemical exchange saturation transfer (CEST) technique, poor overall sensitivity has appeared to be one of its strongest limitations for future applications. Research has therefore focused on designing systems, such as supramolecular and nanosized agents, that contain a high number of magnetically equivalent mobile spins. However, the number of mobile spins offered by these systems is still limited by their composition and surface/volume ratio. The design of compartmentalized agents, that is, systems where an aqueous inner core is separated from the MRI-detected bulk pool via a semipermeable barrier/membrane, is very much a step forward for the technique. These vesicular systems can (i) act as biocompatible and versatile carriers for dia-, para-, and hetero-nuclear CEST probes, thus offering new application options; and (ii) act as CEST probes themselves via the encapsulation of a suitable agent (e.g., a paramagnetic shift reagent) that can change the resonance frequency of the spin pool in the inner compartment only. LipoCEST agents were the pioneers in the latter category, as they are able to grant picomolar sensitivity (in terms of nanoparticle concentration), and paved the way for new applications for CEST agents, especially in the theranostic research area. The use of larger, natural vesicular systems, such as yeasts and cells, in which the huge number of intravesicular spins lowers the detection threshold to a femtomolar limit, is a further step forward in the development of compartmentalized CEST agents. Finally, interesting combinations of nanovesicular and cellular compartmentalized systems have been proposed, thus highlighting how the approach has the potential to drive CEST agents towards completing their journey to mature clinical translation.

Novel Mn(II)-based nanoprobes were rationally designed as high contrast enhancing agents for magnetic resonance imaging (MRI) and obtained by anchoring a Mn(II)-CDTA derivative to the surface of organo-modified silica nanoparticles (SiNPs). Large payloads of paramagnetic metal-chelates have been immobilized on biocompatible SiNPs with spherical shape and narrow size distribution of 80-90 nm, resulting in a relaxivity gain of 250% at clinical fields (0.5 T) as compared to the free chelate. Such substantial efficacy enhancement of the nanoprobes is mainly attributed to the restriction of the rotational dynamics of the conjugated complex, as revealed by comprehensive 1H-NMR relaxometric investigations. The paramagnetic nanospheres exhibit good colloidal stability over time in biological matrices, allowing for MRI applications. High image contrast was found in T1w-MRI images collected at 1 T on phantoms containing relatively small amounts of contrast agent (CA), for which low cellular toxicity was observed on three different cell lines. Preliminary in vivo studies on healthy mice demonstrated the efficiency of the novel Mn-based silica nanoparticle as T1w-MRI probes, resulting in significant contrast enhancement in the liver. These findings demonstrate that these novel Mn-SiNPs are high efficacy CAs suitable for preclinical MRI applications.

Rationale: The tireless research for effective drug delivery approaches is prompted by poor target tissue penetration and limited selectivity against diseased cells. To overcome these issues, various nano- and micro-carriers have been developed so far, but some of them are characterized by slow degradation time, thus hampering repeated drug administrations. The aim of this study was to pursue a selective delivery of magnetic biodegradable polyelectrolyte capsules in a mouse breast cancer model, using an external magnetic field. Methods: Four different kinds of magnetic polyelectrolyte capsules were fabricated via layer-by-layer assembly of biodegradable polymers on calcium carbonate templates. Magnetite nanoparticles were embedded either into the capsules' shell (sample S) or both into the shell and the inner volume of the capsules (samples CnS, where n is the number of nanoparticle loading cycles). Samples were first characterized in terms of their relaxometric and photosedimentometric properties. In vitro magnetic resonance imaging (MRI) experiments, carried out on RAW 264.7 cells, allowed the selection of two lead samples that proceeded for the in vivo testing on a mouse breast cancer model. In the set of in vivo experiments, an external magnet was applied for 1 hour following the intravenous injection of the capsules to improve their delivery to tumor, and MRI scans were acquired at different time points post administration. Results: All samples were considered non-cytotoxic as they provided more than 76% viability of RAW 264.7 cells upon 2 h incubation. Sample S appeared to be the most efficient in terms of T2-MRI contrast, but the less sensitive to external magnet navigation, since no difference in MRI signal with and without the magnet was observed. On the other side, sample C6S was efficiently delivered to the tumor tissue, with a three-fold T2-MRI contrast enhancement upon the external magnet application. The effective magnetic targeting of C6S capsules was also confirmed by the reduction in T2-MRI contrast in spleen if compared with the untreated with magnet mice values, and the presence of dense and clustered iron aggregates in tumor histology sections even 48 h after the magnetic targeting. Conclusion: The highlighted strategy of magnetic biodegradable polyelectrolyte capsules' design allows for the development of an efficient drug delivery system, which through an MRI-guided externally controlled navigation may lead to a significant improvement of the anticancer chemotherapy performance.

Nanogels (NGs) obtained by electrostatic interactions between chitosan and hyaluronic acid and comprising paramagnetic Gd chelates are gaining increasing attention for their potential application in magnetic resonance bioimaging. Herein, the macrocyclic complexes [Gd(DOTP)]

Water 1H relaxivity profiles of macrocyclic complexes [Gd(DOTP)]5−, lacking metal-bound water molecules (q = 0), confined or used as a cross-linker in nanogels (NGs) obtained by electrostatic interactions between chitosan and hyaluronic acid.

Simple Summary Lung cancer and in particular non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related death. The development of new therapeutic approaches, including immunotherapy, has led to substantial improvement in survival time and quality of life. However, the clinical benefit of immunotherapy-based strategies is still limited to a minority of patients, reflecting the need to identify predictive biomarkers of response, which are any substance, structure, or process or its products that can be measured in the body and that can influence or predict clinical response. In this work, we provide an overview of the approved and the most promising investigational biomarkers, which have been assessed in vitro/ex vivo and in vivo, to identify patients who could benefit the most from immunotherapy-based treatment. Abstract Lung cancer remains the leading cause of cancer-related death, and it is usually diagnosed in advanced stages (stage III or IV). Recently, the availability of targeted strategies and of immunotherapy with checkpoint inhibitors (ICI) has favorably changed patient prognosis. Treatment outcome is closely related to tumor biology and interaction with the tumor immune microenvironment (TME). While the response in molecular targeted therapies relies on the presence of specific genetic alterations in tumor cells, accurate ICI biomarkers of response are lacking, and clinical outcome likely depends on multiple factors that are both host and tumor-related. This paper is an overview of the ongoing research on predictive factors both from in vitro/ex vivo analysis (ranging from conventional pathology to molecular biology) and in vivo analysis, where molecular imaging is showing an exponential growth and use due to technological advancements and to the new bioinformatics approaches applied to image analyses that allow the recovery of specific features in specific tumor subclones.

D-Glucose and 3-O-Methyl-D-glucose (3OMG) have been shown to provide contrast in magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) images. However, a systematic comparison between these two molecules has yet to be performed. The current study deals with the assessment of the effect of pH, saturation power level (B1 ) and magnetic field strength (B0 ) on the MRI-CEST contrast with the aim of comparing the in vivo CEST contrast detectability of these two agents in the glucoCEST procedure. Phosphate-buffered solutions of D-Glucose or 3OMG (20 mM) were prepared at different pH values and Z-spectra were acquired at several B1 levels at 37°C. In vivo glucoCEST images were obtained at 3 and 7 T over a period of 30 min after injection of D-Glucose or 3OMG (at doses of 1.5 or 3 g/kg) in a murine melanoma tumor model (n = 3-5 mice for each molecule, dose and B0 field). A markedly different pH dependence of CEST response was observed in vitro for D-Glucose and 3OMG. The glucoCEST contrast enhancement in the tumor region following intravenous administration (at the 3 g/kg dose) was comparable for both molecules: 1%-2% at 3 T and 2%-3% at 7 T. The percentage change in saturation transfer that resulted was almost constant for 3OMG over the 30-min period, whereas a significant increase was detected for D-Glucose. Our results show similar CEST contrast efficiency but different temporal kinetics for the metabolizable and the nonmetabolizable glucose derivatives in a tumor murine model when administered at the same doses.

Breast cancer is the most common cancer among women worldwide. For high-risk women, contrast enhanced (CE)-magnetic resonance imaging (MRI) is recommended as supplemental screening together with mammography. The development of new MRI contrast agents is an active field of research, which requires efficacy tests on appropriate preclinical pathological models. In this work, a refined method to orthotopically induce breast cancer in BALB/c mice was developed using ultrasound (US) as a guide for the precise localisation of the tumour induction site and to improve animal welfare. The method was coupled with CE-MRI to characterise the evolution of the tumoural lesion.

Immunotherapy is an effective therapeutic option for several cancers. In the last years, the introduction of checkpoint inhibitors (ICIs) has shifted the therapeutic landscape in oncology and improved patient prognosis in a variety of neoplastic diseases. However, to date, the selection of the best patients eligible for these therapies, as well as the response assessment is still challenging. Patients are mainly stratified using an immunohistochemical analysis of the expression of antigens on biopsy specimens, such as PD-L1 and PD-1, on tumor cells, on peritumoral immune cells and/or in the tumor microenvironment (TME). Recently, the use and development of imaging biomarkers able to assess in-vivo cancer-related processes are becoming more important. Today, positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) is used routinely to evaluate tumor metabolism, and also to predict and monitor response to immunotherapy. Although highly sensitive, FDG-PET in general is rather unspecific. Novel radiopharmaceuticals (immuno-PET radiotracers), able to identify specific immune system targets, are under investigation in pre-clinical and clinical settings to better highlight all the mechanisms involved in immunotherapy. In this review, we will provide an overview of the main new immuno-PET radiotracers in development. We will also review the main players (immune cells, tumor cells and molecular targets) involved in immunotherapy. Furthermore, we report current applications and the evidence of using [18F]FDG PET in immunotherapy, including the use of artificial intelligence (AI).

PurposeTriple-negative breast cancer (TNBC) patients have usually poor outcome after chemotherapy and early prediction of therapeutic response would be helpful. [18F]F-FDG-PET/CT acquisitions are often carried out to monitor variation in metabolic activity associated to response to the therapy, despite moderate accuracy and radiation exposure limit its application. The glucoCEST technique relies on the use of unlabelled D-glucose to assess glucose uptake with conventional MRI scanners and is currently under active investigations at clinical level. This work aims at validating the potential of MRI-glucoCEST in monitoring early therapeutic responses in a TNBC tumor murine model.ProceduresBreast tumor (4T1) bearing mice were treated with doxorubicin or dichloroacetate for one week. PET/CT with [18F]F-FDG and MRI-glucoCEST were performed at baseline and after 3 cycles of treatment. Metabolic changes measured with [18F]F-FDG-PET and glucoCEST were compared and evaluated with changes in tumor volumes.ResultsDoxorubicin treated mice showed a significant decrease in tumor growth when compared to the control group. GlucoCEST imaging provided early metabolic response after three cycles of treatment, conversely, no variations were detect by in [18F]F-FDG uptake. Dichloroacetate treated mice did not show any decrease either in tumor volume or in tumor metabolic activity as assessed by both glucoCEST and [18F]F-FDG-PET.ConclusionsEarly metabolic changes during doxorubicin treatment can be predicted by glucoCEST imaging that appears more sensitive than [18F]F-FDG-PET in reporting on early therapeutic response. These findings support the view that glucoCEST may be a sensitive technique for monitoring metabolic response, but future studies are needed to explore the accuracy of this approach in other tumor types and treatments.

PurposeD-Glucose and 3-O-Methyl-D-glucose (3OMG) have been shown to provide contrast in MRI-CEST images. However, a systematic comparison between these two molecules has not yet been performed. This study dealt with the assessment of the effect of pH, saturation power level (B1) and magnetic field strength (B0) on the MRI-CEST contrast with the aim of comparing the in vivo CEST contrast detectability of these two agents in the glucoCEST procedure.MethodsPhosphate buffered solutions of D-Glucose or 3OMG (20 mM) were prepared at different pH values and Z-spectra acquired at several B1 levels and at 37°C. In vivo glucoCEST images were obtained at 3 T and 7 T over a period of 30 min after injection of D-Glucose or 3OMG (at the doses of 1.5 and 3 g/kg) in a murine melanoma tumour model.ResultsA markedly different pH dependence of CEST response was observed in vitro for D-Glucose and 3OMG. The glucoCEST contrast enhancement in the tumour region following the intravenous administration (at the dose 3 g/kg) resulted to be comparable for both the molecules: 1-2% at 3 T and 2-3% at 7 T. The ST% resulted almost constant for 3OMG over the 30 min period, whereas a significant increase in the case of D-Glucose was detected.ConclusionOur results show similar CEST contrast efficiency but different temporal kinetics for the metabolizable and the non-metabolizable glucose derivatives in tumour murine models when administered at the same doses.

Triple-negative breast cancer (TNBC) patients have usually poor outcome after chemotherapy and early prediction of therapeutic response would be helpful. [Breast tumor (4T1)-bearing mice were treated with doxorubicin or dichloroacetate for 1 week. PET/CT with [Doxorubicin-treated mice showed a significant decrease in tumor growth when compared to the control group. GlucoCEST imaging provided metabolic response after three cycles of treatment. Conversely, no variations were detected in [Metabolic changes during doxorubicin treatment can be predicted by glucoCEST imaging that appears more sensitive than [

Immunotherapy is an effective therapeutic option for several cancers. In the last years, the introduction of checkpoint inhibitors (ICIs) has shifted the therapeutic landscape in oncology and improved patient prognosis in a variety of neoplastic diseases. However, to date, the selection of the best patients eligible for these therapies, as well as the response assessment is still challenging. Patients are mainly stratified using immunohistochemical analysis of the expression of anti-gens on biopsy specimens, such as PD-L1 and PD-1, on tumor cells, on peritumoral immune cells, and/or in the tumor microenvironment (TME). Recently, the use and development of imaging biomarkers able to assess in-vivo cancer-related processes are becoming more important. Today, positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) is used routinely to evaluate tumor metabolism, and also to predict and monitor response to immunotherapy. Although highly sensitive, FDG-PET, in general, is rather unspecific. Novel radiopharmaceuticals (immuno-PET radiotracers) able to identify specific immune system targets are under investigation in pre-clinical and clinical settings. In this review, we will provide an overview of the main new immuno-PET radiotracers in development. We will also review the main players (immune cells, tumor cells, and molecular targets) involved in immunotherapy. Furthermore, we report current applications and the evidence of using [18F]FDG PET in immunotherapy, including the use of artificial intelligence (AI).

Sonodynamic Therapy (SDT) is a new anticancer strategy based on ultrasound (US) technique and is derived from photodynamic therapy (PDT), SDT is still, however, far from clinical application. In order to move this therapy forward from bench to bedside, investigations have been focused on treatment selectivity between cancer cells and normal cells. As a result, the effects of the porphyrin activation by SDT on cancer (HT-29) and normal (HDF 106-05) cells were studied in a co-culture evaluating cell cytotoxicity, reactive oxygen species (ROS) production, mitochondrial function and plasma membrane fluidity according to the bilayer sonophore (BLS) theory. While PDT induced similar effects on both HT-29 and HDF 106-05 cells in co-culture, SDT elicited significant cytotoxicity, ROS production and mitochondrial impairment on HT-29 cells only, whereas HDF 106-05 cells were unaffected. Notably, HT-29 and HDF 106-05 showed different cell membrane fluidity during US exposure. In conclusion, our data demonstrate a marked difference between cancer cells and normal cells in co-culture in term of responsiveness to SDT, suggesting that this different behavior can be ascribed to diversity in plasma membrane properties, such as membrane fluidity, according to the BLS theory.

Molecular Imaging (MI) protocols require the availability of tailored probes that are specific for reporting on the selected event. The design of imaging probes relies on the setup of synthetic procedures for the preparation of the imaging reporters and the targeting vectors, as well as efficient bioconjugation routes to link the targeting and reporting moieties, eventually organized in nanosized macromolecular and supramolecular systems. The design of molecular imaging probes is dictated by the chosen imaging modality. Several chemical factors must be optimized in order to affect their in vivo behavior such as (i) the intravascular half-life (to allow the agents to accumulate at the target site), (ii) the binding affinity eventually through the design of multivalency systems, (iii) the capacity to carry a high payload of probe molecules able to generate strong contrast effects (particularly relevant for imaging modalities endowed with low sensitivity or for targeting procedures involving a low number of targeting epitopes); (iv) to modulate biodistribution, excretion, and toxicity properties. Special attention must be devoted to search for efficient conjugation procedures that occur under mild experimental conditions and selected examples of activated procedures (including the application of click chemistry applications) are reported. The chapter surveys also agents and procedures for cellular labeling including endosomal uptake by pinocytosis and electroporation, receptor-mediated endocytosis, phagocytosis and labeling at the outer cell surface.

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a−/− mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a−/− basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a−/− Lyn−/− showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a−/− hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.

Medulloblastoma is the most common malignant brain tumor in children. Current treatment for medulloblastoma consists of surgery followed by irradiation of the whole neuraxis and high-dose multiagent chemotherapy, a partially effective strategy associated with highly invalidating side effects. Therefore, identification and validation of novel target molecules capable of contrasting medulloblastoma growth without disturbing brain development is needed. Citron kinase protein (CITK), encoded by primary microcephaly gene MCPH17, is required for normal proliferation and survival of neural progenitors. Constitutive loss of CITK leads to cytokinesis failure, chromosome instability, and apoptosis in the developing brain, but has limited effects on other tissues. On this basis, we hypothesized that CITK could be an effective target for medulloblastoma treatment. In medulloblastoma cell lines DAOY and ONS-76, CITK knockdown increased both cytokinesis failure and DNA damage, impairing proliferation and inducing cell senescence and apoptosis via TP53 or TP73. Similar effects were obtained in the NeuroD-SmoA1 transgenic mouse model, in which CITK deletion increased apoptotic cells and senescence markers such as P21CIP1, P27KIP1, and P16INK4A. Most importantly, CITK deletion decreased tumor growth and increased overall survival in these mice, with no apparent side effects. These results suggest that CITK can be a useful molecular target for medulloblastoma treatment. Significance: In vitro and in vivo proof of concept identifies citron kinase protein as a suitable target for medulloblastoma treatment. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4599/F1.large.jpg. Cancer Res; 78(16); 4599–612. ©2018 AACR.

Magnetic Resonance Imaging (MRI) enables to provide anatomical, functional and molecular information of pathological angiogenesis when used with properly tailored imaging probes. Functional studies have been the domain of Dynamic Contrast Enhancement (DCE) -MRI protocols from which it is possible to extract quantitative estimations on key parameters such as the volumes of vascular and extracellular compartments and the rates of the bidirectional exchange of the imaging reporters across the endothelial barrier. Whereas paramagnetic Gd-complexes able to reversibly bind to serum albumin act better than the clinically used small-sized, hydrophilic species, new findings suggest that an accurate assessment of the vascular volume is possible by analyzing images acquired upon the i.v. administration of Gd-labelled Red Blood Cells (RBCs). As far as it concerns molecular MRI, among the many available biomarkers, αvβ3 integrins are the most investigated ones. The low expression of these targets makes mandatory the use of nano-sized systems endowed with the proper signal enhancing capabilities. A number of targeted nano-particles have been investigated including micelles, liposomes, iron oxides and perfluorocarbon containing systems. Finally, a growing attention is devoted to the design and testing of "theranostic" agents based on the exploitation of MRI to monitor drug delivery processes and therapeutic outcome.

Although nowadays there is a renewed and growing interest in Mn-based contrast agents, there are only few studies dealing with Mn-based lipophilic nanoparticles and how they may be optimized as MRI contrast agents. Three amphiphilic paramagnetic Mn(II) complexes based on derivatives of EDTA and 1,4-DO2A were used for the preparation of lipidic nanoparticles. The length and position of the aliphatic chains were found to control whether either vesicular liposomes, nonvesicular bicelles, or a mixture of both was produced as well as the size and morphology of phospholipid-based self-assembling nanoaggregates. These differences determine whether hydrophilic Gd-based contrast agents or fluorescent dyes can be entrapped in the aqueous core of the nanoaggregate. Structural characterization was performed by cryo-TEM. Detailed

One of the most challenging tasks of the cancer research is the enhancement of the amount of the chemotherapeutic agent that can reach the target site. To achieve this goal, nanovectors capable of encapsulating the drug and releasing it following a specific stimulus have been developed. In light of this, a key point is the necessity to monitor the effective drug release through a safe and highly performing imaging technology such as Magnetic Resonance Imaging (MRI). Liposomes are highly biocompatible nanovesicles that consist of bilayered phospholipid-based membrane encompassing an aqueous core. Almost 20 drug-loaded liposomes are currently approved for clinical use in USA and EU countries. If a liposomal nanomedicine is loaded with an MRI agent whose contrast is sensitive to the microenvironment and with a release kinetics similar to the co-transported drug, the system can act as an imaging reporter of drug release. This Perspective will offer a critical and brief overview of using MRI not only to verify and monitor the release process but also as a valuable tool to predict the therapeutic outcome. In particular, it will be presented representatives preclinical studies illustrating the in vivo potential of MRI-guided drug release protocols triggered by thermal and mechanical ultrasound-induced effects. Considering the therapeutic advantages of this approach, the possible benefits in reducing the side effects and the good results reported at preclinical level, there is a reasonable hope that the near future could witness the entrance in clinical routine of MRI-guided procedures supporting ultrasound-induced drug release protocols.

Different classes of small- or nano-sized calcium-sensitive probes for magnetic resonance imaging (MRI) have been proposed in the last two decades. These compounds have been developed mainly for functional MRI purposes and tested in vivo in different animal models. Most of them are paramagnetic systems that change their relaxivity in the presence of the divalent ion calcium, resulting in increased T-1 or T-2 contrast. In this work, we report the investigation of their relaxometric behavior at low magnetic fields, specifically the comparison of the monomeric Ca-sensitive probe and the corresponding dendrimer conjugates of generations 0, 1 and 2 (G0, G1 and G2, respectively). As a result, a relaxivity hump between 10 and 100 MHz of the Larmor frequency progressively appeared with an increase in the size of the investigated contrast agent, indicative of a restricted rotational motion of the complexes as long as the size of the molecule increases. The same trend with a more pronounced effect was detectable in the presence of calcium. The relaxivity enhancement for the Ca2+ adducts, primarily caused by an increase of the hydration state of Gd3+, went from ca. 130% for the monomeric probe to ca. 310% for the G2 dendrimer conjugate at 0.5 T and 25 degrees C. T-1 weighted magnetic resonance images acquired at 1 T displayed the strong ability of these systems to change their contrast according to the presence of calcium at this field, thus laying the basis for promising future in vivo applications.

Prostate cancer (PCa), the most widespread male cancer in western countries, is generally eradicated by surgery, especially if localized. However, during surgical procedures, it is not always possible to identify malignant tissues by visual inspection. Among the possible consequences, there is the formation of positive surgical margins, often associated with recurrence. In this work, the gastrin-releasing peptide receptor (GRPR), overexpressed in the prostatic carcinoma and not in healthy tissues or in benign hyperplasia (BPH), is proposed as target molecule to design a novel near-infrared fluorescent (NIRF) probe for image-guided prostatectomy. The NIRF dye Sulfo-Cy5.5 was conjugated to a Bombesin-like peptide (BBN), targeting GRPR. The final product, called BBN-Cy5.5, was characterized and tested in vitro on PC-3, DU145, and LnCAP cell lines, using unconjugated Sulfo-Cy5.5 as control. In vivo biodistribution studies were performed by optical imaging in PC-3 tumor-bearing and healthy mice. Finally, simulation of the surgical protocol was carried out. BBN-Cy5.5 showed high water solubility and a good relative quantum yield. The ability of the probe to recognize the GRPR, highly expressed in PC-3 cells, was tested both in vitro and in vivo, where a significant tumor accumulation was achieved 24 h post-injection. Furthermore, a distinguishable fluorescent signal was visible in mice bearing PCa, when the surgery was simulated. By contrast, low signal was found in healthy or BPH-affected mice. This work proposes a new NIRF probe ideal to target GRPR, biomarker of PCa. The promising data obtained suggest that the dye could allow the real-time intraoperative visualization of prostate cancer.

The synthesis and characterization of a novel HPDO3A-based ligand having a C16 alkyl chain and its Eu3+, Gd3+ and Yb3+ complexes are reported. These amphiphilic paramagnetic complexes can form micelles with very good stability both in phosphate buffer and in human serum. A high number of Ln-complexes (ca. 200 molecules) are present in the micelle, providing this system with good sensitivity (μM in terms of micelle concentration) for MRI detection. Moreover, it has been found that the cell toxicity of the micelles may be reduced by adding DSPE-PEG2000 in the formulation. Both relaxometric and CEST properties of the micelles were investigated in detail. The micelles loaded with Eu- and Yb-HPDO3A complexes, similar to what was reported for the water-soluble analogs, act as pH-sensors and appear to be suitable for CEST multicolor experiments.

Amphiphilic Janus-dendrimers are able to self-assemble into nanosized vesicles named dendrimersomes. We recently synthesized the 3,5-C12-EG-(OH)4 dendrimer that generates dendrimersomes with very promising safety and stability profiles, that can be loaded with different contrast agents for in vivo imaging. In this contribution, nanovesicles were loaded with both the Magnetic Resonance Imaging (MRI) reporter GdDOTAGA(C18)2 and the glucocorticoid drug Prednisolone Phosphate (PLP), in order to test their effective potential as theranostic nanocarriers on murine melanoma tumour models. The incorporation of GdDOTAGA(C18)2 into the membrane resulted in dendrimersomes with a high longitudinal relaxivity (r1=39.1mM-1s-1, at 310K and 40MHz) so that, after intravenous administration, T1-weighted MRI showed a consistent contrast enhancement in the tumour area. Furthermore, the nanovesicles encapsulated PLP with good efficiency and displayed anti-tumour activity both in vitro and in vivo, thus enabling their practical use for biomedical theranostic applications.

In this study, murine Mesenchymal Stem Cells (MSCs) labeled with the clinically approved MRI agent Gadoteridol through a procedure based on the hypo-osmotic shock were successfully tracked in vivo in a murine model of Spinal Cord Injury (SCI). With respect to iso-osmotic incubations, the hypo-osmotic labeling significantly increased the Gd(3+) cellular uptake, and enhanced both the longitudinal relaxivity (r1) of the intracellular Gadoteridol and the Signal to Noise Ratio (SNR) measured on cell pellets, without altering the biological and functional profile of cells. A substantial T1 Contrast Enhancement after local transplantation of 3.0×10(5) labeled cells in SCI mice enabled to follow their migratory dynamics in vivo for about 10days, and treated animals recovered from the motor impairment caused by the injury, indicating unaltered therapeutic efficacy. Finally, analytical and histological data corroborated the imaging results, highlighting the opportunity to perform a precise and reliable monitoring of the cell-based therapy.

The work aimed at developing a novel MRI-based theranostic protocol for improving the anticancer efficacy of a Doxil-like liposomal formulation. The goal was achieved stimulating the intratumor release of the drug from the nanocarrier and favoring its diffusion in the lesion by the sequential application of low-intensity pulsed ultrasound. The protocol was tested on mice bearing a syngeneic breast cancer model. The combination of acoustic waves with different characteristics allowed for: i) the release of the drug and the co-encapsulated MRI agent (Gadoteridol) from the liposomes in the vessels of the tumor region, and ii) the extravasation of the released material, as well as intact liposomes, in the tumor stroma. The MR-T1 contrast enhancement measured in the tumor reported on the delivery and US-triggered release of Doxorubicin. The developed protocol resulted in a marked increase in the intratumor drug concentration that, in turn, led to the complete regression of the lesion. The protocol has a good clinical translatability because all the components of the theranostic agent (Doxorubicin, liposomes, Gadoteridol) are approved for human use.

Two-dimensional (2D) cell cultures, in which cells grow in flat layers on plastic surfaces, are considered the standard model for use in drug screening and for biological assays. However, these models do not accurately represent in vivo cell organization due to a lack in cell-cell/matrix interactions and in tissue and microenvironment structure. For that reason, three-dimensional (3D) cell cultures have been introduced as an innovative platform in recent years, allowing cells to grow and interact with each other in all three dimensions thanks to an artificial environment. In a 3D model cells show more interesting aspects from a physiological point of view, demonstrating several improvements in viability, morphology, proliferation and differentiations, response to external and internal stimuli, drug metabolism and efficacy and in vivo relevance. This review explores recent techniques in the development of 3D cell models with a particular focus on their application from a pharmacological point of view, starting from the concept of spheroid models generated by scaffold-free or scaffold-based techniques. Finally, special attention is paid to the concept of organoids, 3D constructs that replicate the 3D architecture of intact organs and the technology involved.

Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34

Meningioma in vivo research is hampered by the difficulty of establishing an easy and reproducible orthotopic model able to mimic the characteristics of a human meningioma. Moreover, leptomeningeal dissemination and high mortality are often associated with such orthotopical models, making them useless for clinical translation studies. An optimized method for inducing meningiomas in nude mice at two different sites is described in this paper and the high reproducibility and low mortality of the models are demonstrated. Skull base meningiomas were induced in the auditory meatus and convexity meningiomas were induced on the brain surface of 23 and 24 nude mice, respectively. Both models led to the development of a mass easily observable by imaging methods. Dynamic contrast enhanced MRI was used as a tool to monitor and characterize the pathology onset and progression. At the end of the study, histology was performed to confirm the neoplastic origin of the diseased mass.