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Glioblastoma multiforme is generally recalcitrant to current surgical and local radiotherapeutic approaches. Moreover, systemic chemotherapeutic approaches are impeded by the blood–tumor barrier. To circumvent limitations in the latter area, we developed a multicomponent, chain-like nanoparticle that can penetrate brain tumors, composed of three iron oxide nanospheres and one drug-loaded liposome linked chemically into a linear chain-like assembly. Unlike traditional small-molecule drugs or spherical nanotherapeutics, this oblong-shaped, flexible nanochain particle possessed a unique ability to gain access to and accumulate at glioma sites. Vascular targeting of nanochains to the αvβ3 integrin receptor resulted in a 18.6-fold greater drug dose administered to brain tumors than standard chemotherapy. By 2 hours after injection, when nanochains had exited the blood stream and docked at vascular beds in the brain, the application of an external low-power radiofrequency field was sufficient to remotely trigger rapid drug release. This effect was produced by mechanically induced defects in the liposomal membrane caused by the oscillation of the iron oxide portion of the nanochain. In vivo efficacy studies conducted in two different mouse orthotopic models of glioblastoma illustrated how enhanced targeting by the nanochain facilitates widespread site-specific drug delivery. Our findings offer preclinical proof-of-concept for a broadly improved method for glioblastoma treatment. Cancer Res; 75(7); 1356–65. ©2015 AACR.

Supplementary Fig. S1. Representative in vivo and ex vivo images using a Maestro FLEX In Vivo Imaging System. Supplementary Fig. S2. Effect of the RF field on the temperature of brain tissues. Supplementary Fig. S3. Bright field microscopy and fluorescence was performed on an entire histological section of the brain stained with Hematoxylin-Eosin (green: CNS-1 glioma cells (GFP)). The same histological section shown in Fig. 4A was used (left panel). Higher magnification of the same section shows the H&E stain of an invasive site (right panel). Supplementary Fig. S4. Cytotoxicity of nanochain particles on CNS-1 cells. Supplementary Fig. S5. Weight progression of animals bearing orthotopic (A) CNS-1 or (B) 9L glioma tumors after DOX treatments.

IntroductionPlacenta accreta spectrum (PAS) occurs when the placenta is pathologically adherent to the myometrium. An intact retroplacental clear space (RPCS) is a marker of normal placentation, but visualization with conventional imaging techniques is a challenge. In this study, we investigate use of an FDA-approved iron oxide nanoparticle, ferumoxytol, for contrast-enhanced magnetic resonance imaging of the RPCS in mouse models of normal pregnancy and PAS. We then demonstrate the translational potential of this technique in human patients presenting with severe PAS (FIGO Grade 3C), moderate PAS (FIGO Grade 1), and no PAS.MethodsA T1-weighted gradient recalled echo (GRE) sequence was used to determine the optimal dose of ferumoxytol in pregnant mice. Pregnant Gab3-/-mice, which demonstrate placental invasion, were then imaged at day 16 of gestation alongside wild-type (WT) pregnant mice which do not demonstrate invasion. Signal-to-noise ratio (SNR) was computed for placenta and RPCS for all fetoplacental units (FPUs) with ferumoxytol-enhanced magnetic resonance imaging (Fe-MRI) and used for the determination of contrast-to-noise ratio (CNR). Fe-MRI was also performed in 3 pregnant subjects using standard T1 and T2 weighted sequences and a 3D magnetic resonance angiography (MRA) sequence. RPCS volume and relative signal were calculated in all three subjects.ResultsFerumoxytol administered at 5 mg/kg produced strong T1 shortening in blood and led to strong placental enhancement in Fe-MRI images. Gab3-/-mice demonstrated loss of hypointense region characteristic of the RPCS relative to WT mice in T1w Fe-MRI. CNR between RPCS and placenta was lower in FPUs of Gab3-/-mice compared to WT mice, indicating higher degrees of vascularization and interruptions throughout the space. In human patients, Fe-MRI at a dose of 5 mg/kg enabled high uteroplacental vasculature signal and quantification of the volume and signal profile in severe and moderate invasion of the placenta relative to a non-PAS case.DiscussionFerumoxytol, an FDA-approved iron oxide nanoparticle formulation, enabled visualization of abnormal vascularization and loss of uteroplacental interface in a murine model of PAS. The potential of this non-invasive visualization technique was then further demonstrated in human subjects. Diagnosis of placental invasion using Fe-MRI may provide a sensitive method for clinical detection of PAS.

Prior preclinical studies established the utility of liposomal nanoparticle blood-pool contrast agents in visualizing the retroplacental clear space (RPCS), a marker of normal placentation, while sparing fetuses from exposure because the agent does not cross the placental barrier. In this work, we characterized RPCS disruption in a mouse model of placenta accreta spectrum (PAS) using these agents.Contrast-enhanced MRI (CE-MRI) and computed tomography (CE-CT) using liposomal nanoparticles bearing gadolinium (liposomal-Gd) and iodine were performed in pregnant Gab3Pregnant Gab3Imaging of the Gab3

IntroductionPrior preclinical studies established the utility of liposomal nanoparticle blood-pool contrast agents in visualizing the retroplacental clear space (RPCS), a marker of normal placentation, while sparing fetuses from exposure because the agent does not cross the placental barrier. In this work we characterized RPCS disruption in a mouse model of placenta accreta spectrum (PAS) using these agents.MethodsContrast-enhanced MRI (CE-MRI) and computed tomography (CE-CT) using liposomal nanoparticles bearing gadolinium (liposomal-Gd) and iodine were performed in pregnant Gab3-/- and wild type (WT) mice at day 16 of gestation. CE-MRI was performed on a 1T scanner using a 2D T1-weighted sequence (100×100×600 µm3 voxels) and CE-CT was performed at a higher resolution (70×70×70 µm3 voxels). Animals were euthanized post-imaging and feto-placental units (FPUs) were harvested for histological examination. RPCS conspicuity was scored through blinded assessment of images.ResultsPregnant Gab3-/- mice show elevated rates of complicated pregnancy. Contrast-enhanced imaging demonstrated frank infiltration of the RPCS of Gab3-/- FPUs. RPCS in Gab3-/- FPUs was smaller in volume, demonstrated a heterogeneous signal profile, and received lower conspicuity scores than WT FPUs. Histology confirmed in vivo findings and demonstrated staining consistent with a thinner RPCS in Gab3-/- FPUs.DiscussionImaging of the Gab3-/- mouse model at late gestation with liposomal contrast agents enabled in vivo characterization of morphological differences in the RPCS that could cause the observed pregnancy complications. An MRI-based method for visualizing the RPCS would be valuable for early detection of invasive placentation.

MYCN is a major driver for neuroblastoma (NB) and the tyrosine hydroxylase (TH)-MYCN transgenic mouse model is extensively used for preclinical NB studies. However, spatio-temporal NB progression in the TH-MYCN model has not been studied, and questions remain about the value of implanted models as a surrogate for transgenic mice. In this work, we used magnetic resonance imaging (MRI) to study tumor progression and nanoparticle contrast-enhanced computed tomography (n-CECT) to assess tumor vascular architecture in TH-MYCN transgenic mice (2–7 weeks of age) and TH-MYCN+/+-derived orthotopic allograft and syngeneic mice (2–5 weeks post-tumor implantation). Tumors in TH-MYCN transgenic mice became evident in the abdominal paraspinal region at week 5. A delayed thoracic paraspinal mass became evident at week 6 and most mice succumbed by week 7. In allograft and syngeneic mice, single mass tumor growth was restricted to the peritoneal cavity. N-CECT revealed a predominantly microvascular network in TH-MYCN tumors while implanted tumors exhibited heterogeneous and tortuous vessels. N-CECT quantitative analysis demonstrated high vascularity (tumor fractional blood volume ~ 0.12) in all models. Multi-modal imaging of TH-MYCN transgenic and implanted models revealed differences in growth patterns and vascular architecture that should be considered in designing preclinical studies.

Atheromatous lesions are characterized by intrusion into the vascular lumen, resulting in morphological changes to the blood compartment and into the vessel wall, resulting in characteristic molecular and cellular signatures in the solid tissue of the intima, tunica media, adventitia and surrounding tissue. Nanoprobes can be easily formulated to provide long blood-pool residence and molecular targeting, facilitating the imaging of atheromatous changes. Detection of nanoprobes can be accomplished by a variety of methods. We focus in this chapter on the use of cross-sectional imaging techniques, computed tomography (CT) and magnetic resonance imaging (MRI), that facilitate in vivo, noninvasive imaging of the vascular morphology and molecular/cellular signatures of the atheroma. The methods described are suitable for use in animal models, although versions of the probes are being readied for clinical trials, potentially facilitating clinical use in the future.

Background: Sporadic aortic aneurysm and dissection (AAD), caused by progressive aortic smooth muscle cell (SMC) loss and extracellular matrix degradation, is a highly lethal condition. Identifying mechanisms that drive aortic degeneration is a crucial step in developing an effective pharmacologic treatment to prevent disease progression. Recent evidence has indicated that cytosolic DNA and abnormal activation of the cytosolic DNA sensing adaptor STING (stimulator of interferon genes) play a critical role in vascular inflammation and destruction. Here, we examined the involvement of this mechanism in aortic degeneration and sporadic AAD formation. Methods: The presence of cytosolic DNA in aortic cells and activation of the STING pathway were examined in aortic tissues from patients with sporadic ascending thoracic AAD. The role of STING in AAD development was evaluated in Sting -deficient ( Sting gt/gt ) mice in a sporadic AAD model induced by challenging mice with a combination of a high-fat diet and angiotensin II. We also examined the direct effects of STING on SMC death and macrophage activation in vitro. Results: In human sporadic AAD tissues, we observed the presence of cytosolic DNA in SMCs and macrophages and significant activation of the STING pathway. In the sporadic AAD model, Sting gt/gt mice showed significant reductions in challenge-induced aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions. Single-cell transcriptome analysis revealed that aortic challenge in wild-type mice induced the DNA damage response, the inflammatory response, dedifferentiation and cell death in SMCs, and matrix metalloproteinase expression in macrophages. These changes were attenuated in challenged Sting gt/gt mice. Mechanistically, nuclear and mitochondrial DNA damage in SMCs and the subsequent leak of DNA to the cytosol activated STING signaling, which induced cell death through apoptosis and necroptosis. In addition, DNA from damaged SMCs was engulfed by macrophages in which it activated STING and its target interferon regulatory factor 3, which directly induced matrix metalloproteinase-9 expression. We also found that pharmacologically inhibiting STING activation partially prevented AAD development. Conclusions: Our findings indicate that the presence of cytosolic DNA and subsequent activation of cytosolic DNA sensing adaptor STING signaling represent a key mechanism in aortic degeneration and that targeting STING may prevent sporadic AAD development.

Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Patients with high-risk, treatment refractory, or relapse disease have a survival rate of less than 50%. The development of clinically relevant models of these aggressive tumors will facilitate studies to identify drugs that target these cells.Methods: Fresh, whole primary tumor samples were implanted into the livers of immunocompromised mice. Tumor growth was monitored with MRI and ELISA to measure serum human Alpha-fetoprotein (AFP), which is detectable in the blood of tumor-bearing animals. Tumors were validated with immunohistochemistry (IHC) for HB markers Glypican-3 (GPC3) and Beta-catenin; short tandem repeat (STR) DNA validation; next generation sequencing-based mutation profiling of 124 genes involved in pediatric solid tumors; RNA sequencing (RNA-seq), and single cell RNA-seq (scRNA-seq). Lung metastasis was also detected in models with serial sectioning and H&E staining. Cells derived from tumors were grown in vitro in adherent and spheroid conditions and used for high throughput drug screening of candidate agents. Tumors were serially passaged in animals for further in vivo drug testing of novel targeted agents.Results: Nine patient-derived xenograft (PDX) models were generated that represent low- and high-risk tumors, treatment refractory cases, and relapsed tumors. Passaging of these models showed consistent implantation rates at or above 80% with tumors detectable in 2 to 4 weeks. Eight of nine models secrete human serum AFP. All models mimic gene expression and histological patterns of their primary tumor counterparts as well as identical STR DNA profiles. The models also show gene expression consistent with an HB2/high-risk profile according to the Sumazin HB expression signature. Interestingly, two models represent unique sub-clones of a very aggressive HB relapse with different AFP secretion and transcriptomic expression. scRNA-seq of these two models indicated outgrowth of disparate disease sub-clones. The nine models also demonstrate a range of DNA mutations with three or four mutations per tumor; all variants present in the original clinical samples were conserved in the PDX models. Lung metastasis was evident in six of nine models. Two stable patient-derived cell lines (PDCLs) were developed from models, and these cell lines show expression of HB markers and secrete AFP with growth in culture. Drug screening of adherent and spheroid tumor cells support the efficacy of novel targeted agents and indicate a spectrum of sensitivity to cisplatin, a frontline standard chemotherapy agent. Importantly, the models replicate the chemotherapy responses of the corresponding patients. Additional in vitro and in vivo work showed the efficacy of a histone deacetylase inhibitor, panobinostat.Conclusions: These novel orthotopic PDX models of HB fully recapitulate the primary tumors and represent a platform for clinically relevant drug screening and testing. Citation Format: Sarah E Woodfield, Roma H Patel, Andres F Espinoza, Richard S Whitlock, Jessica Epps, Andrew Badachhape, Samuel R Larson, Rohit K Srivastava, Aayushi P Shah, Saiabhiroop R Govindu, Barry Zorman, Brandon J Mistretta, Kevin E Fisher, Ilavarasi Gandhi, Jacquelyn Reuther, Martin Urbicain, Aryana M Ibarra, Sakuni Rankothgedera, Kimberly R Holloway, Stephen F Sarabia, Andras Heczey, Ketan B Ghaghada, Kalyani R Patel, Dolores Lopez-Terrada, Angshumoy Roy, Preethi H Gunaratne, Pavel Sumazin, Sanjeev A Vasudevan. Patient-derived xenograft mouse models of hepatoblastoma for a personalized medicine pipeline [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO013.

Half of high-risk neuroblastoma patients have MYCN amplification. Here, the authors show that MYCN induces fatty acid uptake and synthesis to support neuroblastoma and inhibition of a fatty acid transporter impairs tumor progression in preclinical models.Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. This article has an associated First Person interview with the first author of the paper.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB.Summary StatementIn this work, we developed and thoroughly characterized several unique models of hepatoblastoma derived from the HepT1 cell line that show high-risk features.

Deposition of nanoparticles to tumors often can be enhanced by targeting receptors overexpressed in a tumor. However, a tumor may exhibit a finite number of a biomarker that is accessible and targetable by nanoparticles, limiting the available landing spots. To explore this, we selected two different biomarkers that effectively home nanoparticles in brain tumors. Specifically, we used either an α(v)β(3) integrin-targeting peptide or a fibronectin-targeting peptide as a ligand on nanoparticles termed RGD-NP and CREKA-NP, respectively. In mouse models of glioblastoma multiforme, we systemically injected the nanoparticles loaded with a cytotoxic drug at different doses ranging from 2-8 mg/kg drug. The upper dose threshold of RGD-NP is ~2 mg/kg. CREKA-NP reached its upper dose threshold at 5 mg/kg. For both targeted nanoparticle variants, higher dose did not ensure higher intratumoral drug levels but it contributed to elevated off-target deposition and potentially greater toxicity. A cocktail combining RGD-NP and CREKA-NP was then administered at a dose corresponding to the upper dose threshold for each formulation resulting in a 3-fold higher intratumoral deposition than the individual formulations. The combination of the two different targeting schemes at the appropriate dose for each nanoparticle variant facilitated remarkable increase in intratumoral drug levels that was not achievable by a sole targeting nanoparticle alone.

Introduction Visualization of the retroplacental clear space (RPCS) may provide critical insight into the development of abnormally invasive placenta (AIP). In this pre-clinical study, we characterized the appearance of the RPCS on magnetic resonance imaging (MRI) during the second half of gestation using a liposomal gadolinium contrast agent (liposomal-Gd). Materials and methods Studies were performed in fifteen pregnant C57BL/6 mice at 10, 12, 14, 16, and 18 days of gestation. MRI was performed on a 1T permanent magnet scanner. Pre-contrast and post-contrast images were acquired using T1-weighted gradient-recalled echo (T1w-GRE) and T2-weighted fast spin echo (T2w-FSE) sequences. Animals were euthanized after imaging and feto-placental units harvested for histological examination. Visualization of the RPCS was scored by a maternal-fetal radiologist and quantified by measuring the contrast-to-noise ratio (CNR) on T1w images. Feto-placental features were segmented for analysis of volumetric changes during gestation. Results Contrast-enhanced T1w images enabled the visualization of structural changes in placental development between days 10–18 of gestation. Although the placental margin on the fetal side was clearly visible at all time points, the RPCS was partially visible at day 10 of gestation, and clearly visible by day 12. Hematoxylin and eosin (H&E) staining of the placental tissue corroborated MRI findings of structural and morphological changes in the placenta. Conclusions Contrast-enhanced MR imaging using liposomal-Gd enabled adequate visualization of the retroplacental clear space starting at day 12 of gestation. The agent also enabled characterization of placental structure and morphological changes through gestation.

Objective: Early detection of aortic degeneration is critical for improving outcomes in patients with aortic aneurysm and dissection. We investigated nanoparticle contrast-enhanced computed tomography for the early detection of aortic injury. Approach and Results: In a mouse model of sporadic aortic aneurysm and dissection, C57BL/6J mice were challenged with a high-fat diet and Ang II (angiotensin II) infusion (n=20). Unchallenged control mice received a standard laboratory diet and saline infusion (n=19). Computed tomography angiography (CTA) was performed to evaluate aortic enlargement, and delayed nanoparticle contrast-enhanced computed tomography (CTD) imaging was performed to detect wall signal enhancement indicative of aortic wall degeneration. Aortic segments that exhibited CTD findings but appeared normal on CTA were termed preclinical aortic disease. Aortic aneurysm and dissection development was determined upon the gross examination of excised aortas. Aortic degeneration and inflammation were examined by performing histological and immunofluorescence analyses. Leakage of Evans blue dye into the aortic wall was used to validate changes in vascular permeability. In challenged mice, gross findings of aortic disease were found in 41% of aortic segments. CTA findings of mild disease (dilatation) and advanced disease (aortic aneurysm and dissection with the presence of false lumen) were seen in 33% of aortic segments. CTD findings of wall signal enhancement were seen in 63% of aortic segments. Of those, 48% appeared normal on CTA. Aortic segments with CTD findings showed aortic wall degeneration and inflammation on histological and immunofluorescence analyses. Immunofluorescence analysis and Evans blue dye uptake suggested passive leakage of nanoparticle contrast agent due to endothelial injury as a potential mechanism underlying the detection of aortic disease on CTD. Conclusions: In mice, CTD imaging exhibits high sensitivity for detecting aortic wall degeneration and inflammation before vessel enlargement becomes evident on CTA.

Hepatoblastoma (HB) is the most common pediatric liver malignancy. High-risk patients have poor survival, and current chemotherapies are associated with significant toxicities. Targeted therapies are needed to improve outcomes and patient quality of life. Most HB cases are TP53 wild-type; therefore, we hypothesized that targeting the p53 regulator Murine double minute 4 (MDM4) to reactivate p53 signaling may show efficacy. MDM4 expression was elevated in HB patient samples, and increased expression was strongly correlated with decreased expression of p53 target genes. Treatment with NSC207895 (XI-006), which inhibits MDM4 expression, or ATSP-7041, a stapled peptide dual inhibitor of MDM2 and MDM4, showed significant cytotoxic and antiproliferative effects in HB cells. Similar phenotypes were seen with short hairpin RNA (shRNA)-mediated inhibition of MDM4. Both NSC207895 and ATSP-7041 caused significant upregulation of p53 targets in HB cells. Knocking-down TP53 with shRNA or overexpressing MDM4 led to resistance to NSC207895-mediated cytotoxicity, suggesting that this phenotype is dependent on the MDM4-p53 axis. MDM4 inhibition also showed efficacy in a murine model of HB with significantly decreased tumor weight and increased apoptosis observed in the treatment group. This study demonstrates that inhibition of MDM4 is efficacious in HB by upregulating p53 tumor suppressor signaling.

Objective. Tumor-associated macrophages (TAMs) within the tumor immune microenvironment (TiME) of solid tumors play an important role in treatment resistance and disease recurrence. The purpose of this study was to investigate if nanoradiomics (radiomic analysis of nanoparticle contrast-enhanced images) can differentiate tumors based on TAM burden. Materials and Methods. In vivo studies were performed in transgenic mouse models of neuroblastoma with low (N = 11) and high (N = 10) tumor-associated macrophage (TAM) burden. Animals underwent delayed nanoparticle contrast-enhanced CT (n-CECT) imaging at 4 days after intravenous administration of liposomal-iodine agent (1.1 g/kg). CT imaging-derived conventional tumor metrics (tumor volume and CT attenuation) were computed for segmented tumor CT datasets. Nanoradiomic analysis was performed using a PyRadiomics workflow implemented in the quantitative image feature pipeline (QIFP) server containing 900 radiomic features (RFs). RF selection was performed under supervised machine learning using a nonparametric neighborhood component method. A 5-fold validation was performed using a set of linear and nonlinear classifiers for group separation. Statistical analysis was performed using the Kruskal–Wallis test. Results. N-CECT imaging demonstrated heterogeneous patterns of signal enhancement in low and high TAM tumors. CT imaging-derived conventional tumor metrics showed no significant differences ( p > 0.05 ) in tumor volume between low and high TAM tumors. Tumor CT attenuation was not significantly different ( p > 0.05 ) between low and high TAM tumors. Machine learning-augmented nanoradiomic analysis revealed two RFs that differentiated ( p < 0.002 ) low TAM and high TAM tumors. The RFs were used to build a linear classifier that demonstrated very high accuracy and further confirmed by 5-fold cross-validation. Conclusions. Imaging-derived conventional tumor metrics were unable to differentiate tumors with varying TAM burden; however, nanoradiomic analysis revealed texture differences and enabled differentiation of low and high TAM tumors.

Glioblastomas (GBMs) remain highly lethal. This partially stems from the presence of brain tumor initiating cells (BTICs), a highly plastic cellular subpopulation that is resistant to current therapies. In addition to resistance, the blood-brain barrier limits the penetration of most drugs into GBMs. To effectively deliver a BTIC-specific inhibitor to brain tumors, we developed a multicomponent nanoparticle, termed Fe@MSN, which contains a mesoporous silica shell and an iron oxide core. Fibronectin-targeting ligands directed the nanoparticle to the near-perivascular areas of GBM. After Fe@MSN particles deposited in the tumor, an external low-power radiofrequency (RF) field triggered rapid drug release due to mechanical tumbling of the particle resulting in penetration of high amounts of drug across the blood-brain tumor interface and widespread drug delivery into the GBM. We loaded the nanoparticle with the drug 1400W, which is a potent inhibitor of the inducible nitric oxide synthase (iNOS). It has been shown that iNOS is preferentially expressed in BTICs and is required for their maintenance. Using the 1400W-loaded Fe@MSN and RF-triggered release, in vivo studies indicated that the treatment disrupted the BTIC population in hypoxic niches, suppressed tumor growth and significantly increased survival in BTIC-derived GBM xenografts.

Rationale: Brain arteriovenous malformations (AVMs) are abnormal tangles of vessels where arteries and veins directly connect without intervening capillary nets, increasing the risk of intracerebral hemorrhage and stroke. Current treatments are highly invasive and often not feasible. Thus, effective noninvasive treatments are needed. We previously showed that AVM-brain endothelial cells (BECs) secreted higher VEGF (vascular endothelial growth factor) and lower TSP-1 (thrombospondin-1) levels than control BEC; and that microRNA-18a (miR-18a) normalized AVM-BEC function and phenotype, although its mechanism remained unclear. Objective: To elucidate the mechanism of action and potential clinical application of miR-18a as an effective noninvasive treatment to selectively restore the phenotype and functionality of AVM vasculature. Methods and Results: The molecular pathways affected by miR-18a in patient-derived BECs and AVM-BECs were determined by Western blot, RT-qPCR (quantitative reverse transcription polymerase chain reaction), ELISA, co-IP, immunostaining, knockdown and overexpression studies, flow cytometry, and luciferase reporter assays. miR-18a was shown to increase TSP-1 and decrease VEGF by reducing PAI-1 (plasminogen activator inhibitor-1/SERPINE1) levels. Furthermore, miR-18a decreased the expression of BMP4 (bone morphogenetic protein 4) and HIF-1α (hypoxia-inducible factor 1α), blocking the BMP4/ALK (activin-like kinase) 2/ALK1/ALK5 and Notch signaling pathways. As determined by Boyden chamber assays, miR-18a also reduced the abnormal AVM-BEC invasiveness, which correlated with a decrease in MMP2 (matrix metalloproteinase 2), MMP9, and ADAM10 (ADAM metallopeptidase domain 10) levels. In vivo pharmacokinetic studies showed that miR-18a reaches the brain following intravenous and intranasal administration. Intranasal co-delivery of miR-18a and NEO100, a good manufacturing practices-quality form of perillyl alcohol, improved the pharmacokinetic profile of miR-18a in the brain without affecting its pharmacological properties. Ultra-high-resolution computed tomography angiography and immunostaining studies in an Mgp − /− AVM mouse model showed that miR-18a decreased abnormal cerebral vasculature and restored the functionality of the bone marrow, lungs, spleen, and liver. Conclusions: miR-18a may have significant clinical value in preventing, reducing, and potentially reversing AVM.

Data mining of nanoparticle contrast–enhanced 3D images reveals phenotypes indicative of tumor response to cellular immunotherapy., Immunotherapies, including cell-based therapies, targeting the tumor microenvironment (TME) result in variable and delayed responses. Thus, it has been difficult to gauge the efficacy of TME-directed therapies early after administration. We investigated a nano-radiomics approach (quantitative analysis of nanoparticle contrast–enhanced three-dimensional images) for detection of tumor response to cellular immunotherapy directed against myeloid-derived suppressor cells (MDSCs), a key component of TME. Animals bearing human MDSC-containing solid tumor xenografts received treatment with MDSC-targeting human natural killer (NK) cells and underwent nanoparticle contrast–enhanced computed tomography (CT) imaging. Whereas conventional CT-derived tumor metrics were unable to differentiate NK cell immunotherapy tumors from untreated tumors, nano-radiomics revealed texture-based features capable of differentiating treatment groups. Our study shows that TME-directed cellular immunotherapy causes subtle changes not effectively gauged by conventional imaging metrics but revealed by nano-radiomics. Our work provides a method for noninvasive assessment of TME-directed immunotherapy potentially applicable to numerous solid tumors.

Recent advancements in unravelling elements of cancer biology involved in disease progression and treatment resistance have highlighted the need for a holistic approach to effectively tackle cancer. Stimuli-responsive nanotheranostics based on iron oxide nanoparticles are an emerging class of versatile nanomedicines with powerful capabilities to "seek, sense, and attack" multiple components of solid tumors. In this work, the rationale for using iron oxide nanoparticles and the basic physical principles that impact their function in biomedical applications are reviewed. Subsequently, recent advances in the integration of iron oxide nanoparticles with various stimulus mechanisms to facilitate the development of stimuli-responsive nanotheranostics for application in cancer therapy are summarized. The integration of an iron oxide core with various surface coating mechanisms results in the generation of hybrid nanoconstructs with capabilities to codeliver a wide variety of highly potent anticancer therapeutics and immune modulators. Finally, emerging future directions and considerations for their clinical translation are touched upon.

In these preclinical studies, we describe ADx-001, an Aβ-targeted liposomal macrocyclic gadolinium (Gd) imaging agent, for MRI of amyloid plaques. The targeting moiety is a novel lipid-PEG conjugated styryl-pyrimidine. An MRI-based contrast agent such as ADx-001 is attractive because of the lack of radioactivity, ease of distribution, long shelf life, and the prevalence of MRI scanners. Dose-ranging efficacy studies were performed on a 1 T MRI scanner using a transgenic APP/PSEN1 mouse model of Alzheimer's disease. ADx-001 was tested at 0.10, 0.15, and 0.20 mmol Gd/kg. Gold standard post-mortem amyloid immunostaining was used for the determination of sensitivity and specificity. ADx-001 toxicity was evaluated in rats and monkeys at doses up to 0.30 mmol Gd/kg. ADx-001 pharmacokinetics were determined in monkeys and its tissue distribution was evaluated in rats. ADx-001-enhanced MRI demonstrated significantly higher (p 0.05) brain signal enhancement in transgenic mice relative to wild type mice at all dose levels. ADx-001 demonstrated high sensitivity at 0.20 and 0.15 mmol Gd/kg and excellent specificity at all dose levels for in vivo imaging of β amyloid plaques. ADx-001 was well tolerated in rats and monkeys and exhibited the slow clearance from circulation and tissue biodistribution typical of PEGylated nanoparticles.

High-Z based nanoparticles (NP) are emerging as promising agents for both cancer radiotherapy (RT) and CT imaging. NPs can be delivered to tumors via the enhanced permeability and retention (EPR) effect and they preferentially accumulate in tumor’s perivascular region. Both gold and iodine NPs produce low-energy, short-range photoelectrons during RT, boosting radiation dose. Using spectral CT imaging, we sought to investigate (1) if iodine nanoparticles augmentation of RT increases vascular permeability in solid tumors, and (2) if iodine-RT induced changes in tumor vascular permeability improves delivery of nanoparticle-based chemotherapeutics. In vivo studies were performed in a carcinogen-induced and genetically engineered primary mouse model of soft tissue sarcoma. Tumor-bearing mice in test group were intravenously injected with liposomal-iodine (Lip-I) (1.32 g I/kg) on day 0. On day 1, both test (with Lip-I) and control (without Lip-I) mice received RT (single dose, 10 Gy). One day post-RT (day 2), all mice were intravenously injected with liposomal gadolinium (Lip-Gd) (0.32 g Gd/kg), a surrogate of nanoparticle chemotherapeutic agent. Three days later (day 5) mice were imaged on our hybrid spectral micro-CT system. A dual source pre-clinical CT prototype system that combines a photon counting detector (PCD) and an energy integrating detector (EID) in a single hybrid system served as our imaging device. The results demonstrate that Lip-I augmented RT, resulting in increased tumor vascular permeability compared to control mice treated with RT alone. Consequently, Lip-I +RT treated mice demonstrated a 4- fold higher intra-tumoral accumulation of Lip-Gd compared to RT alone treated mice. In conclusion, our work suggests that Lip-I augments RT-induced effects on tumor vasculature, resulting in increased vascular permeability and higher intratumoral deposition of chemotherapeutic nanoparticles.

Fluorine-19 (19F) magnetic resonance imaging (MRI) has the potential for a wide range of in vivo applications but is limited by lack of flexibility in exogenous probe formulation. Most 19F MRI probes are composed of perfluorocarbons (PFCs) or perfluoropolyethers (PFPEs) with intrinsic properties which limit formulation options. Hydrophilic organofluorine molecules can provide more flexibility in formulation options. We report herein a hyperfluorinated hydrophilic organoflourine, ET1084, with ∼24 wt. % 19F content. It dissolves in water and aqueous buffers to give solutions with ≥8 M 19F. 19F MRI phantom studies at 9.4T employing a 10-minute multislice multiecho (MSME) scan sequence show a linear increase in signal-to-noise ratio (SNR) with increasing concentrations of the molecule and a detection limit of 5 mM. Preliminary cytotoxicity and genotoxicity assessments suggest it is safe at concentrations of up to 20 mM.

Non-invasive methods for estimating placental fractional blood volume (FBV) are of great interest for characterization of vascular perfusion in placentae during pregnancy to identify placental insufficiency that may be indicative of local ischemia or fetal growth restriction (FGR). Nanoparticle contrast-enhanced magnetic resonance imaging (CE-MRI) may enable direct placental FBV estimation and may provide a reliable, 3D alternative to assess maternal-side placental perfusion. In this pre-clinical study, we investigated if placental FBV at 14, 16, and 18 days of gestation could be estimated through contrast-enhanced MRI using a long circulating blood-pool liposomal gadolinium contrast agent that does not penetrate the placental barrier. Placental FBV estimates of 0.47 ± 0.06 (E14.5), 0.50 ± 0.04 (E16.5), and 0.52 ± 0.04 (E18.5) were found through fitting pre-contrast and post-contrast T1 values in placental tissue using a variable flip angle method. MRI-derived placental FBV was validated against nanoparticle contrast-enhanced computed tomography (CE-CT) derived placental FBV, where signal is directly proportional to the concentration of iodine contrast agent. The results demonstrate successful estimation of the placental FBV, with values statistically indistinguishable from the CT derived values.

Glioblastomas are highly lethal cancers defined by resistance to conventional therapies and rapid recurrence. While new brain tumor cell-specific drugs are continuously becoming available, efficient drug delivery to brain tumors remains a limiting factor. We developed a multicomponent nanoparticle, consisting of an iron oxide core and a mesoporous silica shell that can effectively deliver drugs across the blood-brain barrier into glioma cells. When exposed to alternating low-power radiofrequency (RF) fields, the nanoparticle's mechanical tumbling releases the entrapped drug molecules from the pores of the silica shell. After directing the nanoparticle to target the near-perivascular regions and altered endothelium of the brain tumor via fibronectin-targeting ligands, rapid drug release from the nanoparticles is triggered by RF facilitating wide distribution of drug delivery across the blood-brain tumor interface.

Advances in computed tomography (CT) hardware have propelled the development of novel CT contrast agents. In particular, the spectral capabilities of x-ray CT can facilitate simultaneous imaging of multiple contrast agents. This approach is particularly useful for functional imaging of solid tumors by simultaneous visualization of multiple targets or architectural features that govern cancer development and progression. Nanoparticles are a promising platform for contrast agent development. While several novel imaging moieties based on high atomic number elements are being explored, iodine (I) and gadolinium (Gd) are particularly attractive because of their existing approval for clinical use. In this work, we investigate the in vivo discrimination of I and Gd nanoparticle contrast agents using both dual energy micro-CT with energy integrating detectors (DE-EID) and photon counting detector (PCD)-based spectral micro-CT. Simulations and phantom experiments were performed using varying concentrations of I and Gd to determine the imaging performance with optimized acquisition parameters. Quantitative spectral micro-CT imaging using liposomal-iodine (Lip-I) and liposomal-Gd (Lip-Gd) nanoparticle contrast agents was performed in sarcoma bearing mice for anatomical and functional imaging of tumor vasculature. Iterative reconstruction provided high sensitivity to detect and discriminate relatively low I and Gd concentrations. According to the Rose criterion applied to the experimental results, the detectability limits for I and Gd were approximately 2.5 mg ml−1 for both DE-EID CT and PCD micro-CT, even if the radiation dose was approximately 3.8 times lower with PCD micro-CT. The material concentration maps confirmed expected biodistributions of contrast agents in the blood, liver, spleen and kidneys. The PCD provided lower background signal and better simultaneous visualization of tumor vasculature and intratumoral distribution patterns of nanoparticle contrast agent compared to DE-EID decompositions. Preclinical spectral CT systems such as this could be useful for functional characterization of solid tumors, simultaneous quantitative imaging of multiple targets and for identifying clinically-relevant applications that benefit from the use of spectral imaging. Additionally, it could aid in the development nanoparticles that show promise in the developing field of cancer theranostics (therapy and diagnostics) by measuring vascular tumor biomarkers such as fractional blood volume and the delivery of liposomal chemotherapeutics.

The advent of genomic, proteomic, and high-throughput screening technologies has made available many new targets for brain tumor imaging; however, target availability and accessibility need to be carefully considered when designing imaging probes. Nanoparticles, although largely still only used in preclinical studies, are a versatile tool for targeted imaging of physiologic and molecular aspects of brain tumors through many clinically used modalities. Liposomes can be used to transport diverse payloads in vivo, including contrast agents and drugs, and may be functionalized to increase circulation half-life and achieve targeting specificity. Polymeric, gold, and iron oxide nanoparticles have been used for diverse applications in preclinical studies; however, the utility of other methods, such as quantum dots and self-assembling DNA molecules, is yet to be established.