Abstract Resistance to cisplatin and low response rates to immune checkpoint blockade (ICB) limit survival outcomes in oral cavity squamous cell carcinoma (OCSCC). Anthracyclines, including liposomal doxorubicin (Doxil®), possess immunomodulatory properties and can induce immunogenic cell death (ICD), providing a rationale for combination with radiation and immunotherapy. We therefore hypothesized that Doxil would enhance anti-tumor immunity in multimodal treatment of OCSCC. To test this hypothesis, we evaluated anti-tumor efficacy and modulation of the tumor immune microenvironment (TIME) using HPV-negative (MOC2, ROC1) and HPV-positive (mEER) syngeneic murine models of OCSCC. C57BL/6 mice bearing subcutaneous flank tumors were treated with radiation therapy (RT; 8 Gy × 3 fractions), Doxil (5 mg/kg IV weekly × 4), and anti-CTLA-4 (100 µg IP twice weekly × 4), alone or in combination. For immune profiling, tumors were harvested approximately 21 days post-inoculation after mice had received one dose of Doxil, one fraction of RT, and/or two doses of anti-CTLA-4, then analyzed using full-spectrum flow cytometry and single nuclear RNA sequencing (snRNA-seq). Doxil demonstrated modest single-agent activity in both HPV-positive and HPV-negative models but showed enhanced efficacy when combined with RT and/or anti-CTLA-4. The greatest therapeutic benefit was observed with the triple combination of Doxil, RT, and anti-CTLA-4 (DRC), which resulted in complete response rates of 43.5-69.6% and prolonged median survival of up to 173 days in the mEER model. Flow cytometric analysis of the TIME of Doxil- and DRC-treated mice revealed a 1.9- to 7.6-fold decrease in regulatory T cells and 1.4- to 1.7-fold decrease in proliferating M2-like macrophages. snRNA-seq demonstrated increased expression of ICD-associated genes in Doxil- or DRC-treated tumor cells. Notably, DRC treatment induced robust upregulation of Cxcl10, Cxcl9, and Ccl5 with mean expression fold changes of 5.6, 26.8, and 4.7, respectively. These tumor-intrinsic changes were associated with a 3.1- and 3.4-fold increase in mean Cxcr3 expression on CD8+ T cells in Doxil- and DRC-treated tumors, respectively. Doxil and DRC treatment also increased proportions of activated dendritic cells. Additionally, snRNA-seq analysis demonstrated approximately 50% and 75% reductions in mean expression of epithelial-to-mesenchymal transition (EMT)-related genes including Vim, Sparc, and Spp1 in Doxil- and DRC-treated tumor cells, respectively, suggesting Doxil may inhibit EMT. In summary, combining Doxil with radiation and CTLA-4 blockade induces ICD, reshapes the TIME toward a pro-inflammatory and antigen-presenting state, and promotes durable tumor control in both HPV-positive and HPV-negative OCSCC models. These findings support the translational potential of incorporating liposomal doxorubicin into multimodal immunotherapeutic strategies for OCSCC. Citation Format: Jennifer L. Anderson, Fabio H. Brasil Da Costa, Allison Nipper, Nicolas Oltean, Laxman Devkota, Rohan Bhavane, Ratna Veeramachaneni, Sofia Cortes, Neeraja Dharmaraj, Sarah L. Latka, Andrew Badachhape, Renuka T. Menon, Prajwal Bhandari, Sujuan Yang, Ansam Sinjab, Humam Kadara, Roberto Rangel, Faye M. Johnson, Jeffrey N. Myers, Ketankumar B. Ghaghada, Simon Young, Ananth V. Annapragada, Andrew G. Sikora. Liposomal doxorubicin potentiates anti-tumor immune response in oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB151.

Intratumoral immunotherapy presents a promising approach for enhancing cancer treatment; however, its effectiveness is limited by heterogeneous intratumoral drug distribution and rapid drug leakage following direct injection. To address these limitations, we developed a biodegradable nanofibrous drug-eluting seed (b-NDES), a reservoir-based implant designed for sustained, localized diffusive delivery of immunotherapeutics. The b-NDES reduces systemic exposure and eliminates the necessity for surgical removal through gradual biodegradation. Implant bodies were fabricated by electrospinning polymeric formulations comprising varying ratios of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and barium sulfate to provide radiopacity. Surface modifications were implemented to adjust the porous structure, allowing for tailored drug elution rates. Comparative comprehensive evaluations of morphology, in vitro release profiles, and degradation kinetics were performed. The optimized 1:4 PCL:PLGA formulation reduced permeable porosity from 18.99 ± 1.26% to 2.74 ± 1.04%, effectively decreasing the rhodamine delivery rate from 162.58 ± 16.11 μg/h to 30.68 ± 11.60 μg/h in vitro. The 1:4 PCL:PLGA structure achieved controlled diffusive drug release profile that extended intratumoral drug persistance in a 4 T1 triple-negative breast cancer (TNBC) murine model, with negligible systemic off-target exposure. Further, long-term degradation studies showed an overall mass loss of 46.32 ± 12.01% at 6 months. When loaded with a combination of CD40 agonist antibody (α-CD40) and a STING agonist (STINGa) and paired with stereotactic radiotherapy, the b-NDES platform achieved complete tumor eradication in 60% of animals. Importantly, no systemic adverse effects were observed with the intratumoral administration of the immunotherapeutic combination via b-NDES. By providing a minimally invasive, sustained-release strategy that naturally degrades to eliminate the need for surgical removal, the b-NDES represents a versatile platform for delivering potent immunotherapeutic combinations against aggressive malignancies.

Abstract Despite advances in the treatment of head and neck cancer, squamous cell carcinoma of the oral cavity remains primarily a surgical disease with few effective systemic therapies. Oral cavity tumors harbor both innate and acquired resistance mechanisms to cytotoxic chemotherapy. Additionally, the tumor microenvironment (TME) has limited immune cell infiltration, resulting in low response rates to immune checkpoint inhibitors. Given the need for novel systemic therapies for oral cavity squamous cell carcinoma, we investigated the role of liposomal doxorubicin (Doxil®) as both a cytotoxic agent and an immunomodulatory agent. We utilized the MOC2 syngeneic murine model of oral cavity squamous cell carcinoma, an aggressive tumor model with resistance to immune checkpoint blockade. We demonstrated that Doxil has moderate activity as a single agent in vivo for C57BL/6J mice harboring MOC2 flank tumors. We next performed flow cytometric analysis to characterize the changes in immune cell populations in the TME after treatment with Doxil. We found significantly increased numbers of innate immune cells including NK cells and myeloid cells. Multiplex immunofluorescence was also used to confirm the increase in myeloid cell tumor infiltration upon Doxil treatment. Given the changes seen in the tumor immune microenvironment, we hypothesized Doxil may improve response to immune checkpoint blockade. Therefore, we treated C57BL/6J mice inoculated with MOC2 flank tumors with Doxil alone or in combination with radiation therapy (RT) and/or anti-CTLA-4 therapy. While RT or anti-CTLA-4 alone had modest anti-tumor activity, combining either RT or anti-CTLA-4 with Doxil significantly reduced tumor growth. Moreover, the triple combination therapy of Doxil, RT, and anti-CTLA-4 had an increased effect compared to the dual therapy of Doxil and anti-CTLA-4. This included several complete responses, which resulted in a significant improvement in survival. Triple combination therapy also reduced both local and distant metastatic burden compared to single agent and dual combination therapy. Next, we selectively inhibited CD8+ T cells, myeloid cells, or NK cells to determine which immune cell populations contribute to the immunomodulatory activity of Doxil. We observed that inhibition of NK cells resulted in increased tumor growth as well as decreased survival, suggesting that Doxil-mediated infiltration of NK cells into the TME contributes to response to anti-CTLA-4 therapy. Taken together, these data provide a rationale for combining liposomal doxorubicin with anti-CTLA-4 therapy for the treatment of oral cavity squamous cell carcinoma. Citation Format: Jennifer L Anderson, Fabio H Brasil Da Costa, Allison Nipper, Laxman Devkota, Rohan Bhavane, Ratna Veeramachaneni, Sofia Cortes, Neeraja Dharmaraj, Sarah Latka, Andrew Badachhape, Renuka TR Menon, Prajwal Bhandari, Ketankumar Ghaghada, Simon Young, Ananth Annapragada, Andrew Sikora. Liposomal doxorubicin improves response to immune checkpoint blockade by enhancing innate immunity in a murine model of oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A119.

ACTA2 pathogenic variants altering arginine 179 cause childhood-onset strokes due to moyamoya disease (MMD)-like occlusions of the distal internal carotid arteries, but the mechanisms of pathogenesis are unknown and no preventive treatments exist. Here we show that Acta2R179C/+ smooth muscle cells (SMCs) fail to fully differentiate and maintain stem cell-like features, including increased migration and glycolytic flux compared to wildtype (WT) SMCs. Increasing mitochondrial respiration with nicotinamide riboside (NR) drives differentiation and decreases migration of Acta2R179C/+ SMCs. Carotid artery injury of Acta2SMC-R179C/+ mice leads to premature death, intraluminal SMC accumulation leading to MMD-like occlusive lesions, neurologic symptoms, and neuron loss, whereas injured WT mice have none of these phenotypes, and all are prevented by NR treatment in the Acta2SMC-R179C/+ mice. These data show that driving differentiation and quiescence of Acta2R179C/+ SMCs by altering cellular metabolism attenuates MMD-like disease in the Acta2SMC-R179C/+ mice, highlighting a role of immature and highly migratory SMCs in the pathogenesis of MMD.

Introduction Placenta 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. In this study, we investigate use of the FDA-approved iron supplement ferumoxytol for contrast-enhanced MRI of the RPCS in mouse models of normal pregnancy and PAS. We then demonstrate the translational potential of this technique in human patients (n=6) presenting with severe PAS (FIGO Grade 3C), moderate PAS (FIGO Grade 1), and no PAS. Methods T1-weighted sequences were used to determine the optimal dose of ferumoxytol in pregnant mice. Pregnant Gab3-/- mice which demonstrate adherent placentation were imaged alongside wild-type (WT) pregnant mice with non-adherent placentation. Fe-MRI was also performed in 6 pregnant subjects using standard T1 and T2 weighted sequences and a 3D magnetic resonance angiography (MRA) sequence. Results Ferumoxytol administered at 5 mg/kg led to strong placental enhancement in Fe-MRI images. Gab3-/- mice demonstrated loss of the hypointense region characteristic of the RPCS relative to WT mice. In human patients, Fe-MRI enabled high uteroplacental vasculature signal and quantification of the volume and signal profile in severe and moderate invasion of the placenta relative to non-PAS cases. Discussion Ferumoxytol, an FDA-approved iron oxide nanoparticle formulation, enabled T1w MRI 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 and suggests the possibility of PAS diagnosis using contrast enhanced MRI.

Background: Aortopathies, such as aortic aneurysm and dissection (AAD), are associated with enhanced aortic wall permeability and endothelial dysfunction. We previously demonstrated that nanoparticle contrast-enhanced computed tomography (nCECT), which detects enhanced aortic wall permeability, could enable non-invasive detection of early AAD before its progresses to fatal aortic rupture. This study investigated the effect of nanoparticle contrast agent (NPCA) size and dose on detection of aortopathy by nCECT. Methods: In vivo studies were performed in a mouse model of sporadic AAD induced by challenging animals with high fat diet (5 weeks) and angiotensin II infusion (last one week). The effects of NPCA size (80, 150, and 240 nm) and NPCA dose (300, 600, and 1200 mg I/kg) on detection of aortopathy were studied. To examine temporal changes in aortic wall NPCA signal at sites of AAD, mice underwent longitudinal CT. To investigate changes in aortic wall integrity, mice underwent follow-up nCECT at 6 months after initial challenge. Imaging findings were compared with gross and histologic examination of the aorta. Fluorescence microscopy was used to confirm presence or absence of intramural NPCA. Results: nCECT using all three sizes of NPCAs demonstrated significantly higher sensitivity (p< 0.05) for the detection of aortopathy compared to gross examination. Histologic analysis showed excellent correlation between the nCECT finding of intramural signal and the presence of aortopathy. The absolute improvements in detection rates were 16%, 20%, and 17% for 80 nm, 150 nm, 250 nm NPCA respectively. Sensitivity of nCECT for detection of aortic injury improved with increasing NPCA dose compared to gross exam (-17% at 300 mg I/kg to 14% at 1200 mg I/kg). Temporal analysis of aortic wall NPCA signal at sites of AAD demonstrated a peak in aortic wall CT attenuation at day 3-5 post-contrast followed by gradual return to baseline by day 120. Follow-up nCECT at 6 months demonstrated absence of wall signal enhancement compared to baseline, suggesting resolution of the altered aortic wall permeability and injury. Histologic analysis demonstrated remodeling and healing of the aortic wall. Conclusions: Nanoparticle contrast-enhanced CT using all three studied nanoparticle sizes demonstrated higher sensitivity than gross examination for the detection of aortopathy. A dose-dependent effect on sensitivity was observed with only high NPCA dose (1200 mg I/kg) demonstrating superior performance compared to gross examination for detecting early stages of aortic injury. Nanoparticle contrast-enhanced CT enabled in vivo interrogation of changes in aortic wall integrity.

The clinical availability of photon-counting computed tomography (PCCT) has ushered in a new era of CT imaging. Spectral imaging coupled with superior contrast resolution, and ultrahigh spatial resolution (200 μm) offered by PCCT has the potential to revolutionize value-driven imaging. The potential of multicolor PCCT has generated excitement, and renewed interest, in novel contrast agent development for comprehensive disease interrogation, prediction and monitoring of treatment outcomes. Nanoparticles provide a versatile and powerful platform for the development of next generation contrast agents for spectral PCCT. In this article, we review recent developments and use of nanoparticle contrast agents for PCCT. We also discuss future research and translational opportunities for nanoparticle-based CT contrast agents enabled by the advent of PCCT and describe key considerations for their clinical translation.

Revascularization plays a critical role in the successful engraftment of transplanted pancreatic islets, which are inherently rich in capillaries to meet their high metabolic demands. Innovative islet encapsulation strategies such as the NICHE (neovascularized implantable cell homing and encapsulation), generate a prevascularized transplantation site that allows for direct integration of the graft with the systemic circulation. Timing the transplantation is key to maximizing islet engraftment and survival, especially in diabetic individuals, who exhibit impaired wound healing. Therefore, in this study, we explored different methods to assess vascular development within NICHE in vivo in a non-invasive fashion. We effectively tracked neoangiogenesis using nanoparticle contrast-enhanced computed tomography (nCECT), observing a steady increase in vascularization over an 8-week period, which was confirmed histologically. Next, we estimated relative vascularization changes via T2 mapping with magnetic resonance imaging (MRI) before and after islet transplantation. On the first day post-transplantation, we measured a slight decrease in T2 values followed by a significant increase by day 14 attributable to islet revascularization. Our findings underscore the potential of non-invasive imaging techniques to provide insightful information on the readiness of the transplant site within cell encapsulation systems to support cell graft transplantation.

Background: Aortic pathologies, such as aortic aneurysm and dissection (AAD), are characterized by enhanced endothelial permeability and dysfunction. Non-invasive monitoring of changes in endothelial permeability could enable early detection and prediction of AAD progression. Herein, we investigated nanoparticle contrast-enhanced computed tomography (nCECT) to probe endothelial permeability changes in a mouse model of sporadic AAD. Methods: In vivo studies were performed in a mouse model of AAD challenged with high fat diet (5 weeks) and angiotensin II infusion (last one week). The effects of nanoparticle contrast agent (NPCA) size (80, 150, and 250 nm) and dose (300, 600, and 1200 mg I/kg) on detecting endothelial permeability changes were studied. In vivo and ex vivo nCECT imaging were performed 4 days post-contrast and findings were compared with gross and histologic examination. A subset of mice was followed-up with longitudinal nCECT up to 6 months. Results: nCECT showed a higher incidence of aortic injury associated with enhanced endothelial permeability compared to gross examination findings. The absolute improvements in detection rates were 21%, 17% and 16% for 80 nm, 150 nm, 250 nm NPCA respectively (Figure 1B). Sensitivity of nCECT for detection of aortic injury improved with increasing NPCA dose compared to gross exam (-17% at 300 mg I/kg to 14% at 1200 mg I/kg, Figure 1C). Follow-up nCECT at 6 months did not show significant increase in wall signal enhancement compared to baseline (Figure 2), suggesting resolution of inflammation and endothelial dysfunction, and progressive repair and remodeling of aortic wall which were confirmed by the microscopic analysis. Conclusion: nCECT detected enhanced endothelial permeability before pathological changes were evident on gross examination in a mouse model of sporadic AAD. Furthermore, this approach enabled non-invasive monitoring of changes in endothelial permeability at sites of aortic degeneration.

Amyloid plaques are a pathological hallmark of Alzheimer’s disease (AD). In a recent pre-clinical study, we showed an amyloid-targeted liposomal macrocyclic gadolinium (Gd) contrast agent, ADx-001, for in vivo MRI-based detection of amyloid plaques in mouse. Although ADx-001 showed high sensitivity at a high dose (0.2 mmol Gd/kg), the performance was sub-optimal (<70%) at lower doses (0.1 and 0.15 mmol Gd/kg). In this work, we investigated if nano-radiomics (radiomic analysis of nanoparticle contrast-enhanced images) would increase sensitivity of ADx-001 at lower dose levels. In vivo studies were performed in the APP/PSEN1 mouse model of amyloid pathology. The efficacy of ADx-001- enhanced MRI was studied at three dose-levels: 0.10, 0.15, and 0.20 (mmol Gd/kg). Pre- and post-contrast MRI was performed in transgenic (n = 6/dose) and wild-type mice (n = 6/dose) using a T1-weighted spin-echo sequence. Semi-automatic 3D segmentation of hippocampal and cortical regions was performed using a mouse brain MR atlas (Fig.1). Radiomic analysis was executed on the hippocampus and cortex regions of ADx-001-enhanced MR images 900 radiomic features (RFs). RF selection was completed using a non-parametric neighborhood component method. 5-fold cross-validation was performed using a set of linear and non-linear classifiers to confirm the accuracy of group separation. Seven RFs (three RFs for cortex and four RFs for hippocampus) were identified that differentiated amyloid-positive transgenic mice from amyloid-negative wild-type mice based on ADx-001-enhanced MRI. The best performing nearest-neighbor classification model was trained simultaneously on all ADx-001 dose groups. Nano-radiomic analysis of ADx-001-enhanced MRI demonstrated 100% accuracy, specificity, and sensitivity for dose levels of 0.2 and 0.15 mmol Gd/kg. For low dose level (0.1 mmol Gd/kg), radiomics achieved accuracy of 91.6% and sensitivity of 83.3%, while maintaining specificity at 100%. The new results were superior when compared to previously reported results based on global signal enhancement analysis. Nano-radiomic analysis of ADx-001-enhanced MRI improved sensitivity of ADx-001 at lower dose levels for the detection of amyloid pathology and demonstrated excellent for intermediate and high dose. Our study demonstrates that radiomic analysis of contrast-enhanced MR images could boost the performance of targeted molecular imaging agents for early detection of AD.

Three dimensional (3D) printed perfused tissue models are gaining attention due to promise of reproducing patient-specific pathology. We present here the design and characterization of a novel multimodal imaging compatible micro-physiological system (MicMPS) that contains three innovative features: (1) a perfused 4.5 mL enclosure volume for generating large tissue-mimicking 3D cellular systems comparable to rodent sizes, (2) ability to take ‘biopsy-like’ samples for non-destructive longitudinal interrogation of tissue composition, and (3) compatibility with cross-sectional, high-resolution imaging modalities, including computed tomography (CT), magnetic resonance imaging (MRI) and ultrasound (US), for longitudinal and functional assessments of MicMPS and enclosed tissue architecture. Contrast-enhanced micro-CT imaging was employed to investigate MicMPS architecture and demonstrate reproducibility in fabrication process. MicMPS perfusion was examined using dynamic contrast-enhanced MRI performed on a low-cost permanent MR magnet scanner. As a proof-of-concept, fibroblasts-laden MicMPS was developed and maintained under perfusion for 5 days. Evaluation of cell viability and perfusion was performed across the entire MicMPS. US image-guided biopsy was performed to obtain engineered tissue for microscopic analysis, thus demonstrating non-destructive capabilities for interim MicMPS interrogation. The newly designed MicMPS bridges the gap between microfluidic systems and animal studies, providing the controlled environment of a cell culture system while enabling imaging investigations that are routinely performed in animal studies.

Introduction: The immunosuppressive tumor microenvironment (TME) in oral cancer inhibits response to conventional treatment (surgery +/- chemoradiotherapy) and check-point inhibitor (αPD1) immunotherapy. The chemotherapeutic agent doxorubicin and its PEGylated liposomal nanoparticle formulation (PLD) have been shown to target myeloid derived suppressor cells (MDSCs) in the TME. In this preclinical work, we investigated whether PLD improves response to radio-immunotherapy in a highly aggressive and immunologically ‘cold’ oral cancer model. Experimental Procedure: Studies were performed in the MOC2 syngeneic mouse model of oral cancer. Mice were randomized to one of seven treatment groups: αPD1, Radiation (XRT), αPD1+XRT, PLD, PLD+αPD1, PLD+XRT, PLD+αPD1+XRT. High-resolution nanoparticle contrast-enhanced computed tomography (nCECT) imaging was performed, and whole-body T2-weighted magnetic resonance imaging (MRI) used to monitor primary tumor response and development of metastases. Response evaluation criteria in solid tumors (RECIST) were used to evaluate treatment outcomes. Results: Tumors in all non-PLD groups were treatment resistant and grew progressively, consistent with the immunologically ‘cold’ MOC2 model. In contrast, all PLD groups showed tumor regression with 25-38% complete responders (CR) and 12-88% partial responders (PR) while all animals in non-PLD groups showed progressive disease (PD). MR imaging identified a high incidence of regional and distant metastases in the non-PLD groups while the PLD groups showed decreased regional and no distant metastases (Table 1). Presence of metastases was confirmed by nCECT imaging and histopathological analysis. Conclusion: PLD improved response to radio-immunotherapy in a highly aggressive immunologically ‘cold’ mouse model of oral cancer while simultaneously preventing disease progression and metastasis. Citation Format: Laxman Devkota, Rohan Bhavane, Andrew Badachhape, Ratna Veeramachaneni, Renuka Menon, Prajwal Bhandari, Sofia Cortes, Fabio Henrique Brasil Da Costa, Ketan Ghaghada, Simon Young, Andrew G. Sikora, Ananth V. Annapragada. PEGylated liposomal doxorubicin improves oral cancer response to radio-immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5174.

Introduction: Placenta 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. Methods: A 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. Results: Ferumoxytol 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. Discussion: Ferumoxytol, 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.

Introduction: Endothelium forms a protective barrier and maintains vascular hemostasis. However, the role of endothelial injury in aortic aneurysms and dissections (AAD) remains poorly understood. Receptor-interacting protein kinase 3 (RIP3)-mediated necroptosis and gasdermin D (GSDMD)-mediated pyroptosis trigger necrotic cell death. We hypothesize endothelial cell (EC) death induced by necroptosis and pyroptosis contributes to AAD formation. Methods: Endothelial integrity and gene expression were examined in ascending aortic tissues from ascending thoracic aortic aneurysm (ATAA) patients (n=9) and organ donor controls (n=8) by single-cell transcriptome analysis. Effects of EC death on AAD formation were determined in EC-specific Rip3 knockout (EC-Rip3 -/- , n=26), EC-specific Gsdmd knockout (EC-Gsdmd -/- , n=21), and necroptosis/pyroptosis inhibitor necrosulfonamide (NSA) treated mice (n=15) in sporadic AAD models induced by angiotensin II (Ang II) infusion. Evans blue staining and nanoparticle-mediated contrast-enhanced CT (n-CECT) detected endothelial hyperpermeability Results: Single-cell transcriptome and immunostaining analyses revealed significant upregulation of pro-death genes (e.g., RIP3 and GSDMD), but downregulation of cell junction genes (e.g., TJP1 and GJA1) in ECs of ATAA patients compared with controls. In the mouse AAD model, endothelial injury and hyperpermeability were detected 2-5 days after Ang II infusion and were associated with intramural nanoparticle accumulation, elastic fiber fragmentation, and macrophage infiltration. Importantly, EC-Rip3 -/- mice and EC-Gsdmd -/- mice showed preserved EC barrier function, reduced nanoparticle accumulation and elastic fiber fragmentation, and reduced AAD incidence (including aneurysm, dissection, and rupture) compared to their littermate controls (EC-Rip3 -/- mice: 52% vs 23.1%, P=0.033; EC-Gsdmd -/- mice: 52.6% vs 14.3%, P=0.01). Blocking necroptosis/pyroptosis by NSA treatment reduced the incidence (65% vs 27%; P=0.04) and severity of AAD. Conclusions: Endothelial injury and subsequent barrier dysfunction and infiltration are key features of AAD formation. Prevention of EC necrotic cell death can be a potential AAD therapeutic target.

ACTA2 pathogenic variants altering arginine 179 cause childhood-onset strokes due to moyamoya disease (MMD)-like occlusion of the distal internal carotid arteries. A smooth muscle cell (SMC)-specific knock-in mouse model (Acta2 SMC-R179C/+ ) inserted the mutation into 67% of aortic SMCs, whereas explanted SMCs were uniformly heterozygous. Acta2 R179C/+ SMCs fail to fully differentiate and maintain stem cell-like features, including high glycolytic flux, and increasing oxidative respiration (OXPHOS) with nicotinamide riboside (NR) drives the mutant SMCs to differentiate and decreases migration. Acta2 SMC-R179C/+ mice have intraluminal MMD-like occlusive lesions and strokes after carotid artery injury, whereas the similarly treated WT mice have no strokes and patent lumens. Treatment with NR prior to the carotid artery injury attenuates the strokes, MMD-like lumen occlusions, and aberrant vascular remodeling in the Acta2 SMC-R179C/+ mice. These data highlight the role of immature SMCs in MMD-associated occlusive disease and demonstrate that altering SMC metabolism to drive quiescence of Acta2 R179C/+ SMCs attenuates strokes and aberrant vascular remodeling in the Acta2 SMC-R179C/+ mice.

Women exhibit higher prevalence of Alzheimer’s disease (AD) with faster cognitive decline compared to men, but the mechanisms of this dimorphism remain understudied. Astrocyte function and morphology is distinct in some regions of male and female adult brains which may reflect different courses taken by the neurodegenerative process. Additionally, the p-tau levels are higher in females than in males. The 3xTg AD mouse model, which manifests amyloid and tau pathology, has been reported to exhibit similar sexual dimorphism. In this work, we investigated whether molecular MRI using a targeted agent that detects hyperphosphorylative cells expressing cell-surface vimentin predominantly on neurons and the disease associated astrocytes in early states of tau pathology can be used for in vivo imaging of sexual dimorphism in AD pathology. Liposomal Withaferin nanoparticles (WNP) that contain Gd/DOTA for an MRI readout were injected in 3xTg AD mice, at 2-,5-,7- 9- and 12- months of age. Animals (n = 14/timepoint, 7 males and 7 females) underwent contrast enhanced magnetic resonance imaging (MRI) using T1 weighted sequences to visualize hyperphosphorylative cells expressing cell surface vimentin. Animals were euthanized after the final time point and brains were harvested for histology and immunofluorescence analysis. CE-MR images were analyzed for signal enhancement. In this study, we report the sexually dimorphic nature of the cell surface vimentin expressed by the hyperphosphorylative cells in the brain using the 3xTg AD mice. Both sexes were imaged at 2-,5-,7- and 9- months of age to reveal significant differences in the expression of VIM in males and . Signal enhancement in post-contrast MRI scans over the baseline scans showed significantly higher enhancement in the females compared with the males (p<0.05) Simultaneously, we also investigate the plasma concentrations of total tau, ptau181, amyloid beta 40 and 42. Female mice expressed higher vimentin in comparison with males, thus reflecting changes in the neurodegenerative processes in the brains of females and males of this strain. Sexually dimorphic molecular changes were successfully visualized by molecular MRI.

Introduction: Prior preclinical studies established the utility of liposomal nanoparticle blood-pool contrast agents in visualizing the retro-placental 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. Materials and Methods: Contrast-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 & POUND;100 & POUND;600 mm3 voxels) and CE-CT was performed at a higher resolu-tion (70 & POUND;70 & POUND;70 mm3 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. Results: Pregnant Gab3-/-mice showed elevated rates of complicated pregnancy. Contrast-enhanced imaging demonstrated frank infil-tration 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 thin-ner RPCS in Gab3-/-FPUs. Discussion: Imaging 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.

The abnormal phosphorylation of tau is a necessary precursor to the formation of tau fibrils, a marker of Alzheimer's disease. We hypothesize that hyperphosphorylative conditions may result in unique cell surface markers. We identify and demonstrate the utility of such surrogate markers to identify the hyperphosphorylative state. Methods: Cell SELEX was used to identify novel thioaptamers specifically binding hyperphosphorylative cells. Cell surface vimentin was identified as a potential binding target of the aptamer. Novel molecular magnetic resonance imaging (M-MRI) probes using these aptamers and a small molecule ligand to vimentin were used for in vivo detection of this pre-pathological state. Results: In a mouse model of pathological tau, we demonstrated in vivo visualization of the hyperphosphorylative state by M-MRI, enabling the identification at a pre-pathological stage of mice that develop frank tau pathology several months later. In vivo visualization of the hyperphosphorylative state by M-MRI was further validated in a second mouse model (APP/PS1) of Alzheimer's disease again identifying the mutants at a pre-pathological stage. Conclusions: M-MRI of the hyperphosphorylative state identifies future tau pathology and could enable extremely early-stage diagnosis of Alzheimer's disease, at a pre-patholgical stage.