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[Abstract] We present a quantitative analysis of the thermodynamic stabilities of Mn(II) complexes, defined by the equilibrium constants (log KMnL values) and the values of pMn obtained as −log[Mn]free for total metal and ligand concentrations of 1 and 10 μM, respectively. We used structural descriptors to analyze the contributions to complex stability of different structural motifs in a quantitative way. The experimental log KMnL and pMn values can be predicted to a good accuracy by adding the contributions of the different motifs present in the ligand structure. This allowed for the identification of features that provide larger contributions to complex stability, which will be very helpful for the design of efficient chelators for Mn(II) complexation. This issue is particularly important to develop Mn(II) complexes for medical applications, for instance, as magnetic resonance imaging (MRI) contrast agents. The analysis performed here also indicates that coordination number eight is more common for Mn(II) than is generally assumed, with the highest log KMnL values generally observed for hepta- and octadentate ligands. The X-ray crystal structure of [Mn2(DOTA)(H2O)2], in which eight-coordinate [Mn(DOTA)]2– units are bridged by six-coordinate exocyclic Mn(II) ions, is also reported. C.P.-I. and D.E.-G. (PID2019-104626GB-I00) and A.R.-R. (Grant PID2019-108352RJ-I00) thank Ministerio de Ciencia e Innovación and Xunta de Galicia (Grant ED431B 2020/52) for generous financial support. R.U.-V. thanks the Xunta de Galicia (Grant ED481A-2018/314) for funding her Ph.D. contract. L.V is indebted to CACTI (Universidade de Vigo) for X-Ray measurements. P.C. acknowledges funding from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK121789) Xunta de Galicia; ED431B 2020/52 Xunta de Galicia; ED481A-2018/314 Estados Unidos de América. National Institute of Diabetes and Digestive and Kidney Diseases; R01DK121789

PURPOSE: New generation of receptor tyrosine kinase inhibitors (RTKIs) have shown to improve survival in many solid tumors. However, an imaging biomarker is needed for patient selection and prediction of treatment response. This study evaluates the use of quantitative changes of HER3 on (68) Ga-NOTA-HER3P1 PET/MRI for prediction of early response to pan-RTKIs in gastric cancer (GCa). PROCEDURES: GCa cell lines were evaluated for expression of RTKs, and downstream signaling pathways (AKT and MAPK). Cell viability was assessed following 24–72 h of treatment with 0.01–1 μmol/L of afatinib, a pan-RTKI. HER3-expressing afatinib-sensitive (NCI-N87) and resistant cells (SNU16) were selected for evaluation of changes in RTKs expression and downstream pathways, with 24–72 h of 0.1 μmol/L afatinib treatment. (68) Ga-NOTA-HER3P1 PET/MRI was performed in subcutaneous NCI-N87 and SNU16 xenografts (nu:nu, n = 12/group) at baseline and 4 days after afatinib treatment (10 mg/kg, PO, daily). Temporal changes in PET measures were correlated to HER3 expression in tumors, tumor growth rate, and treatment response. RESULTS: With afatinib therapy, NCI-N87 cells showed increased total HER3 expression, and reduction of other RTKs and downstream nodes within 72 h, while SNU16 cells showed no significant change in total HER3 and downstream nodes. (68) Ga-HER3P1 PET/MRI showed increased uptake in NCI-N87 and no significant change in SNU16 tumors (day 4 vs. baseline SUV(mean): 3.8 ± 0.7 vs. 1.6 ± 0.6, p < 0.05 in NCI-N87, and 1.5 ± 0.7 vs. 1.7 ± 0.7, p > 0.05 in SNU16). These findings were in concordance with HER3 expression in histopathological analyses and tumor growth over 3 weeks of treatment (mean tumor volume in treated vs. control: 11 ± 17 mm(3) vs. 293 ± 79 mm(3), p < 0.001 in NCI-N87, and 238 ± 91 mm(3) vs. 282 ± 35 mm(3), p > 0.05 in SNU16). CONCLUSIONS: Quantitative changes in HER3 PET could be used to predict response to pan-RTKI within few days after initiation of treatment and can help with personalizing GCa management.

To compare positron emission tomography (PET)/magnetic resonance imaging (MRI) to the standard of care imaging (SCI) for the diagnosis of peritoneal carcinomatosis (PC) in primary abdominopelvic malignancies.Identifying PC impacts prognosis and management of multiple cancer types.Adult subjects were prospectively and consecutively enrolled from 4/2019 to 1/2021. Inclusion criteria were: (a) acquisition of whole-body contrast-enhanced (CE) 18F-fluorodeoxyglucose PET/MRI, (b) pathologically confirmed primary abdominopelvic malignancies. Exclusion criteria were: (a) greater than 4 weeks interval between SCI and PET/MRI, (b) unavailable follow-up. SCI consisted of whole-body contrast-enhanced (CE) PET/computed tomography (CT) with diagnostic quality CT, and/or CE-CT of the abdomen and pelvis, and/or CE-MRI of the abdomen±pelvis. If available, pathology or surgical findings served as the reference standard, otherwise, imaging follow-up was used. When SCI and PET/MRI results disagreed, medical records were checked for management changes. Follow-up data were collected until 8/2021.One hundred sixty-four subjects were included, eighty-five (52%) were female, and the median age was 60 years (IQR 50-69). At a subject level, PET/MRI had higher sensitivity (0.97, 95% CI 0.86-1.00) than SCI (0.54, 95% CI 0.37-0.71), p0.001, without a difference in specificity, of 0.95 (95% CI 0.90-0.98) for PET/MRI and 0.98 (95% CI 0.93-1.00) for SCI, p = 0.250. PET/MRI and SCI results disagreed in 19 cases. In 5/19 (26%) of the discordant cases, PET/MRI findings consistent with PC missed on SCI led to management changes.PET/MRI improves detection of PC compared to SCI which frequently changes management.

SNIO–CBP, a single-nanometer iron oxide (SNIO) nanoparticle functionalized with a type I collagen-binding peptide (CBP), was developed as a T 1 -weighted MRI contrast agent with only endogenous elements for fast and noninvasive detection of liver fibrosis. SNIO–CBP exhibits 6.7-fold higher relaxivity compared to a molecular gadolinium-based collagen-binding contrast agent CM-101 on a per CBP basis at 4.7 T. Unlike most iron oxide nanoparticles, SNIO–CBP exhibits fast elimination from the bloodstream with a 5.7 min half-life, high renal clearance, and low, transient liver enhancement in healthy mice. We show that a dose of SNIO–CBP that is 2.5-fold lower than that for CM-101 has comparable imaging efficacy in rapid (within 15 min following intravenous injection) detection of hepatotoxin-induced liver fibrosis using T 1 -weighted MRI in a carbon tetrachloride–induced mouse liver injury model. We further demonstrate the applicability of SNIO–CBP in detecting liver fibrosis in choline-deficient L -amino acid-defined high-fat diet mouse model of nonalcoholic steatohepatitis. These results provide a platform with potential for the development of high relaxivity, gadolinium-free molecular MRI probes for characterizing chronic liver disease.

During fibroproliferation, protein-associated extracellular aldehydes are formed by the oxidation of lysine residues on extracellular matrix proteins to form the aldehyde allysine. Here we report three Mn(II)-based, small molecule magnetic resonance (MR) probes that contain α-effect nucleophiles to target allysine in vivo and report on tissue fibrogenesis. We used a rational design approach to develop turn-on probes with a 4-fold increase in relaxivity upon targeting. The effects of aldehyde condensation rate and hydrolysis kinetics on the performance of the probes to detect tissue fibrogenesis noninvasively in mouse models were evaluated by a systemic aldehyde tracking approach. We showed that for highly reversible ligations, off-rate was a stronger predictor of in vivo efficiency, enabling histologically validated, three-dimensional characterization of pulmonary fibrogenesis throughout the entire lung. The exclusive renal elimination of these probes allowed for rapid imaging of liver fibrosis. Reducing the hydrolysis rate by forming an oxime bond with allysine enabled delayed phase imaging of kidney fibrogenesis. The imaging efficacy of these probes, coupled with their rapid and complete elimination from the body, make them strong candidates for clinical translation.

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Purpose:To evaluate the prognostic value of posttreatment fibrosis in human PDAC patients, and to compare a type I collagen targeted MRI probe, CM-101, to the standard contrast agent, Gd-DOTA, for their abilities to identify FOLFIRINOX-induced fibrosis in a murine model of PDAC.Experimental Design:Ninety-three chemoradiation-treated human PDAC samples were stained for fibrosis and outcomes evaluated. For imaging, C57BL/6 and FVB mice were orthotopically implanted with PDAC cells and FOLFIRINOX was administered. Mice were imaged with Gd-DOTA and CM-101.Results:In humans, post-chemoradiation PDAC tumor fibrosis was associated with longer overall survival (OS) and disease-free survival (DFS) on multivariable analysis (OS P = 0.028, DFS P = 0.047). CPA increased the prognostic accuracy of a multivariable logistic regression model comprised of previously established PDAC risk factors [AUC CPA (−) = 0.76, AUC CPA (+) = 0.82]. In multiple murine orthotopic PDAC models, FOLFIRINOX therapy reduced tumor weight (P < 0.05) and increased tumor fibrosis by collagen staining (P < 0.05). CM-101 MR signal was significantly increased in fibrotic tumor regions. CM-101 signal retention was also increased in the more fibrotic FOLFIRINOX-treated tumors compared with untreated controls (P = 0.027), consistent with selective probe binding to collagen. No treatment-related differences were observed with Gd-DOTA imaging.Conclusions:In humans, post-chemoradiation tumor fibrosis is associated with OS and DFS. In mice, our MR findings indicate that translation of collagen molecular MRI with CM-101 to humans might provide a novel imaging technique to monitor fibrotic response to therapy to assist with prognostication and disease management.

Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ’s microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of Vegfr2 (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of Il6 or Vcan (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes.

Liver fibrogenesis is accompanied by upregulation of lysyl oxidase enzymes, which catalyze oxidation of lysine ε-amino groups on the extracellular matrix proteins to form the aldehyde containing amino acid allysine (Lys

Non-alcoholic steatohepatitis (NASH) is an increasing cause of chronic liver disease characterized by steatosis, inflammation, and fibrosis which can lead to cirrhosis, hepatocellular carcinoma, and mortality. Quantitative, noninvasive methods for characterizing the pathophysiology of NASH at both the preclinical and clinical level are sorely needed. We report here a multiparametric magnetic resonance imaging (MRI) protocol with the fibrogenesis probe Gd-Hyd to characterize fibrotic disease activity and steatosis in a common mouse model of NASH. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) to induce NASH with advanced fibrosis. Mice fed normal chow and CDAHFD underwent MRI after 2, 6, 10 and 14 weeks to measure liver T1, T2*, fat fraction, and dynamic T1-weighted Gd-Hyd enhanced imaging of the liver. Steatosis, inflammation, and fibrosis were then quantified by histology. NASH and fibrosis developed quickly in CDAHFD fed mice with strong correlation between morphometric steatosis quantification and liver fat estimated by MRI (r = 0.90). Sirius red histology and collagen quantification confirmed increasing fibrosis over time (r = 0.82). Though baseline T1 and T2* measurements did not correlate with fibrosis, Gd-Hyd signal enhancement provided a measure of the extent of active fibrotic disease progression and correlated strongly with lysyl oxidase expression. Gd-Hyd MRI accurately detects fibrogenesis in a mouse model of NASH with advanced fibrosis and can be combined with other MR measures, like fat imaging, to more accurately assess disease burden.

Objectives Mn-PyC3A is an experimental manganese (Mn)-based extracellular fluid magnetic resonance imaging (MRI) contrast agent that is being evaluated as a direct replacement for clinical gadolinium (Gd)-based contrast agents. The goals of this study were to use simultaneous positron emission tomography (PET)-MRI to (1) compare the whole-body pharmacokinetics, biodistribution, and elimination of Mn-PyC3A with the liver-specific contrast agent mangafodipir (Mn-DPDP), (2) determine the pharmacokinetics and fractional excretion of Mn-PyC3A in a rat model of renal impairment, and (3) compare whole-body elimination of Mn-PyC3A to gadoterate (Gd-DOTA) in a rat model of renal impairment. Methods Mn-PyC3A and Mn-DPDP were radiolabeled with the positron emitting isotope Mn-52 via Mn2+ exchange with 52MnCl2. Dynamic simultaneous PET-MRI was used to measure whole-body pharmacokinetics and biodistribution of Mn-52 immediately and out to 7 days after an intravenous 0.2 mmol/kg dose of [52Mn]Mn-PyC3A to normal or to 5/6 nephrectomy rats or a 0.01 mmol/kg dose of [52Mn]Mn-DPDP to normal rats. The fractional excretion and 1- and 7-day biodistribution in rats after the injection of 2.0 mmol/kg [52Mn]Mn-PyC3A (n = 11 per time point) or 2.0 mmol/kg Gd-DOTA (n = 8 per time point) were quantified by gamma counting or Gd elemental analysis, respectively. Comparisons of Mn-PyC3A pharmacokinetics and in vivo biodistribution in normal and 5/6 nephrectomy rats and comparisons of ex vivo Mn versus Gd biodistribution data in 5/6 nephrectomy were made with an unpaired t test. Results Dynamic PET-MRI data demonstrate that both [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP were eliminated by mixed renal and hepatobiliary elimination but that a greater fraction of [52Mn]Mn-PyC3A was eliminated by renal filtration. Whole-body PET images show that Mn-52 from [52Mn]Mn-PyC3A was efficiently eliminated from the body, whereas Mn-52 from [52Mn]Mn-DPDP was retained throughout the body. The blood elimination half-life of [52Mn]Mn-PyC3A in normal and 5/6 nephrectomy rats was 13 ± 3.5 minutes and 23 ± 12 minutes, respectively (P = 0.083). Area under the curve between 0 and 60 minutes postinjection (AUC0-60) in the bladder of normal and 5/6 nephrectomy rats was 2600 ± 1700 %ID/cc*min and 750 ± 180 %ID/cc*min, respectively (P = 0.024), whereas AUC0-60 in the liver of normal and 5/6 nephrectomy rats was 33 ± 13 %ID/cc*min and 71 ± 16 %ID/cc*min, respectively (P = 0.011), indicating increased hepatobiliary elimination in 5/6 nephrectomy rats. The %IDs of Mn from [52Mn]Mn-PyC3A and Gd from Gd-DOTA recovered from 5/6 nephrectomy rats 1 day after injection were 2.0 ± 1.1 and 1.3 ± 0.34, respectively (P = 0.10) and 7 days after injection were 0.14 ± 0.11 and 0.41 ± 0.24, respectively (P = 0.0041). Conclusions Mn-PyC3A has different pharmacokinetics and is more efficiently eliminated than Mn-DPDP in normal rats. Mn-PyC3A is efficiently eliminated from both normal and 5/6 nephrectomy rats, with increased fractional hepatobiliary excretion from 5/6 nephrectomy rats. Mn-PyC3A is more completely eliminated than Gd-DOTA from 5/6 nephrectomy rats after 7 days.

MRI has emerged as the most comprehensive noninvasive diagnostic tool for liver diseases. In recent years, the value of MRI in the liver has been significantly enhanced by a wide range of contrast agents, both clinically available and under development, that add functional information to the anatomically detailed morphological images, or increase the distinction between normal and pathological tissues by targeting molecular and cellular events. Several classes of contrast agents are available for contrast-enhanced hepatic MRI, including 1) conventional nonspecific extracellular fluid (ECF) contrast agents for assessing tissue perfusion; 2) hepatobiliary-specific contrast agents that are taken up by functioning hepatocytes and excreted through the biliary system for evaluating hepatobiliary function; 3) Superparamagnetic iron oxide particles that accumulate in Kuppfer cells; and 4) novel molecular contrast agents that are biochemically targeted to specific molecular/cellular processes for staging liver diseases or detecting treatment responses. The use of different functional and molecular MRI methods allows noninvasive assessment of disease burden, progression, and treatment response in a variety of liver diseases. The multiparametric capability of MR imaging provides the opportunity for high diagnostic performance by combining imaging biomarkers.

RATIONALE AND OBJECTIVES Liver inflammation is associated with nonalcoholic steatohepatitis and other pathologies, but noninvasive methods to assess liver inflammation are limited. Inflammation causes endothelial disruption and leakage of plasma proteins into the interstitial space and can result in extravascular coagulation with fibrin deposition. Here we assess the feasibility of using the established fibrin-specific magnetic resonance probe EP-2104R for the noninvasive imaging of fibrin as a marker of liver inflammation. METHODS Weekly 100 mg/kg diethylnitrosamine (DEN) dosing was used to generate liver fibrosis in male rats; control animals received vehicle. Magnetic resonance imaging at 1.5 T with EP-2104R, a matched non-fibrin-binding control linear peptide, or the collagen-specific probe EP-3533 was performed at 1 day or 7 days after the last DEN administration. Imaging data were compared with quantitative histological measures of fibrosis and inflammation. RESULTS After 4 or 5 DEN administrations, the liver becomes moderately fibrotic, and fibrosis is the same if the animal is killed 1 day (Ishak score, 3.62 ± 0.31) or 7 days (Ishak score, 3.82 ± 0.25) after the last DEN dose, but inflammation is significantly higher at 1 day compared with 7 days after the last DEN dose (histological activity index from 0-4, 3.54 ± 0.14 vs 1.61 ± 0.16, respectively; P < 0.0001). Peak EP-2104R signal enhancement was significantly higher in animals imaged at 1 day post-DEN compared with 7 days post-DEN or control rats (29.0% ± 3.2% vs 22.4% ± 2.0% vs 17.0% ± 0.2%, respectively; P = 0.017). Signal enhancement with EP-2104R was significantly higher than control linear peptide at 1 day post-DEN but not at 7 days post-DEN indicating specific fibrin binding during the inflammatory phase. Collagen molecular magnetic resonance with EP-3533 showed equivalent T1 change when imaging rats 1 day or 7 days post-DEN, consistent with equivalent fibrosis. CONCLUSIONS EP-2104R can specifically detect fibrin associated with inflammation in a rat model of liver inflammation and fibrosis.

Introduction: Interleukin 12 (IL-12) is an important bridge between the innate and adaptive immune systems. It is mainly produced by macrophages and can bind to its receptor (IL12R) on T cells. It affects several aspects of T cell activation, including IFN-γ and perforin production. Tumor-infiltrating CD8+ T cells perform important anti-tumoral actions, which need to be overcome to allow cancer cell growth. This work studied IL-12 administration as an HCC preventive strategy in cirrhotic mice. We investigated whether IL-12 can boost the recruitment of macrophages to the liver, increase their antigen-presenting ability, and activate cytolytic T cells to inhibit HCC development in cirrhotic livers. Method: 8 weeks old mice were fed a choline-deficient High fat diet (CDAHFD) for 16 weeks to create NAFLD-induced HCC. A subset of CDAHFD mice was injected intravenously with IL-12 (0.5mg/gr) weekly from 12 to 16 weeks. Mice were euthanized after 16 weeks, and liver and serum were collected for further analysis. Results: After tissue collection, we observed that 50% of untreated animals developed HCC lesions, while IL-12-treated mice did not have any HCC. After treatment with IL12, qPCR and western blot on total liver tissue showed up-regulation in the IL12 receptor and activation of the IL-12 pathway, evidenced by the increase in Stat4 expression and phosphorylation. Additionally, we detected an increase in IFN-γ expression by qPCR in IL-12-treated mice. Histological analysis showed a significant decrease in fat and collagen deposition in IL-12-treated livers compared to untreated livers. IL-12-treated mice had lower serum aminotransferase enzymes (AST & ALT), indicating improved liver function. Immunostaining and flow cytometry of F4/80 (macrophage marker) showed an increase in the number of macrophages in the livers treated with IL-12. Flow cytometry data showed that the majority of macrophages present in the IL-12 livers were CCR2+, a marker for recruited macrophages. Notably, IL-12-treated macrophages expressed more MHCII (antigen presentation marker) and less B7H4 (inhibitory marker). In addition, we observed a significant increase in the number of cytolytic T cells (CD8+) in the IL-12 treated livers, which had higher expression of the T cell activation marker (CD25) and elevated levels of perforin and IFN-γ that are nessacery for their cytotoxic activity. Conclusion: There is a lack of substantial evidence to describe the underlying mechanisms supporting the idea of cirrhosis regression and HCC prevention. Our study shows that the exogenous administration of IL-12 causes an increase in migrating profibrolysis macrophages to the liver and elevates cytolytic T-cell activity. IL-12 immunotherapy can trigger cirrhosis regression and prevent HCC development. Citation Format: Mozhdeh Sojoodi, Stephen C. Barrett, Derek J. Erstad, Veronica Clavijo Jordan, Eric M. Gale, Shadi Salloum, Yongtao Wang, Michael Lanuti, Lawrence Zukerberg, Peter Caravan, Georg M. Lauer, Kenneth K. Tanabe. IL-12 immunotherapy prevents hepatocellular carcinoma in a murine NAFLD induced cirrhosis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB278.

Purpose: Cu-64 labeled TTX-MC138, a microRNA-10b (miR-10b) inhibitor, will be evaluated in a Phase 0 clinical study conducted under an Exploratory IND to evaluate delivery of the molecule to metastatic lesions in subjects with advanced solid tumors by using positron emission tomography-magnetic resonance imaging (PET-MRI). Background: miR10b is a master regulator of the viability of metastatic tumor cells and promotes capacity of tumor cells to migrate and invade surrounding tissue. These findings prompted design of a miR10b-targeted therapeutic consisting of an anti-miR10b antagomir conjugated to ultrasmall iron oxide nanoparticles (MN), termed TTX-MC138. In mouse models of cancer, TTX-MC138 caused durable regressions of established metastases with no systemic toxicity. Clinical development will investigate if TTX-MC138 would accumulate in clinical metastases. TTX-MC138 is radiolabeled with isotope Cu-64 to generate the radiopharmaceutical TTX-MC138-NODAGA-Cu64. PET-MRI will assess pharmacokinetics (PK) of the radiolabeled product in humans and determine uptake in metastatic lesions. Methods: This open-label, single-center, single-arm, phase 0, microdose study in subjects with advanced solid tumors and radiographically confirmed metastases will evaluate delivery of TTX-MC138-NODAGA-Cu64 by using PET-MRI; establish PK of TTX-MC138-NODAGA-Cu64; and assess safety of TTX-MC138-NODAGA-Cu64. Approximately 12 subjects age ≥ 18 years are expected to be enrolled in the study. Eligible subjects will have confirmed diagnosis of at least 1 metastatic solid tumor (that is not amenable to curative therapy unless participation in the study would delay standard therapy), Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1, and at least 1 target lesion per Response Evaluation Criteria in Solid Tumors version 1.1 (at least 10 mm per MRI from fludeoxyglucose [FDG] PET-MRI). Study duration is up to 46 days and includes a screening visit, dosing period (2 visits), and a follow-up visit. On Day 1, subjects receive 1 microdose of TTX-MC138-NODAGA-Cu64 by intravenous bolus. Days 1 and 2 include whole body PET-MRI imaging, ECG and blood collection. Safety data are collected at all visits. A Data Review Committee may review specific study data, including safety data. The primary endpoint will measure percent injected dose per cubic centimeter (%ID/cc) tissue of TTX-MC138-NODAGA-Cu64 delivered to metastatic lesions. Secondary endpoints may include evaluation of PK of key components of TTX-MC138-NODAGA-Cu64, metabolite analysis, and target engagement. Safety data, including incidence of adverse events, clinical lab data, ECGs, vital signs and concomitant medications, will be summarized. Enrollment is expected to start in 2023. Citation Format: Andreas Varkaris, Peter Caravan, Neil Robertson, Subrata Ghosh, Michael Warren, Zdravka Medarova, Susan Duggan. An open-label, single-center, phase 0, microdose study to demonstrate delivery of TTX-MC138-NODAGA-Cu64 to radiographically confirmed metastases in subjects with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT246.

The recent past has seen impressive progress in the treatment of various malignancies using immunotherapy. One of the most promising approaches involves immune checkpoint inhibition, mainly relying on monoclonal antibodies or small molecules. With specific relevance to pancreatic cancer, however, checkpoint inhibitors are largely ineffective, in part because of poor T cell infiltration, low tumor mutational burden, an immunosuppressive tumor microenvironment, and the presence of a dense desmoplastic stroma. To overcome these challenges, here we describe the preclinical testing of a new checkpoint inhibitor named MN-siPDL1 in combination with gemcitabine in an aggressive model of PDAC. MN-siPDL1 utilizes the RNA interference mechanism (siRNA) to block the synthesis of the critical immune checkpoint mediator, PD-L1 by tumor cells. The siRNA is delivered by a nanoparticle carrier (MN) that is optimized for oligonucleotide delivery to solid tumors. The efficacy and preliminary safety of MN-siPDL1 were tested in an orthotopic syngeneic model of pancreatic ductal adenocarcinoma generated by implanting Hy15549 murine PDAC cells into the pancreas of C57BL/6J mice. Our studies demonstrated that MN-siPDL1 was successfully delivered and effective even in the highly desmoplastic and hypovascular Hy15549 murine model of PDAC, which has been deemed nonresponsive to antibody-based immune checkpoint blockade. Anatomic MRI showed that in the animals treated with MN-siPDL1 plus gemcitabine, tumor growth rates were lower than in the vehicle control. After two weekly treatments with MN-siPDL1 plus gemcitabine, tumor volumes were four times smaller than in untreated animals. Importantly, animal survival was improved dramatically in animals treated with MN-siPDL1 plus gemcitabine compared to all other groups. Among the animals treated with MN-siPDL1 plus gemcitabine, the hazard ratio for overall survival (OS) relative to PBS was 0.08. Interestingly, even in the absence of gemcitabine, MN-siPDL1 as monotherapy improved survival more dramatically than gemcitabine (HR, 0.24 for MN-siPDL1 vs. 0.42 for gemcitabine). Immunohistology on the tumor tissues post-necropsy indicated that the treatment inhibited PD-L1 and induced a cytotoxic immune response. Finally, as an initial measurement of tissue damage due to the treatment, we analyzed major organs by histopathology and saw no differences from the vehicle-treated controls. Considering the aggressive and fibrous nature of the Hy15549 model and its resistance to traditional checkpoint inhibitors, the described RNAi-based therapeutic approach could be promising against PDAC and could make an impact on one of the most intractable cancers which has long evaded the power of modern medicine to deliver long-term survival. Citation Format: Byunghee Yoo, Mozhdeh Sojoodi, Veronica Clavijo Jordan, Pamela Pantazopoulos, Subrata Ghosh, Peter Caravan, Zdravka Medarova. Cancer immunotherapy based on the RNAi-based PD-L1 inhibitor, MN-siPDL1, demonstrates efficacy in preclinical pancreatic adenocarcinoma. [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 5069.

Background and Purpose: High-risk atherosclerosis is an underlying cause of cardiovascular events, yet identifying the specific patient population at immediate risk is still challenging. Here, we used a rabbit model of atherosclerotic plaque rupture and human carotid endarterectomy specimens to describe the potential of molecular fibrin imaging as a tool to identify thrombotic plaques. Methods: Atherosclerotic plaques in rabbits were induced using a high-cholesterol diet and aortic balloon injury (N=13). Pharmacological triggering was used in a group of rabbits (n=9) to induce plaque disruption. Animals were grouped into thrombotic and nonthrombotic plaque groups based on gross pathology (gold standard). All animals were injected with a novel fibrin-specific probe 68 Ga-CM246 followed by positron emission tomography (PET)/magnetic resonance imaging 90 minutes later. 68 Ga-CM246 was quantified on the PET images using tissue-to-background (back muscle) ratios and standardized uptake value. Results: Both tissue-to-background (back muscle) ratios and standardized uptake value were significantly higher in the thrombotic versus nonthrombotic group ( P 68 Ga-CM246 PET agreed with gross pathology assessment (85%). In ex vivo surgical specimens obtained from patients undergoing elective carotid endarterectomy (N=12), 68 Ga-CM246 showed significantly higher binding to carotid plaques compared to a D-cysteine nonbinding control probe. Conclusions: We demonstrated that molecular fibrin PET imaging using 68 Ga-CM246 could be a useful tool to diagnose experimental and clinical atherothrombosis. Based on our initial results using human carotid plaque specimens, in vivo molecular imaging studies are warranted to test 68 Ga-CM246 PET as a tool to stratify risk in atherosclerotic patients.

Background Fibrin deposition is a fundamental pathophysiological event in the inflammatory component of various CNS disorders, such as multiple sclerosis (MS) and Alzheimer’s disease. Beyond its traditional role in coagulation, fibrin elicits immunoinflammatory changes with oxidative stress response and activation of CNS-resident/peripheral immune cells contributing to CNS injury. Purpose To investigate if CNS fibrin deposition can be determined using molecular MRI, and to assess its capacity as a non-invasive imaging biomarker that corresponds to inflammatory response and barrier impairment. Materials and methods Specificity and efficacy of a peptide-conjugated Gd-based molecular MRI probe (EP2104-R) to visualise and quantify CNS fibrin deposition were evaluated. Probe efficacy to specifically target CNS fibrin deposition in murine adoptive-transfer experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for MS (n = 12), was assessed. Findings were validated using immunohistochemistry and laser ablation inductively coupled plasma mass spectrometry. Deposition of fibrin in neuroinflammatory conditions was investigated and its diagnostic capacity for disease staging and monitoring as well as quantification of immunoinflammatory response was determined. Results were compared using t-tests (two groups) or one-way ANOVA with multiple comparisons test. Linear regression was used to model the relationship between variables. Results For the first time (to our knowledge), CNS fibrin deposition was visualised and quantified in vivo using molecular imaging. Signal enhancement was apparent in EAE lesions even 12-h after administration of EP2104-R due to targeted binding (M ± SD, 1.07 ± 0.10 (baseline) vs. 0.73 ± 0.09 (EP2104-R), p = .008), which could be inhibited with an MRI-silent analogue (M ± SD, 0.60 ± 0.14 (EP2104-R) vs. 0.96 ± 0.13 (EP2104-La), p = .006). CNS fibrin deposition corresponded to immunoinflammatory activity (R2 = 0.85, p 2 = 0.81, p M ± SD, 6.6 ± 1 (EP2104-R) vs. 2.7 ± 0.4 (gadobutrol), p = .004) than clinically used contrast media, which increases sensitivity for lesion detection. Conclusions Molecular imaging of CNS fibrin deposition provides an imaging biomarker for inflammatory CNS pathology, which corresponds to pathophysiological ECM remodelling and disease activity, and yields high signal-to-noise ratio, which can improve diagnostic neuroimaging across several neurological diseases with variable degrees of barrier impairment.

During newborn lung injury, excessive activity of lysyl oxidases (LOXs) disrupts extracellular matrix (ECM) formation. Previous studies indicate that TGFβ activation in the O2-injured mouse pup lung increases lysyl oxidase (LOX) expression. But how TGFβ regulates this, and whether the LOXs generate excess pulmonary aldehydes are unknown. First, we determined that O2-mediated lung injury increases LOX protein expression in TGFβ-stimulated pup lung interstitial fibroblasts. This regulation appeared to be direct; this is because TGFβ treatment also increased LOX protein expression in isolated pup lung fibroblasts. Then using a fibroblast cell line, we determined that TGFβ stimulates LOX expression at a transcriptional level via Smad2/3-dependent signaling. LOX is translated as a pro-protein that requires secretion and extracellular cleavage before assuming amine oxidase activity and, in some cells, reuptake with nuclear localization. We found that pro-LOX is processed in the newborn mouse pup lung. Also, O2-mediated injury was determined to increase pro-LOX secretion and nuclear LOX immunoreactivity particularly in areas populated with interstitial fibroblasts and exhibiting malformed ECM. Then, using molecular probes, we detected increased aldehyde levels in vivo in O2-injured pup lungs, which mapped to areas of increased pro-LOX secretion in lung sections. Increased activity of LOXs plays a critical role in the aldehyde generation; an inhibitor of LOXs prevented the elevation of aldehydes in the O2-injured pup lung. These results reveal new mechanisms of TGFβ and LOX in newborn lung disease and suggest that aldehyde-reactive probes might have utility in sensing the activation of LOXs in vivo during lung injury.