The predictive impact of dual somatostatin receptor/fluorodeoxyglucose (FDG) positron emission tomography (PET) in metastatic gastroenteropancreatic neuroendocrine tumors (GEP-NETs): review of literature and a single institution experience.

Treatment with radiolabelled somatostatin analogs, a form of peptide receptor radionuclide therapy (PRRT), has changed the management of patients with advanced gastroenteropancreatic neuroendocrine tumors (GEP-NETs). There is a subgroup of patients who have suboptimal benefit and rapidly progress on PRRT, indicating that accurate prognostic and predictive markers are urgently needed. Currently, most of the literature concentrate on the prognostic impact of the dual positron emission tomography (PET) scan with very few information regarding the predictive value. We report a case series and review the literature to summarizes the predictive value of combined somatostatin receptor (SSTR) and fluorodeoxyglucose (FDG) PET in metastatic GEP-NETs. We conducted a review of the literature for data published from 2010 to 2021 in MEDLINE, Embase, the National Institutes of Health trial registry, Cochrane CENTRAL, and published proceedings from major gastrointestinal and neuroendocrine cancer meetings. Our main criteria included all published prospective and retrospective data in which the predictive value of dual PET scans using SSTR and FDG was correlated with PRRT response in patients with metastatic GEP-NETs. We summarized clinical outcomes including progression-free survival (PFS), overall survival (OS), and post-therapy complications associated with PRRT according to FDG avidity. We excluded studies that did not include FDG PET scan, GEP patients, studies with no clear predictive value of the FDG PET scan, and studies that did not report a direct correlation between FDG avidity and primary outcome. Additionally, we summarized our institutional experience in eight patients who progressed during or within the first year of PRRT treatment. Our search identified 1306 articles; most of them showed only the prognostic value of Integrated SSTR/FDG PET imaging biomarker in GEP-NETs. Only three studies (n=75 patients) met our inclusion criteria and retrospectively investigated the predictive value of dual SSTR and FDG imaging in subjects being considered for PRRT. The results confirmed that FDG avidity correlates with advanced NET grades. Lesions that are both SSTR and FDG avid had early disease progression. In one study, at multivariate analysis, FDG PET results were independently predictive of lower PFS for PRRT. In our case series, there were eight patients with metastatic well-differentiated GEP-NETs (grades 2 and 3) who progressed within one year of PRRT. Seven of them had positive FDG PET scan at the time of progression. In conclusion, Dual SSTR/FDG PET imaging has a potential predictive impact for PRRT in GEP-NETs. It permits the capturing of the disease complexity and aggressiveness, which correlates with PRRT response. Therefore, prospective future trials should validate the predictive value of dual SSTRs/FDG PET for better PRRT stratification.

PET Imaging of Hepatocellular Carcinoma Using ZD2-(68Ga-NOTA).

Purpose: We tested a recently developed short peptide radioligand for PET imaging of hepatocellular carcinoma (HCC) by targeting an oncoprotein, extra-domain B fibronectin (EDB-FN) in the tumor microenvironment. Methods: The radioligand consists of a small linear peptide ZD2 with 68Ga-NOTA chelator, and specifically binds to EDB-FN. PET images were acquired dynamically for 1 hour after intravenously (i.v.) injecting 37 MBq (1.0 mCi) of the radioligand into the woodchuck model of naturally occurring HCC. Woodchuck HCC originated from chronic viral hepatitis infection, which recapitulates the corresponding human primary liver cancer. The animals were euthanized post-imaging for tissue collection and validation. Results: For ZD2 avid liver tumors, the radioligand accumulation plateaued a few minutes after injection, while the liver background uptake stabilized 20 min post-injection. The status of EDB-FN in woodchuck HCC was confirmed by histology and validated by PCR and western blocking. Conclusion: We have showed the viability of using the ZD2 short peptide radioligand targeting EDB-FN in liver tumor tissue for PET imaging of HCC, which can potentially impact the clinical care for HCC patients.

Imaging of Tumor-Associated Vascular Prostate-Specific Membrane Antigen in Woodchuck Model of Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Radiolabeled short peptide ligands targeting prostate-specific membrane antigen (PSMA) were developed initially for imaging and treatment of prostate cancers. While many nonprostate solid tumors including hepatocellular carcinoma (HCC) express little PSMA, their neovasculature expresses a high level of PSMA, which is avid for Gallium-68-labeled PSMA-targeting radio-ligand (68Ga-PSMA-11) for positron emission tomography (PET). However, the lack of a spontaneous animal model of tumor-associated vascular PSMA overexpression has hindered the development and assessment of PSMA-targeting radioligands for imaging and therapy of the nonprostatic cancers. We identified detectable indigenous PSMA expression on tumor neovascular endothelia in a naturally occurring woodchuck model of HCC. METHODS: Molecular docking was performed with 3 bait PSMA ligands and compared between human and woodchuck PSMA. Initially, PET images were acquired dynamically after intravenously injecting 37 MBq (1.0 mCi) of 68Ga-PSMA-11 into woodchuck models of HCC. Subsequently, 10-minute static PET scans were conducted for other animals 1-hour after injection due to HCC and liver background uptake stabilization at 30-45 minutes after injection. Liver tissue samples were harvested after imaging, fresh-frozen for quantitative reverse transcription polymerase chain reaction and western blot for validation, or fixed for histology for correlation. RESULTS: Our preclinical studies confirmed the initial clinical findings of 68Ga-PSMA-11 uptake in HCC. The agents (ligands and antibodies) developed against human PSMA were found to be reactive against the woodchuck PSMA. CONCLUSION: This animal model offers a unique opportunity for investigating the biogenesis of tumor-associated vascular PSMA, its functional role(s), and potentials for future treatment strategies targeting tumor vascular PSMA using already developed PSMA-targeting agents.

The Role of the Microbiome in Gastroentero-Pancreatic Neuroendocrine Neoplasms (GEP-NENs).

Gut microbiome balance plays a key role in human health and maintains gut barrier integrity. Dysbiosis, referring to impaired gut microbiome, is linked to a variety of diseases, including cancers, through modulation of the inflammatory process. Most studies concentrated on adenocarcinoma of different sites with very limited information on gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). In this study, we have analyzed the gut microbiome (both fungal and bacterial communities) in patients with metastatic GEP-NENs. Fecal samples were collected and compared with matched healthy control samples using logistic regression distances utilizing R package MatchIt (version 4.2.0, Daniel E. Ho, Stanford, CA, USA). We examined differences in microbiome profiles between GEP-NENs and control samples using small subunit (SSU) rRNA (16S), ITS1, ITS4 genomic regions for their ability to accurately characterize bacterial and fungal communities. We correlated the results with different behavioral and dietary habits, and tumor features including differentiation, grade, primary site, and therapeutic response. All tests are two-sided and p-values ≤ 0.05 were considered statistically significant. Gut samples of 34 patients (12 males, 22 females, median age 64 years) with metastatic GEP-NENs (22 small bowel, 10 pancreatic, 1 gall bladder, and 1 unknown primary) were analyzed. Twenty-nine patients had well differentiated GEP-neuroendocrine tumors (GEP-NETs), (G1 = 14, G2 = 12, G3 = 3) and five patients had poorly differentiated GEP-neuroendocrine carcinomas (GEP-NECs). Patients with GEP-NENs had significantly decreased bacterial species and increased fungi (notably Candida species, Ascomycota, and species belonging to saccharomycetes) compared to controls. Patients with GEP-NECs had significantly enriched populations of specific bacteria and fungi (such as Enterobacter hormaechei, Bacteroides fragilis and Trichosporon asahii) compared to those with GEP-NETs (p = 0.048, 0.0022 and 0.034, respectively). In addition, higher grade GEP-NETs were associated with significantly higher Bacteroides fragilis (p = 0.022), and Eggerthella lenta (p = 0.00018) species compared to lower grade tumors. There were substantial differences associated with dietary habits and therapeutic responses. This is the first study to analyze the role of the microbiome environment in patients with GEP-NENs. There were significant differences between GEP-NETs and GEP-NECs, supporting the role of the gut microbiome in the pathogenesis of these two distinct entities.

Staphylococcus aureus and Acinetobacter baumannii Inhibit Osseointegration of Orthopedic Implants.

Bacterial infections routinely cause inflammation and thereby impair osseointegration of orthopedic implants. Acinetobacter spp., which cause osteomyelitis following trauma, on or off the battlefield, were, however, reported to cause neither osteomyelitis nor osteolysis in rodents. We therefore compared the effects of Acinetobacter strain M2 to those of Staphylococcus aureus in a murine implant infection model. Sterile implants and implants with adherent bacteria were inserted in the femur of mice. Bacterial burden, levels of proinflammatory cytokines, and osseointegration were measured. All infections were localized to the implant site. Infection with either S. aureus or Acinetobacter strain M2 increased the levels of proinflammatory cytokines and the chemokine CCL2 in the surrounding femurs, inhibited bone formation around the implant, and caused loss of the surrounding cortical bone, leading to decreases in both histomorphometric and biomechanical measures of osseointegration. Genetic deletion of TLR2 and TLR4 from the mice partially reduced the effects of Acinetobacter strain M2 on osseointegration but did not alter the effects of S. aureus. This is the first report that Acinetobacter spp. impair osseointegration of orthopedic implants in mice, and the murine model developed for this study will be useful for future efforts to clarify the mechanism of implant failure due to Acinetobacter spp. and to assess novel diagnostic tools or therapeutic agents.

Human multipotent adult progenitor cells effectively reduce graft-vs-host disease while preserving graft-vs-leukemia activity.

Graft-vs-host disease (GvHD) limits successful outcomes following allogeneic blood and marrow transplantation (allo-BMT). We examined whether the administration of human, bone marrow-derived, multipotent adult progenitor cells (MAPCs™) could regulate experimental GvHD. The immunoregulatory capacity of MAPC cells was evaluated in vivo using established murine GvHD models. Injection of MAPC cells on day +1 (D1) and +4 (D4) significantly reduced T-cell expansion and the numbers of donor-derived, Tumor Necrosis Factor Alpha (TNFα) and Interferon Gamma (IFNγ)-producing, CD4+ and CD8+ cells by D10 compared with untreated controls. These findings were associated with reductions in serum levels of TNFα and IFNγ, intestinal and hepatic inflammation and systemic GvHD as measured by survival and clinical score. Biodistribution studies showed that MAPC cells tracked from the lung and to the liver, spleen, and mesenteric nodes within 24 hours after injection. MAPC cells inhibited mouse T-cell proliferation in vitro and this effect was associated with reduced T-cell activation and inflammatory cytokine secretion and robust increases in the concentrations of Prostaglandin E2 (PGE2) and Transforming Growth Factor Beta (TGFß). Indomethacin and E-prostanoid 2 (EP2) receptor antagonism both reversed while EP2 agonism restored MAPC cell-mediated in vitro T-cell suppression, confirming the role for PGE2. Furthermore, cyclo-oxygenase inhibition following allo-BMT abrogated the protective effects of MAPC cells. Importantly, MAPC cells had no effect on the generation cytotoxic T lymphocyte activity in vitro, and the administration of MAPC cells in the setting of leukemic challenge resulted in superior leukemia-free survival. Collectively, these data provide valuable information regarding the biodistribution and regulatory capacity of MAPC cells, which may inform future clinical trial design.

Small Molecule-Based Prodrug Targeting Prostate Specific Membrane Antigen for the Treatment of Prostate Cancer.

Metastatic castration-resistant prostate cancer poses a serious clinical problem with poor outcomes and remains a deadly disease. New targeted treatment options are urgently needed. PSMA is highly expressed in prostate cancer and has been an attractive biomarker for the treatment of prostate cancer. In this study, we explored the feasibility of targeted delivery of an antimitotic drug, monomethyl auristatin E (MMAE), to tumor tissue using a small-molecule based PSMA lig-and. With the aid of Cy5.5, we found that a cleavable linker is vital for the antitumor activity of the ligand-drug conjugate and have developed a new PSMA-targeting prodrug, PSMA-1-VcMMAE. In in vitro studies, PSMA-1-VcMMAE was 48-fold more potent in killing PSMA-positive PC3pip cells than killing PSMA-negative PC3flu cells. In in vivo studies, PSMA-1-VcMMAE significantly inhibited tumor growth leading to prolonged animal survival in different animal models, including metastatic prostate cancer models. Compared to anti-PSMA antibody-MMAE conjugate (PSMA-ADC) and MMAE, PSMA-1-VcMMAE had over a 10-fold improved maximum tolerated dose, resulting in improved therapeutic index. The small molecule-drug conjugates reported here can be easily synthesized and are more cost efficient than anti-body-drug conjugates. The therapeutic profile of the PSMA-1-VcMMAE encourages further clin-ical development for the treatment of advanced prostate cancer.

Liver background uptake of [18F]FLT in PET imaging.

High liver uptake presents a problem for 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) as a radiotracer for imaging cellular proliferation in the liver with positron emission tomography (PET). This investigation re-visited some issues related to the high liver background uptake of [18F]FLT with an animal model of woodchucks. Several enzymes involved in the hepatic catabolism of FLT, thymidine phosphorylase (TP, TYMP), uridine 5'-diphospho-glucuronosyl-transferases (UDP-GTs, short for UGTs), and ß-glucuronidase (GUSB), their homology as well as hepatic expression between the human and the woodchuck was examined. Inhibitors of these enzymes, TP inhibitor (TPI) tipiracil hydrochloride, UGT inhibitor probenecid, ß-glucuronidase inhibitor L-aspartate, were administered to the animals at human equivalent doses either intravenously (i.v.) and orally before the injection of tracer-dose [18F]FLT for PET imaging to examine any changes in liver uptake. Liver tissue samples were harvested from the animals after PET imaging and used to perform polymerase chain reaction (PCR) for TP expression or assays for enzymatic activities of TP and ß-glucuronidase. Non-radiolabeled (cold) FLT was also applied for enzyme saturation. Animals administered with TPI displayed lower radioactivity in the liver in comparison with the baseline scan. The application of probenecid did not change [18F]FLT liver uptake even though it reduced renal uptake. L-aspartate reduced the liver background uptake of [18F]FLT slightly. The application of cold FLT reduced overall uptake of [18F]FLT including the liver background. Therefore, the combined application of cold FLT and [18F]FLT merits further clinical investigation for reducing liver background uptake of [18F]FLT.

A pilot clinical trial of the cytidine deaminase inhibitor tetrahydrouridine combined with decitabine to target DNMT1 in advanced, chemorefractory pancreatic cancer.

DNA methyltransferase 1 (DNMT1) is scientifically validated as a molecular target to treat chemo-resistant pancreatic ductal adenocarcinoma (PDAC). Results of clinical studies of the pyrimidine nucleoside analog decitabine to target DNMT1 in PDAC have, however, disappointed. One reason is high expression in PDAC of the enzyme cytidine deaminase (CDA), which catabolizes decitabine within minutes. We therefore added tetrahydrouridine (THU) to inhibit CDA with decitabine. In this pilot clinical trial, patients with advanced chemorefractory PDAC ingested oral THU ~10 mg/kg/day combined with oral decitabine ~0.2 mg/kg/day, for 5 consecutive days, then 2X/week. We treated 13 patients with extensively metastatic chemo-resistant PDAC, including 8 patients (62%) with ascites: all had received ≥ 1 prior therapies including gemcitabine/nab-paclitaxel in 9 (69%) and FOLFIRINOX in 12 (92%). Median time on THU/decitabine treatment was 35 days (range 4-63). The most frequent treatment-attributable adverse event was anemia (n=5). No deaths were attributed to THU/decitabine. Five patients had clinical progressive disease (PD) prior to week 8. Eight patients had week 8 evaluation scans: 1 had stable disease and 7 PD. Median overall survival was 3.1 months. Decitabine systemic exposure is expected to decrease neutrophil counts; however, neutropenia was unexpectedly mild. To identify reasons for limited systemic decitabine effect, we measured plasma CDA enzyme activity in PDAC patients, and found a > 10-fold increase in those with metastatic vs resectable PDAC. We concluded that CDA activity is increased not just locally but also systemically in metastatic PDAC, suggesting a need for even higher CDA-inhibitor doses than used here.

Peritumoral/vascular expression of PSMA as a diagnostic marker in hepatic lesions.

BACKGROUND: The differential diagnosis between primary cholangiocarcinoma and metastatic pancreatobiliary adenocarcinoma is histologically challenging due to lack of distinct morphological features and reliable molecular markers. Prostate-specific membrane antigen (PSMA) is expressed in prostate epithelium and upregulated on the surface of prostatic adenocarcinoma cells. Studies have shown PSMA enzymatic activity is involved in malignancy-driven neoangiogenesis in the endothelium of tumor-associated neovasculature in breast, lung, thyroid, hepatocellular carcinoma (HCC) and urothelial cancer. Recently, PSMA-targeted imaging technology (PSMA PET-CT) detected the presence of PSMA in primary cholangiocarcinoma. However histological correlation with PSMA expression other mass lesions in the liver has not yet been studied. METHODS: 72 cases of liver mass resection were collected at a tertiary hospital from 2011 to 2019. Immunohistochemical stains for PSMA and CD34 were performed. The expression of PSMA in tumor cells and associated neovascular endothelium were analyzed separately and the locations of vascular structures were confirmed by CD34 expression. RESULTS: Among 72 cases, 28 cases (22/72, 38.9%) showed PSMA peritumoral/vascular expression only, 3 cases (3/72, 4.2%) showed tumor cell expression only, and 2 cases (2/72, 2.8%) showed both tumor cell and peritumoral/vascular expression. The remainder (39/72, 54.2%) showed no expression. Particularly, most of primary cholangiocarcinoma showed PSMA vascular expression (13/15, 86.7%), while none of the 18 cases of metastatic pancreatobiliary adenocarcinoma were positive for PSMA (0/18, 0%) (p < 0.01). Outside of pancreatobiliary adenocarcinoma, none of the metastatic tumors, including colon and lung cancers, expressed PSMA. In 8 cases of metastatic prostate carcinoma, 3 showed PSMA expressions in tumor cells only (3/8, 37.5%) and 2 expressed PMSA in both tumor cells and neovasculature (2/8, 25.0%). Out of 22 HCC cases, 15 (15/22, 68.2%) were positive for PSMA in tumor vasculature. None of the 5 hepatic adenoma expressed PSMA (0/5, 0%). CONCLUSION: Significantly enhanced tumor-associated neovascular PSMA expression was identified in primary cholangiocarcinoma, compared to metastatic pancreatobiliary adenocarcinoma. Our findings potentially provide a sensitive marker in differential diagnosis between otherwise morphologically indistinguishable cases.

Delayed response to proton beam treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has become one of the leading causes of cancer death worldwide. There has been anecdotal report regarding the effectiveness of proton beam treatment for HCC. In this pre-clinical investigation, the woodchuck model of viral hepatitis infection-induced HCC was used for proton beam treatment experiment. The radiopaque fiducial markers that are biodegradable were injected around the tumor under ultrasound guidance to facilitate positioning in sequential treatments. An α cradle mode was used to ensure reproducibility of animal positioning on the treatment couch. A CT scan was performed first for contouring by a radiation oncologist. The CT data set with contours was then exported for dose planning. Three fractionations, each 750 CcGyE, were applied every other day with a Mevion S250 passive scattering proton therapy system. Multiphase contrast-enhanced CT scans were performed after the treatment and at later times for follow-ups. 3 weeks post-treatment, shrinking of the HCC nodule was detected and constituted to a partial response (30% reduction along the long axis). By week nine after treatment, the nodule disappeared during the arterial phase of multiphase contrast-enhanced CT scan. Pathological evaluation corroborated with this imaging response. A delayed, but complete imaging response to proton beam treatment applied to HCC was achieved with this unique and clinically relevant animal model of HCC.

[18F] Clofarabine for PET Imaging of Hepatocellular Carcinoma.

Clinical diagnosis of hepatocellular carcinoma (HCC) relies heavily on radiological imaging. However, information pertaining to liver cancer treatment such as the proliferation status is lacking. Imaging tumor proliferation can be valuable in patient management. This study investigated 18F-labeled clofarabine ([18F]CFA) targeting deoxycytidine kinase (dCK) for PET imaging of dCK-dependent proliferation in HCC. Since clinical PET scans showed a high liver background uptake of [18F]CFA, the aim of this study was to reduce this liver background uptake. A clinically relevant animal model of spontaneously developed HCC in the woodchucks was used for imaging experiments. Several modifiers were tested and compared with the baseline PET scan: Forodesine, probenecid, and cold clofarabine, all applied before the hot [18F]CFA injection to evaluate the reduction in liver background uptake. Application of forodesine before hot [18F]CFA injection did not reduce the background uptake. Instead, it increased the background by 11.6-36.3%. Application of probenecid also increased the liver background uptake by 16.6-32.1%. Cold CFA application did reduce the liver background uptake of [18F]CFA, comparing to the baseline scan. Combining cold CFA with [18F]CFA for PET imaging of liver cancers is a promising strategy, worthy of further clinical evaluation.

Synthesis and assessment of ZD2-(68Ga-NOTA) specific to extradomain B fibronectin in tumor microenvironment for PET imaging of pancreatic cancer.

Patients diagnosed with pancreatic cancer at a late stage have a dismal survival rate. Accurate early detection of pancreatic cancer with a size of 10 mm or less could dramatically improve patient survival after timely treatments. We have developed a new PET probe ZD2-(68Ga-NOTA) specific to extradomain-B fibronectin (EDB-FN), an oncoprotein in tumor microenvironment, for sensitive molecular imaging and early diagnosis of pancreatic cancer. A targeted ligand ZD2-NOTA is synthesized by conjugation of a macrocyclic ligand NOTA via a 6-aminohexanoic acid spacer to a linear ZD2 peptide (Thr-Val-Arg-Thr-Ser-Ala-Asp). ZD2-(68Ga-NOTA) is synthesized by relabeling of ZD2-NOTA with 68GaCl3 in a high purity under GMP conditions. The expression of EDB-FN is demonstrated in BxPC3 and Capan-1 human pancreatic cancer cells and tumor xenografts in mice. ZD2-(68Ga-NOTA) results in significantly higher uptake in the both BxPC3 and Capan-1 tumor xenografts than normal organs and tissues, including the brain, heart, liver and muscle, at 1 hr postinjection in mice. The tumor to muscle uptake ratio is at least 5 folds for the tracer in both tumors. ZD2-(68Ga-NOTA) is able to clearly delineate the PaCa tumors with a size of 10 mm or less with minimal background noise in normal tissues, including the liver. Substantial tumor uptake is still visible at 2 hr post-injection. The results suggest that the ZD2 peptide targeted PET probe has a potential for sensitive molecular imaging of EDB-FN and early detection of pancreatic cancer to improve healthcare of the patients diagnosed with the disease.

PET imaging of hepatocellular carcinoma with anti-1-amino-3-[18F]fluorocyclobutanecarboxylic acid in comparison with L-[S-methyl-11C]methionine.

PURPOSE: [11C]methionine ([11C]Met) was used for cancer imaging based on upregulated amino acid transport and protein synthesis in different tumor types. However, the short half-life of 11C decay limited further clinical development of [11C]Met. Synthetic amino acid analog anti-1-amino-3-[18F]fluoro-cyclobutyl-1-carboxylic acid ([18F]FCABC) was developed and FDA-approved for PET imaging of recurrent prostate cancer. This study investigated "repurposed" [18F]FACBC for PET imaging of primary liver cancer such as hepatocellular carcinoma (HCC) in comparison with [11C]Met. METHODS: [11C]Met was synthesized in the lab, and [18F]FACBC was purchased from a commercial outlet. A clinically relevant animal model of spontaneously developed HCC in the woodchucks was used for PET imaging. Bioinformatics analysis was performed for the expression of amino acid transporters responsible for radiotracer uptake and validated by PCR. Dynamic PET scans of [11C]Met and [18F]FACBC were acquired within 1 week. Standardized uptake value (SUV) was calculated for regions of interest (ROIs) defined over HCC and a liver background region. H&E staining and immunohistochemical (IHC) staining were performed with harvested tissues post-imaging. RESULTS: Higher expression of ACST2 and LAT1 was found in HCC than in the surrounding liver tissues. PCR validated this differential expression. [11C]Met and [18F]FACBC displayed some differences in their uptake and retention in HCC. Both peaked in HCC with an SUV of 3.5 after 10 min post-injection. Met maintained a plateaued contrast uptake in HCC to that in the liver while [18F]FCABC declined in HCC and liver after peak uptake. The pathological assessment revealed the liver tumor as moderately differentiated similar to the human HCC and proliferative. CONCLUSION: Both [18F]FACBC and [11C]Met showed uptake in HCC through the use of a clinically relevant animal model of woodchuck HCC. The uptake and retention of [18F]FACBC and [11C]Met depend on their metabolism and also rely on the distribution of their principal amino acid transporters.

Senescence cell-associated extracellular vesicles serve as osteoarthritis disease and therapeutic markers.

Senescent cells (SnCs) are increasingly recognized as central effector cells in age-related pathologies. Extracellular vesicles (EVs) are potential cellular communication tools through which SnCs exert central effector functions in the local tissue environment. To test this hypothesis in a medical indication that could be validated clinically, we evaluated EV production from SnCs enriched from chondrocytes isolated from human arthritic cartilage. EV production increased in a dose-responsive manner as the concentration of SnCs increased. The EVs were capable of transferring senescence to nonsenescent chondrocytes and inhibited cartilage formation by non-SnCs. microRNA (miR) profiles of EVs isolated from human arthritic synovial fluid did not fully overlap with the senescent chondrocyte EV profiles. The effect of SnC clearance was tested in a murine model of posttraumatic osteoarthritis. miR and protein profiles changed after senolytic treatment but varied depending on age. In young animals, senolytic treatment altered expression of miR-34a, -30c, -125a, -24, -92a, -150, and -186, and this expression correlated with cartilage production. The primary changes in EV contents in aged mice after senolytic treatment, which only reduced pain and degeneration, were immune related. In sum, EV contents found in synovial fluid may serve as a diagnostic for arthritic disease and indicator for therapeutic efficacy of senolytic treatment.

Preparation and Evaluation of ZD2 Peptide 64Cu-DOTA Conjugate as a Positron Emission Tomography Probe for Detection and Characterization of Prostate Cancer.

Positron emission tomography (PET) is a sensitive modality for cancer molecular imaging. We aim to develop a PET probe for sensitive detection and risk stratification of prostate cancer by targeting an abundant microenvironment oncoprotein, extradomain-B fibronectin (EDB-FN). The probe consists of a small ZD2 peptide specific to EDB-FN and a 64Cu-DOTA chelate. The probe was synthesized using standard solid-phase peptide chemistry and chelated to 64Cu prior to imaging. PET images were acquired at 4 and 22 h after intravenously injecting a 200 µCi probe into mice bearing human PC3 and LNCaP tumors, which represent highly aggressive and slow-growing prostate tumors, respectively. At 4 and 22 h postinjection, tumors could be clearly identified in the PET images. A significant higher signal was observed in PC3 tumors than in LNCaP tumors at 22 h (p = 0.01). Probe accumulation was also higher in PC3 tumors at 24 h. These data demonstrated that PET molecular imaging of EDB-FN in the tumor microenvironment of prostate cancer allows efficient differentiation of PC3 and LNCaP tumors in vivo. The ZD2 peptide-targeted PET probe shows potential in the detection and characterization of high-risk prostate cancer to improve the clinical management of prostate cancer.

Analysis of -5p and -3p Strands of miR-145 and miR-140 During Mesenchymal Stem Cell Chondrogenic Differentiation.

The chondrogenic differentiation of mesenchymal stem cells (MSCs) is mediated by transcription factors and small noncoding RNAs such as microRNAs (miRNAs). Each miRNA is initially transcribed as a long transcript, which matures to produce -5p and -3p strands. It is widely believed that the mature and functional miRNA from any given pre-miRNA, usually the -5p strand, is functional, while the opposing -3p strand is degraded. However, recent cartilage literature started to show functional -3p strands for a few miRNAs. This study aimed at examining both -5p and -3p strands of two key miRNAs miR-140 and miR-145, known to be involved in the chondrogenic differentiation of MSCs. The level (copy number) of both -5p and -3p strands of miR-145 and miR-140 along the time line of MSC chondrogenic differentiation was determined by polymerase chain reaction. The gene expression profiles of several genes related to MSC chondrogenesis were compared with these miRNA profiles along the same timeline. While miR-145-3p is declining in step with miR-145-5p in pellet cultures during the process, the -3p strand is only 1-2% of the total miR-145 products. In contrast, the mature -3p and -5p products of miR-140 are found to increase with near-equal molar expression throughout chondrogenic differentiation. Numerous genes are expressed by cartilage progenitor cells during development. One such target gene, Sox9, is a regulatory target of the dominant miR-145-5p, consistent with the data. Further experimental validations are warranted to confirm that ACAN, FOXO1, and RUNX3 as direct targets of miR-145-5p in the context of MSC chondrogenesis. Similarly, TRSP1 and ACAN are worth further validation as direct targets of miR-145-3p. For miR-140, SOX4 shall be further validated as a direct target of miR-140-5p, while KLF4, PTHLH, and WNT5A can be validated as direct targets of miR-140-3p.

Loss of dihydrotestosterone-inactivation activity promotes prostate cancer castration resistance detectable by functional imaging.

Androgens such as testosterone and dihydrotestosterone are a critical driver of prostate cancer progression. Cancer resistance to androgen deprivation therapies ensues when tumors engage metabolic processes that produce sustained androgen levels in the tissue. However, the molecular mechanisms involved in this resistance process are unclear, and functional imaging modalities that predict impending resistance are lacking. Here, using the human LNCaP and C4-2 cell line models of prostate cancer, we show that castration treatment-sensitive prostate cancer cells that normally have an intact glucuronidation pathway that rapidly conjugates and inactivates dihydrotestosterone and thereby limits androgen signaling, become glucuronidation deficient and resistant to androgen deprivation. Mechanistically, using CRISPR/Cas9-mediated gene ablation, we found that loss of UDP glucuronosyltransferase family 2 member B15 (UGT2B15) and UGT2B17 is sufficient to restore free dihydrotestosterone, sustained androgen signaling, and development of castration resistance. Furthermore, loss of glucuronidation enzymatic activity was also detectable with a nonsteroid glucuronidation substrate. Of note, glucuronidation-incompetent cells and the resultant loss of intracellular conjugated dihydrotestosterone were detectable in vivo by 18F-dihydrotestosterone PET. Together, these findings couple a mechanism with a functional imaging modality to identify impending castration resistance in prostate cancers.