RNA-Seq Analysis of Extradomain A and Extradomain B Fibronectin as Extracellular Matrix Markers for Cancer.

Alternatively spliced forms of fibronectin, called oncofetal fibronectin, are aberrantly expressed in cancer, with little to no expression in normal tissue, making them attractive biomarkers to exploit for tumor-targeted therapeutics and diagnostics. While prior studies have explored oncofetal fibronectin expression in limited cancer types and limited sample sizes, no studies have performed a large-scale pan-cancer analysis in the context of clinical diagnostics and prognostics to posit the utility of these biomarkers across multiple cancer types. In this study, RNA-Seq data sourced from the UCSC Toil Recompute project were extracted and analyzed to determine the correlation between the expression of oncofetal fibronectin, including extradomain A and extradomain B fibronectin, and patient diagnosis and prognosis. We determined that oncofetal fibronectin is significantly overexpressed in most cancer types relative to corresponding normal tissues. In addition, strong correlations exist between increasing oncofetal fibronectin expression levels and tumor stage, lymph node activity, and histological grade at the time of diagnosis. Furthermore, oncofetal fibronectin expression is shown to be significantly associated with overall patient survival within a 10-year window. Thus, the results presented in this study suggest oncofetal fibronectin as a commonly upregulated biomarker in cancer with the potential to be used for tumor-selective diagnosis and treatment applications.

Effective gene therapy of Stargardt disease with PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles.

Stargardt disease (STGD) is the most common form of inherited retinal genetic disorders and is often caused by mutations in ABCA4. Gene therapy has the promise to effectively treat monogenic retinal disorders. However, clinically approved adeno-associated virus (AAV) vectors do not have a loading capacity for large genes, such as ABCA4. Self-assembly nanoparticles composed of (1-aminoethyl)iminobis[N-(oleoylcysteinyl-1-amino-ethyl)propionamide (ECO; a multifunctional pH-sensitive/ionizable amino lipid) and plasmid DNA produce gene transfection comparable with or better than the AAV2 capsid. Stable PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles produce specific and prolonged expression of ABCA4 in the photoreceptors of Abca4 -/- mice and significantly inhibit accumulation of toxic A2E in the eye. Multiple subretinal injections enhance gene expression and therapeutic efficacy with an approximately 69% reduction in A2E accumulation in Abca4 -/- mice after 3 doses. Very mild inflammation was observed after multiple injections of the nanoparticles. PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles are a promising non-viral mediated gene therapy modality for STGD type 1 (STGD1).

Magnetic resonance molecular imaging of extradomain B fibronectin enables detection of pancreatic ductal adenocarcinoma metastasis.

Extradomain-B Fibronectin (EDB-FN) is an oncomarker that can be visualized with magnetic resonance molecular imaging (MRMI) to detect pancreatic ductal adenocarcinoma (PDAC) metastasis. In this study, we sought to assess the expression of EDB-FN in clinical samples of PDAC and to evaluate MRMI of PDAC metastasis with an EDB-FN-specific gadolinium-based contrast agent (MT218) in an orthotopic KPC-GFP-Luc mouse model. EDB-FN expression was evaluated in PDAC tissue samples through immunohistochemistry. RNA-Seq data obtained from the GEPIA2 project was evaluated to demonstrate EDB-FN expression in large patient cohorts. FLASH-3D MRI at 3 T of the KPC-GFP-Luc metastasis model was performed following injection of MT218. Tumor enhancement in MR images was correlated to postmortem distribution of KPC-GFP-Luc tumors using fluorescent and bright-field cryo-imaging and anatomical landmarks. EDB-FN immunohistochemical staining scores of human metastatic tumor stroma, (2.17 ± 0.271), metastatic tumor parenchyma (2.08 ± 0.229), primary tumor stroma (1.61 ± 0.26), and primary tumor parenchyma (1.61 ± 0.12) were significantly (p < 0.0001) higher than normal pancreas stroma (0.14 ± 0.10) and normal pancreas parenchyma (0.14 ± 0.14). EDB-FN mRNA expression in tumors is 4.98 log2(TPM + 1) and 0.18 log2(TPM + 1) in normal tissue (p < 0.01). A mouse model of EDB-FN rich PDAC metastasis exhibited T1-weighted contrast to noise (CNR) changes of 21.80 ± 4.34 in perimetastatic regions and 8.38 ± 0.79 in metastatic regions identified through cryo-imaging, significantly higher (p < 0.05) than CNR changes found in normal liver (-6.43 ± 0.92), mesentery (2.24 ± 0.92), spleen (-3.06 ± 2.38) and intestine (1.08 ± 2.15). We conclude that EDB-FN is overexpressed in metastatic and primary PDAC tumors and MRMI with MT218 enables the detection of metastatic and perimetastatic tissues.

Efficacy of Targeted ECO/miR-200c Nanoparticles for Modulating Tumor Microenvironment and Treating Triple Negative Breast Cancer as Non-invasively Monitored by MR Molecular Imaging.

PURPOSE: To investigate the effectiveness of targeted ECO/miR-200c in modulating tumor microenvironment and treating triple negative breast cancer (TNBC) using non-invasive magnetic resonance molecular imaging (MRMI) of extradomain B fibronectin (EDB-FN) with a targeted MRI contrast agent. METHODS: MDA-MB-231 and Hs578T TNBC cells were transfected with RGD-PEG-ECO/miR-200c. Invasive and migratory potential was evaluated using transwell, scratch wound, and spheroid formation assays. Athymic nude mice bearing orthotopic MDA-MB-231 and Hs578T xenografts were treated with weekly i.v. injection of RGD-PEG-ECO/miR-200c nanoparticles at 1.0 mg/kg/week RNA for 6 weeks. MRMI of EDB-FN was performed using a targeted contrast agent MT218 [ZD2-N3-Gd(DO3A)] on a 3 T MRS 3000 scanner. T1-weighted images were acquired following intravenous injection of MT218 at dose of 0.1 mmol/kg using a fast spin echo axial sequence with respiratory gating. RESULTS: Systemic administration of RGD-PEG-ECO/miR-200c nanoparticles in mice bearing orthotopic TNBC xenografts significantly suppressed tumor progression without toxic side-effects. MRMI with MT218 revealed that the treatment significantly suppressed tumor proliferation as compared to the control. MRMI also showed that the miR-200c treatment altered tumor microenvironment by reducing EDB-FN expression, as evidenced by decreased contrast enhancement in both MDA-MB-231 and Hs578T tumors. The reduction of EDB-FN was confirmed by immunohistochemistry. CONCLUSIONS: Targeted delivery of miR-200c with RGD-PEG-ECO/miR-200c nanoparticles effectively modulates tumor microenvironment and suppresses TNBC proliferation in animal models. MRMI of tumor EDB-FN expression is effective to non-invasively monitor tumor response and therapeutic efficacy of RGD-PEG-ECO/miR-200c nanoparticles in TNBC.

Preclinical Assessment of the Effectiveness of Magnetic Resonance Molecular Imaging of Extradomain-B Fibronectin for Detection and Characterization of Oral Cancer.

PURPOSE: Oral squamous cell carcinoma (OSCC) has not seen a substantial improvement in patient survival despite therapeutic advances, making accurate detection and characterization of the disease a clinical priority. Here, we aim to demonstrate the effectiveness of magnetic resonance imaging (MRI) with the targeted MRI contrast agent MT218 specific to extradomain-B fibronectin (EDB-FN) in the tumor microenvironment for detection and characterization of aggressive OSCC tumors. PROCEDURES: EDB-FN expression was evaluated in human normal tongue and OSCC specimens with immunohistochemistry. Invasiveness of human CAL27, HSC3, and SCC4 OSCC cells was analyzed with spheroid formation and transwell assays. EDB-FN expression in the cells was analyzed with semiquantitative real-time PCR, western blotting, and a peptide binding study with confocal microscopy. Contrast-enhanced MRI with MT218 was performed on subcutaneous OSCC mouse models at a dose of 0.04 mmol/kg, using gadoteridol (0.1 mmol/kg) as a control. RESULTS: Strong EDB-FN expression was observed in human untreated primary and metastatic OSCC, reduced expression in treated OSCC, and little expression in normal tongue tissue. SCC4 and HSC3 cell lines demonstrated high invasive potential with high and moderate-EDB-FN expression, respectively, while CAL27 showed little invasive potential and low-EDB-FN expression. In T1-weighted MRI, MT218 produced differential contrast enhancement in the subcutaneous tumor models in correlation with EDB-FN expression in the cancer cells. Enhancement in the high-EDB-FN tumors was greater with MT218 at 0.04 mmol/kg than gadoteridol at 0.1 mmol/kg. CONCLUSIONS: The results suggest EDB-FN has strong potential as an imageable biomarker for aggressive OSCC. MRMI results demonstrate the effectiveness of MT218 and the potential for differential diagnostic imaging of oral cancer for improving the management of the disease.

Effective MR Molecular Imaging of Triple Negative Breast Cancer With an EDB-Fibronectin-Specific Contrast Agent at Reduced Doses.

MR molecular imaging (MRMI) of abundant oncogenic biomarkers in tumor microenvironment has the potential to provide precision cancer imaging in high resolution. Extradomain-B fibronectin (EDB-FN) is an oncogenic extracellular matrix protein, highly expressed in aggressive triple negative breast cancer. A targeted macrocyclic gadolinium-based contrast agent (GBCA) ZD2-N3-Gd(HP-DO3A) (MT218), specific to EDB-FN, was developed for MRMI of aggressive breast cancer. The effectiveness of different doses of MT218 for MRMI was tested in MDA-MB-231 and Hs578T human triple negative breast cancer models. At clinical dose of 0.1 and subclinical dose of 0.04 mmol Gd/kg, MT218 rapidly bound to the extracellular matrix EDB-FN and produced robust tumor contrast enhancement in both the tumor models, as early as 1-30 min post-injection. Substantial tumor enhancement was also observed in both the models with MT218 at doses as low as 0.02 mmol Gd/kg, which was significantly better than the clinical agent Gd(HP-DO3A) at 0.1 mmol Gd/kg. Little non-specific enhancement was observed in the normal tissues including liver, spleen, and brain for MT218 at all the tested doses, with renal clearance at 30 min. These results demonstrate that MRMI with reduced doses of MT218 is safe and effective for sensitive and specific imaging of aggressive breast cancers.

Formulation of Biocompatible Targeted ECO/siRNA Nanoparticles with Long-Term Stability for Clinical Translation of RNAi.

Nanoparticle based siRNA formulations often suffer from aggregation and loss of function during storage. We in this study report a frozen targeted RGD-polyethylene glycol (PEG)-ECO/siß3 nanoparticle formulation with a prolonged shelf life and preserved nanoparticle functionality. The targeted RGD-PEG-ECO/siß3 nanoparticles are formed by step-wised self-assembly of RGD-PEG-maleimide, ECO, and siRNA. The nanoparticles have a diameter of 224.5 ± 9.41 nm and a zeta potential to 45.96 ± 3.67 mV in water and a size of 234.34 ± 3.01 nm and a near neutral zeta potential in saline solution. The addition of sucrose does not affect their size and zeta potential and substantially preserves the integrity and biological activities of frozen and lyophilized formulations of the targeted nanoparticles. The frozen formulation with as low as 5% sucrose retains nanoparticle integrity (90% siRNA encapsulation), size distribution (polydispersity index [PDI] ≤20%), and functionality (at least 75% silencing efficiency) at -80°C for at least 1 year. The frozen RGD-PEG-ECO/siß3 nanoparticle formulation exhibits excellent biocompatibility, with no adverse effects on hemocompatibility and minimal immunogenicity. As RNAi holds the promise in treating the previously untreatable diseases, the frozen nanoparticle formulation with the low sucrose concentration has the potential to be a delivery platform for clinical translation of RNAi therapeutics.

Abuse of supraphysiologic doses of anabolic steroids.

The following article is a literature review of supraphysiologic doses of anabolic-androgenic steroids (AAS). This article contains a brief review of the history of AAS, the chemistry of the varying forms of AAS, and proposed mechanisms of action. The article then focuses on how AAS are used in an illicit manner by the general population. Terms such as "stacking" and "pyramiding" are discussed. The article concludes by looking at the major detrimental side effects, such as liver damage and cardiovascular changes, which physicians may encounter when treating AAS abusers.

Atypical neuroleptic malignant syndrome presenting as fever of unknown origin in the elderly.

A 79-year-old nursing home resident who was taking olanzapine presented to multiple hospitals with fevers of unknown origin. After an extensive workup to rule out infection, the patient was diagnosed as having neuroleptic malignant syndrome (NMS). This patient's presentation was atypical because of the lack of rigidity or tremor at the time of admission. This case highlights the importance of including NMS in the differential diagnosis of fever of unknown origin in medical patients. A review of the medical literature is taken, focusing on diagnoses, prevalence, predisposing factors, pathophysiology, and current treatment approaches for NMS.