Precision Medicine to Treat Urothelial Carcinoma-The Way Forward.

The treatment of urothelial carcinoma (UC) is challenging given its molecular heterogeneity and variable response to current therapies. To address this, many tools, including tumor biomarker assessment and liquid biopsies, have been developed to predict prognosis and treatment response. Approved therapeutic modalities for UC currently include chemotherapy, immune checkpoint inhibitors, receptor tyrosine kinase inhibitors, and antibody drug conjugates. Ongoing investigations to improve the treatment of UC include the search for actionable alterations and the testing of novel therapies. An important objective in recent studies has been to increase efficacy while decreasing toxicity by taking into account unique patient and tumor-related factors-an endeavor called precision medicine. The aim of this review is to highlight advancements in the treatment of UC, describe ongoing clinical trials, and identify areas for future study in the context of precision medicine.

Utility of Handheld Ultrasound Performed by Cardiology Fellows in Patients Presenting with Suspected ST-Elevation Myocardial Infarction.

Background: In academic hospitals, cardiology fellows may be the first point of contact for patients presenting with suspected ST-elevation myocardial infarction (STEMI) or acute coronary syndrome (ACS). In this study, we sought to determine the role of handheld ultrasound (HHU) in patients with suspected acute myocardial injury (AMI) when used by fellows in training, its association with the year of training in cardiology fellowship, and its influence on clinical care. Methods: This prospective study's sample population comprised patients who presented to the Loma Linda University Medical Center Emergency Department with suspected acute STEMI. On-call cardiology fellows performed bedside cardiac HHU at the time of AMI activation. All patients subsequently underwent standard transthoracic echocardiography (TTE). The impact of the detection of wall motion abnormalities (WMAs) on HHU in regard to clinical decision-making, including whether the patient would undergo urgent invasive angiography, was also evaluated. Results: Eighty-two patients (mean age: 65 years, 70% male) were included. The use of HHU by cardiology fellows resulted in a concordance correlation coefficient of 0.71 (95% confidence interval: 0.58-0.81) between HHU and TTE for left ventricular ejection fraction (LVEF), and a concordance correlation coefficient of 0.76 (0.65-0.84) for wall motion score index. Patients with WMA on HHU were more likely to undergo invasive angiogram during hospitalization (96% vs. 75%, P < 0.01). The time interval between the performance of HHU to initiation of cardiac catheterization (time-to-cath) was shorter in patients with abnormal versus normal HHU examinations (58 ± 32 min vs. 218 ± 388 min, P = 0.06). Finally, among patients who underwent angiography, those with WMA were more likely to undergo angiography within 90 min of presentation (96% vs. 66%, P < 0.001). Conclusion: HHU can be reliably used by cardiology fellows in training for measurement of LVEF and assessment of wall motion abnormalities, with good correlation to findings obtained via standard TTE. HHU-identified WMA at first contact was associated with higher rates of angiography as well as sooner angiography compared to patients without WMA.

Preclinical Evaluation of the Safety and Efficacy of Cryopreserved Bone Marrow Mesenchymal Stromal Cells for Corneal Repair.

Purpose: Mesenchymal stromal cells (MSCs) have been shown to enhance tissue repair as a cell-based therapy. In preparation for a phase I clinical study, we evaluated the safety, dosing, and efficacy of bone marrow-derived MSCs after subconjunctival injection in preclinical animal models of mice, rats, and rabbits. Methods: Human bone marrow-derived MSCs were expanded to passage 4 and cryopreserved. Viability of MSCs after thawing and injection through small-gauge needles was evaluated by vital dye staining. The in vivo safety of human and rabbit MSCs was studied by subconjunctivally injecting MSCs in rabbits with follow-up to 90 days. The potency of MSCs on accelerating wound healing was evaluated in vitro using a scratch assay and in vivo using 2-mm corneal epithelial debridement wounds in mice. Human MSCs were tracked after subconjunctival injection in rat and rabbit eyes. Results: The viability of MSCs after thawing and immediate injection through 27- and 30-gauge needles was 93.1% ± 2.1% and 94.9% ± 1.3%, respectively. Rabbit eyes demonstrated mild self-limiting conjunctival inflammation at the site of injection with human but not rabbit MSCs. In scratch assay, the mean wound healing area was 93.5% ± 12.1% in epithelial cells co-cultured with MSCs compared with 40.8% ± 23.1% in controls. At 24 hours after wounding, all MSC-injected murine eyes had 100% corneal wound closure compared with 79.9% ± 5.5% in controls. Human MSCs were detectable in the subconjunctival area and peripheral cornea at 14 days after injection. Conclusions: Subconjunctival administration of MSCs is safe and effective in promoting corneal epithelial wound healing in animal models. Translational Relevance: These results provide preclinical data to support a phase I clinical study.

Activating the Intrinsic Pathway of Apoptosis Using BIM BH3 Peptides Delivered by Peptide Amphiphiles with Endosomal Release.

Therapeutic manipulation of the BCL-2 family using BH3 mimetics is an emerging paradigm in cancer treatment and immune modulation. For example, peptides mimicking the BIM BH3 helix can directly target the full complement of anti- and pro-apoptotic BCL-2 proteins to trigger apoptosis. This study has incorporated the potent BH3 α-helical death domain of BIM into peptide amphiphile (PA) nanostructures designed to facilitate cellular uptake and induce cell death. This study shows that these PA nanostructures are quickly incorporated into cells, are able to specifically bind BCL-2 proteins, are stable at physiologic temperatures and pH, and induce dose-dependent apoptosis in cells. The incorporation of a cathepsin B cleavable linker between the BIM BH3 peptide and the hydrophobic tail resulted in increased intracellular accumulation and mitochondrial co-localization of the BIM BH3 peptide while also improving BCL-2 family member binding and apoptotic reactivation. This PA platform represents a promising new strategy for intracellular therapeutic peptide delivery for the disruption of intracellular protein:protein interactions.

Effect of Human Corneal Mesenchymal Stromal Cell-derived Exosomes on Corneal Epithelial Wound Healing.

Purpose: Mesenchymal stromal cells (MSCs) have been used therapeutically to modulate inflammation and promote repair. Extracellular vesicles, including exosomes, have been identified as one of the important mediators. This study investigated the effect of human corneal MSC-derived exosomes on corneal epithelial wound healing. Methods: Corneal MSCs (cMSCs) were isolated from human cadaver corneas. The secretome was collected after 72 hours and exosomes were isolated using differential ultracentrifugation. Morphology and size of exosomes were examined by electron microscopy and dynamic light scattering. Expression of CD9, CD63, and CD81 by cMSC exosomes was evaluated by western blotting. Cellular uptake of exosomes was studied using calcein-stained exosomes. The effect of exosome on wound healing was measured in vitro using a scratch assay and in vivo after 2-mm epithelial debridement wounds in mice. Results: cMSC exosomes were morphologically round and main population ranged between 40 and 100 nm in diameter. They expressed CD9, CD63, and CD81, and did not express GM130, Calnexin, and Cytochrome-C. Stained cMSC exosomes were successfully taken up by human cMSCs, human corneal epithelial cells (HCECs), and human macrophages in vitro and by corneal epithelium in vivo. In scratch assay, after 16 hours, cMSC exosome treated HCECs had 30.1% ± 14% remaining wound area compared to 72.9% ± 8% in control (P < 0.005). In vivo, after 72 hours, cMSC exosome-treated corneas had 77.5% ± 3% corneal wound healing compared to 41.6% ± 7% in the control group (P < 0.05). Conclusions: Human cMSC exosomes can accelerate corneal epithelial wound healing, and thus, may provide a therapeutic approach for ocular surface injuries.

Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation.

Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein-protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a Förster resonance energy transfer (FRET)-based tracking system. Using this platform, we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

miR-206 Inhibits Stemness and Metastasis of Breast Cancer by Targeting MKL1/IL11 Pathway.

Purpose: Effective targeting of cancer stem cells is necessary and important for eradicating cancer and reducing metastasis-related mortality. Understanding of cancer stemness-related signaling pathways at the molecular level will help control cancer and stop metastasis in the clinic.Experimental Design: By analyzing miRNA profiles and functions in cancer development, we aimed to identify regulators of breast tumor stemness and metastasis in human xenograft models in vivo and examined their effects on self-renewal and invasion of breast cancer cells in vitro To discover the direct targets and essential signaling pathways responsible for miRNA functions in breast cancer progression, we performed microarray analysis and target gene prediction in combination with functional studies on candidate genes (overexpression rescues and pheno-copying knockdowns).Results: In this study, we report that hsa-miR-206 suppresses breast tumor stemness and metastasis by inhibiting both self-renewal and invasion. We identified that among the candidate targets, twinfilin (TWF1) rescues the miR-206 phenotype in invasion by enhancing the actin cytoskeleton dynamics and the activity of the mesenchymal lineage transcription factors, megakaryoblastic leukemia (translocation) 1 (MKL1), and serum response factor (SRF). MKL1 and SRF were further demonstrated to promote the expression of IL11, which is essential for miR-206's function in inhibiting both invasion and stemness of breast cancer.Conclusions: The identification of the miR-206/TWF1/MKL1-SRF/IL11 signaling pathway sheds lights on the understanding of breast cancer initiation and progression, unveils new therapeutic targets, and facilitates innovative drug development to control cancer and block metastasis. Clin Cancer Res; 23(4); 1091-103. ©2016 AACR.

A rapid, automated surface protein profiling of single circulating exosomes in human blood.

Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids.

Alterations of Ca²âº-responsive proteins within cholinergic neurons in aging and Alzheimer's disease.

The molecular basis of selective neuronal vulnerability in Alzheimer's disease (AD) remains poorly understood. Using basal forebrain cholinergic neurons (BFCNs) as a model and immunohistochemistry, we have demonstrated significant age-related loss of the calcium-binding protein calbindin-D(28K) (CB) from BFCN, which was associated with tangle formation and degeneration in AD. Here, we determined alterations in RNA and protein for CB and the Ca(2+)-responsive proteins Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), growth-associated protein-43 (GAP43), and calpain in the BF. We observed progressive downregulation of CB and CaMKI RNA in laser-captured BFCN in the normal-aged-AD continuum. We also detected progressive loss of CB, CaMKIδ, and GAP43 proteins in BF homogenates in aging and AD. Activated µ-calpain, a calcium-sensitive protease that degrades CaMKI and GAP43, was significantly increased in the normal aged BF and was 10 times higher in AD BF. Overactivation of µ-calpain was confirmed using proteolytic fragments of its substrate spectrin. Substantial age- and AD-related alterations in Ca(2+)-sensing proteins most likely contribute to selective vulnerability of BFCN to degeneration in AD.