A high-content, multiplexed screen in human breast cancer cells identifies profilin-1 inducers with anti-migratory activities

Profilin-1 (Pfn-1) is a ubiquitously expressed actin-binding protein that is essential for normal cell proliferation and migration. In breast cancer and several other adenocarcinomas, Pfn-1 expression is downregulated when compared to normal tissues. Previous studies from our laboratory have shown that genetically modulating Pfn-1 expression significantly impacts proliferation, migration, and invasion of breast cancer cells in vitro, and mammary tumor growth, dissemination, and metastatic colonization in vivo. Therefore, small molecules that can modulate Pfn-1 expression could have therapeutic potential in the treatment of metastatic breast cancer. The overall goal of this study was to perform a multiplexed phenotypic screen to identify compounds that inhibit cell motility through upregulation of Pfn-1. Screening of a test cassette of 1280 compounds with known biological activities on an Oris™ Pro 384 cell migration platform identified several agents that increased Pfn-1 expression greater than two-fold over vehicle controls and exerted anti-migratory effects in the absence of overt cytotoxicity in MDA-MB-231 human breast cancer cells. Concentration-response confirmation and orthogonal follow-up assays identified two bona fide inducers of Pfn-1, purvalanol and tyrphostin A9, that confirmed in single-cell motility assays and Western blot analyses. SiRNA-mediated knockdown of Pfn-1 abrogated the inhibitory effect of tyrphostin A9 on cell migration, suggesting Pfn-1 is mechanistically linked to tyrphostin A9's anti-migratory activity. The data illustrate the utility of the high-content cell motility assay to discover novel targeted anti-migratory agents by integrating functional phenotypic analyses with target-specific readouts in a single assay platform. © 2014 Joy et al

Gadoxetate-enhanced abbreviated MRI is highly accurate for hepatocellular carcinoma screening.

The primary objective was to compare the performance of 3 different abbreviated MRI (AMRI) sets extracted from a complete gadoxetate-enhanced MRI obtained for hepatocellular carcinoma (HCC) screening. Secondary objective was to perform a preliminary cost-effectiveness analysis, comparing each AMRI set to published ultrasound performance for HCC screening in the USA. This retrospective study included 237 consecutive patients (M/F, 146/91; mean age, 58 years) with chronic liver disease who underwent a complete gadoxetate-enhanced MRI for HCC screening in 2017 in a single institution. Two radiologists independently reviewed 3 AMRI sets extracted from the complete exam: non-contrast (NC-AMRI: T2-weighted imaging (T2wi)+diffusion-weighted imaging (DWI)), dynamic-AMRI (Dyn-AMRI: T2wi+DWI+dynamic T1wi), and hepatobiliary phase AMRI (HBP-AMRI: T2wi+DWI+T1wi during the HBP). Each patient was classified as HCC-positive/HCC-negative based on the reference standard, which consisted in all available patient data. Diagnostic performance for HCC detection was compared between sets. Estimated set characteristics, including historical ultrasound data, were incorporated into a microsimulation model for cost-effectiveness analysis. The reference standard identified 13/237 patients with HCC (prevalence, 5.5%; mean size, 33.7 ± 30 mm). Pooled sensitivities were 61.5% for NC-AMRI (95% confidence intervals, 34.4-83%), 84.6% for Dyn-AMRI (60.8-95.1%), and 80.8% for HBP-AMRI (53.6-93.9%), without difference between sets (p range, 0.06-0.16). Pooled specificities were 95.5% (92.4-97.4%), 99.8% (98.4-100%), and 94.9% (91.6-96.9%), respectively, with a significant difference between Dyn-AMRI and the other sets (p < 0.01). All AMRI methods were effective compared with ultrasound, with life-year gain of 3-12 months against incremental costs of US$ < 12,000. NC-AMRI has limited sensitivity for HCC detection, while HBP-AMRI and Dyn-AMRI showed excellent sensitivity and specificity, the latter being slightly higher for Dyn-AMRI. Cost-effectiveness estimates showed that AMRI is effective compared with ultrasound. • Comparison of different abbreviated MRI (AMRI) sets reconstructed from a complete gadoxetate MRI demonstrated that non-contrast AMRI has low sensitivity (61.5%) compared with contrast-enhanced AMRI (80.8% for hepatobiliary phase AMRI and 84.6% for dynamic AMRI), with all sets having high specificity. • Non-contrast and hepatobiliary phase AMRI can be performed in less than 14 min (including set-up time), while dynamic AMRI can be performed in less than 17 min. • All AMRI sets were cost-effective for HCC screening in at-risk population in comparison with ultrasound

Urinary Albumin Excretion is Associated with Pulmonary Hypertension in Sickle Cell Disease: Potential Role of Soluble Fms-Like Tyrosine Kinase-1

Pulmonary hypertension (PHT) is reported to be associated with measures of renal function in patients with sickle cell disease (SCD). The purpose of this exploratory study was to determine the relationship between albuminuria and both clinical and laboratory variables in SCD

Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines

yesWe describe a novel protocol to quantitatively and simultaneously compare the chemotactic responses of cells towards different chemokines. In this protocol, droplets of agarose gel containing different chemokines are applied onto the surface of a Petri dish, and then immersed under culture medium in which cells are suspended. As chemokine molecules diffuse away from the spot, a transient chemoattractant gradient is established across the spots. Cells expressing the corresponding cognate chemokine receptors migrate against this gradient by crawling under the agarose spots towards their centre. We show that this migration is chemokine-specific; meaning that only cells that express the cognate chemokine cell surface receptor, migrate under the spot containing its corresponding chemokine ligand. Furthermore, we show that migration under the agarose spot can be modulated by selective small molecule antagonists present in the cell culture medium

Collective cell migration of smooth muscle and endothelial cells: impact of injury versus non-injury stimuli

BACKGROUND: Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration - some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces. RESULTS: Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour. CONCLUSION: For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]