Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling.

Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets.
ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin.
Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model.
Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.

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Does Antihypertensive Drug Class Affect Day-to-Day Variability of Self-Measured Home Blood Pressure? The HOMED-BP Study.

Recent literature suggests that blood pressure variability (BPV) predicts outcome beyond blood pressure level (BPL) and that antihypertensive drug classes differentially influence BPV. We compared calcium channel blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockade for effects on changes in self-measured home BPL and BPV and for their prognostic significance in newly treated hypertensive patients.
We enrolled 2484 patients randomly allocated to first-line treatment with a calcium channel blocker (n=833), an angiotensin-converting enzyme inhibitor (n=821), or angiotensin receptor blockade (n=830). Home blood pressures in the morning and evening were measured for 5 days off treatment before randomization and for 5 days after 2 to 4 weeks of randomized drug treatment. We assessed BPL and BPV changes as estimated by variability independent of the mean and compared cardiovascular outcomes. Home BPL response in each group was significant (P≤0.0001) but small in the angiotensin-converting enzyme inhibitor group (systolic/diastolic: 4.6/2.8 mm Hg) compared with the groups treated with a calcium channel blocker (systolic/diastolic: 8.3/3.9 mm Hg) and angiotensin receptor blockade (systolic/diastolic: 8.2/4.5 mm Hg). In multivariable adjusted analyses, changes in home variability independent of the mean did not differ among the 3 drug classes (P≥0.054). Evening variability independent of the mean before treatment significantly predicted hard cardiovascular events independent of the corresponding home BPL (P≤0.022), whereas BPV did not predict any cardiovascular outcome based on the morning measurement (P≥0.056). Home BPV captured after monotherapy had no predictive power for cardiovascular outcome (P≥0.22).
Self-measured home evening BPV estimated by variability independent of the mean had prognostic significance, whereas antihypertensive drug classes had no significant impact on BPV changes. Home BPL should remain the primary focus for risk stratification and treatment.
URL: View Source Unique identifier: C000000137.
© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

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CD133 does not enrich for the stem cell activity in vivo in adult mouse prostates.

CD133 is widely used as a marker for stem/progenitor cells in many organ systems. Previous studies using in vitro stem cell assays have suggested that the CD133-expressing prostate basal cells may serve as the putative prostate stem cells. However, the precise localization of the CD133-expressing cells and their contributions to adult murine prostate homeostasis in vivo remain undetermined. We show that loss of function of CD133 does not impair murine prostate morphogenesis, homeostasis and regeneration, implying a dispensable role for CD133 in prostate stem cell function. Using a CD133-CreER(T2) model in conjunction with a fluorescent report line, we show that CD133 is not only expressed in a fraction of prostate basal cells, but also in some luminal cells and stromal cells. CD133(+) basal cells possess higher in vitro sphere-forming activities than CD133(-) basal cells. However, the in vivo lineage tracing study reveals that the two cell populations possess the same regenerative capacity and contribute equally to the maintenance of the basal cell lineage. Similarly, CD133(+) and CD133(-) luminal cells are functionally equivalent in maintaining the luminal cell lineage. Collectively, our study demonstrates that CD133 does not enrich for the stem cell activity in vivo in adult murine prostate. This study does not contradict previous reports showing CD133(+) cells as prostate stem cells in vitro. Instead, it highlights a substantial impact of biological contexts on cellular behaviors.
Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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STUMP un"stumped": anti-tumor response to anaplastic lymphoma kinase (ALK) inhibitor based targeted therapy in uterine inflammatory myofibroblastic tumor with myxoid features harboring DCTN1-ALK fusion.

Recurrent, metastatic mesenchymal myxoid tumors of the gynecologic tract present a management challenge as there is minimal evidence to guide systemic therapy. Such tumors also present a diagnostic dilemma, as myxoid features are observed in leiomyosarcomas, inflammatory myofibroblastic tumors (IMT), and mesenchymal myxoid tumors. Comprehensive genomic profiling was performed in the course of clinical care on a case of a recurrent, metastatic myxoid uterine malignancy (initially diagnosed as smooth muscle tumor of uncertain malignant potential (STUMP)), to guide identify targeted therapeutic options. To our knowledge, this case represents the first report of clinical response to targeted therapy in a tumor harboring a DCTN1-ALK fusion protein.
Hybridization capture of 315 cancer-related genes plus introns from 28 genes often rearranged or altered in cancer was applied to >50 ng of DNA extracted from this sample and sequenced to high, uniform coverage. Therapy was given in the context of a phase I clinical trial ClinicalTrials.gov Identifier: ( NCT01548144 ).
Immunostains showed diffuse positivity for ALK1 expression and comprehensive genomic profiling identified an in frame DCTN1-ALK gene fusion. The diagnosis of STUMP was revised to that of an IMT with myxoid features. The patient was enrolled in a clinical trial and treated with an anaplastic lymphoma kinase (ALK) inhibitor (crizotinib/Xalkori) and a multikinase VEGF inhibitor (pazopanib/Votrient). The patient experienced an ongoing partial response (6+ months) by response evaluation criteria in solid tumors (RECIST) 1.1 criteria.
For myxoid tumors of the gynecologic tract, comprehensive genomic profiling can identify clinical relevant genomic alterations that both direct treatment targeted therapy and help discriminate between similar diagnostic entities.

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A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening.

Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work.
© 2015 Society for Laboratory Automation and Screening.

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