Development and Analytical Validation of a 6-Plex Reverse Transcription Droplet Digital PCR Assay for the Absolute Quantification of Prostate Cancer Biomarkers in Circulating Tumor Cells of Patients with Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Gene expression in circulating tumor cells (CTCs) can be used as a predictive liquid biopsy test in metastatic castration-resistant prostate cancer (mCRPC). We developed a novel 6-plex reverse transcription droplet digital PCR (RT-ddPCR) assay for the absolute quantification of 4 prostate cancer biomarkers, a reference gene, and a synthetic DNA external control (DNA-EC) in CTCs isolated from mCRPC patients. METHODS: A novel 6-plex RT-ddPCR assay was developed for the simultaneous absolute quantification of AR-FL, AR-V7, PSA, and PSMA, HPRT (used as a reference gene), and a synthetic DNA-EC that was included for quality control. The assay was optimized and analytically validated using DNA synthetic standards for each transcript as positive controls. Epithelial cellular adhesion molecule (EpCAM)-positive CTC fractions isolated from 90 mCRPC patients and 11 healthy male donors were analyzed, and results were directly compared with reverse transcription quantitative PCR (RT-qPCR) for all markers in all samples. RESULTS: Linear dynamic range, limit of detection, limit of quantification, intra- and interassay precision, and analytical specificity were determined for each marker. Application of the assay in EpCAM-positive CTC showed positivity for AR-FL (71/90; 78.9%), AR-V7 (28/90; 31.1%), PSA (41/90; 45.6%), PSMA (38/90; 42.2%), and HPRT (90/90; 100%); DNA-EC concentration was constant across all samples. Direct comparison with RT-qPCR for the same markers in the same samples revealed RT-ddPCR to have superior diagnostic sensitivity. CONCLUSIONS: Our 6-plex RT-ddPCR assay was highly sensitive, specific, and reproducible, and enabled simultaneous and absolute quantification of 5 gene transcripts in minute amounts of CTC-derived cDNA. Application of this assay in clinical samples gave diagnostic sensitivity and specificity comparable to, or better than, RT-qPCR.

Caveolar uptake and endothelial-protective effects of nanostructured lipid carriers in acid aspiration murine acute lung injury.

PURPOSE: Nanostructured lipid carriers (NLC), nanosized phospholipids/triglyceride particles developed for drug delivery, are considered biologically inactive. We assessed the efficacy of unloaded NLC as experimental treatment for acute lung injury (ALI). METHODS: To induce ALI, C57Black/6 male mice received intratracheal injections of HCl or saline; A single dose of 16 mg/Kg NLC or saline was injected intravenously concomitantly with HCl challenge. NLC uptake mechanisms and effects on endothelial permeability and signaling were studied in cultured endothelial cells and neutrophils. RESULTS: NLC pre-treatment attenuated pulmonary microvascular protein leak, airspace inflammatory cells, thrombin proteolytic activity and histologic lung injury score 24 h post insult. Using fluorescence measurements and flow cytometry in mouse lung microvascular endothelial cell culture homogenates, we determined that NLC rendered fluorescent by curcumin labeling are taken up by endothelial cells from mice expressing caveolin-1, the coat protein of caveolar endocytic vesicles, but not from caveolin-1 gene-disrupted mice, which lack caveolae. In contrast, conventional emulsions (CE), consisting of larger particles, were not incorporated. In addition, NLC pre-treatment of cultured human lung microvascular endothelial cells abrogated thrombin-induced activation of p44/42, albumin permeability response, actin cytoskeletal remodeling and interleukin-6 production. Finally, NLC but not CE abrogated lipopolysaccharide-triggered interleukin-8 release. CONCLUSIONS: NLC are engulfed by endothelial caveolae and possess endothelial-protective effects. These novel properties may be of potential utility in ALI.

Acid-induced acute lung injury in mice is associated with P44/42 and c-Jun N-terminal kinase activation and requires the function of tumor necrosis factor α receptor I.

Aspiration of hydrochloric acid (HCl)-containing gastric juice leads to acute lung injury (ALI) and hypoxemic respiratory failure due to an exuberant inflammatory response associated with pulmonary edema from increased vascular and epithelial permeability. The aim of this study was to determine the role and signaling mechanisms of tumor necrosis factor α (TNF-α) in experimental ALI from HCl aspiration using a combination of genetic animal models and pharmacologic inhibition strategies. To this end, HCl was instilled intratracheally to mice, followed by respiratory system elastance measurement, bronchoalveolar lavage, and lung tissue harvesting 24 h after injection. Hydrochloric acid instillation induced an inflammatory response in the lungs of wild-type mice, evidenced as increased bronchoalveolar lavage total cells, neutrophils, and total protein; histologic lung injury score; and respiratory system elastance, whereas TNF-α receptor I mRNA levels were maintained. These alterations could be prevented by pretreatment with etanercept or genetic deletion of the 55-kd TNF-α receptor I, but not by deletion of the TNF-α gene. Hydrochloric acid induced a 6-fold increase in apoptotic, caspase 3-positive cells in lung sections from wild-type mice, which was abrogated in mice lacking TNF-α receptor I. In immunoblotting and immunohistochemistry studies, HCl stimulated signaling via p44/42 and c-Jun N-terminal kinase, which was blocked in TNF-α receptor I knockout mice. In conclusion, ALI induced by HCl requires TNF-α receptor I function and associates with activation of downstream proinflammatory signaling pathways p44/42 and c-Jun N-terminal kinase.