Trypan Blue Dye Enters Viable Cells Incubated with the Pore-Forming Toxin HlyII of Bacillus cereus

Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability

Cells have normal metabolic activity following incubation with HlyII.

<p>Macrophages were incubated with increasing concentrations of HlyII (0 to 0.5 µg/mL) for the times indicated. Cellular metabolic activity was evaluated by measuring absorbance at 490 nm, which is proportional to NADH- or NADPH-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) for up to 4 h after incubation with the toxin. The values were normalized to those for untreated cells (no toxin).</p

HlyII induces transient membrane permeability.

<p>Macrophages were incubated with increasing concentrations of HlyII (0 to 0.5 µg/mL) for 2 h, and membrane permeability was assessed by trypan blue dye exclusion (black scale). A representative image is shown for non-treated cells (B), and cells incubated with HlyII at 0.2 µg/mL (C) and 0.5 µg/mL (D). Alternatively, macrophages were incubated with HlyII (0 to 0.5 µg/mL) for 2 h, and washed to remove the toxin. The macrophages were then allowed to recover in fresh medium supplemented with FBS for 24 h. After recovering, membrane permeability was assessed by trypan blue dye exclusion (grey scale). A representative image is shown of recovered cells after incubation with HlyII at 0.2 µg/mL and 24 h recovery (E).</p

Circulating Tumor Cells with Aberrant ALK-Copy Number Predicts Progression-Free Survival to Crizotinib in ALK-Rearranged Non-Small-Cell Lung Cancer Patients

Abstract The duration and magnitude of clinical response are unpredictable in ALK-rearranged non–small cell lung cancer (NSCLC) patients treated with crizotinib, although all patients invariably develop resistance. Here, we evaluated whether circulating tumor cells (CTC) with aberrant ALK-FISH patterns [ALK-rearrangement, ALK-copy number gain (ALK-CNG)] monitored on crizotinib could predict progression-free survival (PFS) in a cohort of ALK-rearranged patients. Thirty-nine ALK-rearranged NSCLC patients treated with crizotinib as first ALK inhibitor were recruited prospectively. Blood samples were collected at baseline and at an early time-point (2 months) on crizotinib. Aberrant ALK-FISH patterns were examined in CTCs using immunofluorescence staining combined with filter-adapted FISH after filtration enrichment. CTCs were classified into distinct subsets according to the presence of ALK-rearrangement and/or ALK-CNG signals. No significant association between baseline numbers of ALK-rearranged or ALK-CNG CTCs and PFS was observed. However, we observed a significant association between the decrease in CTC number with ALK-CNG on crizotinib and a longer PFS (likelihood ratio test, P = 0.025). In multivariate analysis, the dynamic change of CTC with ALK-CNG was the strongest factor associated with PFS (HR, 4.485; 95% confidence interval, 1.543–13.030, P = 0.006). Although not dominant, ALK-CNG has been reported to be one of the mechanisms of acquired resistance to crizotinib in tumor biopsies. Our results suggest that the dynamic change in the numbers of CTCs with ALK-CNG may be a predictive biomarker for crizotinib efficacy in ALK-rearranged NSCLC patients. Serial molecular analysis of CTC shows promise for real-time patient monitoring and clinical outcome prediction in this population. Cancer Res; 77(9); 2222–30. ©2017 AACR.</jats:p

Method for semi-automated microscopy of filtration-enriched circulating tumor cells

BACKGROUND: Circulating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients. METHODS: Spiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner. RESULTS: Two combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45(−) cells, cytomorphological staining, then scanning and analysis of CD45(−) cell phenotypical and cytomorphological characteristics. CD45(−) cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 μm(2). The second assay sequentially combined fluorescent staining, automated selection of CD45(−) cells, FISH scanning on CD45(−) cells, then analysis of CD45(−) cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 μm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals. CONCLUSIONS: The semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-016-2461-4) contains supplementary material, which is available to authorized users

Abstract B174: Baseline and procedural characteristics influencing tumor cell content on on-purpose tumor biopsies: Results from a prospective molecular triage trial (MOSCATO).

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The Clinical Utility of Liquid Biopsy by Digital Droplet PCR in Patients with Advanced NSCLC [P34.06]

INTRODUCTION : EGFR mutations occur in 15% of Caucasian and up to 50% of Asian patients with advanced NSCLC. Tissue genomic profiling is the gold standard but the liquid biopsy is a good surrogate of the tissue for molecular diagnosis. The digital droplet PCR (ddPCR) is a rapid and low-cost liquid biopsy technique for genomic analyses. We aimed to evaluate the clinical utility of the ddPCR for genomic profiling of advanced NSCLC with EGFR mutations. [...