High-recovery visual identification and single-cell retrieval of circulating tumor cells for genomic analysis using a dual-technology platform integrated with automated immunofluorescence staining.

BACKGROUND: Circulating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers. There is great interest in using CTCs to monitor response to therapies, to identify clinically actionable biomarkers, and to provide a non-invasive window on the molecular state of a tumor. Here we characterize the performance of the AccuCyte®--CyteFinder® system, a comprehensive, reproducible and highly sensitive platform for collecting, identifying and retrieving individual CTCs from microscopic slides for molecular analysis after automated immunofluorescence staining for epithelial markers. METHODS: All experiments employed a density-based cell separation apparatus (AccuCyte) to separate nucleated cells from the blood and transfer them to microscopic slides. After staining, the slides were imaged using a digital scanning microscope (CyteFinder). Precisely counted model CTCs (mCTCs) from four cancer cell lines were spiked into whole blood to determine recovery rates. Individual mCTCs were removed from slides using a single-cell retrieval device (CytePicker™) for whole genome amplification and subsequent analysis by PCR and Sanger sequencing, whole exome sequencing, or array-based comparative genomic hybridization. Clinical CTCs were evaluated in blood samples from patients with different cancers in comparison with the CellSearch® system. RESULTS: AccuCyte--CyteFinder presented high-resolution images that allowed identification of mCTCs by morphologic and phenotypic features. Spike-in mCTC recoveries were between 90 and 91%. More than 80% of single-digit spike-in mCTCs were identified and even a single cell in 7.5 mL could be found. Analysis of single SKBR3 mCTCs identified presence of a known TP53 mutation by both PCR and whole exome sequencing, and confirmed the reported karyotype of this cell line. Patient sample CTC counts matched or exceeded CellSearch CTC counts in a small feasibility cohort. CONCLUSION: The AccuCyte--CyteFinder system is a comprehensive and sensitive platform for identification and characterization of CTCs that has been applied to the assessment of CTCs in cancer patient samples as well as the isolation of single cells for genomic analysis. It thus enables accurate non-invasive monitoring of CTCs and evolving cancer biology for personalized, molecularly-guided cancer treatment.

Cell cycle progression and de novo centriole assembly after centrosomal removal in untransformed human cells.

How centrosome removal or perturbations of centrosomal proteins leads to G1 arrest in untransformed mammalian cells has been a mystery. We use microsurgery and laser ablation to remove the centrosome from two types of normal human cells. First, we find that the cells assemble centrioles de novo after centrosome removal; thus, this phenomenon is not restricted to transformed cells. Second, normal cells can progress through G1 in its entirety without centrioles. Therefore, the centrosome is not a necessary, integral part of the mechanisms that drive the cell cycle through G1 into S phase. Third, we provide evidence that centrosome loss is, functionally, a stress that can act additively with other stresses to arrest cells in G1 in a p38-dependent fashion.

Immunofluorescent and molecular characterization of effusion tumor cells reveal cancer site-of-origin and disease-driving mutations.

BACKGROUND: Effective cancer treatment relies on precision diagnostics. In cytology, an accurate diagnosis facilitates the determination of proper therapeutics for patients with cancer. Previously, the authors developed a multiplexed immunofluorescent panel to detect epithelial malignancies from pleural effusion specimens. Their assay reliably distinguished effusion tumor cells (ETCs) from nonmalignant cells; however, it lacked the capacity to reveal specific cancer origin information. Furthermore, DNA profiling of ETCs revealed some, but not all, cancer-driver mutations. METHODS: The authors developed a new multiplex immunofluorescent panel that detected both malignancy and pulmonary origin by incorporating the thyroid transcription factor-1 (TTF-1) biomarker. Evaluation for TTF-1-positive ETCs (T-ETCs) was performed on 12 patient samples. T-ETCs and parallel ETCs from selected patients were collected and subjected to DNA profiling to identify pathogenic mutations. All samples were obtained with Institutional Review Board approval. RESULTS: Malignancy was detected in all samples. T-ETCs were identified in 9 of 10 patients who had clinically reported TTF-1 positivity (90% sensitivity and 100% specificity). Furthermore, DNA profiling of as few as five T-ETCs identified pathogenic mutations with equal or greater sensitivity compared with profiling of ETCs, both of which showed high concordance with clinical findings. CONCLUSIONS: The findings suggest that the immunofluorescent and molecular characterization of tumor cells from pleural effusion specimens can provide reliable diagnostic information, even with very few cells. The integration of site-specific biomarkers like TTF-1 into ETC analysis may facilitate better refined diagnosis and improve patient care.

The good, the bad and the ugly: the practical consequences of centrosome amplification.

Centrosome amplification (the presence of more than two centrosomes at mitosis) is characteristic of many human cancers. Extra centrosomes can cause the assembly of multipolar spindles, which unequally distribute chromosomes to daughter cells; the resulting genetic imbalances may contribute to cellular transformation. However, this raises the question of how a population of cells with centrosome amplification can survive such chaotic mitoses without soon becoming non-viable as a result of chromosome loss. Recent observations indicate that a variety of mechanisms partially mute the practical consequences of centrosome amplification. Consequently, populations of cells propagate with good efficiency, despite centrosome amplification, yet have an elevated mitotic error rate that can fuel the evolution of the transformed state.

Abrogation of the postmitotic checkpoint contributes to polyploidization in human papillomavirus E7-expressing cells.

High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint.

RareCyte® CTC Analysis Step 2: Detection of Circulating Tumor Cells by CyteFinder® Automated Scanning and Semiautomated Image Analysis.

The RareCyte CyteFinder instrument is an automated scanner that allows rapid identification of circulating tumor cells (CTCs) on microscope slides prepared by the AccuCyte process (see Chapter 13 ) and stained by immunofluorescence. Here, we present the workflow for CyteFinder scanning, analysis, and CyteMapper scan review which includes CTC confirmation and report generation.

RareCyte® CTC Analysis Step 1: AccuCyte® Sample Preparation for the Comprehensive Recovery of Nucleated Cells from Whole Blood.

The RareCyte platform addresses important technology limitations of current circulating tumor cell (CTC) collection methods, and expands CTC interrogation to include advanced phenotypic characterization and single-cell molecular analysis. In this respect, it represents the "next generation" of cell-based liquid biopsy technologies. In order to identify and analyze CTCs, RareCyte has developed an integrated sample preparation, imaging and individual cell retrieval process. The first step in the process, AccuCyte®, allows the separation, collection, and transfer to a slide the nucleated cell fraction of the blood that contains CTCs. Separation and collection are based on cell density-rather than size or surface molecular expression-and are performed within a closed system, without wash or lysis steps, enabling high CTC recovery. Here, we describe our technique for nucleated cell collection from a blood sample, and the spreading of these nucleated cells onto glass slides permitting immunofluorescent staining, cell identification, and individual cell picking described in subsequent chapters. In addition to collection of rare cells such as CTCs, AccuCyte also collects cells of the circulating immune system onto archivable slides as well as plasma from the same sample.

RareCyte® CTC Analysis Step 3: Using the CytePicker® Module for Individual Cell Retrieval and Subsequent Whole Genome Amplification of Circulating Tumor Cells for Genomic Analysis.

The CytePicker module built into the RareCyte CyteFinder instrument allows researchers to easily retrieve individual cells from microscope slides for genomic analyses, including array CGH, targeted sequencing, and next-generation sequencing. Here, we describe the semiautomated retrieval of CTCs from the blood processed by AccuCyte (see Chapter 13) and amplification of genomic DNA so that molecular analysis can be performed.

Microscope basics.

This chapter provides information on how microscopes work and discusses some of the microscope issues to be considered in using a video camera on the microscope. There are two types of microscopes in use today for research in cell biology-the older finite tube-length (typically 160mm mechanical tube length) microscopes and the infinity optics microscopes that are now produced. The objective lens forms a magnified, real image of the specimen at a specific distance from the objective known as the intermediate image plane. All objectives are designed to be used with the specimen at a defined distance from the front lens element of the objective (the working distance) so that the image formed is located at a specific location in the microscope. Infinity optics microscopes differ from the finite tube-length microscopes in that the objectives are designed to project the image of the specimen to infinity and do not, on their own, form a real image of the specimen. Three types of objectives are in common use today-plan achromats, plan apochromats, and plan fluorite lenses. The concept of mounting video cameras on the microscope is also presented in the chapter.

Practical aspects of adjusting digital cameras.

This chapter introduces the adjustment of digital camera settings using the tools found within image acquisition software and discusses measuring gray-level information such as (1) the histogram, (2) line scan, and (3) other strategies. The pixel values in an image can be measured within many image capture software programs in two ways. The first is a histogram of pixel gray values and the second is a line-scan plot across a selectable axis of the image. Understanding how to evaluate the information presented by these tools is critical to properly adjusting the camera to maximize the image contrast without losing grayscale information. This chapter discusses the 0-255 grayscale resolution of an 8-bit camera; however, the concepts are the same for cameras of any bit depth. This chapter also describes camera settings, such as exposure time, offset, and gain, and the steps for contrast stretching such as setting the exposure time, adjusting offset and gain, and camera versus image display controls.

DNA damage-induced cell death is enhanced by progression through mitosis.

Progression through the G(2)/M transition following DNA damage is linked to cytokinesis failure and mitotic death. In four different transformed cell lines and two human embryonic stem cell lines, we find that DNA damage triggers mitotic chromatin decondensation and global phosphorylation of histone H2AX, which has been associated with apoptosis. However, extended time-lapse studies in HCT116 colorectal cancer cells indicate that death does not take place during mitosis, but 72% of cells die within 3 days of mitotic exit. By contrast, only 11% of cells in the same cultures that remained in interphase died, suggesting that progression through mitosis enhances cell death following DNA damage. These time-lapse studies also confirmed that DNA damage leads to high rates of cytokinesis failure, but showed that cells that completed cytokinesis following damage died at higher rates than cells that failed to complete division. Therefore, post-mitotic cell death is not a response to cytokinesis failure or polyploidy. We also show that post-mitotic cell death is largely independent of p53 and is only partially suppressed by the apical caspase inhibitor Z-VAD-FMK. These findings suggest that progression through mitosis following DNA damage initiates a p53- and caspase-independent cell death response that prevents propagation of genetic lesions.

A sealed preparation for long-term observations of cultured cells.

INTRODUCTIONThe continuous long-term observation of cultured cells on the microscope has always been a technically demanding undertaking. This protocol describes a sealed preparation that allows the continuous long-term observation of cultured mammalian cells on upright or inverted microscopes without environmental CO(2) control. The preparation allows for optical conditions consistent with high-quality imaging and good cell viability for at least 100 hours. The preparation is an aluminum support slide with a square aperture cut in its center. The coverslip bearing the cells is attached to the top of the slide with a thin layer of silicone grease, and the bottom of the slide is similarly covered with a clean coverslip of the same size. The thickness of the slide is intended to coordinately maximize the volume of the medium while maintaining optical properties that allow Koehler illumination with standard condensers. The chamber is filled in equal parts with HEPES-buffered media containing fetal calf serum and a low-viscosity fluorocarbon oil. These oils have a high solubility for atmospheric gases. The inclusion of the oil in the preparations is intended to provide a source of oxygen and perhaps a sink for some of the CO(2) produced by the cells. Although the inclusion of fluorocarbon oil in the preparation may not be necessary for short-term (~24 hr) observations, particularly with cells that are sparsely plated, long-term cell viability is ensured when the oil is present.