Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

Summary

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Abstract

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Introduction

In 2013 it is estimated that 580,350 individuals will die from cancer and that 1,660,290 new cases of this disease will be diagnosed in the United States alone¹. The majority of these deaths occur subsequent to the development of metastatic disease². The current lack of effective therapies in treating metastases and a limited understanding of the metastatic cascade makes this stage of disease highly lethal. The presence of circulating tumor cells (CTCs) within the bloodstream have been demonstrated to correlate with metastatic disease³. These cells are extremely rare and their detection is indicative of overall survival in metastatic breast⁴, prostate⁵, and colorectal⁶ cancer. In these patients, the presence of ≥5 (breast and prostate) or ≥3 (colorectal) CTCs in 7.5 ml of blood is indicative of poorer prognosis when compared to those patients with fewer or no detectable CTCs in the same blood volume. In addition, the change in CTC number during or after therapeutic intervention has been demonstrated to be useful as a predictor of treatment response, often sooner than currently utilized techniques^7-10.

It has been estimated that, in metastatic cancer patients, CTCs occur at a frequency of approximately 1 CTC per 10⁵-10⁷ blood mononuclear cells and in patients with localized disease, this frequency may be even lower (~1 in 10⁸). The rare nature of these cells can make it difficult to accurately and reliably detect and analyze CTCs¹¹. Several methods (reviewed previously^12-14) have been utilized to enrich and detect these cells by exploiting properties that differentiate them from surrounding blood components. In general, CTC enumeration is a two-part process that requires both an enrichment step and a detection step. Traditionally, enrichment steps rely on differences in physical properties of CTCs (cell size, density, deformability) or on protein marker expression (i.e. epithelial cell adhesion molecule [EpCAM], cytokeratin [CK]). Following enrichment, CTC detection can be performed in a number of different ways, the most common of which are nucleic acid-based assays and/or cytometric approaches. Each of these strategies are unique, having distinct advantages and disadvantages, however they all lack standardization; a necessity for entrance into the clinical setting. The CellSearch system (CSS) was therefore developed to provide a standardized method for the detection and enumeration of rare CTCs in human blood using fluorescence microscopy and antibody-based techniques^4-6. This platform is currently considered the gold standard in CTC enumeration and is the only technique approved by the U.S. Food and Drug Administration (FDA) for use in the clinic¹⁵.

The CSS is a two component platform consisting of, (1) the CellTracks AutoPrep system (hereafter referred to as the preparation instrument), which automates the preparation of human blood samples, and (2) the CellTracks Analyzer II (hereafter referred to as the analysis instrument), which scans these samples following preparation. To distinguish CTCs from contaminating leukocytes the preparation instrument employs an antibody mediated, ferrofluid-based magnetic separation approach and differential fluorescence staining. Initially, the system labels CTCs using anti-EpCAM antibodies conjugated to iron nanoparticles. The sample is then incubated in a magnetic field, and all unlabeled cells are aspirated. Selected tumor cells are resuspended, and incubated in a differential fluorescence stain, consisting of fluorescently-labeled antibodies and a nuclear staining reagent. Finally, the sample is transferred to a magnetic cartridge, called a MagNest (hereafter referred to as the magnetic device), and scanned using the analysis instrument.

The analysis instrument is used to scan prepared samples using different fluorescence filters, each optimized to the appropriate fluorescent particle, using a 10X objective lens. CTCs are identified as cells that are bound by anti-EpCAM, anti-pan-CK-phycoerythrin (PE) (CK8, 18, and 19), and the nuclear stain 4',6-diamidino-2-phenylindole (DAPI). Conversely, contaminating leukocytes are identified as cells that are bound by anti-CD45-allophycocyanin (APC) and DAPI. Following scanning, computer-defined potential tumor cells are presented to the user. From these images, the user must employ qualitative analysis using the defined parameters and differential staining discussed above to determine which events are CTCs.

In addition to providing a standardized method for CTC enumeration, the CSS allows for molecular characterization of CTCs based on protein markers of interest. This interrogation can be performed at the single-cell level, using a fluorescein isothiocyanate (FITC) fluorescence channel not required for CTC identification¹⁶. Although this platform provides the capacity for molecular characterization, the detailed process of protocol development and optimization is not well-defined. Three commercially available markers have been developed by the manufacturer for use with the CSS, including epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and insulin-like growth factor 1 receptor (IGF-1R). HER2 analysis, in combination with the CSS, has been utilized by several groups to illustrate the potential for CTC characterization to inform clinical decision-making and to potentially change existing treatment guidelines. For example, Fehm et al.¹⁷ demonstrated that approximately one third of breast cancer patients with HER2-primary tumors had HER2⁺ CTCs. In addition, Liu et al.¹⁸ recently reported that up to 50% of patients with HER⁺ metastatic breast cancer did not have HER2⁺ CTCs. Herceptin, a HER2 receptor interfering monoclonal antibody demonstrated to greatly benefit patients whose tumors express sufficient levels of HER2, is a commonly utilized treatment for patients with HER2⁺ primary tumors^19-21. However, these studies suggest that Herceptin may be being sub-optimally utilized and that CTC characterization may aid in predicting treatment response. Ultimately, CTC characterization may have the potential to improve personalized care.

CTC research is unique in that it has largely utilized a bedside-to-benchtop approach. This method, unlike benchtop-to-bedside research, which can often take years to impact patient care, has allowed CTCs quick entry into the clinical setting. However, physicians are hesitant to use results from CTC analysis in patient treatment decision-making due to a lack of understanding of their underlying biology. Therefore appropriate preclinical mouse models of metastasis and complementary CTC analysis techniques must be utilized in order to investigate these outstanding questions. In general, there are two types of preclinical models used to study the metastatic cascade, (1) spontaneous metastasis models, which allow for the study of all the steps in the metastatic cascade, and (2) experimental metastasis models, which only allow for the study of later steps in the metastatic process such as extravasation and secondary tumor formation²². Spontaneous metastasis models, involve tumor cell injections into appropriate orthotopic locations (e.g. injection of prostate cancer cells into the prostate gland for the study of prostate cancer). Cells are then given time to form primary tumors and spontaneously metastasize to secondary sites such as the bone, lung, and lymph nodes. In contrast, experimental metastasis models involve direct injection of tumor cells into the bloodstream (e.g. via tail vein or intracardiac injection to target cells to specific locations) and therefore skip the initial steps of intravasation and dissemination to secondary organs²². Thus far the majority of CTC analysis in in vivo model systems has been performed using either cytometry-based²³ or adapted human-based CTC techniques (e.g. AdnaTest)²⁴. Although useful, none of these techniques adequately reflect CTC enumeration using the gold standard CSS. Based on the clinical approval, standardized nature, and widespread usage of the CSS, the development of a CTC capture and detection technique for in vivo modeling that utilizes equivalent sample preparation, processing, and identification criteria would be advantageous as results would be comparable to those obtained from patient samples. However, due to the volume requirements of the preparation instrument it is not possible to process small volumes of blood using this automated platform. In addition, previous work by Eliane et al.²⁵ has demonstrated that contamination of samples with mouse epithelial cells (which also meet the standard CTC definition [EpCAM⁺CK⁺DAPI⁺CD45^-]) can lead to misclassification of mouse squamous epithelial cells as CTCs. To address these issues an adapted technique that allows the utilization of the CSS CTC kit reagents combined with a manual isolation procedure was developed. The addition of a FITC labeled human leukocyte antigen (HLA) antibody to the assay allows human tumor cells to be distinguished from mouse squamous epithelial cells.

This manuscript describes the standard, commercially developed and optimized CSS protocol for processing patient blood samples and common pitfalls that may be encountered, including discrepant items that can lead to CTC misclassification errors. In addition, customization of the CSS assay to examine user-defined protein characteristics of captured CTCs and a comparable adapted CSS technique that allows for the enrichment and detection of CTCs from small volumes of blood in preclinical mouse models of metastasis are described.

Protocol

All human studies described in this manuscript were carried out under protocols approved by Western University's Human Research Ethics Board. All Animal studies were conducted in accordance with the recommendations of the Canadian Council on Animal Care, under protocols approved by the Western University Animal Use Subcommittee.

1. Standard CTC Enumeration from Patient Blood Samples Using the CSS

1. Human Blood Sample Collection and Preparation for Processing on the Preparation Instrument

1. Using standard aseptic phlebotomy techniques, draw a minimum of 8.0 ml of human blood into a 10 ml CellSave tube (hereafter referred to as the CTC preservative tube), which contains ethylene diaminetetraacetic acid (EDTA) and a proprietary cellular preservative. Invert the tube 5x to prevent blood from clotting. Samples may be processed immediately or stored at room temperature for up to 96 hr.
2. Remove CSS reagents from the fridge and allow them to warm to room temperature before using.
3. Using a disposable 10 ml pipette and automated pipettor, collect 7.5 ml of blood from the CTC preservative tube and slowly dispense blood into an appropriately labeled preparation instrument processing tube.
4. Add 6.5 ml of dilution buffer to each sample. Mix by inverting sample 5x. Centrifuge sample at 800 x g for 10 min with the brake in the "off" position. Follow the on-screen instructions on the preparation instrument to load all patient samples onto the system for processing. Samples must be processed within 1 hr of preparation.

2. Control Preparation for Processing on the Preparation Instrument

1. Gently vortex the control vial and invert 5x to mix.
2. Carefully remove the cap from the control vial and place an inverted preparation instrument processing tube on top of the uncapped vial. In one swift motion, invert the control vial, and pour the contents into the processing tube. While inverted, gently flick the sides of the control vial to release any remaining contents.
3. Carefully remove the inverted control vial from the processing tube, ensuring that no liquid is lost and place aside. Using a 1,000 μl pipette, collect any remaining contents from the vial and lid and gently pipette into the processing tube.
4. Follow the on-screen instructions on the preparation instrument to load the control onto the system for processing.

3. Sample Scanning on the Analysis Instrument

1. Follow the on-screen instructions on the preparation instrument to unload all samples from the system. Loosely cap each magnetic device cartridge and tap the magnetic device using hands or lab bench to release any bubbles that are stuck to the edges of the cartridge. Once all the bubbles have been removed, firmly cap the cartridge, lay the magnetic device flat, and incubate in the dark for at least 20 min at room temperature. Samples must be scanned within 24 hr of preparation.
2. Turn on the analysis instrument and initialize the lamp. Once warmed (~15 min), load the system verification cartridge onto the analysis instrument and select the QC Test tab. Follow the on-screen instructions to perform the necessary quality control measures.
3. Load a sample onto the analysis instrument and select the Patient Test tab. All saved information from the preparation instrument will be displayed. Click Start to initialize sample scanning. The system will perform a coarse focus and edge detection on the magnetic device cartridge.
4. Adjust all edges as necessary using the directional keys. Select Accept. The system will perform a fine focus and begin sample scanning.
5. Following control scanning the results should be validated using the defined criteria for cells spiked at high (CK⁺DAPI⁺CD45^-APC⁺) and low (CK⁺DAPI⁺CD45^-FITC⁺) concentrations. Following patient sample scanning the results should be reviewed for captured CTCs using the defined CTC criteria (CK⁺DAPI⁺CD45^-).

2. CTC Characterization for User-defined Markers using the CSS

1. Preparation of User-defined Markers and Instrument Initialization

1. Dilute the antibody of interest using Bond Primary Antibody Diluent to the desired concentration in a marker reagent cup using the following formula, where the working concentration is the concentration of the antibody after addition to the sample and the stock concentration is the concentration of antibody in the reagent cup. For multiple samples, adjust the antibody volumes as described in Table 1. Place the marker reagent cup into position 1 in the reagent cartridge and load the cartridge onto CSS.
   Stock Concentration = (Working Concentration x 850 μl) / 150 μl
   
2. Collect blood, prepare samples, and load the preparation instrument as described in the above Standard CTC Enumeration from Patient Blood Samples using the CSS protocol. To enable custom marker addition, select User Defined Assay when prompted by the preparation instrument. Input the marker name and select Save. As samples are loaded onto the instrument, the operator will be prompted to indicate which should receive custom marker by selecting Yes or No as necessary.

2. Sample Scanning of User-Defined Markers on the Analysis Instrument

1. Turn on the analysis instrument, initialize the lamp, and perform quality control and system verification as described in Section 3.2 of the Standard CTC Enumeration from Patient Blood Samples using the CSS protocol.
2. Load a sample onto the analysis instrument and select the Setup tab. To initialize the FITC channel, select CellSearch CTC as the Kit ID under the Test Protocols section. From this menu, select CTC Research, click the Edit button and set the exposure time as desired. It is recommended that an exposure time of 1.0 sec not be exceeded when using the CSS CTC kit as this can increase bleed-through into other fluorescent channels utilized for CTC identification.

3. Adaptation of the Standard CSSProtocol for use in Preclinical Mouse Models

**Adapted from Veridex Mouse/Rat CellCapture Kit (no longer commercially available)

1. Mouse Blood Collection and Storage

1. Prior to blood collection, run ~30 μl of 0.5 M EDTA back and forth through a 22 G needle, leaving a small amount of EDTA in the hub.
2. Collect a minimum of 50 μl of mouse blood from mice previously injected with human tumor cells via orthotopic, tail vein, or intracardiac routes. Collect blood from the saphenous vein (for serial CTC analysis) or by cardiac puncture (for terminal CTC analysis). Remove needle and dispense blood into a 1 ml EDTA microtainer blood collection tube. Mix by inversion or gently flick tube to prevent blood from clotting. Blood may be processed immediately or stored at room temperature for up to 48 hr following the addition of an equal volume of CytoChex cellular preservative.

2. CTC Enrichment

1. Remove CSS reagents from the fridge and allow them to warm to room temperature before using.
2. Transfer the equivalent of 50 μl of whole blood to a 12 mm x 75 mm flow cytometry tube. Add 500 μl of dilution buffer to each sample, washing down any blood that remains on the sides of the tube. If necessary, a short centrifuge spin can be used to collect any remaining blood.
3. Gently vortex the anti-EpCAM ferrofluid and add 25 μl to each sample by placing the tip of the pipette directly into the sample. Add 25 μl of Capture Enhancement reagent and vortex gently to mix. Incubate samples at room temperature for 15 min.
4. Place sample tubes into the magnet and incubate for 10 min. While the sample tubes are still in the magnet, use a glass pipette to carefully aspirate the residual liquid without touching the wall of the tube next to the magnet and discard.

3. CTC Staining

1. Remove the sample tubes from the magnet and resuspend in 50 μl of Nucleic Acid Dye, 50 μl of Staining Reagent, 1.5 μl of anti-mouse CD45-APC, 5.0 μl of anti-human HLA-Alexa Fluor 488, and 100 μl of Permeabilization Reagent. For multiple samples, these reagents may be premixed and 206.5 μl of mixture may be added to each tube. Vortex gently to mix and incubate for 20 min at room temperature in the dark.
2. Add 500 μl of dilution buffer, vortex gently, place sample tubes into the magnet, and incubate for 10 min. While the sample tubes are still in the magnet, use a glass pipette to carefully aspirate the residual liquid without touching the wall of the tube next to the magnet and discard. Remove the sample tubes from the magnet and resuspend in 350 μl of dilution buffer. Vortex gently to mix.

4. Magnetic Device Loading

1. Using a gel loading tip, carefully transfer the entire volume from the sample tube into a cartridge in the magnetic device. Start at the bottom of the cartridge and slowly withdraw the tip as the sample is dispensed.
2. Once the entire sample has been transferred, loosely cap the magnetic device cartridge and tap the magnetic device using hands or lab bench to release any bubbles that are stuck to the edges of the cartridge as described in Section 3.1 of the Standard CTC Enumeration from Patient Blood Samples using the CSS.
3. Pop any bubbles using a sterile 22 G needle by trapping them between the bevel and the edge of the cartridge. Once all the bubbles have been removed, firmly cap the cartridge, lay the magnetic device flat, and incubate in the dark for at least 10 min. Samples must be scanned within 24 hr of preparation.

5. Scanning of Manually Separated Samples on the Analysis Instrument

1. Turn on the analysis instrument, initialize the lamp, and perform quality control and system verification as described in Section 3.2 of the Standard CTC Enumeration from Patient Blood Samples using the CSS protocol.
2. Load the sample onto the analysis instrument and select the Setup tab. Clear any existing data on the magnetic device data button by clicking the Format Sample button. Enable the FITC channel and set the exposure time to 1.0 sec as described in Section 2.2 of the CTC Characterization for User-Defined Markers using the CSS.
3. Click on the Patient Test tab and select Edit to input the sample information. Select CellSearch CTC as the Kit ID and CTC Research as the Test Protocol. Input the remaining necessary information as indicated as the asterisk. Save the sample information and click Start.

Results

Standard CTC Enumeration Assay

The sensitivity and specificity of the CSS has been well documented in the literature. However, to validate equivalent CTC recovery, spiked (1,000 LNCaP human prostate cancer cells) and unspiked human blood samples from healthy volunteer donors were processed on the CSS using the standard CSSCTC protocol. As expected, unspiked samples were free of CTCs, 0.00±0.00%, and CTC recovery was demonstrated to be 86.9±4.71% for spiked samples (Figure 1A). CS...

Discussion

Despite the development of many new CTC technologies since the introduction of the CSS in 2004, this technique is still the only clinically approved technology on the market today and therefore it is considered the current gold standard for CTC detection and enumeration. This manuscript has demonstrated that although the CSS has rigorous quality control standards it can be subject to interpretation bias and that CTC identification in patient samples is much different from identification in spiked samples. Six categories ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 M EDTA
Anti-human CD44-FITC	BD Pharmigen	555478
Anti-human CD44-PE	BD Pharmigen	555479
Anti-human HLA-Alexa Fluor 488	BioLegend	311415
Anti-mouse CD45-APC	eBioscience	17-0451-82
Bond Primary Antibody Diluent	Leica	AR9352
CellSave Preservative Tubes	Veridex	952820 (20 pack) 79100005 (100 pack)
CellSearch CTC Control Kit	Veridex	7900003
CellSearch CTC Kit	Veridex	7900001
CellSearch CXC Control Kit	Veridex	7900018RUO
CellSearch CXC Kit	Veridex	7900017RUO
Instrument Buffer	Veridex	7901003
Streck Cell Preservative (aka CytoChex)	Streck	213350
1 ml Syringe
10 ml Serological pipette
1,000 µl Pipette
1,000 µl Pipette tips
12 mm x 75 mm Flow tubes
200 µl Gel loading tips
200 µl Pipette
22 G Needle
5 3/4 in Disposible Pasteur pipet	VWR	14672-200
5 ml Serological pipette
Automated pipettor
Capillary Blood Collection Tube (EDTA)	BD Microtainer	365974
CellSearch Analyzer II	Veridex	9555	Includes magnests and verification cartirdges
CellSearch AutoPrep System	Veridex	9541
Centrifuge
MagCellect Magnet	R&D Systems	MAG997
Small Latex Bulb	VWR	82024-550
Vortex