This work was supported by grants from the Ontario Institute of Cancer Research (#08NOV230), the Canada Foundation for Innovation (#13199), Prostate Cancer Canada, Janssen Oncology, the London Regional Cancer Program, and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). L.E.L. is supported by a Frederick Banting and Charles Best Canada Graduate Scholarship Doctoral Award. A.L.A is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.

All human studies described in this manuscript were carried out under protocols approved by Western University&#39;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
<ol>
	<li>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.</li>
	<li>Remove CSS reagents from the fridge and allow them to warm to room temperature before using.</li>
	<li>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.</li>
	<li>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 &#34;off&#34; 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.</li>
</ol>
2. Control Preparation for Processing on the Preparation Instrument
<ol>
	<li>Gently vortex the control vial and invert 5x to mix.</li>
	<li>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.</li>
	<li>Carefully remove the inverted control vial from the processing tube, ensuring that no liquid is lost and place aside. Using a 1,000 &#956;l pipette, collect any remaining contents from the vial and lid and gently pipette into the processing tube.</li>
	<li>Follow the on-screen instructions on the preparation instrument to load the control onto the system for processing.</li>
</ol>
3. Sample Scanning on the Analysis Instrument
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Adjust all edges as necessary using the directional keys. Select Accept. The system will perform a fine focus and begin sample scanning.</li>
	<li>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-).</li>
</ol>
2. CTC Characterization for User-defined Markers using the CSS
1. Preparation of User-defined Markers and Instrument Initialization
<ol>
	<li>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 &#956;l) / 150 &#956;l 
	</li>
	<li>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.</li>
</ol>
2. Sample Scanning of User-Defined Markers on the Analysis Instrument
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
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
<ol>
	<li>Prior to blood collection, run ~30 &#956;l of 0.5 M EDTA back and forth through a 22 G needle, leaving a small amount of EDTA in the hub.</li>
	<li>Collect a minimum of 50 &#956;l 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.</li>
</ol>
2. CTC Enrichment
<ol>
	<li>Remove CSS reagents from the fridge and allow them to warm to room temperature before using.</li>
	<li>Transfer the equivalent of 50 &#956;l of whole blood to a 12 mm x 75 mm flow cytometry tube. Add 500 &#956;l 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.</li>
	<li>Gently vortex the anti-EpCAM ferrofluid and add 25 &#956;l to each sample by placing the tip of the pipette directly into the sample. Add 25 &#956;l of Capture Enhancement reagent and vortex gently to mix. Incubate samples at room temperature for 15 min.</li>
	<li>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.</li>
</ol>
3. CTC Staining
<ol>
	<li>Remove the sample tubes from the magnet and resuspend in 50 &#956;l of Nucleic Acid Dye, 50 &#956;l of Staining Reagent, 1.5 &#956;l of anti-mouse CD45-APC, 5.0 &#956;l of anti-human HLA-Alexa Fluor 488, and 100 &#956;l of Permeabilization Reagent. For multiple samples, these reagents may be premixed and 206.5 &#956;l of mixture may be added to each tube. Vortex gently to mix and incubate for 20 min at room temperature in the dark.</li>
	<li>Add 500 &#956;l 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 &#956;l of dilution buffer. Vortex gently to mix.</li>
</ol>
4. Magnetic Device Loading
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Scanning of Manually Separated Samples on the Analysis Instrument
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+from+patients+with+advanced+prostate+and+breast+cancer+display+both+epithelial+and+mesenchymal+markers.">Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers.</a> Mol. Cancer Res. 9 (8), 997-1007 (2011).</li></ol>

The author (A.L.A.) received funding that was provided by Janssen Oncology, whose parent company Johnson &amp; Johnson also owns Janssen Diagnostics LLC, which produces reagents and instruments used in this article.

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 ...

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 &#8805;5 (breast and prostate) or &#8805;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 techniques7-10.
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 previously12-14) 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 techniques4-6. 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&#39;,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&#160;demonstrated that approximately one third of breast cancer patients with HER2-primary tumors had HER2+ CTCs. In addition, Liu et al.18&#160;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 tumors19-21. 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&#160;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&#160;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.

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			<td height="21" style="height:21px;width:267px;">REAGENTS</td>
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			<td height="20" style="height:20px;width:267px;">0.5M EDTA</td>
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			<td height="20" style="height:20px;width:267px;">Anti-human CD44-FITC</td>
			<td style="width:175px;">BD Pharmigen&#160;</td>
			<td style="width:161px;">555478</td>
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			<td height="20" style="height:20px;width:267px;">Anti-human CD44-PE</td>
			<td style="width:175px;">BD Pharmigen&#160;</td>
			<td style="width:161px;">555479</td>
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			<td height="20" style="height:20px;width:267px;">Anti-human HLA-AlexaFluor488</td>
			<td style="width:175px;">BioLegend</td>
			<td style="width:161px;">311415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Anti-mouse CD45-APC</td>
			<td style="width:175px;">eBioscience</td>
			<td style="width:161px;">17-0451-82</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:267px;">Bond Primary Antibody Diluent</td>
			<td style="width:175px;">Leica</td>
			<td style="width:161px;">AR9352</td>
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			<td height="60" style="height:60px;width:267px;">CellSave Preservative Tubes</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">952820 (20 pack)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 79100005 (100 pack)</td>
			<td></td>
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			<td height="20" style="height:20px;width:267px;">CellSearch CTC Control Kit</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">7900003</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:267px;">CellSearch CTC Kit</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">7900001</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:267px;">CellSearch CXC Control Kit</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">7900018RUO</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:267px;">CellSearch CXC Kit</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">7900017RUO</td>
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			<td height="20" style="height:20px;width:267px;">Instrument Buffer</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">7901003</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Streck Cell Preservative (aka CytoChex)</td>
			<td style="width:175px;">Streck</td>
			<td>213350</td>
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			<td height="20" style="height:20px;width:267px;">1 ml syringe</td>
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			<td height="20" style="height:20px;width:267px;">10 ml serological pipette</td>
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			<td style="width:161px;"></td>
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			<td height="21" style="height:21px;width:267px;">1000&#181;l pipette</td>
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			<td style="width:161px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:267px;">1000&#181;l pipette tips</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:267px;">12 x 75mm flow tubes</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">200&#181;l gel loading tips</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:267px;">200&#181;l pipette</td>
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			<td style="width:161px;"></td>
			<td></td>
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			<td height="20" style="height:20px;width:267px;">22 gauge needle</td>
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			<td height="20" style="height:20px;width:267px;">5 3/4&#34; disposible pasteur pipet</td>
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			<td height="40" style="height:40px;width:267px;">Automated pipettor</td>
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			<td style="width:161px;"></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Capillary Blood Collection Tube (EDTA)</td>
			<td style="width:175px;">BD Microtainer</td>
			<td style="width:161px;">365974</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">CellSearch Analyzer II</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">9555</td>
			<td rowspan="2" style="width:188px;">Includes magnests and verification cartirdges</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:267px;">CellSearch AutoPrep System</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">9541</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:267px;">MagCellect Magnet&#160;</td>
			<td style="width:175px;">R&#38;D Systems</td>
			<td style="width:161px;">MAG997</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Small Latex Bulb</td>
			<td style="width:175px;">VWR</td>
			<td>82024-550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Vortex</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
	</tbody>
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adaptation of semiautomated circulating tumor cell (ctc) assays for clinical and preclinical research applications

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&#160;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.

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&#177;0.00%, and CTC recovery was demonstrated to be 86.9&#177;4.71% for spiked samples (Figure 1A)</s...

Watch this Scientific Journal Video about Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications at JoVE.com

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

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&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

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

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

adaptation-semiautomated-circulating-tumor-cell-ctc-assays-for

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JoVE Journal

Medicine

15.8K Views.  London Health Sciences Centre. 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.

Video: Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of radiation, the in vivo properties of photon and proton beams differ greatly and maybe exploited to optimize clinical outcomes. In particular, proton particles lose energy in a predictable manner as they pass through the body. This property is used clinically to control the depth at which the proton beam is terminated, and to limit radiation dose beyond the target region. This strategy can allow for substantial reductions in radiation dose to normal tissues located just beyond a tumor target. However, the degradation of proton energy in the body remains highly sensitive to tissue density. As a consequence, any changes in tissue density during the course of treatment may significantly alter proton dosimetry. Such changes may occur through alterations in body weight, respiration, or bowel filling/gas, and may result in unfavorable dose deposition. In this manuscript, we provide a detailed method for the delivery of proton therapy using both passive scatter and pencil beam scanning techniques for prostate cancer. Although the described procedure directly pertains to prostate cancer patients, the method may be adapted and applied for the treatment of virtually all solid tumors. Our aim is to equip readers with a better understanding of proton therapy delivery and outcomes in order to facilitate the appropriate integration of this modality during cancer therapy.

The fundamentals of radiation planning and delivery for proton therapy using prostate cancer as a model are presented. The application of these principles to other selected disease sites highlights how proton radiotherapy may enhance clinical outcomes for cancer patients.

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...