We thank all patients that donated blood for our study, as well as all involved clinicians and study nurses. We thank Jens Eberhardt, Uwe Birke, and Dr. Katharina Uhlig from ALS Automated Lab solutions GmbH for continuous support. We thank all members of the Aceto lab for feedback and discussions. Research in the Aceto lab is supported by the European Research Council, the European Union, the Swiss National Science Foundation, the Swiss Cancer League, the Basel Cancer League, the two Cantons of Basel through the ETH Z&#252;rich, and the University of Basel.

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</ol>

N.A. and B.M.S. are listed as inventors in patent applications that relate to circulating tumor cells and the treatment of cancer. N.A. is a paid consultant for pharmaceutical and insurance companies with an interest in liquid biopsy.

The molecular characterization of CTCs holds the promise to improve our understanding of the metastatic process and guide the development of new anti-metastasis therapies. Here we provide a detailed description of those protocols that enable CTC micromanipulation and downstream analysis, including both single cell-based functional assays, gene expression analysis and in vivo transplantation for metastatic potential assessment20.
Among the most critical steps of...

The clinical manifestation of metastasis in distant organs represents the final stage of cancer progression and accounts for more than 90% of cancer-related deaths1. The transition from localized to metastatic disease is a multi-step process, often mediated by circulating tumor cells (CTCs)2,3,4. These cells are shed from the primary tumor into the blood circulation and are transported to distant organs, where they may extravasate and establish metastatic lesions5,6. Although solid tumors can release a relatively high number of CTCs, most CTCs are destined to die, owing to high shear forces in circulation, anoikis-mediated cell death, immune attack or limited capabilities to adapt to a foreign microenvironment7. Therefore, it is pivotal to establish tools that enable the dissection of the molecular features of those CTCs that are endowed with metastasis-seeding ability. Recent preclinical and clinical studies suggest that the presence and quantity of single CTCs and CTC clusters is associated with a worse outcome in patients with various types of solid tumors8,9,10,11,12,13,14. CTC clusters are groups of two or more CTCs attached to each other during circulation and are more efficient in forming metastasis compared to single CTCs3,15,16. Cells within a cluster maintain strong cell-cell adhesion through desmosomes and adherens junctions, which may help to overcome anoikis17,18. Recently, we observed that clustering of CTCs is linked to hypomethylation of binding sites for stemness- and proliferation-associated transcription factors, leading to an increased ability to successfully initiate metastasis19. CTC cluster dissociation results in remodeling of key binding sites, and consequently, the suppression of their metastatic potential19. Additionally to clusters of cancer cells, CTCs can also associate to white blood cell (most frequently neutrophils) to maintain high proliferation levels in circulation and increase their metastatic capability20. However, the biology of CTCs is understood only in part and several questions remain open, including the underlying molecular features and vulnerabilities of single and clustered cells.
In recent years, several strategies have been established that exploit cell-surface expression patterns as well as physical properties of CTCs for their isolation21,22,23,24,25. Antigen-dependent isolation methods rely mostly on the expression of cell surface Epithelial Cell Adhesion Molecule (EpCAM)26. The most frequently used and (at present) the only FDA-approved platform for CTC enumeration, is the CellSearch system, which is based on a two-step procedure to isolate CTCs21. In the first step, plasma components are removed by centrifugation, while CTCs are captured with magnetic ferrofluids coupled to anti-EpCAM antibodies. In the second step, the CTC-enriched solution is stained for nucleated (DAPI-positive) cells expressing cytokeratin (CK)8,18,19, while white blood cells (WBCs) are identified using the pan-leukocyte marker CD45. Finally, captured cells are placed on an integrated screening platform and CTCs are identified through the expression of EpCAM, CKs, and DAPI while being negative for CD45. Although this is considered to be the gold standard for CTC enumeration, downstream molecular analysis is challenging with this technology due to inherent constraints in CTC retrieval. Additionally, given its isolation procedure, CellSearch&#160;may favor the enrichment of CTCs with higher EpCAM levels compared to CTCs with lower EpCAM expression, due for instance to cancer heterogeneity27 or downregulation of epithelial markers28,29. To overcome these limitations, antigen-independent technologies for the enrichment of CTCs have emerged. For example, the CTC-iChip integrates hydrodynamic separation of nucleated cells, including CTCs and WBCs from remaining blood components, followed by an immunomagnetic depletion of antibody-tagged WBCs, allowing purification of untagged and viable CTCs in solution25. Additionally, the fact that most CTCs are slightly bigger than red blood cells (RBCs) or WBCs led to the development of size-based CTC enrichment technologies23,30 (e.g., the Parsortix system (ANGLE)) which makes use of a microfluidic-based technology, comprising a narrowing channel across the separation cassette, leading cells to a terminal gap of either 10, 8, 6.5 or 4.5 &#181;m (different sizes are available depending upon the expected diameter of target cancer cells). Most of the blood cells pass through the narrow gap, while CTCs get trapped due to their size (but also due to their lower deformability) and are, therefore, retained in the cassette. Reverting the flow direction enables the release of captured CTCs, which are in a viable state and suitable for downstream analysis. Independently of the chosen protocol for CTC isolation, however, typical post-enrichment procedures still yield CTCs that are mixed with a relatively small number of RBCs and WBCs, making the analysis of pure single or bulk CTCs challenging. To address this issue, we established a workflow that allows CTC manipulation without potential bias introduced by blood cell contaminants. The addition of immunostaining beforehand, with variable antibody-combinations, distinguishes CTCs from blood cells and even allows to identify CTC subgroups with distinct surface-marker expression profiles. This highly customizable procedure can be then further combined with specific downstream applications.
Here, we describe a workflow that starts from a CTC-enriched product (obtained with any CTC enrichment technology of choice) and combines several approaches to gain insight into CTC biology at single-cell resolution. In a nutshell, our workflow enables the identification of single CTCs, CTC clusters and CTC-WBC clusters by live immunostaining, followed by single-cell micromanipulation and downstream analysis using ex vivo culturing protocols, single cell sequencing, and in vivo metastasis assays.

<table><tbody><tr><td>Anti-human EpCAM-AF488</td><td>Cell Signaling Technology</td><td>CST5198</td><td>clone: VU1D9</td></tr><tr><td>1X DPBS</td><td>Invitrogen</td><td>14190169</td><td>no calcium, no magnisium</td></tr><tr><td>6-wells Ultra-low attachment plate</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>Anti-human CD45-BV605</td><td>Biolegend</td><td>304041</td><td>clone: HI30</td></tr><tr><td>Anti-human EGFR-FITC&amp;nbsp;</td><td>GeneTex</td><td>GTX11400</td><td>clone: ICR10</td></tr><tr><td>Anti-human HER2-AF488&amp;nbsp;</td><td>Biolegend</td><td>324410</td><td>clone: 24D2</td></tr><tr><td>Anti-mouse CD45-BV605</td><td>Biolegend</td><td>103139</td><td>clone: 30-F11</td></tr><tr><td>BD Vacutainer K2EDTA</td><td>BD</td><td>366643</td><td>for human blood collection</td></tr><tr><td>Cell Celector</td><td>ALS</td><td>CC1001</td><td>core unit&amp;nbsp;</td></tr><tr><td>CellD software</td><td>ALS</td><td></td><td>version 3.0</td></tr><tr><td>Cultrex PathClear Reduced Growth Factor BME, Type 2</td><td>R&amp;amp;D Systems</td><td>3533-005-02</td><td></td></tr><tr><td>Micro tube 1.3 mL K3EDTA</td><td>Sarstedt</td><td>41.3395.005</td><td>for mouse blood collection</td></tr><tr><td>PCR tubes</td><td>Corning</td><td>PCR-02-L-C</td><td></td></tr><tr><td>RLT Plus</td><td>Quiagen</td><td>1053393</td><td></td></tr><tr><td>SUPERase&amp;nbsp; In RNase Inhibitor</td><td>Thermo Fisher</td><td>AM2696</td><td>&amp;nbsp;1 U/&amp;micro;L&amp;nbsp;</td></tr></tbody></table>

micromanipulation of circulating tumor cells for downstream molecular analysis and metastatic potential assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

The presented workflow allows the preparation of individual CTCs, either from single CTCs or separated from CTC clusters. CTCs from patients or tumor-bearing mice are enriched from whole blood with available CTC-enrichment methods and then stained with antibodies against cancer-associated&#160;markers (e.g., EpCAM, green) and WBC-specific markers (e.g., CD45, red) (Figure 1A). The stained CTC product is then transferred to the micromanipulation station were i...

Watch this Scientific Journal Video about Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment at JoVE.com

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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Cancer Research

8.6K Views.  University of Basel and University Hospital Basel. Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Video: Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bioengineering

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

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.

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.

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

Medicine

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 ...