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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	<li>Talmadge, J. E., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AACR+centennial+series:+the+biology+of+cancer+metastasis:+historical+perspective.">AACR centennial series: the biology of cancer metastasis: historical perspective.</a> Cancer Research. 70 (14), 5649-5669 (2010).</li><li>Lambert, A. W., Pattabiraman, D. R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Biological+Principles+of+Metastasis.">Emerging Biological Principles of Metastasis.</a> Cell. 168 (4), 670-691 (2017).</li><li>Aceto, N., Toner, M., Maheswaran, S., Haber, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=En+Route+to+Metastasis:+Circulating+Tumor+Cell+Clusters+and+Epithelial-to-Mesenchymal+Transition.">En Route to Metastasis: Circulating Tumor Cell Clusters and Epithelial-to-Mesenchymal Transition.</a> Trends in Cancer. 1 (1), 44-52 (2015).</li><li>Hong, Y., Fang, F., Zhang, Q. <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+cell+clusters:+What+we+know+and+what+we+expect+(Review).">Circulating tumor cell clusters: What we know and what we expect (Review).</a> International Journal of Oncology. 49 (6), 2206-2216 (2016).</li><li>Nguyen, D. X., Bos, P. 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M., 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=Clinical+significance+and+molecular+characteristics+of+circulating+tumor+cells+and+circulating+tumor+microemboli+in+patients+with+small-cell+lung+cancer.">Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer.</a> Journal of Clinical Oncology. 30 (5), 525-532 (2012).</li><li>Long, E., 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=High+expression+of+TRF2,+SOX10,+and+CD10+in+circulating+tumor+microemboli+detected+in+metastatic+melanoma+patients.+A+potential+impact+for+the+assessment+of+disease+aggressiveness.">High expression of TRF2, SOX10, and CD10 in circulating tumor microemboli detected in metastatic melanoma patients. A potential impact for the assessment of disease aggressiveness.</a> Cancer Medicine. 5 (6), 1022-1030 (2016).</li><li>Wang, C., 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=Longitudinally+collected+CTCs+and+CTC-clusters+and+clinical+outcomes+of+metastatic+breast+cancer.">Longitudinally collected CTCs and CTC-clusters and clinical outcomes of metastatic breast cancer.</a> Breast Cancer Research and Treatment. 161 (1), 83-94 (2017).</li><li>Mu, Z., 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=Prospective+assessment+of+the+prognostic+value+of+circulating+tumor+cells+and+their+clusters+in+patients+with+advanced-stage+breast+cancer.">Prospective assessment of the prognostic value of circulating tumor cells and their clusters in patients with advanced-stage breast cancer.</a> Breast Cancer Research and Treatment. 154 (3), 563-571 (2015).</li><li>Zhang, D., 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+microemboli+(CTM)+and+vimentin++circulating+tumor+cells+(CTCs)+detected+by+a+size-based+platform+predict+worse+prognosis+in+advanced+colorectal+cancer+patients+during+chemotherapy.">Circulating tumor microemboli (CTM) and vimentin+ circulating tumor cells (CTCs) detected by a size-based platform predict worse prognosis in advanced colorectal cancer patients during chemotherapy.</a> Cancer Cell International. 17, 6 (2017).</li><li>Zheng, X., 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=Detection+of+Circulating+Tumor+Cells+and+Circulating+Tumor+Microemboli+in+Gastric+Cancer.">Detection of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer.</a> Translational Oncology. 10 (3), 431-441 (2017).</li><li>Chang, M. 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J., 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=Polyclonal+breast+cancer+metastases+arise+from+collective+dissemination+of+keratin+14-expressing+tumor+cell+clusters.">Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (7), E854-E863 (2016).</li><li>Giuliano, M., 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=Perspective+on+Circulating+Tumor+Cell+Clusters:+Why+It+Takes+a+Village+to+Metastasize.">Perspective on Circulating Tumor Cell Clusters: Why It Takes a Village to Metastasize.</a> Cancer Research. 78 (4), 845-852 (2018).</li><li>Gkountela, S., Aceto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-like+features+of+cancer+cells+on+their+way+to+metastasis.">Stem-like features of cancer cells on their way to metastasis.</a> Biology Direct. 11, 33 (2016).</li><li>Gkountela, 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+Cell+Clustering+Shapes+DNA+Methylation+to+Enable+Metastasis+Seeding.">Circulating Tumor Cell Clustering Shapes DNA Methylation to Enable Metastasis Seeding.</a> Cell. 176 (1-2), 98-112 (2019).</li><li>Szczerba, B. M., 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=Neutrophils+escort+circulating+tumour+cells+to+enable+cell+cycle+progression.">Neutrophils escort circulating tumour cells to enable cell cycle progression.</a> Nature. , (2019).</li><li>Beije, N., Jager, A., Sleijfer, S. <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+cell+enumeration+by+the+CellSearch+system:+the+clinician's+guide+to+breast+cancer+treatment?.">Circulating tumor cell enumeration by the CellSearch system: the clinician's guide to breast cancer treatment?.</a> Cancer Treatment Reviews. 41 (2), 144-150 (2015).</li><li>Sarioglu, A. F., 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=A+microfluidic+device+for+label-free,+physical+capture+of+circulating+tumor+cell+clusters.">A microfluidic device for label-free, physical capture of circulating tumor cell clusters.</a> Nature Methods. 12 (7), 685-691 (2015).</li><li>Xu, L., 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=Optimization+and+Evaluation+of+a+Novel+Size+Based+Circulating+Tumor+Cell+Isolation+System.">Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation System.</a> PLoS One. 10 (9), e0138032 (2015).</li><li>Stott, S. 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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 border="0" cellpadding="0" cellspacing="0" style="width:1003px;" width="1004">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:333px;">Anti-human EpCAM-AF488</td>
			<td style="width:145px;">Cell Signaling Technology</td>
			<td style="width:353px;">CST5198</td>
			<td style="width:171px;">clone: VU1D9</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1X DPBS</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td>no calcium, no magnisium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">6-wells Ultra-low attachment plate</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-human CD45-BV605</td>
			<td>Biolegend</td>
			<td>304041</td>
			<td>clone: HI30</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-human EGFR-FITC&#160;</td>
			<td>GeneTex</td>
			<td>GTX11400</td>
			<td>clone: ICR10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-human HER2-AF488&#160;</td>
			<td>Biolegend</td>
			<td>324410</td>
			<td>clone: 24D2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-mouse CD45-BV605</td>
			<td>Biolegend</td>
			<td>103139</td>
			<td>clone: 30-F11</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD Vacutainer K2EDTA</td>
			<td>BD</td>
			<td>366643</td>
			<td>for human blood collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell Celector</td>
			<td>ALS</td>
			<td>CC1001</td>
			<td>core unit&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CellD software</td>
			<td>ALS</td>
			<td></td>
			<td>version 3.0</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cultrex PathClear Reduced Growth Factor BME, Type 2</td>
			<td>R&#38;D Systems</td>
			<td>3533-005-02</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micro tube 1.3 mL K3EDTA</td>
			<td>Sarstedt</td>
			<td>41.3395.005</td>
			<td>for mouse blood collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PCR tubes</td>
			<td>Corning</td>
			<td>PCR-02-L-C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RLT Plus</td>
			<td>Quiagen</td>
			<td>1053393</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SUPERase&#160; In RNase Inhibitor</td>
			<td>Thermo Fisher</td>
			<td>AM2696</td>
			<td>&#160;1 U/&#181;L&#160;</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

8.5K 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

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 ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

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 ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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 ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

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 ...

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 ...

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed to mechanical stress due to hemodynamic flow. One of these stresses that circulating tumor cells (CTCs) experience is fluid shear stress (FSS). While cancer cells may experience low levels of FSS within the tumor due to interstitial flow, CTCs are exposed, without extracellular matrix attachment, to much greater levels of FSS. Physiologically, FSS ranges over 3-4 orders of magnitude, with low levels present in lymphatics (&#60;1 dyne/cm2) and the highest levels present briefly as cells pass through the heart and around heart valves (&#62;500 dynes/cm2). There are a few in vitro models designed to model different ranges of physiological shear stress over various time frames. This paper describes a model to investigate the consequences of brief (millisecond) pulses of high-level FSS on cancer cell biology using a simple syringe and needle system.

Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells.

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...