This research project in the Pannequin lab was supported by research grant from the SIRIC: Grant &#171; INCa-DGOS-Inserm 6045 &#187;. The PhD theses of Guillaume Belthier and Zeinab Homayed were supported by the anti-cancer league/Ligue contre le Cancer. C&#233;line Bouclier salary was financed by &#34;region Occitanie&#34;. Thanks to Julian Venables for English editing.

All in vivo protocols were approved by the animal ethical agencies.
1. CTC Amplification in 3D Culture Conditions
<ol>
	<li>To culture CTCs in suspension, first seed CTCs in wells of an Ultra-Low Attachment (ULA) 24-well plate at the maximal concentration of 5 cells/&#181;L and into 1 mL of M12 medium (i.e., advanced DMEM-F12 supplemented with 2&#8197;mM l-glutamine, 100&#8197;Unit/mL penicillin and streptomycin, N2 supplement, 20&#8197;ng/mL epidermal growth factor and 10&#8197;ng/mL fibroblast growth factor10).</li>
	<li>To allow sphere formation and cell expansion, incubate the cells in hypoxic conditions (2% O2) between 6 to 10 days.</li>
	<li>Once the spheres are large enough (100 &#181;m), to prevent necrosis, dissociate them for amplification.
	<ol>
		<li>Place the cell suspension in a 2 mL tube or a 15 mL tube and centrifuge at 300 x g for 5 minutes.</li>
		<li>Remove the supernatant and add 500 &#181;L of gentle dissociation reagent (e.g., Accumax) to the pellet.</li>
		<li>Vortex gently and incubate the cell suspension with gentle dissociation reagent for 20 to 30 minutes at 37 &#176;C. 
		NOTE: Exceeding the incubation time in the gentle dissociation reagent (Accumax) may cause abnormal increased cell mortality.</li>
		<li>Add 1.5 mL of Phosphate Buffered Saline (PBS) and centrifuge the tube for 5 minutes at 300 x g.</li>
		<li>Pour off the supernatant and resuspend the pellet in fresh M12 medium to reach the density of 1-5 cells per &#181;L.</li>
		<li>Seed the cell suspension in new wells of an Ultra-Low Attachment plate: 10 mL for T75 flasks, 2 mL for 6 well plates and 100 &#181;L for 96 well plates</li>
	</ol>
	</li>
</ol>
2. Immuno-Fluorescence (IF) Staining on CTC Sphere Sections
<ol>
	<li>Centrifuge CTC spheres into a 15 mL tube at 300 x g for 5 min, aspirate the supernatant, and wash the pellet twice with PBS.</li>
	<li>Resuspend the pellet with 500 &#181;L of 4% paraformaldehyde (PFA) and incubate for 20 min on ice. Centrifuge the suspension at 300 x g and wash twice with 5 mL of PBS.</li>
	<li>Resuspend the pellet with (i.e., 15 to 20 &#181;L) hot and liquid Histogel. With a pipette, take all the suspension and make a droplet on a precooled surface at 4 &#176;C and let it polymerize for 5 to 10 minutes. 
	NOTE: Work fast to avoid the polymerization of the suspension into the tip.</li>
	<li>Detach the solid droplet with the blade of a scalpel and insert it into a compartmented embedding cassette.</li>
	<li>Perform the following steps: paraffin inclusion, paraffin sectioning, section rehydration, antigen retrieval and antibody incubation. These steps are the same as for any tumor or organ embedded in paraffin and processed for immunofluorescence staining12. 
	NOTE: The cassette can be stored in 70% ethanol at 4 &#176;C until paraffin embedding.</li>
</ol>
3. Cytometry Analysis
<ol>
	<li>Centrifuge CTC spheres into a 15 mL tube at 300 x g for 5 min and wash the pellet twice with PBS.</li>
	<li>Aspirate the supernatant and add 500 &#181;L of the gentle dissociation reagent to the pellet.</li>
	<li>Vortex gently and incubate the cell suspension with the gentle dissociation reagent for 20 to 30 min at 37 &#176;C. 
	NOTE: Exceeding 40 min of incubation in the gentle dissociation reagent (Accumax) may result in an abnormal increased cell mortality.</li>
	<li>Centrifuge the cell suspension for 5 min at 300 x g, eliminate the supernatant and resuspend the pellet in 5 mL of blocking buffer (i.e., PBS with 1% bovine serum albumin (BSA)).</li>
	<li>Pass the cell suspension through a 40 &#181;m cell strainer to eliminate the remaining spheres.</li>
	<li>After counting, put 200,000 cells into three fluorescence-activated cell sorting (FACS) tubes, centrifuge at 300 x g for 5 min and remove the supernatant. Use one of the three FACS tube as a control that will not receive any staining reagents.</li>
	<li>According to the datasheet, add the appropriate antibody into the two remaining FACS tubes (i.e., one conjugated antibody against the protein of interest, one conjugated isotype control).</li>
	<li>After the recommended incubation time, add 300 &#181;L of the blocking buffer to wash the cells, centrifuge the 3 tubes at 300 x g and aspirate the supernatant. Repeat this step twice. Then, resuspend the pellet with the blocking buffer with a cell viability staining reagent (except for the FACS tube control without any staining).</li>
	<li>Keep the tubes on ice until FACS analysis. Use first the tube without staining to identify the gate for the cell viability and the antibody staining. Then analyze the FACS tube with the conjugated antibody against the protein of interest, to quantify the percentage of CTC expressing the protein of interest. The FACS tube with the conjugated isotype control should not indicate a shift. This confirms that the shift is specific to the targeted protein of interest. 
	&#8203;NOTE: If possible, use a cell line expressing a high level of protein of interest as a positive control and a cell line that does not express the protein as a negative control.</li>
</ol>
4. Luminescence Viability Assay (CellTiter-Glo)
<ol>
	<li>Resuspend dissociated CTCs (as described in section 1.4.5) in M12 medium. Count the cells and adapt the cell concentration to 200,000 cells/mL.</li>
	<li>In a ULA 96-well plate, seed 10,000 dissociated CTCs per well, in 50 &#181;L, and incubate the plate in hypoxia (2% O2) for 24 h.</li>
	<li>The following day, add 50 &#181;L of the tested drug to the CTCs. It is previously recommended to perform a titration of the drug concentration, to determine the half maximal inhibitory concentration (IC50). Treat some wells with vehicle only to determine basal cell viability. 
	NOTE: The number of cells seeded may differ between CTC lines, depending on their doubling time. In addition, cells should be seeded in triplicates to minimize results variability.</li>
	<li>Incubate plates in hypoxia for another 48 h.</li>
	<li>At day 3, place the cultured plates and the luminescence cell viability buffer and substrate at room temperature and reconstitute the substrate with an appropriate volume of buffer, according to manufacturer protocol.</li>
	<li>Add 70 &#181;L of the luminescence viability mix into each cultured plate well. Put plates on an orbital shaker for 20 min to induce cell lysis and then transfer 100 &#181;L of the mix into an opaque-walled multi-well plate, compatible with the luminometer.</li>
	<li>Allow the plate to rest at room temperature, cover it with aluminum foil in order to stabilize the luminescent signal. 
	&#8203;NOTE: Luminescent signal is stable for up to 3 hours.</li>
	<li>Record luminescence using a microplate reader (i.e., Tecan Infinite 200 Pro).</li>
	<li>For data analysis, calculate the mean of the triplicate relative light unit RLU per condition. To assess the percentage of viability according to each drug concentration needed: (RLU of treated cells/RLU of untreated cells) x 100.</li>
</ol>
5. Subcutaneous Injections
<ol>
	<li>In a microcentrifuge tube, resuspend cells in 100 &#181;L of sterile PBS. If the number of injected cells is low, resuspend cells in 1:1 PBS/Matrigel, reduced in growth factors, to concentrate cells together and promote tumor initiation. 
	NOTE: Cell density can be from 10 cells (to challenge the tumor initiation capacity) to 1 million of cells depending on the scientific question and the aim of the experiment.</li>
	<li>Pull up the full volume in an insulin syringe.</li>
	<li>On an immune-deficient mouse, pinch the skin of the flank between the index and the ring finger and gently insert the needle at the base of the skin. 
	NOTE: This procedure is not painful and is done on conscious mice.</li>
	<li>Slowly inject the volume in the same spot to prevent the spreading of the cells. This will create a small bleb beneath the skin.</li>
	<li>Apply gentle pressure on the injection site to prevent the backflow of the volume.</li>
	<li>Monitor the tumor growth, every other day, using a caliper.</li>
	<li>Sacrifice the animal if the tumor exceeds 1500 mm3. Depending on the material availability, euthanize the mice by carbon dioxide or anaesthetic gases inhalation or by cervical dislocation.</li>
</ol>
6. Intrasplenic Injection
<ol>
	<li>Before starting, autoclave the surgical instruments (scissors and forceps) at 124 &#176;C for 15 min and clean out all surfaces of the working space with 70% ethanol.</li>
	<li>In a microcentrifuge tube, resuspend cells in 50 &#181;L of sterile PBS and store them on ice. Cell density can go from 0.5 to 1 million cells. A larger number of cells can cause a clots development into the blood circulation.</li>
	<li>Prior to injecting the cells, inject 100 &#181;L of 0.015 mg/mL buprenorphine subcutaneously, for pain relief.</li>
	<li>Place the mouse in an induction chamber, where isoflurane is maintained at 3%, for 2-3 min. When the animal is no longer responsive to movement, put it on its right-hand side on a heating pad and maintain the anaesthesia using a breathing circuit face mask that delivers a gas mixture of O2 and isoflurane.</li>
	<li>Apply eye lubricant over each eye to avoid eye drying during the surgery. Disinfect the thoracic area with&#160;Povidone-iodine solution (i.e. Betadine).</li>
	<li>On the dorsoventral side, make a small incision of 0.5 cm using scissors below the 10th false rib.</li>
	<li>Gently lift the spleen out and lay it on sterile gauze. Using an insulin syringe, inject the 50 &#181;L of CTCs in the tip of the spleen. Make sure to maintain the syringe upright and wait 3-5 min to avoid backflow, and then remove the needle.</li>
	<li>Ligate the splenic vessels from the two sides (arteries and veins) and remove the spleen by cutting directly above the two ligatures. The spleen is removed to prevent unwanted tumor formation in this organ.</li>
	<li>Stitch the abdominal peritoneum and then the skin using 5.0 degradable sterile sutures.</li>
	<li>Keep the mouse warm until its full recovery from anesthesia.</li>
	<li>Monitor the mouse 3 days per week for abnormal bodily function, abnormal movement and posture and for weight loss.</li>
	<li>Sacrifice the animal when humane end points reach (approximately 6 weeks after the surgery) to analyze liver metastasis.</li>
</ol>

<ol>
	<li>Wittekind, C., Neid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+invasion+and+metastasis.">Cancer invasion and metastasis.</a> Oncology. 69, 14-16 (2005).</li><li>Eger, A., Mikulits, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+epithelial-mesenchymal+transition.">Models of epithelial-mesenchymal transition.</a> Drug Discovery Today: Disease Models. 2, 57-63 (2005).</li><li>Palmirotta, R., 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=Liquid+biopsy+of+cancer:+a+multimodal+diagnostic+tool+in+clinical+oncology.">Liquid biopsy of cancer: a multimodal diagnostic tool in clinical oncology.</a> Therapeutic Advances in Medical Oncology. 10, (2018).</li><li>Hong, B., Zu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+circulating+tumor+cells:+current+challenges+and+new+trends.">Detecting circulating tumor cells: current challenges and new trends.</a> Theranostics. 3, 377-394 (2013).</li><li>vander Toom, E. E., Verdone, J. E., Gorin, M. A., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+challenges+in+the+isolation+and+analysis+of+circulating+tumor+cells.">Technical challenges in the isolation and analysis of circulating tumor cells.</a> Oncotarget. 7, 62754-62766 (2016).</li><li>Jin, 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=Evaluation+of+the+diagnostic+value+of+circulating+tumor+cells+with+CytoSorter&#174;+CTC+capture+system+in+patients+with+breast+cancer.">Evaluation of the diagnostic value of circulating tumor cells with CytoSorter&#174; CTC capture system in patients with breast cancer.</a> Cancer Medicine. 9, 1638-1647 (2020).</li><li>Huang, 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=Relationship+between+circulating+tumor+cells+and+tumor+response+in+colorectal+cancer+patients+treated+with+chemotherapy:+a+meta-analysis.">Relationship between circulating tumor cells and tumor response in colorectal cancer patients treated with chemotherapy: a meta-analysis.</a> BMC Cancer. 14, 976 (2014).</li><li>Yu, 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=Ex+vivo+culture+of+circulating+breast+tumor+cells+for+individualized+testing+of+drug+susceptibility.">Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility.</a> Science. 345, 216-220 (2014).</li><li>Aceto, N., 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+Clusters+are+Oligoclonal+Precursors+of+Breast+Cancer+Metastasis.">Circulating Tumor Cell Clusters are Oligoclonal Precursors of Breast Cancer Metastasis.</a> Cell. 158, 1110-1122 (2014).</li><li>Grillet, 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=Circulating+tumour+cells+from+patients+with+colorectal+cancer+have+cancer+stem+cell+hallmarks+in+ex+vivo+culture.">Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture.</a> Gut. 66, 1802-1810 (2017).</li><li>Lee, W. Y., Hong, H. K., Ham, S. K., Kim, C. I., Cho, Y. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Colorectal+Cancer+in+Differentially+Established+Liver+Metastasis+Models.">Comparison of Colorectal Cancer in Differentially Established Liver Metastasis Models.</a> Anticancer Research. 34, 3321-3328 (2014).</li><li>Giraud, 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=Progastrin+production+transitions+from+Bmi1+/Prox1++to+Lgr5high+cells+during+early+intestinal+tumorigenesis.">Progastrin production transitions from Bmi1+/Prox1+ to Lgr5high cells during early intestinal tumorigenesis.</a> Translational Oncology. 14, (2020).</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, 0138032 (2015).</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, 144-150 (2015).</li><li>Karabacak, N. 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=Microfluidic,+marker-free+isolation+of+circulating+tumor+cells+from+blood+samples.">Microfluidic, marker-free isolation of circulating tumor cells from blood samples.</a> Nature Protocols. 9, 694-710 (2014).</li><li>Ozkumur, 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=Inertial+Focusing+for+Tumor+Antigen-Dependent+and+-Independent+Sorting+of+Rare+Circulating+Tumor+Cells.">Inertial Focusing for Tumor Antigen-Dependent and -Independent Sorting of Rare Circulating Tumor Cells.</a> Science Translational Medicine. 5, (2013).</li></ol>

The authors have no competing interests to disclose.

The protocol described above was used initially for colorectal CTC functional characterization, but it can be used for other types of cancer such as breast cancer and can be adapted for mouse models.
The real limiting factor is the number of CTCs present in the blood sample and the efficiency of the technique used to isolate and expand them. Several CTC isolation technics have been described based on specific CTC properties such as the Parsortix, a microfluidic device, that allows the isolatio...

Despite recent improvements in early cancer diagnosis and in therapeutic strategy, more than ninety percent of cancer morbidity is still due to metastasis1. The metastatic process is a multi-step cascade that starts with the local detachment of cells from the primary tumor and their entrance into the bloodstream where they become circulating tumor cells (CTCs) to finally colonize distant sites such as liver and lungs, in the case of colorectal cancer (CRC)2. Recently, there has been growing attention to liquid biopsies, which are a non-invasive tool to notably detect and enumerate CTCs from patient blood samples. Intratumor genetic heterogeneity is a major cause of drug resistance; thus, isolating representative cells from tumor material constitutes a promising tool for personalized medicine3.
Despite low frequency of CTCs in the blood (1 CTC per 106 - 107 leukocytes)4, several detection and isolation techniques were developed based on property differences between CTCs and other components of the blood5. The number of CTCs in patient blood samples, alone, can provide information about the stage of malignancy, treatment response and disease progression6,7. Thus, CTC isolation is a crucial tool for translational studies to assess genetic heterogeneity or perform drug screening, as well as for fundamental studies to characterize these invasive cells, as they are the key actors of metastatic induction8,9. Indeed, compared to commercially established cancer cell lines that have accumulated thousands of mutations over time, fresh CTCs share the main features of the original primary tumor including a potent capacity to metastasize, and they are a better reflection of the disease. These features make them a robust tool for fundamental studies, especially in knockout experiments of predicted key factors involved in metastasis. The outcome of these experiments can be validated in vivo, on mice, as described below.
Once CTCs are isolated, they can be expanded in non-adherent culture conditions and then, they can be manipulated just as any available cancer cell line, i.e. they can equally be cultured in adherent conditions or embedded in Matrigel, depending on the scientific question10. For example, to test the expression and localization of a protein of interest, CTC spheres can be grown in suspension condition and be embedded in Histogel to perform immunofluorescence on sphere sections. In addition, if the protein is membranous, its expression on living cells can be measured by cytometry.
For functional studies, to test the role of a protein of interest that may play a role in liver colonization, CTCs with genes edited, by shRNA or CRISPR/Cas9, can be injected into the spleen of immunodeficient mice. This latter experiment is a powerful model to mimic liver metastasis colonization11.
The ability of CTCs to initiate tumors can be assessed by injecting a very small number of cells into immuno-deficient mice. As tumor initiation is a hallmark of cancer stem cells (CSCs) this assay will indicate the percentage of CSCs within CTC lines. This stem cell phenotype makes circulating tumor cell lines resistant to some gold-standard cancer therapies. Expanded CTCs can therefore be used to screen drugs and pinpoint the best potential efficient treatment for the patient; CTC response to treatment can be tested in vitro using a luminescence viability assay, for example.
In a long-term perspective, drug screening on freshly isolated and amplified CTCs could be used as a new tool for personalized medicine to aid in choosing the most efficient and adapted treatment for patients.
In the present paper, protocols to culture CTC lines, to stain specific proteins via immunostaining and cytometry, to perform cytotoxicity assays as well as in vivo xenograft experiments with CTC are detailed.

<table><tbody><tr><td>Accumax solution</td><td>Sigma-Aldrich</td><td>A7089</td><td></td></tr><tr><td>Advanced DMEM/F-12</td><td>Gibco</td><td>12634028</td><td></td></tr><tr><td>CellTiter-Glo Luminescent Cell Viability Assay</td><td>Promega</td><td>G7570</td><td></td></tr><tr><td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Costar 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates,</td><td>Corning</td><td>3473</td><td></td></tr><tr><td>Histiogel Specimen Medium</td><td>LabStorage</td><td>HG-4000</td><td></td></tr><tr><td>Human EGF, premium grade</td><td>Miltenyi Biotec</td><td>130-097-751</td><td></td></tr><tr><td>Human FGF-2, premium grade</td><td>Miltenyi Biotec</td><td>130-093-564</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>N-2 Supplement</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>Penicillin-Streptomycin (5,000 U/mL)</td><td>Gibco</td><td>15070063</td><td></td></tr></tbody></table>

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.

Both EpCAM and CD26 expressions observed by IF (Figure 1A right panel) and FACS (Figure 1B) respectively, indicate that the CTC line is epithelial and display one of the CSC hallmarks10. This epithelial trait can be further characterized by staining with antibodies directed against other epithelial and mesenchymal markers. Thereby, it could be possible to approximately know where the CTC line is along the epithelial-mesen...

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

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 ...

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4.0K Views.  Montpellier University. Metastase is een belangrijke oorzaak van kankergerelateerde sterfgevallen. Dit fenomeen treedt op wanneer kankercellen zich losmaken van de primaire tumor, in de bloedcirculatie gaan om uiteindelijk een ver doelorgaan te bereiken. Zodra deze losgemaakte cellen in het bloed gaan, noemen we ze secundaire tumorcellen.Ze vertegenwoordigen een variabel materiaal omdat ze aan de ene kant de belangrijkste boosdoener zijn van metastasevorming en aan de andere kant zijn ze gemakkelijker dan tum...

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

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 ...

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 ...

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

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.

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 ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicine

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 ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

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 ...

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

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