Name: circulating tumor cell lines: an innovative tool for fundamental and translational research
Uploaded: 2021-12-25T14:00:00
Description: Watch this Scientific Journal Video about Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research at JoVE.com

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&#...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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...

Watch this Scientific Journal Video about Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research at JoVE.com

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 ...

Metastasis is a leading cause of cancer-related deaths. This phenomenon occurs when cancer cells detach from the primary tumor, go into the blood circulation to finally reach a distant target organ. Once these detached cells go into the blood, we call them secondary tumor cells.They represent a variable material because on one hand, they are the main culprit of metastasis formation and in other hand, they are easier than tumor biopsies. Indeed, they can be collected by a simple blood puncture. Despite the low number of these cells in the blood sample, we are the first to establish several circulating tumor cell line from the blood of patient with greater concern using the 3D controlled condition.This cell line can be used at any commercial cell line for routine experiment. For example, for genetic drug screening, to protein of interest, but also for in vivo experiment to test the tumor initiation capacity and their metastatic potential. CTC, circulating tumor cell lines, should be cultured in 3D conditions in hypoxia.Once CTC has formed spheres, we can put it in the medium and centrifuge it for five minutes at 300 RCF. Discard the supernatant and add 500 microliters of Accumax. We suspend one and incubate the solution 20 minutes at 37 degrees.Then wash dissociated spheres with PBS and pellet the cells. Resuspend the pellet with a freshly supplemented DMEM/F12 and see the cells at the density of five cells per microliter in a new 24 Low Attachment plate. Centrifuge CTCs spheres eliminate the supernatant and wash the pellet twice with PBS.At 500 microliter of PFE mix well and incubate the tube on ice for 20 minutes. Then, centrifuge the fixed spheres and wash the pellet twice with PBS, eliminating the maximum volume of PBS and add 20 microliter of formed HistoGel. Take it to suspension and make a droplet on the culture vase, let it dry for 10 minutes and detach it using a scalpel.You can now process for any desired in order to CTCs targeting a desired protein of interest. Collect CTCs and disassociate the spheres with Accumax as previously described. Eliminate the Accumax and resuspend the pellet with three mL of blocking buffer and transfer the suspension through a 14 micrometers cell strainer to obtain single cell solution.Count the cells and dispatch into tubes and add a volume of 200, 000 cells, centrifuge the tubes and discard the supernatant. According to the antibody data sheet, add in a tube, the desired volume of the antibody and in another tube its isotype and continue the steps following manufacturer's guidance. The remaining tube will not be labeled and the use of a viability market is strongly suggested in this experiment.On the cytometer, start with the non-labeled tube to set the voltage in the negatively labeled area. Following this, place the isotype tube. The isotype would serve as a negative control to distinguish between the positive signal and non-specific signal.And finish the experiment with the labeled cells with the antibody of interest where you should see a shift in the positively labeled gate, if the protein of interest is expressed. This suspend dissociated CTCs in supplemented medium at the density of 200, 000 cells per mL. In an Ultra Low attachment 96, well plate, seal the volume of 50 microliter and incubate the plate 24 hours in hypoxia.The following day, add 50 microliter of different concentration of the tested drug and add the same volume of DMEM/F12 only to further determine the base of cellular viability and incubate the plate for another 48 hours. At day three, reconstitute the agent and add 70 microliter of the mix into each well. Put the plate on an orbital shaker for 20 minutes and then measure the luminescence.This assay is a useful tool to test a large number of drugs in order to evaluate their effects on CTC's cell growth. In Eppendorf, resuspend cells in 100 microliter of PBS pinch the skin on the back of the mouse and insert the needle beneath the skin. Inject slowly the whole volume in the same spot and monitor tumor growth every second day and sacrificed the mice if the tumor exceeded a size of 1.5 cubic centimeters.Prior to the surgery, inject subcutaneously a pain reliever, at the dorsal ventral side of the mouse, do a small incision below the 10th rib. Gently lift the spleen out and lay it on a sterile gauze. Using an insulin syringe, inject 50 microliter of resuspended CTCs in PBS and wait for three minutes without removing the needle.Ligate the arterial vessel from one side and the venal vessel from the other side. Then remove the spleen by cutting directly above the two ligatures. Stitch the abdominal peritoneum and the skin and disinfect well the area.The aim of this experiment is to test the capacity of CTCs derived from colorectal cancer patient to colonize the liver by inducing, visible metastatic foresight. Isolating and establishing of the clinic tumor cell line is an emergent tool for both fundamental and translational studies. For example, if you have a gene of interest where the in vivo studies demonstrated that this gene is involved in migration and invasion capacity of colorectal cancer cell lines, knocking out this gene in circulating tumor cell lines prior to the injection in the spleen of mice, to let them colonize the liver might be a good tool to confirm your result in vivo.For translational studies, our main perspective of isolating circulating tumor cell lines is to use this precious material in personalized medicine. From patient's blood samples, we would isolate and amplify CTCs in culture for two or three weeks in order to have enough material to screen the panel of drugs that is available and assess its cytotoxicity to finally attribute

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