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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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. 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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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Cancer 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 ...

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 ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

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 ...

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 ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.