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 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.
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.
All in vivo protocols were approved by the animal ethical agencies.
1. CTC Amplification in 3D Culture Conditions
2. Immuno-Fluorescence (IF) Staining on CTC Sphere Sections
3. Cytometry Analysis
4. Luminescence Viability Assay (CellTiter-Glo)
5. Subcutaneous Injections
6. Intrasplenic Injection
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-mesenchymal axis. Expression of other CSC markers could be also tested by both immunostaining and cytometry. Otherwise, functional tests as drug resistance and tumor initiating assay in vivo can validate this CSC phenotype10. Here for example, luminescence viability assay shows that the CTC IC50 is only reached at high drug concentration highlighting the high resistance of these cells (Figure 2). Moreover, subcutaneous CTC injection shows that this CTC line has tumorigenic ability (Figure 3A). These latter results confirm the CSC phenotype of this CTC line. Challenging the tumor initiation capacity by injecting from 10 to 10,000 cells can refine the estimation of the tumorigenic ability. Finally, liver metastasis formation by mimicking dissemination through intrasplenic injection shows that this CTC line can survive in a distant organ and has a metastatic potential (Figure 3B). This metastatic potential can be compared to other cell lines or challenged by inhibiting specific gene expression or upon chemotherapy administration in mice after intrasplenic injection.
Figure 1: Microscopy and Flow cytometry analysis of CTC spheres show that they expressed both epithelial and CSC markers. (A) Left panel: CTC spheres cultured for 6 days before embedding. Right panel: epifluorescence microscopy analysis of embedded CTC spheres in Histogel. 5 µm sections of embedded CTC spheres were stained by an antibody against the epithelial marker Epithelial cell adhesion molecule (EPCAM) (in red) and the nucleus is labelled with DAPI (in blue). Scale bar is 20 µm. (B) Flow cytometry analysis of alive single CTCs. Upper left FACS plot shows CTCs without labelling to gate cells based on their size and granularity. Upper right FACS plot shows CTCs with a viability marker to gate alive cells only. Bottom left FACS plot shows CTCs labelled with APC-conjugated isotype control to gate positively labelled cells. Bottom right FACS plot shows CTCs labelled by APC-conjugated CD26 antibody to assess the percentage of cells expressing this CSC marker (67%). Please click here to view a larger version of this figure.
Figure 2: Representative result of the cell growth assay using a luminescence viability assay. 10,000 CTCs were seeded per well and were then treated with an increasing concentration of the molecule of interest for 72 hours. The IC50 is reached at x µM. Please click here to view a larger version of this figure.
Figure 3: CTCs have a tumorigenic and metastatic potential. (A) Subcutaneous injection of 200,000 CTCs on the right flank of Nude mice. Photos were taken 1 month after the injection and tumor size was measured 3 times per week. (B) Intrasplenic injection of CTCs to mimic liver metastatic colonization. Right panel: representative photo of mouse liver dissected 4 weeks after intrasplenic injection of 300,000 CTCs. Left panel: mouse liver dissected 4 weeks after intrasplenic injection of PBS. Please click here to view a larger version of this figure.
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 isolation of CTCs based on the cell size and its compressibility13. In addition, there is the CellSearch, an immunobead based assay that is the only FDA-approved method to enumerate CTCs in blood samples based on a negative CD45 labeling to eliminate contaminating lymphocytes combined with positive epithelial marker labelling such as EpCAM and cytokeratin 8/18/1914. The iChip is another size-based technology but it also combines the CTC enrichment using either an EpCAM based positive selection or CD45 negative depletion15,16. Finally, we have recently used the fast and easy immunodensity procedure, Rosette sep kit, to isolate and amplify CTC from the colorectal cancer patient blood samples10.
If working on a mouse model, it is also possible to pool mouse blood samples from the same cohort to increase the number of CTCs and the chance of amplifying them in culture. In the case of murine CTCs, it is not mandatory to use immunodeficient mice to test the liver colonization by intrasplenic injection. Control mice with the same genetic background can be used as recipient mice without any impact of the immune response.
Once CTCs are amplified in suspension, they can be compared with other cancer cell lines from the same type of cancer using each technique described here. In this case, different cancer cell lines must be cultivated in the same conditions. Particular attention must be paid to innate auto-fluorescence, for the immunofluorescence staining and cytometry analysis. The CTCs and each cancer cell line used must be tested without any staining in the different fluorescent channels.
If the CTCs under study can adhere and proliferate in adherent conditions like any classical cell line, then the techniques described above can be adapted for cells expanded in these conditions. The resulting readouts will likely thus be more relevant where the cells are from more differentiated cancer cell populations as cells tend to be less differentiated in suspension.
In conclusion, CTC lines are very precious tools to deeply characterize mechanisms involved in metastatic processes leading cancer lethality, and in the future as culture success rates improve, CTCs could be used to propose the best therapeutic strategy adapted to each patient.
The authors have nothing to disclose.
This research project in the Pannequin lab was supported by research grant from the SIRIC: Grant « INCa-DGOS-Inserm 6045 ». The PhD theses of Guillaume Belthier and Zeinab Homayed were supported by the anti-cancer league/Ligue contre le Cancer. Céline Bouclier salary was financed by "region Occitanie". Thanks to Julian Venables for English editing.
Accumax solution | Sigma-Aldrich | A7089 | |
Advanced DMEM/F-12 | Gibco | 12634028 | |
CellTiter-Glo Luminescent Cell Viability Assay | Promega | G7570 | |
Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | 354230 | |
Costar 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, | Corning | 3473 | |
Histiogel Specimen Medium | LabStorage | HG-4000 | |
Human EGF, premium grade | Miltenyi Biotec | 130-097-751 | |
Human FGF-2, premium grade | Miltenyi Biotec | 130-093-564 | |
L-Glutamine (200 mM) | Gibco | 25030081 | |
N-2 Supplement | Gibco | 17502048 | |
Penicillin-Streptomycin (5,000 U/mL) | Gibco | 15070063 |