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Abstract

Introduction

Protocol

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Cancer Research

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

Published: December 25th, 2021

DOI:

10.3791/62329

1Department of Physiology and Cancer, Institute for Functional Genomics, Montpellier University
* These authors contributed equally

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. Intratu....

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All in vivo protocols were approved by the animal ethical agencies.

1. CTC Amplification in 3D Culture Conditions

  1. 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/µL and into 1 mL of M12 medium (i.e., advanced DMEM-F12 supplemented with 2 mM l-glutamine, 100 Unit/mL penicillin and streptomycin, N2 supplement, 20 ng/mL epidermal growth factor and 10&#.......

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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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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.......

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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.

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Name Company Catalog Number Comments
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

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