Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Summary

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.

Abstract

Blood-borne metastasis accounts for 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 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.

Introduction

The clinical manifestation of metastasis in distant organs represents the final stage of cancer progression and accounts for more than 90% of cancer-related deaths¹. The transition from localized to metastatic disease is a multi-step process, often mediated by circulating tumor cells (CTCs)^2,3,4. These cells are shed from the primary tumor into the blood circulation and are transported to distant organs, where they may extravasate and establish metastatic lesions^5,6. Although solid tum....

Protocol

All the procedures involving blood samples from patients were performed upon signed informed consent of the participants. Procedures were run according to protocols EKNZ BASEC 2016-00067 and EK 321/10, approved by the ethical and institutional review board (Ethics Committee northwest/central Switzerland [EKNZ]), and in compliance with the Declaration of Helsinki.

All the procedures concerning animals were performed in compliance with institutional and cantonal guidelines (approved mouse protoc.......

Discussion

The molecular characterization of CTCs holds the promise to improve our understanding of the metastatic process and guide the development of new anti-metastasis therapies. Here we provide a detailed description of those protocols that enable CTC micromanipulation and downstream analysis, including both single cell-based functional assays, gene expression analysis and in vivo transplantation for metastatic potential assessment²⁰.

Among the most critical steps of.......

Materials

Name	Company	Catalog Number	Comments
Anti-human EpCAM-AF488	Cell Signaling Technology	CST5198	clone: VU1D9
1X DPBS	Invitrogen	14190169	no calcium, no magnisium
6-wells Ultra-low attachment plate	Corning	3471
Anti-human CD45-BV605	Biolegend	304041	clone: HI30
Anti-human EGFR-FITC	GeneTex	GTX11400	clone: ICR10
Anti-human HER2-AF488	Biolegend	324410	clone: 24D2
Anti-mouse CD45-BV605	Biolegend	103139	clone: 30-F11
BD Vacutainer K2EDTA	BD	366643	for human blood collection
Cell Celector	ALS	CC1001	core unit
CellD software	ALS		version 3.0
Cultrex PathClear Reduced Growth Factor BME, Type 2	R&D Systems	3533-005-02
Micro tube 1.3 mL K3EDTA	Sarstedt	41.3395.005	for mouse blood collection
PCR tubes	Corning	PCR-02-L-C
RLT Plus	Quiagen	1053393
SUPERase  In RNase Inhibitor	Thermo Fisher	AM2696	1 U/µL