Protocol for the creation and utilization of 3D pancreatic cancer models from circulating tumor cells

Abstract

We introduce a protocol for generating 3D organoids from circulating tumor cells (CTCs), enabling longitudinal functional and molecular analyses in pancreatic cancer patients, including those with unresectable disease, which constitutes the majority of cases. We outline the process for isolating and characterizing CTCs from the blood of pancreatic cancer patients and provide detailed instructions for initiating, passaging, and phenotyping CTC-derived organoids. Additionally, we describe techniques for utilizing these organoids in drug screening with a focus on stemness-related pathways. For complete details on the use and execution of this protocol, please refer to Tang et al.¹.

Keywords: Cancer; Cell-based Assays; Single Cell.

Graphical abstract

Figure 1

M2-like macrophages differentiated from PBMCs ready for the production of MCM

Figure 2

Schematic illustration of the CTC isolation process using the VAR2-Isoflux platform

Figure 3

Representative image of retrieved CTCs

Figure 4

Schematic for the initiation of CTC in vitro organoid cultures (A) Retrieval of CTCs from magnetic beads. (B) Establishing CTC in vitro organoid cultures.

Figure 5

Representative images of CTC-derived cultures (A) CTC-derived cultures in bright field in low magnification (5×). (B) CTC-derived cultures show organoid-like structures at high magnification (20×). (C) CTC-derived cultures exhibit positive signals for cytokeratin and negative signals for CD45 in immunofluorescent confocal staining.

Figure 6

Expansion of CTC-derived cultures (A) Organoids produce adherently growing monolayer primary cultures. (B) Culture of CTC-derived organoids as 3D sphere models in ultralow-adhesion plates.