In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Summary

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

Abstract

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche. The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines. Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions. Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs. Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions. However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly. CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs. Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques. In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system. This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

Introduction

Metastasis is a complex multi-step process that remains poorly understood. The E-selectin/selectin ligand axis has been shown to play an important role in tumor metastasis by promoting primary adhesive interactions between the vascular endothelium and cancer cells^1,2. Endothelial (E)-selectin is a transmembrane protein expressed by activated endothelial cells, while different E-selectin ligand(s) are expressed by tumor cells³. Numerous in vitro approaches have been successfully employed to model E-selectin/selectin ligand interactions between tumor cells and endothelial cells (ECs)¹. To study these interact....

Protocol

1. Culturing HUVECs on Microslides for Observing CTC-endothelial Interactions

1. Under the tissue culture hood, first rinse the microslide, channel width of 1 mm with PBS. Gently coat the microslide with 200 µl of 50 µg/ml fibronectin (dissolved in PBS) using a 1-ml Luer-lock syringe.
2. Cover the microslide with the lid and keep it inside the tissue culture hood for 30 min. Slow dispensing of the liquid in the microslide prevents bubble formation in the channel.
3. Perfuse 200 µl of warm (37 °C) HUVEC growth medium (M199 media, 1M HEPES, 20% FBS, 5 mg/ml heparin, 100 µg/ml endothelial cell growth factor, and L-glutamine....

Results

Figure 1 shows an O/N culture of a monolayer of ECs on the microslide. The scalings on Figure 1A shows that 100% of the microslide is visible using 5X objective while 70% is visible using a 10X objective (Figure 1B). For E-selectin mediated interactions, cells rolling at the edges are not considered which makes more than 70% of the microslide available for video-recording and playback analysis. In our experience, initially this set-up assembly needs some practice to cult.......

Discussion

Due to the low number of CTCs among blood cells, it is difficult to isolate CTCs as a pure population of cells. In order to study CTC/EC interactions, the rare and impure population of CTCs poses two major challenges: a) Identification of CTCs among blood cells; b) Observation of CTC/EC interactions.

To overcome the first limitation of identifying prostate CTCs among blood cells, we took advantage of the fact that virtually all prostate tumor cells express PSM.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microslide	Ibidi	80331
Fibronectin	Millipore	FC010
Plastic tubing	Cole Parmer	EW-96115-08
Male Luer adapter	GlycoTech	31-001
Female Luer adapter	GlycoTech	31-001
Syringe pump	Chemyx Inc	Fusion 100
Luer-lock syringe	BD Biosciences	309628
M199 medium	Sigma	M7653
Endothelial Mitogen	Biomedical Technologies	BT-203
HBSS	Sigma	H9269
Anti-PSMA J591-488	Weill Cornell Medical College-Lab of Urologic Oncology
Interleukin-1 beta	Peprotech	200-01B
Trypsin	Millipore	SM-2002-C
Heparin	Sigma	H-3149
HUVECs	Weill Cornell Medical College-Department of Surgery		provided by Marco Seandel
VE-Cadherin	Santa Cruz	sc-5648
10x objective	Zeiss Plan Neofluar
Enzyme free cell dissociation reagent	Millipore	S-004-C
RPMI-1640	Lonza	12-702-F
Ficoll-paque plus	GE healthcare	17-1440-02