Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

Summary

Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells.

Abstract

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed to mechanical stress due to hemodynamic flow. One of these stresses that circulating tumor cells (CTCs) experience is fluid shear stress (FSS). While cancer cells may experience low levels of FSS within the tumor due to interstitial flow, CTCs are exposed, without extracellular matrix attachment, to much greater levels of FSS. Physiologically, FSS ranges over 3-4 orders of magnitude, with low levels present in lymphatics (<1 dyne/cm²) and the highest levels present briefly as cells pass through the heart and around heart valves (>500 dynes/cm²). There are a few in vitro models designed to model different ranges of physiological shear stress over various time frames. This paper describes a model to investigate the consequences of brief (millisecond) pulses of high-level FSS on cancer cell biology using a simple syringe and needle system.

Introduction

Metastasis, or the spread of cancer beyond the initial tumor site, is a major factor underlying cancer mortality¹. During metastasis, cancer cells utilize the circulatory system as a highway to disseminate to distant sites throughout the body^2,3. While en route to these sites, circulating tumor cells (CTCs) exist within a dynamic fluid microenvironment unlike that of their original primary tumor^3,4,5. It has been proposed that this fluid microenvironment is one of many barriers to meta....

Protocol

1. Cell preparation

1. Release cells from tissue culture dish when 70-90% confluent by following the recommended guidelines for the cell line in use.
   1. For example, aspirate the growth medium for PC-3 cells, and wash the 10 cm dish of cells with 5 mL of calcium- and magnesium-free phosphate-buffered saline (PBS).
   2. Aspirate the PBS before adding 1 mL of 0.25% trypsin using manufacturer's protocol.
   3. After observing the detachment of the cells under an inverted microscope, add 5 mL of DMEM:F12 medium containing 10% fetal bovine serum to inhibit the trypsin.
2. Place the cell suspension into a conical tube.

Results

Elevated resistance to FSS-induced mechanical destruction has been previously shown to be a conserved phenotype across multiple cancer cell lines and cancer cells freshly isolated from tumors relative to non-transformed epithelial cell comparators^15,24. Here, additional cancer cell lines from a variety of tissue origins (Table 2) were tested to demonstrate that the majority of these cells display viability ≥ 20% after 10 pulses of FSS at 25.......

Discussion

This paper demonstrates the application of FSS to cancer cells in suspension using a syringe and needle. Using this model, cancer cells have been shown to be more resistant to brief pulses of high-level FSS relative to non-transformed epithelial cells^15,22,24. Furthermore, exposure to FSS using this model results in a rapid increase in cell stiffness, activation of RhoA, and increased cortical F-actin and myosin II-based contrac.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin	Gibco	25200-056
14 mL round bottom tubes	Falcon - Corning	352059
30 G 1/2" Needle	BD	305106
5 mL syringe	BD	309646
96-well black bottom plate	Costar - Corning	3915
Bioluminescence detector	AMI	AMI HTX
BSA, Fraction V	Sigma	10735086001
Cell Titer Blue	Promega	G8081
crystal violet	Sigma	C0775
D-luciferin	GoldBio	D-LUCK
DMEM	Gibco	11965-092
FBS	Atlanta Biologicals	S11150
PBS	Gibco	10010023
Plate Reader	BioTek	Synergy HT
Sodium Azide (NaN3)	Sigma	S2002
Syringe Pump	Harvard Apparatus	70-3005