A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

Summary

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Abstract

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

Introduction

It is established that cancer cell motility is predictive of metastatic potential, given that such behavior facilitates invasion through the basement membrane and entry into the circulatory system¹. Most research on motility has focused on how cells behave in a two-dimensional (2D) setting, though it is becoming widely recognized that the movement of cells in a three-dimensional (3D) matrix is more representative of how these same cells will actually behave in vivo². 3D culture systems are increasingly used to study cellular behaviors that range from cell morphology, to growth kinetics and drug sensitivity³. The desire to moni....

Protocol

1. Generation of Spheroids

1. Prepare single-cell suspension for hanging drop cultures by detaching adherent cancer cell cultures of ~70% confluence using a PBS wash followed by exposure to 0.05% trypsin-EDTA solution.
   1. Neutralize the trypsin solution with cell culture media and count the cells using an aliquot of the cell suspension. Note: The specific cell culture media will depend on the cell line being tested. Follow ATCC media recommendations, where the cell culture media is typically DMEM + 10% FBS. More information can be found in the Materials Table.
   2. Perform a dilution to allow for the seeding of 500-1,000 cells per 20 µl dr....

Results

Using the spheroid invasion assay (Figure 1), a panel of cancer cell lines was tested for their ability to form spheroids, as well as for the amount of cell egress exhibited after implantation in a 3D matrix consisting of basement membrane materials and type I collagen (Table 1). These results demonstrate that not every cancer cell line will create well-formed spheroids, where cell lines possessing an epithelial morphology in vitro tended to produce regular and smooth aggregates.......

Discussion

This study evaluated the performance of a panel of cancer cell lines in a spheroid invasion assay (Table 1). Generally, we find that spheroid formation is enhanced in the more epithelial-looking cell lines, where the presence of cell-cell junctions promotes the formation of a spheroid-like architecture. Known cell lines that have undergone an epithelial-mesenchymal transition, like MDA-MB-231 cells do not form spheroids in the hanging drop culture most likely due to their reduced E-, N-, and P-cadherin e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Matrigel Growth Factor Reduced	Corning	CB-40234
Collagen Type I, Rat Tail, 100 mg	Millipore	08-115
DMEM	Life Technologies	11995-065
RPMI 1640 Medium	Life Technologies	11875-093
PBS	Life Technologies	10010-023
0.05% Trypsin-EDTA	Life Technologies	25300-054
Fetal Bovine Serum, Heat Inactivated	Omega Scientific	FB-12
100 mm TC-treated Dishes	Corning Incorporated	430167
24-well TC-treated Plates	NEST Biotechnlology	702001
Olympus IX-71 Inverted Microscope
Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.