Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Summary

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Abstract

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Introduction

Migration and invasion of individual or collective cells are two hallmarks of cancer, and required for the metastatic spread of cancer cells^1-4. The ability of cancers cells to initiate metastasis depends on their capability to migrate and invade into the neighboring tissue using invadopodia to degrade the basement membrane of the cells. Invadopodia are dynamic actin-rich matrix degradation protrusions that enable degradation of the extracellular matrix through the release of matrix-degrading proteases⁵. Cancer cell invasion involves the degradation of the matrix followed by the migration of the cancer cells and this is accompanied by a reorganiz....

Protocol

1. Three-dimensional Culture of Breast Cancer Cells in Basement Membrane Matrix (The Embedment Technique)

1. Handling Matrigel basement membrane matrix: Thaw on ice overnight at 4 °C. Basement membrane matrix is liquid at low temperature but solidifies at room temperature. Keep basement membrane matrix on ice (Figures 1A-B).
2. Cover the Confocal No.1 glass-bottom dish with 50 μl of basement membrane matrix by means of spreading the matrix using the tip of a P-200 Pipetman in a spiral pattern (Figure 1C). Use caution when spreading the basement membrane matrix to avoid the formation of air bubbles. Likewise, avoid spre....

Results

An example of the MDA-MB-231 cells invading in 3D matrix is illustrated in Figure 3C. The cells are embedded in matrix (Day 1), and start forming invasive (stellate) structures by Day 3, and completely invade into the matrix by Day 5 (Figure 3C). The number of stellate colonies formed are counted, and expressed as a percentage of total number of colonies per dish (invasive and non-invasive). Additionally, since measurements are done daily for the five days, the rate of invasion can also .......

Discussion

The development of 3D cell culture techniques has allowed researchers to study the transformation of breast epithelial cells, allowing us to visualize the dramatic morphological changes. Besides analyzing cell invasion, the single or multicellular mammary epithelial spheroids can be used to assess changes in cellular adhesion, proliferation, size, and basal-apical polarity. In contrast to previously reported methodologies where the cells are overlaid with ECM⁸, our method embeds the cells in ECM^18,19,24

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 ml tubes	VWR	CA10011-700	Sterile, disposable
100-mm culture dish BD353003	VWR	CABD353003	Sterile, disposable
15 ml Falcon tube	VWR	CA21008-918	Sterile, disposable
1 ml filtered tips	VWR	10011-350	Sterile, disposable
200 μl filtered tips	VWR	22234-016	Sterile, disposable
20 μl filtered tips	VWR	22234-008	Sterile, disposable
35-mm glass-bottomed Confocal No.1 culture dishes	MatTek Corporation	P35G-1.0-14-C	Precooled before use
Bovine serum albumin (BSA)	BioShop	ALB003.100	Used at 3% for IF
Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed	Life Technologies	A11029	1:250 for IF
Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody	Life Technologies	A11011	1:1,200 for IF
Anti-Beta-Catenin	BD Transduction Laboratories	610153	Mouse-monoclonal; Used at 1:100 for IF
Fetal bovine serum (FBS)	Sigma	F1051	Used at 10% (v/v)
Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg⁄ml Solution in Water	Life Technologies	H3569	Used at 0.1% (1:10,000 dilution)
InVivo Analyzer Suite	Media Cybernetics		Used for 3D culture imaging (DIC images at 10X and 40X)
Kisspeptin-10 (KP-10)	EZ Biolabs	PT0512100601	Used at 100 nM
Anti-Laminin	Cedarlane	AB19012(CH)	Rabbit-polyclonal full length human; Used at 1:100 for IF
LSM-510 META laser scanning microscope	Zeiss		Used at 63X objective; oil immersion lens
Matrigel phenol red free (BD356237)	VWR	CACB356237	Lot No.2180819; 10.4 mg/ml
Olympus IX-81 microscope	Olympus		Used for 3D culture imaging (DIC images at 10X and 40X)
Penicillin-streptomycin (10,000 U/ml)	Life Technologies	15140-122	Antibiotic (added to media; used at 0.01%)
10 ml pipette	VWR	CA53300-523	Sterile, disposeable
RPMI 1640 Medium with Glutamine	Life Technologies	11875-119	Used for culturing of MDA-MB-231 cells
0.25% Trypsin-EDTA (1x), Phenol Red	Life Technologies	25200-072	Used to trypsinize MDA-MB-231 cells
MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)	Lonza	CC-3150	Used for culturing of MCF10A cells