Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Summary

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Abstract

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1β) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Introduction

Tumor biology is far more intricate than previously thought. Mounting evidence shows that tumor cells are more than a mere bulk of uncontrolled proliferating cells; rather, different neoplastic cells seem to perform different functions displaying high organization and hierarchy within the tumor¹. Tumor cells are also in intimate communication with non-transformed cells: macrophages, fibroblasts, lymphocytes, adipocytes, endothelial cells, among other cells are all immersed in the scaffold proteins and polysaccharides that constitute the ECM. Numerous direct and indirect interactions are established between transformed and non-transformed cells ....

Protocol

Samples from BrC patients were obtained from the tissue bank of the Unidad de Investigación en Virología y Cáncer, Hospital Infantil de México Federico Gómez. This study was approved by the scientific, ethical, and biosecurity review boards of the Hospital Infantil de México Federico Gómez: Comité de Investigación, Comité de Ética en Investigación and Comité de Bioseguridad. All patients were prospectively enrolled and were informed about the nature of the study: those willing to participate signed a written informed consent prior to specimen collection and were treated according to the ethical guideline....

Results

Morphological Analysis of BrC Cells in 3D Cultures:

The morphology of primary BrC cells growing in 3D cultures at low and high densities was studied over 5 days. During the first 48 h, cells adhere to the ECME and maintain a low density. At this time point, it can clearly be appreciated that cells present an elongated spindle-like shape, some with two or more long cytoplasmic projections and without showing appa.......

Discussion

Epithelial cells grow in a 3D spatial conformation and their interaction with the ECM proteins is pivotal for tissue homeostasis. Many cancer studies have been based on cells grown in monolayers (2D) and although they have been critical to understanding many aspects of tumor formation and progression, monolayers do not recapitulate the characteristics that the ECM imposes on cells, for instance: limiting proliferation, adhesion-dependent cell survival, apical-basolateral polarity, ECM remodeling, cell differentiation,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
U937	American Type Culture Collection ATCC	CRL-1593.2	Monocytic cell line/histiocytic lymphoma
THP-1	American Type Culture Collection ATCC	TIB-202	Monocytic cell line/acute monocytic leukemia
MCF-10A	American Type Culture Collection ATCC	CRL-10317	Non-transformed breast cell line
MCF-7	American Type Culture Collection ATCC	HTB-22	Breast Cancer Cell line
T47D	American Type Culture Collection ATCC	HTB-133	Breast Cancer Cell line
HS578T	American Type Culture Collection ATCC	HTB-126	Breast Cancer Cell line
MDA-MB-231	American Type Culture Collection ATCC	HTB-26	Breast Cancer Cell line
RPMI 1640 medium	GIBCO BRL Life Technologies	11875-093
DMEM High Glucose	GIBCO BRL Life Technologies	11964-092	4.5 g/L glucose
DMEM/F12	GIBCO BRL Life Technologies	11039-021
Antibiotic/Antimycotic	GIBCO BRL Life Technologies	15240-062	100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL Fungizone
Fetal Bovine Serum	GIBCO BRL Life Technologies	16000-044
Horse serum	GIBCO BRL Life Technologies	16050114
0.05% Trypsin-EDTA 1X	GIBCO BRL Life Technologies	25300-062
PBS 1X (Phosphate Buffered Saline	GIBCO BRL Life Technologies	20012-027
Epidermal Growth Factor (EGF)	PeproTech	AF-100-15 (1.00mg)
Insulin	SIGMA-ALDRICH	I1882-100MG
Hydrocortisone	SIGMA-ALDRICH	H088-5G
Cholera toxin Vibrio cholerae	SIGMA-ALDRICH	C8052-1MG
Matrigel	Corning Inc	356237	Engelbreth-Holm-Swarm (EHS) mouse sarcoma, extracellular matrix extract, Store at -20°C until use at 4°C
GM-CSF	PeproTech	300-03
MCP-1	PeproTech	300-04
RANTES	PeproTech	300-06
IL-8	PeproTech	200-08
IL-1β	PeproTech	200-01B
Crystal-violet	Hycel México	541
Paraformaldehyde	SIGMA-ALDRICH	P6148
Transwell permeable supports (inserts)	Corning Inc	3422	6.5 mm diameter, 8 µm pore size/24-well plates
Transwell permeable supports (inserts)	Thermofisher Scientific	140620	6.5 mm diameter, 0.4 µm pore size/24-well plates
Monocyte Isolation Kit II Human	Miltenyi Biotec	130-091-153
LS Columns	Miltenyi Biotec	130-042-401
Human Cytokine/Chemokine Magnetic Bead Panel Kit 96 Well Plate Assay	EMD Millipore	HCYTOMAG-60K
Magpix with xPonent software (laser based fluorescent analytical test instrumentation)	Luminex Corporation	40-072
Lab-Tek chamber slide with cover (8 well)	Nalge Nunc International	177402
Glass bottom microwell 35mm petri  dishes	MatTek Corp.	P35G-1.5-14-C