Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Summary

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Abstract

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Introduction

Glioblastoma multiforme is an aggressive human brain cancer with a median survival of approximately 12 months from the time of diagnosis ^1,2. Glioblastoma is one of the most deadly and clinically challenging cancers as it is refractory to standard chemotherapy and surgical resection. The diffuse nature of glioblastoma enables tumor cells to spread throughout the normal brain making the advanced tumor practically impossible to surgically resect completely. This highly invasive aspect is a hallmark feature of glioblastoma and other advanced astrocytomas. Therefore, the focus of much research has been on the molecular mechanism of glioblastoma cell invasion. T....

Protocol

1. Fluorescent Labeling of Cells

NOTE: Label glioblastoma cell lines and microglia with fluorescent dyes ⁹. Alternatively, generate cell lines that constitutively express fluorescent proteins such as GFP/RFP as described in ¹⁴.

1. Plate cells on a 6 well plate such that they will be 70 - 80% confluent on day of staining. For the murine glioblastoma cell line GL261 and human glioblastoma cell line U87, plate 1 x 10⁶ and 1.5 x 10⁶ cells, respectively on a 6 cm dish 24 hr before staining.
2. Prepare fluorescent cell stain dye solution in DMSO and add 5 µM dye to media, either Roswell Park Memorial In....

Results

Using the methods outlined here, we have shown that microglia and macrophages can substantially stimulate glioblastoma cell invasion. Two different invasion assays are employed and are depicted in Figure 1. In Figure 2, GL261 cells that constitutively express the fluorescent protein mCherry were plated on pre-coated chambers with and without microglia for 48 hr. GL261 cells were minimally invasive on their own however when cultured with microglia the inva.......

Discussion

The highly invasive nature of high grade astrocytomas and glioblastoma make these brain cancers very deadly. It is therefore of paramount importance to understand the molecular and cellular mechanisms of glioblastoma invasion. Much has been learned about the process of glioblastoma invasion already ¹⁷. Using the assay formats detailed in this paper, our laboratory has shown in both mouse and human models that tumor associated macrophages can stimulate glioma cell invasion by 5 - 10 fold. This coculture model f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Corning BioCoat Matrigel Invasion Chamber: With BD Matrigel Matrix	Corning/Fisher Scientific	Cat: 354481
Macrophage Serum Free Media (MSFM) (500 ml)	Life Technologies	12065-074
CellTracker Red CMTPX Dye	Life Technologies/Molecular Probes	C34552
CellTracker Green CMFDA Dye	Life Technologies/Molecular Probes	C2925
GL261 cell line	National Cancer Institute (NCI)
U87 cell line	American Tissue Type Culture Collection	HTB-14
THP-1 cell line	American Tissue Type Culture Collection	ATCC TIB-202
RPMI 1640 Medium (500 ml)	Life Technologies/Gibco	11875-093
Formaldehyde solution	Sigma Aldrich	F1635
Corning Transwell polycarbonate membrane cell culture inserts (8 µM pore) 48 per pack.	Corning	CLS3422
Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, PathClear	Trevigen	3445-005-01
Fetal Calf Serum (FBS)	Life Technologies	Cat: 10500064
Bovine Serum Albumin, Fraction V, Heat Shock Treated	Fisherscientific	BP1600-100
0.5 M EDTA	ThermoFisher Scientific	15575-020
phorbol 12-myristate 13-acetate (PMA)	Sigma Aldrich	P8139-1MG