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. The glioblastoma tumor microenvironment plays vital roles in establishing malignancy ^3-6. Tumor associated macrophages/microglia were shown to be responsible for promoting glioblastoma invasion ^7,8. Most of these studies however measured the effect of macrophages/microglia using assays which physically separate them from the glioblastoma cells. Our laboratory has set out to generate improved assays which allow us to study how glioblastoma invasion is dependent on macrophages/microglia in cocultures and enable us to image the physical interaction between them during invasion.

Classic assays to measure cell invasion include the "standard" Boyden chamber chemotaxis and chemoinvasion formats. Here the cells to be studied are plated in a plastic chamber which contains a polycarbonate filter on the bottom that has pores of a specified size (generally between 0.4 and 8 µM in diameter). The process of cell invasion involves a physical barrier, usually composed of extracellular matrix protein. In the chemoinvasion assay, the preferred matrix used is Matrigel (hereafter referred to as "matrix"), an extracellular matrix protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and consists largely of collagen type IV and laminin. The chambers are then placed in a tissue culture well which contains cell culture media with or without growth factors that are suspected to stimulate invasion. Cells which have a higher invasive capacity will invade through the extracellular matrix coated filter at a higher frequency and adhere to the underside of the filter. We have modified this assay in order to assess the role of microglia and tumor associated macrophages on glioblastoma cell invasion.

We have been able to determine using coculture assays described within this paper that microglia can stimulate the invasion of two glioblastoma cell lines by 5 - 10 fold ^9,10. This reflects what is observed in animal models of glioblastoma. Furthermore, we developed a three dimensional invasion assay where the interactions between glioblastoma cells and macrophages/microglia can be examined more directly. The extent of glioblastoma cell invasion stimulated by macrophages/microglia in the 3D assay is comparable to what is seen using the matrix coated chamber approach. Similar assays were previously developed to study breast carcinoma interactions with macrophages during invasion ^11-13. Both methods described in this paper should aid in the ability to dissect the molecular mechanism(s) of macrophage/microglia-stimulated invasion of glioblastoma cells.

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 Institute medium (RPMI) or Macrophage Serum Free Medium (MSFM), vortex well.
3. Incubate cells with dye for 30 min at 37 °C and 5% CO₂.
4. Remove dye containing media and add fresh media (RPMI or MSFM) to the cells.
5. Incubate cells for 30 min. Note: Cells are now stained and ready to be used in experiment.

2. Pre-coated Matrix Chamber Coculture Invasion Assay

1. Differentiate THP-1 cells using phorbol myristate acetate (PMA)¹⁵.
   1. Plate 2 - 3 x 10⁵ THP-1 cells in 1.5 ml of RPMI/10% FBS in a 6 well culture plate. Immediately after plating add 100 nM PMA and incubate at 37 °C, 5% CO₂ for 48 hr.
      NOTE: After 48 hr, THP-1 cells that have successfully undergone differentiation will be adherent and spread to the bottom of the well taking on a round morphology.
   2. Remove PMA by washing once with 1x PBS and replace with fresh RPMI/10% FBS. Wait another 48 hr until cells are ready to use in assay.
2. Equilibrate matrix pre-coated chambers by placing them in a well containing 500 µl of media alone (no serum) and adding 500 µl of media alone to the top. Incubate chambers for 2 hr at 37 °C and 5% CO₂.
   NOTE: Pre-coated chambers were purchased from the manufacturer; see Table of Materials/Equipment.
3. To gently detach cells (fluorescently labeled glioblastoma cell lines and the microglia/macrophages), remove media from cells by aspiration. Wash cells on dish with 2 mM EDTA/PBS. Add 500 µl of 2 mM EDTA/PBS and incubate for 5 - 10 min in a cell incubator at 37 °C and 5% CO₂.
4. Resuspend cells in 5 ml of media containing 0.3% Bovine Serum Albumin (BSA).
5. Centrifuge for 5 min at 120 x g.
6. Aspirate supernatant and resuspend pellet in MSFM/0.3% BSA media (for GL261 cells and microglia) or RPMI/0.3% BSA (for U87 and THP-1 cells) such that the concentration of cells is equal to 1 x 10⁶ cells/ml. Use a hemocytometer to count the cells.
7. Add 500 µl media/0.3% BSA per well of 24 well plate.
8. Remove media from chambers that have been equilibrated for at least 2 hr (step 2.2). Place chambers in well containing 500 µl media/0.3% BSA (step 2.7).
9. If inhibitors are being used to study their effect on macrophage/microglia stimulated glioma invasion, add the appropriate concentration to both the bottom and top portions of the chamber.
   NOTE: The concentrations of CSF-1R inhibitor used to fully inhibit microglia stimulated GL261 glioblastoma invasion can be found in reference ⁹.
10. Depending on the cell lines used, add cells to the top chamber as described in the next two steps: mouse glioblastoma (2.10.1) and human glioblastoma (2.10.2).
    1. Mix 1.5 x 10⁵ GL261 (glioblastoma) cells (150 µl) and 5 x 10⁴ mouse microglia (50 µl) (see step 2.6 for media details). Bring volume to 500 µl by adding 300 µl MSFM/0.3% BSA. Add cell mixture to the top chamber and incubate at 37 °C , 5% CO₂ for 48 hr.
       ​NOTE: Murine microglia are isolated from neonatal mice as described in ¹⁶.

Mix 7.5 x 10⁴ U87 (glioblastoma) cells (75 µl) and 2.5 x 10⁴ THP1 (macrophage) cells (25 µl). Bring volume in 500 µl by adding 400 µl RPMI/0.3% BSA. Add the cell mixture to the top chamber. Incubate cells at 37 °C, 5% CO₂ for 24 hr.

1. After incubation, remove cells from top of chamber by gentle aspiration and fix chambers by placing in 3.7% formaldehyde in PBS. Allow chambers to remain in fixative for 15 min at room temperature (or overnight at 4 °C). Remove fixative by gentle aspiration and replace with 500 µl 1x PBS. Proceed to section 4 for image analysis.
   ​NOTE: Experiment can be paused at this moment and saved for imaging in 1x PBS. Fluorescent dye signals can be detected for up to 2 weeks after fixation.

3. Invasion Assay "3D"-embedding Glioma and Macrophages/Microglia in Matrix

1. Thaw matrix mix at 4 °C overnight. Perform all further manipulations using matrix on ice in the hood.
2. Prepare 10 mg/ml concentration of matrix in media (MSFM or RPMI) with 0.3% BSA on ice.
   NOTE: Stock concentration of matrix is typically around 15 mg/ml.
3. To prepare cells (labelled in step 1) for suspension in matrix, remove media from cells by aspiration. Wash cells on dish with 2 mM EDTA/PBS. Add 500 µl of 2 mM EDTA/PBS to cells and incubate for 5 - 10 min in incubator at 37 °C and 5% CO₂.
4. Resuspend cells in 5 ml of media containing 0.3% BSA.
5. Centrifuge for 5 min at 120 x g.
6. Aspirate supernatant from pellet. Resuspend pellet in media/0.3% BSA such that the concentration of cells is equal to 1 x 10⁶ cells/ml. Use a hemocytometer to count the cells.
7. Add 1.5 x 10⁵ GL261 cells (in 150 µl) + 5 x 10⁴ microglia cells (in 50 µl) into a 1.5 ml microfuge tube. Add 2 x 10⁵ GL261 cells (200 µl) into a separate 1.5 ml microfuge tube.
   NOTE: GL261 cells alone without microglia serves as a control.
8. Spin cells in microfuge for 5 min at 120 x g.
9. Resuspend cells in 200 µl cold matrix on ice.
10. Plate 50 µl (50,000 cells) onto the center of the top compartment of 8 µM pore size chamber insert.
11. Incubate at 37 °C for 30 min for polymerization to occur.
12. Add 200 µl serum-free medium to the upper chamber and 700 µl serum containing cell growth medium to the lower well.
13. Incubate at 37 °C, 5% CO₂ for 48 hr.
14. After incubation, remove cells from top of chamber by gentle aspiration and fix chambers by placing in 3.7% formaldehyde in PBS. Allow chambers to remain in fixative for 15 min at room temperature (or overnight at 4 °C). Remove fixative by gentle aspiration and replace with 500 µl 1x PBS. Proceed to section 4 for image analysis.

4. Imaging Assays

1. Remove chambers from 3.7% formaldehyde and wash in well containing fresh 500 µl 1x PBS.
   NOTE: Do not allow chambers to dry.
2. Using a cotton tipped applicator, gently clean the non-invasive cells from the top portion of the filter (side facing the inside of the chamber) by scraping the filter surface. Start gently and increase pressure gradually. Do this at least 3 times per chamber.
3. Place chambers in either a glass-bottomed dish or leave in a 24 well plate.
4. Place sample on the stage of an epifluorescent or laser confocal fluorescent microscope equipped with camera and image capture software ^9,10.
5. Take several 10X or 20X images of representative fields.
6. Using an image analysis software, such as ImageJ, count the number of glioblastoma cells that have crossed the filter to the underside ^9,10.
7. If imaging the "3D" invasion assay (described in section 3), generate a Z stack series using a fluorescent laser confocal microscope ^5-6. To image the invasive cell population on the underside of the filter, follow the protocol steps 4.2 -  4.6 described above.

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