In Vitro Assay to Study Tumor-macrophage Interaction

Summary

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Abstract

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

Introduction

Macrophages are immune cells with high phenotypic and functional heterogeneity¹. They play important roles in the host defense systems, tissue repair, development and tumor progression¹. TAMs are macrophages in the microenvironment of solid tumors. Certain TAMs can promote tumor growth through inhibiting T cell-mediated cytotoxic activity, modulating the tumor microenvironment (TME), promoting angiogenesis, invasion and metastasis^2,3,4,5. TAMs are among the most abundant cell types in the TME a....

Protocol

1. Medium Preparation

1. Preparation of the serum-free stem cell medium
   1. Thaw the 50x B27 supplement, the epidermal growth factor (EGF, 20 μg/mL in 10 mM acetic acid with 0.1% BSA), and the fibroblast growth factor (FGF, 20 μg/mL in 10 mM acetic acid with 0.1% BSA).
   2. Add 500 μL of EGF (final concentration 20 ng/mL), 500 μL of FGF (final concentration 20 ng/mL), 10 mL of 50x B27 to 500 mL of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) and 5 mL of 100x Penicillin/Streptomycin (Pen/Strep). Invert the bottle 4-6 times to mix, filter sterilize through a 500 mL 0.1 μm filtratio....

Results

The results are usually showed via bar graphs (example shown in Figure 1). Samples with high 480/520 values indicate that the conditioned media has high capacity to recruit macrophages. Depending on experimental need, additional controls can be included. For example, one can use neutralizing antibodies to treat the conditioned media to abolish the macrophage chemotaxis, and perform the same assay. One can also add extra chemokines (i.e., CCL2) to the conditio.......

Discussion

In this protocol, there are several key steps: 1) selection of the Transwell insert. For the MV-4-11 cell line, 5 μm Transwell inserts work well. However, for other cell lines such as the commonly used monocyte cell line THP-1, a different pore size might work better. 2) As different cell lines grow at different speeds, it is important to adjust the volume of the conditioned media according to cell numbers. For this purpose, cell-free media incubated under the same experimental conditions can be used to dilute the c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1 μm filtration cup	Thermo fisher	566-0010
0.45 μm filter unit	Millipore	SLHA033SS
10 mL serological pipettes	Olympus plastics	12-104
15 mL sterile centrifuge tubes	Olympus plastics	28-103
1 mL pipette tip	ART molecular bioproducts	2779-RI
2 mL aspirating pipet	Falcon	357558
24-well plate	Millipore	ECM507	Part of ECM507, or can be purchased separately
4x lysis buffer	Millipore	ECM507	Part of ECM507, or can be purchased separately
5 μm Transwell insert	Millipore	ECM507	Part of ECM507, or can be purchased separately
75 cm2 flask	Corning	430641U
Accutase	Innovative cell technologies	AT-104
B27	Gibco	12587-010
CyQuant Dye	Millipore	ECM507	Part of ECM507, or can be purchased separately
DMEM	Gibco	11965-092
DMEM:F12	Gibco	10565-018
EGF	Peprotech	AF-100-15
FBS	Gibco	26140
FGF	Peprotech	100-18B
IMDM	Gibco	12440-053
PBS	Gibco	14190-144
Pen Strep	Gibco	15140-122
Trypan blue	Biorad	1450021