This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.
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.
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 metastasis2,3,4,5. TAMs are among the most abundant cell types in the TME and higher number of TAMs generally correlates with worse patient survival in many types of solid tumors6. The distinct genetic signatures of the tumor cells affect their ability to recruit macrophages. In GBM, an aggressive brain tumor with no cure, macrophages can represent up to a half of the tumor mass7. Co-amplification of the epidermal growth factor receptor (EGFR) and its truncation mutant EGFRvIII is frequently observed in GBM, which confers tumor growth advantages8. Cells co-expressing EGFR and EGFRvIII attract more macrophages compared to cells expressing EGFR or EGFRvIII singly7.
Chemokines are a family of small cytokines that play significant roles in regulating immune composition in the TME6,9. Human cells express more than 50 cytokines10. Immune infiltration in the tumors is largely realized by the interaction between cytokines and cytokine receptors11. Each type of immune cells expresses distinct chemokine receptors/chemokines and can be recruited by cells secreting specific chemokines/chemokine receptors12. Cancer cells can increase expression of certain chemokines to recruit immune cells such as TAMs, regulatory T cells and myeloid-derived suppressor cells (MDSCs)6. Blockade of specific chemokine secreted by the tumors can be a promising way in inhibiting infiltration of immune cells into the tumor mass.
Here, we describe a protocol that allows in vitro evaluation of tumor-macrophage interaction, using conditioned media from the tumor cells containing chemokines and macrophage cell lines.
1. Medium Preparation
2. Cell Preparation
Day 1:
Day 2:
Day 3:
3. In Vitro Macrophage Attraction Assay
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 conditioned media, which serves as a positive control.
Figure 1: Conditioned media from U87 cells co-expressing EGFR and EGFRvIII attract more macrophages than U87 cells expressing a control vector, or EGFR/EGFRvIII singly. This figure has been modified from An, et al.7. Please click here to view a larger version of this figure.
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 conditioned media. 3) FBS contains cytokines. It is important to use serum-free medium to seed MV-4-11 cells in the upper chamber. If FBS is used here, sometimes cell migration cannot be observed.
Researchers can modify this assay for different applications. For example, other tumor cells, either primary patient-derived xenograft culture, or mouse cells can substitute the brain tumor cell line used in the above-mentioned example. The macrophage cell line can be substituted by primary macrophages or monocytes from patients or mice depending on the specific need. One important point is that the researchers need to select cells from the same species to ensure best result. Although the macrophage chemotaxis pathway is highly conserved, the variation of protein sequence of structure between different species may add layers of complication to interpret experimental results.
TAMs are attracting increasingly more attention in cancer immunology13,14. How different genetic changes in the tumors affect recruitment of TAMs and other immune cells in the tumor microenvironment still awaits further studies. By changing pore sizes of the Transwell inserts, this assay can be used to study interaction between tumor and other immune cells such as T cells and NK cells. One advantage of using the Transwell assay to evaluate tumor-immune interaction is that it is easy to demonstrate how specific oncogenes/mutations affect recruitment of a certain type of immune cells. Moreover, this assay is easy to scale-up for genome-wide screens. This assay opens up additional possibilities for researchers to study tumor-immune cell interactions. Additionally, this assay can be used to study how the conditioned media from tumor cells affect the transcription profile of the macrophages/other immune cells.
All assays have their limitations. For this assay, the conditioned medium is from tumor cells cultured in vitro. The secreted chemokines here might be different from those secreted by tumors grown in vivo. Additionally, the macrophage cell line is different from tumor infiltrating macrophages, which are more phenotypically and transcriptionally diverse. Therefore, further confirmation of the in vitro findings using other in vitro and in vivo methods is necessary.
The authors have nothing to disclose.
Grant Support: Z. An received support from Alex’s Lemonade Stand Foundation, American Brain Tumor Association, NIH T32CA108462 and Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation. W. Weiss was supported by NIH grants R01CA221969, R01NS091620, P50CA097257, U01CA217864, P30CA82103; the Samuel G. Waxman Cancer Research Foundation; and the Evelyn and Mattie Anderson Chair.
0.1 μm filtration cup | Thermo fisher | 566-0010 | |
0.45 μm filter unit | Millipore | SLHA033SS | |
10 mL serological pipettes | Olympus plastics | 12-104 | |
15mL 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 |
75cm2 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 |