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Markers of Tumor-Associated Macrophages

Garrett Heinrich
Tags: Immunity, Cancer, Successful Research Tips

What are Tumor-Associated Macrophages?

Using the body’s immune system to fight various types of cancer has seen rapid advancements in recent years. The tumor microenvironment (TME), the immediate niche surrounding a tumor, is composed of several cell types. Blood vessels supply oxygen and nutrients and are responsible for removing waste products; in contrast, stromal and immune cells modulate tumor growth through the secretion of signaling molecules and extracellular matrix (ECM) components.

Within the TME, tumor-associated macrophages (TAMs) account for a large percentage of the cell population. These TAMs are not all alike; some can be pro-inflammatory and some are anti-inflammatory. The pro-inflammatory TAMs are considered to have an M1-like polarization while the anti-inflammatory TAMs have an M2-like polarization, while some cells within the TME co-express markers of both. This polarization determines whether the immune cells are trying to fight the tumor cells (M1) or help the tumor cells by supporting tissue repair and remodeling (M2). The polarization status of the TAMs is not static and the polarization of the TAMs contributes to the metabolism and development of the tumor cells. So, TAMs have become an interesting target of immunotherapy to help fight cancer.

Myeloid cells are the progenitors of bone-derived macrophages, while some tissues express resident macrophages such as microglia in the brain, Kupffer cells in the liver, and alveolar macrophages in the lungs. Myeloid cells circulating in the peripheral blood are attracted to tumors by secreted chemokines such as C-C motif chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), and vascular endothelial growth factor A (VEGFA). These can be quantitatively measured by Enzo’s MCP-1 (rat) ELISA and VEGF (human) ELISA kits on the compact Absorbance 96 plate reader. Elevated CCL2 has a high association with a number of TAMs in a tumor and overall negative outcomes. These chemokines promote immunosuppression and vascularization that allow for tumor growth.

What affects M1, M2 polarization?

What determines whether a macrophage is M1- or M2-polarized? Signaling through receptor kinases of the TAM receptor family is thought to play a part. Signaling through these TAM receptors, which include Tyro3, Axl, and MerTK, induces M2-like polarization and the secretion of immunosuppressive cytokines. Axltide is a protein kinase substrate of Axl derived from mouse insulin receptor 1 and can be used to investigate modulators of signaling through these TAM receptor pathways. The phosphoinositide 3 kinase (PI3K)/Akt is one of the pathways affected by TAM receptor signaling and plays a major part in many other metabolic processes. Activation of PI3K-gamma by Ras stimulates a pathway through the mechanistic target of rapamycin complex 1 (mTORC1), ribosomal protein S6 kinase (S6K), and the transcription factor CCAAT enhancer binding protein beta (C/EBPb) that leads to up-regulation of TAM-associated genes arginase 1 (ARG1), transforming growth factor beta (TGF-Β), and interleukin-10 (IL-10) (Figure 1).
PI3K signaling

Figure 1. PI3K signaling promotes tumor growth.


Amino acids such as leucine and arginine can also activate mTORC1 and stimulate growth. In addition, PI3K-gamma activation decreases proinflammatory factors such as interleukin-12 (IL-12), interferon gamma (IFN-γ), and nitrogen oxide synthase 2 (iNOS). Therefore, inhibition of PI3K-gamma in myeloid cells (e.g. with Wortmannin) has been studied as a mechanism to repolarize macrophages by reducing immunosuppressive cytokines and increasing proinflammatory cytokines, thus reducing tumor growth. Enzo has a number of tools that can be used to investigate this PI3K pathway (Table 1).

Cytokines Secreted by M2 Macrophages
Target Product Description
PI3K Inhibitor Wortmannin
mTORC1 Inhibitor Ku-0063794
S6K Assay P70 S6K activity kit
Arg1 Antibody Arginase I (human), recombinant
IL-10 ELISA IL-10 (human) ELISA
TGF-Β ELISA TGF-Β1 ELISA
IL-12 ELISA IL-12p70 (human) ELISA
IFN-γ ELISA IFN-gamma (human) ELISA
iNOS ELISA iNOS (human), recombinant, His-tag

Table 1. Tools to investigate the PI3K-gamma pathway

Increased activation of signal transducer and activator of transcription 6 (STAT6) also induces M2-type macrophage polarization. Activation of the interleukin-4 receptor (IL-4R) by interleukin-4 (IL-4) leads to tyrosine phosphorylation of STAT6 and the beginning of transcription of downstream targets. Circulating IL-4 in mice can be measured with the IL-4 (mouse) ELISA kit. This colorimetric assay can detect as little as 4.34 pg/mL IL-4 in serum, plasma, or culture supernatants. STAT6 can also be induced by fatty acid oxidation (FAO), a result of the limited nutritional resources in the TME.

How to identify an M2 macrophage?

When investigating the macrophage phenotypes in the TME, how do you identify if a macrophage is M1 or M2? M2 macrophages secrete cytokines such as IL-6, IL-8, IL-10, and TGF-Β that promote immunosuppression and tumor growth. Enzo has a number of ELISAs to measure these cytokines (Table 2), and our colorimetric assays can be performed on the Absorbance 96 plate reader. M2 macrophages also secrete prostaglandin E2 (PGE2) and matrix metalloproteinase-7 (MMP-7) that can prohibit T cells from recognizing the antigens found on tumor cells and further contribute to immunosuppression (Table 3).

Cytokines Secreted by M2 Macrophages
Target Enzo Kit Sensitivity (pg/mL)
IL-6 IL-6 (mouse) ELISA 1.01
IL-6 IL-6 (human), high sensitivity ELISA 0.057
IL-8 IL-8 (human) ELISA 0.72
IL-10 IL-10 (human) ELISA 3.75
TGF-Β TGF-Β1 ELISA 3.3

Table 2. Enzo ELISAs for detecting M2 macrophage-related cytokines and chemokines.


PGE2 and MMP7 Assays Available
Target Enzo Kit Sensitivity
PGE2 PGE2 ELISA 13.4 pg/mL
PGE2 PGE2 High Sensitivity ELISA 8.26 pg/mL
MMP7 MMP7 colorimetric drug discovery kit
MMP7 MMP7 fluorometric drug discovery kit
MMP7 MMP7 fluorometric drug discovery kit, RED

Table 3. Assays available for prostaglandin and matrix metalloproteinase markers of M2 macrophages.

By identifying the mechanisms of polarization and markers of these growth-related TAMs, researchers are working to target these cells with anti-cancer treatments.

How can Enzo help guide your cancer research?

Do you have more questions on markers of tumor associated macrophages and how to find the best assays and molecules for your research? Reach out to our Technical Support Team. We will be happy to assist!

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