Immunohistochemistry Staining for Tumor-associated Macrophage Polarization in Murine Subcutaneous Colon Tumor Allografts

Background

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME) in most cancers (Noy and Pollard, 2014), and also have the most substantial and pervasive effect of any immune cell in the TME (Chen et al., 2005; Allavena et al., 2008; Erreni et al., 2011; Marech et al., 2016). TAMs have been shown to have both anti-tumor and pro-tumor functions, depending on whether they are polarized more towards an M1 (classical) or M2 (alternative) phenotype (Chen et al., 2005), respectively, and their physical location within a tumor (Marech et al., 2016). In reality, M1 and M2 TAMs are the extremes of a continuum of intermediate cells which may have both anti-tumor and pro-tumor functions. Broad anti-tumor functions of classically activated M1-polarized TAMs include inflammation and immune response (Sugita et al., 2002; Funada et al., 2003; Zhou et al., 2010). On the other hand, pro-tumor functions of alternatively activated M2-polarized TAMs include tumor growth, angiogenesis, immunosuppression, and matrix remodeling (Liu and Cao, 2015). In most cancers, TAMs are skewed more towards the pro-tumor M2-phenotype (Yang and Zhang, 2017); therefore, TAMs have gained clinical momentum as immunotherapy targets for cancer. Currently, there are over 30 clinical trials targeting TAMs; strategies include reducing monocyte recruitment to the tumor and reprogramming M2-TAMs to M1-TAMs. Yang and Zhang provide a comprehensive review of TAM immunotherapy strategies (Yang and Zhang, 2017). Still many other groups are actively investigating TAMs in the basic science and pre-clinical realms (Cassetta et al., 2016; He et al., 2017; Jarosz-Biej et al., 2018; Suarez-Lopez et al., 2018). However, TAMs in some cancers, like colorectal cancer (CRC), simultaneously have both detrimental and beneficial effects on the patient (Zhong et al., 2018).

The dual-role of TAMs in CRC has yet to be fully understood. It has recently been shown that macrophages induce resistance to fluorouracil-based chemotherapy, and that this TAM-induced resistance may contribute to the poor chemotherapy response in some CRC patients (Zhang et al., 2008; Zhang et al., 2016). Currently, a gap in colon cancer research is how therapy affects TAMs in the colorectal TME, and, in turn, how altering TAM population and polarization affects tumor therapeutic response. Therefore, this immunohistochemistry staining protocol was developed to stain for TAMs in murine subcutaneous colon tumor allografts, although this protocol can be used for other murine tissue. In this article, Balb/c mice were subcutaneously injected in the left flank with CT26 murine colon carcinoma cells. Tumors grew until they reached 75 mm³. Following three days of additional tumor growth, untreated tumors were dissected, flash-frozen in isopentane and liquid nitrogen, and stored at -80 °C until cryosectioning, and then sectioned at a thickness of 5 μm. The scope of this article is on macrophage immunohistochemistry of murine tumors, not on the tumor model itself. Therefore, this protocol can be readily modified to stain any murine tissue in allograft, xenograft, or orthotopic tumor models.

This direct immunohistochemistry staining method uses three primary-conjugated macrophage antibodies: anti-CD68, anti-CD80, and anti-CD206. CD68 is a pan-macrophage surface marker (Gordon et al., 2014), CD80 is a cell surface marker for M1-type macrophages (Zhou et al., 2017), and CD206 is a cell surface marker for M2-type macrophages (Kigerl et al., 2009). CD68, CD80, and CD206 are markers for other cell types as well but were chosen such that (CD68⁺/CD80⁺)-cells were considered M1-TAMs, (CD68⁺/CD206⁺)-cells were considered M2-TAMs, and (CD80⁺/CD206⁺)-cells were considered dendritic cells (DCs). Cells expressing all macrophage surface markers were considered to have a mixed M1-M2 phenotype (Figure 1).

With this simple and reproducible method, we are able to accurately stain for M1 and M2 macrophages. To ensure long-term relevancy and usability of this protocol, this work also provides an easy-to-follow mathematical analysis of antibody concentrations so that readers can easily modify and optimize this protocol for their specific test system. This work is relevant for investigators developing and/or testing TAM-targeting cancer immunotherapies in mice.


Figure 1. A double stain technique was used to distinguish M1- and M2- polarized macrophages. CD68⁺/CD80⁺ cells were considered M1 TAMs and CD68⁺/CD206⁺ cells were considered M2 TAMs. Additionally, CD68⁺/CD80⁺/CD206⁺ TAMs were considered to have a mixed phenotype.