Background

Evasion of anti-tumor immunity is now a well-established hallmark of cancer progression (Hanahan and Weinberg, 2011). Translation of fundamental concepts in tumor immunity has resulted in the development of immune checkpoint inhibitors that are highly efficacious in select oncological types (Xin et al., 2019; Robert, 2020). However, several types of malignancies do not respond, have intrinsic resistance, or develop secondary adaptive resistance to these therapies (Sharma et al., 2017; Jenkins et al., 2018; Schoenfeld and Hellmann, 2020). These issues have led to an outpouring of basic science and translational projects aimed at identifying additional molecular mechanisms by which cancer cells or infiltrating immune cells evade anti-tumor immunity, with the goal of developing novel immunotherapeutics (Fares et al., 2019; Karasarides et al., 2022). Here, we detail a published tumor/immune cell–admixture model (Liu et al., 2020; Noe et al., 2021), using tumor-associated macrophages as an example, that can serve as a useful tool to investigate the relative contributions of tumor-infiltrating immune cells in promoting cancer progression.

Tumor-associated macrophages (TAMs) are myeloid-derived immune cells that infiltrate into the tumor microenvironment to affect tumorigenesis (Mantovani et al., 1992; Noy and Pollard, 2014; Pan et al., 2020; Pittet et al., 2022). TAMs have both pro- and anti-tumor effector functions—their phagocytosis of cancer cells (Kamber et al., 2021), presentation of tumor-associated antigens (Asano et al., 2011), and activation of anti-tumor adaptive immunity play a critical role in the elimination of cancer cells (Modak et al., 2022). In contrast, TAMs can also promote tumorigenesis by suppressing phagocytic and antigenic innate immune responses (Barkal et al., 2019; Demaria et al., 2019; Zhou et al., 2020), as well as by inducing adaptive immune cell exhaustion (DeNardo and Ruffell, 2019; Singhal et al., 2019). Understanding the TAM-dependent contribution to tumor progression or regression is critical for identifying novel tumor immunotherapeutic strategies.

Several different tumor models have been developed to interrogate the effects of specific immune cell types in tumorigenesis. These protocols traditionally include the targeted depletion (Xiao and Jiang, 2014) or transgenic manipulation (McCubbrey et al., 2017) of the desired immune cell in the context of orthotopic or heterotopic tumors in xenograft or allograft models. The tumor-admixture model described herein is a heterotopic, allograft murine model using ex vivo differentiated/polarized bone marrow–derived macrophages (BMDMs) injected with tumor cell lines into congenic recipient mice. This admixture model has been shown to be an advantageous strategy to relatively easily determine gene-specific contributions to TAM-dependent tumor progression (Liu et al., 2020; Noe et al., 2021).

While this protocol was developed to investigate the ability of ex vivo interleukin-4 (IL-4) polarized pro-tumor TAMs to suppress anti-tumor immunity leading to enhanced tumor growth (Liu et al., 2020; Noe et al., 2021), this model can be used with other TAM phenotypes, different immune cell types, and additional experimental manipulations. A potential critique of this model includes the relative contribution of host-derived, tumor-infiltrating TAMs, which can be addressed by incorporating a targeted depletion strategy (Xiao, and Jiang, 2014). Adapting this model using anti-tumor TAMs (Liu et al., 2020; Noe et al., 2021) offers an interesting approach to understanding the mechanisms by which TAMs promote tumor regression. With rigorous optimization, other immune cell types that are critical regulators of tumorigenesis, such as neutrophils (Shaul, and Fridlender, 2019), dendritic cells (Wculek et al., 2020), or myeloid-derived suppressor cells (Veglia et al., 2021), could be adapted for use. Lastly, this model is highly amendable to the post-injection administration of small molecules or biologic inhibitors to further validate their importance as a clinically viable therapeutic (Zanoni et al., 2012; Noe and Mitchell, 2020). Altogether, this tumor-admixture model is a relatively straightforward, highly adaptable protocol to investigate immune cell–dependent cancer progression.