A new study by Folkert et al. (https://doi.org/10.1084/jem.20230420) defines an “iron-rich” subset of tumor-associated macrophages (iTAMs). The metabolism of heme leads to the degradation of the transcriptional repressor Bach1 and shapes the transcriptional profile of iTAMs. The endothelin receptor B in iTAMs signals tumor-supportive functions.

Tumor-associated macrophages (TAMs) promote tumor progression as well as therapy resistance and contribute to the maintenance of an immunosuppressive environment. Intratumoral hemorrhage is common in solid tumors and is associated with invasive tumor growth, abnormal vascular architecture, and necrosis (Hashizume et al., 2000). A key function of macrophages is the phagocytosis of damaged or extravasated red blood cells (RBCs). This process prevents the extracellular release of iron-containing hemoglobin and heme, thereby playing a critical role in iron homeostasis (Humar et al., 2022). In a new study (Folkert et al., 2024), an iron-rich subset of TAMs (iTAMs) is characterized by their uptake and metabolism of heme, and by the expression of endothelin receptor B (Ednrb). How heme uptake and metabolism regulate macrophage functions in cancer was not well characterized until now. This new study characterizes iTAMs, identifies a key role for the transcriptional repressor Bach1 in regulating iTAMs gene expression, and elucidates iTAM functions related to the promotion of tumor angiogenesis and immunosuppression.

Lijuan Sun and Mikala Egelbad.

The phagocytosis of RBC and the resulting uptake of the iron-containing hemoglobin and heme are critical for inducing the gene expression and phenotypes of iTAMs: intratumoral injection of RBCs directly induced the iTAM phenotype in a fibrosarcoma mouse model. Furthermore, iTAMs express high levels of heme-oxygenase 1 (Hmox1), a key enzyme in heme degradation. Other recent studies support these findings: circulating HMOX1+ monocytes and HMOX1hi TAMs are associated with poor prognosis in melanoma patients (Consonni et al., 2021). Additionally, bone marrow–derived macrophages exposed to heme develop into iTAM-like macrophages, named heme-TAM, which express the markers Hmox1hiArg1hiSpp1hi. These heme-TAMs enhance MC38 colon cancer growth and invasiveness as well as confer resistance to immunotherapy in mice (Schaer et al., 2023). The striking similarities in heme metabolism, gene expression, and functions of HMOX1hi TAMs, heme-TAMs, and the newly identified iTAMs support the broad relevance of the effects of hemorrhage via RBC uptake on macrophage function—and on cancer progression.

In hemorrhagic tumors, the ability of Hmox1 to metabolize heme has previously been proposed to contribute to the immunosuppressive properties of macrophages (Alaluf et al., 2020; Consonni et al., 2021). Deletion of the Hmox1 gene in the myeloid cell compartment enhanced the therapeutic effects of an experimental antitumor vaccine (ovalbumin antigen with poly [I:C]) by restoring CD8+ T cell proliferation and cytotoxicity in a thymoma mouse model (Alaluf et al., 2020). Additionally, deletion or enzymatic inhibition of Hmox1 in F4/80hi TAMs promotes a phenotypic switch from tumor-promoting to tumoricidal macrophages and reduces metastasis and angiogenesis in fibrosarcoma and melanoma mouse models (Consonni et al., 2021). These data suggest that Hmox1 could be a therapeutic target in cancer. However, Hmox1’s ability to convert heme into biliverdin, carbon monoxide, and iron is critical for the reutilization of iron in erythropoiesis and in restoring physiological homeostasis (Vijayan et al., 2018). Furthermore, cells lacking Hmox1 activity become exceedingly vulnerable to heme exposure (Vallelian et al., 2015). Thus, identifying a safe pharmaceutical window to target Hmox1 could be difficult.

The new study on iTAMs offers potential strategies to target iTAM functions while also preserving the roles of macrophages and Hmox1 in heme scavenging and iron recycling: Ednrb is shown to play a critical role in mediating tumor-promoting functions of iTAMs. Endothelin signaling has been implicated in various aspects of tumor progression (Bhalla et al., 2009). Among the endothelins, endothelin-1 has been studied the most in clinical context and exerts its effects through the activation of two distinct receptors: endothelin receptor A (Ednra) and Ednrb. While numerous preclinical studies have focused on the role of Ednra in promoting tumor growth in a cancer cell-intrinsic manner (Said et al., 2011; Yin et al., 2003), the role of Ednrb in macrophage function and cancer biology has not been explored as much. The new study by Folkert et al. (2024) finds that Ednrb is a marker of iTAMs in sarcoma mouse models and in synovial sarcoma and melanoma patient samples. However, Ednrb is more than just a marker: deletion of Ednrb in macrophages resulted in reduced tumor growth and angiogenesis, as well as increased surface MHC-II expression on TAMs. Importantly, the deletion did not affect intracellular iron levels in iTAMs. Nevertheless, whether Ednrb targeting also affects the immunosuppressive functions of macrophages, including via effects on T cell functions, is unclear. The fibrosarcoma cells and the conditional Ednrb macrophage knockout mice used in the study have different genetic backgrounds and are immunologically incompatible, and T cells had to be depleted to assess macrophage Ednrb function in the mouse cancer model. An important future step is to develop and use cancer models to determine whether targeting Ednrb could overcome immune suppression in solid tumors. In contrast to the positive effect of deleting Ednrb in the fibrosarcoma mouse model, one earlier study found that decreased Ednrb expression in large primary uveal melanomas is associated with metastasis and shorter survival (Smith et al., 2002). Thus, although the new study offers compelling evidence that Edrnb in iTAMs promotes tumor progression, the mechanisms by which iTAMs exert their tumor-supportive functions need further characterization, and more work is needed to determine if Ednrb functions similarly in human cancer as in the mouse model.

An interesting finding of the new study is the critical role of the transcriptional repressor Bach1 in regulating the iTAM phenotype. When Bach1 was knocked out in mice, the immune cells increased their expressions of Hmox1 and Ednrb. These data are supported by prior studies showing that Bach1 acts as a transcriptional repressor of Hmox1 (Vijayan et al., 2018), and that Hmox1 plays a crucial role in regulating endothelin signaling (Rios-Arrabal et al., 2021; Saurage et al., 2022). In samples from colorectal cancer patients, there is a significant correlation between HMOX1 and endothelin-converting enzyme-1 expression. Furthermore, in colorectal cancer cells, Hmox1 overexpression induces endothelin-converting enzyme-1 expression and endothelin-1 production (Ríos-Arrabal et al., 2021). Together, these data support the hypothesis that Bach1 regulates Ednrb signaling through Hmox1, but this relationship needs further validation. Additionally, the correlation between HMOX1 and endothelin signaling in macrophages across various human cancers still has to be investigated.

Another interesting finding of the study is that iTAMs comprise two distinct subsets: perivascular iTAMs (pviTAMs) and stromal iTAMs (stiTAMs). Both subsets express genes involved in heme metabolism, but they are distinguished by unique markers and transcriptional profiles. The pviTAMs highly express previously published markers of perivascular macrophages, including Lyve-1 and Cd163 (Chakarov et al., 2019; Anstee et al., 2023), and exhibit higher expression of Ednrb. The stiTAMs show elevated levels of Hmox1 and Cd36. Furthermore, Ednrb+ pviTAMs predominantly locate next to blood vessels (murine sarcoma tumors), while Hmox1+ stiTAMs are uniformly distributed throughout the tumor stroma. Based on their location, the authors suggest that pviTAMs are likely the first to encounter and phagocytose extravasated RBCs. However, spatial transcriptomic analyses of human melanomas showed that EDNRB+ pviTAMs are not close to hemorrhagic areas within the tumor. The macrophages surrounding hemorrhagic areas (not EDNRB+ pviTAMs) express CD163, which is identified as a marker of mouse Ednrb+ pviTAMs. The new study offers important insights into iTAMs, but translating these findings from preclinical models to human cancers will require determining whether there are species differences in iTAM gene expression, functions, and behaviors.

The identification of two iTAMs subsets, pviTAMs and stiTAMs, highlights how regional tumor variations, such as hemorrhage, alter gene expression and cellular phenotypes. Important future directions include (1) exploring the molecular pathways and transcriptional networks responsible for the development and function of pviTAMs and stiTAMs, and (2) elucidating their interactions with other cell types, such as endothelial cells and T cells. Such work could yield new strategies to uniquely targeting each subset. Future research should also determine whether the presence of iTAMs or specific iTAM subsets correlates with poor prognosis and whether evaluating the presence of iTAMs provides advantages over evaluating the presence of more general TAMs.

In conclusion, the study sheds light on a newly characterized subset of TAMs, the iTAMs, which are distinguished by Ednrb expression and negatively regulated by the transcriptional repressor Bach1. The widespread presence of iTAMs across various tumor types and their clear effect on tumor growth in mouse models support that they play a pivotal role in cancer biology. By focusing on the overlooked effects of heme uptake on macrophage gene expression and phenotype, the research also highlights how regional variations in the composition of the tumor microenvironment—e.g., the presence of heme from leaked RBCs—shape other parts of the tumor microenvironment, e.g., macrophages. Both the specific insights on the effects of heme metabolism within macrophages and the broader concept of the microenvironment evolving in a regional manner provide new understanding of tumor progression and may open novel avenues for therapeutic strategies in cancer treatment.

In the hemorrhagic areas of tumors, iTAMs phagocytose RBCs. The heme from these RBCs binds to the transcriptional repressor Bach1, leading to its degradation and allowing the expression of Hmox1. Hmox1 metabolizes heme into iron and other products. Additionally, Ednrb expression is increased following Bach1 knockout. Ednrb signaling enhances the tumor-supportive functions of iTAMs. RBCs: red blood cells. Hmox1: heme-oxygenase 1. Edn: endothelin. Ednrb: endothelin receptor B. Created with https://BioRender.com by Celia Hallinan.

We thank Celia Hallinan for help with the design of the figure.

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