Metastatic triple-negative breast cancer secretes heme metabolites to alter macrophage immune suppression and function

A promising approach to improve immunotherapy options for metastatic breast cancer is therapeutic inhibition of metabolic pathways that regulate both pro-tumorigenic and immune suppressive signals. The heme breakdown, or catabolism, pathway is often elevated in aggressive cancers; however, the effects of this pathway on the breast tumor microenvironment (TME) remains understudied. We revealed that the heme catabolizing enzyme heme oygenase-1 (HO-1/HMOX1) is elevated in aggressive triple-negative breast cancer (TNBC) specimens where it positively correlates with immune suppressive macrophages. In mouse TNBC-like mammary tumors, pharmacologic inhibition of HO-1 did not impact primary tumor growth, but knockdown of Hmox1 in tumor cells significantly decreased primary tumor growth and spontaneous lung metastasis. Analysis of tumor interstitial fluid and matching plasma from these animals revealed that levels of the heme metabolite bilirubin were significantly higher intratumorally, but they were depleted with tumor cell Hmox1 knockdown. To test if bilirubin impacts immune cells, mouse bone-marrow derived macrophages (BMDM) were treated with exogenous bilirubin that decreased macrophage efferocytic capacity, or dead cell engulfment, in a dose dependent manner. Conversely, bilirubin treatment significantly increased the ability of BMDM to suppress activation of CD8+ splenocytes. Macrophages isolated from 66Cl-4 mammary tumors treated with SnMP to deplete intratumoral bilirubin were unable to suppress production of cytotoxic molecules Granzyme-B and Perforin in stimulated CD8+ splenocytes. 66Cl-4 mammary tumors harboring HO-1 knockdown had a significant decrease in growth rate and number of pro-metastatic CD206+ macrophages after treatment with αPD-1. Together these results suggest that tumor HO-1 supports macrophage dysfunction and immune suppression during TNBC progression via bilirubin. Further, depletion of intratumoral bilirubin levels has the potential to dynamically impact macrophage populations, particularly in combination with immunotherapy, demonstrating an underappreciated role for this metabolic pathway and metabolite as a pro-metastatic, immunomodulators in cancer.