Background Studies in non-cancer diseases revealed that bilirubin, a metabolite of the heme-degrading enzyme heme oxygenate-1 (HO-1/HMOX1), suppresses normal macrophage function. Although HO-1 expression is often elevated in aggressive cancers, such as triple-negative breast cancer (TNBC), the effects of bilirubin on the tumor microenvironment (TME) remain unknown. We hypothesized that tumor cell-HO-1 activity and subsequent bilirubin secretion enhance TNBC metastasis by supporting immune suppressive, pro-tumor macrophage function.
Methods We tested the impact of bilirubin on human and mouse macrophage immune suppression and efferocytic capacity (engulfment of dead tumor cells) using qRT-PCR, flow cytometry, and live cell imaging (n=6–9). Primary tumor growth, lung metastatic burden, macrophages, and T cells were assessed in syngeneic, immunocompetent mice harboring 66Cl-4 mammary tumors with HO-1 genetic (shHmox1, n=8/group) or pharmacologic inhibition (tin mesoporphyrin or SnMP, 25 mg/kg/daily, n=10/group).
Results Treatment with exogenous bilirubin enhanced macrophage PD-L1 mRNA and protein levels by over 6-fold. In contrast, bilirubin decreased expression of the efferocytosis genes Mertk and Tryo3 by at least 50% and nearly ablated macrophage efferocytic capacity. To assess whether tumor produced bilirubin supports metastatic progression, we evaluated lung metastasis after 66Cl-4-Hmox1 knockdown. Spontaneous lung metastasis from shHmox1 tumors was decreased when compared to shCnt tumors. Global inhibition of HO-1 via SnMP, an FDA approved HO-1 enzymatic inhibitor, decreased tumor cell-bilirubin levels by 82%, lung metastatic burden by 35%, and the number of pro-tumor CD206+ macrophages by 74%. Macrophages isolated from 66Cl-4 mammary tumors treated with SnMP were unable to suppress the expression of cytotoxic molecules granzyme-B and perforin in stimulated CD8 T cells. Interestingly, data mining of KM Plotter showed that high expression of genes required to generate bilirubin predict a poor overall survival of several cancer types treated with Pembrolizumab. To test if HO-1-inhibited tumors are primed for response to T cell directed therapies, 66Cl-4 mammary carcinomas were treated with the combination of SnMP and anti-PD-1. The number of pro-tumor macrophages expressing Arg1 decreased by 35%, while the number of CD8 T cells expressing cytotoxic molecules increased by 63% in 66Cl-4 mammary tumors that received combination treatment compared to anti-PD-1 alone.
Conclusions Tumor cell-HO-1 may support macrophage immune suppression and dysfunction during TNBC progression via bilirubin. Since HO-1 inhibitors including SnMP are FDA approved for treatment of other diseases, these findings could be rapidly translated to provide an alternative immunotherapy approach in aggressive TNBC directed at suppressive, pro-tumor macrophages.
Acknowledgements The authors acknowledge the University of Colorado Cancer Center/NIH/NCI Cancer Core Support Grant P30 CA046934 and use of the Flow Cytometry, Human Immunology and Immunotherapy, Mass Spectrometry, Cell Technologies, Pathology and Genomics shared resources. This work was supported by the following: NIH/NCI-K99/R00 K99CA266748–01A1 (MMW); Cancer League of Colorado AWD#222559 (MMW); NIH/NCI NRSA T32 CA190216–03 (MMW); NIH/NCI NRSA F32 CA239436–01A1 (MMW); DOD BCRP W81XWH-19-BCRP-EA (JKR)
Ethics Approval All animal experiments were performed using humane procedures in accordance with the protocols #00407 and #01056 as approved by the University of Colorado Institutional Animal Care and Use Committee (IACUC).
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