Hemorrhage-activated NRF2 in tumor-associated macrophages drives cancer growth, invasion, and immunotherapy resistance
Dominik J. Schaer, … , Elena Dürst, Florence Vallelian
Dominik J. Schaer, … , Elena Dürst, Florence Vallelian
Published December 7, 2023
Citation Information: J Clin Invest. 2024;134(3):e174528. https://doi.org/10.1172/JCI174528.
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Hemorrhage-activated NRF2 in tumor-associated macrophages drives cancer growth, invasion, and immunotherapy resistance

Abstract

Microscopic hemorrhage is a common aspect of cancers, yet its potential role as an independent factor influencing both cancer progression and therapeutic response is largely ignored. Recognizing the essential function of macrophages in red blood cell disposal, we explored a pathway that connects intratumoral hemorrhage with the formation of cancer-promoting tumor-associated macrophages (TAMs). Using spatial transcriptomics, we found that NRF2-activated myeloid cells possessing characteristics of procancerous TAMs tend to cluster in perinecrotic hemorrhagic tumor regions. These cells resembled antiinflammatory erythrophagocytic macrophages. We identified heme, a red blood cell metabolite, as a pivotal microenvironmental factor steering macrophages toward protumorigenic activities. Single-cell RNA-Seq and functional assays of TAMs in 3D cell culture spheroids revealed how elevated intracellular heme signals via the transcription factor NRF2 to induce cancer-promoting TAMs. These TAMs stabilized epithelial-mesenchymal transition, enhancing cancer invasiveness and metastatic potential. Additionally, NRF2-activated macrophages exhibited resistance to reprogramming by IFN-γ and anti-CD40 antibodies, reducing their tumoricidal capacity. Furthermore, MC38 colon adenocarcinoma–bearing mice with NRF2 constitutively activated in leukocytes were resistant to anti-CD40 immunotherapy. Overall, our findings emphasize hemorrhage-activated NRF2 in TAMs as a driver of cancer progression, suggesting that targeting this pathway could offer new strategies to enhance cancer immunity and overcome therapy resistance.

Authors

Dominik J. Schaer, Nadja Schulthess-Lutz, Livio Baselgia, Kerstin Hansen, Raphael M. Buzzi, Rok Humar, Elena Dürst, Florence Vallelian

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Figure 2

Erythrophagocytic transformation of macrophages in RBC-heme Matrigel plugs.

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Erythrophagocytic transformation of macrophages in RBC-heme Matrigel plu...
(A) Subcutaneously placed FGF-enriched Matrigel plugs were used to study the effect of hemorrhage on the phenotype of invading macrophages. Plugs enriched with intact RBCs (RBC-heme) or RBC-ghosts were used to study the specific effect of hemoglobin-heme. (B) FGF-enriched Matrigel plugs were collected from the subcutaneous (s.c.) injection site 7 days after injection and classified by blinded investigators as non-hemorrhagic or hemorrhagic based on their macroscopic appearance. (C) Flow cytometry contour plots of plug-invading cells, defining F4/80+CD11b+ macrophages with lower MHC class II expression in hemorrhagic plugs. The box plots depict the mean fluorescence intensities of MHC class II expression within the CD11b+F4/80+ population (n = 6–8 plugs per condition; each dot represents 1 plug; t test). (D) RBC-heme and RBC-ghost plugs were collected 7 days after s.c. injection. (E) Flow cytometry contour plots of plug-invading cells defining F4/80+CD11b+ macrophages with lower MHC class II expression in RBC-heme than RBC-ghost plugs. The box plots depict the mean fluorescence intensities of MHC class II expression within the CD11b+F4/80+ population (n = 9 plugs per condition; each dot represents 1 plug; t test). (F) Fluorescence immunohistochemistry images of Matrigel plug sections stained with TER119 (red, RBC-heme or RBC-ghost), anti-F4/80 (yellow, macrophages), and anti-HMOX1 antibodies (cyan). Nuclei were stained with DAPI (white). Images were acquired using a PhenoImager HT (Akoya). Corresponding consecutive sections stained for iron show iron accumulation in infiltrating cells. Scale bars: 500 μm. The inset shows an iron-positive erythrophagocyte containing multiple RBC remnants.