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 3

RBC-heme enforces the transformation of macrophages into heme-TAMs.

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RBC-heme enforces the transformation of macrophages into heme-TAMs. (A) ...
(A) RBC-heme– and RBC-ghost–enriched Matrigel plugs were collected 7 days after s.c. placement and processed for scRNA-Seq analysis. (B) Plug-invading cells were enriched by anti-F4/80 and anti-CD11b magnetic beads from an RBC-heme and an RBC-ghost Matrigel plug before scRNA-Seq analysis. UMAPs are color-coded for condition, Leiden cluster, and the attributed functional class. (C) Proportion of macrophages per functional class stratified for RBC-heme and RBC-ghost. The dot size corresponds to the proportion of cells (%) per functional class. This analysis visualizes that erythrophagocytosis fosters the emergence of oxidative stress–handling macrophages while suppressing cells equipped for antigen presentation. (D) Expression heatmaps of selected signature genes. The dashed line highlights the region of the UMAP containing the oxidative stress cluster. These macrophages have high expression of Arg1, Spp1, and heme-, iron-, and oxidative stress–handling genes, while expression of MHC class II–related genes is low. (E) Expression heatmap and unsupervised hierarchical clustering analysis of heme-TAM marker genes measured by quantitative reverse transcriptase PCR (RT-qPCR) in plug-invading cells after enrichment of F4/80+ cells. Each column represents data from 1 plug collected 7 days after s.c. injection (n = 8 RBC-heme plugs, n = 7 RBC-ghost plugs). (F) A score for the oxidative stress–related macrophages in the RBC plug was calculated based on differentially expressed genes (log2[fold change] 2, P 0.001, n = 114 genes). This score was mapped into the spatial transcriptome data of the MC38 tumor sections reported in Figure 1. The heatmap visualizes that macrophages with an oxidative stress–handling identity accumulate in the perinecrotic tumor microenvironment.