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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Research Article Inflammation Oncology

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 7

Heme-TAM transformation progresses via NRF2 signaling.

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Heme-TAM transformation progresses via NRF2 signaling. (A) Keap1- and Nr...
(A) Keap1- and Nrf2-KO mice generated BMDMs with locked NRF2-on and NRF2-off states, independent of heme exposure. (B) Factorial experiment defining the effect of heme treatment and MC38 cell culture supernatant on the expression of Arg1 mRNA measured by RT-qPCR in Nrf2-WT and Nrf2-KO BMDMs. Color and size of the dots indicate the normalized gene expression per sample (n = 4 per condition). The data demonstrate that NRF2 is required to leverage the synergistic effect of heme and tumor cell supernatant. (C) Live-cell microscopy analysis of spheroids of GFP-MC38 cancer cells mixed with Nrf2-KO and Nrf2-WT BMDMs that were untreated or pretreated with heme. Data represent the GFP fluorescence intensity integrated across the object area. Data are mean ± 95% CI of 15 replicates analyzed within 1 representative experiment. (D) Representative fluorescence images of spheroids. Scale bar: 0.5 mm. (E) Multiplexed scRNA-Seq experiment of untreated 2D cultured WT BMDMs, heme-treated WT BMDMs, and untreated Keap1-KO BMDMs. The UMAP visualizes that the interaction of genotype and treatment defines distinct gene expression patterns. The violin plots visualize the expression of canonical myeloid marker genes, NRF2-regulated genes, and heme metabolism genes as log10 (normalized count + 1) values. (F) PCA of the transcriptome data described in E. The genes defining PC1 and PC2 were analyzed for driver transcription factors by EnrichR using the TRRUST Transcription Factors 2019 data set. This analysis indicates that heme-treated BMDMs and Keap1-KO macrophages share activated NRF2 as a driver of their phenotype.