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 6

Heme-TAMs resist tumoricidal transformation by IFN-γ.

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Heme-TAMs resist tumoricidal transformation by IFN-γ. (A) Integrated sph...
(A) Integrated spheroid GFP fluorescence obtained by live-cell microscopy. Data are mean ± 95% CI of 10 replicates from 1 representative experiment. (B) Expression heatmap and clustering of differentially expressed genes (log2[fold change] 0.5, P 0.005, n = 3). Right: Normalized count data for Cxcl9, Cxcl10, and Cxcl11. Each point represents 1 replicate. (C) Spheroids identical to those in A were grown in microwell plates for scRNA-Seq and scanned with a fluorescence microscope on day 9. Violin plots depict GFP fluorescence integrated across the object area for ≥1,400 spheroids per condition (ANOVA with Tukey-Kramer post-test corrected with P 0.001 for each comparison). (D) scRNA-Seq workflow. (E) GSEA-defined functional attributes for the 3 tumor cell clusters. Dot plot visualizes the fraction of tumor cells within each functional state. (F) Tumor cell densities normalized by the mean integrated fluorescence of the input. Bubbles beneath the UMAPs depict the mean spheroid size. (G) Gene expression score intensities for GSEA categories. (H) Approximately 750 spheroids (GFP-MC38 cancer cells + BMDMs) were collected from microwell plates on day 4 after spheroid formation and injected i.v. into Rag2−/−γc−/− mice. Lungs were collected 20 days after injection. Paraffin sections of lung tissue visualize metastatic disease. Scale bar: 5 mm. GFP fluorescence of whole-lung fluorescence images (see Supplemental Figure 4E) was integrated across the imaged lung area and quantified for n = 3–4 animals per condition. Each dot represents 1 mouse (lung). ANOVA with Tukey-Kramer post-test corrected for multiple comparisons, IFN-γ vs. heme + IFN-γ P = 0.0028, heme + IFN-γ vs. control P = 0.0046, heme vs. IFN-γ P = 0.0030, heme vs. control P = 0.0051, control vs. IFN-γ P = 0.98, heme vs. heme + IFN-γ P = 0.97.