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 9

NRF2 signaling in hematopoietic cells promotes resistance to immunotherapy.

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NRF2 signaling in hematopoietic cells promotes resistance to immunothera...
(A) Experimental workflow of Matrigel plug experiments. Macrophages were enriched using F4/80 magnetic beads. (B) Relative mRNA expression of Hmox1, Cxcl9, and Cxcl10 in macrophages. Each dot represents 1 plug (n = 8–11); ANOVA with Tukey-Kramer post-test corrected for multiple comparisons. (C) Identical experiments in conditional Nrf2-WT and -KO mice. (D) Experimental workflow of MC38 tumor cell experiments. (E) Top: Bright-field and GFP fluorescence images visualizing MC38 tumors in situ. Scale bar: 5 mm. Bottom: Tumor sections. Scale bar: 2 mm. (F) GFP fluorescence intensity integrated across the tumor area. Each dot represents 1 tumor grown on the right and left flank of a mouse; color indicates mouse of origin (n = 12); Wilcoxon’s test. (G) Live-cell microscopy of spheroids of GFP-MC38 cells mixed with Nrf2-KO and Nrf2-WT BMDMs that were untreated or pretreated with heme with or without cross-linked anti-CD40 antibody. Data represent GFP fluorescence intensity integrated across object area. Data are mean ± 95% CI of 15 replicates analyzed within 1 representative experiment. (H) Spheroids were collected on day 4 after formation and approximately 750 spheroids were injected i.v. per mouse. Lungs were collected after 23 days. Metastatic foci were manually counted on GFP fluorescence whole-lung images. Each dot represents the number of metastatic lesions in 1 mouse (n = 4–6). Wilcoxon’s test was used to test for the anti-CD40 antibody effect in the 3 experiments. (I) MC38 tumor cells were injected s.c. into conditional Nrf2-WT or -KO mice. On day 7, mice were treated with 2 sequential doses of anti-CD40 antibodies, and tumors were imaged 2 days later. Each dot represents 1 tumor (integrated GFP fluorescence intensity across the tumor area, n = 6); Wilcoxon’s test.