Heme-signaling progresses via NRF2 to educate heme-TAM transformation, tumor cell growth, and invasiveness

Heme-activation of NRF2 is a strong anti-inflammatory signal in macrophages, and analyses in this study indicated that the expressed transcriptome in heme-TAMs was consistently enriched for NRF2 target genes. We have therefore delineated the role of NRF2 in a series of experiments with Nrf2 knockout BMDMs, leading to a locked NRF2-inactive state, and macrophages with a knockout of the cytoplasmic NRF2 capture protein KEAP1, leading to a locked NRF2-active state, irrespective of the presence or absence of heme. To demonstrate that activated NRF2 is sufficient to drive heme-TAM transformation, we analyzed Keap1 knockout macrophages. BMDMs from one Keap1 KO mouse (control) and one WT littermate mouse (control and heme-treated) were resuspended and transferred into 15 ml falcon tubes. 1 x 106 cells per condition were taken for cell staining using lipid tags following 10X Genomics protocol (CG00391; Demonstrated Protocol Cell Multiplexing Oligo Labeling for Samples with >80% Viable Cells; Rev B). Cell Multiplexing Oligos (CMO) labels B308 - B311 (3’ CellPlex Kit Set A) were used to label the 4 samples. After labeling, cell suspensions were counted again and the samples were pooled according to the pooling calculations in appendix of the labeling protocol (CG00391) in equivalent ratios.