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

血红素（heme）对NRF2（核因子E2相关因子2，Nuclear Factor Erythroid 2-related Factor 2）的激活是巨噬细胞中强效的抗炎信号通路，本研究的分析结果显示，血红素处理的肿瘤相关巨噬细胞（heme-Tumor-associated macrophages, heme-TAMs）的表达转录组始终富集有NRF2靶基因。
为此，我们通过一系列实验明确了NRF2的功能：分别使用Nrf2基因敲除的骨髓来源巨噬细胞（Bone Marrow-derived Macrophages, BMDMs）以构建稳定的NRF2失活状态，以及敲除胞质NRF2捕获蛋白KEAP1（Kelch样ECH关联蛋白1，Kelch-like ECH-associated protein 1）的巨噬细胞以构建稳定的NRF2激活状态，该状态不受血红素存在与否的影响。
为验证激活的NRF2足以介导血红素相关肿瘤相关巨噬细胞的表型转化，我们对Keap1基因敲除的巨噬细胞进行了分析。
整体实验设计：将来自1只Keap1基因敲除（Knockout, KO）小鼠（对照组）以及1只同窝野生型（Wild Type, WT）小鼠（分为对照组与血红素处理组）的骨髓来源巨噬细胞重悬后转移至15 mL Falcon管中。按每实验条件1×10^6个细胞的量，参照10X Genomics官方方案（CG00391；《细胞多重标记寡核苷酸标记示范方案：适用于活细胞占比>80%的样本；修订版B》）使用脂质标签进行细胞染色。本实验使用细胞多重标记寡核苷酸（Cell Multiplexing Oligos, CMO）标签B308-B311（3' CellPlex Kit Set A）对4个样本进行标记。标记完成后，再次对细胞悬液进行计数，并参照标记方案附录（CG00391）中的混合计算方法，按等比例将样本混合。