Stat1-induced NAMPT sustains glycolysis to regulate gene expression in classically activated macrophages

Single Cell RNA Sequencing is used to profile in vivo responses by immune cells lacking the Nampt NRE1 region to melanoma challenge, finding multiple defects in macrophages and other myeloid APCs. We analyzed B16F10 melanoma tumor infiltrating immune cells from mice specifically lacking NRE1 in hematopoietic cells, finding inflammatory and metabolic defects in APC populations within tumors, correlating with trends toward defective control of malignant growth. 500,000 B16F10 cells (from Mingnan Chen lab) injected subcutaneously into NRE1 fl/fl VavCre(-) or NRE1 fl/fl VavCre(+) male mice. Tumors were excised at the endpoint, pooled and digested with Accumax to liberate single cells. Cells were stained with APC-CD45 (1:200) for 15 min on ice and sorted via flow cytometer (HCI Aria) after spiking 1:100 DAPI (1x final). CD45+DAPI- cells were obtained (860,000 cells from WT, 1,0260,00 cels from KO), and subjected to Single Cell RNA Sequencing.

本研究采用单细胞RNA测序（Single Cell RNA Sequencing），对缺失Nampt NRE1区域的免疫细胞在黑色素瘤（melanoma）攻击下的体内应答反应进行表征，发现巨噬细胞及其他髓系抗原呈递细胞（antigen-presenting cells, APCs）存在多种功能缺陷。我们分析了造血细胞（hematopoietic cells）中特异性缺失NRE1的小鼠体内的B16F10黑色素瘤肿瘤浸润免疫细胞，发现肿瘤内抗原呈递细胞群体存在炎症与代谢功能缺陷，该缺陷与恶性生长调控受损的趋势相关。将50万个B16F10细胞（源自陈明楠实验室）皮下接种至NRE1 flox/flox VavCre(-)或NRE1 flox/flox VavCre(+)雄性小鼠体内。实验终点处摘取肿瘤组织，混合后使用Accumax细胞解离液进行消化以获取单个细胞悬液。以1:200的稀释比例将APC-CD45抗体加入细胞悬液，冰上孵育15分钟；随后以1:100的比例加入终浓度为1×的DAPI（DAPI）染料，通过流式细胞仪（flow cytometer, HCI Aria）进行细胞分选。最终获取CD45阳性且DAPI阴性的细胞（野生型[wild type, WT]小鼠样本获取86万个细胞，敲除型[knockout, KO]小鼠样本获取102.6万个细胞），并对其进行单细胞RNA测序分析。