Induction of antitumor activity of CD8+ T cells by inactivation of ATPIF1 through F1Fo-ATPase-mediated metabolic reprogramming

In this study, CD45+ leukocytes were sorted from the mixed tumor samples, which were collected from the C57BL/6 wild type mice and ATPIF1 knock-out mice, respectively. The live CD45+ cells were used for single cell RNA sequencing and TCR sequencing. B16 melanoma cells were implanted in the right flank of male WT and KO mice (n=6). On day 12, the tumors were dissected from the surrounding fascia, mechanically minced to approximately 1 mm3 pieces, then the tumor tissue suspension from KO mice was mixed together, so did the tumor tissue suspension from WT mice. After that, the mixed tumor tissue suspension was stored in a solution provided by the Beijing Analytical Biosciences Technology Co., Ltd, a company specialize at single cell RNA sequencing and data analysis, which is founded by the Professor Zhang Zemin in Peking University, Beijing, China. The samples of WT and KO mice were sent to this company for CD45+ T cell sorting immediately and single cell RNA-seq. The preparation of tumor single cell suspension, the CD45+ tumor-infiltrating leukocytes sorting, reverse transcription, amplification, sequencing, the quality control, scRNA-seq data processing, cluster analysis, trajectory analysis, STARTRAC analysis and RNA velocity-based cell fate tracing were all performed in the Beijing Analytical Biosciences Technology Co., Ltd, which was described in detail in two studies