肿瘤微生物组的研究日益受到关注。我们开发了一个计算管道 (CSI-Microbes)，用于从单细胞 RNA 测序 (scRNA-seq) 数据中识别微生物读数并分析类群的差异丰度。通过一系列对照实验和分析，我们对通过多种 scRNA-seq 技术恢复微生物独特分子标识符的功效进行了首次系统评估，确定较新的 10x 化学方法（3' v3 和 5'）是最适合的方法。我们分析了食管癌和结直肠癌患者，发现来自不同属的读数往往在同一宿主细胞中同时出现，证明了可能的细胞内多种微生物相互作用。骨髓细胞内的微生物读数异常丰富，可上调 IL1B 和 CXCL8 等促炎细胞因子，而受感染的肿瘤细胞则上调抗原加工和呈递途径。这些结果表明，被细菌吞噬的骨髓细胞是肿瘤微环境 (TME) 内细菌 RNA 的主要来源，可能会导致 TME 发炎并影响免疫治疗反应。

The study of the tumor microbiome has been garnering increased attention. We developed a computational pipeline (CSI-Microbes) for identifying microbial reads from single-cell RNA sequencing (scRNA-seq) data and for analyzing differential abundance of taxa. Using a series of controlled experiments and analyses, we performed the first systematic evaluation of the efficacy of recovering microbial unique molecular identifiers by multiple scRNA-seq technologies, which identified the newer 10x chemistries (3' v3 and 5') as the best suited approach. We analyzed patient esophageal and colorectal carcinomas and found that reads from distinct genera tend to co-occur in the same host cells, testifying to possible intracellular polymicrobial interactions. Microbial reads are disproportionately abundant within myeloid cells that up-regulate proinflammatory cytokines like IL1Β and CXCL8, while infected tumor cells up-regulate antigen processing and presentation pathways. These results show that myeloid cells with bacteria engulfed are a major source of bacterial RNA within the tumor microenvironment (TME) and may inflame the TME and influence immunotherapy response.