Gut Dysbiosis Impacts Brain Metastasis Outgrowth and Immune Response Dynamics [CITE-seq]

We used CITE-seq (10x Gemoics-based) to profile and compare the transcriptomes and cell surface expression of a set of immune markers of brain immune cells from young-adult C57BL/6 mice with and without antibiotics induced gut microbiota depletion (ABX) during brain metastasis. Brain immune cells were collected from naive brains (D00) and at different time points throughout brain metastasis outgrowth (4 (D04), 8 (D08) and 14 (D14) days post injection). We sequenced a total of 24 different mouse brains (3 biological replicates per group: (1) D00 control, (2) D00 ABX, (3) D04 control, (4) D04 ABX, (5) D08 control, (6) D08 ABX, (7) D14 control, (8) D14 ABX). We created an antibody pool consisting of 31 different antibodies (CCR2/CD192, C117/c-kit, CD11b, CD11c, CD172a/SIRPα, CD25, CD3, CD38, CD4, CD44, CD45, CD45R/B220, CD86, CD8a, CD90.1, Cx3cr1, F4/80, I-A/I-E, Ly6C, Ly6G, NK1.1, PD-1, PD-L1, CD169/Siglec-1, Siglec-H, TMEM119, XCR1, CD24, CD103, CD64, CD83) and stained each brain individually with this antibody pool. Then, we stained each brain with it's own unique hashing antibody so that we could subsequently pool samples. Two groups were collapsed into one to generate four samples, each loaded into one well on the 10x Chromium (D00 control + D00 ABX, D04 control + D04 ABX, D08 control + D08 ABX, D14 control + D14 ABX). Sequencing libraries were prepared for each of the four collapsed groups (6 samples per group). Following sequencing, each sample is separated in silico by it's unique hashing antibody. Naive brains were obtained from ~12 week old female C57BL/6 mice with vehicle or ABX treatment. Brains with metastases were collected from ~12 week old female C57BL/6 mice with vehicle or ABX treatment at 4, 8 and 14 days post intra-carotid injection. Cell suspensions were prepared by percoll gradient centrifugation to obtain suspensions enriched for immune cells. We created an antibody pool consisting of 31 different antibodies (CCR2/CD192, C117/c-kit, CD11b, CD11c, CD172a/SIRPα, CD25, CD3, CD38, CD4, CD44, CD45, CD45R/B220, CD86, CD8a, CD90.1, Cx3cr1, F4/80, I-A/I-E, Ly6C, Ly6G, NK1.1, PD-1, PD-L1, CD169/Siglec-1, Siglec-H, TMEM119, XCR1, CD24, CD103, CD64, CD83) and stained each brain individually with this antibody pool. Then, we stained each brain with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium. Two groups were collapsed into one to generate four samples, each loaded into one well on the 10x Chromium (D00 control + D00 ABX, D04 control + D04 ABX, D08 control + D08 ABX, D14 control + D14 ABX). Sequencing libraries were prepared for each of the four collapsed groups (6 samples per group).

本研究采用基于10x Genomics平台的CITE-seq技术，对脑转移过程中经抗生素诱导肠道菌群耗竭（ABX，antibiotics-induced gut microbiota depletion）与未处理的年轻成年C57BL/6小鼠的脑免疫细胞转录组及一组免疫标志物的细胞表面表达水平进行分析与比较。脑免疫细胞采集自未荷瘤的正常脑组织（D00），以及脑转移灶形成过程中的不同时间点（注射后4天（D04）、8天（D08）及14天（D14））。本研究共对24份不同的小鼠脑组织样本进行测序，每组设置3次生物学重复，共分为8组：(1) D00对照组、(2) D00 ABX组、(3) D04对照组、(4) D04 ABX组、(5) D08对照组、(6) D08 ABX组、(7) D14对照组、(8) D14 ABX组。
我们制备了包含31种不同抗体的抗体组合，具体包括CCR2/CD192、C117/c-kit、CD11b、CD11c、CD172a/SIRPα、CD25、CD3、CD38、CD4、CD44、CD45、CD45R/B220、CD86、CD8a、CD90.1、Cx3cr1、F4/80、I-A/I-E、Ly6C、Ly6G、NK1.1、PD-1、PD-L1、CD169/Siglec-1、Siglec-H、TMEM119、XCR1、CD24、CD103、CD64、CD83，并用该抗体组合对每一份脑组织样本单独进行染色。随后，我们使用各自专属的哈希抗体对每一份脑组织样本进行染色，以便后续进行样本混合。我们将两组样本合并为一个样本，最终得到4组混合样本，每组分别上样至10x Chromium系统的一个反应孔中，分别为：D00对照组+D00 ABX组、D04对照组+D04 ABX组、D08对照组+D08 ABX组、D14对照组+D14 ABX组。为这4组混合样本分别构建测序文库，每组对应6份原始样本。测序完成后，通过每一样本专属的哈希抗体，采用生物信息学方法对样本进行拆分。
未荷瘤脑组织采集自约12周龄的雌性C57BL/6小鼠，这些小鼠分别经赋形剂或ABX处理。荷脑转移灶的脑组织则采集自约12周龄的雌性C57BL/6小鼠，这些小鼠经颈动脉注射肿瘤细胞后，分别于4天、8天、14天接受赋形剂或ABX处理。采用Percoll密度梯度离心法制备细胞悬液，以获得免疫细胞富集的细胞悬液。
我们制备了包含31种不同抗体的抗体组合，具体包括CCR2/CD192、C117/c-kit、CD11b、CD11c、CD172a/SIRPα、CD25、CD3、CD38、CD4、CD44、CD45、CD45R/B220、CD86、CD8a、CD90.1、Cx3cr1、F4/80、I-A/I-E、Ly6C、Ly6G、NK1.1、PD-1、PD-L1、CD169/Siglec-1、Siglec-H、TMEM119、XCR1、CD24、CD103、CD64、CD83，并用该抗体组合对每一份脑组织样本单独进行染色。随后，我们使用各自专属的哈希抗体对每一份脑组织样本进行染色，以便后续混合样本并上样至10x Chromium系统。我们将两组样本合并为一个样本，最终得到4组混合样本，每组分别上样至10x Chromium系统的一个反应孔中，分别为：D00对照组+D00 ABX组、D04对照组+D04 ABX组、D08对照组+D08 ABX组、D14对照组+D14 ABX组。为这4组混合样本分别构建测序文库，每组对应6份原始样本。