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/SIRPa, 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. Overall design: 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/SIRPa, 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).