CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10

We used CITE-seq (10x Genomics-based) to profile and compare the transcriptomes and cell surface expression of a set of immune markers of naïve brain myeloid cells from wild type C57BL/6 mice to brain metastasis-associated myeloid cells (Br.MAM) from C57BL/6 mice bearing E0771 brain metastases. In each sequencing round, we sequenced a total of four different mouse brains (2 naive and 2 metastasis-burdened). We created an antibody pool consisting of 6 different antibodies (CD45, CD25, CD3, CD4, CD11b, and CD86) 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 and later prepare one library consisting of all six samples and finally separate each sample in silico by it's unique hashing antibody. Overall design: Naïve brains were obtained from 2-3 month old female C57Bl/6 mice without brain metastases. Brains bearing metastases initiated by carotid injection of E0771 cells were obtained from 2-3 month old female mice (C57Bl/6) with established brain metastases (~2 weeks post injection). Cell suspensions were prepared by percoll gradient centrifugation enrichment to obtain suspensions enriched for immune cells. We created an antibody pool consisting of 6 different ADT antibodies (CD45, CD25, CD3, CD4, CD11b, and CD86) 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 Genomics Chromium and later prepare one library consisting of all four samples and finally separate each sample in silico by it's unique hashing antibody. Please note that the ADT-HTO-tags.csv is the bar code table used for CITE-seq-Count software (CITE-seq-Count v1.3.4; https://github.com/Hoohm/CITE-seq-Count).