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

本研究采用基于10x Genomics平台的细胞索引转录组和表位测序（CITE-seq），对野生型C57BL/6小鼠的未致敏脑髓系细胞，以及携带E0771脑转移灶的C57BL/6小鼠的脑转移相关髓系细胞（Br.MAM）的转录组与细胞表面免疫标志物表达谱进行了分析与比较。每一轮测序中，我们共对4份不同的小鼠脑组织样本进行测序，其中2份来自未致敏小鼠，2份来自携带脑转移灶的小鼠。我们构建了包含CD45、CD25、CD3、CD4、CD11b及CD86六种抗体的混合液，对每份脑组织样本分别进行染色；随后，为每份样本标记专属的哈希抗体，以便后续可将所有样本混合后上样至10x Chromium系统，构建包含全部4份样本的单一测序文库，最终通过计算机分析基于各样本专属的哈希抗体完成样本拆分。
实验整体设计如下：未致敏脑组织取自2~3月龄的雌性C57BL/6小鼠，无脑转移灶。携带脑转移灶的脑组织则取自经颈动脉注射E0771细胞后造模成功的2~3月龄雌性C57BL/6小鼠，造模时长约2周。通过Percoll密度梯度离心富集免疫细胞，制备细胞悬液。我们构建了包含CD45、CD25、CD3、CD4、CD11b及CD86六种抗体衍生标签（Antibody-Derived Tags, ADT）的抗体混合液，对每份脑组织样本分别进行染色；随后，为每份样本标记专属的哈希抗体，以便后续可将所有样本混合后上样至10x Genomics Chromium系统，构建包含全部4份样本的单一测序文库，最终通过计算机分析基于各样本专属的哈希抗体完成样本拆分。
请注意，ADT-HTO-tags.csv为用于CITE-seq-Count软件（CITE-seq-Count v1.3.4；https://github.com/Hoohm/CITE-seq-Count）的条形码对照表。