CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10 [R07_Cx3cr1]. CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10 [R07_Cx3cr1]

We performed CITE-seq (10x Genomics-based) to profile and compare the transcriptomes and cell surface expression of immune epitopes in the brains of Cx3cr1+/- and Cx3cr1-/- mice during homeostasis or brain metastasis. We sequenced a total of eight different samples. We created an antibody pool consisting of 35 different antibodies and stained each sample individually with this antibody pool. Then, we stained each sample 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 sight samples and finally separate each sample in silico by it's unique hashing antibody. The samples are as follows: (1) HTO1: brain of naïve Cx3cr1+/- mouse; (2) HTO2: brain of naïve Cx3cr1+/- mouse; (3) HTO3: brain of metastasis-burdened Cx3cr1+/- mouse; (4) HTO4: brain of metastasis-burdened Cx3cr1+/- mouse; (5) HTO5: brain of naïve Cx3cr1-/- mouse; (6) HTO6: brain of naïve Cx3cr1-/- mouse; (7) HTO7: brain of metastasis-burdened Cx3cr1-/- mouse; (8) HTO8: brain of metastasis-burdened Cx3cr1-/- mouse. The following sample comparisons were made: HTO1 and HTO2 versus HTO5 and HTO6; HTO3 and HTO4 versus HTO7 and HTO8. Overall design: Naïve brains were obtained from 2-3 month old female Cx3cr1+/- or Cx3cr1-/- mice without brain metastases. Brains from brain metastasis-bearing mice initiated by carotid injection of E0771 cells were obtained from 2-3 month old female mice of the aforementioned genotypes with established brain metastases (~2 weeks post injection). Cell suspensions were prepared by percoll gradient centrifugation enrichment to obtain suspensions enriched for immune cells from the brain. We created an antibody pool consisting of 35 different antibodies and stained each sample individually with this antibody pool. Then, we stained each sample 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 eight samples and finally separate each sample in silico by it's unique hashing antibody.

本研究采用基于10x Genomics平台的CITE-seq技术，对稳态或脑转移状态下Cx3cr1+/-与Cx3cr1-/-小鼠脑内的转录组以及免疫表位的细胞表面表达情况进行分析与比较，共完成8份不同样本的测序。

首先构建包含35种抗体的抗体池，将每份样本单独用该抗体池进行染色；随后为每份样本添加专属的哈希抗体（hashing antibody），以便后续可将所有样本混合后上样至10x Chromium系统，仅需制备一份包含全部8份样本的测序文库，最终通过计算机分析根据各样本专属的哈希抗体完成样本的虚拟拆分。

本研究的8份样本具体如下：(1) HTO1：未造模的Cx3cr1+/-小鼠脑组织；(2) HTO2：未造模的Cx3cr1+/-小鼠脑组织；(3) HTO3：携带脑转移瘤的Cx3cr1+/-小鼠脑组织；(4) HTO4：携带脑转移瘤的Cx3cr1+/-小鼠脑组织；(5) HTO5：未造模的Cx3cr1-/-小鼠脑组织；(6) HTO6：未造模的Cx3cr1-/-小鼠脑组织；(7) HTO7：携带脑转移瘤的Cx3cr1-/-小鼠脑组织；(8) HTO8：携带脑转移瘤的Cx3cr1-/-小鼠脑组织。

本次研究设置如下样本比较组：HTO1与HTO2 对比 HTO5与HTO6；HTO3与HTO4 对比 HTO7与HTO8。

总体实验设计如下：未发生脑转移的正常脑组织取自2~3月龄的雌性Cx3cr1+/-或Cx3cr1-/-小鼠；脑转移瘤模型通过颈动脉注射E0771细胞构建，取接种后约2周、已建立脑转移瘤的上述基因型雌性（2~3月龄）小鼠的脑组织作为转移组样本。通过Percoll密度梯度离心富集法制备细胞悬液，以富集脑内免疫细胞。后续实验流程同前：先构建包含35种抗体的抗体池，将每份样本单独染色，再添加专属哈希抗体以实现混合上样、统一建库及虚拟拆分样本。