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.

我们采用基于10x Genomics的细胞转录组与表位联合测序（CITE-seq）技术，对稳态或脑转移状态下，Cx3cr1+/-与Cx3cr1-/-小鼠大脑内的免疫表位转录组与细胞表面表达谱进行组学表征分析。本研究共对8份不同样本完成测序。

我们构建了包含35种不同抗体的抗体池，使用该抗体池对每份样本分别进行染色；随后为每份样本添加其专属的细胞哈希抗体（hashing antibody），以便后续将样本混合后上样至10x Chromium系统，随后构建包含全部8份样本的单一测序文库，最终通过各样本专属的细胞哈希抗体在计算机中完成样本拆分。

样本信息如下：
(1) HTO1：未荷瘤稳态Cx3cr1+/-小鼠脑组织；
(2) HTO2：未荷瘤稳态Cx3cr1+/-小鼠脑组织；
(3) HTO3：脑转移荷瘤Cx3cr1+/-小鼠脑组织；
(4) HTO4：脑转移荷瘤Cx3cr1+/-小鼠脑组织；
(5) HTO5：未荷瘤稳态Cx3cr1-/-小鼠脑组织；
(6) HTO6：未荷瘤稳态Cx3cr1-/-小鼠脑组织；
(7) HTO7：脑转移荷瘤Cx3cr1-/-小鼠脑组织；
(8) HTO8：脑转移荷瘤Cx3cr1-/-小鼠脑组织。

本研究设置如下对照组合：HTO1与HTO2组 vs HTO5与HTO6组；HTO3与HTO4组 vs HTO7与HTO8组。

实验整体设计：未荷瘤稳态脑组织取自2-3月龄的雌性Cx3cr1+/-或Cx3cr1-/-小鼠，无脑转移灶。脑转移荷瘤小鼠的脑组织则取自经颈动脉注射E0771细胞造模的上述基因型雌性小鼠，造模后约2周采集，此时已形成明确脑转移灶。

采用Percoll密度梯度离心法对脑组织进行免疫细胞富集，制备细胞悬液。

我们构建了包含35种不同抗体的抗体池，使用该抗体池对每份样本分别进行染色；随后为每份样本添加其专属的细胞哈希抗体，以便后续将样本混合后上样至10x Chromium系统，构建包含全部8份样本的单一测序文库，最终通过各样本专属的细胞哈希抗体在计算机中完成样本拆分。