Chemotherapy-mediated changes in the immune cell landscape of murine triple negative breast cancer.. Chemotherapy-mediated changes in the immune cell landscape of murine triple negative breast cancer.

We performed CITE-seq (10x Genomics-based) to profile and compare the transcriptomes and cell surface expression of immune epitopes in the primary breast tumors of transgenic mice (C3-1-TAg) with or without MCT chemotherapy. We sequenced a total of six different tumor 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: tumor of PBS treated C3-1-TAg mouse; (2) HTO2: tumor of PBS treated C3-1-TAg mouse; (3) HTO3: tumor of PBS treated C3-1-TAg mouse; (4) HTO4: tumor of MCT treated C3-1-TAg mouse; (5) HTO5: tumor of MCT treated C3-1-TAg mouse; (6) HTO6: tumor of MCT treated C3-1-TAg mouse. The following sample comparisons were made: HTO1, HTO2, and HTO3 versus HTO4, HTO5, and HTO6. Overall design: Breast tumors were isolated from C3-1-TAg mice treated with either MCT or PBS for 4 weeks. These mice were started on treatment when the tumors were around 5 mm* 5 mm in size. Tumors were dissociated using collagenase enzyme and the single cells were enriched with CD45+ immune cells using MACS magnetic beads (Miltenyi). 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，对经MCT化疗与未经MCT化疗的转基因小鼠（C3-1-TAg）原发性乳腺肿瘤的转录组及免疫表位细胞表面表达特征进行表征与比较。本研究共对6份不同的肿瘤样本开展测序。
我们构建了包含35种不同抗体的抗体池，并使用该抗体池对每份样本分别进行染色。随后，我们使用各自专属的哈希抗体（hashing antibody）对每份样本进行染色，以便后续可将样本混合后上样至10x Chromium系统，随后制备包含全部6份样本的单一测序文库，最终通过各样本专属的哈希抗体在计算机虚拟环境中完成样本拆分。
样本信息如下：
(1) HTO1：经PBS处理的C3-1-TAg小鼠肿瘤样本；
(2) HTO2：经PBS处理的C3-1-TAg小鼠肿瘤样本；
(3) HTO3：经PBS处理的C3-1-TAg小鼠肿瘤样本；
(4) HTO4：经MCT化疗的C3-1-TAg小鼠肿瘤样本；
(5) HTO5：经MCT化疗的C3-1-TAg小鼠肿瘤样本；
(6) HTO6：经MCT化疗的C3-1-TAg小鼠肿瘤样本。
本研究设置如下分组对照：HTO1、HTO2与HTO3组 vs HTO4、HTO5与HTO6组。
实验整体设计：
从经MCT化疗或PBS处理4周的C3-1-TAg小鼠体内分离乳腺肿瘤；当肿瘤体积约为5 mm×5 mm时，开始对小鼠进行相应处理。使用胶原酶对肿瘤组织进行解离，并通过美天旎（Miltenyi）公司的MACS磁珠富集CD45阳性免疫细胞。
我们构建了包含35种不同抗体的抗体池，并使用该抗体池对每份样本分别进行染色。随后，我们使用各自专属的哈希抗体（hashing antibody）对每份样本进行染色，以便后续可将样本混合后上样至10x Chromium系统，随后制备包含全部6份样本的单一测序文库，最终通过各样本专属的哈希抗体在计算机虚拟环境中完成样本拆分。