Single cell transcriptomes of brain metastases and micrometastases derived from triple negative human breast cancer cell line in mouse model.. Single cell transcriptomes of brain metastases and micrometastases derived from triple negative human breast cancer cell line in mouse model.

In order to gainan understanding of potential mechanisms by which dystroglycan could effect DTC quiescence signaling in brain, we took an unbiased approach. We inoculated a series of mice with a tdTomato-positive, brain metastatic variant of T4-2 cells, and examined mice after 6-weeks in order to identify those with distinct brain metastases. Metastatic lesions were surgically dissected, digested and flow sorted to purify tdTomato+ cells (“Metastases”). In parallel, the remaining uninvolved brain was digested, and residual tdTomato+tumour cells were purified from this digest via flow sorting (“Micromets”). These two populations were indexed, and the libraries were prepared following standard 10X Genomics protocols. Overall design: In total, 1,779 micromet-derived DTCs (including both mVenus-p27+ and p27- cells from uninvolved brain) were subjected to single cell RNAseq. 4,032 mVenus-p27- and 251 mVenus-p27+ (quiescent) cells from metastatic lesions were profiled.

为阐明肌营养不良蛋白聚糖 (dystroglycan) 调控大脑内播散肿瘤细胞 (Disseminated Tumor Cells, DTC) 静息信号传导的潜在机制，本研究采用无偏倚研究策略。我们将一批小鼠接种表达tdTomato荧光蛋白的T4-2细胞脑转移变体，并于接种6周后对小鼠开展检测，以筛选出存在明确脑转移灶的个体。将转移灶经手术剥离、酶解消化后，通过流式分选纯化得到tdTomato阳性细胞，命名为"转移灶来源细胞 (Metastases)"。与此同时，对剩余未受肿瘤累及的脑组织进行酶解消化，并通过流式分选从消化产物中纯化残留的tdTomato阳性肿瘤细胞，命名为"微转移灶来源细胞 (Micromets)"。对上述两群细胞完成索引标注后，我们按照标准10X基因组学 (10X Genomics) 实验流程构建测序文库。

实验整体设计：本研究共纳入1779个微转移灶来源的播散肿瘤细胞（包括来源于未受累脑组织的mVenus-p27阳性及p27阴性细胞）进行单细胞RNA测序 (single cell RNA sequencing, scRNA-seq)。同时对4032个来源于转移灶的mVenus-p27阴性细胞，以及251个mVenus-p27阳性（静息型）细胞开展测序分析。