4T1这个TNBC小鼠肿瘤模型的CAFs异质性

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前面提到了：肿瘤样品的单细胞需要提取上皮细胞继续细分，就是感兴趣的fibroblasts等细胞亚群占比非常少，所以研究者们做了第二次单细胞转录组数据，见：什么，你想要的单细胞亚群比例太少了？，其实这样的策略屡见不鲜。

让我们一起再看看另外一个示例，关于小鼠模型的！

文章：《Single-Cell Transcriptomic Analysis of Tumor-Derived Fibroblasts and Normal Tissue-Resident Fibroblasts Reveals Fibroblast Heterogeneity in Breast Cancer》，在这个研究里面，作者 investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors.

之所以选择 这个小鼠模型，就是因为它 high stromatogenic response and immune exclusion.

区分了 six CAF subpopulations，详见文末！

这个研究是常规的10x单细胞转录组数据，10x Genomics Chromium platform ，质控后是 6420 cells ，第一层次降维聚类分群是：

  • 免疫细胞（66.4% ）：Cells in clusters 0, 2, 3, 5, 7, 8 and 9 expressed CD45 (Ptprc)，包括淋巴系和髓系
  • 上皮细胞（24.5% ）：Epcam (clusters 1 and 6)
  • 基质细胞，主要是内皮细胞和成纤维细胞和周细胞
    • CAFs（535 cells ） ：Cells in cluster 4 had high levels of Thy1, Pdpn and Pdgfra
    • 内皮细胞：Cells in cluster 10 expressed high levels of Pecam1 and Mcam
    • a small population of pericytes (cluster 11)

如下所示：

常规的降维聚类分群

可以看到， 虽然CAFs的细胞数量有点少，但是其功能很清晰，主要是下面的3个功能，符合逻辑：

  • collagens (Col1a1, Col1a2, Col3a1, Col5a1-3, Col6a1-3 etc.),
  • proteoglycans (Dcn, Lum, Bgn, Prg4 etc.)
  • glycoproteins (Postn, Dpt, Tnc, Fbln, Fbn1 etc.)

值得注意的是并不是使用的collagens家族基因都应该是在成纤维细胞高表达哦 ：

  • Col4a1 and Col4a2, the highest expression for these two genes was detected in pericytes and endothelial cells.

为了更好的研究CAFs，重新做单细胞数据，这次首先去除免疫细胞，这样捕获了4000多个细胞，主要是 epithelial (Epcam+), endothelial (Pecam1+), pericytes (Mcam+, Rgs5+) 这3类细胞需要继续排除，剩下 ~1600 cells 是 待研究CAFs，可以区分成为6群：

  • Cells in cluster 0 expressed high levels of lymphocyte antigen 6 complex, locus C1 (Ly6c1)
  • while cells in cluster 1 were highly enriched for alpha smooth muscle actin (α-SMA/Acta2) (Figure 2C);
  • Cells in cluster 2 expressed high levels of cyclin-dependent kinase 1 (Cdk1) and other cell cycle genes including Cenpa and Cenpf and were identified as ‘dividing cells’ (Figure 2C).
  • Leukocyte surface antigen Cd53 was highly enriched in cluster 3 and was used as a marker for this cluster (Figure 2C).
  • Cells in cluster 4 showed significant enrichment for cellular retinoic acid-binding protein 1 (Crabp1),
  • while cells in cluster 5 showed enrichment for Cd74 (Figure 2C).

分群，并且进行比较好的描述：

进行比较好的描述

其实挺容易看懂的，如果你是现在才入坑单细胞的，可以先看看基础10讲：

最基础的往往是降维聚类分群，参考前面的例子：人人都能学会的单细胞聚类分群注释

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