4T1这个TNBC小鼠肿瘤模型的CAFs异质性

生信技能树jimmy

发布于 2022-01-17 17:33:37

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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：