【原】CAFs到底是两个亚群还是三个呢？

健明 2021-07-14

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前面我们在推文细胞亚群的特异性标记基因也许真的很难提到的Cancer-associated fibroblasts (CAFs)是比较难以精确的细分亚群。而且我们讨论了T细胞可以简单分成4类，其实CAFs的分类也值得深入讨论！

首先，在小鼠里面可以看到，myofibroblast (myCAF), inflammatory fibroblast (iCAF), and antigen-presenting fibroblast (apCAF)，但是在人类里面并没有很明显的apCAF，也就是说通常是分2类，很有意思的，值得分享！比如，于AUGUST 2019发表在 CANCER DISCOVERY 杂志的文章：《Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts》，链接是：https://cancerdiscovery./content/9/8/1102.long

最后得到了两个CAFs亚群，分别是：

CAFs expressing high levels of αSMA, which we therefore named “myofibroblastic CAFs” (myCAF),
CAFs expressing low levels of αSMA but high levels of cytokines and chemokines, which we named “inflammatory CAFs” (iCAF;)

而且全文就是重点讨论了CAFs的两个亚群的功能，就是myofibroblast (myCAF) 和 inflammatory fibroblast (iCAF)。这个课题的单细胞转录组数据共有2万多个细胞，The human RNA-seq data are available at NCBI dbGaP under the accession number phs001840.v1.p1. 不方便下载。样品来源于：Six tumors from patients with untreated PDAC and adjacent-normal pancreas tissues from two of these patients，分成15个细胞亚群，其中962 fibroblasts仍然是可以分成两个亚群，都表达COL1A1, FAP等CAFs的标志基因，但是这两群又有所差异，如下所示：

CAFs的两个亚群

两个亚群各自的标志基因是：

两个亚群各自的标志基因

差异还是很明显的！

在肿瘤小鼠模型的单细胞数据里面可以分3群

肿瘤小鼠模型数据是：We combined the sequencing data from the fibroblast-enriched populations isolated from the four KPC tumors into one data set consisting of 8,443 cells and analyzed them together.

数据在：https://www.ncbi.nlm./geo/query/acc.cgi?acc=GSE129455

GSM3713177 KPC1_Viable
GSM3713178 KPC2_Viable
GSM3713179 KPC1_FibroblastEnriched
GSM3713180 KPC2_FibroblastEnriched
GSM3713181 KPC3_Viable
GSM3713182 KPC4_Viable
GSM3713183 KPC3_FibroblastEnriched
GSM3713184 KPC4_FibroblastEnriched

多了的这个Cancer-associated fibroblasts (CAFs)亚群命名为：“antigen-presenting CAFs” (apCAF).

使用ARACNe and VIPER algorithms.，清晰可见：

另外一种可视化方法如下：

确实是各自的marker基因很清晰，“antigen-presenting CAFs” (apCAF) 就特异性表达：

GSEA分析发现这3群也有各自的通路激活情况：

upregulation of inflammatory pathways in mouse iCAFs (e.g., JAK/STAT signaling, cytokine interactions with their receptors and coagulation;
The pathways specifically upregulated in mouse myCAFs were EMT, myogenesis, ECM receptor interaction, and focal adhesion
而apCAF subtype,独特的通路包括：antigen presentation and processing, fatty-acid metabolism, MYC targets, and MTORC1 signaling

但是为什么前面在人类单细胞数据里面没有发现apCAF呢

前面提到962 fibroblasts仍然是可以分成两个亚群，都表达COL1A1, FAP等CAFs的标志基因，那我们检查看看apCAF subtype的标志基因，如下：

消失的apCAF亚群

只能说姑且认为是两个物种的差异？或者说小鼠模型本来就不可能是一模一样的模拟人类的肿瘤微环境。

看看Cancer-associated fibroblasts (CAFs)在原位肿瘤和转移瘤的差异情况

2020年9月的文章：《Single-cell transcriptome analysis of tumor and stromal compartments of pancreatic ductal adenocarcinoma primary tumors and metastatic lesions》，链接是：https://genomemedicine./articles/10.1186/s13073-020-00776-9

总共是 16个10x单细胞数据：https://www.ncbi.nlm./geo/query/acc.cgi?acc=GSE154778

GSM4679532 P01: Primary tumor 01 (10x 3' v1)
GSM4679533 P02: Primary tumor 02 (10x 3' v2)
GSM4679534 P03: Primary tumor 03 (10x 3' v2)
GSM4679535 P04: Primary tumor 04 (10x 3' v2)
GSM4679536 P05: Primary tumor 05 (10x 3' v2)
GSM4679537 P06: Primary tumor 06 (10x 3' v2)
GSM4679538 P07: Primary tumor 07 (10x 3' v2)
GSM4679539 P08: Primary tumor 08 (10x 3' v2)
GSM4679540 P09: Primary tumor 09 (10x 3' v2)
GSM4679541 P10: Primary tumor 10 (10x 3' v2)
GSM4679542 MET01: Metastatic lesion 01 (10x 3' v2)
GSM4679543 MET02: Metastatic lesion 02 (10x 3' v2)
GSM4679544 MET03: Metastatic lesion 03 (10x 3' v2)
GSM4679545 MET04: Metastatic lesion 04 (10x 3' v2)
GSM4679546 MET05: Metastatic lesion 05 (10x 3' v2)
GSM4679547 MET06: Metastatic lesion 06 (10x 3' v2)

同样的，可以进行基本的降维聚类分群，参考前面的例子：人人都能学会的单细胞聚类分群注释，第一次分群就非常漂亮！图例是：

c Three major clusters are formed by CAFs from primary tumors.
d CAFs from different patients are mixed in the different clusters

描述是：

Figure 2c shows the unsupervised clustering of 1753 CAF cells identified in Fig. 1a. The CAFs formed 3 major clusters: c0, c1, and c2 (Fig. 2c). Unlike the tumor cells, the CAFs did not cluster by patient.

这篇文章也提到了：Elyada and colleagues previously described 3 subtypes of CAFs identified in PDAC tumors: myofibroblasts (myCAFs), inflammatory fibroblasts (iCAFs), and antigen-presenting fibroblasts (apCAFs)

参考：

Semin Cancer Biol . 2020 May; 《Cancer-associated fibroblasts in desmoplastic tumors: emerging role of integrins 》， https://pubmed.ncbi.nlm./31415910/
https://cancerdiscovery./content/9/2/173
https://www./journals/ebiom/article/PIIS2352-3964(20)30030-X/fulltext
Am J Physiol Cell Physiol . 2020 Aug 1; 《Recent advances in understanding cancer-associated fibroblasts in pancreatic cancer》，https://pubmed.ncbi.nlm./32432930/
J Exp Med. 2017 Mar 6; 《Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer》
https://onlinelibrary./doi/epdf/10.1111/cas.14537
GSE125588
GSE129455
Yin & Yang: The Duality of Cancer-Associated Fibroblasts in Pancreatic Cancer