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2.2.12 研究 | 2021-单细胞转录组揭示乳头状甲状腺癌起始与发展

刘小泽写于2021.10.26

前言

题目:Single-cell transcriptomic analysis of the tumor ecosystems underlying initiation and progression of papillary thyroid carcinoma
日期:2021-10-18
期刊:nature communications

一句话概括

文章关注肿瘤微环境:先初步分群,然后依次看了几个重要组成部分:thyrocytes、immune细胞群、基质细胞(fibroblasts 、endothelial cells)

使用的数据

11个病人的23个组织,得到158,577个细胞
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image-20211026100416227

初步分群结果

得到6个主要的细胞群:
  • T/natural killer (NK) cells (CD3D, CD3E, CD3G, CD247)
  • B cells (CD79A, CD79B, IGHM, IGHD)
  • thyrocytes (TG, EPCAM, KRT18, KRT19)
  • myeloid cells (LYZ, S100A8, S100A9, CD14)
  • fibroblasts (COL1A1, COL1A2, COL3A1, ACTA2)
  • endothelial cells (PECAM1, CD34, CDH5, VWF)
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image-20211026100646905
接下来重点关注免疫细胞(T, NK, B, and myeloid cell )的细分,得到22个亚群
  • T细胞可以根据CD4和CD8的表达量继续细分,得到7 clusters for CD4+ cells & 4 clusters for CD8+ cells
  • NK, B, and myeloid cells同样细分得到 2, 3, and 6 subsets
并且每个亚群都有各自的组织偏好性(图e)
其中,CD4-c6中的FOXP3上调,CD8-c4中的PDCD1上调,分别对应了 regulatory CD4+ T cells (Treg) 和 exhausted CD8+ T cells (Tex),它们主要存在于皮下和肿瘤组织中;另外CD8-c3 中的GNLY上调,对应了效应T细胞,主要存在于肿瘤组织
这几种免疫细胞均在肿瘤组织中富集,说明了宿主免疫应答与肿瘤免疫逃逸共存
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image-20211026101100992

看甲状腺细胞群的异质性

拿thyrocytes这群细胞继续细分,得到9个亚群,其中1、2亚群主要是para-tumor,可以代表non- malignant thyrocytes;3-9则是malignant thyrocytes
image-20211026102825082
接下来就用3种方法证明这样划分是对的:
image-20211026103431062
  • 首先是相关性(图c),发现1、2最相关
  • 然后(图d)计算每个亚群的thyroid differentiation score (TDS)。TDS用来评价分化程度,其中1、2最高,并且照应了para-tumor最高的情况
  • 最后使用TCGA的PTC样本bulk转录组,构建一个分类器,达到97% sensitivity and 96% specificity ;再将这个分类器用在这个单细胞数据,发现与推测的组织类型相似度达到了:95%, 97% and > 98% of c01, c02, and c03–09 cells
于是,下面就认为malignant (c03-c09) and non-malignant (c01, c02) thyrocytes
探索了恶性vs非恶性、恶性肿瘤 vs恶性淋巴、恶性肿瘤 vs恶性皮下,发现:TMSB4X as a suggestive biomarker that potentially involves in PTC initiation and progression
image-20211026103921664

既然分开了恶性和非恶性,那就先研究非恶性的两个亚群

之前图d看到,c1和c2的分化指标虽然比其他群高,但二者还是有差距,于是就开始探索这两群非恶性细胞的差异
看到c1到c2会有中间态,然后看到c1=》c2=》c3-c9的过程中,TMSB4X也确实逐渐升高
推测:c2这个群可能不是完全的正常状态,是出于正常和恶性之间的过渡态
image-20211026104550993
通过实验验证了c2的属性是:cancer-primed nature of these outwardly normal but transcriptionally altered premalignant cells, which provide both seeds and soil for the eruption of malignant growths,将c2归结为premalignant
最后对比了c1和c2,对PKHD1L1这个基因感兴趣,推测 PKHD1L1 might function as a tumor suppressor gene in multiple solid tumors
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接下来重点看恶性的细胞——发育轨迹

正常的c1和premalignant的c2在右上角,作为发育起源(normal-cell-initiated State 1),看看三个发育状态有什么区别:
image-20211026110343493
  • state1有一个明显的转录因子SOX9,作用是branching folliculogenesis(卵泡生成) of normal thyroid gland,于是state1的名称是follicular-like thyrocyte
image-20211026110630820
  • State 2 was basically a half-half mixture of tumor and LN-metastatic thyrocytes
  • State 3, featured by the lowest TDS score and RAS score, and highest BRAF score, contained the vast majority of RAIR subcutaneous metastatic thyrocytes;另外state3高表达GATA2, MYC, SOX4(去分化相关的转录因子)

拿发育轨迹的结果做点什么

得到了480 pseudotime-associated genes (PAGs),因为它们主要就是在甲状腺上皮细胞中,再结合2个bulk转录组数据,可以用来更新BRAF-/RAS-like 分子亚型方法。发现和之前的分型方法一样,BRAF-like tumors也是存在很强的异质性,将其分成2个亚型(BRAF-like-A and BRAF-like-B),发现BRAF-like-B更加特别:associated with TCV pathology ; lower TDS scores;advanced staging;significantly compromised DFS;
image-20211026123054006
GSEA结果发现BRAF-like-B在免疫相关通路活性更高
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image-20211026123531415
并且三个亚型RAS-like, BRAF-like-A, BRAF-like-B分别对应了三种表型:follicular-like, p-EMT-like, dediff-like
image-20211026123856784

接下来看基质细胞——CAFs

其中重点关注cancer-associated fibroblasts (CAFs)
初步分群的fibroblasts这群,表达了CAF的marker:VIM, S100A4, ACTA2 (α-SMA), and PDGRFA
因此可以直接把fibroblasts成为CAF,然后把CAF分群,一开始分成4群,其中cluster 0, 1 and 3可以当成myofibroblastic CAFs (myoCAF);cluster 2是inflammatory subtype (iCAF) ,它的marker基因是CFD, PLA2G2A, CCDC80
image-20211026124527891
正式有了iCAF和myoCAF,再看和分型的关系,发现CAFs主要存在于BRAF-like中
接着做了iCAF和myoCAF的细胞通讯,发现:
  • regulation of cellular immunity is an important function for iCAFs in PTC
  • myoCAFs tends to exert mechanical and chemical influence on tumor progression rather than through direct cell communications, as described in other solid tumors
image-20211026124310210

再看基质细胞——内皮细胞(endothelial cells, ECs)

根据已有的marker,将EC分成5个类型:arterial, venous, lymphatic, immature and tip
  • arterial 高表达 arterial development and remodeling (FBLN5, GJA5, JAG1) and smooth muscle contraction (PPP1R14A)
  • venous 高表达 VWF (von Willebrand factor) and genes associated with leukocyte recruitment (ACKR1) or adhesion (SELE)
  • Lymphatic 高表达 canonical marker LYVE1 and a chemokine ligand CCL21
  • immature 高表达 Notch signaling and target genes (JAG1, HES1, ID1, ID2, ID3), and genes involving in barrier integrity (ENG, PLVAP, HSPG2, APLNR), which may resemble the stalk-like cells
  • tip 高表达 cell migration (NRP1, ENPP2), adhesion (THY1) and vessel formation (FLT1, also called VEGFR1; KDR, also called VEGFR2; NRP1, also called VEGF165R);并且tip高表达一些转录因子,比如 endothelial migration and sprouting, such as ZEB1, HOXB5 and STAT family
图h看到,只有lymphatic在正常的甲状腺组织中富集,其他均存在于原发或转移癌中
最后的细胞通讯看了EC和免疫细胞,发现:
  • lymphatic 和免疫细胞通过atypical chemokine receptor 2 (ACKR2)进行关联【它用来调控chemokine availability】(在附图)
  • venous, immature and arterial ECs 和免疫细胞通过 Intercellular Adhesion Molecule 1 (ICAM1) 关联 (在附图)
  • tip 和免疫细胞通过key angiogenic VEGF-VEGFR signalings 关联(图i)
image-20211026125434470