单细胞交响乐
搜索文档…
⌃K
Links

2.2.13 研究 | 2021-解析食管鳞癌化疗病人的单细胞转录组

刘小泽写于2021.11.01

前言

题目:Dissecting the single-cell transcriptome network in patients with esophageal squamous cell carcinoma receiving operative paclitaxel plus platinum chemotherapy
日期:2021-10-26
期刊:Oncogenesis

一句话概括

分段式作文:5个常规治疗病人和5个化疗病人得到的单细胞数据,先初步分成上皮、基质和免疫,然后每一类继续细分,分别探讨

首先是初步的分群

三大群:上皮、基质和免疫,然后基质又细分一下:内皮和成纤维细胞;免疫又细分为T、NK、B、髓细胞、肥大细胞
选择的样本:5个病人underwent surgery alone (SA-ESCC) + 5个病人underwent preoperative paclitaxel plus platinum chemotherapy (NACT-ESCC),总共得到113,581个细胞(63,837 SA-ESCC derived cells and 49,744 NACT-ESCC derived cells)
使用SingleR结合CellMarker的数据库以及其它文献的marker基因进行注释,得到了epithelial, stromal (fibroblasts and endothelial cells), and immune cells (T, NK, B, Myeloid, and Mast cells)
图D是细胞比例分布:NACT-ESCC 病人的免疫细胞(尤其是T、B细胞)比例很高,但它的上皮细胞和基质细胞比较少,说明NACT病人的宿主抗肿瘤免疫应答反应被激活
图E展示了几个通路的关键基因的热图:
  • cytokines和NF-kB相关基因(TNFSF13B, IL6, and NKFB1)在monocytes上调,说明monocytes与食管癌发生相关
  • hypoxia genes (e.g., LDHA, HIF1A, and EPO)在SA-ESCC病人中高于NACT-ESCC,说明化疗影响缺氧微环境
Cytokines, nuclear factor-kB (NF-kB), and hypoxia signaling pathways play an essential role in tumorigenesis
image-20211101101344645

然后看上皮

上皮细胞细分得到7个亚群:
  • Tumor epithelial cell markers (EPCAM, MDK, and SOX4) 在cluster1、6中高表达
  • non-malignant epithelial cell markers (KRT5 and KRT14)在cluster0、2、4、5高表达
  • cluster3同时表达tumor和normal的上皮细胞marker
同时CNV也显示tumor epi的malignant scores要比normal epi高(图C
另外,scpredR包发现基本没有误判的情况:
  • 86% (3662/4245) of malignant epithelial cells were not identified as healthy epithelial cells
  • 95% (6977/7324) of non-malignant epithelial cells were not identified as tumor epithelial cells
再次证明细胞分群注释的正确性
Could not load image
image-20211101102815761
然后是差异分析:
  • 预后差相关基因(SOX4 and MDK)在SA-ESCC-malignant epithelial cells富集
  • 免疫相关基因(IGLC2, FABP5, and S100A2)在non-malignant epithelial cells上调
  • SPRR3 and CEACAM6在NACT-malignant epithelial cells高表达
另外,non-malignant clusters也是存在异质性的,可以按起源继续分成2个cluster
然后是拟时序分析:
  • 最开始是non-malignant epithelial cells derived from SA-ESCC patients
  • 然后是 NACT-derived non-malignant epithelial cells
  • 然后分叉:一个是SA-ESCC-malignant epithelial cell,一个是epithelial cells from different sources (NACT-ESCC or SA-ESCC patients)
最后看不同cluster的通路活性差异
  • TME formation related pathways (e.g., hypoxia and angiogenesis)主要在non-malignant epithelial cells
  • ESCC development and progression (e.g., G2M-checkpoint and DNA-repair pathways)主要在 SA-ESCC malignant epithelial cell
  • inflammatory response, and IL2/IL6 related signaling pathways 在NACT-ESCC malignant epithelial cells和non-malignant epithelial都有表达

SA-ESCC和NACT-ESCC的细胞通讯

在SA-ESCC中,fibroblasts与其他类型细胞联系密切
在NACT-ESCC中,免疫细胞(尤其是T细胞和单核细胞)作用明显
针对这两种类型,然后分别对SA-ESCC的基质细胞和NACT-ESCC的免疫细胞取top50差异基因做通路,发现:
  • SA-ESCC的基质细胞主要调控extracellular matrix
  • NACT-ESCC的T和单核细胞主要抑制肿瘤发展
image-20211101104446457
接着做了代谢分析,发现:
  • Oxidative phosphorylation, glycolysis/gluconeogenesis, and citrate cycle (TCA cycle) pathways在SA-ESCC病人的恶性与非恶性细胞都上调
  • 而在NACT-ESCC中,只在非恶性细胞中上调
  • NACT-ESCC has a unique TME compared to SA-ESCC
  • mitochondria is a potential therapeutic target for SA-ESCC
image-20211101103900818

接着看基质细胞(内皮+成纤维)

基质细胞包括了19,977 and 6588 fibroblasts and endothelial cells (EDCs)
  • 内皮细胞的maker选取了CLDN5 and RAMP2,又细分为8个cluster,整合为4个子细胞类型:immune EDCs (CCL5 and CXCL13), lymphatic EDCs (PDPN and PROX1), tumor EDCs (ACKR1 and POSTN), and vascular EDCs (PLVAP and SLC9A3R2)【图A】
  • 成纤维细胞marker选取了C1R and COL1A2,得到11个cluster,整合为3个子细胞类型:COL14A1 matrix fibroblasts (COL14A1 and GSN), myofibroblasts (CTHRC1 and MMP11), and vascular smooth muscle cells (DES and MYH11)【图B】

看内皮细胞

在图A发现:NACT-ESCC的tumor EDC虽然少于SA-ESCC,但它的immune EDCs却高,于是接下来做了tumor EDC和immune EDC的GSVA和差异分析【图C、D】,发现:
  • metabolic pathways, including cholesterol homeostasis and fatty acid metabolism在NACT-ESCC的tumor EDCs中更高
  • cancer-promoting genes (e.g., S100A family genes) were highly expressed in SA-ESCC
  • HSP family genes were significantly elevated in the NACT-ESCC
  • marker genes for SA-ESCC derived immune EDCs were implicated in biological regulation and cell growth
  • marker genes for immune EDCs in the NACT-ESCC group were mainly involved in cellular and immune responses
HSP family genes promote cancer growth and metastasis in several tumors

看成纤维细胞

图B看到:
  • COL14A1+ matrix fibroblasts 主要在 NACT-ESCC
  • myofibroblasts 主要在 SA-ESCC
图F看到:
  • POSTN and WNT5A 在 myofibroblasts高表达,和Wnt and Notch signaling pathways相关
  • ACTA2在myofibroblasts and smooth muscle cells高表达
另外图G的GSVA也看到,Wnt and Notch signaling也是在myofibroblasts富集
image-20211101105031858

接着看髓细胞

基于CD68 and LYZ基因,拿到16,305 myeloid cells,继续细分为monocytes, macrophages, dendritic cells, and undefined myeloid cells
NACT-ESCC的macrophage细胞相对少,但monocyte多
图B选了一个marker基因Secreted phosphoprotein1 (SPP1),它与预后差相关,发现它主要在SA-ESCC的macrophage
图C想看从单核到巨噬细胞的转变过程:State 1 and 2包括了NACT-ESCC and SA-ESCC的巨噬细胞;但3和4主要就是NACT-ESCC
另外,GSVA看到anti-tumor (e.g., Interferon-gamma (IFN-gamma) and Interferon-alpha (IFN-alpha)) response pathways主要在NACT-ESCC富集;pro-tumor responses (e.g., KRAS and EMT pathways)在SA-ESCC富集,TNF-α-NF-κB pathway也是在SA-ESCC富集
图ESCENIC做了转录因子分析:
  • SA-ESCC中,NFIA, TWIST1, and MAFB是在macrophages中top3上调;RXRA, TGIF1, and XBP1在monocytes中上调
  • NACT- ESCC中,SPI1 and KLF4在macrophages中上调
图F中显示:PDCD1LG2 (PD1-L2), CD274 (PD-L1), CD276 (B7-H3), and LAG3这些与抑制CD8+ T 细胞活性相关的基因,在macrophages中上调;促炎相关免疫检查点基因(IL1A and IL6)在monocytes上调
Could not load image
image-20211101110603940

最后看B细胞和T细胞

根据CD79A and IGKC,拿到2999个B细胞,分成3个cluster,整合成2个亚型:Plasma B cells和follicular B cells
发现 plasma B cells在NACT-ESCC更多
图C做了富集分析,发现:
  • NACT-ESCC的plasma B cells会富集 interferon(干扰素) response-related pathways, MYC, and allograft(异源移植) rejection pathways
  • SA-ESCC的plasma B cells会富集unfolded protein responses and angiogenesis pathways
同样是转录因子分析:
  • STAT3 were elevated in follicular B cells
  • ATF3 were observed in plasma B cells
Could not load image
image-20211101111834562
对于T细胞,根据CD3D and CD3E拿到36,706 T cells,细分为10个cluster,整合成6个亚型,发现CD8+ T在NACT-ESCC中更多,不过NKT cells, CD8/CD4 mixed Th, and CD4+ T cells数量减少
图C发现:
  • naïve and co-stimulatory molecule markers在CD4+ T细胞上调
  • Cytokines, effector molecules and inhibitory receptors 在CD8+ and NK T上调
图D计算了Bhattacharyya距离,用于估计NACT-ESCC and SA-ESCC的T细胞亚型相似性,发现它们的差异主要来自CD8+ T细胞(图D)
然后将 CD8+ T 继续细分(图E),得到10个cluster,整合成3个亚型:cytotoxic CD8+ T cells, exhausted CD8+ T cells, and naïve CD8+ T cells。在NACT-ESCC中,exhausted CD8+ T cells and naïve CD8+ T占比明显升高,表示化疗改变了ESCC的免疫微环境
Could not load image
image-20211101112607678
之后又对 CD8+ T cells进行了拟时序分析,轨迹从naïve CD8+ T & cytotoxic CD8+ T cell开始,分叉之一是cytotoxic CD8+ T cells,另一个分叉是exhausted CD8+ T cells;
轨迹热图发现了3个时期:
  • phase 1主要是Naïve CD8+ and cytotoxic CD8+ T cells,GZMK and IL7R高表达,HAVCR2 and LAG3 低表达;
  • phase2 高表达STMN1, CENPE, and GNLY,包含了exhausted CD8+ and cytotoxic CD8+ T cel
  • phases 3 主要包括exhausted CD8+ T cells
另外,发现了SA-ESCC和NACT-ESCC免疫检查点相关的差异:PDCD1LG2基因在NACT-ESCC的exhausted CD8+ T cells高表达,而在SA-ESCC中是在cytotoxic CD8+ T中高表达,说明SA-ESCC and NACT-ESCC在免疫治疗反应方面存在差异
image-20211101124629150