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
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】
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E用SCENIC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
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
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
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