第1部分基于空间转录组和单细胞转录组的肝内胆管癌肿瘤微环境探索

肿瘤内免疫浸润是影响肝内胆管癌（intrahepatic cholangiocarcinoma，ICC）中免疫效果的重要因素。然而，其表型及相关的空间结构仍不明确。为了解决这些局限性，我们开展了一项综合研究，结合了空间数据（来自6个样本的29,632个点）和单细胞数据（来自35个样本的21,158个细胞）。我们识别了两种不同的浸润模式：巨噬细胞阳性（标志为CD68和MARCO）和浆细胞阳性（标志为IGHG1和JCHAIN）。巨噬细胞阳性和浆细胞阳性标志分别显示了对ICC患者生存的不利和有利作用。值得注意的是，MARCO阳性肿瘤相关巨噬细胞（tumor-associated macrophage，TAM）被识别为巨噬细胞阳性样本中的主要细胞类型，表明了免疫抵抗性微环境。在MARCO+TAM中观察到了上调的上皮-间质转化活性、血管生成和缺氧反应。空间转录组学和整体转录组数据均显示了MARCO+TAM和CTSE+肿瘤细胞的共定位，并通过对20个ICC样本进行多重免疫荧光实验得到了验证。共定位区域展现出类似的促肿瘤通路和抑制的免疫反应。CTSE与肝内转移和血管侵犯存在关联。MARCO+TAM和CTSE+肿瘤细胞均与较差的生存率相关，并且在两种细胞类型高度浸润的患者中生存率最差。在共定位区域中，半乳糖凝集素信号通路在细胞-细胞通讯中最为活跃，主要的配体-受体对为LGALS9-CD44。本研究识别出巨噬细胞阳性和浆细胞阳性肿瘤内免疫浸润模式，以及MARCO+TAM和CTSE+肿瘤细胞的共定位是免疫抵抗的重要贡献因素。

第2部分基于空间转录组和单细胞转录组的肝内胆管癌肿瘤微环境探索

第1章DKK1阳性肿瘤细胞抑制CCL19+成纤维细胞和浆细胞的浸润导致HCC患者较差的免疫治疗反应

肿瘤内免疫浸润在肝细胞癌（hepatocellular carcinoma，HCC）中与肿瘤细胞的相互作用中起着关键作用。然而，其表型及相关的空间结构尚未明确。为解决这些局限性，我们进行了结合空间数据（来自8个样本的38,191个点）和单细胞数据（来自20个样本的56,022个细胞）的综合研究。我们的分析揭示了两种不同的浸润模式：免疫排斥型和免疫激活型。浆细胞在肿瘤内免疫亚群中作为主要细胞类型出现。值得注意的是，我们观察到CCL19+成纤维细胞与浆细胞的共定位，这些细胞分泌趋化因子并促进T细胞激活和白细胞迁移。相反，在免疫排斥样本中，此种共定位主要出现在相邻的正常区域内。通过对20个HCC样本进行多重免疫荧光验证，发现这种共定位与T细胞浸润和三级淋巴结构的形成相关。CCL19+成纤维细胞和浆细胞均与良好的生存预后相关。在免疫治疗队列中，对治疗反应良好的HCC患者表现出较高的CCL19+成纤维细胞和浆细胞浸润。此外，我们观察到在免疫排斥样本中，DKK1阳性肿瘤细胞在肿瘤区域内特别是肿瘤边缘处的聚集，抑制了CCL19+成纤维细胞和浆细胞向肿瘤区域的浸润。此外，在免疫排斥样本中，SPP1信号通路在肿瘤与免疫亚群之间的通讯中表现出最高的活性，而CCL19-CCR7在免疫亚群的自我通讯中发挥了关键作用。本研究揭示了HCC中的免疫排斥和免疫激活模式，并识别了导致免疫抵抗的相关因素。

第2章单细胞和空间分析揭示癌症干细胞与SPP1阳性巨噬细胞在缺氧区域的共定位决定了HCC的不良预后

在肝细胞癌（hepatocellular carcinoma，HCC）中，经典的癌症干细胞（cancer stem cells, CSCs）标志物与正常干细胞标志物相同，对其进行靶向治疗可能会阻碍肝脏再生并导致肝功能衰竭。此外，CSC相关的空间结构尚不清晰。为解决这些问题，我们结合单细胞数据（来自20个样本的56,022个细胞）和空间数据（来自8个样本的38,191个点）进行了综合研究，以获得CSC特征并揭示其空间结构。利用CytoTRACE算法，我们准确地识别了CSC，其表现出由缺氧诱导因子1A（hypoxia inducible factor 1A，HIF1A）调控的增殖通路上调。通过加权基因共表达网络分析（weighted gene co-expression network analysis，WGCNA），我们开发了由107个基因组成的CSC特征。值得注意的是，CSC水平高的HCC患者表现出富集了表达金属蛋白酶（MMP9、MMP12和MMP7）的SPP1阳性巨噬细胞（Macro_SPP1），这些金属蛋白酶的表达受HIF1A调控，表明存在连接Macro_SPP1和CSC的缺氧肿瘤区域。CSC和Macro_SPP1均与较差的预后和不良的免疫治疗反应相关。空间分析揭示了CSC和Macro_SPP1的共定位，同时CD8阳性T细胞被排除在肿瘤区域之外。该共定位区域及边界的非肿瘤区域表现出高度缺氧，同时在该区域中高表达HAVRC2探索点。在共定位区域内，SPP1信号通路在细胞间通信中最为活跃，SPP1-CD44和SPP1-ITGA/ITGB被确定为主要的配体-受体对。本研究成功构建了CSC特征，并在缺氧区域中展示了CSC与Macro_SPP1的共定位，加剧了HCC的肿瘤微环境恶化。

Part 1: Exploration of the tumor microenvironment in intrahepatic cholangiocarcinoma based on spatial transcriptomics and single-cell transcriptomics

Intra-tumor immune infiltration is a crucial element interacting with tumor cells in intrahepatic cholangiocarcinoma (ICC). However, its phenotype and related spatial structure remained elusive. To address these limitations, we undertook a comprehensive study combining spatial data (29,632 spots from six samples) and single-cell data (21,158 cells from 35 samples). We identified two distinct infiltration patterns: macrophage+ (characterized by CD68 and MARCO) and plasma cell+ (characterized by IGHG1 and JCHAIN). The macrophage+ and plasma cell+ signatures showed adverse and favorable roles in ICC patients’ survival, respectively. Notably, MARCO+ tumor-associated macrophage (TAM) was recognized as the main cell type in macrophage+ samples, indicating an immune-resistant microenvironment. Increased epithelial-mesenchymal transition activities, angiogenesis, and hypoxia were observed in MARCO+ TAM. The co-location of MARCO+ TAM and CTSE+ tumor cells was observed in spatial transcriptomics and bulk transcriptomics data, validated by multiplex immunofluorescence performed on twenty ICC samples. The co-location area exhibited similar protumorigenic pathways and suppressed immune response. CTSE exhibited associations with intrahepatic metastasis and vascular invasion. Both MARCO+ TAM and CTSE+ tumor cells were associated with worse survival and patients with high infiltration of two cell types displayed the worst survival. Within the co-location area, the galectin signaling pathway was most active in cell-cell communication, with LGALS9-CD44 identified as the main ligand-receptor pair. This study identified macrophage+ and plasma cell+ intra-tumor immune infiltration patterns and the co-location of MARCO+ TAM and CTSE+ tumor cells as contributors to immune resistance.

Part 2: Exploration of the tumor microenvironment in hepatocellular carcinoma based on spatial transcriptomics and single-cell transcriptomics

Chapter 1: DKK1+ tumor cells inhibited the infiltration of CCL19+ fibroblasts and plasma cells contributing to worse immunotherapy response in hepatocellular carcinoma

Intra-tumor immune infiltration plays a pivotal role in the interaction with tumor cells in hepatocellular carcinoma (HCC) However, its phenotype and related spatial structure remained elusive. To address these limitations, we conducted a comprehensive study combining spatial data (38,191 spots from eight samples) and single-cell data (56,022 cells from 20 samples). Our analysis revealed two distinct infiltration patterns: immune exclusion and immune activation. Plasma cells emerged as the primary cell type within intra-tumor immune clusters. Notably, we observed the co-location of CCL19+ fibroblasts with plasma cells, which secrete chemokines and promote T cell activation and leukocyte migration. Conversely, in immune-exclusion samples, this co-location was primarily observed in the adjacent normal area. This co-localization correlated with T cell infiltration and the formation of tertiary lymphoid structures, validated by multiplex immunofluorescence conducted on twenty HCC samples. Both CCL19+ fibroblasts and plasma cells were associated with favorable survival outcomes. In an immunotherapy cohort, HCC patients who responded favorably exhibited higher infiltration of CCL19+ fibroblasts and plasma cells. Additionally, we observed the accumulation of DKK1+ tumor cells within the tumor area in immune-exclusion samples, particularly at the tumor boundary, which inhibited the infiltration of CCL19+ fibroblasts and plasma cells into the tumor area. Furthermore, in immune-exclusion samples, the SPP1 signaling pathway demonstrated the highest activity in communication between tumor and immune clusters, and CCL19-CCR7 played pivotal role in the self-communication of immune clusters. This study elucidates immune exclusion and immune activation patterns in HCC and identifies relevant factors contributing to immune resistance. 

Chapter 2: Single-cell and spatial analyses revealed the co-location of cancer stem cells and SPP1+ macrophage in hypoxic region that determines the poor prognosis in hepatocellular carcinoma 

In hepatocellular carcinoma (HCC), classical cancer stem cells (CSC) markers were shared by normal stem cells, targeting which may hinder hepatic regeneration and cause liver failure. Additionally, the spatial structure of CSC still remained elusive. To address these limitations, we undertook a comprehensive study combining single-cell data (56,022 cells from 20 samples) and spatial data (38,191 spots from eight samples) to obtain CSC signature and uncover its spatial structure. Utilizing the CytoTRACE algorithm, we discretely identified CSC, which displayed upregulated proliferation pathways regulated by HIF1A. A CSC signature of 107 genes was then developed using Weighted Gene Co-expression Network Analysis (WGCNA). Notably, HCC patients with high CSC levels exhibited an accumulation of SPP1+ macrophages (Macro_SPP1) expressing metalloproteinases (MMP9, MMP12, and MMP7) regulated by HIF1A, suggesting a hypoxic tumor region connecting Macro_SPP1 and CSC. Both CSC and Macro_SPP1 correlated with worse prognosis and undesirable immunotherapy response. Spatial analysis revealed the co-location of CSC and Macro_SPP1, with CD8 T cells excluded from the tumor region. The co-location area and non-tumor area of boundary exhibited a high level of hypoxia, with the HAVRC2 checkpoint highly expressed. Within the co-location area, the SPP1 signaling pathway was most active in cell-cell communication, with SPP1-CD44 and SPP1-ITGA/ITGB identified as the main ligand-receptor pairs. This study successfully constructed a CSC signature and demonstrated the co-location of CSC and Macro_SPP1 in a hypoxic region that exacerbates the tumor microenvironment in HCC.