Spatial transcriptomic profiling of precursor lesions of gallbladder adenocarcinoma. Spatial transcriptomic profiling of precursor lesions of gallbladder adenocarcinoma

Genomic and epigenomic studies support that adenocarcinomas of the gallbladder develop according to a metaplasia-biliary intraepithelial neoplasia (BilIN)-adenocarcinoma histogenic sequence, in which metaplasia and BilIN are non-cancerous lesions of the epithelium. Moreover, recent genomic data suggest that adenocarcinoma can develop in a BilIN-dependent or -independent way, pointing toward patient-specific tumourigenic processes. Spatial transcriptomic data addressing these processes are still missing. Here, using GeoMx digital spatial profiling (NanoString), we characterized the spatial transcriptome of normal gallbladder epithelium, BilINs and adenocarcinoma coexisting within the same samples. To address intra-patient variability we profiled the whole transcriptome of a high number of regions of interest (ROIs) per sample in two patients (Patient #1, 81 year old woman: 24 ROIs; Patient #2, 53-year old man:32 ROIs), with each ROI covering approximately 200 cells of normal epithelium, low-grade BilIN, high-grade BilIN or adenocarcinoma. Our results showed that each type of lesion displayed little transcriptomic variability within the same patient, but differed significantly between patients. They also suggested that adenocarcinoma can derive from high-grade BilIN or from low-grade BilIN, with co-existing high-grade BilIN evolving via a distinct process in the latter case. We also provide strong evidence for patient-specific tumourigenic mechanisms, characterized by distinct sequences of signalling pathway activation. Among those pathways, we functionally investigated SEMAPHORIN 4A (SEMA4A) and provided evidence that repression of SEMA4A expression, as is observed in the gallbladder samples of the two patients, can enhance cell migration and survival, and perturb polarisation of the epithelial cells. In conclusion, our results support that gallbladder adenocarcinoma develops according to patient-specific processes that can be promoted by repression of SEMA4A. They underscore the need to gain gene expression data in addition to histological information to evaluate the risk of low-grade preneoplastic lesions. Overall design: We investigated formalin-fixed paraffin-embedded sections from two patients. Patient #1, gallbladder section: normal epithelium, 4 regions of interest (ROIs); Low-grade BilIN, 8 ROIs; High-grade BilIN, 6 ROIs; Adenocarcinoma, 6 ROIs. Patient #2, gallbladder section 1: normal epithelium, 8 ROIs. Patient #2, gallbladder section 2: Low-grade BilIN, 6 ROIs; High-grade BilIN, 12 ROIs; Adenocarcinoma, 6 ROIs. *** FASTQ raw data files have been requested. ***

基因组与表观基因组研究证实，胆囊腺癌遵循化生-胆道上皮内瘤变（biliary intraepithelial neoplasia, BilIN）-腺癌的组织发生序列发展，其中化生与BilIN均为上皮源性非癌性病变。此外，近期基因组学数据显示，腺癌可通过依赖或不依赖BilIN的途径发生，提示肿瘤发生过程存在显著的患者特异性。目前针对该过程的空间转录组学研究数据仍较为匮乏。本研究借助GeoMx数字空间分析（NanoString公司）技术，对同一样本中共存的正常胆囊上皮、BilIN及腺癌的空间转录组进行了表征分析。为解析患者内部的异质性，本研究对两名患者的每一样本设置了大量感兴趣区域（regions of interest, ROIs）并开展全转录组分析：患者1为81岁女性，共设置24个ROIs；患者2为53岁男性，共设置32个ROIs。每个ROI覆盖约200个细胞，对应正常上皮、低级别BilIN、高级别BilIN或腺癌组织。研究结果显示，同一患者体内的各类病变组织内部转录组异质性较低，但不同患者间的转录组特征存在显著差异。此外结果提示，腺癌可起源于高级别BilIN或低级别BilIN；当腺癌起源于低级别BilIN时，共存的高级别BilIN会通过独特的演化进程进展。本研究还为患者特异性的肿瘤发生机制提供了有力证据，这类机制以信号通路激活的独特序列为核心特征。在上述通路中，我们对信号素4A（SEMAPHORIN 4A, SEMA4A）开展了功能研究，结果证实，如两名患者胆囊样本中所观察到的那样，SEMA4A表达抑制可增强细胞迁移与存活能力，并扰乱上皮细胞的极化状态。综上，本研究结果表明，胆囊腺癌的发生遵循患者特异性的进程，且该进程可因SEMA4A表达抑制而得到促进。本研究同时强调，除组织学信息外，还需获取基因表达数据以评估低级别癌前病变的风险。研究整体设计：本研究对两名患者的福尔马林固定石蜡包埋（FFPE）胆囊组织切片进行分析。患者1的胆囊切片：正常上皮4个ROIs、低级别BilIN 8个ROIs、高级别BilIN 6个ROIs、腺癌6个ROIs。患者2的胆囊切片1：正常上皮8个ROIs；患者2的胆囊切片2：低级别BilIN 6个ROIs、高级别BilIN 12个ROIs、腺癌6个ROIs。*** FASTQ原始数据文件已申请获取。***