Kisakol Batuhan, Sturrock Anna, Cho Sanghee, Azimi Mohammadreza, Lindner Andreas U, Salvucci Manuela, McDonough Elizabeth, Fay Joanna, O'Grady Tony, McCawley Niamh, Burke John P, McNamara Deborah A, Graf John, McDade Simon, Longley Daniel B, Ginty Fiona, Prehn Jochen H M
Department of Physiology and Medical Physics & RCSI Centre for Systems Medicine, RCSI University of Medicine and Health Sciences, 123 St Stephen's Green, Dublin 2, Ireland.
GE HealthCare Technology & Innovation Center (Formerly GE Research Center), 1 Research Circle, Niskayuna, NY, 12309, U.S.A.
bioRxiv. 2025 Jul 8:2025.05.07.652478. doi: 10.1101/2025.05.07.652478.
Identification of the consensus molecular subtypes (CMS) opened significant potential for understanding the tumor biology and intertumoral heterogeneity of colorectal cancer (CRC). However, molecular subtyping in CRC traditionally relies on bulk transcriptomics, therefore, lacks spatial and single-cell level aspect.
We constructed tissue microarrays using tumor cores from 222 CRC patients. Arrays were stained and imaged using 54 cell identity and cancer hallmark markers, delivering spatially resolved protein profiles of >2 million cells. RNA sequencing data and CMS classification were also available for these patients. After segmentation of cancer, stromal and immune cells, we investigated intratumoral heterogeneity within CMS subtypes using spatially resolved single-cell protein profiling (>2 million cells). We compared cell types, their spatial organization and their expression of cancer hallmark-related proteins in CMS 1-4 subtypes.
We revealed tissue atlases illustrating the cell types/states, spatial heterogeneity, cellular neighborhoods, cellular network, and single-cell protein profiles of CMS tumors. CMS1 tumors had more CD3, CD8, and PD1 immune cells that were found in the epithelial layer frequently. CMS1 was also associated with higher levels of metabolic reprogramming markers such as upregulated glycolysis. CMS2 showed immune segregation, reactive stroma patterns and higher levels of apoptotic and proliferative signaling proteins. CMS3 exhibited clustered cancer cells with high RIP3 levels, suggesting a pro-inflammatory microenvironment. CMS4 displayed stromal-centric and immune-evasive tumors characterized by decreased HLA-1 levels and regulatory T-cell exclusion from epithelium.
We present a spatial protein atlas of CRC at single-cell resolution and demonstrate novel aspects of CMS tumour structures.
共识分子亚型（CMS）的鉴定为理解结直肠癌（CRC）的肿瘤生物学和肿瘤间异质性开辟了巨大潜力。然而，CRC的分子亚型传统上依赖于批量转录组学，因此缺乏空间和单细胞水平的研究。
我们使用222例CRC患者的肿瘤核心构建了组织微阵列。使用54种细胞标志物和癌症特征标志物对阵列进行染色和成像，获得超过200万个细胞的空间分辨蛋白质谱。这些患者也有RNA测序数据和CMS分类。在对癌症、基质和免疫细胞进行分割后，我们使用空间分辨单细胞蛋白质谱（超过200万个细胞）研究了CMS亚型内的肿瘤内异质性。我们比较了CMS 1-4亚型中的细胞类型、它们的空间组织以及它们与癌症特征相关蛋白的表达。
我们展示了组织图谱，阐明了CMS肿瘤的细胞类型/状态、空间异质性、细胞邻域、细胞网络和单细胞蛋白质谱。CMS1肿瘤有更多的CD3、CD8和PD1免疫细胞，这些细胞经常在上皮层中发现。CMS1还与更高水平的代谢重编程标志物相关，如糖酵解上调。CMS2显示出免疫隔离、反应性基质模式以及更高水平的凋亡和增殖信号蛋白。CMS3表现出具有高RIP3水平的聚集癌细胞，提示促炎微环境。CMS4表现为以基质为中心和免疫逃逸的肿瘤，其特征是HLA-1水平降低以及调节性T细胞被排除在上皮之外。
我们展示了单细胞分辨率的CRC空间蛋白质图谱，并揭示了CMS肿瘤结构的新方面。
