Table_1_Spatially resolved transcriptomics revealed local invasion-related genes in colorectal cancer.xlsx

ObjectiveLocal invasion is the first step of metastasis, the main cause of colorectal cancer (CRC)-related death. Recent studies have revealed extensive intertumoral and intratumoral heterogeneity. Here, we focused on revealing local invasion-related genes in CRC. MethodsWe used spatial transcriptomic techniques to study the process of local invasion in four CRC tissues. First, we compared the pre-cancerous, cancer center, and invasive margin in one section (S115) and used pseudo-time analysis to reveal the differentiation trajectories from cancer center to invasive margin. Next, we performed immunohistochemical staining for RPL5, STC1, AKR1B1, CD47, and HLA-A on CRC samples. Moreover, we knocked down AKR1B1 in CRC cell lines and performed CCK-8, wound healing, and transwell assays to assess cell proliferation, migration, and invasion.ResultsWe demonstrated that 13 genes were overexpressed in invasive clusters, among which the expression of CSTB and TM4SF1 was correlated with poor PFS in CRC patients. The ribosome pathway was increased, while the antigen processing and presentation pathway was decreased along CRC progression. RPL5 was upregulated, while HLA-A was downregulated along cancer invasion in CRC samples. Pseudo-time analysis revealed that STC1, AKR1B1, SIRPA, C4orf3, EDNRA, CES1, PRRX1, EMP1, PPIB, PLTP, SULF2, and EGFL6 were unpregulated along the trajectories. Immunohistochemic3al staining showed the expression of STC1, AKR1B1, and CD47 was increased along cancer invasion in CRC samples. Knockdown of AKR1B1 inhibited CRC cells’ proliferation, migration, and invasion.ConclusionsWe revealed the spatial heterogeneity within CRC tissues and uncovered some novel genes that were associated with CRC invasion.

本研究旨在揭示结直肠癌（CRC）中与局部侵袭相关的基因。方法上，我们运用空间转录组技术对四例CRC组织的局部侵袭过程进行研究。首先，在S115切片中，我们对癌前病变、癌中心以及侵袭边缘进行了比较，并采用伪时间分析技术揭示从癌中心至侵袭边缘的分化轨迹。接着，我们对CRC样本进行了RPL5、STC1、AKR1B1、CD47和HLA-A的免疫组化染色。此外，我们在CRC细胞系中敲低AKR1B1，并通过CCK-8、伤口愈合和Transwell实验评估细胞增殖、迁移和侵袭能力。结果显示，侵袭簇中过表达13个基因，其中CSTB和TM4SF1的表达与CRC患者的无病生存期（PFS）不良相关。在CRC进展过程中，核糖体通路增强，而抗原处理和呈递通路减弱。在CRC样本中，RPL5表达上调，而HLA-A表达下调。伪时间分析显示，STC1、AKR1B1、SIRPA、C4orf3、EDNRA、CES1、PRRX1、EMP1、PPIB、PLTP、SULF2和EGFL6沿轨迹未发生调控。免疫组化染色显示，STC1、AKR1B1和CD47的表达随CRC侵袭过程增加。敲低AKR1B1抑制了CRC细胞的增殖、迁移和侵袭。结论部分，我们揭示了CRC组织内部的时空异质性，并发现了与CRC侵袭相关的一些新型基因。