| Background:Colorectal cancer is one of the most common malignancies of the digestive tract.According to the latest epidemiological data,colorectal cancer ranks third in incidence of all cancers and second in mortality.Although the prognosis of colorectal cancer patients has been significantly improved with the continuous development of diagnosis and treatment technology,their mortality rate is still rising.Metastasis is the leading cause of death in colorectal cancer patients,and the liver is the main target organ for colorectal cancer metastasis.Once it occurs,the patient’s prognosis drops dramatically.There are still no effective means to reduce the occurrence of liver metastases and improve the prognosis of patients with liver metastases.Exploring metastatic mechanisms is of great significance in both clinical and basic colorectal cancer research.Colorectal cancer is a highly heterogeneous disease,in addition to the differences between different individuals,there is also heterogeneity inside the same tumor,which is closely related to tumor recurrence,drug resistance,metastasis,etc.Tumor stem cells are one of the main causes of heterogeneity formation within tumors.The study of intratumoral heterogeneity and tumor stem cells is of great significance for exploring the mechanism of liver metastasis in colorectal cancer.The development of various sequencing techniques in recent years has greatly changed the pattern of tumor research.Analyzing the gene expression profile of patients through high-throughput sequencing can help clinicians better carry out personalized and precise treatment.However,conventional sequencing methods cannot combine the expression results of genes with the original spatial information of the cell.The spatial position information of cells is of great significance for the differentiation of cells and the interaction between cells during tumorigenesis and development.Spatial transcriptome sequencing is an emerging sequencing technique that provides "tissue spatial information" for gene expression to understand the relative positional relationship between specific cells and tissue sections,providing a new perspective on tumor research.In this study,we intend to use the emerging technology of spatial transcriptome sequencing to detect liver metastasis of colorectal cancer,and conduct in-depth research on the mechanism of liver metastasis through bioinformatics analysis and cell biology experiments.MethodWe collected 2 tissues each from the surgical samples of the primary and liver metastases of colorectal cancer,and the above 4 tissues were measured by 10 x Genomics through transcriptome technology.The obtained sequencing data were first data-based,followed by principal component analysis of all detected spots for primary dimensionality reduction and unified manifold approximation and projection for secondary dimensionality reduction and analysis of gene expression profiling to explore heterogeneity within primary and metastatic lesions of colorectal cancer.The cell cycle,EMT and other biological behaviors of Clusters were analyzed to explore the changes of biological characteristics and key differential genes in Cluster.Pseudo-timing analysis of each Cluster was then performed to explore the cell development trajectory during tumor metastasis and identify key genetic changes.Cellphone DB algorithm was used to analyze the cell-to-cell interactions in primary and metastatic foci,and to explore the patterns of cell-to-cell communication.Combined with the TCGA database and clinical samples from our department,the key genes identified above were prognostically analyzed and the above findings were validated using cell lines.Research result:First of all,we collected 2 pieces of primary and metastatic tissue that were removed at the same time as liver metastasis of colorectal cancer.The above 4 tissues were measured by 10 x Genomics for spatial transcriptomic sequencing,and after the data obtained were quality controlled and clustered,the 4 tissues were clustered into 19 Clusters,of which 19 Clusters could be divided into 6 categories,namely primary colonic carcinoma,hepatic metastasis colonic carcinoma,paracancerous enterocyte,paracancerous hepatic tissues,mesenchymal cells and lymphatic follicles,and each Cluster has its characteristic gene expression and unique biological function.The number of genes detected in Cluster,where the tumor tissue is located,is significantly higher than that of other tissues.Subsequently,we analyzed the cell cycle and partial epithelial mesenchymal transformation status of each Cluster,compared the gene expression differences of different Clusters,and found that the tumor stem cell-related markers in the metastases were enriched in Cluster12 and accompanied by elevated FOXD1 expression.Subsequently,all spots were traced and pseudo-time found that all tumor spots showed 2 differentiation trajectories,combined with all the spatial information of spot,it was found that in the process of metastatic foci formation,tumor cells first underwent dedifferentiation to form metastases,and then underwent re-differentiation in metastases to colonize them in metastases.Among them,Cluster12 showed the lowest differentiation state,which was in line with the characteristics of high expression of its tumor stem cell marker.Further analysis of the gene expression of Cluster12 showed that the Hippo pathway and WNT/β-catenin showed activation,suggesting that FOXD1,Hippo pathway and WNT/β-catenin pathway were associated in maintaining tumor stemness.Subsequently,cell-to-cell interactions were analyzed using Cellphone DB and it was found that CD74-MIF had significant interactions between tumor cells in metastatic tissue.Based on the above research and found that FOXD1 is expressed in cluster12 in metastatic foci,we first used the TCGA database to find that foxd1 high expression patients had poor prognosis,and combined FOXD1 with clinical pathological information,the prognostic prediction model constructed had good predictive efficacy.We then constructed a cell line with FOXD1 knockdown and overexpression,and found that FOXD1 overexpression caused increased YAP and β-catenin expression,while knocking down FOXD1 reduced the expression of YAP and β-catenin,and FOXD1 could regulate the secretion of MIF by tumor cells.In summary,we conducted in-depth research on colorectal cancer liver metastasis using spatial transcription sequencing technology,and the results showed that tumor stem cells were enriched in liver metastases,and the tumors underwent the process of decentralization and re-differentiation during metastasis.In addition,we found that the transcription factor FOXD1 regulates hippo and WNT/β-catenin pathways involved in colorectal cancer metastasis.The above results provide a new research direction for the study of liver metastasis in colorectal cancer. |
