| Background and significance:Colorectal cancer is one of the most common malignant tumors in the digestive tract.In 2020,the latest global cancer burden data released by the International Agency for Research on Cancer(IARC)of the World Health Organization(WHO)shows that the number of new cases of colon cancer in 2020 will be the third in the world,and China will be the second.Clinical data show that the number of colon cancer patients has increased significantly in the past 20 years.How to carry out early screening and inhibit the progression of colon cancer has become an urgent challenge for medical science to overcome.Therefore,it is of great clinical significance to study how to carry out early screening and control of colon cancer.Colorectal cancer is one of the most common gastrointestinal malignancies,and the latest global cancer burden data released by the World Health Organization’s International Agency for Research on Cancer(IARC)in 2020 indicates that the number of new cases of colon cancer will rank third in the world and second in China in 2020.Clinical data show that the number of colon cancer patients has increased significantly in the past 20 years,and how to screen colon cancer at an early stage and inhibit the deterioration of the disease has become an urgent challenge for today’s medicine.Therefore,it is of great clinical importance to study how to screen and control colon cancer at an early stage.In this project,we focus on single exosome clustering of plasma exosomes from normal subjects,patients with in situ colon cancer(stage Ⅱ)and patients with liver metastases from colon cancer(stage Ⅳ).The single exosome clustering is based on some protein molecular markers on the exosome membrane,and the plasma exosomes with different markers are clustered by machine learning to trace which exosomes are changed in colon cancer patients relative to normal subjects.We found that cancer cell-derived ITGB3rich exosomes were significantly increased in the plasma of metastatic colon cancer patients,while monocyte/macrophage-derived ITGAMrich exosomes were largely absent in the plasma of colon cancer patients.We then verified the functions of these two sources of exosomes and found that ITGAMrich exosomes inhibited the proliferation,migration and invasion of colon cancer cells while ITGB3rich exosomes promoted the proliferation,migration and invasion of colon cancer cells.Further study found that the expression of ITGAM in plasma exosomes decreased sequentially in healthy subjects,adenoma patients and patients with in situ colon cancer,which provided a new idea for early screening of colon cancer,and plasma exosome ITGB3 expression was significantly higher in patients with liver metastasis,suggesting that the expression level of ITGB3rich exosomes found new markers and therapeutic measures for the metastasis and treatment of colon cancer.Methods:Using single exosome fractionation technique,exosomes from different sources in human plasma were fractionated to identify the subpopulations of exosomes that changed in the plasma of healthy individuals and colon cancer patients.Western blot was then used to validate the results.PKH26 stained exosomes and DAPI stained nuclei to verify the uptake of exosomes.Plasma exosomes from normal subjects,plasma exosomes from colon cancer patients with liver metastases,THP-1 and mononuclear macrophage-derived exosomes and lentivirus-transfected ITGB3 overexpressed exosomes were extracted and then incubated with HCT116 and SW480 human colon cancer cell lines to observe the proliferation,migration and invasion changes of cancer cellsResults:The proportion of ITGAMrich exosome subsets in the plasma of normal subjects was 22.79%,but it was significantly lower in the plasma of patients with liver metastases from in situ and colon cancer at 0.79%and 1.28%,respectively.In contrast,the proportion of ITGB3rich exosome subpopulation in the plasma of patients with liver metastases from colon cancer was 76.34%,which was much higher than that of normal subjects and patients with in situ colon cancer,27.71%and 32.76%,respectively.western blot results also confirmed that the expression of ITGAM in plasma exosomes of normal subjects was indeed higher than that of patients with colon cancer,and in plasma exosomes of patients with liver metastases from colon cancer The expression of ITGB3 was higher in plasma exosomes of patients with liver metastases than in normal subjects and patients with colon cancer in situ.Cell function assays including colony formation assays and cell migration and invasion assays showed that normal human plasma exosomes and THP-1 macrophage and monocyte-derived exosomes inhibited the proliferation,migration and invasion of colon cancer,while cancer cell-derived exosomes overexpressing ITGB3 significantly promoted the proliferation,migration and invasion of cancer cellsConclusion:1.The expression of ITGAMnch and ITGB3rich exosomes in the plasma exosomes of normal individuals and patients with colon cancer in situ and colon cancer liver metastases differed significantly.The percentage of plasma ITGAM-positive exosome subsets was significantly higher in normal subjects than in patients with colon cancer,whereas the percentage of ITGB3-positive exosome subsets was significantly increased in the plasma of patients with liver metastases from colon cancer.2.ITGAM-positive exosome subsets of mononuclear macrophage origin significantly inhibited the proliferation,migration and invasion of colon cancer cells,while ITGB3-positive exosome subsets of cancer cell origin promoted the proliferation migration and invasion of colon cancer cells.3.The expression level of ITGAM in plasma exosomes gradually decreased in normal subjects,precancerous lesions(adenoma patients)and patients with in situ colon cancer,suggesting that it may be used as an early cancer marker for clinical primary screening of colon cancer. |
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