The Role Of VEGFR1Positive Hematopoietic Progenitor Cells In Liver Metastasis Of Colorectal Cancer

Background and objectives:Colorectal cancer is the one of the most common malignancies in thedigestive tract with its incidence increasing every year. Distant metastasesare the most important causes of patients’ death. Liver is the major targetorgan of metastasis for colorectal cancer. About20%to25%of patientswere found to have hepatic metastasis when the primary colorectal tumorwas first detected. Even after radical resection, about8%to30%ofpatients will eventually develop liver metastases, and8%to10%ofpatients will have lung metastases. Therefore, early detection, proactivelyprevention and treatment of distant metastases has been one of the keymeasurements to improve long survival of patients with colorectal cancer.Researches have shown that the metastasis of tumor is a complex processwith multiple steps tumorigenesis, multiple factors participation, andmultiple links adjustment. Even the researches on tumor metastasis havebeen put forward in-depth, however, the exact molecular mechanisms oftumor metastasis is still unclear up to now. Especially about themechanisms of organ-specific spread of tumor, also known as homingmetastasis has been the focus and still the hard nut of researches. In2005, Kaplan et al first reported that the VEGFR1positivehematopoietic progenitor cells (VEGFR1~+HPC), identified as VEGFR1~+and CD133~+double-positive cells, play an important role in thedirectional transferring of cancer cells. Studies have shown that beforethe occurrence of tumor metastasis, VEGFR1~+HPC have reached the premetastatic sites and change the microenvironments of target organs toadapt to the growth of tumor cells. Further, they direct the transferringof tumor cells to the target organs through a specific ligand-receptorbinding. The current project was designed to observe the expression anddistribution of VEGFR1~+HPC in the primary tumor and liver metastasiswith an aim to explore the relationships between VEGFR1~+HPC andliver metastasis. Further, we establish the liver metastasis animal modelof colorectal cancer cells, In the process of metastases developing, wedynamicly observe the quantity changes of VEGFR1~+HPC in the primarytumor tissue, the liver, and the bone marrow to investigate the role ofVEGFR1~+HPC in directional hepatic spread of colorectal cancer.Methods:1. Clinical research: Thirty-seven specimens resected from patientswith colorectal cancer and liver metastasis, and six specimens of colonfrom patients with non-neoplastic diseases from2006to2011in SichuanCancer Hospital were included in the study. The method ofimmunohistochemical staining was used to detect the expression and distribution of VEGFR1and CD133in colorectal primary tumor, thetumor positive lymph nodes, the negative lymph nodes, and the hepaticmetastatic lesions.2. Animal research: We established the mouse liver metastasismodel of colon cancer HCT-116cell lines by cecal orthotopicimplantation of tumor tissues. Based on that, we detected the expressionof VEGFR1and CD133in the primary tumor and liver metastatic sites(or normal liver tissues) by immunohistochemical staining, andinvestigated the quantity changes of VEGFR1~+HPC (VEGFR1~+/CD133~+)cells in the liver and bone marrow by flow cytometry, before tumorinoculation and2weeks,4weeks,6weeks,8weeks after transplantation,respectively.Results:1. VEGFR1~+HPC cells clusters were found both in primarycolorectal cancer and adjacent tissues, and the positive rate of colorectalcancer tissues was significantly higher than that of the adjacent tissues (P<0.05). Highly expression of VEGFR1~+HPC cells was also observed inthe metastatic lesions of liver, and its positive rate was higher than theadjacent tissues (P <0.05). VEGFR1~+HPC cells were not found in thelymph nodes of six patients with non-neoplastic diseases. Contrary to that,appearance of VEGFR1~+HPC cells was detected in both tumor positiveand negative lymph nodes, which located mainly in the subcapsular region and the marginal sinus. In the metastatic nodes, VEGFR1~+HPCcells appear in flake, while in the non-metastatic nodes, VEGFR1~+HPCcells scatter in foci or cluster. The positive rate of the metastatic ones washigher than that of the non-metastatic ones (P <0.05).2. Tumor formation in the cecum site was found in all the mice2weeks,4weeks,6weeks, and8weeks after tumor block orthotopictransplantation of the HCT-116cells, which ranges from1to3.5cm indiameter and grows invasively along the intestinal tube. Metastasesappear in6weeks after transplantation, which were located mainly in theliver, and could be found in the lung, spleen, and peritoneal cavitysimultaneously.3. Immunohistochemical staining has shown that the VEGFR1~+HPCcell clusters were not found in the liver of the mice before thetransplantation, and on2and4weeks after transplantation, a smallamount of VEGFR1~+HPC cells clusters begin to appear, before theformation of liver metastases, which were in dot, or focal distributionmainly near the portal area. On6and8weeks after transplantation, whenthe hepatic, splenic, and/or pulmonary metastases develop, the quantity ofHPC cells also increase significantly, which appear in flake mainly in themetastatic sites, peri-tumor tissue, and hepatic portal area.4. Flow cytometry has also demonstrated that the quantity ofVEGFR1~+HPC cells in both liver and bone marrow increase successively from the point before tumor inoculation and2weeks,4weeks,6weeks,8weeks after transplantation (P <0.05).Conclusions:1. The expression level and rate of VEGFR1~+HPC in colorectalprimary tumor and liver metastasis foci was significantly higher than thatof the adjacent tissues. VEGFR1~+HPC cells were found both in tumorpositive and negative lymph nodes. However, the number of VEGFR1~+HPC cells increased significantly in the metastatic lymph nodes, whichlocated mainly in the subcapsular region and marginal sinus, commonsites that tumor cells generally metastasize to. These findings suggest thatVEGFR1~+HPC cells probably have gathered in the pre-metastatic foci ofcertain lymph nodes before the occurrence of tumor metastasis.2. The method of tumor block cecum orthotopic transplantation byHCT-116cell lines is compatible with the biology characteristics of thecolorectal cancer cells, which mimics the clinical natural processes ofcolorectal cancer metastasizing, with stable tumorigenecity and highspread incidence, and serve to be an ideal animal model to studycolorectal cancer metastasizing.3. Studies from the animal models have shown that the VEGFR1~+HPC has gathered in the liver before the formation of liver metastases andwith progressing of metastasis, the number of VEGFR1~+HPC increasedaccordingly, which indicates that VEGFR1~+HPC might play a role of orientation and guidance in the process of colorectal cancer metastasizingto liver.4. Researches on the animal have also demonstrated that the numberof VEGFR1~+HPC in the bone marrow increases simultaneously,consistent with its’ changes in the liver, which indicates that VEGFR1~+HPC gathering in the liver might resulted from the bone marrowmobilization.