Establishment Of Tissue-like Hepatocellular Carcinoma Models, Lung-invasive Related Protein Screening

Two Dimensional (2D) cell culture model have good application in Hepatocellular carcinoma (HCC) study and three dimensional (3D)culture model have advanced the existing research for the tissue-like characteristic. There have been short of the experimental models under 3D conditions to study the HCC invasive and metastatic mechanisms. The investigation applied rotating wall vessel (RWV) system and PLGA scaffold material, RWV system has the features of low gravity and low shearing force. PLGA biological material can sustain the tissue-like organism which cells formed. The investigation firstly established a spheroid model derived from high-metastatic potential MHCC97H cells. PLGA material is integrated into the system. MHCC97H cells and the scaffold adhered to each other. Along with the culture, the cells mass had grown compact and got a transparent surface, then the metastatic tissue-like spheroid formed. Histochemistry results displayed compactly arrayed cells. The ultra microscopic construct showed the spheroid had some structures of solid tissue, such as desmosome and tight conjunction. Clinically, the level of glucose, lactate dehydrogenase (LDH), albumin (ALB), glutamyl transpeptidase (γ-GT) and alpha-fetoprotein (AFP) are often used to evaluate the liver function. Compared with the monolayer cells, glucose consumption decreased in the spheroid but LDH secretion increased, which suggest the enhanced glucose decomposition and inhibited glucose oxidation; During the development of the spheroid, the secretion ofγ-GT and AFP are higher than that of the monolayer cells. All the results suggest that the spheroid gained the ability to product and secret the specific HCC protein, similar to serum pathological characteristic of HCC patient. Flow cytometry analysis displayed lower S phase and lower G2-M phase of the spheroid cells, which suggest the inactive cellular growth and proliferation. HCC specific genes (AFP, ALB), adhesive molecular ((ICAM1, CD29) and metastatic genes (CD44, E-cadherin, MMP9, MMP2) of the spheroid, especially the spheroid on day 15 had the differential expression compared with that of monolayer cells. The up-regulation of the adhesive molecular suggest the spheroid gained the obvious cellular interaction and conjunction; Increase of ALB and AFP verified the level of them in culture supernatant and also suggest the spheroid partially has the feature of liver and HCC. The metastatic genes up-regulation and E-cadherin down-regulation have resembled with the HCC characteristic. By immunohistology chemistry, we found ICAM1, CD29 and AFP had higher expression in the spheroid than in the monolayer cells and the expression pattern had conformed to the genes transcription. After inoculation of the spheroid into mouse liver, the spheroid generated tumor and the tumor had the classic HCC tumor morphology, and also occurred dissemination in liver and the lung metastasis. The metastasis-related protein:CK7, CK8, CK19 and MMP9 had higher expression level in the tumor. All the results suggest that the model is a tissue-like HCC model, but also a high metastatic-potential model. So we successfully established a novel metastatic HCC spheroid model. The method of tumor formation after spheroid inoculation may have application in animal tumor model establishment and the model may apply in drug screening. Further, the differential proteomics between 2D cells and 3D spheroid were investigated. The results illustrated that under 3D condition, many characteristics of the cells in spheroid is different from that of the monolayer cells. And also the results suggested cellular 3D morphology and the peripheral tissue structure is important to maintain the malignant function of the tumor cells.The microenvironment of the metastatic target tissue has great effect on the invasion and metastasis of the tumor cells. To establish the experimental model of HCC lung invasion, and to mimic the process of tumor cells adhesion to lung, invasion and formation of caner cells focus. The model is designed to remedy the shortage of HCC experimental models under 3D conditions and the shortage of ideal sample experimental system of HCC lung invasion. Also the model is designed to study the malignant function mechanisms of lung invasion and metastasis. On the basis of the metastatic HCC spheroid model, the investigation analyzed the comparative quantitative proteomics between Hep3B spheroid and MHCC97H spheroid and got some metastatic genes, some of which were detected in the HCC lung-invasive model mentioned below. Further, MHCC97H cells and lung mass were added into RWV system and the HCC lung-invasive model was established. From the comparative quantitative proteomics of the dynamic pathological process of the model's development, a series of proteins related to HCC lung invasion were found. Among these proteins, CORO1B is up regulated in the dynamic process. CORO1B inhibited cells have lower cell motility potential. CORO1B can affect cell motility. After lung extract induction, the cell motility potential increased. The results indicated that invasive protein and the metastatic target tissue microenvironment together dominate the cell motility. The HCC models established in the study is in vitro experimental models easy to detect and observe. Compared with the monolayer models, they have the advantage of tissue-like feature, and compared with the animal model, they have the advantage of easiness of handling and timesaving, also the individual difference of the animal model has been avoided.Part One Establishment of a novel high metastatic potential HCC Spheroid ModelFor the study of biological characteristics and HCC metastatic potential, a rotating wall vessel (RWV) system under three-dimensional culture was successfully established. A series of in vitro & in vivo experiment displayed features of the spheroid which is related to clinical pathological traits of human HCC cell. These experiments showed the spheroid tissue-like morphology and ultra micro morphology, protein production and secretion, glucose metabolism, tissue specific genes expression, cell growth and apoptosis and the tumorigenesis & metastasis after inoculation in the liver of nude mice. All these experiments indicated that the spheroid model can better mimic the main traits of metastatic HCC tumor, especially the ability of tumorigenesis & metastasis. The results may have application in drug screening and establishment of HCC animal model. The study illuminated the three-dimensional morphology is important for maintaining of the function of HCC cell.Part Two Establishment of HCC Lung-invasive Model and Related Protein Screening & AnalysisOn the basis of the establishment of in vitro HCC metastatic spheroid model, we mix the lung mass and MHCC97H cell to be cultured in RWV system. During the culture period, MHCC97H cells adhered to lung mass and invaded into the inner lung mass. Some metastatic genes have changed along with the culture. Quantitative proteomics displayed the differential protein expression at the different time point of the culture. Quantitative PCR tested some of the differential protein expression. One of the differential protein is CORO1B, which is an actin-binding protein. CORO1B-ShRNA MHCC97H cell have lower cell motility and lung extracts can partially resume the cell motility.Conclusions1. A novel in vitro high metastatic potential HCC spheroid model was established. The spheroid, compared with monolayer cells, can better mimic the HCC pathological characteristics in the tissue morphology, specific gene expression, protein secretion and production and the tumorigenesis and metastasis.2. Under 3D conditions, the HCC cells are different from themonolayer cells in morphology, genes expression, protein secretion and tumorigenesis and metastasis.3. Cellular 3D morphology and the peripheral tissue structure are very important to maintain the malignant function of tumor cells.4. Established the lung-invasive model which is easy to detect and observe. The model is to mimic the process of tumor cells adhesion to lung, invasion and formation of caner cells focus. The model is designed to remedy the shortage of HCC experimental models under 3D conditions and the shortage of ideal sample experimental system of HCC lung invasion. Also the model is designed to study the malignant function mechanisms of lung invasion and metastasis.5. A series of differential proteins related to HCC lung-invasive function were got. From the study of CORO1B, we found CORO1B can affect cell motility. After lung extract induction, the cell motility potential increased. The results indicated that invasive protein and the metastatic target tissue microenvironment together dominate the cell motility.Novelty1. Established the novel in vitro 3D high metastatic potential HCC spheroid model. The model has advanced the metastatic mechanisms study of malignant HCC and may found new metastatic mechanisms. The model may resolve the shortage of ideal model in the in vitro study of HCC tumor. Also the model may resolve the shortage of ideal models in radiotherapy and chemotherapy.2. Established the lung-invasive model which is easy to detect and observe. The model is to mimic the process of tumor cells adhesion to lung, invasion and formation of caner cells focus. The model is designed to remedy the shortage of HCC experimental models under 3D conditions and the shortage of ideal sample experimental system of HCC lung invasion. Also the model is designed to study the malignant function mechanisms of lung invasion and metastasis.The potential value of this work1. The high-metastatic potential spheroid model can better mimic the metastatic biological characteristic compared with the monolayer cells and the model is easy to repeat and simple to handle. The model can help the mechanisms study of malignant HCC and the model is ideal for the in vitro study of HCC. The model may resolve the problem of the shortage of ideal evaluating system in clinical HCC drug research. The innovation is simple to apply in these fields.2. During the formation of the lung-invasive model, the dynamic process of the lung invasion was analyzed, including the process related proteins and genes, the dynamic proteomics, the dynamic genes screening and the dynamic molecular pathology.3. During the development of the lung-invasive model, the candidate drug of adhesion and invasion can add into the culture of the model, from the dynamic analysis of the growth and pathology, and the curative effect of the drug can be evaluated in vitro4. As a novel HCC lung-invasive model, the model can be used to investigate the malignant function mechanisms under 3D condition, also the model can be used the study of in vitro experiment in HCC tissue level.