The Effect Of CD3AK Cell On Tumor Metastasis And Recurrence After Radical Hepatectomy In A Patient-like Orthotopic Nude Model Bearing Human Hepatocellular Carcinoma With High Metastatic Potential

Although great progress has been made in the fields of surgery and non-surgery treatments in hepatocellular carcinoma (HCC) recently, the overall survival rate is far from satisfaction because the recurrence and metastasis of tumor. Metastasis and recurrence are quite common, being as high as 50% five year curative resection even for small liver cancers less than 5 cm in diameter. Invasion and metastasis have become the most prominent obstacles to further improvement of clinical treatment efficacy. Many efforts have been made to find a more efficient treatment to inhibit and prevent tumor metastasis. It is well known that the metastasis process is a very complex one, including tumor cells dissociating from the primary locus, invading the surrounding tissue, entering and extravasating from the circulation, and growing in distant organs. A good study model and appropriate methodology are essential for the thorough understanding of the biological behaviors of liver cancer cells. Nude mice model of human hepatocellular carcinoma with highly metastatic potential must be established. The strategy of orthotopic implantation of histologically intact tissue have been widespread used just because it can acquire high metastatic tumor model of hepatoma. Recently, highly metastatic human hepatocellular carcinoma cell line HCCLM3 have been established and extensivly studied in FuDan University liver cancer research institution. With use this cell line, we made highly metastatic nude mice model bearing human-like hepatocellular carcinoma in order to understand the mechanism of metastasis and to explore the effect of killercell induced by anti-CD3 mAbs plus IL-2 on tumor metastasis and recurrence after radical hepatectomy.Part one: Establishment of a high metastatic model of human hepatocellular carcinoma in nude mice via orthotopicimplantation of histologically intact tissue Objective: To set up a high metastatic model of human hepatocellular carcinoma and patient-like metastatic model after radical resection in nude mice. Methods: Human hepatoma cell line HCCLM3 was maintained in RMPI-1640 media and subcutaneous inoculated in 4-week-old male or female BALBA/c nude mice. The orthotopic tumor model was established by implanting histologically intact tissue under the embrane of liver. Comparison of the two kind differently high metastatic model of hepatocellular carcinoma was also performed. Results: The rate for the tumor formation of high metastasis heptocellular carcinoma subcutaneous and orthotopic implanted model is 100% and 92.9%, respectively. There is no death in the period of perioperation. Except for intraheptic metastasis, liver cancer in orthotopic model could metastasize to lung, iliac fossa, inferior kidney, diaphragm, lymph node of mesentery, paraaorta, retroperitoneum, etc. The rate of plumonaiy metastasis is 100%. But high metastasis model via subcutaneous implantation is characterized by 100% lung metastasis and 0% other organ metastasis evaluated by microscope and HE staining. In addition, the orthotopic tumor model from subcutaneous model of human hepatocellular carcinoma in nude mice propagated more stably. Conclusion: Patient-like hepatocellular carcinoma nude mice model with high metastatic potential was successfully made by orthotopic implantating intact tumor tissue under liver embrane. This model is an ideal instrument for us to further study the mechanism of metastasis and recurrence of liver cancer, explore new methods for inhibit the growth metastasisPart two: Expression of E-cadherin and vascular endothelialgrowth factor (VEGF) in human-like hepatocellularcarcinoma with high metastatic potentialObjective: To investigate whether immunohistochemical expression of E-cadheirn in conjunction with VEGF may correlate with the vascular invasion and intrahepatic metastasis of HCC with high metastatic potential. Methods: Two different nude mice heptocellular carcinoma model is Established. Specimens of HCC with high metastatic potential (A group) and common potential (B group) were obtained from 8 nude mice model, respectively. Another 8 nude mice were used as normal controls(C group). Using the antibodies of E-cadherin, VEGF performed immunohistochemical staining (S-P method). Their expressing difference were comparison by combination immunohistochemical staining with image analysis meter with Image Pro Plus software. By PEMS3.1 software, the correlation is analyzed between the expression of E-cadherin and VEGF and MVD (micro vascular density) and the vascular invasion and intrahepatic metastasis of HCC, and the correlation between the expression of E-cadherin and the expression of VEGF. P values less than 0.05 were considered significant. Results: (1) There is stastaticly difference between A, B and C groups in intrahepatic metastasis and lung and other organ's metastasis (PO.01). (2) E-cadherin was normally expressed by hepatocytes and epithelial cells in normal liver tissues. Reduced E-cad expression was noted in A and B groups. Reduced E-cad expression was found more often in A group than in non-invasive ones (12.5%, 37.5% vs 62.5%, P>0.05); A significantly higher IOD (Integrated Optical Density) of A group showed reduced E-cad expression compared with B group and C groups (PO.01). (3) Increased VEGF expression was found more often ininvasive HCC than in non-invasive group. Compared with A, B and C group, the difference of positive rate of VEGF was no significant (87.5%,75% vs 50%, P>0.05); A significantly higher IOD of A group showed increased VEGF expression compared with B group and C groups (PO.01). (4) There was obvious correlation between the expression of MVD and the expression of VEGF (PO.05). Conclusions: (1) Loss or decreased expression of E-cadherin is more frequently seen in highly invasive HCC. E-cadherin exerts a significant inhibitory function on metastasis generation. (2) Increased expression of VEGF is found more often in highly invasive HCC. VEGF promotes metastatasis, but maybe not a mainly metastatic factor in HCC. (3) Changes in E-cad and VEGF expression occur independently during HCC carcinogenesis and tumor progression. (4) Expression of E-cad or VEGF or MVD may be a useful diagnostic parameter for the tumor invasion and metastasis in HCC.Part three: Experimental study on CD3AK's effectson HCCLM3 cell lineObjective: To probe the immunobiological features of CD3 monoclonal antibody activated killer cells (CD3 AK) and its anti-tumor mechanism for live cancer with high metastasis. Methods: CD3 monoclonal antibody (CD3McAb) and recombinant interleukin-2 (rIL-2) costimulated human peripheral blood mononeuclear cells (PBMC) for inducing CD3AK. MTT methods were separately used to investigate the proliferation and cytotoxicity of CD3AK against tumor cell lines of HCCLM3. Morphological change of tumor cells apoptosis was observed by flow cytometric anlaysis and fluorescence microscope. Results: 1 The proliferative ability of CD3AK cells: The proliferative multiple of CD3AK and LAK and NK were similar at thefirst 4 days (P>0.05). With time going on, the proliferative multiple of CD3AK was significantly higher than that of LAK and NK (P<0.01). CD3 AK kept the growth tendency for at least sixteen days. 2 The exprimental result showed that the optimal concentration of CD3McAb activated for CD3AK was 30ng/ml.3 Dynamic observation on Cytotoxicity of CD3 AK: CD3 AK cultured to the 8th day had a strong cytotoxicity and reached to the highest activity at the 10th day. 4 Comparing of CD3AK and LAK and NK on Cytotoxicity: Cytotoxicity of CD3AK against HCCLM3 tumor cell line was significantly higher than that of LAK and NK cells (PO.05). Its cytotoxicity against SMMC-7721 was similar to LAK and NK cells (P>0.05). 5 The morphological changes about HCCLM3 apoptosis: Under fluorescence microscope, there were typical apoptotic cell features, such as: nucleus shrinks, chromatin accumulates under nuclear membrane and taking the shape of star-moon, cell membrane swells, apoptotic bodies, etc. Conclusion: CD3AK cell is easily to be induced and proliferated, and has a strong cytotoxicity against tumor cell lines. It is a new immuno-effect cell better than LAK cell and NK cell. Part Four: Effect of CD3AK on metastasis and recurrence after radical resection , and relationship between expression of MAGE-1 mRNA and AFP- mRNA in Peripheral Blood ofthe nude mice and metastasisObjective: To correlate AFP mRNA and MAGE-lmRNA with recurrence and metastasis after nude mice hepatocellular carcinoma model received curative operation and CD3AK infusion, to investigate the possibility of combination AFP mRNA and MAGE-lmRNA as a marker of recurrence and metastasis after surgery in HCC. Methods: In the base of previous part, the high metastasis nude mice model was established and were divided into 6subgroups depending on the therapy performed: (1) resection in early stage and CD3AK infusion (groupl); (2) resection in early stage (group2); (3) resection in advanced stage and CD3AK infusion (group3); (4) resection in advanced stage(group4); (5) CD3AK infusion(group5); (6) no therapy (group6) . All subgroups were compared by HE staining and routine pathological test. A highly sensitive RT-PCR assay was established. Peripheral blood and tumor tissue of nude mice's total RNA was extracted from the cells by TRizol based on the method of instructions. RNA was purified from the blood of nude mice and liver cancer tissue. Complementary DNA synthesis by reverse transcriptase and polymerase chain reaction amplification was performed with primers specfically for the AFP and MAGE-1 gene. The PCR product was subjected to electrophoresis on 1.5% agarose gel. Data was analyzed using SPSS software package. P <0.05 was considered statistically significant. Results: AFPmRNA in peripheral blood was not detected in all heptocellular carcinoma, and MAGE-mRNA was only detected in HCC. Combining AFPmRNA and MAGE-1 mRNA, the rate of sensitivity and specialty is increased to 92.8% and 66.7%, respectively. The detection of AFPmRNA and MAGE-lmRNA in peripheral blood is higher in detection of metastasis of HCC than not to find metastasis of HCC. Among group 1 and 3, group 5 and 6, there is significantly statisaticly difference (P <0.05) in the expression of AFPmRNA by RT-PCR and half-determination. Among group 1 and 2 and 3, group 2 and 4 and 5, group 3 and 5, and group 4 and 6, there is significantly statisaticly difference (P <0.05) in the expression of MAGE-lmRNA. But there is not statistically significant difference between all of other groups (P >0.05). In 16 cases with AFPmRNA in tumors, AFPmRNA in peripheral blood were also positive. The expression of AFPmRNA and MAGE-lmRNA in tumor are not correlated with the expression in peripheral blood (P >0.05). Conclusions: 1 The positive AFPmRNA and MAGE-lmRNA test results present HCC cells disseminatedin blood. Blood AFPmRNA and MAGE-lmRNA can be a early detection marker for micrometastasis. But the metastatic threshold, quantity-effect relationship or other added parameters should be further investigated and established. 2 The expression of AFP mRNA and MAGE-lmRNA in peripheral blood was accordant, so combined detection may increase the reliablity of results. 3 The AFP mRNA and MAGE-1 mRNA test findings in peripheral blood significantly correlated with recurrence and metastasis. Both of them can be considered as new indicator for prognosis. 4 RT-PCR assay is a sensitive technique for AFP mRNA and MAGE-lmRNA detection in blood of HCC. With convenient sample collection and simple manipulation steps, it can be put into clinical practice. 5 CD3 AK administration is useful method for decreasing metastasis and recurrence in liver cancer after radical resection in early stage.