| Background and purposeLung cancer is one of the leading causes of cancer-related morbidity and mortality in worldwide, more than 1 million people lost their lives due to lung cance in the world each year. Although we have made a huge investment in the treatment of lung cancer research, in the past few decades, the improvement of efficiency and survival period of treatment in lung cancer is very slow, this prompted the researchers there still have important unknown mechanisms can affect the progress and the therapeutic in lung cancer. In the cancer treatment process, we tend to focus on the tumor cells themselves, while ignoring the immune system caused by the tumor immune response and immune surveillance. The clinical study found that infiltration of lymphocytes existed in many types of tumor tissues. It illustrates that the tumor antigen can mediate and stimulate the body’s tumor immunogenicity. The complex relationship between the immune system and tumor has always been puzzled researchers, Studies have demonstrated that tumor-infiltrating lymphocytes is a favorable factor associated with better prognostic, but there are also some scholars raised questions about it. As a result of each patient’s own immune function different is vast, clinical studies are difficult to find good control sample,Therefore it is necessary to find a suitable animalmodel to study the relationship between the immune mechanisms associated with tumor.The tumor animal model has always been one of an important tool to study tumorigenesis and development mechanism of tumor. But due to the inherent genetic and immune system differences in mouse and human organism, it limited the study of human disease in mice model to some extent. Therefore, the establishment of an experimental animal model similar to the human immune environment is especially important. We use a simple definition of humanized mice as human-mouse chimeric model engrafted with functional human peripheral blood mononuclear cells through the peripheral blood circulation or peritoneal injection human cells.Humanized mouse models most commonly chosen the combined immunodeficiency (SCID) mouse and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse strains. It is better to reconstruct the human’s immune system and mimic the fluid environment and cellular immune function of human body in these strains of mice. Humanized mouse has solved a longstanding difficult problem, which were the animal model can not accurately reflect the body’s immune system characteristics and the study of human immune response limitation by the vitro experiments.In vivo imaging technology can take advantage of in vivo bioluminescence or fluorescence imaging to directly detect the behavior of the biology of living cells in vivo, so in vivo imaging technology has become widely used in medicine, biology and drug development and other areas of research on the frontier technology. In recent years, researchers using of green fluorescent protein(GFP) marker target tumor cells, using in vivo fluorescence imaging system to the way of visual images in the tumor-bearing animal models, to obtain the information of tumor growth and metastasis, it also can observe the early effects of tumor treatment. In the previous study, we constructed a lung adenocarcinoma drug resistant cell strain Am1010, which came from a female lung cancer patient’s upper arm muscle metastatic lesions. Before the emergence of this muscle metastatic lesions, this patient had been received four cycles of chemotherapy, radiotherapy and targeted therapy. This strain of the Am1010 cells showed the resistance capability of a varied chemotherapy drugs, radiation therapy, targeted drug Iressa and invasion matrix and blood vessels in vivo. Am1010 descendant sub-cell lines have a unique growth and adhesion ability, this cells line also showed especially strong invasion the ability to the matrix and blood vessels in SCID mice traced by GFP label.So how is the performance of Am010 cells with treatment resistance in the mimic human immune environment humanized mice behaving? The purpose of this research is to observe the invasion and metastasis of green fluorescent labeled the Am1010 cells in humanized mice and to further explore the relationship between tumor invasion and metastasis in vivo with the human’s immune system by constructing a humanized mouse model combined with the animal in vivo imaging system.Method:1.Twenty NOD/SCID mice of ectopic lung cancer were randomly divided into two groups:humanized group (group A, n=10), non-humanized group (group B, n=10); five NOD/SCID mice were tumor-bearing humanized group (group C, n=5).Twenty NOD/SCID mice of orthotopic lung cancer were randomly divided into two groups:humanized group (group D, n=10), non-humanized group (group E, n=10)2.0.2ml Am1010-GFP cell suspension (4×108cells/ml) was injected subcutaneously to the back of a NOD/SCID mouse, the tumor was removed by surgical method and cut into Ix3x3mm pieces until the formation of the subcutaneous transplantation tumor. Inserted one tumor piece to the left side of rib wing subcutaneous of the each mouse of ectopic lung model group, to construct mouse heterotopic lung cancer model (n=20).0.1 ml Am1010-GFP cell suspension (5×108cells/ml) was injected into the right intrathoracic of orthotropic lung cancer mouse, to construct mouse orthotropic lung cancer model (n=20).3.0.2ml (5×108cells/ml) human peripheral blood mononuclear cells were intraperitoneally injected into the mice of group A on the day when were inoculated the tumor, at the same time the mice of group C. The mice of group D intrathoracic inoculation of tumor cells after a week, the same way to build a humanized orthotropic tumor-bearing mouse model.The T lymphocytes’ expression of mice peripheral blood, the proportion of subsets T lymphocytes, and the T lymphocytes’ expression of mice’s spleen, liver, lung tissue after reconstitution detected by using flow cytometry, immunohistochemistry and Western blot analysis.4. After injected the tumor every week (for 4 weeks), the ectopic lung cancer model mice were visualized by animal in vivo fluorescence imaging system to test the tumor growth. Before the death of orthotropic lung cancer model mice, the mice were visualized by animal in vivo fluorescence imaging system to test the primary tumor and metastases.5. The infiltrating situation of tumor tissue of ectopic lung cancer model and orthotropic lung cancer were observed by HE staining, and the expression of human subsets T lymphocytes:CD3+T, CD4+T, CD8+T, Foxp3+Treg were further used by immunohistochemistry and Western blot analysis.6. Statistical Methods:All experimental data the application SPSS 13.0 software to analyze. Statistical data were expressed as mean ± standard deviation (x±s), using t tests and repeated measures analysis of variance.Results:1. After one week intraperitoneal injection PBMCs, the human CD3+T cells could be detect in peripheral blood of all of mice in group A and C, and the distinct population of human CD3+T cells in the peripheral blood is more than 1%. The success rate of immune reconstitution is 100%. In the following three weeks, the rate of human CD3+T cells in the proportion of all nucleated cells in mice peripheral blood had been increased gradually. The repeated measures ANOVA analysis showed that the number of CD3+T at different time point had significant difference (F=855.026, P=0.000). The expression of CD3+T had no significant difference between groups (F=0.165, P=0.692). The expression of CD3+T of two groups had no interaction with time (F=0.139, P=0.745), these suggested that the expression of CD3+T trend is the same with time. After four weeks engraftment with PBMCs, the ratio of human CD3+T cells in mice peripheral blood cells of group A and C’s mice, the ratio of human T lymphocyte subsets CD3+CD4+T/CD3+CD8+T peripheral blood mice of group C could reach the normal range for the proportion of human peripheral blood lymphocytes. The repeated measures ANOVA analysis showed that ratio of CD3+CD4+T/CD3+CD8+T at different time point had significant difference(F=8.382, P=0.000). The ratio of CD3+CD4+T/CD3+CD8+ had no significant difference between groups (F=99.624, P=0.000). The ratio of CD3+CD4+T/CD3+CD8+T of two groups had interaction with time (F=6.528, P=0.001), these suggested that the ratio trend of CD3+CD4+T/CD3+CD8+T is different with time. The number of human CD4+T in peripheral blood mice of group A began to decline after reconstitution in the week, and the number of CD8+T cells increased instead, the ratio of CD3+CD4+T/CD3+CD8+T appeared the trend of inverted and downward.2. Human CD3+T cells were with high expression in spleens of group A and group C mice detection by immunohistochemistry, The expression of CD3+T cell in humanized mice’s spleen was no significant difference between group A and C (t=0.373,P=0.715). A great expression of human CD3 protein in humanized mice’s spleen, liver and lung tissues was detected by Western blot. CD4+T cells, CD8+T cells and Foxp3+Treg were expressed in mice of two group after reconstruction. The expression of CD4+T cells, CD8+T cells and Foxp3+Treg in humanized mice’s spleen were significant difference between group A and C (CD4 t=8.382, P<0.01; CD8 t=10.919, P<0.01; Foxp3 t=2.569, P=0.023). The expression of CD8+T cell in spleen of group A was higher than that in of group C, and the expression of CD4+T cell and Foxp3+Treg in spleen of group A was lower than that in of group C.3. The tumorigenicity of mice subcutaneously engrafted AM1010-GFP tumor pieces in group A and group B were 100%. The repeated measures ANOVA analysis showed that the volume of tumor at different time point had significant difference (F=304.600, P=0.000). The volume of tumor of two groups had interaction with time (F=99.271, P=0.000), these suggested that the volume of tumor was difference with time, the growth of tumor volume in group B was faster. Further analysis by t test at different time point, the volume of tumor had no significant difference between two groups after engraftment for one week. The volume of tumor was significant difference between group A and group B after engrafted tumor pieces in the following week (t=2.252, P=0.043), the volume of tumor in group B was bigger than that of in group A. The volume of tumor in group B was bigger than that of in group A in the later two weeks obviously (P<0.01). The GVHD syndrome was observed in 5 of 10 mice in group A,3 of 5 mice in group C, one of the mouse which had severe GVHD syndrome in group A died on 26th day after reconstruction. As the severe cancer depletion, two mice died on 23th day and 25th day respectively in group B after engraftment tumor pieces. The repeated measures ANOVA analysis showed that mice weight of group A and group B at different time point had significant difference(F=206.212, P=0.000). The mice weight had significant difference between groups (F=0.307, P=0.000). The mice weight of two groups had no interaction with time (F=1.556, P=0.204), these all suggested that the mice weight was the same with time.4. The GFP protein expressed stability and tumor fluorescence illuminated significantly detected by In vivo imaging device after heterotopic lung cancer mice model of two groups of NOD/SCID mice subcutaneously inoculated with Am1010-GFP tumor pieces. The repeated measures ANOVA analysis showed that fluorescence area and photons of tumor blocks of group A and group B at different time point had significant difference (Area F=300.630, P=0.000; Photons F=327.810, P=0.000). The fluorescence area and photons of tumor blocks of two groups had interaction with time (Area F=92.226, P=0.000; Photons F=19.817, P=0.001), these all suggested that the fluorescence area and photons of tumor blocks were different with time. The fluorescence area and photons of tumor blocks in group B was stronger than those in group A. Furthermore, analysis by t test at different time point, the fluorescence area and photons of tumor blocks had no significant difference between group A and group B after engraftment for one week. The fluorescence area and photons of tumor blocks in group B was stronger than those in group A after engraftment in the following week (Area t=2.149, P=0.045, photons t=2.185, P=0.042). More significantly differences of fluorescence area and photons of tumor blocks between two groups in in the later two weeks.5. A large number of lymphocytes were found infiltration in tumor tissue of mice in group A, while there was no obvious tumor infiltration of lymphocytes in tumor tissue of mice in group B. Further analysis antigen labeling staining to T cells subset in tumor tissues, a larger number of CD3+T、CD8+T lymphocyte infiltrated in subcutaneously engraftment tumor tissues of humanized mice. Small amount of CD4+T lymphocyte infiltration and no Foxp3+Treg infiltration were found in tissues mentioned above. A great expression of human T Lymphocyte protein in the two groups (group A and C) humanized mice’ spleen, liver and lung tissues were detected by Western blot. Group A compare with group C, the CD3+T cell protein content of humanized mice’ spleen, liver and lung tissues were likewise, the expression of CD8+T cell protein content was high, while the expression of CD4+T cells and Foxp3 protein content were lower. The expression of CD4+T cells and CD8+T cell protein content were high in tumor tissues of group A, the expression of CD4+T cells and Foxp3 protein content were less yet; There had no expression of human T Lymphocyte cell protein in all organs and tumor tissues of group B.6. After one week by intraperitoneal injection PBMC, the success rate of immune reconstitution is 100% in mice of group D. In the following three weeks, the rate of human CD3+T cells in the proportion of all nucleated cells in mice peripheral blood had been increased gradually. The ratio of CD3+CD4+T/CD3+CD8+T were found with the trend of inverted and downward. A large number of human CD3+T cells expressed in spleen of mice in group D detection by immunohistochemistry.7. All of mice in two orthotopic lung cancer mice model groups survived after intrathoracic inoculation operation. The repeated measures ANOVA analysis showed that mice weight of group A and group B at different time point had significant difference (F=580.095, P=0.000). Mice weight had significant difference between groups (F=4.768, P=0.048). The mice weight of two groups had interaction with time (F=25.868, P=0.000), these suggested that the mice weight was different with time. The mice weight of two groups began to appear decreasing in the trend on 10th days, the mice weight of group E loss faster. The GVHD syndrome was observed in 6 of 10 in group D, and one mouse died on the third week after reconstruction. As the severe cancer depletion, four mice died between 25 and 35 days successively in group E after engraftment tumor pieces. There was significant difference mean value of mice’ weight between group A and group B groups with the time pass by, The weight loss of group E was more obvious than that of group D.8. Primary lesion were generated both in the right side of the chest cavity of mice of two groups observation by in vivo imaging system. The tumorigenicity rate of orthotopic lung cancer mice model was 100%. The pleural metastasis rate in mice of group D was 40%(4/10), and the pleural metastasis rate in mice of group E was 100%(10/10). By fluorescence in vivo imaging analysis, metastases lesions often located in the other lobe of the primary tumor ipsilateral, mediastinal tissue, diaphragm, chest wall, or contralateral lung tissue, diaphragm. The fluorescence area of primary and metastases lesions in the chest cavity of mice had significant difference between groups (Primary t=2.745, P=0.013, Metastasis t=9.691, P<0.01), The fluorescence area of primary and metastases lesions in group D was smaller than those in group E. The photons of primary and metastases lesions in the chest cavity of mice had significant difference between groups (Primary t=2.560, P=0.020, Metastasis t=3.068, P=0.008), The photons of primary and metastases lesions in group D was lower than those in group E. The abdomen and brain of two groups were not found metastatic lesions by fluorescence in vivo imaging analysis.9. A large number of tumor infiltration of lymphocytes were found in tumor tissue of mice of group D, while there was no obvious tumor infiltration of lymphocytes in tumor tissue of group E’mice. Further detection by immunohistochemistry and western blot for T cell subsets showed that CD3+T, CD8+T cells rather than CD4+T were with high expression in tumor tissue of group D’ mice. There was weak expression of Foxp3 protein in tumor tissue of group D.Conclusion1. The humanized mouse model can be established in NOD/SCID mice by the intraperitoneal injection of human peripheral mononuclear cells.2. Lung cancer drug resistance cell lines Am1010 cells can be established subcutaneous heterotopic lung cancer model and orthotopic lung cancer model in NOD/SCID mice.3. By the way of detecting T lymphocyte subsets in the peripheral blood, organ and tumor tissue of tumor-bearing humanized mice, telling us there are strong cellular immune response of human immune system in the mice during the reconstruction process, it could obviously inhibit the process of tumor invasion and metastasis.4. The humanized mouse lung metastasis model is expected to be a new research tool to explore the interaction between human immune system with lung cancer invasion and metastasis. |
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