| BackgroundGlioma is a malignant brain tumor, according to the result of CBTRUS, it accounts for 80% of brain malignant tumors. In China, though there is no large epidemiological study, most clinical study showing the morbidity is from 40%-67%. Although surgery, radiotherapy and chemotherapy can be used to the patients, the result is also not good. Taking Glioblastoma for example, most patients die in 14.6 months. Radiotherapy and Temozolomide (TMZ) may be work after surgery, but the patients will recur sooner or later. The recurrence areas always near the resection area. There may be two reasons for the tumor recurrence. The glioma cells produce some cytokines, like IL-10、TGF-β、 PGE2 to participate the immune escape. The other reason may be the tumor cell react with the stromal cell and induce the tumor recurrence. Therefore, study the interaction between tumor cells and stromal cells, may give us a new way to fight with glioma.Stromal cells associated with glioma include glial cells, endothelial cells, immune cells and neurons, among which glial cells are the most abundant cells. Astrocytes as the main part of glial cells, had been proved up-regulate the survival gene of tumor cells and improve the chemotherapy resistance in brain metastasis of melanoma and breast cancer. However, there is no research about the effect of astrocyte in glioma. In order to study the role of astrocyte, we need to build the appropriate in vitro model. The model needs to mimic the pathological process of glioma. Meanwhile, the glioma cell and astrocytes can be sorted sufficiently and quickly, and then we can learn more about the astrocytes.In vitro model usually indicates the cell culture model. If we want to study the role of stromal cell in the chemotherapy resistance, we first need to make a tumor cell and stromal cell co-culture model. Then add the chemotherapy medicine, compare the cell survival of tumor cell in co-culture models with only the tumor cells model. After the the co-culture model been built, we can examine the tumor cell in the co-culture model and we can also pick up the stromal cell, analyze the change of cytokines or signal pathways. Find the target spots which may paly a role on tumor survival or chemotherapy resistance. Two dimensions (2-D) monolayer co-culture model has been built, but glioma, like other solid tumors, is growing to three dimensions (3-D). The 2-D model can’t mimic the tumor growth appropriate.Animal experiment is a bridge which connects the bench withthe clinic. The immunodificient animals, such as nude rats, nude mice as well as SCID mice can be used in cancer research because they enable xenografting of human tumor cells or biopsies. Glioma cell lines or biopsies can be xenograft in immunodificient animals successfully. In the last decades, some fluorescent nude mice or SCID mice were generated. The glioma cells can be engrafted in the brain successfully. The fluorescent nude rat has the same characters in tumor research as nude mice, but it also has some advantages. The nude rat has a long lifespan, the body is bigger than mice, and it’s easier to operate. What’s more, it can provide more material for further study. However, no one breed the fluorescent nude rats before.There are two parts of the thesis. The first part introduces a new 3-D co-culture model, which is made up of glioma cells and astrocytes. The bioluminescence assay can be used to quantify the tumor cells’ survival in the co-culture spheroids. The second part introduces a novel GFP nude rat model. The GFP nude rat can be xenografted with the tumor cells, and the stromal cell can be sorted sufficiently form tumor tissue. We hope make a new way to study the tumor-stromal interactions with the two new models.Part 1:A co-culture model with brain tumor-specific bioluminescence demonstrates astrocyte-induced drug resistance in gliomaObjectiveTo bulid a 3-D co-culture model which is composed of eGFP-Luc glioma cells and TNC-1 astrocytes. The survival rate of glioma cells in the co-culture model can be measured by the bioluminescence assay. And the model can be used to study the astrocytes-induced chemotherapy resistance.Methods1. Lentiviral vector production and infection of cells by lentiviral vectorsLentivirus was produced by BBS/CaCl2 mediated triple-transfection of 293FT cells with psPAX.2, pMD2.G and eGFP/Luc vectors. Then the glioma cell lines were transfected with the lentivirus. Then use the Fluorescence Activated Cell Sorting (FACS) to sort the eGFP glioma cells.2. Formation of the 3-D co-culture models8000 glioma cells and 5000 TNC-1 astrocytes were seeded into each well of a 96 well V-shape plate. Each well has 100μl culture medium containing concentration of 1.74p.g/mL methylcellulose. Subsequently, the 96-well plates were spun down at 756G at room temperature for 15 minutes. After 48h culture, the spheroid formed at the bottom of the well.3. Test the glioma cell survivial by bioluminescence assayStudy the relationship between bioluminescence and glioma cell number. U251 and A172 cells with different numbers were seeded in the 96-well plate. After 48 h cultures, the bioluminescence intensity of spheroids was examined.Identify the role of the stromal cell in bioluminescence assay.8000 A172 cells with different numbers’ TNC-1 cells were seeded in 96-well plates. After 48 h cultures, the bioluminescence intensity of spheroids was examined.Study the bioluminescence intensity of co-culture spheroids after added TMZ and Doxorubicin. Glioma cells and TNC-1 cells formed spheroids, then added TMZ and Doxorubicin in different concentrations. After 5 days culture, test the bioluminescence intensity of co-culture spheroids.4. Compare the Bioluminescence assay with MTS assay8000 LN18 cells were seed in 2 96-well plates, after 48h culture, the tumor spheroids formed. TMZ was addedin different concentrations. One plate underwent bioluminescence assay and the other underwent MTS assay. Then collected and dissociated the spheroids into single cell suspensions. FACS counted the eGFP glioma cell numbers.8000 LN18 cells and 5000 TNC-1 cells were seeded in 296-well plates, after 48h culture, the tumor spheroids formed. TMZ was addedin different concentrations. One plate underwent bioluminescence assay and the other underwent MTS assay. Then collected and dissociated the spheroids into single cell suspensions. FACS counted the eGFP glioma cell numbers.Using the same method measure U251, A172, C6 glioma cells and TNC-1 cells co-culture spheroids.5. Study the astrocytes-induced chemotherapy resistanceGlioma cells alone or with TNC-1 formed spheroids, then added different concentrations’ TMZ or Doxorubicin. After 5 days culture, bioluminescence assay measure the intensity of bioluminescence.Results1. Generation of eGFP/Luc expression glioma cellsLentivirus with eGFP/Luc gene was transfected into different glioma cell lines. Observe the transfected cells under the fluorescent microscopes. Make sure the glioma expressed eGFP. FACS picked up the eGFP high-expressed glioma cells for the next experiment.2. Formation of the 3-D co-culture spheroidsThe tumorand astrocyte co-culture spheroid model express the eGFP homogeneous, mimic the growth of glioma in vivo.3. Test the glioma cell survival by Bioluminescence assayBioluminescence intensity of the spheroids is reproducible and proportional with the number of tumor cells. Tumor cell specific bioluminescence intensity is independent of the size of the TNC-1 stromal cell compartment. After added the TMZ and Doxorubicin, the bioluminescence assay can also be used to test the cell survival.4. The Bioluminescence assay is more sensitive and accurate than MTS assayTo the glioma cells spheroids, both bioluminescence assay and MTS assay can reflect the tumor cells’proliferation. However, when compared the 2 assays in the 3-D co-culture spheroids, the bioluminescence assay showing more sensitive. Measurement of tumor-specific bioluminescence demonstrated reduction in cell survival rates not detected by the MTS assay (p<0.05).5. TNC-1 cells modulate sensitivity to TMZ and Doxorubicin in different glioma cell linesTNC-1 can enhance U251, C6, A172 and P3 cells’ chemoresistance to TMZ, but has no effect to LN18 and HF66. TNC-1 can enhance U251, LN18 and HF66 cells’ chemoresistance to Doxorubicin, but has no effect to A172, C6 and P3.Conclusion1. Bioluminescence intensity of spheroids is reproducible and proportional with the number of tumor cells.2. Tumor cell specific bioluminescence intensity is independent of the size of the TNC-1 stromal cell compartment.3. Measurement of tumor-specific bioluminescence demonstrated reduction in cell survival rates not detected by the MTS assay (p<0.05).4. TNC-1 can enhance U251, C6, A172 and P3 cells’ chemoresistance to TMZ, but has no effect to LN18 and HF66.5. TNC-1 can enhance U251, LN18 and HF66 cells’ chemoresistance to Doxorubicin, but has no effect to A172, C6 andP3.Part 2:A GFP nude rat model to investigate tumor-stroma interactionsObjectiveModeling a GFP nude rat to study the tumor-stroma interaction, verify the tumor cell lines and primary tumor tissue can be xenografted on GFP nude rat. After the tumor formed, the stromal cells and tumor cells can be separated by FACS efficiently.Methods1. Breeding the GFP nude ratsSD-TG (GFP) 2BalRrrc transgenic rats were crossbred with HsdHanTM:rnu/rnu Rowett nude rats. After each cross, heterozygous GFP rats and parental HsdHanTM: rnu/rnu Rowett nude rats were crossbred again to obtain heterozygous GFP nude rats.Evaluation of GFP expression was performed in two ways. Firstly, fresh internal organs harvested from a heterozygous GFP nude rat were examined grossly under a UV dissecting microscope. Secondly, RT-PCR was used to detect and quantify GFP expression at the mRNA level of different organs.2. Compare the immunophenotype between GFP nude rats and nude ratsPeripheral blood was collected from GFP nude rats and 2 parental rats. White blood cells were isolated, incubated with different fluorescently conjugated antibodies for various immune cells (T, B and NK cells).3. The tumor cells or biopsies can be xenografted on GFP nude ratDsRed 4T1breast cancer cells were injected in the mammary fat pad.6 primary glioblastoma biopsies were transplanted intracranial into GFP nude rats. MRI scanning verified the tumor formation. After the rats were sacrificed, the tumor tissues were made into paraffin block for H&E staining.4. The tumor and stromal cells can be separated efficientlyThe breast cancer tissue was minced into small piece, then enzyme into single cell suspension. FACS can separate red tumor cells from green stromal cells.The glioma tissue was made into single cell suspension; FACS can separate tumor cells from green stromal cells. ICC with a specific antibody for the human nucleus (HuNu) revealed that tumor tissue cell populations were well separated into human glioma cells and rat cells.Results1. GFP nude rats stably express GFP in diverse organsFresh organs under UV dissecting microscope showing green color, RT-PCR revealed all organs expressed GFP at the transcriptional level. GFP expressed at the highest levels in brain and the lowest levels in the heart.2. GFP nude rats exhibit the same immunophenotype as nude ratsGFP nude rats and HsdHanTM:rnu/rnu Rowett nude rats have same immunophenotype. Both of them lack T cells, but harbor B and NK cells.3. The tumor cells or biopsies can be xenografted on GFP nude ratDsRed 4T1 breast cancer cells can be transplanted into the breast pad and formed breast tumor.6 human primary glioma biopsies were transplanted into the brain of GFP nude rats and 5 can form tumor.4. The tumor and stromal cells can be separated efficientlyThe high speed FACS can separate red breast cancer cells and glioma cells from green stromal cells efficiently.Conclusion1. GFP nude rats stably express GFP in diverse organs.2. GFP expressed at the highest levels in brain and the lowest levels in the heart.3. GFP nude rats lack T lymphocytes and exhibit the same immunophenotype as nude rats.4. Murine breast cancer cells and human primary glioma biopsies can be xenografted on GFP nude rat.5. The high speed FACS can separatetumor cells and stromal cells efficiently. |
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