| Part 1 Modified clinically relevant orthotopic xenograft mouse model of human gliomaObjective Animal model of human glioma was mainly established by injecting tumor cell suspensions into nude mice subcutaneouly at the early stage. However the utility of each particular model depends on how close it replicates the original tumor. Though orthotopic models used recently could meet this demand well, they still have some disadvantages including major trauma and low take rate. Here we modified an orthotopic xenograft mouse model establised by ourselves years ago to reduce the operation trauma and elevate the take rate significantly.Methods (1) Screen a suitable i.v.canular from different types of trocars and reassemble to a special trocar system for mouse brain puncture; (2) Design a tissue propeller to push tiny fragments of surgical tumors tissues into the trocar system; (3) Drill a hole on mouse skull using a micro-drill; (4) Tiny fragments of surgical tumors were implanted into the caudatum of mouse brain with the trocar system; (5) Tumor-bearing mice were nursed carefully after operation. Clinical manifestation, survival time and tumorigenicity rates were recorded. Immunohistochemistry and HE staining were performed on tumor tissue sections.Results (1) None of the mice with xenograft developed focal neurological signs in the early stage, however, in the advanced stage, all the tumor-bearing mice presented with reduced food intake, dull response, emaciated figure and cachexia. Tumor-bearing mouse usually died two days after cachexia. A part of tumor-bearing mice seldom had the symptoms of hemiparalysis and hyperspasmia even the tumor grew into a huge mass; (2) The take rate was nearly 50% in the original mice group which were injected by the fresh surgical fragments, however, the tumorigenicity rate was steadily up to 100% from the third generation when the tumor was transferred between the nude mice. Survival time was gradually steady from the 5th generation (23.9+1.7 days); (3) Brain transplants perfectly resembled their original tumors in biological behaviors. CD133 positive or SOX2 positive brain tumor stem cells(BTSCs)could be observed in the tumor tissues, besides the Ki67 positive and GFAP positive tumor cells.Conclusions Internationally, animal model of human glioma transplanted with tumor tissue was made by burying tumor tissue under the subdural space after craniotomy, which was very complicated and had a major trauma. Moreover tumor cells in this model proliferated out of the brain and can not replicate the original tumor well, which deviate the feature of orthotopic transplantation. Our specially designed trocar implantation system made the orthotopic transplantation simpler, easier but more efficient than any other methods described previously. The injected tumor tissues containing stem cells and stromal cells could promise the forming of new tumor mass closely replicating the orignal tumor. Our new method was already published by SCI journal.Part 2 Establishment of a highly invasive cell line of glioma stem cells and progenitor cells and the study of its proliferated and invasive characteristics in different anatomic sites of nude miceObjective (1) Clinically, high invasion is the basic feature of malignant glioma and the invasive degree usually increased with the tumor grade. However, xenograft models of human glioma initiated from cell lines often yield well-circumscribed intracranial tumors which have different pathological features versus the features of clinical tumor. Even transplated with glioma stem cell (GSC) suspensions, invasive degree in transplantation tumors is different between primary and secondary tumor cell suspensions. Here we try to establish a highly invasive model in vitro or in vivo which replicates the biologic phenotypes of clinical glioma and lay the foundation for further research of the role of GSCs in tissue remolding in host tissues. (2) Recently, more attention should be paid on the microecosystem when study the biological properties of glioma. To answer the question of whether the features of glioma cells injected in different anatomic sites affected by different microecosystem of host animal is the second objective of this study.Methods (1) Biopsy specimens were obtained in the highly invasive focus according to the image of MRI. One part was sent to department of pathology for rapid diagnosis; the rest was made into single cell suspensions and cultured in stem cell medium condition for 7 days. After that tumor spheres were collected and digested into single cells. Monoclonal GSCs were obtained by limiting dilution assay. (2) Tumor spheres from limiting dilution assay were collected and stained by related antibodies such as CD133, nestin and GFAP. Spheres with the features of glioma stem cells and progenitor cells (GSCPs) were cultured, proliferated and cryopreserved. (3) Invasion ability of recovered GSCPs was tested by scarification test in vitro. (4) In vivo, GSCPs were injected into different anatomic sites of mouse body including intracalvarium, subcutaeous and abdominal cavity. Tumorigenesis rates were recorded. (5) Transplantation tumors from different place were observed carefully. Immunohistochemistry and HE staining were performed on tumor tissue sections.Results (1) Tumor spheres (SU3) were positive for CD133 and nestin, but weakly expressed GFAP. After cultured in medium containing 10% serum, tumor cells differentiated from SU3 were positive for nestin and GFAP, but negative for CD133. (2) Take rates of GSCPs injected in different sites of mouse and survival time of tumor-bearing mice were as follows: take rate of GSCPs injected in caudate nucleus was 90.48% (19/21)and the survival time was 35.5±0.94 days; take rate of GSCPs injected in abdominal cavity was 87.5% (7/8) and the survival time was 52.20±1.31 days; take rate of GSCPs injected in subcutaeous was 80% (8/10) and the survival time was 50.75±0.75 days. (3) Invasion of SU3 in vivo: in brain, SU3 spreaded into surrounding places, and arrived at striatum, hippocampus, ventricular system, medulla, cortex and menigeal. In some cases, SU3 could invade into contralateral hemisphere, rhinencephalon, even grew out of the brain surface. A few of tumor-bearing mice could have hydrocephalus; huge tumor with bulky shape was formed under subcutaeous. Tumor had a pseudo-envelope at the early stage, invaded skin, muscle and ribs at the mid-stage, and developed disunion ulcer at the advanced stage; tumors developed in abdominal cavity could invade greater omentum,mesentery, peritoneum and almost all the organs, and produced bloody ascites as well. Tumor cells proliferated from bloody ascites could form new tumors when transplanted into nude mice. (4) Histopathology: Preliminary studies showed that SU3 may have the multiple differentiation capacity which can be induced by local microecosystem of host tissue. At least, tumor developed by SU3 on choroid is pathologically similar to choroid plexus carcinoma; tumor generated by SU3 in hepatic tissue is pathologically similar to hepatocellular carcinoma.Conclusions (1) According to the features of biology, histopathology and molecular marker, GSCPs cell line SU3 is a highly invasive cell line, which will contribute to the study of the generation and development of glioma, especially the invasive mechanisms of glioma. (2) The multiple differentiation capacity of SU3 which can be induced by local microecosystem of host tissue indicates that in the progression of tumor tissue remolding, the influence between tumor cells and host cells is mutual. Multiple differentiation capacity of SU3 is not fixed in a regular pattern, and the direct of differentiation of SU3 could be affected by different microecosystem. These findings will challenge the idea that tumor stem cells are totipotential stem cell and have a great significance in analyzing dialectically the theory of“seed and soil”.Part 3 Molecular phenotype analysis of stem cell related markers, stem cell niche and neovascularization in SU3 transplantation tumorObjective As we know, tumor marker detection plays an important role in the study of biocharacteristics of tumors. Protein Bence-Jones was the first specific tumor marker observed by Kahler in multiple myeloma, but in the succedent 20 years, the research progress of tumor-specific markers is unsatisfactory. As far as glioma is concerned, its invasiveness determines the malignant phenotype, refractoriness and poor prognosis. Though many proteins involved in the invasiveness of glioma have been found, their relation with GSCPs is less clear, moreover, new stem cell related markers have emerged one after another, which indicates that more study should be taken. Here we analyzed the molecular phenotype of SU3 xenograft, the relation between SU3 and stem cell niche, and the relation between SU3 and tumor vascularization. Our study will pave the way for the further study of the invassive mechanisms of glioma.Methods As described in part 2, SU3 cell suspensions were injected into intracalvarium and abdominal cavity of nude mice; cell suspensions of U87MG were injected in to mice brain as a control. Orthotopic transplanted tumor-bearing mice were sacrificed when cachexia was occurred, tumor-bearing mice injected via abdominal cavity were sacrificed when abdominal circumference was significant increased. The xenogeneic graft tumor samples of tumor-bearing mice were fixed in 4% phosphate buffered formaldehyde, processed into paraffin blocks, sectioned. Related molecular markers were stained on the tumor sections as well as the clinical specimens by routine immunohistochemistry method. To prove the relation betwwen tumor related markers and neovascularization, or blood transport function, a part of tumor-bearing mice were perfused with activated carbon through systemic circulation and the distribution of activated carbon was observed.Results (1) The expression of stem cell related markers: xenograft formed by SU3 highly expressed CD133 and SOX2, weakly expressed nestin and almost did not express GFAP; xenograft formed by U87MG highly expressed nestin, but weakly expressed CD133, SOX2 and GFAP; (2) The relation between CD133 positive cells and stem cell niche: CD133~+ tumor cell clusters in xenografts are actually stem cell niche which were in close relation with tumor vessels. (3) The relation between CD31 positive cells and tumor vessels: CD31 could express on the tumor cell-like cells, though most CD31 positive cells were vascular endothelial cell-like cells. There were some CD31 positve cells whose phenotype falls in between tumor cell-like cells and vascular endothelial cell-like cells as well. The function of these vascualr-like tubes was tested by activated carbon perfusion. (4) The expression of invasion related markers: Ki67, MMP2 and MMP9 were highly expressed in xenograft formed by SU3, but not in xenograft formed by U87MG.Conclusion Transplantation tumor formed by SU3 which was cultured from the highly invasive focus could keep the feature of invasiveness just as its original tumor presented. The high proliferation and invasion ability of SU3 may relate to the high expression of stem cell markers CD133 and SOX2, and matrix metalloprotein MMP2 and MMP9. Further more, effective regulation of SU3 in the tumor neovascularization and the generation of stem cell niche maybe involved as well. |
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