| Background and objective: Tumor microenvironment is the symbiotic microecosystem between tumor cells and host-tissues and cells. Recently, the research progress on tumor microenvironment is very fast, but these data, mainly derived from animal experimental models not patients due to ethical issue, are much limited. Therefore,researchers of oncology very play attention to highly mimetic animal experimental models.As to the immunoinflammatory microenvironment, the requirements of human tumor animal models are higher than those of general animal models. In the models, the sources of these host immunoinflammatory cells in the tumor tissues and edges remodel by tumor cells should be exactly reflected, and the mutual interactions of these cells in the process of tumor generation and development should be clear as well. Base on these requirements and our established red fluorescent protein(RFP)-human glioma stem cells and green fluorescent protein(GFP)-NC/Balbc nude mice, our present study aimed to investigate the relevant issues of tumor immunoinflammatory microenvironment animal models induced by glioma stem cells, especially in the effect of hyperbaric oxygen on tumor microenvironment.Materials and methods: The human glioma stem cells SU3, RFP-expressed SU3,GFP-expressed NC/Balbc nude mice, trocar with cell inoculations and hyperbaric oxygen therapy-independent ventilated cages(HBOT/IVC) of specific pathogen free(SPF) were established by our laboratory. The inoculation methods of tumor cells were obviously different according to the part-inoculated difference. The intracranial inoculation in situ was performed by very slow injection through cranial hole under help of stereotaxic apparatus. The parts of subcutaneous inoculation were located at the forefoot subaxillary and plantar, and the process of inoculation was performed by using trocar for fragment of tissue or injector of skin test for tumor cell suspension. The intrahepatic inoculation was performed by very slow injection using the micro-pump after incision of right costal edges.The intraperitoneal inoculation was performed by direct injection with injector of penicillin skin test. The inoculated doses of tumor cells were different according to the parts and design parameters. Generally, the dose of subcutaneous inoculation was 1-3 mm3 for tissue fragments or 106-107 cells/0.5 mL for cell suspension, and the doses of intracranial and intrahepatic inoculations were 105 cells/10-15 μL for cell suspension.In order to observe the tumor immunoinflammatory microenvironment, the transplanted tumor tissues including tumor edge tissues were collected, these specimens were examined under general light and fluorescent microscopes after paraffin and frozen sections to determine the distribution of GFP+ cells and its expression level using the IPPs6.0 software. To determine the ratio of immunoinflammatory cells in the GFP+ cells, the cell-marked proteins in the transplanted tumors and edge tissues were determined by immunohistochemical assay, this cell ratio in the tissue-digested suspension might also be determined by fluorescence activated cell sorter(FACS) analysis, or other methods including isolation, collection and cultivation according to the experimental objective.To observe the effects of hyperbaric oxygen on tumors, the nude mice with subcutaneousness-inoculated SU3 were treated with hyperbaric oxygen in the system of HBOT/IVC 100 min per day for 21 days, while the nude mice with alone chemotherapy were intraperitoneally injected ACNU 30 mg/kg weekly for three times. At experimental end, the tumor tissues were taken and weighed. The oxygen content of tumor tissues was determined by using microelectrode.Results and analysis: In the intracranial transplanted tumor tissues, the light microscope observed the bleeding, necrosis, angiogensis and malignant phenotypes of tumor infiltration growth. The RFP+, GFP+ and RFP+/GFP+ cells were clearly observed under fluorescent microscope, the numerous green inflammatory cells were seen in the necrotic foci and edges of tumor tissues.In the subcutaneous transplanted tumor tissues, the markers of tumor microenvironment-related proteins and host myeloid-derived suppressor cells(MDSC)including HIF-1α, TNF-α, Ki67, CD68 and CD11 b were highly expressed, and the GFP+,CD68+ and CD11b+ cells in the transplanted tumor matrixes were obviously increased as well. After treatment of transplanted tumors with hyperbaric oxygen, the tumor weight was reduced, especially in the hyperbaric oxygen plus antitumor drug ACNU group.In the celiac transplanted tumor tissues, the RFP+, GFP+ and RFP+/GFP+ cells in thetumor tubercles were observed under fluorescent microscope. After further cultivation of these tumor tubercles, the GFP+ cells might change to the malignant CD68 cancer cells.In the plantar transplanted tumor tissues, the light microscope examination might observe the nuclear hyperchromatism and karyokinesis, dense cellular arrangement, and obvious cellular heteromorphy of tumor cells in the tumor edge tissues located the heart proximal end. The fluorescent microscope examination might observe the inflammatory cells derived from blood in the transplanted tumor tissues.In the hepatic transplanted tumor tissues, the light microscope examination might observe that the SU3 cells in ectopia livers were dense arrangement, nuclear large and hyperchromatism, and were able to infiltrate into normal hepatic tissues. Under fluorescent microscope, the red tumor cells and green host cells including tumor-associated macrophages(TAM) were cross. The further laser confocal microscope examination found that the both cells might change to yellow cells via the fusion or phagocytic action. The ratios of three-colour cells were 73.65%, 19.56% and 6.79%, respectively. These yellow cells might obtain the unlimited proliferation and translate into B4, B9 and B10 cells after monoclonal treatment. The B4 cells might express the RFP/GFP and biomarkers of dendritic cells, such as CD1 a, CD83 and CD86, whereas B9 and B10 cells only expressed the GFP and biomarkers of macrophages and fibroblasts, such as CD68, F4/80, FAP-α,α-SMA and S100. Pathological diagnoses of them were the malignant tumor cells.The average oxygen content in the tumor tissues was 28.93±8.06 mmol/L for scalp transplanted tumors and 47.46±15.96 mmol/L for intracranial transplanted tumors under ordinary state. After treatment of intracranial transplanted tumors with hyperbaric oxygen,the average oxygen content in the tumor tissues was up to 86.61±10.91 mmol/L, the difference was obvious relative to the ordinary state group(P<0.01), and the immunoinflammatory cells were significantly reduced as well.Conclusion:(1) Compared with the traditionary models of general nude mice inoculated human glioma stem cells, our present established the models with bicolor fluorescent trace(RFP-human glioma stem cells and GFP-host tissue, respectively) had the obvious advantages, it might not only identify the tumor cells and its interstitial cells in the tumor immunoinflammatory microenvironment under fluorescent microscope, but also identify the RFP+/GFP+ yellow co-location cells, namely, the cointegrate cells after tumor-cell and host-cell fusion.(2) Considering that tumor immunoinflammatory responseis a systemic response of local tumors and these involved cells mainly originate from the MDSC in the bone marrow, we thought that the inoculated parts might not be important,the identification and measurement of these immunoinflammatory cells might be more important.(3) The RFP+/GFP+ transplanted tumor models might apply to the studies on the positive or negative effects of tumor immunoinflammatory microenvironment on tumor growth, invasion, dissemination and metastasis, the malignant transform of tumor microenvironment cells themselves and their resistance against chemotherapy and radiotherapy.(4) Our present animal models demonstrated that the HBOT might increase the sensitivity of glioma chemotherapy via the improvement of tumor microenvironment hypoxic state and subsequent inhibition of tumor immunoinflammatory response. |
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