The temozolomide derivative 2T-P400 inhibits glioma growth via administration route of intravenous injection.

The aim of this study is to investigate the inhibitory effects of 2T-P400, a derivative of temozolomide (TMZ), on glioma growth. SHG-44 and U373 human glioblastoma cell lines and SHG-44 cell subcutaneous and intracranial xenograft mouse models were used as the model system for these studies. Cell growth was analyzed using MTT assay. For intracranial glioma xenograft model, mouse brains were obtained and made as paraffin section for immunohistochemical staining. Tumor volume was calculated with this formula: tumor volume = length × width2/ 2. The results showed that 2T-P400 or TMZ significantly inhibits cell growth in a concentration dependent manner with the IC50 values of 12.90 ± 1.05 or 9.73 ± 2.12 µg/ml on SHG-44 cell line and 13.12 ± 0.86 or 10.13 ± 1.02 µg/ml on U373 cell line respectively. In SHG-44 cell subcutaneous xenograft model, the tumor volume of 2T-P400 or TMZ treated group was 1,062.12 ± 204.76 or 803.59 ± 110.32 mm3 respectively, which was significantly smaller than that in physiological saline (with volume of 1,968.85 ± 348.37 mm3) treated group. In intracranial xenograft model, the tumor volume of 2T-P400 or TMZ group was 6.12 ± 1.69 or 5.58 ± 1.45 mm3 respectively, significantly smaller than that in physiological saline group of 33.08 ± 6.88 mm3. Moreover, polyethylene glycol 400 (PEG400) exhibited no significant tumor growth inhibition. Our results indicated that 2T-P400 posses the same growth inhibitory effect as TMZ on glioblastoma cell lines and the subcutaneously and intracranially transplanted gliomas in xenograft mouse models. It may be a suitable alternate of TMZ for the treatment of glioma via intravenous administration route.

[Establishment and characterization of dual-color fluorescence nude mouse models of glioma].

OBJECTIVE: To establish red-green dual-color fluorescence glioma model in nude mice and to explore its practical values. METHODS: CM-DiI-stained rat glioma C6 cells (C6-CM- DiI cells) expressing red fluorescence were inoculated into the brain of athymic nude mice expressing green fluorescence protein (NC-C57BL/6J-EGFP). Then the whole-body dual-color fluorescence imaging was detected dynamically. Finally whole brains of the tumor-bearing mice were removed and 5 µm thick serial frozen slices were made. Light microscopy, fluorescence microscopy and confocal laser scanning microscopy were performed to observe the transplanted tumor tissue structure and fluorescent cells. RESULTS: Tumor mass with red fluorescence increased gradually under continuous in-vivo fluorescence imaging monitoring. Under the fluorescence microscope, cells with red, green and yellow fluorescence were observed in the frozen sections of transplanted tumor tissue and the mutual structural relationship among them could be defined. The tumor cells migration, implantation and cell fusion between transplanted tumor cells and host cells could be observed. It could be distinguished according to the fluorescence, that blood vessels of tumor-origin displayed red fluorescence, blood vessels of host-origin displayed green fluorescence and mosaic blood vessels appeared yellow fluorescence. It was depicted that host innate astrocytes and oligodendrocytes in the microenvironment at the tumor periphery could be activated and dedifferentiated into nestin-positive cells. CONCLUSIONS: In contrast to traditional animal model, the dual-color fluorescence imaging of nude mouse models of glioma possesses enormous advantages in investigating tumor mass in-vivo fluorescence imaging, tumor cells migration and metastasis, tumor angiogenesis and reactive activation of host innate cells in the microenvironment at tumor periphery, thus, has highly practical application value.

[Malignant transformation of glioma stromal cells induced by glioma stem cells heterotopically inoculated in host liver].

OBJECTIVE: Tumor stromal cells have the potential of undergoing malignant transformation induced by glioma stem cells (GSCs) in orthotopic glioma model. The purpose of this study was to explore whether malignant transformation of tumor stromal cells induced by GSCs is dependent on specific local microenvironment. METHODS: Human glioma stem/progenitor cell line SU3 transfected with red fluorescent protein (SU3-RFP) gene were implanted into the liver of nude mice with whole-body expressing green fluorescence protein (GFP). Then hepatic tumors were harvested to prepare single cell suspension and analyzed with routine pathological examinations. GFP cells with high proliferative abilities were obtained from the cultivation of single cell suspension. Immortalized glioma stromal cells only expressing GFP and double expressing GFP/RFP were further monocloned with micro-pipetting techniques and under continuous passages. Cell phenotypic analysis and tumorigenicity tests were also performed. RESULTS: SU3-RFP was transplanted into liver with a tumor formation rate of 83%. Immortalized glioma stromal cells were obtained from re-cultured xenograft tumor tissue. Three monoclonal cell lines B4, B9, B10 were established and proved to be host-derived cells. B4 was found to be a fusion cell co-expressing GFP/RFP and dendritic cell markers CD1 a, CD83 and CD86. Both B9 and B10 were GFP⁺ cells. B9 expressed macrophage markers CD68 and F4/80 while B10 produced fibroblast marker proteins FAP-α, α-SMA and S100. Three cells were all aneuploid with a tumorigenicity rate of 100% in nude mice. CONCLUSION: Tumor stromal cells have the potential of malignant transformation in a heterotopic xenograft glioma model. Malignant transformation may also occur outside the central nervous system and contribute to tumor heterogeneity. Further studies are warranted for elucidating the relationship between tumorigenesis, evolution and tumor microenvironment.

Effect and Blood Flow Parameters of Biomaterials-Based Endovascular Interventional Embolization and Craniotomy Clipping in the Treatment of Cerebral Aneurysms.

Objective: The effects of interventional embolization and craniotomy clipping on the treatment of intracranial aneurysms were investigated in this study, as well as their influence on the hemodynamics of postoperative patients. Methods: 102 patients with intracranial aneurysms were selected as the research objects, and they were rolled into an experimental (group A) and a control group (group B) according to the random number table method, with 51 cases in each group. The group A was treated with intravascular interventional embolization, and the group B received craniotomy clipping. Besides, a biodegradable magnesium titanium alloy biological stent was independently developed in this study, which was applied to endovascular interventional embolization in the group A. The hemodynamic model was established by using three-dimensional (3D) computer hemodynamic numerical simulation technology. Besides, the effects of all the patients before and after treatment were evaluated, in terms of blood pressure (BP), average wall shear stress (WSS) (AWSS), AWSS gradient (AWSSG), oscillatory shear index (OSI), aneurysm formation index (AFI), gradient oscillation number (GON), and intraoperative complication rate. Results: After 3 days of treatment, the BP, AWSS, and AWSSG of patients from the two groups were higher than those before treatment. The index values of the group A were greater markedly than the values of the group B (P < 0.05); the BP of the group A and the group B at the 0th day, 1st day, 3rd day, 5th day, and 7th day after treatment was 21±5.1 Versus 20.1±4.7, 22±4.8 Versus 21.1± 5.17, 26±6.2 Versus 22.31±5.21, 27±5.77 Versus 24.02±5.11, and 30±6.09 Versus 24.99±5.03, respectively; AWSSG values were 120±10.11 Versus 120.1±10.98, 130.1±10.36 Versus 123.3±11.06, 162.5±9.92 Versus 131.31±10.97, 171±8.13 Versus 155.02±8.36, and 200.1±7.22 Versus 180.01±8.98 in turn. GON and OSI were both decreased, and the values of various indexes in the group A were sharply lower than those of the group B (P < 0.05); the values of GON at the 0th day, 1st day, 3rd day, 5th day, and 7th day after treatment in the group A and the group B were 0.077±0.01 Versus 0.08±0.011, 0.07±0.012 Versus 0.073 ± 0.01, 0.051 ± 0.02 Versus 0.071 ± 0.011, 0.045 ± 0.01 Versus 0.069 ± 0.011, and 0.042 ± 0.012 Versus 0.063±0.013, respectively; OSI values were 4.8±0.51 Versus 4.9±0.52, 3.6±0.52 Versus 3.62±0.51, 2.82±0.51 Versus 3.1 ± 0.57, 1.9 ± 0.512 Versus 2.91 ± 0.51, and 0.5 ± 0.51 Versus 1.8 ± 0.501 in turn. By comparing the intraoperative complications and postoperative mortality risk score (MRS) of patients in the two groups, it was found that the incidence of intraoperative complications and postoperative MRS scores in the group A were lower steeply than those of the group B, suggesting that endovascular interventional embolization had a better effect on the treatment of intracranial aneurysms. Conclusion: Endovascular interventional embolization based on biodegradable magnesium alloy coated scaffold could better improve the distribution of shear stress on the vascular wall, stabilize vascular blood flow, and achieve better therapeutic effect for patients.

Alteration of DNA damage signaling pathway profile in radiation-treated glioblastoma stem-like cells.

The present study aimed to investigate the alteration of the DNA damage signaling pathway profile in radiation-treated glioblastoma stem-like cells (GSLCs), and also aimed to explore potential targets for overcoming glioblastoma radioresistance. Serum-free medium was used to isolate and culture GSLCs. Cell growth was detected using a cell counting kit-8 assay and cell sorting analysis was performed by flow cytometry. X-ray irradiation was produced by a Siemens-Primus linear accelerator. Reverse transcription-quantitative polymerase chain reaction (qPCR)was performed to investigate target genes. SPSS 15.0 was used for all statistical analyses. Human glioblastoma U251 and U87 cells were cultured in serum-free medium supplemented with epidermal growth factor and fibroblast growth factor 2, which constitutes tumor sphere medium, and demonstrated sphere formation, with significantly increased the proportion of CD133+ and Nestin+ cells, which are referred to as GSLCs. The present data revealed that treatment with 10 Gy X-ray radiation alters the expression profile of DNA damage-associated genes in GSLCs. The expression levels of 12 genes demonstrated a ≥2-fold increase in the irradiated U87 GSLCs compared with the untreated U87 GSLCs. Three genes, consisting of XPA, RAD50 and PPP1R15A, were selected from the 12 genes by gene functional searching and qPCR confirmatory studies, as these genes were considered to be potential targets for overcoming radioresistance. The expression of XPA, RAD50 and PPP1R15A is significantly increased in U87 and U251 radiation resistant GSLCs, indicating three potential targets for overcoming the radioresistance of GSLCs.

Malignant transformation of host stromal fibroblasts derived from the bone marrow traced in a dual-color fluorescence xenograft tumor model.

Solid tumors are abnormal tissues containing tumor and non-tumor cells, also known as tumor stromal cells. However, the malignant potential of tumor stromal cells remains largely unknown. The aim of the present study was to investigate the malignant potential of host bone marrow­derived stroma cells in transplanted subcutaneous tumors of the glioma stem/progenitor cells (GSPCs) labeled using the dual-color fluorescent tracer technique. The previously established human glioma stem/progenitor cell line SU3 was transfected with red fluorescence protein (SU3-RFP) and transplanted subcutaneously into green fluorescent protein (GFP) transgenic nude mice and chimeric mice in which GFP was only expressed by bone marrow-derived cells (BMDCs). The xenograft tumors were subcultured in vitro and two immortalized GFP-expressing stromal cell lines were cloned from the transplanted tumors. The two cloned cell lines showed an accelerated growth rate, loss of cell contact inhibition, high cloning efficiency, and high DNA content and telocentric (murine) chromosomes with heteroploid characteristics. The tumorigenesis rate (10/10, 1x10(6)) of these host stromal cells was further evidence of malignant transformation. Immunofluorescence assay of the two host cell lines showed that they expressed fibroblast markers such as FAP, S100A4 and α-SMA, as well as mesenchymal cell markers such as CD44 and CD105. In conclusion, bone marrow-derived stromal fibroblasts recruited to tumors have the potential for malignant transformation induced by the tumor microenvironment, which provides new evidence for the role of the stroma in malignant transformation.

Advantages of a dual-color fluorescence-tracing glioma orthotopic implantation model: Detecting tumor location, angiogenesis, cellular fusion and the tumor microenvironment.

Various organs of the body have distinct microenvironments with diverse biological characteristics that can influence the growth of tumors within them. However, the mechanisms underlying the interactions between tumor and host cells are currently not well understood. In the present study, a dual-color fluorescence-tracing glioma orthotopic implantation model was developed, in which C6 rat glioma cells labeled with the red fluorescent dye CM-Dil, and SU3 human glioma cells stably expressing red fluorescence protein, were inoculated into the right caudate nucleus of transgenic female C57BL/6 nude mice expressing enhanced green fluorescent protein. The dual-color tracing with whole-body in vivo fluorescence imaging of xenografts was performed using a live imaging system. Frozen sections of the transplanted tumor were prepared for histological analyses, in order to detect the presence of invading tumor cells, blood vessels and cellular fusion. Dual-color images were able to distinguish between red tumor cells and green host cells. The results of the present study suggested that a dual-color fluorescence-tracing glioma orthotopic implantation model may be convenient for detecting tumor location, angiogenesis, cellular fusion, and the tumor microenvironment.