| Glioma is the most common subtype of primary tumor in central nervous system, about 40~50% of total primary tumors, with the characteristic of fast recurrence and high mortality, and requires an available therapy strategy. In recent years, with the development of molecular biology, pathogenesis and treatment of glioma research, it is found that the standard treatment principle of malignant glioma is radical surgical resection, combined with temozolomide(TMZ) plus radiotherapy after operation. Despite progress in the glioma therapy, the prognosis of malignant glioma remains frustrating, especially the patients with newly diagnosed glioblastoma multiforme represent a median overall survival no more than 18 months. The most difficult question is the treatment failure induced by recurrence and chemotherapeutic resistance. Recent studies have shown that the glioma biology is closely connected to the existence of glioma stem cells(GSCs). They are pluripotent and have the potential to self-renew, and play a crucially important role on the cell growth, recurrence, invasion, and metastasis. They are seed cells, and critical to the progression and recurrence of glioma. At the same time, these seed cells also exhibit stronger resistance to treatment than normal glioma cells, lead to drug resistance with various other factors and ultimately result in treatment failure. Therefore, the study of GSCs and drug resistance mechanisms is becoming the focus of improving the efficacy of glioma therapy. To deal with such study, the priority is to develop a suitable glioma cells culture method in vitro. Because there is a significant drawback, which is difficult to provide a similar glioma physical microenvironment by losing the 3D space and extracellular matrix(ECM) in vivo, in present cell culture technique. Tumor physical microenvironment plays an important role in the growth, invasion, metastasis, stem cell maintenance, intercellular communication, and drug resistance of tumor cells. Traditional 2D culture method has been shown to recapitulate some phenotypic traits observed clinically. However, to model the full range of microenvironmental traits is an impossible mission for it, especially the method cannot avoid losing important cues elicited by 3D cell–cell and cell–ECM. Accelerated development of anti-glioma drugs is critically dependent on preclinical models. Unsuitable culture methods cannot simulate in vivo tumor growth and intracellular signaling as accurately as possible, and lead to some wrong conclusions in preclinical studies. Thus, the in vitro study of tumor cell biology needs to find a better model of cell culture.In recent years, with the development of tissue engineering and biomaterials science, cell culture technology is undergoing a developing process from the 2D to 3D, monolayer to stereoscopic. Firstly, heightened awareness of the importance of 3D tumor cells culture has resulted in the increasing use of tumorsphere culture systems for cancer research. However, these non–adhesion-mediated systems cannot provide good control over the tumor architecture and cell–cell interactions; as a result of culture conditions that prohibit cellular attachment onto surrounding surfaces, cells autonomously aggregate and form the 3D geometry of spheroid. Actually, there are no floating tumor spheroids in solid tumors. To overcome this inherent drawback of spheroid culture, some biomaterials have been developed. They have good biocompatibility and mechanical properties, can better simulate ECM to provide sufficient support for the growth of cells. Then the 3D scaffolding technologies comprised by these biomaterials have been developed for tumor tissue-engineering applications, to guide tumor tissue formation in vitro with controlled architectural complexity. The 3D cell culture approaches(gel systems and spheroid cultures) have dramatically improved our understanding of the role of 3D culture on glioma cells, but there still exists a need for innovative 3D tumor models. The scaffold materials which are capable of recreating distinct glioma niches is lack, the association of the tumor physical microenvironment and the mechanism of GSCs and drug resistance is unclear. It is, therefore, necessary to improve in vitro glioma cell-based testing methods and culture technology for more effective research on GSCs and more informed prediction of anti-glioma drug candidate efficacy and safety.In this study, we developed a porous 3D collagen scaffold to solve the problems mentioned above. We firstly constructed an in vitro 3D culture model of glioma cells(U87 cell line and primary cell) by this collagen scaffold. Morphology, proliferation, apoptosis and cell cycle of glioma cells in 3D collagen scaffolds were examined. Malignant phenotypes, such as drugs resistance-related phenotype expression and GSCs property acquisition, were also studied by quantitative real-time reverse transcription-PCR(qRT-PCR), western blotting and fluorescence activated cell sorting(FACS) in vitro. We further investigated the potential drug resistant mechanisms. Finally, we explored the inhibitory effect of major anti-glioma drugs under different culture models to demonstrate the effectiveness of 3D collagen scaffold method in drug discovery. The main results and conclusions are as follows.Part 1: The construction of in vitro 3D culture model of glioma cells by porous 3D collagen scaffold and general phenotypic observation.Objective: In order to better study the malignant biological behavior of glioma cells, this study aims to establish a 3D culture model of glioma cells in vitro. Taking into account the most important component of the glioma extracellular matrix is collagen, the collagen is chosen as the biomaterials of porous 3D scaffold. Glioma cells were seed into scaffold, and their adhesion capability, several general phenotypes such as morphology, growth kinetics, proliferation and apoptosis were observed. The ultimate aim is to evaluate whether the collagen scaffold can support glioma cells for a good growth, and discussed the changes of general cell phenotype between the collagen scaffold 3D culture model and traditional 2D culture model.Methods: The prepared scaffold was observed by scanning electron microscope(SEM), and the average pore size was calculated. The adhesion, migration and proliferation of glioma cells on the scaffold were observed by fluorescence microscopy and laser scanning confocal microscopy. H&E staining was performed to histological observation in different glioma growth models and human glioma specimens. The ultrastructure and morphology of glioma cells was observed by SEM. Finally, the tumor cell growth rate was evaluated by CCK8 dye reduction method and cell cycle, proliferation, apoptosis and differentiation were detected by flow cytometry.Results: Our collagen scaffold had a 3D aligned microstructure, and it had a suitable pore size for cell culture. The glioma primary cells were round, ovoid shape-like with microvillus in collagen scaffold, but fusiform, flat and epithelioid in 2D culture. The collagen scaffolds provided a good morphology mimic of glioma cells compared with real human glioma tissues. The distribution of cells in the scaffold was stereoscopically and conferted, which reflect the introrsely movements in the process of glioma cells proliferation on the 3D scaffolds. The growth rate of glioma cells in 3D scaffolds was significantly slower compared with in 2D monolayer culture. Furthermore, the new culture model induced an accumulation of glioma cells in the G0/G1 phase fraction with concomitant reduction of cell numbers in S phase, lower Ki-67, similar apoptosis and dedifferentiation compared with 2D culture.Conclusion: The collagen scaffold is a novel, 3D, microporous, aligned scaffold. It is very suitable for the growth of glioma cells on the surface and in the inside. And glioma cells cultured in collagen scaffold exhibit many different phenotypes with cells of traditional culture, but are similar with glioma cells in vivo. Our collagen scaffold has a good application prospect in the construction of tissue-engineered glioma.Part 2: Characterization and analysis of stemness-associated properties in 3D collagen scaffold cultured glioma cells.Objective: The feasibility, biocompatibility and bionic-character of 3D glioma cells culture model with collagen scaffold were verified in previous part study. We found the glioma cells cultured in collagen scaffold exhibit some biological features belonging to GSCs-like cells, such as more quiescent cells and taking place dedifferentiation. However, the present enrichment culture method of GSCs is very limited, and the traditional monolayer cell culture method cannot achieve the GSCs enrichment. Therefore, this part focused on using various experimental methods to explore the characterization of stemness-associated properties of glioma cells cultured in 3D collagen scaffold, and verified its feasibility as a new GSCs enrichment culture method.Methods: The proportion of CD133 positive glioma cells were detected by immunofluorescence and flow cytometry under 2D and 3D culture conditions respectively. Furthermore, the relative mRNA levels and protein abundance of main stemness associated transcription factors Sox2, Oct-4 and Nanog were tested by q RT-PCR and western blotting method. Finally, the important properties of tumor stem cells, strong self-renewal and tumor formation capability, were detected by plate colony formation, tumorspheres formation and subcutaneous xenografts assay in different culture modelsResults: Glioma sells from collagen scaffold culture model possess stem cell properties. More CD133 positive U87 cells were observed in 3D collagen scaffold culture, either in immunofluorescence staining or flow cytometer analysis. Compared with monolayer cells, glioma cells in collagen scaffold highly expressed stemness-related factors Nanog and Sox2 at both the transcriptional and translational levels. Moreover, the colony formation and tumor-spheroid formation assays reflected stronger self-renewal capability, and xenograft assay of nude mice showed that glioma cells in 3D scaffold culture were highly tumorigenic. Those data suggested that the collagen scaffold culture could induce enhanced stem cell properties of glioma cells.Conclusion: The stemness of glioma cells was improved by upregulation of stem cell markers and cancer stem cell transcription factors in our study. It suggested that the collagen scaffold culture method could be used as a new strategy for enrichment of GSCs.Part 3: The enhancement of drug resistance properties of glioma cells in 3D collagen scaffolds and its potential mechanisms.Objective: Previous work had been reported using 3D collagen scaffold culture for better understanding some malignant phenotypes and enriching more GSCs in vitro. On the basis of previous studies, our hypothesis was that in vitro grown cells in 3D scaffold will provide better information on drug susceptibility of glioma cells than the 2D culture. Therefore, this part focused on the cytotoxic evaluation of clinical antitumor drugs to glioma cells under 2D and 3D scaffold culture conditions. The purpose is to clarify whether the 3D collagen scaffold culture model can induce drug resistance, and explore its potential related mechanism. And finally to investigate whether 3D collagen scaffold culture model can be used as better anti-glioma drugs testing platform.Methods: U87 cell line and glioma primary cell were used in this part. The half maximal inhibitory concentration(IC50) value of different chemotherapy drugs(DDP, CCNU, TMZ) to glioma cells was measured by CCK8 kit assay. And the main resistance related genes were tested by q RT-PCR. Then according to the results of PCR, the expression of drug resistance proteins was verified through western blotting assay. Finally, the inhibition rate tests of major anti-glioma drugs(DDP, CCNU, TMZ, CPT-11) on U87 cells at human plasma peak concentrations(PPCs) were performed by CCK8 assay for comparing with clinical response efficiency.Results: In this comparison, both U87 and primary cells cultured in 3D collagen scaffolds demonstrated greater resistance for all three drugs than cells in 2D monolayer culture. The resistance to alkylating agents was more prominent than to DDP. The mRNA and protein levels of CD133 and MGMT were significantly upregulated in both U87 and primary cells in 3D scaffold culture. The results suggested that upregulation of drug resistance in the 3D collagen scaffolds may be attributed to both the improvement of DNA damage repair capacity and the enhanced stemness of tumor cells. A first finding was that the MGMT-negative glioma cell line in traditional culture assay could emerge MGMT positive expression with the changes of growth dimension and ECM microenvironments. The final data suggested the drug sensitivity of glioma cells in 3D collagen scaffold culture but not 2D culture was close to the clinical objective response rates.Conclusion: 3D collagen scaffold culture model can induce glioma chemotherapy resistance in vitro. And it suggested the microenvironment changes of glioma cells lead to drug resistance at least partly induced by GSCs enrichment and enhanced DNA damage repair mechanism. The final results reflect collagen scaffold can be an appropriate and promising research platform for new anti-glioma drugs screening and the mechanism study of tumor microenvironment related resistance.In conclusion, we developed an in vitro 3D glioma culture model using porous collagen scaffold. Through a serious of experiments, the model showed it could provide a good mechanical, attachment, migration, proliferation support for glioma cells. Furthermore, the 3D culture exhibited stark different morphological and biochemical features in contrast with conventional monolayer culture model. Glioma cells in this model contained greater drug-resistance with higher proportion of GSCs and overexpression of MGMT. Significantly, this method of culture produced the similar chemotherapy sensitivity with clinical objective response rates. We believe it should be an appropriate and promising research platform for new anti-glioma drugs screening and its mechanism study. |
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