Xingliang Dai

Glioma initiating cells contribute to malignant transformation of host glial cells during tumor tissue remodeling via PDGF signaling

INTRODUCTION Glioma initiating cells (GICs) play important roles in tumor initiation and progression. However, interactions between tumor cells and host cells of local tumor microenvironment are kept largely unknown. Besides GICs and their progeny cells, whether adjacent normal glial cells contribute to tumorigenesis during glioma tissue remodeling deserves further investigation. METHODS Red fluorescence protein (RFP) gene was stably transfected into human GIC cells lines SU3 and U87, then were transplanted intracerebrally into athymic nude mice with whole-body green fluorescence protein (GFP) expression. The interactions between GICs and host cells in vivo were observed during tissue remodeling processes initiated by hGICs. The biological characteristics of host glial cells with high proliferation capability cloned from the xenograft were further assayed. RESULTS In a SU3 initiated dual-fluorescence xenograft glioma model, part of host cells cloned from the intracerebral tumors were found acquiring the capability of unlimited proliferation. PCR and FISH results indicated that malignant transformed cells were derived from host cells; cell surface marker analysis showed these cells expressed murine oligodendrocyte specific marker CNP, and oligodendrocyte progenitor cells (OPCs) specific markers PDGFR-α and NG2. Chromosomal analysis showed these cells were super tetraploid. In vivo studies showed they behaved with high invasiveness activity and nearly 100% tumorigenic ratio. Compared with SU3 cells with higher PDGF-B expression, GICs derived from U87 cells with low level of PDGF-B expression failed to induce host cell transformation. CONCLUSIONS Primary high invasive GICs SU3 contribute to transformation of adjacent normal host glial cells in local tumor microenvironment possibly via PDGF/PDGFR signaling activation, which deserved further investigation.

Coaxial 3D bioprinting of self-assembled multicellular heterogeneous tumor fibers

Three-dimensional (3D) bioprinting of living structures with cell-laden biomaterials has been achieved in vitro, however, some cell-cell interactions are limited by the existing hydrogel. To better mimic tumor microenvironment, self-assembled multicellular heterogeneous brain tumor fibers have been fabricated by a custom-made coaxial extrusion 3D bioprinting system, with high viability, proliferative activity and efficient tumor-stromal interactions. Therein, in order to further verify the sufficient interactions between tumor cells and stroma MSCs, CRE-LOXP switch gene system which contained GSCs transfected with “LOXP-STOP-LOXP-RFP” genes and MSCs transfected with “CRE recombinase” gene was used. Results showed that tumor-stroma cells interacted with each other and fused, the transcription of RFP was higher than that of 2D culture model and control group with cells mixed directly into alginate, respectively. RFP expression was observed only in the cell fibers but not in the control group under confocal microscope. In conclusion, coaxial 3D bioprinted multicellular self-assembled heterogeneous tumor tissue-like fibers provided preferable 3D models for studying tumor microenvironment in vitro, especially for tumor-stromal interactions.

Experimental research of host macrophage canceration induced by glioma stem progenitor cells

The involvement of tumor-associated macrophages in tumor progression is an indisputable fact. However, whether the growth-promotion effects of macrophages towards tumors in the aggressive stage affect their own canceration remains unknown. In the present study, human glioma stem/progenitor cells transfected with red fluorescent protein gene (SU3-RFP) were seeded inside the abdominal cavity of transgenic nude mice, of which all nucleated cells could express green fluorescent protein (GFP), forming a tumor model with a double-color RFP/GFP fluorescent tracer. Ascites and tumor nodules from tumor-bearing mice were cultured, then the GFP+ cells were separated for clonal culture and further related phenotypic characterization and tumorigenicity tests. It was observed that the GFP+ cells isolated from ascites and solid tumors exhibited unlimited proliferative potential; the monoclonal cells were mouse-original, had a cancer cell phenotype and expressed the macrophage marker protein CD68. Thus, in the abdominal tumor model with double-color fluorescent tracer, macrophages recruited by tumor cells not only promoted tumor cell growth, but also exhibited their own canceration. This discovery is significant for the further study of tumor tissue remodeling and the tumor microenvironment.

Establishment of a green fluorescent protein tracing murine model focused on the functions of host components in necrosis repair and the niche of subcutaneously implanted glioma.

Due to progress in the research of glioma stem cells and the glioma niche, development of an animal model that facilitates the elucidation of the roles of the host tissue and cells is necessary. The aim of the present study was to develop a subcutaneous xenograft green fluorescent protein nude mouse model and use this model to analyze the roles of host cells in tumor necrosis repair. Tumors derived from the human glioma stem/progenitor cell line SU3 were subcutaneously implanted in green fluorescent protein nude mice. The implanted tumors were then passed from animal to animal for 10 generations. Finally, subcutaneous xenografts were assayed with traditional pathology, immunopathological techniques and fluorescence photography. For each generation, the tumorigenicity rate was 100%. Subcutaneous xenografts were rich in blood vessels, and necrotic and hemorrhagic foci, which highly expressed hypoxia-inducible factor-1α, tumor necrosis factor, Ki-67, CD68 and CD11b. In the interstitial tissue, particularly in old hemorrhagic foci, there were numerous cells expressing green fluorescent protein, CD68 and CD11b. Green fluorescent protein nude mouse subcutaneous xenografts not only consistently maintained the high invasiveness and tumorigenicity of glioma stem/progenitor cells, but also consisted of a high concentration of tumor blood vessels and necrotic and hemorrhagic foci. Subcutaneous xenografts also expressed high levels of tumor microenvironment-related proteins and host-derived tumor interstitial molecules. The model has significant potential for further research on tumor tissue remodeling and the tumor microenvironment.

Preliminary analysis of cellular sociology of co-cultured glioma initiating cells and macrophages in vitro

ObjectiveReal-time monitoring of cytokine secretion at the single immunocyte level, based on the concept of immune cells, sociology has been recently reported. However, the relationships between gl...

Fusion of cancer stem cells and mesenchymal stem cells contributes to glioma neovascularization

The ability of tumor cells to autonomously generate tumor vessels has received considerable attention in recent years. However, the degree of autonomy is relative. Meanwhile, the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on tumor neovascularization has not been fully elucidated. The present study aimed to illuminate whether cell fusion between glioma stem cells and BMSC is involved in glioma neovascularization. BMSCs were isolated from transgenic nude mice, of which all nucleated cells express green fluorescent protein (GFP). The immunophenotype and multilineage differentiation potential of BMSC were confirmed. SU3 glioma stem/progenitor cells were transfected with red fluorescent protein (SU3-RFP cells). In a co-culture system of BMSC-GFP and SU3-RFP, RFP+/GFP+ cells were detected and isolated by dual colors using FACS. The angiogenic effect of RFP+/GFP+ cells was determined in vivo and in vitro. Flow cytometry analysis showed that BMSC expressed high levels of CD105, C44, and very low levels of CD45 and CD11b. When co-cultured with SU3-RFP, 73.8% of cells co-expressing RFP and GFP were identified as fused cells in the 5th generation. The fused cells exhibited tube formation ability in vitro and could give rise to a solid tumor and form tumor blood vessels in vivo. In the dual-color orthotopic model of transplantable xenograft glioma, yellow vessel-like structures that expressed CD105, RFP and GFP were identified as de novo-formed vessels derived from the fused cells. The yellow vessels observed in the tumor-bearing mice directly arose from the fusion of BMSCs and SU3-RFP cells. Thus, cell fusion is one of the driving factors for tumor neovascularization.

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.

Prognostic value of NUSAP1 in progression and expansion of glioblastoma multiforme

Nucleolar and spindle-associated protein (NUSAP1) is a microtubule and chromatin-binding protein that stabilizes microtubules to prevent depolymerization, maintains spindle integrity. NUSAP1 could cross-link spindles into aster-like structures, networks and fibers. It has also been found to play roles in progression of several cancers. However, the potential correlation between NUSAP1 and clinical outcome in patients with glioblastoma multiforme (GBM) remains largely unknown. In the current study, we demonstrated that NUSAP1 was significantly up-regulated in GBM tissues compared with adult non-tumor brain tissues both in a validated cohort and a TCGA cohort. In addition, Kaplan–Meier analysis indicated that patients with high NUSAP1 expression had significantly lower OS (P = 0.0027). Additionally, in the TCGA cohort, NUSAP1 expression was relatively lower in GBM patients within the neural and mesenchymal subtypes compared to other subtypes, and associated with the status of several genetic aberrations such as PTEN deletion and wild type IDH1. The present study provides new insights and evidence that NUSAP1 over-expression was significantly correlated with progression and prognosis of GBM. Furthermore, knockdown of NUSAP1 revealed its regulation on G2/M progression and cell proliferation (both in vitro and in vivo). These data demonstrate that NUSAP1 could serve as a novel prognostic biomarker and a potential therapeutic target for GBM.

3D bioprinted glioma cell-laden scaffolds enriching glioma stem cells via epithelial-mesenchymal transition: 3D bioprinted glioma cell-laden scaffolds enriching glioma stem cells via epithelial-mesenchymal transition

Glioma stem cells (GSCs) are thought to be the root cause of tumor recurrence and drug resistance in glioma patients. In-depth study of GSCs is of great significance for developing the treatment strategies of glioma. Unfortunately, it is difficult and takes complicated process to obtain GSCs. Therefore, establishing an ideal in vitro model for enriching GSCs will greatly promote the study of GSCs. In this study, the stemness properties of glioma cells were enhanced in three-dimensional (3D) bioprinted tumor model. Furthermore, the possible molecular mechanism of GSCs enrichment: epithelial-mesenchymal transition (EMT) was explored. Compared with two-dimensional cultured cells, the proportion of GSCs and EMT-related genes in 3D cultured cells were significantly increased. Moreover, the 3D cultured glioma cells with improved stemness properties resulted in higher drug resistance in vitro and tumorigenicity in vivo. Taken together, 3D bioprinted glioma cell-laden scaffold provides a proper platform for the enrichment of GSCs and it is expected to further promote the research on glioma drug resistance.

Bioprinting of glioma stem cells improves their endotheliogenic potential

Glioblastoma (GBM), with rich blood vessels and high invasiveness, is the most common malignant primary brain tumor. The current treatment strategies are less effective, resulting in tumor recurrence. Tumor angiogenesis plays an important role in the occurrence, development and metastasis of GBM. Currently, GBM has been treated by inhibiting tumor angiogenesis. In-depth study of tumor angiogenesis is of great significance for the treatment of GBM. Recent studies have shown that glioma stem cells (GSCs) are involved in tumor vascularization by secreting vascular endothelial growth factor (VEGF). It is necessary to construct an ideal in vitro model to study the mechanism of GSCs in tumor vascularization. Here we used extrusion-based three-dimensional (3D) bioprinting technology to fabricate GSCs tumor model. In this study, the viability of cells after bioprinting was 86.27 ± 2.41%. Furthermore, compared with traditional suspension culture, the proliferation of 3D printed GSCs was more stable. Through the transmission electron microscopy (TEM), numerous long microvilli of cells cultured in 3D bioprinted scaffolds were observed. 3D bioprinted GSCs also have more abundant mitochondria and rough endoplasmic reticulum. Additionally, the stemness properties, the expression of tumor angiogenesis-related genes and vascularization potential of 3D bioprinted GSCs in vitro were higher than that of suspension cultured cells. In summary, 3D bioprinted cell-laden hydrogel scaffolds provide a proper model for investigating the biological characteristics of GSCs and tumor angiogenesis.