Qing Lan

Glioma stem cells are more aggressive in recurrent tumors with malignant progression than in the primary tumor, and both can be maintained long-term in vitro

BackgroundDespite the advances made during decades of research, the mechanisms by which glioma is initiated and established remain elusive. The discovery of glioma stem cells (GSCs) may help to elucidate the processes of gliomagenesis with respect to their phenotype, differentiation and tumorigenic capacity during initiation and progression. Research on GSCs is still in its infancy, so no definitive conclusions about their role can yet be drawn. To understand the biology of GSCs fully, it is highly desirable to establish permanent and biologically stable GSC lines.MethodsIn the current study, GSCs were isolated from surgical specimens of primary and recurrent glioma in a patient whose malignancy had progressed during the previous six months. The GSCs were cryopreserved and resuscitated periodically during long-term maintenance to establish glioma stem/progenitor cell (GSPC) lines, which were characterized by immunofluorescence, flow cytometry and transmission electronic microscopy. The primary and recurrent GSPC lines were also compared in terms of in vivo tumorigenicity and invasiveness. Molecular genetic differences between the two lines were identified by array-based comparative genomic hybridization and further validated by real-time PCR.ResultsTwo GSPC lines, SU-1 (primary) and SU-2 (recurrent), were maintained in vitro for more than 44 months and 38 months respectively. Generally, the potentials for proliferation, self-renewal and multi-differentiation remained relatively stable even after a prolonged series of alternating episodes of cryopreservation and resuscitation. Intracranial transplantation of SU-1 cells produced relatively less invasive tumor mass in athymic nude mice, while SU-2 cells led to much more diffuse and aggressive lesions strikingly recapitulated their original tumors. Neither SU-1 nor SU-2 cells reached the terminal differentiation stage under conditions that would induce terminal differentiation in neural stem cells. The differentiation of most of the tumor cells seemed to be blocked at the progenitor cell phase: most of them expressed nestin but only a few co-expressed differentiation markers. Transmission electron microscopy showed that GSCs were at a primitive stage of differentiation with low autophagic activity. Array-based comparative genomic hybridization revealed genetic alterations common to both SU-1 and SU-2, including amplification of the oncogene EGFR and deletion of the tumor suppressor PTEN, while some genetic alterations such as amplification of MTA1 (metastasis associated gene 1) only occurred in SU-2.ConclusionThe GSPC lines SU-1 and SU-2 faithfully retained the characteristics of their original tumors and provide a reliable resource for investigating the mechanisms of formation and recurrence of human gliomas with progressive malignancy. Such investigations may eventually have major impacts on the understanding and treatment of gliomas.

Glioma Stem/Progenitor Cells Contribute to Neovascularization via Transdifferentiation

ObjectivePrevious studies suggest that tumor cells might be the progenitor for tumor vasculature. Whether vascular tube formation from transdifferentiation of human glioma stem/progenitor cells (hGSPCs) contribute to angiogenesis of gliomas remain largely uncertain.MethodshGSPCs were isolated from thirteen surgical specimens of gliomas and cultured in medium favored for stem cell growth. In vitro transdifferentiation of hGSPCs was performed under hypoxia. Expression of vascular endothelial cells (VECs) markers CD31, CD34, kinase insert domain receptor (KDR), and von Willebrand factor (vWF) were analyzed with real-time quantitative RT-PCR and immunofluorescence techniques. Vasculogenic mimicry of hGSPCs was evaluated in a tumor stem cell xenograft model in vivo. Relationships between content of hGSPCs and expression levels of both VECs markers and proangiogenic factors in large number of clinical specimens were further investigated in glioma tissue microarray.ResultsIn vitro, hGSPCs can transdifferentiate into VECs under hypoxia, they manifested typical “flagstone” pattern when cultivated in medium containing VEGF for a few days; when cultivated on Matrigel they were capable of forming capillary-like structures. Expression of VECs markers including CD31, CD34, KDR, and vWF were significantly up-regulated after transdifferentiation. Human leukocyte antigen (HLA) positively stained vessels were observed inside the xenograft tumors after intracerebral transplantation of hGSPCs in athymic nude mice, implied part of tumor cells with human origin were involved in formation of tumor vessels. In surgical specimens of human glioma, tumor vascular cells coexpressing the markers of early VECs (CD34) and markers of hGSPCs (ABCG2 and nestin) suggest that these vascular cells may stemmed from hGSPCs.ConclusionsOur observations suggest the functional role of hGSPCs as endothelial progenitors, which have properties that give rise to VECs, and have the ability to form vascular endothelial tubes. However, unspecific markers (ABCG2, nestin) that stain for both endothelial as well as glioma stem cells, were found to be expressed in tumor vasculature of human specimen, and limit further interpretation of this finding.

Overexpression of CDC2/CyclinB1 in gliomas, and CDC2 depletion inhibits proliferation of human glioma cells in vitro and in vivo

BackgroundGliomas are the most common and aggressive primary brain tumors for which unfortunately no effective treatment modalities exist despite advances in molecular biology as the knowledge base to unravel the extremely complex molecular mechanisms of tumorigenesis is limited. In this study an attempt has been made to understand the molecular pathological basis of tumorigenesis which led to an identification of an oncogene, CDC2, and an epigenetic strategy has been evaluated to control the tumorigensis by downregulating this oncogene.MethodsTissue microarrays were utilized to investigate the expression of genes in a large number of tumor samples and to identify overexpressed genes which could be potentially causing tumorigenesis. Retroviral vectors expressing short hairpin RNAs (shRNAs) targeted against CDC2 were designed and transducted into human glioma cell line ex vivo in order to downregulate the expression of CDC2. Real-Time PCR was used to determine the level of CDC2 mRNA. Western Blotting was used to determine the level of expression of CDC2 protein as measure to quantify down regulation of CDC2 expression along with use of flow cytometry to investigate effect of shRNAs on cell cycles and detection of apoptosis. Following ex vivo study, viral particles containing small interfering RNA for CDC2 were subsequently injected into xenogeneic graft tumor of nude mice and the weight of human glioma xenografts, survival and resulting phenotypic changes of target gene were investigated.ResultsHuman glioma tissue microarrays indicated the positive expression rates of CDC2/CyclinB1 with a positive correlation with pathologic grades (r = 0.982, r = 0.959, respectively). Retroviral vectors expressing short hairpin RNAs (shRNAs) against CDC2 caused efficient deletion of CDC2, cellular G2/M arrest concluding in apoptosis and inhibition of proliferation in human glioma cells U251 and SHG-44 cell lines ex vivo. And the viral particles containing small interfering RNA for CDC2 were subsequently injected into subcutaneous and intracranial xenogeneic graft tuomrs of nude mice. For subcutaneous tumors, injection of CDC2-shRNA retroviruses significantly decreased tumor weight and volume compared with control. Immunohistochemistry indicated that CDC2 are negative and TUNEL are positive in tumors treated with recombinant retrovirus. For mice implanted with intracranial gliomas, treatment of CDC2-shRNA retroviruses increased survival times compared with control.ConclusionCDC2 gene plays an important role in the proliferation of human gliomas. Downregulation of CDC2 could potentialy inhibit human gliomas cells growth ex vivo and in vivo. From these results, it was suggested that CDC2 might be a potential target on gene therapy of human gliomas.

3D bioprinted glioma stem cells for brain tumor model and applications of drug susceptibility

Glioma is still difficult to treat because of its high malignancy, high recurrence rate, and high resistance to anticancer drugs. An alternative method for research of gliomagenesis and drug resistance is to use in vitro tumor model that closely mimics the in vivo tumor microenvironment. In this study, we established a 3D bioprinted glioma stem cell model, using modified porous gelatin/alginate/fibrinogen hydrogel that mimics the extracellular matrix. Glioma stem cells achieved a survival rate of 86.92%, and proliferated with high cellular activity immediately following bioprinting. During the in vitro culture period, the printed glioma stem cells not only maintained their inherent characteristics of cancer stem cells (Nestin), but also showed differentiation potential (glial fibrillary acidic protein and β-tubulin III). In order to verify the vascularization potential of glioma stem cells, tumor angiogenesis biomarker, vascular endothelial growth factor was detected by immunohistochemistry, and its expression increased from week one to three during the culture period. Drug-sensitivity results showed that 3D printed tumor model was more resistant to temozolomide than 2D monolayer model at TMZ concentrations of 400-1600 μg ml-1. In summary, 3D bioprinted glioma model provides a novel alternative tool for studying gliomagenesis, glioma stem cell biology, drug resistance, and anticancer drug susceptibility in vitro.

Glioma stem cells involved in tumor tissue remodeling in a xenograft model.

OBJECTnAlthough tissue remodeling plays a crucial role in the tumorigenesis and progression of human gliomas, its mechanisms remain largely uncertain. In the current study, the authors investigated the potential role of human glioma stem cells (hGSCs) in the tissue remodeling of gliomas.nnnMETHODSnTransgenic nude mice with ubiquitous green fluorescent protein (GFP) expression were obtained by crossing nontransgenic NC athymic nude mice with the GFP transgenic C57BL/6J mice. As a result, GFP was expressed in essentially all tissues in the offspring. Human glioma stem cells were then orthotopically implanted into the GFP nude mice in an effort to assess the hGSC-host brain interactions and thereby elucidate the roles of tissue remodeling during tumorigenesis and progression of human gliomas.nnnRESULTSnAll of the essential tissues in the GFP transgenic nude mice, including the brain, fluoresced green under an excitation light; therefore, tumor remodeling by hGSCs can be unambiguously distinguished from a bright green background composed of adjacent host GFP-expressing components. This technique enabled the authors to address the following concerns: 1) hGSCs were involved in the invasiveness of gliomas and adjacent stroma degradation of the host. 2) An in vivo study demonstrated that cell fusion occurred between hGSCs and host cells. 3) Vasculogenic mimicry--the formation of patterned, tubular networks of vascular channels by transdifferentiated hGSCs--could be observed. 4) Differentiation mimicry--namely, the differentiation direction of hGSCs bearing multidifferentiation potentials--seemed to be decided by the local host cellular microenviroment.nnnCONCLUSIONSnThe results of this study indicated that the GFP transgenic nude mice model with GFP expression in essentially all tissues could be obtained by crossing nontransgenic athymic nude mice with transgenic GFP mice. This model should greatly expand our knowledge of glioma-host interactions. The data indicated that hGSCs might play a decisive role in tissue remodeling of gliomas as well.

Development of Three-Dimensional Printed Craniocerebral Models for Simulated Neurosurgery

OBJECTIVEnTo use three-dimensional (3D) printed craniocerebral models to guide neurosurgery and design the best operative route preoperatively.nnnMETHODSnComputed tomography, magnetic resonance imaging, computed tomography angiography, and functional magnetic resonance images of the patients were collected as needed, reconstructed to form multicolor 3D craniocerebral images, and printed to form solid 3D models. The hollow aneurysm model was printed with rubberlike material; craniocerebral models were printed with resin or gypsum.nnnRESULTSnThe 3D printed hollow aneurysm model was highly representative of what was observed during the surgery. The model had realistic texture and elasticity and was used for preoperative simulation of aneurysm clipping for clip selection, which was the same as was used during the surgery. The craniocerebral aneurysm model clearly showed the spatial relation between the aneurysm and surrounding tissues, which can be used to select the best surgical approach in the preoperative simulation, to evaluate the necessity of drilling the anterior clinoid process, and to determine the feasibility of using a contralateral approach. The craniocerebral tumor and anatomic model showed the spatial relation between tumor and intracranial vasculatures, tractus pyramidalis, and functional areas, which was helpful 1) when selecting the optimal surgical approach to avoid damage to brain function, 2) for learning the functional anatomy of the craniocerebral structure, and 3) for preoperative selection of surgical spaces in the sellar region.nnnCONCLUSIONSn3D printing provides neurosurgeons with solid craniocerebral models that can be observed and operated on directly and effectively, which further improves the accuracy of neurosurgeries.

Are Morphologic Parameters Actually Correlated with the Rupture Status of Anterior Communicating Artery Aneurysms

OBJECTIVEnThe aim of this study was to identify whether computed tomography-based morphologic parameters actually are correlated with the rupture status of anterior communicating artery aneurysms (ACoAAs).nnnMETHODSnA total of 167 patients with ACoAAs were treated in our neurosurgery department from May 2010 to May 2015, and the morphologic and clinical characteristics of 80 of them (50 ruptured and 30 unruptured) were analyzed retrospectively. Morphologic parameters were evaluated on the basis of 3-dimensional computed tomography angiograms and included neck diameter, maximum height, perpendicular height, aspect ratio, size ratio, aneurysm angle, vessel angle, flow angle, parent-daughter angle, aneurysm shape, number of aneurysms, variation of the A1 segment, and the direction of the aneurysm dome.nnnRESULTSnThe χ(2) test revealed that the anterior direction was associated with ACoAA rupture. The independent sample t-tests revealed that the parent-daughter angle and the size ratio were associated with ACoAA rupture. However, the binary logistic regression revealed that the size ratio was the strongest factor associated with ACoAA rupture.nnnCONCLUSIONSnThe anterior direction, parent-daughter angle, and size ratio between ruptured and unruptured ACoAAs were found to be statistically significant; they may be implicated in the rupture of ACoAAs, but the size ratio was the strongest factor that was correlated with rupture of ACoAAs based on binary logistic regression.

Local delivery of slow-releasing temozolomide microspheres inhibits intracranial xenograft glioma growth

BackgroundCurrently, treatment of malignant gliomas with temozolomide in addition to surgical resection and radiotherapy remains the foundation of glioma therapy. In an effort to develop new therapeutic choices to treat malignant gliomas, we have designed slow-releasing microspheres that deliver temozolomide (P-TMZ). The local continuous release of temozolomide at the intracranial tumor site may overcome many obstacles associated with systemic delivery, which will help to further improving the therapeutic effects against malignant gliomas.MethodsSlow-releasing microspheres containing 10xa0% temozolomide were prepared, the antitumor efficacy in vitro was evaluated with MTT assay, and the therapeutic efficacy in vivo against gliomas was assessed in human glioma (SGH44) nude mice s.c. and orthotopic xenograft models.ResultsA single local injection of P-TMZ led to significant reduction both in s.c. and orthotopic human SHG44 glioma xenografts. P-TMZ, BCNU and TMZ had significant antiglioma effect (Pxa0<xa00.01), their IC50 value was all less than 10xa0μg/ml. Tumor inhibition ratio of P-TMZ, BCNU and TMZ in vivo was higher than empty microspheres P0 (Pxa0<xa00.01); P-TMZ and BCNU showed higher antitumor efficacy than TMZ (Pxa0<xa00.05).ConclusionsOur present results suggest that local delivery of slow-releasing temozolomide microspheres is effective for malignant gliomas. P-TMZ retained good antitumor activity and had better therapeutic effect against glioma both in vitro and in vivo, which provide a new choice for future clinical interstitial chemotherapy.

Ultrastructural Studies of Glioma Stem Cells/Progenitor Cells

Although the ultrastructural features of several brain tumor cells have been studied in details, the ultrastructure of glioma stem cells/progenitors cells (GSPC) has rarely been reported. In this paper, the authors describe the ultrastructural features of GSPCs isolated from both a glioma tissue and the human glioma cell SHG-44 cell line. The ultrastructural features of the two kinds of GSPCs were similar, with relatively developed mitochondria, Golgi apparatuses, ribosomes, undeveloped rough endoplasmic reticula, seldom lysosomes and no typical autophagosomes, and high nuclear–cytoplasmic ratio. Their nuclei, frequently containing huge amounts of euchromatin and a small quantity of heterochromatin, were mostly globular; and the majority of the nuclei had only one nucleole. Typical apoptotic cells could hardly be found in tumor spheres, and between adjacent cells there were cell junctions, which probably were incompletely developed desmosomes or intermediate junctions. In conclusion, their ultrastructural features showed that GSPCs were at the primary stage of differentiation, and could even partially reveal the underlying reasons for the malignant proliferation and differential inhibition of GSPCs.

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.