The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Abstract

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.

Figure 1

GBM stem-like cell plasticity properties in GBM02 and GBM02-SF cells. The expression of Cx43, Cx46, and SOX2 in the GBM and the respective serum-free cell lines was evaluated by WB (A) and immunofluorescence (B). (A) GBM02 cells were maintained in DMEM-F12 supplemented with serum for 2 weeks (GBM02 T0). At the end of this time, part of the cells was isolated and the other part was maintained in culture with NS34, a serum-free medium, for 2 more weeks (GBM02-SF T2). Next, part of the cells was also isolated and the other part was again maintained in culture with DMEM/F12 supplemented with serum for a further 2 weeks (GBM02 T4). At the end of this time, cells were collected, and the expression of Cx43, Cx46, and SOX2 was quantified by WB as previously described. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA). Each value represents the mean ± SEM from three independent experiments; *P < .05, **P < .01. (B) Immunofluorescence staining of Cx43, Cx46, and SOX2 was performed in parallel. Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health). (C) The expression of Cx43 and SOX2 in the GBM02 and GBM02-SF cells was evaluated by qPCR. Total RNA was extracted using a PureLink RNA Mini Kit following the manufacturer's instructions. One microgram of total RNA, oligo(dT) primer, and High-Capacity cDNA Reverse Transcription Kit were used to perform cDNA synthesis. The qPCR reaction was done in duplicate using SOX2, Cx43, and GAPDH primers and Power SybrGreen Master Mix. To calculate the relative fold variation in mRNA expression, GBM02 cells (T0) were applied as control at the 2^−ΔΔCT method. Each value represents the ± SEM, *P < .05.

Figure 2

GBM stem-like cell plasticity properties in OB1 and OB1-SF cells. The expression of Cx43, Cx46, and SOX2 was analyzed in the GBM, and the respective serum-free cell lines were evaluated by WB (A) and immunofluorescence (B). (A) OB1-SF cells were maintained in culture with the serum-free medium NS34 for 2 weeks (OB1-SF T0). At the end of this time, part of the cells was isolated and the other part was maintained in DMEM-F12 supplemented with serum for 2 more weeks (OB1 T2). After this time, part of the cells was also isolated and the other part was again maintained in culture with NS34, a serum-free medium, for a further 2 weeks (OB1-SF T4). At the end of this time, cells were collected, and the expression of Cx43, Cx46, and SOX2 was quantified by WB as previously described. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA). Each value represents the mean ± SEM from three independent experiments; *P < .05, **P < .01. B. Immunofluorescence staining of Cx43, Cx46, and SOX2 was performed in parallel. Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health). (C) The expression of Cx43 and SOX2 in OB1-SF and OB1 cells was evaluated by qPCR. Total RNA was extracted using PureLink RNA Mini Kit following the manufacturer's instructions. One microgram of total RNA, oligo(dT) primer, and High-Capacity cDNA Reverse Transcription Kit were used to achieve cDNA synthesis. The qPCR reaction was done in duplicate using SOX2, Cx43, and GAPDH Taqman Probes. To calculate the relative fold variation in mRNA expression, OB1 cells (T2) were used as control at the 2^−ΔΔCT method. Each value represents the mean ± SEM; *P < .05, **P < .01, ****P < .0001.

Figure 3

The GSC maintenance (A) and clonogenic Assay (B). (A) The human tumor cell lines were established in our laboratory. The use of patients' surgical specimens for the establishment of cell lines for in vitro and in vivo studies had written informed consent from the patients and was approved by the Brazilian Ministry of Health Ethics Committee under Institutional Review Board (Research Ethics Committee of Hospital Universitário Clementino Fraga Filho) consent, CEP-HUCFF no. 002/01. Cells were grown and maintained in DMEM-F12 supplemented with 10% FBS. Culture flasks were maintained at 37°C in a humidified 5% CO₂ and 95% air atmosphere. GSCs were maintained as tumor-sphere cultures in NeuroBasal medium supplemented with sodium pyruvate, glutamine, B27 supplement, EGF, basic FGF, penicillin, and streptomycin. The OB1 differentiated cell line was obtained by the removal of EGF and bFGF and the addition of 10% fetal bovine serum. (B) To estimate stem cell frequency in the cell population, tumor-sphere formation was assayed with 2 cells per well in in 96-well plates in NeuroBasal medium supplemented with sodium pyruvate, glutamine, B27 supplement, EGF, basic FGF, penicillin, and streptomycin. During 4 weeks of culture, sphere-positive wells were scored by observation under an inverted microscope with phase-contrast optics. The image is representative of three independent experiments. Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).

Figure 4

Stem-like cell marker expression in two GBM cell lines isolated from patients in DMEM/F12 supplemented with serum by WB (A) and immunofluorescence (B). (A) The expression of SOX2, OCT-4A, and Nanog in both the GBM02 and GBM11 cell lines, isolated from GBM diagnosed patients, and the respective serum-free GBM cells, as previously described, was quantified by WB. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA). Each value represents the mean ± SEM from three independent experiments; *P < .05, **P < .01. (B) Immunofluorescence staining of SOX2, OCT-4A, and Nanog as well as Nestin and GFAP was performed in both the GBM02 and GBM11 cell lines and the respective serum-free GBM cells, GBM02-SF and GBM11-SF. Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).

Figure 5

Stem-like cell marker expression in the U87 ATCC GBM cell line and the OB1, a GBM cell line isolated from a patient directly in NS34 serum-free medium, by WB (A) and immunofluorescence (B). (A) The expression of SOX2, OCT-4A, and Nanog was quantified in both the OB1 cell line isolated from a GBM diagnosed patient directly in NS34 serum-free medium and in U87, a GBM cell line acquired by ATCC. The respective OB1 differentiated cells were isolated from OB1-SF, and the serum-free GBM cells were isolated from the U87 cell line, U87-SF, as previously described, by WB. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA). Each value represents the mean ± SEM from three independent experiments; *P < .05, **P < .01. B. Immunofluorescence staining of SOX2, OCT-4A, and Nanog as well as Nestin and GFAP was performed in both the OB1 and U87 cell lines and the respective serum-free GBM cells, OB1-SF and U87-SF. Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).

Figure 6

Cx43 and Cx46 expression analysis in the GBM and the respective serum-free cell lines (GSCs) by WB (A) and immunofluorescence (B). (A) The expression of Cx43 and Cx46 in the GBM02, GBM11, OB1, and U87 cell lines and the respective serum-free cells, GBM02-SF, GBM11-SF, OB1-SF, and U87-SF, as previously described, was quantified by WB. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA). Each value represents the mean ± SEM from three independent experiments; *P < .05, **P < .01. (B) Immunofluorescence staining of Cx43 and Cx46 was also performed in all cell lines. Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).

Figure 7

Capability of GBM cell lines to form tumors in vivo. (A) Schematic depiction of GBM11 cell injection in striatum region of immunocompetent Swiss mouse brain. (B) MRI 14 days after GBM11 cell transplantation into a representative mouse brain. Coronal, sagittal, and axial cuts of tumor site in control and GBM11-injected mice. The asterisk (*) indicates the tumor mass. (C) Histopathological characteristics of the tumor mass in mouse brain parenchyma 14 days after implantation of GBM11 cells, revealed by hematoxylin-eosin staining. I: Neoplastic cells forming a circumscribed solid tumor mass in the brain tissue (black asterisk). II: Glomeruloid vessels (black asterisk). Microscopic analysis showed anaplastic cells and tumoral necrosis. (D) Immunohistochemical characteristics of the tumor revealed the expression of human vimentin (hVim). hVim staining (green) at the core of the tumor mass (red asterisk) and SOX2 (red), depicted by cell nuclei atypia (DAPI counterstaining in cyan blue, inset), and at the border of the tumor mass. Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).

Figure 8

Analysis of SOX2 and Cx46 expression in glioblastoma, astrocytoma grade II, and normal human brain tissues by immunohistochemistry. Patients with clinical evidence of disease, MRI- and histologically confirmed diagnosis of astrocytoma grade II and astrocytoma grade IV (GBM) based on the World Health Organization (WHO) 2007 criteria , and who gave written informed consent to participate in the study were included. To compare the SOX2 and Cx46 expression, normal brain samples from patients that underwent an epilepsy surgery with mesial sclerosis were also analyzed. The study was approved by the University Hospital of Coimbra Ethics Committee, according to the Declaration of Helsinki protocol. (A) Histopathological characteristics of the tumor mass were evaluated by hematoxylin-eosin staining. The normal brain picture was acquired from the white matter. The astrocytoma grade II showed a nuclear atypia and microcysts, and the glioblastoma showed nuclear atypia, microvascular proliferation (+), and necrosis (*). (B) Immunohistochemistry was performed using the diaminobenzidine method with hematoxylin counterstaining to evaluate the percentage of cells expressing SOX2 and Cx46. (C) Tumor samples were ranked for SOX2 or Cx46 expression as high (60% positive cells), moderate (between 20% and 60% positive cells), and low (below 20% cells). Each image was acquired at 10× magnification (×10/0.25 PH1 NPLAN) and amplified 40× (×40/0.55 CORR PH2) (scale bar = 100 μm) with a Leica DMI3000B light microscope (Leica, Germany) and acquired with the Leica Application Suite v 4.3 (Leica, Switzerland).

Figure 9

Plasticity of GBM stem-like cells. Interactions of GBM cells with the tumor microenvironment can dictate the dynamic balance between self-renewal and differentiation properties through growth factors present in serum-free stem-like cell medium, NS34, or through bovine-serum supplement in DMEM/F12 medium, respectively. The stem-like cell markers SOX2, OCT-4A, and Nanog differ in expression according to the stem cell state. Also, the connexins, important for cell-to-cell communication processes, demonstrate the plasticity of stem-like cells. As such, SOX2, OCT-4A, Nanog, and Cx46 are overexpressed in GSCs (green arrow), whereas Cx43 is downregulated (red arrow). In contrast, in non-GSCs, SOX2, OCT-4A, Nanog, and Cx46 are downregulated (red arrow), whereas Cx43 is overexpressed in GSCs (green arrow). Cells were imaged at 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LA SAF Lite. Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).

Supplementary Data 1

Deregulation of EMT markers in OB1 cells. The SLUG and vimentin expression was evaluated in OB1 cells in T0, T2, and T4 time points by WB. There was a significant downregulation in expression of SLUG and vimentin in cells cultured on differentiation conditions (OB1-T2) (P < .05) when compared to OB1-SF (T0). SLUG and vimentin levels were restored when OB1 cells were cultured back on serum-free media (OB1-T4), P < .05. However, the vimentin expression was not statistically significant. Statistical analysis was performed in GraphPad Prism 5 for Windows (version 5.00; GraphPad Software, Inc., San Diego, CA). Each value represents the mean ± SEM from three independent experiments, *P < .05.