A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells

Abstract

Glioblastoma multiforme (GBM) is among the most aggressive tumor types and is essentially an incurable malignancy characterized by resistance to chemo-, radio-, and immunotherapy. GBM is maintained by a hierarchical cell organization that includes stem-like, precursor, and differentiated cells. Recurrence and maintenance of the tumor is attributed to a small population of undifferentiated tumor-initiating cells, defined as glioblastoma stem-like cells (GSLCs). This cellular hierarchy offers a potential treatment to induce differentiation of GSLCs away from tumor initiation to a more benign phenotype or to a cell type more amenable to standard therapies. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth and differentiation. In vitro, a BMP7 variant (BMP7v) decreased primary human GSLC proliferation, endothelial cord formation, and stem cell marker expression while enhancing neuronal and astrocyte differentiation marker expression. In subcutaneous and orthotopic GSLC xenografts, which closely reproduce the human disease, BMP7v decreased tumor growth and stem cell marker expression, while enhancing astrocyte and neuronal differentiation compared with control mice. In addition, BMP7v reduced brain invasion, angiogenesis, and associated mortality in the orthotopic model. Inducing differentiation of GSLCs and inhibiting angiogenesis with BMP7v provides a potentially powerful and novel approach to the treatment of GBM.

Figure 1

BMP7v reduced GSLC proliferation. (a) U-87-MG cells and GSLCs isolated from three separate patients were treated with 100 ng/ml BMP7v and analyzed over a time course of at least three cell doublings and compared with their respective PBS-treated controls. Results represent mean plus S.E.M. for three independent experiments. (b and c) Whole-cell protein extracts were isolated following 15 min (b) or 7 day (c) PBS (−) or 100 ng/ml BMP7v (+) treatment and subjected to western blot analysis using antiserum directed against Smad1,5,8-phosphorylation (pSMAD1,5,8), Ki67, PARP, cleaved PARP (CL PARP), and β-actin as a loading control. Results shown are representative of three independent experiments. (d) Individual cells were plated for high-content analysis following 7 days of PBS or 100 ng/ml BMP7v treatment. Cells were detected using Hoescht dye (blue) to stain the nuclei, and phospho-histone H3 (pHH3) expression (green) was compared between PBS and BMP7v-treated cells using the percentage of cells expressing a minimum total amount of pHH3 for each cell line (X10 magnification). Results represent mean plus S.E.M. for three independent high-content experiments. Asterisks denote statistically significant (Student t-test; *P<0.05; **P<0.01) differences compared with PBS controls. Whole-cell protein extracts were isolated following 7 days of PBS (−) or 100 ng/ml BMP7v (+) treatment from the indicated cell lines and subjected to western blot analysis using antiserum directed against pHH3 and total histone H3 (HH3) as a loading control. Results shown are representative of three independent experiments

Figure 2

BMP7v-induced differentiation of GSLCs. (a) Light micrographs represent cell morphology (X10 magnification, scale bars are 100 μm) following 7 days of PBS or 100 ng/ml BMP7v treatment. (b) Whole cell protein extracts were isolated following 7 days of PBS (−) or 100 ng/ml BMP7v (+) treatment and subjected to western blot analysis using antiserum directed against Sox2, Nestin, Olig2, Nanog, GFAP, βIII-tubulin, and β-actin as a loading control. (a and b) Results shown are representative of three independent experiments. (c) Individual cells were plated for high-content analysis following 7 days of PBS or 100 ng/ml BMP7v treatment. Cells were detected using Hoescht dye (blue) to stain the nuclei, GFAP (red), and βIII-tubulin (green). To determine relative levels of expression, staining intensities were compared with PBS controls and represented as heatmaps that clustered the data from individual cells and sorted each cluster based on the total DNA intensity of the nucleus. The numbers on the left side of each heatmap denote the fraction of cells in each cluster. The parameters measured are listed below each column: total DNA intensity (Total), average DNA intensity (Ave), variation of DNA intensity (Var), nuclear area (Area), total GFAP (GFAP), and total βIII-tubulin (βIII-tubulin). Images and heatmaps shown are representative of three independent experiments

Figure 3

BMP7v inhibited GSLC-induced cord formation. GSLCs were plated into a permeable trans-well system with a co-culture of ADSCs and ECFCs in serum-free media. Following a 4-day treatment with PBS or 100 ng/ml BMP7v, ADSCs and ECFCs were detected using Hoescht dye (blue) to stain the nuclei, and cord formation was assessed by immunofluorescence for CD31 (green) and smooth muscle actin (red) (X5 magnification). Numbers represent mean total cord area±S.E.M. as a percent of the PBS control, and images shown are representative of three independent experiments. Asterisks denote statistically significant (Student t-test; ***P<0.001) differences compared with PBS controls

Figure 4

BMP7v reduced the proliferation and invasion of intracerebral GSLC xenografts. (a) Mice were injected intracerebrally with GFP-expressing GSLCs without BMP7v (control, CNTR) or mixed with 1 ng BMP7v. At 2 weeks after grafting, the tumor cells were found at the injection site, homolateral striatum, and piriform cortex (photomontage of two adjacent coronal brain sections 120 μm apart). (b) Schematic drawings of the experimental paradigm in specimens with 2-week survival. (c) Coronal sections through the grafted striatum at 8 weeks after grafting (photomontages; CC, corpus callosum; St, striatum; PC, piriform cortex). (d) In BMP7v-treated mice with 2 (n=3), 4 (n=3), and 8 (n=4) week survival, both the cranio-caudal extension and volume of the brain region invaded by the GFP-expressing GSLCs were significantly reduced (Student t test; *P<0.05; **P<0.01; results shown represent mean plus S.E.M.) compared with matched control mice (CNTR)

Figure 5

BMP7v reduced the proliferation and induced differentiation of intracerebral GSLC xenografts. (a) Tumor cell proliferation was assessed by counting the mitotic figures of GFP-expressing cells (upper panel; scale bars, upper left, 100 μm; upper right, 10 μm). The proliferating tumor cell fraction did not express GFAP (red, lower panel; arrows indicate mitotic figures; scale bars, lower left, 100 μm; lower right, 10 μm). (b) Eight weeks after grafting, the mitotic index (MI) was significantly lower (Student t-test; *P<0.05; results represent mean plus S.E.M.) in BMP7v-treated tumors (BMP7v; n=4) than in controls (CNTR; n=4). (c) Both in BMP7v-treated and in control tumors, astrocytic differentiation was absent at 2 weeks (upper panel; scale bar, 30 μm) and was first recognized 4 weeks after grafting (lower panel; scale bars, 10 μm). (d) By 8 weeks after grafting, immunofluorescence revealed that BMP7v treatment reduced the expression of Nestin (red, Nest) and Ki67 (red), whereas increasing GFAP (red) and βIII tubulin (red, Tub). The arrows indicate GFP-expressing tumor cells that express Nestin, GFAP, βIII tubulin, or Ki67. The arrowhead indicates a mitotic figure (scale bars, 20 μm). (e) Graph represents the expression of the stem cell marker Nestin, astrocytic marker GFAP, neuronal marker βIII tubulin, and proliferation marker Ki67 in GFP-expressing tumor cells 8 weeks after grafting in either BMP7v-treated tumors (n=4) or controls (CNTR, n=4) (Student t-test; ***P<0.001; **P<0.01, *P<0.05; results represent mean plus S.E.M.)

Figure 6

BMP7v reduced angiogenesis in the striatum and increased survival following intracerebral GSLC xenografts. (a) Vascular structures in the striatum of BMP7v-treated and control xenografts (H&E; anti-CD31 immunostaining; arrows indicate tumor capillaries; arrowheads indicate vascular glomeruli; scale bars, 50 μm). (b) By 8 weeks after grafting, the density of CD31-positive capillaries and vascular glomeruli was significantly lower in the striatum of BMP7v-treated tumors (n=4) compared with controls (n=4) (Student t-test; *P<0.05, **P<0.01, results represent mean plus S.E.M.). (c) The graph represents the survival probability curves in mice harboring brain tumor xenografts, which were either treated with 1 ng BMP7v (n=4) or untreated (CNTR, n=4). There is a significant difference between the curve for BMP7v-treated and untreated (CNTR) mice (log rank test; P=0.018)