Fig 2: Stable knockdown of Spp1 expression in C6 glioma cells reduces sphere formation and stemness factor expression(A–B) Efficient stable knockdown of Spp1 expression in rat C6 glioma cells was confirmed by qPCR and ELISA. Control shNeg cells expressed Spp1 and SPP1/osteopontin at the similar level as parental C6 cells (WT). (C) Stable knockdown of Spp1 expression in C6 glioma cells inhibited formation of spheres. Cells were seeded at a low density (4000 cells/ml) onto non-adherent plates and cultured in DMEM/F-12 medium, supplemented with B27, 20 ng/ml bFGF, 20 ng/ml EGF and antibiotics. Representative images show the reduced formation of spheres (100–200 μm in size) derived from the shSpp1 glioma cells 14 days after seeding. (D) Quantification of resulting spheres derived from shNeg and shSpp1 glioma cells was performed 14 days after seeding; data are means ± s.d., n = 3. (E) Reduced expression of Nanog and Oct3/4 in spheres derived from shSpp1 glioma cells. Spheres were collected by centrifugation 14 days after seeding and the expression of Nanog and Oct3/4 in cultures was determined by qPCR; data are presented as means ± s.d., n = 3.

Fig 3: SPP1 expression is up-regulated in glioma spheres and reduced after forced differentiation(A) Human LN18 glioma spheres express the higher levels of SPP1, OCT3/4 and NANOG than adherent cells. For sphere forming assay, cells were seeded at a low density (4000 cells/ml) onto non-adherent plates and cultured in DMEM/F-12 medium, supplemented with B27, 20 ng/ml bFGF, 20 ng/ml EGF and antibiotics; inset shows a representative sphere, 40× magnification. After 14 days resulting spheres and adherent cultures were collected by centrifugation and lysed in Qiagen RLT lysis buffer. Gene expression was determined with qPCR; data are presented as means ± s.d. T-test analysis was performed, P values were considered significant when *P ≤ 0.05 and **P ≤ 0.01; n = 3. (B) Western blot analysis of stemness factors NANOG, OCT4A, SOX2 in adherent (ctrl) and LN18 spheres grown for 14 days. The expression of stemness markers at the protein level was additionally evaluated in human embryonic teratoma Ntera2 cells expressing those stemness markers at very high levels. Samples were run on the same blots to determine the correct band size of proteins. (C) Spheres cultured for 8 days were differentiated for 5 days in the presence of 100 ng/ml BMP4 or 2% FBS. Light microscopy shows morphological alterations of LN18 sphere cultures, in particular spreading of cells and attachment to the bottom of plates when spheres were cultured in the medium containing 2% FBS. (D) Representative images of cells stained for TUB-β III (a neuronal marker) and co-stained with DAPI to visualize cell nuclei. (E) Addition of BMP4 and 2% serum resulted in reduction of the expression of SPP1 in LN18 cells. Spheres cultured for 8 days were differentiated for 5 days in the presence of 100 ng/ml BMP4 or 2% FBS. BMP4 treatment reduced significantly NANOG expression, while serum addition resulted in the reduction of OCT3/4 mRNA level. Data are means ± s.d., n = 5.

Fig 4: SPP1 expression is up-regulated in glioma initiating cells(A) Representative examples of dot plots and histograms of Rhod 123(–) subpopulations from 5 glioma cell lines were acquired by flow cytometry. Glioma cells (1 × 107) were trypsinized and incubated with 0.1 μg/ml Rhod123 for 20 min in 37°C. After washing the cells were placed in 37°C for 90 min for Rhod123 exclusion in a dark compartment. Cells kept on ice to inhibit exclusion of Rhod123 were used as a positive control for gating in flow cytometry. Fractions of Rhod123(+) and Rhod123(–) cells were sorted using FACS Aria and a left panel shows a gating strategy. (B) Analysis of SPP1, OCT3/4 and NANOG gene expression in Rhod 123(–) subpopulations sorted from four human glioma cell lines. Total RNA was isolated from sorted cells using Qiagen RNeasy kit and the levels of SPP1, NANOG and OCT3/4 mRNA were determined by qPCR in Rhod 123(+) and Rhod 123(–) subpopulations; their expression in Rhod 123(+) subpopulations was taken as 1 (a red line). P values were calculated with a t-test and considered significant when *P ≤ 0.05 and **P ≤ 0.01. (C) Quantification of the expression of Spp1, Oct3/4 and Nanog in Rhod 123(–) subpopulations isolated from rat C6 glioma cells versus their levels in Rhod 123(+) subpopulations taken as 1 (a red line); n = 3.

Fig 5: The CD44 binding domain of Spp1 is critical for sphere formation(A) C6 glioma cells stably expressing shSpp1 were transfected with various constructs: a control pEGFP, a shRNA resistant, wild type Spp1 (WtSpp1-R) or a shRNA resistant Spp1 lacking a CD44 binding domain (Spp1ΔC-R). Twenty four hours after transfection cells were seeded (8000 cells/ml) under sphere forming conditions (DMEM/F-12 medium with B27, 20 ng/ml bFGF, 20 ng/ml EGF and antibiotics). Reconstitution of Spp1 expression in cells transfected with WtSpp1-R or Spp1ΔC-R was determined by qPCR in respective cultures and related to values obtained for shSpp1 cells. Data are presented as means ± s.d., n = 3. (B) Reconstitution of Spp1expression in cells transfected with a control pEGFP, WtSpp1-R or Spp1ΔC-R had no influence cell viability (as determined by MTT metabolism assay 24 h after transfection). (C) Only reconstitution of Spp1 expression in glioma cells transfected with a WtSpp1-R restored cell capacity to form spheres. Data are presented as means ± s.d., P values were considered significant when *P ≤ 0.05 and **P ≤ 0.01, n = 3. (D) Representative images show the reduced formation of spheres derived from the shSpp1 glioma cells transfected with pEGFP 7 days after seeding. Overexpression of the construct coding for a wild type (WtSpp1-R) restored sphere forming capacity of glioma cells; overexpression of Spp1ΔC-R did not restore sphere forming capacity.