Additional file 1: Figure S2. of Endothelial cells induce cancer stem cell features in differentiated glioblastoma cells via bFGF
Creators: name: Fessler, Evelyn
name: Borovski, Tijana
name: Medema, Jan
Publisher: name: Figshare
Year: 2015
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Biotechnology
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: ECCM and HCM increase the clonogenic potential of non-stem GBM cells and increase the expression of CD133. (A) Shown is the clonogenic potential of the SSEA-1− fraction of the G073 culture in control medium or ECCM scored 2 weeks after sorting (n = 2). (B) Indicated is the clonogenic potential of CD133− G408 cells in control medium or ECCM and of CD133+ cells in control medium, scored 2 weeks after sorting (n = 2). (C) G408 CD133− cells were plated in control medium or ECCM directly after sorting. Quantification and FACS profiles depict CD133 expression 72 h after sorting (n = 2). (D) Displayed is the clonogenic potential of CD133− G602 cells in control medium or HCM and of CD133+ cells in control medium, scored 2 weeks after sorting (n = 2). (E) G062 CD133− cells were plated in control medium or HCM directly after sorting. Quantification and FACS profiles depict CD133 expression 72 h after sorting (n = 4). Figure S3. BMP4 differentiation leads to downregulation of SSEA-1 and Nestin and to a decreased clonogenic potential which can be reverted by ECCM and bFGF. (A) Representative FACS plots of SSEA-1 staining on BMP4-differentiated G073 cells and the parental population (+ GFs). The bar plot shows % SSEA-1+ cells upon BMP4-induced differentiation (+ BMP4) compared to the parental population (+ GFs) (n =5). (B) 7 day BMP4 differentiation leads to the downregulation of Nestin expression in G073 (left) and G062 (right) cells as compared to cells plated in CSC medium + GFs (scale bars 20 μm; n = 2). (C) The clonogenic potential of BMP4-differentiated G073 (left) and G062 (right) cells upon plating in control medium, ECCM or medium containing bFGF was determined using clonogenic assays (n = 2). (D) GFAP and βIII-tubulin expression in 7 day BMP4-differentiated G062 cells and in spheres formed in the bFGF condition shown in (C). Depicted is the fold change compared to cells plated in CSC medium + GFs. 1 representative of 3 independent experiments is shown. Figure S4. bFGF is secreted by tMVECs and induces CD133 expression on BMP4-differentiated CD133− cells. (A) Quantification of the growth factor arrays shown in Fig. 3a. Graph depicts quantification of 3 growth factor arrays using different batches of ECCM. Shown is the intensity of each spot minus the intensity of the respective spot on the control array. (B) BMP4-differentiated CD133− G062 cells were plated in the indicated condition directly after sorting. 5 days after sorting CD133 expression was analyzed by FACS (n = 4). Figure S5. Neurospheres derived from CD133− G062 cells stimulated with bFGF recapitulate the differentiation pattern of the parental population. (A) Plating G062 cells for 7 days in BMP4 containing medium leads to morphological differentiation as well as upregulation of GFAP, indicating the differentiation to the astrocytic lineage as compared to cells plated in GF-containing medium (scale bars 20 μm). (B) A culture derived from BMP4-differentiated CD133− G062 cells stimulated with bFGF after sorting was plated in medium containing GFs or BMP4. These cells displayed the same differentiation pattern as the parental population, demonstrated by the expression of GFAP and βIII-tubulin (n = 2, scale bars 20 μm). Figure S6. Differentiated GBM oligodendrocytes can be phenotypically and functionally redirected to the CSC state by bFGF. (A) Differentiation for 7 days by addition of 2 % FCS (+2 % FCS). G073 cells differentiate into all 3 lineages, indicated by the expression of GFAP, βIII-tubulin, and O4 (scale bars 20 μm). (B) Analysis of differentiation and CSC markers by qRT PCR. Depicted is the fold change compared to cells plated in CSC medium + GFs. 1 representative of 3 independent experiments is shown. (C) Differentiated GBM cells express the oligodendrocyte marker O4 on their surface as determined by FACS. (D) CD133−O4+ cells were sorted and plated in the indicated condition. 5 days after sorting CD133 and O4 expression were analyzed by FACS (n = 3). (E) The clonogenic potential of differentiated CD133−O4+ cells was determined using clonogenic assays (n = 4). (F) CD133 and O4 expression of spheres formed in (E) in the bFGF condition compared to the non-differentiated parental GBM spheroid culture (n = 2). Figure S7. Nestin expression is downregulated upon differentiation in G073 cells. 7 day differentiation of G073 cells using GF withdrawal (−GF) or addition of 2 % FCS (+2 % FCS) leads to downregulation of Nestin expression as compared to cells plated in CSC medium + GFs (scale bars 20 μm; n = 2). Figure S8. Gain of CD133 and loss of O4 expression on differentiated cells induced by bFGF is not due to expansion of a contaminating CD133+O4− fraction. (A) Differentiation and sorting of G073 cells as described in (Fig. 5 and Additional file 1: Figure S6). The purity of the sort was analyzed immediately afterwards by FACS. (B) After differentiation using GF withdrawal, CD133−O4+ cells were sorted twice and plated in low adherence for 5 days. Subsequently the expression of CD133 and O4 was analyzed by FACS. Induction of CD133 and loss of O4 were as efficient as after a single sort (n = 1). Figure S9. Neurospheres derived from CD133−O4+ cells stimulated with bFGF recapitulate the differentiation pattern of the parental population. Cultures derived from G073 spheres that formed in a clonogenic assay in bFGF-containing medium (as described in Fig. 5e) were differentiated using the depicted conditions. These cells displayed the same differentiation pattern as the parental population, demonstrated by the expression of GFAP, βIII-tubulin, and O4 (n = 3; scale bars 20 μm). Figure S10. Differentiated GBM oligodendrocytes can be phenotypically and functionally redirected to the CSC state by ECCM. (A,B) Differentiation of G073 cells for 7 days by GF withdrawal (− GFs) (A) or addition of 2 % FCS (B) and subsequent sorting of CD133−O4+ cells in the indicated condition. 5 days after sorting CD133 and O4 expression were reanalyzed by FACS (n = 4 for - GF and n = 2 for + 2 % FCS) (C,D) The clonogenic potential of differentiated CD133−O4+ cells was determined using clonogenic assays (n = 2 for - GF and n = 3 for + 2 % FCS). Figure S1. Gating on cell populations for sorting was performed using an unstained control as reference. (A-C) Shown are exemplary FACS plots for the identification of (A) the CD133−O4+ (− GF and + 2 % FCS) and CD133− (+ BMP4) fraction of G073 cells, (B) the SSEA-1− population of G073 cells, and (C) the CD133− fraction of BMP4-differentiated G062 cells.