Additional file 1 of Inhibition of exosome biogenesis affects cell motility in heterogeneous sub-populations of paediatric-type diffuse high-grade gliomas
Creators: name: Pericoli, Giulia
name: Galardi, Angela
name: Paolini, Alessandro
name: Petrilli, Lucia Lisa
name: Pepe, Gerardo
name: Palma, Alessandro
name: Colletti, Marta
name: Ferretti, Roberta
name: Giorda, Ezio
name: Levi Mortera, Stefano
name: Burford, Anna
name: Carai, Andrea
name: Mastronuzzi, Angela
name: Mackay, Alan
name: Putignani, Lorenza
name: Jones, Chris
name: Pascucci, Luisa
name: Peinado, Hector
name: Helmer-Citterich, Manuela
name: de Billy, Emmanuel
name: Masotti, Andrea
name: Locatelli, Franco
name: Di Giannatale, Angela
name: Vinci, Maria
Publisher: name: figshare
Year: 2024
Subjects: Biophysics
Cell Biology
Molecular Biology
Biotechnology
Developmental Biology
Cancer
Hematology

description: Additional file 1: Figure S1. Overall Survival analysis of HGG patients using TCGA gene expression data. (A-B) The patient cohort was divided into two groups based on the median expression of OPBG-DIPG002 2B4 (A) and OPBG-GBM002 5E2 (B) specific gene signatures. The red curve represents patients with higher expression of this signature, while the blue curve represents patients with lower expression. Figure S2. Motility of single-cell-derived clones in mono and co-culture condition. Individual clones derived from OPBG-DIPG002 (A-D) and OPBG-GBM002 (G) were cultured either alone or co-cultured at an equal cell/cell ratio. Representative brightfield images are shown for the invasion (A) and migration (D-G) assays after 96 hours. The extent of invasion and migration is marked with coloured segmentation: co-culture (light blue); mono-cultures of 1C5 and 5E2 (fuchsia); mono-cultures of 2B4 and 1D3 (yellow). The total area covered by invading and migrating cells was quantified and normalised by the NS area determined at the time t0 (B-E-H). Scale bar = 500μm. Representative fluorescent images of OPBG-DIPG002 single-cell-derived clones 1C5 (Venus) and 2B4 (m-Orange2) 3D invasion (A) and migration (D) and of OPBG-GBM002 single-cell-derived clones 5E2 (Venus) and 1D3 (m-Orange2) migration (G) are shown as mono-culture and as co-culture (overlay of Venus and m-Orange2). Fluorescent images were acquired on a Leica TCS AOBS-SP8X confocal microscope. The percentage of each clone in the invasion (C) and migration (F-I) at 96 hours was analysed with Harmony software. Data are mean ± SD, n = 3. (****) p&lt;0.0001; (***) p&lt;0.001; (**) p&lt;0.01; (*) p&lt;0.05. Figure S3. The exosomal education of single-cell-derived clones does not affect their migratory/invasive phenotype. Exosomal education of the clones was performed. Exosomes isolated from donor clones were used to educate the recipient clone (10 μg/mL of media) every day, over 7 days. The effect on migration (A left panel and B) and invasion (A right panel) was quantified as the total area in the field of view covered by migrating and invading cells was normalized to the size of the NS at t0. Automated image acquisition was performed on a Celigo imaging cytometer. Scale bar = 500μm. Data were derived, and representative images were obtained from n=3 independent experiments. Data are mean ± SD, n = 3. Figure S4. Effect of the inhibition of exosome biogenesis on cell viability. (A) Effect of exosome biogenesis inhibition by GW4869 on the viability of the cells for the clones derived from OPBG-DIPG002 (left panel) and OPBG-GBM002 (right panel) cell lines. Cells were treated with GW4869 or vehicle control. Cell viability was measured with CellTiter Glo assay and dose-response curves are shown. Results are mean ± SD; n = 3. Figure S5. Analysis of exosome biogenesis inhibition upon GW4869 treatment on single-cell-derived clones. (A and C) Plots showing the size distribution of the nanoparticles resulting from the NanoSight particle-tracking analysis performed with the exosomes obtained from OPBG-DIPG002 and OPBG-GBM002 derived-clones CM in the presence or absence of 10μM and 20μM of GW4869 respectively or DMSO as vehicle control (representative experiment is shown, n=2). (B and D) Determination of the exosomal protein concentration. The quantification of the total exosomal protein obtained from OPBG-DIPG002 and OPBG-GBM002 derived-clones in untreated and treated with GW4869 conditions, is shown (n=3). (E) Determination of the exosomal RNA concentration. The quantification of total exosomal RNA obtained from OPBG-DIPG002 2B4 clone, in the presence or absence of 10μM of GW4869. Figure S6. Effect of the inhibition of exosome biogenesis on migration of single-cell-derived clones. (A and B) The mean accumulated distance, mean displacement, and speed of OPBG-DIPG002 (A) and OPBG-GBM002 (B) clone migrating cells in mono- and co-culture are shown. Cells were treated with 10μM and 20μM GW4869 or vehicle control, for 96 hours. Analysis was performed with Harmony software. Data are mean ± SD, n = 3. (****) p&lt;0.0001; (***) p&lt;0.001; (**) p&lt;0.01; (*) p&lt;0.05. Figure S7. Effect of the combination GW4869+exosome treatment on migration of single-cell-derived clones. OPBG-DIPG002 1C5 and 2B4 single-cell-derived clones were treated with GW4869 (10μM) or vehicle control (CTRL), in the presence or absence of exosomes (10 μg/mL of media) isolated from donor clones. The extent of migration is marked with coloured segmentation (white). The effect on migration (A) was quantified as the total area in the field of view covered by migrating cells, normalized to the size of the NS at T0. Automated image acquisition was performed on a Celigo imaging cytometer. Scale bar = 200μm. Data were derived, and representative images were obtained from n=2 independent experiments. Data are mean ± SD. (****) p&lt;0.0001; (***) p&lt;0.001; (**) p&lt;0.01; (*) p&lt;0.05. Figure S8. Proteomic analysis of single-cell-derived clones. Volcano plot of the mono-culture/co-culture ratio values measured in the label-free quantitative analysis performed on single-cell-derived clones of OPBG-DIPG002 (A and B) and OPBG-GBM002 (C and D). The red and green boxes include significant up- and down-regulated proteins respectively, based on the thresholds set for abundance ratio and p-value. Identified proteins related to the miRNA-predicted target genes are highlighted with sky-blue dots and tagged with the UniProt entry name.