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Supplementary Figure Legends:
Supplementary Figure 1. Analysis of GSC EV proteome heterogeneity reveals coherent molecular
modules.
A. 10-gene signature distinguishes P from M GSCs. Gene sets that vary coherently between GSC
were used for subtype classification validated by qPCR data (upper panels) and identified by
Principal Component Analysis (bottom).
B. The GSC EV profile indicated heterogeneous population of EVs secreted by GSCs. Analysis
frequency and size of GSC-derived EVs by NanoSight is shown.
C. The proteomic analysis of GSC EVs indicated diverse molecular modules present in GSC
subtypes. The GSC EV proteome profile was assessed based on the value of signal intensity (left).
Significantly deregulated proteome molecules in EVP and EVM were analyzed by Gene Ontology
enrichment in indicated subcategories (right).
D. The GSC EV proteome profile stratified P from M GSCs. Proteome sets that vary coherently
between GSC subtypes were analyzed by top 6 expressed genes by class (left), and expression
correlation (right).
E. The GSC EV subtype proteomic signature selected for validation (see Figure 1 D) were identified
by clustering.
Supplementary Figure 2. Presence of EVM supports pro-oncogenic traits of P GSCs in vitro.
A-C.
EVM promote growth of P GSCs. Quantification of spheroids frequency (A), size (B), cell viability
(C) and spheroid formation (D); representative micrographs (left). Data, mean ± SD; *, P < 0.05
E.
EV mediates transduction of signaling pathways in GSCs. Western blot analysis of kinase activity
in P and M GSCs (left) and P GSCs treated with EVM (right). Relative densitometry of phospho-SRC is
shown.
F.
EV proteome cargo is transferred by EVs between GSC subtypes. Representative micrographs of
EGFR immunostaining, DAPI and fluorescent EVs (upper panels), qPCR analysis of EGFR mRNA in both
subtypes (bottom left) and IHC quantification (bottom right) are shown. Data, mean ± SD; *, P < 0.05
Supplementary Figure 3. Presence of P GSC supports pro-oncogenic traits of M GSCs in vivo.
A. P GSCs promote proliferation of M GSCs in vivo. Representative micrographs of M GSCoriginated tumors (non-consecutive sections) 9 days after co-implantation with P GSC (left), and
quantification of M GSC tumor volume (right). Data, mean ± SD; *, P < 0.05
B. EVs are transferred intra-tumorally in vivo. Representative micrographs of co-implanted tumors
are shown.
C. Terminal heterogeneous tumors are dominated by M GSCs. Representative micrographs of M
GSCs and P GSCs are shown.
D. Heterogeneous GSC-originated tumors are rapidly taken over by M GSCs. Percentage of M and P
GSCs in time course is shown. Data, mean ± SD; *, P < 0.05
Supplementary Figure 4. GSC EV proteome predicts patients’ outcome.
A-C.
Heatmap clustering and survival analysis based on the impact of the prognostic index of multiple
protein-coding genes from all GSC EVs (A), EVM (B) and EVP (C).
Supplementary Tables:
Supplementary Table 1.
List of the 89 proteins from GSC EV proteome profile distinguishing P from M GSC subtypes; signal
intensity.
Supplementary Table 2.
Genes coding for protein sets that vary coherently between GSC subtypes within 89- protein signature,
were retrieved from TCGA GBM dataset and identified by subtype prediction.
Supplementary Movie:
M and P GSCs display distinct intra-spherical organization in vitro. Representative time lapse movie.