Supplementary information

Supplementary table 1. Information of nine glioma specimens.

Supplementary table 2. The selected 116 abundant proteins in hEC-EVs

(GhEC-EVs and NhEC-EVs) compared with GCs-EVs or GSCs-EVs.

“R1”, “R2” “R3”and “R4” represent the relative protein content from GhEC-

EVs/GC-EVs, NhEC-EVs/GC-EVs, GhEC-EVs/GSC-EVs and NhEC-

EVs/GSC-EVs, “+/-” indicates detected/undetected with mass spectrum.

Supplementary figure 1. Characterization of GhEC-EVs, NhEC-EVs, GC-

EVs and GSC-EVs. (a) NTA analysis of diameter distribution of NhEC-EVs,

GC-EVs and GSC-EVs. (b) Western blot showed the common EV markers in

NhEC-EVs, GC-EVs and GSC-EVs: Flotillin-1, CD81, syntenin-1, CD63, and

Calnexin and Calreticulin for endoplasmic reticulum, and VDAC1 for

mitochondria, 8 µg protein was loaded in each lane. (c) Silver staining showed

the protein characteristic in GhECs and GhEC-EVs. (d) Analysis of the RNA

differences between GhECs and GhEC-EVs with capillary electrophoresis.

Upper figure showed the RNA of GhEC, lower figure showed the RNA of

GhEC-EVs. (e) Analysis of the EVs number in one microgram in GhEC-EVs,

NhEC-EVs, GC-EVs and GSC-EVs.

Supplementary figure 2. GhEC-EVs inhibit GCs proliferation in vitro. (a)

MTS showed the effect of GhEC-EVs on cell viability of three GC lines.

Dunnett test was used for statistical analysis, data are presented as the mean

± SD. *P < 0.05, **P < 0.01 and ***P < 0.001. (b) Immunofluorescence
analysis of EdU incorpation in U87MG, T98G and U251 treated with GhEC-

EVs for 2d (3x103 EVs/target cell, scale bar=50 µm). (c) Analysis of EdU-

positive ratio of U87MG, T98G and U251 treated with GhEC-EVs. Data are

presented as the mean ± SD. *P <0.05, **P <0.01.

Supplementary figure 3. Five alternative proteins expressed in different EVs

and CD9 in different EVs was normalized by CD63 and CD81. (a) Western

blot detected the NOS3, FRZD4, PLAT, CD151 and TB4 expression in ECs.

(b) Analysis of NOS3, FRZD4, PLAT, CD151 and TB4 content in EVs of

GhECs, NhECs, GCs and GSCs. (c, d) Western blot shown relative

expression of Flotillin-1/CD63(c) and Flotillin-1/CD81 (d) in GhEC-EVs, NhEC-

EVs, GC-EVs and GSC-EVs. (e, f) CD9 expression was normalized by CD63

(e) and CD81 (f) in GhEC-EVs, NhEC-EVs, GC-EVs and GSC-EVs.

Supplementary Figure 4. High CD9 expression in gliomas and correlated

with poor patient survival. (a) The CD9 mRNA expression level in whole-

genome gene profiling of 676 gliomas samples from the TCGA; CD9

expression was significantly higher in high grade gliomas than in low-grade

gliomas (**P<0.01, unpaired t-test). (b) High CD9 expression correlated with

poor survival of patients, as described in Supplementary Figure 4 (a) (Mantel-

Cox test).

Supplementary Figure 5. CD9 in GhEC-EVs can be transferred to recipient

cells. Immunofluorescence showed CD9 change in GSC5 (left), GSC5

(middle) and T98G (right) treated with EVs of GCs, GSCs and GhECs
respectively (scale bar=50 µm).

Supplementary Figure 6. Reduction of CD9 produced no significant effect on

the size distribution and number of GhEC-EVs. (a) NTA analysis the fraction

of GhEC-derived EVs, showing the particles distribution of each group. (b)

Number of EVs particles (mean ± SD) were calculated per µg of EVs

released. EVs were derived from negative control GhECs or siRNA infected

GhECs (siCD9-1#GhEC, siCD9-2#GhEC). Dunnett test was used for

statistical analysis, data are presented as the mean ± SD.

Supplementary Figure 7. CD9 inhibited T98G proliferation and the EGFR

phosphorylation. (a, b) Overexpression of CD9 in T98G (a) and the effect of

CD9 on T98G cell viability (b). (c, d) Western blot showed the effect of GhEC-

EVs on phosphorylation of BMX/STST3 (c) and EGFR (d) in T98G. The

relative band intensity values were measured with ImageJ software.

Supplementary Figure 8. CD9 in HUVEC and AC16 had a similar expression

pattern with GhECs and HUVEC-EVs and AC16-EVs enhanced GSCs

tumoursphere formation in vitro. (a, b) Western blot showed CD9 expression

in GhECs, NhECs, HUVECs (a) and EVs from GhECs, NhECs, hUVECs (b).

(c) Phase contrast photomicrographs of GSC2 cells treated with HUVEC-EVs

(scale bar=100 μm). (d) The tumorsphere formation analysis shown the

number and diameter change of GSC2 treated with HUVEC-EVs (3x103

EVs/target cell). (e, f ) Western blot showed CD9 expression in GhECs and

AC16 (e) and EVs from GhECs and AC16 cells (f). (g) Phase contrast
photomicrographs of GSC2 cells treated with AC16-EVs (scale bar=100 μm).

(h) The tumorsphere formation analysis shown the number and diameter

change of GSC2 treated with AC16-EVs (3x103 EVs/target cell).

Supplementary Figure 9. GhEC-EVs could not promote breast cancer stem

like cells and gastric cancer stem like cells proliferation. (a) Western blot

showed the relative CD9 expression of GhECs, MDA-MB-231-SLC, SUN-5-

SLC and BGC-823-SLC. The relative band intensity values were measured

with ImageJ software. (b) MTS showed the MAD-MB-231-SLC, SUN-5-SLC

and BGC-823-SLC treated with GhEC-EVs proliferation change (3x103

EVs/target cell). Data are presented as the mean ± SD. **P <0.01.