Gene Sequence Fragment size ΔEGFR F 5' GGGCTCTGGAGGAAAAGAAAG GT 3' 116 bp R 5' CTTCTTACACTTGCGGACGC 3'

Transcription

1 Supplementary Table 1: Real-time PCR primer sequences for ΔEGFR, wtegfr, IL-6, LIF and GAPDH. Gene Sequence Fragment size ΔEGFR F 5' GGGCTCTGGAGGAAAAGAAAG GT 3' 116 bp R 5' CTTCTTACACTTGCGGACGC 3' wtegfr F 5' TTTGCCAAGGCACGAGTAACA 3' 14 bp R 5' ATTCCCAAGGACCACCTCACA 3' hil 6 F 5' AGCCACTCACCTCTTCAGAACGAA 3' 121 bp R 5' AGTGCCTCTTTGCTGCTTTCACAC 3' mil 6 F 5' ATCCAGTTGCCTTCTTGGGAC 3' 134 bp R 5' TAAGCCTCCGACTTGTGAAGT 3' hlif F 5' TATCACCATCTGTGCCTTTGCTGC 3' 187 bp R 5' TCTGCCAGATTGTTCCTATGCCCA 3' mlif F 5' ATACAGCCCAGCACCAGCTAGAAA 3' 138 bp R 5' GCTTGTTCATACACCCAGCAAGCA 3' hgapdh F 5' CCACATGGCCTCCAAGGAGTAAGAC 3' 131 bp R 5' AGGAGGGGAGATTCAGTGTGGTGGG 3' mgapdh F R h:human; m:mouse 5' CTGACGTGCCGCCTGGAGAAA 3' 5' AGCCCCGGCATCGAAGGTGG 3' 165 bp

2 Supplementary Figure 1: Orthotopic co-injection of U87wt and U87Δ cells results in reduced survival. (a-c) Survival analysis after intracranial injection of U87wt (wt), U87Δ (Δ) or U87wt mixed with U87Δ at indicated ratios (wt + Δ) (1%= 5x1 5 cells). (d) Survival analysis summary table indicating Kruskal-Wallis p values. (e) H&E staining of brain sections obtained at day 12 after intracranial injection of U87DK (DK) 1%, U87Δ (Δ) 1% or U87DK mixed with U87Δ at a ratio of 9:1 respectively (1%= 5x1 5 cells). (f) X-Gal stain quantification of intracranial tumors obtained at day 22 after injection of U87wt cells or U87Δ alone or U87wt + U87Δ-LacZ, at a 9:1 ratio, respectively. Supplementary Figure 2: U87DK cells do not enhance U87wt tumorigenicity after intracranial injection. H&E staining of brain sections obtained 14 days after intracranial injection into nude mice of U87wt cells mixed with U87DK at a ratio of 9:1. Supplementary Figure 3: Tumor growth kinetics of mixed glioma cell injections. (a) Tumor growth kinetics after subcutaneous injection into nude mice of U87Δ or U87wt cells injected alone or co-injected with U87Δ, U87DK or U87Par cells. (b) Detailed view of tumor growth kinetics from the lower part of the graph. Error bars represent mean ± s.e.m. Supplementary Figure 4: Orthotopic co-injection of mouse Ink4/Arf -/- -astrocytes engineered with wtegfr and ΔEGFR. (a) H&E of mouse brains 16 or 19 days after intracranial injection of mastr-ink4/arf -/- astrocytes engineered with wtegfr (mastr-wt), ΔEGFR (mastr-δ), or co-injection of mastr-wt plus mastr-δ mixed at a 9:1 ratio, respectively (mastr-wt+δ) (1%= 5x1 5 cells). (b) H&E staining of brain sections after injection of mastr-δ 1% (Δ 1%) or mastr-wt cells mixed with mastr-δ at a ratio of 9:1 respectively (wt + Δ 9-1%). High magnification illustrating perivascular invasive nature of tumor cells (1%= 5x1 5 cells). Supplementary Figure 5: wtegfr and ΔEGFR cells tend to establish equilibrium in vivo. (a) Tumor volume at day 22 after subcutaneous injection of U87wt-LacZ or U87Δ 1% alone, or mixed at ratios 9:1, 5:5 or 1:9 respectively (1%= 2x1 5 cells). (b) Proportion representation of U87wt cells at the injection time and at the end of the experiment (day 22). (c) Analysis of tumor composition by flow cytometry using Ab-1 (FITC) and Ab-5 (APC) antibodies to stain tumors formed after subcutaneous injection of U87wt mixed with U87Δ at a ratio of 9:1. Left, example of tumor composition analysis by flow cytometry. Right, composition of the mixed tumors according to the results obtained by flow cytometry. Error bars represent mean ± s.e.m.

3 Supplementary Figure 6: Analysis of EGFR and STAT3 phosphorylation by western blot analysis of tumor lysates obtained after subcutaneous injection of indicated mixtures of U87 cell line derivatives at indicated ratios. Each lane represents a different tumor sample. Supplementary Figure 7: ΔCM treatment activates wtegfr and downstream pathways in U178 and U373 glioma cells. (a) Analysis of EGFR (pegfr) and STAT3 (pstat3) phosphorylation by western blot of U178wt lysates non treated (-) or treated with U178Δ CM (ΔCM), or U178Δ CM pre-treated with anti-il-6 (ΔCM+IL-6n) or anti-gp13 (ΔCM+gp13n) neutralizing antibodies. (b) Analysis of EGFR (pegfr) and STAT3 (pstat3) phosphorylation by western blot of U373wt lysates non treated (-) or treated with U373Δ CM (ΔCM), or U373Δ CM pre-treated with anti-lif (ΔCM+LIFn) or anti-gp13 (ΔCM+gp13n) neutralizing antibodies. Total protein and actin were used as loading control in all experiments. Supplementary Figure 8: Quantification of EGF, TGF-α, amphiregulin (AR), HB-EGF and betacellulin (BTC) by ELISA in supernatants of U87Par, U87wt, U87Δ and U87DK cells. Error bars represent mean ± s.e.m. Supplementary Figure 9: Quantification of IL-6 and LIF in U178 and U373 glioma cells. Real-time PCR for IL-6 (a) and LIF (b) expression in U178 and U373 (Par) or engineered to over-express wtegfr (wt), ΔEGFR (Δ) or ΔEGFR with a dead kinase domain (DK). (c) ELISA quantification of IL-6 in supernatants of U178 and U373 cell line derivatives after 48 h of starvation. Error bars in all experiments represent mean ± s.e.m. One-way ANOVA and two-tail t-test were used for statistical analysis. **p<.1. Supplementary Figure 1: Quantification of IL-6 family cytokines in U87 cell line derivatives. Real time PCR of the IL-6 family members Cardiotrophin-like cytokine-1 (CLC- 1), Ciliary Neurotrophic Factor (CTNF), Cardiotrophin-1 (CT-1), IL-6. IL-11, IL-27, Leukemia Inhibitory Factor (LIF), IL-11, and Oncostatin M (OSM), in U87Par (Par), U87wt (wt), U87Δ (Δ) and U87DK (DK). Error bars represent mean ± s.e.m. One-way ANOVA and two-tail t-test were used for statistical analysis. **p<.1. Supplementary Figure 11: Blockade of STAT3 activation by IL-6 and LIF neutralizing antibodies. (a) Western blot analysis of EGFR (pegfr) and STAT3 (pstat3) phosphorylation in U87wt lysates non-treated (-), treated with recombinant IL-6 or with U87Δ CM (ΔCM) pre-treated or not with anti-il-6 neutralizing antibody (IL-6n). (b) Western blot analysis of EGFR (pegfr) and STAT3 (pstat3) phosphorylation in U87wt lysates nontreated (-), treated with recombinant LIF or with U87Δ CM (ΔCM) pre-treated or not with anti- LIF neutralizing antibody (LIFn).

4 Supplementary Figure 12: sirna knock-down of gp13, IL-6, and LIF (c) in vitro. (a) Quantification of gp13 levels by ELISA in lysates of U87wt cells non transfected (Neg) or transfected with 25 nm of a negative control sirna against luciferase (Luc sirna) or two different sirnas against gp13 (gp13 sirna#1 and gp13 sirna#2). (b) ELISA quantification of IL-6 secretion in U87Δ cells supernatants transfected with indicated concentrations of a negative control sirna against luciferase (Neg) or two different sirnas against IL-6 (IL6 sirna#1 and IL6 sirna#2). (c) Relative LIF expression after transfection with 25 nm of luciferase sirna (Luc) or LIF sirna by real-time PCR. cdna from non transfected cells was used as a second negative control. Normalization with GAPDH expression was performed. Error bars in all experiments represent mean ± s.e.m. Supplementary Figure 13: sirna knock-down of gp13, IL-6, and LIF in vivo. (a) Tumor growth kinetics after subcutaneous injection into nude mice of U87wt transfected with luciferase sirna (wt-luc sirna) or sirna targeting gp13 (wt-gp13 sirna) alone or mixed with U87Δ at a ratio 9:1 respectively. (b) Tumor growth kinetics after subcutaneous injection into nude mice of U87wt cells injected alone or with U87Δ transfected with luciferase sirna (Δ-Luc sirna) or sirna targeting IL-6 (Δ-IL6-siRNA). (c) Tumor growth kinetics after subcutaneous injection into nude mice of U87wt cells injected alone or with U87Δ transfected with luciferase sirna (Δ-Luc sirna) or sirna targeting LIF (Δ-LIF sirna). Error bars represent mean ± s.e.m. Supplementary Figure 14: Analysis of wtegfr and IL-6 (top) or LIF (bottom) expression by real-time PCR in 19 human GBM samples. Error bars in all experiments represent mean ± s.e.m.

5 Inda_Suppl.Fig1 a b 12% 1% wt 1% 1% 12% 1% wt 1% 1% wt % % survival 8% 6% 4% wt + 9-1% % survival 8% 6% 4% 2% 2% % days after IC injection % days after IC injection c d % survival 12% 1% 8% 6% 4% 2% wt 1%,1% wt + 99,9-,1% Group U87wt 1% 38 Median survival (days) p value 1 p value 2 U87delta 1% 24,7547 U87wt+U87delta 9-1% 23 <,1 U87delta 1% 27 *,482 U87wt+U87delta 99-1% 24,5 <,1 U87delta,1% 61,5 *,114 U87wt+U87delta 99,9-,1% 3 <,1 p value 1 : comparison between U87delta and U87wt+U87wt+delta; p value2: comparison between U87wt and U87wt+U87wt+delta % e days after IC injection DK1% -LacZ 1% DK+ -LacZ 9-1% f 12% 1% % of tumor area 8% 6% 4% 2% LacZ negative LacZ positive % wt 1% 1% wt + 9-1%

6 Inda_Suppl. Fig 2 U87wt 1% U87DK1% U87wt + U87DK 9-1%

7 a 22 Inda_Suppl. Fig 3 Tumor volume (mm 3 ) WT 1% -LacZ 1% -LacZ 1% WT + -LacZ 9-1% -LacZ 1% WT + -LacZ 99-1% WT + DK-LacZ 9-1% WT + DK-LacZ 99-1% WT + Parental-LacZ 9-1% Days post-sq injection b 1 WT 1% Tumor volume (mm3) WT + DK-LacZ 9-1% WT + DK-LacZ 99-1% WT + Parental-LacZ 9-1% Days post-sq injection

8 a Inda_Suppl.Fig 4 mastr-wt 1% mastr-wt+ 9-1% mastr- 1% Day 16 Day 19 b 1% wt + 9-1% 4X 4X 2X

9 a b Inda_Suppl. Fig % Tumor volume (mm 3 ) % of tumor 1% 8% 6% 4% wt 1% wt + 9-1% wt + 5-5% wt + 1-9% 2% % injection end c Δ wt Tumor volume (mm 3 ) wt U87wt 1% U87 1% U87wt + U87 9-1%

10 Inda_Suppl. Fig 6 wt pegfr EGFR wt pstat3 STAT3 Actin

11 Inda_Suppl. Fig 7 a b pegfr pegfr EGFR pakt Akt perk ERK pstat3 STAT3 Actin EGFR pakt Akt perk ERK pstat3 STAT3 Actin

12 Inda_Suppl. Fig 8

13 Inda_Suppl. Fig 9 a Relative expression U178 ** b Relative expression U178 Par wt DK Par wt DK Relative expression 4 2 U373 Par wt DK Relative expression U373 ** Par wt DK c pg/ml U178 ** Par wt DK pg/μl U373 Par wt DK

14 Inda_Suppl. Fig 1 Relative expression Par wt DK ** ** 5 CLC-1 CNTF-1 CT-1 IL-6 IL-11 IL-27 LIF OSM

15 Inda_Suppl. Fig 11 a b Stim.: Ab: - - IL-6 - IL-6 CM CM Stim.: IL-6n - IL-6n Ab: - - LIF - CM - LIF LIFn CM LIFn pegfr pegfr EGFR EGFR pstat3 pstat3 STAT3 STAT3 Actin

16 a 1 Inda_Suppl. Fig 12 pg/5 μg lysate b Neg Luc sirna gp13 sirna#1 gp13 sirna#2 [IL-6] (pg/ml) nM 2nM 4nM.8nM Luc sirna IL-6 sirna#1 IL-6 sirna#2 c 1,2 Relative expression 1,8,6,4,2 (-) Luc sirna LIF sirna

17 Inda_Suppl. Fig 13 a 5 tumor volume (mm3) wt-luc sirna 9% wt-gp13 sirna 9% 1% wt-luc sirna + 9-1% wt-gp13 sirna + 9-1% b c tumor volume (mm 3 ) Tumor volume (mm3) days post SQ wt 9% -Luc sirna 1% -IL6siRNA wt + -Luc sirna 9-1% wt + -IL6 sirna 9-1% days post SQ wt 9% -Luc sirna 1% -LIF sirna 1% wt + -Luc sirna 9-1% wt + -LIF sirna 9-1% days post SQ

18 Inda_Suppl. Fig 14 a nº of human GBM wtegfr + wtegfr - IL-6 high IL-6 low nº of human GBM wtegfr + wtegfr - LIF high LIF low