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1 DOI: /ncb3021 Supplementary figure 1 Characterisation of TIMPless fibroblasts. a) Relative gene expression of TIMPs1-4 by real time quantitative PCR (RT-qPCR) in WT or ΔTimp fibroblasts (mean ± s.d. (n=3 independent isolates of primary fibroblasts). b) Immunoblot of TIMP3 and β-actin in fibroblasts. Arrows indicate 24 kda and 27 kda (glycosylated) TIMP3 13. c) α-smooth muscle actin (α-sma) immunostained mammary gland and lung sections of WT or ΔTimp mice with the highlighted area magnified to the right. Scale bars, 100 μm. d) Growth curves of WT and ΔTimp fibroblasts. The number of cells per 10 cm dish was quantified every other day (mean ± s.d. (n=3 dishes)). e) Growth crisis experiment using WT and ΔTimp fibroblasts. Isolated WT and ΔTimp fibroblasts were grown in a 10 cm dish and passaged when they reached confluence. Note that ΔTimp fibroblasts grew faster than WT fibroblasts at the beginning of culture, however, both WT and ΔTimp fibroblasts underwent a growth crisis marked by failure to proliferate around 40 passages. Results between the two independent groups were determined by Student s t-test. P values smaller than 0.05 are indicated on respective plots. 1
2 Supplementary figure 2. Altered metalloproteinase regulation in TIMPless fibroblasts. a) RT-qPCR analysis of α-sma gene expression in fibroblasts. Expression of α-sma in the presence of TGF-β (mean ± s.d. (WT n=3; ΔTimp n=4 independent isolates of primary fibroblasts)). b) Relative gene expression of collagens, type I a1 (Col1a1), type I a2 (Col1a2) and type IV a1 (Col4a1) by RT-qPCR in WT or ΔTimp fibroblasts (mean ± s.d. (n=3 independent isolates of primary fibroblasts). c) Analysis of gene expression of MMP2, MMP9, MMP13, MMP14, ADAM10, ADAM12 and ADAM17 in WT or ΔTimp fibroblasts by relative RT-qPCR (mean ± s.d. (n=4 independent isolates of primary fibroblasts)). d) Immunoblot of WT and ΔTimp fibroblasts for ADAM10 and ADAM17. Pro-form (p) and mature form (m) of ADAM10 are indicated. e) RT-qPCR analysis of TGF-b2 gene expression in WT or single or compound Timp deficient fibroblasts (mean ± s.d. (n=3 independent isolates of primary fibroblasts)). f) Gelatin zymography of conditioned medium from fibroblasts stimulated with 50 μg/ml of concanavalin (con) A as indicated (+). Arrows indicate forms of MMP2 and MMP9. g) RT-qPCR analysis of SDF-1 and HGF gene expression in WT or ΔTimp fibroblasts in the presence with or without broad metalloproteinase inhibitor BB94 (mean ± s.d. (n=3 dishes)). Results between the two independent groups were determined by Student s t-test, and comparisons among three or more groups were determined by oneway ANOVA followed by Bonferroni s post-hoc testing. P values smaller than 0.05 are indicated on respective plots. 2
3 Supplementary figure 3 TIMPless fibroblasts augment tumour cell xenografts. a) Tumour weight of human cell line xenografts at time of sacrifice (day 35 for MDA-MB231 xenografts, day 28 for A549 xenografts and day 51 for SCC4 xenografts) (mean ± s.d. (MDA-MB231: n=14 tumours per group; A549: cancer cells alone n=12 tumours, cancer + WT fibroblasts n=10 tumours, cancer +ΔTimp fibroblasts n=10 tumours; SCC4: n=6 tumours per group). Tumor cells (1 x 10 6 ) were implanted with or without fibroblasts (3 x 10 6 ) as indicated. b) Tumour volume measurements of 786-O xenografts subcutaneously injected alone (1 x 10 6 ) or with WT or ΔTimp fibroblasts (3 x 10 6 )(mean ± s.d. (n=6 tumours per group)). Note that 786-O xenografts did not grow c) Hematoxylin and eosin (HE) staining and von Willebrand factor (vwf) immunostaining of sections from representative MDA-MB231 tumours. Arrows indicate vwf-positive vessels, inset: high-power view. Scale bars, 200 μm. d) HE staining of sections from representative A549 xenograft tumours. The borders between tumour and stroma are indicated with dotted lines with the highlighted area magnified to the right. Note that xenograft tumours with TIMPless fibroblasts tend to invade the surrounding tissue by forming small cancer cell islands, while xenograft tumours with WT fibroblasts or control tumours had welldemarcated borderlines with the surrounding tissue. Scale bars, 500 μm. e) Ki-67-immunostained tumor sections (A549 and MDA-MB231). Human cancer cells are labelled with human-specific vimentin (green) and mousespecific Ki-67 clone Tec3 that stains stromal cells only (red) as indicated by arrows and quantitated per high powered field (HPF) (mean ± s.d. (WT n=5; ΔTimp n=5 tumours)). Scale bars, 50 μm. Note that there are very few Ki-67 positive stromal cells in the stroma of these tumour xenografts and there are no significant differences between the number of Ki-67 positive cells in TIMPless and WT groups for both A549 and MDA-MB231 xenografts. P values are indicated on respective plots. Comparisons among three or more groups were determined by one-way ANOVA followed by Bonferroni s post-hoc testing. P values smaller than 0.05 are indicated on respective plots. 3
4 Supplementary figure 4 Effects of conditioned medium or purified exosomes from ΔTimp fibroblasts on cancer cell behavior. a) The relative cell viability of MDA-MB231 cells treated with control (DMEM), WT or ΔTimp medium for 72 h was evaluated by a CellTiter-Glo proliferation assay (mean ± s.d. (n=3 wells)). b) Average migration speed of MDA-MB231 cells incubated with DMEM, WT or ΔTimp medium over 15 h (DMEM: n=20; WT media: n=27; ΔTimp media: n=30 individual cells). c) Relative cell migration was determined by the number of migrating MDA-MB231 cells in the presence of DMEM, WT or ΔTimp medium in a transwell migration assay (mean ± s.d. (n=6 wells)). d) Representative images of A549 cells treated with DMEM (control), WT_exo or ΔTimp_exo. Scale bars, 100 μm. e) Representative images of migrated A549 cells and quantification of migration in a transwell migration assay (A549 cells in the presence of DMEM, WT_exo or ΔTimp_ exo; mean ± s.d. (n=4 wells)). Scale bars, 100 μm. f) Internalisation of fibroblast-derived exosomes by MDA-MB231 cells. Exosomes were purified from WT or ΔTimp fibroblasts, labelled with PKH67 (green) and incubated either with living or with fixed MDA-MB231 cells (vimentin, red) for 12 h at 37 C. Scale bars, 50 μm. Note that PKH67-labelled exosomes from WT or ΔTimp fibroblasts can transfer the PKH67 dye into living, but not fixed MDA-MB231 cells showing that transfer is not due to passive diffusion, but to an active uptake process. g) Z-stack image of MDA-MB231 cells incubated with PKH67-labelled exosomes from ΔTimp fibroblasts. Scale bars, 20 μm. Note that PKH67 dye is detected inside of the cells. h) Transwell migration of MDA-MB231 cells in the presence of exosomes from WT or single, compound or complete Timp-deficient fibroblasts (mean ± s.d. (n=3 wells)). Comparisons among three or more groups were determined by one-way ANOVA followed by Bonferroni s post-hoc testing. P values smaller than 0.05 are indicated on respective plots. 4
5 Supplementary figure 5 Summary of detected proteins in WT- or ΔTimpexosomes by mass spectrometry. a) Representative scanning electronmicroscope images of whole-mounted exosomes purified from WT or ΔTimp media and their average size (n=55 vesicles per group). Scale bars, 100 nm. b) Venn diagrams depicting overlap of proteins identified in three replicate proteomic analyses of purified exosomes. A total of 280 (ΔTimp) and 269 (WT) proteins were identified from the three trials. 272 proteins in ΔTimp-exosomes and 263 proteins in WT-exosomes were identified with high confidence in at least two trials. c) Ratio of differentially expressed extracellular matrix proteins (GO: ) as analyzed by Gene Ontology (GO) in ΔTimp- versus WT-exosomes. d) Intracellular signalling pathways or biological processes representing differentially expressed proteins in exosomes. FDR (%): ECM-receptor interaction (mmu04512) = , Focal adhesion (mmu04510) = , Cytoskeletal regulation by Rho GTPase (P00016) = , Integrin signalling pathway (P00034) = , Huntington disease (P00029) = , Hedgehog signalling pathway (P00025) = Proteasome (mmu03050) = e) Representative images of MDA-MB231 cells treated with control media (DMEM), WT_exo or ΔTimp_exo in the presence of metalloproteinase inhibitors for 15 h of culture, high-power view inset. Scale bars, 50 μm. Note that the morphological change of ΔTimp_exo-treated MDA-MB231 cells was completely inhibited in the presence of ADAM10-specific inhibitor GI254023, combined ADAM17/ADAM10 inhibitor GW280264, and broad metalloproteinase inhibitor BB94. Results between the two independent groups were determined by Student s t-test. 5
6 Supplementary figure 6 Effects of TIMPless-exosomes on cancer cell stemness a) Relative RT-qPCR analysis of aldehyde dehydrogenase1a1 (ALDH1A1) gene expression in MDA-MB231 cells treated with exosomes in the presence of metalloproteinase or γ-secretase inhibitors (mean ± s.d. (n=4 dishes)). b) Relative RT-qPCR analysis of CD44 gene expression in MDA-MB231 cells treated with exosomes in the presence of metalloproteinase inhibitors (mean ± s.d. (n=3 dishes)). c) Mammosphere culture and self-renewal assay. Representative images of primary mammosphere formation of MDA-MB231 cells in the presence of WT- or ΔTimp-exosomes for 5 days are shown in the left panels. Scale bars, 50 μm. Mammosphere self-renewal activity is calculated as described in Methods (mean ± s.d. (n=6 dishes)). d) Immunoblot of RhoA before, and after GTP pull-down to isolate GTP-bound active RhoA in MDA-MB231 and A549 cells treated as indicated. Results between the two independent groups were determined by Student s t-test, and comparisons among three or more groups were determined by one-way ANOVA followed by Bonferroni s post-hoc testing. P values smaller than 0.05 are indicated on respective plots. 6
7 Supplementary figure 7 Establishment of shadam10-knockdown in WT and TIMPless fibroblasts and gene expression analysis on human breast cancer stroma. a) RT-qPCR analysis showing relative expression of ADAM10 after shrna knockdown of ADAM10 (sha10) in WT and ΔTimp fibroblasts compared to scrambled shrna control treated (shctrl) and non-shrna treated parental cells (WT and ΔTimp) (mean ± s.d. (n=3 dishes)). b) RTqPCR analysis showing relative expression of ADAM9,12,17 in ΔTimpshCtrl or ΔTimpshA10 fibroblasts (mean ± s.d. (n=3 dishes)). c) Relative cell viability of WTshCtrl, WTshA10, ΔTimpshCtrl or ΔTimpshA10 fibroblasts (mean ± s.d. (n=4 wells)). d) Log2 expression ratio of TIMPs in murine dysplastic skin fibroblasts (DSFs) from K14-HPV16 mice versus normal dermal fibroblasts (NDFs) from GSE Average of log2 expression ratio of single probes against TIMP1 (1 probe), TIMP2 (6 probes), TIMP3 (4 probes) and TIMP4 (2 probes) is shown. e) Gene expression level of ADAM8, ADAM12 and MMP11 in human breast cancer stroma adjacent to invasive ductal carcinoma (tumour, n=51 patients) versus normal breast reduction tissue (normal, n=6 patients) from GSE9014. P values smaller than 0.05 are indicated on respective plots. Results between the two independent groups were determined by Student s t-test. P values smaller than 0.05 are indicated on respective plots. 7
8 Supplementary figure 8 Uncropped gel images. Uncropped gel images corresponding to Figure 1d, Figure 4d, Figure 5g, Figure 6a, Figure 6d, Figure 7b, Figure 7d, Figure 7f, Supplementary figure 1b, Supplementary figure 2d and Supplementary figure 6d. 8
9 Supplementary Table legends Supplementary Table 1 List of proteins detected in WT- or TIMPless-exosomes by mass spectrometry, providing the Gene name, Swiss port ID, as well as the ratio of spectral counts detected in TIMPless- versus WT-exosomes and its t-test. Supplementary Table 2 Primary antibodies used in this study Supplementary Table 3 Primer sequences and catalogue numbers used for RT-qPCR analysis 9
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