Loss of the Timp gene family is sufficient for the acquisition of the CAF-like cell state

Journal name:
Nature Cell Biology
Volume:
16,
Pages:
889–901
Year published:
DOI:
doi:10.1038/ncb3021
Received
Accepted
Published online

Abstract

Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ​ADAM10. Exosomal ​ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase ​RhoA. Moreover, ​ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ​ADAM10-rich exosomes that promote cell motility and activate ​RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression.

At a glance

Figures

  1. TIMPless fibroblasts mimic CAFs.
    Figure 1: TIMPless fibroblasts mimic CAFs.

    (a) ​Haematoxylin and ​eosin (H&E)-stained or ​α-SMA-immunostained skin sections. Scale bars, 50 μm.​α-SMA-positive cells are indicated by arrows and quantified per high-powered field (HPF; mean ± s.d.; WT n = 5; ΔTimp n = 3 skin tissues from different mice). (b) Mallory-stained skin sections of WT and ΔTimp mice. Scale bars, 100 μm. Note that increased collagen fibres of the connective tissues in ΔTimp skin are easily identified by their blue colour. (c) ​Vimentin or ​α-SMA immunofluorescently stained fibroblasts show that TIMPless fibroblasts are ​vimentin positive and stain stronger for ​α-SMA than WT. Scale bars, 20 μm. (d) Immunoblot of ​α-SMA in fibroblasts and plot of densitometry relative to β-actin (mean ± s.d.; n = 3 independent isolates of primary fibroblasts). (e) Image and quantification of collagen gel contraction induced by WT or ΔTimp fibroblasts show the greater contractility of TIMPless fibroblasts (mean ± s.d.; n = 3 wells). (f) Relative gene expression of ​stroma-derived factor-1 (​SDF-1), ​hepatocyte growth factor (​HGF), and ​transforming growth factor (TGF)-β1, 2, 3 by RT-qPCR in WT or ΔTimp fibroblasts (mean ± s.d.; n = 4 independent isolates of primary fibroblasts). (g) RT-qPCR analysis of ​α-SMA gene expression in WT and single or compound Timp-deficient fibroblasts (mean ± s.d.; n = 3 independent isolates of primary fibroblasts). (h) Fibroblast metalloproteinase activity was assayed by activation of fluorogenic substrate (SMO-3670-PI) with or without the indicated metalloproteinase inhibitors ​GI254023 (​ADAM10), GW280264 (​ADAM10+​ADAM17), ​TAPI-1 (ADAMs) or ​BB94 (ADAMs+MMPs) (mean ± s.d.; n = 3 dishes). (i) RT-qPCR analysis of ​α-SMA gene expression in WT or ΔTimp fibroblasts in the presence or absence of the metalloproteinase inhibitors ​TAPI-1 or ​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 one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc testing. P values smaller than 0.05 are indicated on respective plots. Uncropped images of blots are shown in Supplementary Fig. 8.

  2. TIMPless fibroblasts augment tumour cell xenografts and spontaneous metastasis.
    Figure 2: TIMPless fibroblasts augment tumour cell xenografts and spontaneous metastasis.

    (a) Tumour volume measurements of human cancer cell xenografts grown orthotopically in mammary fat pads or subcutaneously alone (1 × 106), with WT or with ΔTimp fibroblasts (3 × 106) (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). (b) ​Vimentin (human-specific), ​α-SMA and Collagen type IV immunostained A549 xenograft tumours. Scale bars, 100 μm. (c) Haematoxylin and eosin (H&E) staining and ​von Willebrand factor (​vWF) immunostaining of sections from representative A549 tumours. Arrows indicate ​vWF-positive vessels, inset: high-power view. Scale bars, 200 μm. The quantification of ​vWF-positive vessels per high-powered field (HPF; mean ± s.d.; MDA-MB231: n = 14 tumours per group; A549: cancer cells n = 12 tumours, cancer + WT fibroblasts n = 10 tumours, cancer + ΔTimp fibroblasts n = 10 tumours) is shown on the right. (d) Representative H&E-stained sections of lungs from tumour-bearing mice. Metastases are indicated by arrows. Metastatic cancer cells were also confirmed by staining of human ​vimentin in lungs; high-power view inset. Scale bars, 200 μm. (e) Quantification of metastatic lesions throughout the entire lung (mean ± s.d.; MDA-MB231: n = 7 lung tissues per group; A549: cancer cells alone n = 6 lung tissues, cancer + WT fibroblasts n = 5 lung tissues, cancer + ΔTimp fibroblasts n = 5 lung tissues). 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. Exosomes secreted from ΔTimp fibroblasts increase cancer cell motility.
    Figure 3: Exosomes secreted from ΔTimp fibroblasts increase cancer cell motility.

    (a) Relative cell viability of MDA-MB231 cells treated with control medium (DMEM), WT (WT_exo) or TIMPless exosomes (ΔTimp_exo) (mean ± s.d.; n = 5 wells). (b) Representative images of MDA-MB231 cells treated with DMEM, WT_exo or ΔTimp_exo with the highlighted area magnified to the right. Scale bars, 100 μm. (c) Internalization of fibroblast-derived exosomes by MDA-MB231 cells. Exosomes were purified from WT or ΔTimp fibroblasts, labelled with PKH67 (green) and incubated with MDA-MB231 cells (​vimentin, red) for 12 h at 37 °C. Note the PKH67 dye incorporated into living MDA-MB231 cells from PKH67-labelled exosomes. Scale bars, 50 μm. (d) Number of MDA-MB231 cell divisions during 15 h treatment with DMEM, WT_exo or ΔTimp_exo. Data represent the mean ± s.d. of 7 independent experiments. (e) Cell area of MDA-MB231 cells after incubation with DMEM, WT_exo or ΔTimp_exo (DMEM: n = 33; WT_exo: n = 32; ΔTimp_exo: n = 31 individual cells). (f) Average migration speed of MDA-MB231 cells incubated with DMEM, WT_exo or ΔTimp_exo (DMEM: n = 33; WT_exo: n = 32; ΔTimp_exo: n = 31 individual cells). Lines represent the median value. (g) Representative images of migrated MDA-MB231 cells and quantification of migration in a Transwell migration assay (MDA-MB231 cells in the presence of DMEM, WT_exo or ΔTimp_exo; mean ± s.d.; n = 4 wells). Scale bar, 100 μm. 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. TIMPless fibroblasts produce proteomically distinct exosomes.
    Figure 4: TIMPless fibroblasts produce proteomically distinct exosomes.

    (a) Quantification of the total amount of exosome protein produced by WT or ΔTimp fibroblasts (mean ± s.d. (n = 6 independent exosome isolates)). (b) Protein composition of WT and ΔTimp exosomes. The schematic depicts mass spectrometry analysis of ΔTimp exosomes (ΔTimp_exo) versus WT (WT_exo). Exosome-associated proteins previously identified in ExoCarta are highlighted in bold, ΔTimp exosome proteins with >10-fold increase in red, and >10-fold decrease in blue. (c) Representative electron microscope image of whole-mounted exosomes purified from ΔTimp medium. An electron micrograph of ΔTimp exosomes immunolabelled using anti-​ADAM10 antibody is shown in the inset. Scale bars, 50 nm. (d) Silver staining of conditioned media and exosomes, and immunoblots of WT_exo and ΔTimp_exo for flottilin-1, ​CD81, α/β-tubulin, and ​ADAM10 (pro-form (p) and mature form (m) of ​ADAM10 are indicated). (e) Exosome metalloproteinase activity by quantification of fluorogenic signalling on co-incubation of exosomes with fluorogenic substrate (SMO-3670-PI), with or without inhibitors ​GI254023 (​ADAM10), GW280264 (​ADAM10 + ​ADAM17), or ​TAPI-1 (ADAMs) (mean ± s.d.; n = 4 independent exosome isolates). (f) Average cell area of MDA-MB231 cells after incubation with control medium (DMEM), WT_exo or ΔTimp_exo and ​GI254023, GW280264 or ​BB94 (ADAMs + MMPs) inhibitors (DMEM: n = 36; WT_exo: n = 29; ΔTimp_exo: n = 25; ΔTimp_exo+ ​GI254023: n = 20; ΔTimp_exo+ GW280264: n = 22; ΔTimp_exo+ ​BB94: n = 28 individual cells). (g) Average migration speed of MDA-MB231 cells incubated with exosomes and treated with the indicated inhibitors (DMEM: n = 36; WT_exo: n = 29; ΔTimp_exo: n = 25; ΔTimp_exo+ ​GI254023: n = 20; ΔTimp_exo+ GW280264: n = 22; ΔTimp_exo+ ​BB94: n = 28 individual cells). Lines represent the median value. 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. Uncropped images of blots are shown in Supplementary Fig. 8.

  5. Exosomal ADAM10 mediates Notch activation to induce ALDH.
    Figure 5: Exosomal ​ADAM10 mediates Notch activation to induce ALDH.

    (a) RT-qPCR analysis of target gene expression of Notch (Hes1, Hey1, Hey2), canonical WNT (Axin2, Myc, Ccnd1, Lef1), and Hedgehog (Gli2, Hhip, Ptch1, Ptch2) signalling in MDA-MB231 cells treated with control medium (DMEM), WT (WT_exo) or ΔTimp exosomes (ΔTimp_exo) (mean ± s.d.; n = 3 dishes). (b) Relative RT-qPCR analysis of ​HES1 expression in MDA-MB231 cells treated with exosomes in the presence of the inhibitors ​GI254023 (​ADAM10), GW280264 (​ADAM10 + ​ADAM17), ​TAPI-1 (ADAMs), ​BB94 (ADAMs + MMPs) or ​DAPT (γ-secretase) (mean ± s.d.; n = 3 dishes). (c) Relative RT-qPCR analysis of ​aldehyde dehydrogenase 1A3 (Aldh1a3) gene expression in MDA-MB231 cells treated with exosomes in the presence of metalloproteinase or γ-secretase inhibitors (mean ± s.d.; n = 6 dishes). (d) Relative RT-qPCR analysis of ​integrin α6 (Itga6) gene expression in MDA-MB231 cells treated with exosomes in the presence of metalloproteinase inhibitors (mean ± s.d.; n = 3 dishes). (e) Average migration speed of MDA-MB231 cells incubated with exosomes and ​GI254023 or ​DAPT (DMEM n = 22; WT_exo n = 26; ΔTimp_exo n = 24; ΔTimp_exo + ​GI254023 n = 25; ΔTimp_exo + ​DAPT n = 26 individual cells). (f) Average cell area of MDA-MB231 cells after incubation with exosomes and ​GI254023 or ​DAPT (DMEM n = 22; WT_exo n = 26; ΔTimp_exo n = 24; ΔTimp_exo + ​GI254023 n = 25; ΔTimp_exo + ​DAPT n = 26 individual cells). Lines represent the median value. (g) Immunoblot of total or active GTP-bound ​RhoA in MDA-MB231 cells treated with exosomes in the presence of metalloproteinase inhibitors or ​DAPT. (h) Transwell migration of MDA-MB231 cells in the presence of control medium (DMEM), WT_exo or ΔTimp_exo with or without ROCK inhibitor ​HA-1077 (mean ± s.d.; n = 4 dishes). Results between the two independent groups were determined by the 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. Uncropped images of blots are shown in Supplementary Fig. 8.

  6. Disrupting ADAM10 expression in TIMPless fibroblasts abrogates CAF functions.
    Figure 6: Disrupting ​ADAM10 expression in TIMPless fibroblasts abrogates CAF functions.

    (a) ​ADAM10 immunoblot of exosomes from WT or ΔTimp fibroblasts expressing either control or ​ADAM10-specific shRNA. The pro-form (p) and mature form (m) of ​ADAM10 are indicated. (b) Metalloproteinase activity in exosomes derived from WT or ΔTimp fibroblasts expressing control or ​ADAM10-specific shRNAs using fluorogenic peptides (mean ± s.d.; n = 4 independent exosomes). (c) Transwell migration of MDA-MB231 cells in the presence of control medium (DMEM), WTshCtrl_exo (WT_exo Ctrl shRNA), WTshA10_exo (WT_exo ​ADAM10 shRNA), ΔTimpshCtrl_exo (ΔTimp_exo Ctrl shRNA) or ΔTimpshA10_exo (ΔTimp_exo ​ADAM10 shRNA) (mean ± s.d.; n = 4 wells). (d) Immunoblot of total or active ​GTP-bound ​RhoA in MDA-MB231 cells in the presence of control medium (DMEM), WTshCtrl_exo, WTshA10_exo, ΔTimpshCtrl_exo or ΔTimpshA10_exo. (e) Relative RT-qPCR analysis of Hes1 expression in MDA-MB231 cells in the presence of control medium (DMEM), WTshCtrl_exo, WTshA10_exo, ΔTimpshCtrl_exo or ΔTimpshA10_exo (mean ± s.d.; n = 3 dishes). (f) Tumour volume measurements of human cancer cell xenografts orthotopically injected into mammary fat pads alone (1 × 106) or with WTshCtrl, WTshA10, ΔTimpshCtrl or ΔTimpshA10 fibroblasts (3 × 106) (mean ± s.d.; n = 12 tu- mours per group; P < 0.05,∗∗P < 0.01) (∗∗A: MDA + ΔTimpshCtrl versus MDA alone, B: MDA + ΔTimpshCtrl versus MDA+WTshCtrl,C: MDA + ΔTimpshCtrl versus MDA + ΔTimpshA10). (g) The number of metastatic colonies per lung (mean ± s.d.; n = 6 lung tissues per group). 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. Uncropped images of blots are shown in Supplementary Fig. 8.

  7. CAFs secrete ADAM10-rich exosomes.
    Figure 7: CAFs secrete ​ADAM10-rich exosomes.

    (a) RT-qPCR analysis of ​α-Sma gene expression in hCAFs (hCAF1 and hCAF2) and their control hNAFs (hNAF1 and hNAF2) (mean ± s.d.; n = 3 dishes). (b) Immunoblots of exosomes from hCAFs from two different patients (CAF1 and CAF2) for ​CD81, ​flotillin-1 and ​ADAM10. (c) Mass spectrometry analysis on hCAF-derived exosomes from four different patients; shown are the results of label-free quantification (LFQ) values of ​CD81, ​flotillin-1 (​FLOT1) and ​ADAM10. Lines represent the median value. (d) Immunoblots of exosomes from hCAFs (hCAF1 and hCAF2) and hNAFs (hNAF1 and hNAF2) for ​flotillin-1 (​FLOT1) and ​ADAM10 with their densitometry ratio (​ADAM10/​FLOT1). (e) Transwell migration of MDA-MB231 cells in the presence of control medium (DMEM) or hCAF_exo with representative images of migrated MDA-MB231 cells (mean ± s.d.; n = 4 wells). Scale bar, 100 μm. (f) Immunoblot of ​RhoA before, and after ​GTP pulldown to isolate ​GTP-bound active ​RhoA in MDA-MB231 cells treated with hCAF_exo with or without ​GI254023 (​ADAM10 inhibitor). (g) Relative RT-qPCR analysis of Hes1 expression in MDA-MB231 cells treated with hCAF_exo in the presence of ​GI254023 or ​BB94 (ADAMs + MMPs inhibitor) (mean ± s.d.; n = 3 dishes). (h) Gene expression level of Adam10 and Timp3 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. Lines represent the median value. (i) Gene expression of Timp1, Timp2, Timp3 and Timp4 in hCAFs versus NAFs isolated from lung cancer patients (n = 3 dishes). Results between the two independent groups were determined by Student’s t-test. P values smaller than 0.05 are indicated on respective plots. Uncropped images of blots are shown in Supplementary Fig. 8.

  8. A schematic model representing TIMPless fibroblasts as microenvironment modifiers for tumour progression.
    Figure 8: A schematic model representing TIMPless fibroblasts as microenvironment modifiers for tumour progression.

    Fibroblastic loss of the Timp family is sufficient to cause the acquisition of hallmark CAF functions. TIMPless fibroblasts produce ​ADAM10-rich exosomes, which induce ALDH expression in cancer cells through Notch activation, as well as increase cancer cell motility through ​RhoA activation. TIMPless fibroblasts also contain various ECM proteins, which may provide a CSC or pre-metastatic niche for cancer cells.

  9. Characterisation of TIMPless fibroblasts.
    Supplementary Fig. 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 (ref. 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.

  10. Altered metalloproteinase regulation in TIMPless fibroblasts.
    Supplementary Fig. 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 α1 (​Col1a1), ​type I α2 (​Col1a2) and ​type IV α1 (​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-β2 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−1 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 one-way ANOVA followed by Bonferroni’s post-hoc testing. P values smaller than 0.05 are indicated on respective plots.

  11. TIMPless fibroblasts augment tumour cell xenografts.
    Supplementary Fig. 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). Tumour cells (1 × 106) were implanted with or without fibroblasts (3 × 106) as indicated. (b) Tumour volume measurements of 786-O xenografts subcutaneously injected alone (1 × 106) or with WT or ΔTimp fibroblasts (3 × 106)(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 well-demarcated borderlines with the surrounding tissue. Scale bars, 500 μm. (e) ​Ki-67-immunostained tumour sections (A549 and MDA-MB231). Human cancer cells are labelled with human-specific ​vimentin (green) and mouse-specific ​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.

  12. Effects of conditioned medium or purified exosomes from ΔTimp fibroblasts on cancer cell behavior.
    Supplementary Fig. 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.

  13. Summary of detected proteins in WT- or ΔTimp-exosomes by mass spectrometry.
    Supplementary Fig. 5: Summary of detected proteins in WT- or ΔTimp-exosomes by mass spectrometry.

    (a) Representative scanning electron-microscope 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:0031012) as analysed 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) = 0.0004, Focal adhesion (mmu04510) = 0.0007, Cytoskeletal regulation by Rho GTPase (P00016) = 0.7714, Integrin signalling pathway (P00034) = 1.9656, Huntington disease (P00029) = 6.4997, Hedgehog signalling pathway (P00025) = 6.5084. Proteasome (mmu03050) = 7.2460. (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.

  14. Effects of TIMPless-exosomes on cancer cell stemness.
    Supplementary Fig. 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.

  15. Establishment of shADAM10-knockdown in WT and TIMPless fibroblasts and gene expression analysis on human breast cancer stroma.
    Supplementary Fig. 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) RT-qPCR 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 17817. 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.

  16. Uncropped gel images.
    Supplementary Fig. 8: Uncropped gel images.

    Uncropped gel images corresponding to Figs 1d, 4d, 5g, 6a, d, 7b, d, f and Supplementary Figs 1b, 2d and 6d.

Accession codes

Referenced accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Ontario Cancer Institute, University Health Network, Toronto, M5G 2M9, Canada

    • Masayuki Shimoda,
    • Simona Principe,
    • Hartland W. Jackson,
    • Hui Fang,
    • Sam D. Molyneux,
    • Yang W. Shao,
    • Alison Aiken,
    • Paul D. Waterhouse,
    • Christina Karamboulas,
    • Laurie Ailles,
    • Thomas Kislinger &
    • Rama Khokha
  2. Centre for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, M5G 1X5, Canada

    • Valbona Luga &
    • Jeffrey L. Wrana
  3. Institute of Pharmacology and Toxicology, RWTH Aachen University, 52074 Aachen, Germany

    • Franz M. Hess &
    • Andreas Ludwig
  4. Keio University, School of Medicine, Tokyo 160-8582, Japan

    • Takashi Ohtsuka &
    • Yasunori Okada

Contributions

M.S. and R.K. designed the study; M.S., H.F. and A.A. performed TIMPless fibroblast isolation and its characterization; V.L. and J.L.W. provided technical assistance of exosome isolation and performed single-cell motility assay; S.P., S.D.M., Y.W.S. and T.K. conducted mass spectrometry analysis on exosomes; Y.W.S. performed microarray analysis; H.W.J., C.K. and L.A. contributed to xenograft experiments; C.K., L.A., T.O. and Y.O. contributed to the isolation of hCAFs; F.M.H. and A.L. provided metalloproteinase inhibitors and shRNA vectors; all other experiments were carried out by M.S.; R.K. directed the study; M.S., H.W.J. and R.K. wrote the manuscript; and S.D.M. and P.D.W. conceptualized the importance of stromal TIMPs and edited the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Characterisation of TIMPless fibroblasts. (944 KB)

    (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 (ref. 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.

  2. Supplementary Figure 2: Altered metalloproteinase regulation in TIMPless fibroblasts. (380 KB)

    (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 α1 (​Col1a1), ​type I α2 (​Col1a2) and ​type IV α1 (​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-β2 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−1 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 one-way ANOVA followed by Bonferroni’s post-hoc testing. P values smaller than 0.05 are indicated on respective plots.

  3. Supplementary Figure 3: TIMPless fibroblasts augment tumour cell xenografts. (926 KB)

    (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). Tumour cells (1 × 106) were implanted with or without fibroblasts (3 × 106) as indicated. (b) Tumour volume measurements of 786-O xenografts subcutaneously injected alone (1 × 106) or with WT or ΔTimp fibroblasts (3 × 106)(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 well-demarcated borderlines with the surrounding tissue. Scale bars, 500 μm. (e) ​Ki-67-immunostained tumour sections (A549 and MDA-MB231). Human cancer cells are labelled with human-specific ​vimentin (green) and mouse-specific ​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.

  4. Supplementary Figure 4: Effects of conditioned medium or purified exosomes from ΔTimp fibroblasts on cancer cell behavior. (966 KB)

    (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.

  5. Supplementary Figure 5: Summary of detected proteins in WT- or ΔTimp-exosomes by mass spectrometry. (647 KB)

    (a) Representative scanning electron-microscope 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:0031012) as analysed 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) = 0.0004, Focal adhesion (mmu04510) = 0.0007, Cytoskeletal regulation by Rho GTPase (P00016) = 0.7714, Integrin signalling pathway (P00034) = 1.9656, Huntington disease (P00029) = 6.4997, Hedgehog signalling pathway (P00025) = 6.5084. Proteasome (mmu03050) = 7.2460. (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.

  6. Supplementary Figure 6: Effects of TIMPless-exosomes on cancer cell stemness. (698 KB)

    (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.

  7. Supplementary Figure 7: Establishment of shADAM10-knockdown in WT and TIMPless fibroblasts and gene expression analysis on human breast cancer stroma. (283 KB)

    (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) RT-qPCR 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 17817. 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.

  8. Supplementary Figure 8: Uncropped gel images. (687 KB)

    Uncropped gel images corresponding to Figs 1d, 4d, 5g, 6a, d, 7b, d, f and Supplementary Figs 1b, 2d and 6d.

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