Stiff matrix induces exosome secretion to promote tumour growth

Abstract

Tissue fibrosis and extracellular matrix (ECM) stiffening promote tumour progression. The mechanisms by which ECM regulates its contacting cells have been extensively studied. However, how stiffness influences intercellular communications in the microenvironment for tumour progression remains unknown. Here we report that stiff ECM stimulates the release of exosomes from cancer cells. We delineate a molecular pathway that links stiff ECM to activation of Akt, which in turn promotes GTP loading to Rab8 that drives exosome secretion. We further show that exosomes generated from cells grown on stiff ECM effectively promote tumour growth. Proteomic analysis revealed that the Notch signalling pathway is activated in cells treated with exosomes derived from tumour cells grown on stiff ECM, consistent with our gene expression analysis of liver tissues from patients. Our study reveals a molecular mechanism that regulates exosome secretion and provides insight into how mechanical properties of the ECM control the tumour microenvironment for tumour growth.

Extended Data Fig. 1.. Characterization of exosomes derived from different cells.

a, Representative TEM image of exosomes purified from the conditioned media of Huh7 cells. Scale bar: 100 nm. b, Purified exosome proteins were quantitated by Bradford assay and the mean was normalized to 1 for exosomal proteins from soft (0.5 kPa) matrix. Exosomes were collected from equal numbers of cells. Values are presented as Mean ± S.D. n=3. c, The mean diameters of exosomes purified from the conditioned media of Huh7 cells on soft or stiff matrix. Values are presented as mean ± S.D. 3 independent experiments were performed. At least 10⁶ of purified exosomes were measured by NTA for each experiment. d, Exosomes (10 μg) derived from Huh7 cells on soft or stiff matrix were diluted with 1 ml PBS. The particle concentration was determined by NTA. Values are presented as Mean ± S.D. n=3. e, Exosomes released from the same number of primary hepatocytes grown on matrix with different stiffness were quantified, and the concentration of exosomes released from cells were normalized to the 0.5 kPa group. Values are presented as Mean ± S.D. n=3. F, Exosomes released from the same number of MCF10A, MCF10AT, and MCF10CA cells grown on matrix with different stiffness were quantified. The amounts of exosomes released from the cells were normalized to the 0.5 kPa for each cell line. Mean ± S.D. n=3. Source numerical data and unprocessed blots are available in source data. n represents the number of independent experiments.

Extended Data Fig. 2.. Exosome secretion from Huh7 cells treated with Akt and FAK inhibitors.

a, Volcano plot of RPPA data displaying the pattern of protein expression for Huh7 cells cultured on stiff (10 kPa) matrix relative to soft (0.5 kPa) matrix. Significantly up- and down-regulated proteins are indicated by red and blue dots, respectively (cut-off p<0.05). All the data points were normalized for protein loading and transformed to Log2 values (labeled “NormLog2” on X-axis). b, Western blots showing the up-regulation of p-Akt but not p-ERK in Huh7 cells grown on stiff ECM. Molecular weights (in kDa) are shown to the right. c, Huh7 cells on soft or stiff matrix were treated with DMSO or Akt inhibitor GDC-0068. The conditioned media were collected and proceeded for NTA. Exosome concentration from the cells treated with DMSO on soft matrix was normalized to 1. Values are presented as Mean ± S.D., n=3. d-f, Huh7 cells were treated with DMSO or various concentrations of Akt inhibitors MK-2206, GDC-0068 or FAK inhibitor PND-1168. Cell viability was examined by CCK-8 assay and normalized to the value of DMSO treated group. Values are presented as Mean ± S.D. n=3. Source numerical data and unprocessed blots are available in source data. n represents the number of independent experiments.

Extended Data Fig. 3.. Characterization of Rab8 and Rabin8 in cells grown on different matrix.

a, Huh7 cells were transfected with GFP or GFP-Rab8 and grown on soft matrix. Exosomes in the conditioned media were purified by ultracentrifugation, and exosomes from the same number of cells were loaded for western blotting with antibodies against exosome markers HRS and CD63. b, Conditioned media from cells expressing GFP or GFP-Rab8 and treated with DMSO or MK-2206 were collected and proceeded for NTA. Exosome concentration was normalized to those from GFP expressing cells treated with DMSO. Values are presented as Mean ± S.D. n=3. c, Conditioned media from cells with control or Rab8 shRNA treated with DMSO or MK-2206 were collected and proceeded for NTA. Exosome concentration was normalized to those from cells with control shRNA and DMSO. Values are presented as Mean ± S.D. n=3. d, Sequence alignment of Rabin8 from different species. The phosphorylation site Serine 149 was shown in red. e, Schematic diagram showing the use of BRET in analyzing Rabin8 conformation. NanoLuc (BRET donor) and HaloTag (BRET acceptor) were fused to the N and C terminus of Rabin8, respectively. When Rabin8 is adopted in a “closed” conformation and autoinhibited, BRET will occur owing to the close proximity between the donor and acceptor. If Rabin8 switches to an “open” conformation induced by Akt phosphorylation on S149, the BRET signal will significantly decrease. f, Huh7 cells were transfected with different Rabin8 variants (WT, S149A, and S149D). Conditioned media were collected and proceeded for NTA. Exosome concentration was normalized to those from cells expressing wild type Rabin8 on soft matrix (n=3). Values are presented as Mean ± S.D. Source numerical data and unprocessed blots are available in source data. n represents the number of independent experiments.

Extended Data Fig. 4.. Hepa1-6 cells secreted more exosomes when grown on stiff Matrix.

a, Hepa1-6 cells were cultured on soft (0.5 kPa) or stiff (10 kPa) matrix. Exosomes in the conditioned media were purified. Quantification of the exosomal proteins by Bradford assay. The amounts of exosomal proteins were normalized to those from soft matrix. Values are presented as Mean ± S.D. n=3. b, Exosomes from the same number of cells were analyzed by immunoblotting using antibodies against indicated exosome markers. c, Quantification of the levels of HRS, Syntenin-1, CD63, Alix and Tsg101 is presented. Mean ± S.D. n=3. d, Exosomes released from the same number of Hepa1-6 cells grown on matrix with different stiffness were quantified, and the concentration of exosomes released from cells grown on 0.5 kPa matrix were normalized as 1. Values are presented as Mean ± S.D. n=3. n represents the number of independent experiments. e, Growth curves of Hepa 1-6 tumors in C57L/J mice injected with PBS or the same amounts of exosomes derived from Hepa1-6 cells treated with DMSO or MK-2206 (n=5 mice). Values are presented as Mean ± S.D. Source numerical data and unprocessed blots are available in source data.

Extended Data Fig. 5.. Expression of the Notch pathway proteins in Huh7 cells grown on soft or stiff matrix.

Heatmap of RPPA data showing the levels of the Notch pathway proteins in Huh7 cells grown on soft (0.5 kPa) or stiff (10 kPa) matrix. Source numerical data is available in source data.

Extended Data Fig. 6.. Gene expression analysis of liver tissues from patients.

a, Box and whisker plot (bars represent 10–90 percentile, dots represent outliers) of albumin levels in the serum of 226 HCC patients with high (n=113 patients) or low (n=113 patients) Notch gene expression (p value is from Mann-Whitney test (two-tailed). b, Box and whisker plot (bars represent 10–90 percentile, dots represent outliers) of alanine transaminase (ALT) levels in the serum of 226 HCC patients with high (n=113 patients) or low (n=113 patients) Notch gene expression (p value is from Mann-Whitney test (two-tailed). c, Analysis of Notch-high and Notch-low patients with HSC or without (nHSC) the HSC gene signature (Fishers Exact test). d, Notch-high (n=113 patients) and Notch-low (n=113 patients) patients with or without cirrhosis (Fishers Exact test). The proportion of patients with high Notch genes expression in each group were analyzed (Fishers Exact test). e, Survival risk prediction analysis based on the expression data of four Notch associated genes, HEY1, HEY2, HES1, and SOX9 in non-tumor tissues derived from 226 HCC patients (Kaplan-Meier Cox Log Rank test between two groups and leave one out permutation analyses (1000X)). f, Top enriched signaling pathways associated with Notch activation. Ingenuity Pathway Analysis was performed on 1,872 differentially expressed genes (p<0.001) between high-Notch) and low-Notch patient non-tumor samples (see Methods for detail). -log(p-value) were calculated using Fisher’s Exact test with Bonferroni correction. g, Box and whisker plot (bars represent 10–90 percentile, dots represent outliers) analysis of Jagged1 expression in 226 HCC patients with Notch-high (n=113 patients) and Notch-low (n=113 patients) expression.

Extended Data Fig. 7.. RPPA of the exosomal proteins collected from Huh7 cells grown on soft or stiff matrix.

Volcano plot of RPPA data displaying the pattern of protein expression in exosomes derived from Huh7 cells cultured on stiff relative to soft matrix. Significantly up- and down-regulated proteins are indicated by red and blue dots, respectively (cut-off p<0.05). All the data points were normalized for protein loading and transformed to Log2 values on X axis. Source numerical data is available in source data.

Fig. 1.. Stiff ECM promotes exosome secretion.

a, The conditioned media from Huh7 cells growing on soft (0.5 kPa) or stiff (10 kPa) matrix were collected. After depletion of cell debris and large vesicles by centrifugation, the supernatant containing exosomes was proceeded for NTA. A representative NTA analysis is shown. The X-axis represents the diameters of the vesicles and the Y-axis represents the concentration (particles/ml) the vesicles (n=3 independent captures). Values are presented as Mean ± S.E.M. b, Immunoblotting of exosome markers (HRS, Syntenin-1, Alix, Tsg101, and CD63) in the whole cell lysate (“WCL”) and purified exosomes from 10⁷ Huh7 cells on soft and stiff matrix. Grp94 (ER marker) was used as a negative control. GAPDH was used as a cell lysate loading control. Molecular weights (in kDa) are shown to the right. c, Quantification of the levels of HRS, Syntenin-1, CD63, Alix, and Tsg101. Proteins on exosomes released from cells grown on 0.5 kPa matrix was normalized as 1. Values are presented as Mean ± S.D. n=3 independent experiments. d-f, Exosomes released from the same number of cells (Huh7, Panc1, and MCF7) grown on matrix with different stiffness were quantified and the amounts of exosomes released from cells grown on 0.5 kPa matrix were normalized as 1. Values are presented as Mean ± S.D. n=3 independent experiments. See METHODS for statistical analyses of all the figures. Source numerical data and unprocessed blots are available in source data.

Fig. 2.. Akt promotes exosome secretion from cells grown on stiff matrix.

a, Heatmap of RPPA data showing the levels of phospho-Akt and its downstream signaling proteins p-4E-BP1 and pGSK-3 in Huh7 cells grown on soft vs. stiff matrix. b, Exosomes from Huh7 cells expressing GFP control or myr-Akt on soft or stiff matrix were analyzed by NTA. Exosomes from cells expressing GFP on soft matrix was normalized to 1. Relative values are presented as Mean ± S.D., n=3.c, Huh7 cells grown on soft or stiff matrix were treated with DMSO or Akt inhibitor MK-2206. Exosome concentration from the cells treated with DMSO on soft matrix was normalized to 1. Values are presented as Mean ± S.D., n=3. d, Exosomes were purified from the media of equal amounts of Huh7 cells treated with DMSO or MK-2206. The levels of HRS, Syntenin-1, and CD63 in whole cell lysates (“WCL”) and purified exosomes were examined by immunoblotting. Total Akt (t-Akt) and p-Akt in WCL was also examined. GAPDH was used as WCL loading control. e, Quantification of the levels of HRS, Syntenin-1, and CD63. Values are presented as Mean ± S.D. n=3. f, Huh7 cells were treated with MK-2206 and immunostained for CD63 and LAMP1. Scale Bar: 10 μm. Cell peripherals were indicated with dashed lines and the perinuclear region was indicated with solid lines. g, Quantification of the percentage of perinuclear CD63 or LAMP1. Values are presented as Mean ± S.D. n=3. h, An equal amount of Huh7 cells grown on stiff matrix were treated with FAK inhibitor PND-1168, and exosomes in the conditioned media were purified. Exosome markers (HRS, Syntenin-1 and CD63) were examined. The levels of t-Akt and p-Akt in cell lysates were also examined. i, Quantification of the levels of HRS, Syntenin-1 and CD63. Mean ± S.D. n=3. j, Huh7 cells growing on soft or stiff matrix were treated with FAK inhibitor PND-1168. Exosome concentration from cells treated with DMSO on soft matrix was normalized to 1. Values are presented as Mean ± S.D. n=3. Source numerical data and unprocessed blots are available in source data. n represents the number of independent experiments.

Fig. 3.. Rab8 regulates stiff ECM-mediated exosome secretion.

a, The same amounts of lysates from cells grown on 0.5 kPa or 10 kPa matrix were incubated with GST-JFC1 RBD fusion protein. Rab8 and Rab27 in cell lysates or the activated form of Rab8 and Rab27 bound to GST-JFC1 RBD (“GTP-Rab8” and “GTP-Rab27” pulled down) were analyzed by immunoblotting. b, GTP-Rab8 quantified by ImageJ and normalized to levels for cells on soft matrix. Values are presented as Mean ± S.D., n=3. c, Cells grown on stiff matrix were treated with DMSO or MK-2206, and lysed for GST-JFC1 RBD pulldown. Proteins were analyzed by western blotting. d, GTP-Rab8 and GTP-Rab27 were quantified by Image J and normalized to levels of cells treated with DMSO. Values are presented as Mean ± S.D., n=3. e, Huh7 cells expressing GFP or GFP-Rab8 were grown on soft or stiff matrix. The conditioned media were collected for NTA. Exosome concentrations were normalized to those from cells expressing GFP on soft matrix. Values are presented as Mean ± S.D., n=3. f, Conditioned media from Huh7 cells transfected with control shRNA (“shScramble”) or Rab8 shRNA on soft or stiff matrix were collected for NTA. The concentrations of exosomes were normalized to those from cells on soft matrix with control shRNA. Values are presented as Mean ± S.D., n=3. g, Exosomes in the conditioned media of Huh7 cells grown on stiff matrix with or without Rab8 knockdown were purified by ultracentrifugation, and the exosomes from the same number of cells were loaded for immunoblotting for HRS, Syntenin-1, and CD63. h, Quantification of the levels of HRS, Syntenin-1, and CD63. Values are presented as Mean ± S.D. n=3. i, Huh7 cells were treated with control or Rab8 shRNA, and then immunostained for CD63 and LAMP1. Nuclei were stained with DAPI. Rab8 knockdown led to the clustering of CD63 and LAMP1 to the perinuclear region (solid circle). Scale Bar: 10 μm. j, Quantification of the percentage of perinuclear CD63 and LAMP1 signals. Values are presented as Mean ± S.D. n=3. Source numerical data and unprocessed blots are available in source data. n represents the number of independent experiments.

Fig. 4.. Rabin8 is phosphorylated and activated by Akt in cells grown on stiff ECM.

a, Huh7 cells grown on stiff matrix were transfected with control or Rabin8 shRNA. Exosomes in cell media were purified by ultracentrifugation, and the exosomes from the same number of cells were loaded for immunoblotting with antibodies against HRS, CD63, and Syntenin-1. b, Exosomal HRS, CD63, and Syntenin-1 were quantified. Values are presented as Mean ± S.D. n=3. c, Conditioned media from cells treated with control or Rabin8 shRNA on soft and stiff matrix were collected for NTA. Exosome concentrations were normalized to those from cells transfected with control shRNA on soft matrix. Values are presented as Mean ± S.D., n=3. d, Flag-tagged wild-type Rabin8 (WT) or the phospho-deficient mutant Rabin8 (S149A) were co-expressed with myr-Akt or control vector into cells. Flag-Rabin8 was isolated with anti-Flag M2 beads. The phosphorylation of Rabin8 by Akt was examined by phospho-Akt substrate antibody. e, Purified GST-Rabin8 was incubated with or without purified myr-Akt. in vitro phosphorylation of Rabin8 was examined by an Akt phospho-substrate antibody. GST-Rabin8 proteins inputs were shown by Ponceau S. staining. f, Huh7 cells expressing Flag-Rabin8 were grown on soft or stiff matrix. Flag-Rabin8 was pulled down by anti-Flag M2 beads, and its phosphorylation was examined by Akt phospho-substrate antibody. Treatment of cells with MK-2206 inhibited Rabin8 phosphorylation. g, The exchange of GDP-BODIPY bound to Rab8 catalyzed by the wild-type Rabin8, phospho-deficient Rabin8 mutant (S149A), and phospho-mimetic Rabin8 mutant (S149D) was measured. h, Quantification of GEF activities at 7 mins. Values are presented as Mean ± S.D., n=3. Coomassie blue staining shows the protein samples used in the GEF exchange assay. i, Purified Rabin8-NLHT proteins were first incubated with active Akt with or without ATP and then used in the BRET assay. BRET ratios (620nm/460nm) compared. Values are presented as Mean ± S.D., n=3. Immunoblotting of Rabin8 proteins used in BRET assay and their phosphorylation in the presence of ATP were shown below. Source numerical data and unprocessed blots are available in source data. n represents the number of independent experiments.

Fig. 5.. Exosomes induced by stiff matrix promote tumor growth.

a, Growth curves of Hepa1-6 tumors in C57L/J mice injected with PBS and indicated exosomes (n=5). Values are presented as Mean ± S.D. A diagram indicating the time points of exosome infusion is shown at the top. b, The weights of Hepa1-6 tumors in C57L/J mice after treatment with PBS, Exo^soft or Exo^stiff (n=5). Values are presented as Mean ± S.D. c, Immunohistochemistry of Ki67, PCNA and cleaved Caspase3 on xenograft of mice treated with Exo^soft or Exo^stiff. d, Quantification of cells stained positive with these marker proteins were shown to the right. Scale Bar: 100μm. For all figures above, values are presented as Mean ± S.D., n=5. Source numerical data are available in source data. n represents the number of animals.

Fig. 6.. Notch signaling is activated in cells grown on stiffness ECM or treated with Exo^stiff.

a, Huh7 cells grown on 0.5 or 10 kPa matrix were lysed and the cell lysates were subject to immunoblotting for Notch signaling proteins Sox9, c-Myc, Hes1, and Jagged1. GAPDH was used as a loading control. Molecular weights (in kDa) are shown to the right. b, Huh7 cells grown on the soft matrix were treated with Exo^soft or Exo^stiff. Cell lysates were subjected to immunoblotting with antibodies against Notch pathway proteins. GAPDH was used as a loading control. c, Immunohistochemistry of tumors from mice treated with Exo^soft or Exo^stiff. Tumor tissues were harvested at Day 27 and stained with H&E, Sox9, or Hes1. Scale Bar: 100μm. The percentages of Sox9 and Hes1-positive cells in mouse xenografts were shown at the right. Values are presented as Mean ± S.D., n=5. Source numerical data and unprocessed blots are available in source data. n represents the number of animals.

Fig. 7.. Jagged1 is enriched in Exo^stiff.

a, b, Exosomes were purified from the conditioned media of Huh7 (a) or Hepa1-6 cells (b) on soft or stiff matrix. The same amounts of exosomes were loaded for immunoblotting of Jagged1, Syntenin-1, HRS, CD63, Tsg101. The quantification is shown at the right. (n=3 independent experiments) c, Jagged1 in Huh7 cells grown on soft and stiff matrix were co-immunostained with antibodies against CD63 or Flag-tagged HRS for fluorescence imaging. Scale Bar: 10μm. Zoom-in view Scale bar: 1μm.d, Pearson’s correlation coefficient of Jagged1 colocalized with CD63 (n=15) or HRS (n=17). n represents the number of cells. e, HA-tagged Jagged1 was co-transfected with Flag-tagged HRS or vector into cells. The cell lysates were subjected to immunoprecipitation with anti-Flag M2 beads. Isotype IgG and anti-Myc epitope antibodies were used as negative controls. For all figures above, values are presented as Mean ± S.D. Source numerical data and unprocessed blots are available in source data.