Extracellular vesicles modulate integrin signaling and subcellular energetics to promote pulmonary lymphangioleiomyomatosis metastasis

Figures

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  Fig. 1. LAM EVs have modified cargo compared to normal EVs. a, b Immunoblots of EVs from plasma of LAM patients (LAM EVs) (n = 5) and healthy donors (Normal EVs) (n = 5). a EV-associated proteins and JEV controls. b Targeted analysis of EV cargo. The same EV-associated protein controls were used in panels (a, b), as data were split between these panels. c–h Proteome of LAM EVs (n = 7) and Normal EVs (n = 5). c Protein expression pattern in different sample groups by sample cluster analysis. d Hierarchical clustering heat map of differentially expressed proteins in EVs. e Volcano plot. f Top 40 enriched pathways by KEGG. g Circular bar plot showcasing protein dataset of enriched pathways in LAM EVs vs. Normal EVs. h Nodes of significant pathway relationships.

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  Fig. 2. Loss of TSC1/2 increases EV biogenesis. a Immunoblots of EVs isolated from conditioning media of 621-101 and TSC2 addback cells. (a1) Representative immunoblots of EV-associated proteins and JEV controls. (a2) Quantification of (a1), relative expression by densitometry (n = 3). b, c Immunofluorescence of adherent 621-101 cells expressing pHluo_M153-CD63-mScarlet (n = 3). (b1) Red fluorescence indicates CD63 MVB expression. (b2) Quantification of (b1). (c1) Green fluorescence indicates CD63+ EVs deposited on ECM. (c2) Quantification of (c1) (n = 3). d Western immunoblotting of EVs isolated from conditioning media of 621-101 and TSC2 addback cells. (d1) Representative immunoblots of targeted EV protein expression and JEV controls. (d2) Quantification of (d1), relative expression by densitometry (n = 3). The same EV-associated protein controls were used in panels (a1) and (d1) as data were split between these panels. (1–2 SEC fractions 5 and 2, respectively; 3-conditioned media, 4- EV-depleted media). Scatter plots with bars showcase mean ± SEM. Two-tailed unpaired t test. *P < 0.05, **P < 0.01, ****P < 0.0001.

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  Fig. 3. TSC-null EVs enhance CSCs and metastable phenotypes of 621-101 cells. a, b Sphere assay (n = 3). a Primary and b secondary 621-101 spheres exposed to indicated EV subtypes. (a1, b1) Representative images. (a2, b2) Quantification of (a1) and (b2) (n = 3) (one-way ANOVA). c, d BrdU by immunofluorescence (c) and ALDEFLUOR assay by FACS (d) in 621-101 sphere cells exposed to indicated EV subtypes. (c1) Representative images. (c2) Quantification of (c1) (n = 3) (one-way ANOVA). Images are from a single Z stack of 1 µm, respectively. (d1) Gating strategy and FACS analysis. (d2) Absolute count of ALDH-positive cells (n = 5 for PBS, n = 3 for all remaining) (one-way ANOVA). e, f Transwell (e) migration (n = 4) and (f) invasion (n = 3) assay of 621-101 spheres exposed to indicated EV subtypes. (e1, f1) Representative images and (e2, f2) quantification of (e1) and (f1) as a fold change in number of cells relative to PBS (one-way ANOVA). g–i 621-101 spheres exposed to indicated EV subtypes, or not to, in the presence of DMSO; or (g, h) EV uptake inhibitor, Dyngo4a (10 µM); or (i) c-Src inhibitor, Bosutinib (1 µM). (g1-i1) Representative images. (g2-i2) Quantification of (g1-i1) (n = 3)…

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  Fig. 4. Tumor TSC-null EVs promote pseudopodia-localized ATP synthesis to increase TSC-null sphere cell migration. a–g RNA-Seq analysis of 621-101 spheres exposed to tumor TSC-null EVs in comparison to TSC2 EVs (n = 3). a Volcano plot representing downregulated and upregulated genes in 621-101 spheres exposed to tumor TSC-null EVs, in comparison to TSC2 EVs. b–d KEGG pathway and e–g GO analyses for upregulated genes in 621-101 spheres exposed to tumor TSC-null EVs in comparison to TSC2 EVs. Top enriched pathways (b), BP, CC, and MF (e). Circular bar plot containing genes with enriched pathways (c), BP and CC (f). d, g Nodes of significant pathway relationships. h OXPHOS and mitochondria inner membrane related genes by RT-qPCR (n = 3) (two-tailed unpaired t test). i Cellular ATP level in 621-101 spheres exposed to indicated EV (n = 4) (two-tailed unpaired t test). j, k Immunoblots of (j) tumor TSC-null EVs, compared to TSC2 EVs (n = 3); and (k) LAM EV, compared to Normal EV (n = 5). (j1, k1) Representative images. The same EV-associated protein control from panel (k1) was also used in Figs. (1a, b, as data were split between these panels. (j2, k2) Quantification of (j1) and (k1), relative…

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  Fig. 5. Metastasis TSC-null EVs promote IAC formation, thereby increasing sphere cell migration. a–g RNA-Seq analysis of 621-101 spheres exposed to metastasis TSC-null EVs, in comparison to TSC2 EVs (n = 3). a Volcano plot representing downregulated and upregulated genes in 621-101 spheres exposed to metastasis TSC-null EVs, in comparison to TSC2 EVs. b–d GO and (e–g) Reactome analyses for upregulated genes in 621-101 spheres exposed to metastasis TSC-null EVs, in comparison to TSC2 EVs. Top enriched BP, CC, and MF (b), and significant pathways (e). Circular bar plot containing genes with enriched BP and CC (c), and significant pathways (f). d, g Nodes of significant BP, CC, and pathway relationships. h ECM receptor related genes by RT-qPCR (n = 3) (one-tailed unpaired t test). i Immunoblot of 621-101 spheres exposed to PBS or metastasis EVs (n = 3). j, k Immunoblot of (j) IAC isolated from 621-101 spheres exposed to indicated EVs, or (k) migrated spheres exposed to indicated EVs. (j1, k) Representative images. (j2, j3) Quantification of (j1), relative expression by densitometry (n = 3) (unpaired t test). l, m IAC protein expression in (l) metastasis TSC-null EVs, compared to TSC2 EVs (n…

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  Fig. 6. TSC-null EVs promote lung seeding by LAM cells. a Rat DNA by qPCR. (n = 3) (two-tailed unpaired t test). b–e RNA-Seq analyses (n = 3). b Heat map. c Volcano plot. d Top enriched Reactome pathways. e Nodes of significant pathway relationships. f Expression of ECM genes by RT-qPCR. (n = 3) (two-tailed unpaired t test). g Masson-Trichrome stain for collagen (blue). h Expression of S100A4 by (h1) immunohistochemistry and (h2) RT-qPCR (n = 3) (two-tailed unpaired t test). i In vivo bioluminescent reporter imaging. (i1) Bioluminescence imaging of the lungs from mice injected i.v. with 621-L9 cells, 72 h. after i.v. injection of 621L9-plasma EVs, TSC2-plasma EVs, or controls. (i2) Quantification of relative luciferase unit at different time points, normalized to time zero (TSC-null EVs n = 6, TSC2 EVs n = 5, TSC-null EV-depleted plasma [EDP] n = 4, TSC2 EDP n = 3) (two-way ANOVA, significance for “column factor”). j ECM, epithelial, and fibroblast-related genes by RT-qPCR (TSC-null EVs n = 6, TSC2 EVs n = 5 for all except for S100A4; TSC-null EVs n = 6, TSC2 EVs n = 3 for S100A4) (two-tailed unpaired t test). k Plasma FAP ELISA (TSC-null EVs n = 4, TSC2 EVs n = 5) (two-tailed unpaired t…

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  Fig. 7. Signaling triggered by distinct EV subtypes in the recipient tumor cells.