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Extracellular vesicle tetraspanin-8 level predicts distant metastasis in non–small cell lung cancer after concurrent chemoradiation

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Science Advances  11 Mar 2020:
Vol. 6, no. 11, eaaz6162
DOI: 10.1126/sciadv.aaz6162
  • Fig. 1 Characterization of 393P and 344SQ cell phenotype and EV protein expression.

    (A) Representative images and (B) graph of the migration and invasion phenotypes of the murine 393P (nonmetastatic) and 344SQ (metastatic) NSCLC cell lines. Scale bar, 100 μm. Data are means ± SEM from three independent experiments; ns, not significant; **P < 0.01. (C) Western blot of EV markers TSG101, HSP70, and CD9 and the Golgi (cytosol) marker GM130 in EVs or whole-cell lysates (WCLs) of 393P and 344SQ cells. (D) Coomassie-stained SDS-PAGE of 393P or 344SQ cell EV isolates; IntDen, relative mean and SD of the integrated lane densitometry from three replicates. N/A, not applicable. (E) Venn diagram of EV proteins identified by LC-MS/MS. (F) Western blot of proteins in EVs, WCLs, and EV-depleted medium. BP, binding protein. (G) Heat map of 393P versus 344SQ EV Western blot expression from low (light red) to high (dark red) optical density.

  • Fig. 2 Tspan8 is selectively recruited to 344SQ EVs by ITSN2.

    (A) Representative Western blots and (B) quantification of 393P and 344SQ WCL expression of ITSN2, CD49d, Tspan8, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (C) Representative Western blot and (D) quantification of Western blots of Tspan8, ITSN2, TSG101, HSP70, and GAPDH expression in EVs and WCLs of 393P and 393P cells overexpressing ITSN2 (393PItsn2+). Data are means ± SEM from three independent experiments. **P < 0.01.

  • Fig. 3 EV-Tspan8 promotes invasion of mouse 393P and human A549 cells.

    Migration and invasion of (A) 393P cells treated with 393P or 393PItsn2+ EVs (50 μg/ml) for 24 hours and (B) A549 cells treated with A549 or A549ITSN2+ EVs (50 μg/ml) for 24 hours. Scale bars, 100 μm. Representative images and percentage of invasion to migration of (C) 393P and (D) A549 cells in response to increasing concentrations of 393PItsn2+ EVs or A549ITSN2+ EVs. Data indicate means ± SEM from three replicate experiments. Scale bars, 100 μm. ***P < 0.005; ****P < 0.001.

  • Fig. 4 EV-Tspan8 level correlates with distant metastasis in patients with NSCLC.

    Kaplan-Meier plots of freedom from distant metastasis (DM) in 106 patients with stage III NSCLC segregated by Tspan8Low and Tspan8High serum levels (Tspan8 cutoff = 0.08), with P values calculated with log-rank tests. NR, not reached; m, months.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaaz6162/DC1

    Fig. S1. Schematic of the procedures for isolating EVs from conditioned cell culture medium.

    Fig. S2. Characterization of EVs isolated from murine NSCLC cell lines.

    Fig. S3. Gene Ontology analysis of proteins up-regulated or uniquely detected in EVs from 344SQ cells.

    Fig. S4. Tspan8 enriched in EVs derived from A549ITSN2+ cells.

    Fig. S5. Schematic of the procedures for analyzing the influence of EV-Tspan8 on cell metastasis.

    Fig. S6. EV-Tspan8 induced invasion in a concentration-dependent manner.

    Table S1. Patient and treatment characteristics.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Schematic of the procedures for isolating EVs from conditioned cell culture medium.
    • Fig. S2. Characterization of EVs isolated from murine NSCLC cell lines.
    • Fig. S3. Gene Ontology analysis of proteins up-regulated or uniquely detected in EVs from 344SQ cells.
    • Fig. S4. Tspan8 enriched in EVs derived from A549ITSN2+ cells.
    • Fig. S5. Schematic of the procedures for analyzing the influence of EV-Tspan8 on cell metastasis.
    • Fig. S6. EV-Tspan8 induced invasion in a concentration-dependent manner.
    • Table S1. Patient and treatment characteristics.

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