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

Abstract

Non-small cell lung cancer (NSCLC) is the most commonly diagnosed cancer and the leading cause of cancer death worldwide. More than half of patients with NSCLC die after developing distant metastases, so rapid, minimally invasive prognostic biomarkers are needed to reduce mortality. We used proteomics to identify proteins differentially expressed on extracellular vesicles (EVs) of nonmetastatic 393P and metastatic 344SQ NSCLC cell lines and found that tetraspanin-8 (Tspan8) was selectively enriched on 344SQ EVs. NSCLC cell lines treated with EVs overexpressing Tspan8 also exhibited increased Matrigel invasion. Elevated Tspan8 expression on serum EVs of individuals with stage III premetastatic NSCLC tumors was also associated with reduced distant metastasis-free survival, suggesting that Tspan8 levels on serum EVs may predict future metastasis. This result suggests that a minimally invasive blood test to analyze EV expression of Tspan8 may be of potential value to guide therapeutic decisions for patients with NSCLC and merits further study.

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 (393P^Itsn2+). 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 393P^Itsn2+ EVs (50 μg/ml) for 24 hours and (B) A549 cells treated with A549 or A549^ITSN2+ 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 393P^Itsn2+ EVs or A549^ITSN2+ 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 Tspan8_Low and Tspan8_High serum levels (Tspan8 cutoff = 0.08), with P values calculated with log-rank tests. NR, not reached; m, months.