Isolation and characterization of extracellular vesicles from EGFR mutated lung cancer cells

Abstract

The epidermal growth factor receptor (EGFR) signaling pathway is essential for cellular processes such as proliferation, survival, and migration. Dysregulation of EGFR signaling is frequently observed in non-small cell lung cancer (NSCLC) and is associated with poor prognosis. This study aims to isolate and characterize extracellular vesicles (EVs) released by mutant EGFR lung cancer cell line PC9 and compare them with wild-type EGFR lung cancer cell line A549, while also evaluating the effect of gefitinib treatment on EV secretion and cargo composition. The two lung cancer cell lines were cultured with 2% EV-free serum, and EVs were subsequently isolated by differential ultra centrifugation. EVs were characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) for quantification size and shape determination. Western blot analysis confirmed the enrichment and purity of isolated EVs. Results showed that EGFR mutation significantly increased EV release and altered their size, compared to EVs released by wild-type EGFR cells. In addition to classical EV markers such as CD81, Flotillin- 1, and TSG101, Western blot analysis also detected phosphorylated EGFR (p-EGFR) selectively packaged into EVs from PC9 cells. Gefitinib treatment significantly reduced EV secretion in PC9 cells and led to a marked decrease in p-EGFR incorporation into EVs, indicating that EV biogenesis and compostion are modulated by active EGFR signaling. In conclusion, this study highlights the significant influence of EGFR activation on EV secretion and cargo composition while demonstrating that EGFR inhibition via gefitinib alters EV-mediated signaling in lung cancer cells. These findings provide insights into tumor behavior, EV-mediated oncogenic communication, and the potential use of EVs as biomarkers and therapeutic targets in NSCLC.

Keywords: EGFR; Extracellular vesicles; Lung cancer.

Fig. 1

Isolation of EVs by ultracentrifugation. The isolation of EVs by ultracentrifugation involves several key steps. First, cell culture supernatant is collected and subjected to a series of centrifugation steps to remove cellular debris and large particles. Next, the clarified supernatant undergoes ultracentrifugation at high speeds, to pellet EVs. The pellet containing EVs is then carefully resuspended and washed with buffer to further remove contaminants. Finally, the isolated EVs can be resuspended in an appropriate buffer or media without FCS for downstream analyses, such as characterization or functional studies

Fig. 2

Detection of the activation status of EGFR signaling in A549 and PC9 cells by Western blot analysis. Immunoblots were incubated with EGFR, p-EGFR, AKT, p-AKT, ERK1/2 and p-ERK1/2. Phosphorylation of p-EGFR, p-AKT, and p-ERK1/2 was detected in PC9 cells (middle blot) but not in A549 cells (left blot). PC9 cells treated with 200 nM gefitinib for 24 h significantly inhibited p-EGFR and its downstream signaling (right blot). Expression levels normalized to total β-actin are depicted in the right graphs. All data represent mean ± SD of three independent experiments

Fig. 3

Isolation and characterization of EVs from lung cancer cell lines. A. NTA results show the median size of EVs from both PC9 and A549 cell lines from three independent EV isolations. T test analysis reveals a significantly bigger size of PC9-EVs compared to A549. P ≤ 0.02. B. Quantitative analysis reveals a significantly higher concentration of EVs from EGFR-mutant PC9 cells compared to EGFRwt A549 cells. The size distribution of the isolated EVs from A549 C and PC9 D predominantly ranges between 30 and 200 nm, consistent with typical EV sizes. TEM analyses revealed a majority of EVs from A549 E and PC9 F are cup-shaped morphology ranging from70 to 150 nm in size. C-F representative data from one of three independent experiments.

Fig. 4

Identification of EV markers in isolated EVs. Western blot analysis of EVs derived from A549 and PC9 cells. The EV markers CD81, Flotillin- 1, and TSG101 were detected in EV samples (left blot), confirming the presence of EV-associated proteins. Calnexin, an endoplasmic reticulum (ER) marker, was absent in the EV samples but present in whole-cell lysates, indicating the successful isolation of pure EVs without significant cellular contamination

Fig. 5

p-EGFR is selectively packaged into EVs from PC9 cells. Western blot analysis of EVs isolated from A549 (EGFRwt) and PC9 (EGFR-mutant) cells. p-EGFR was prominently detected in EVs from PC9 cells (right blot), whereas it was undetectable in A549-derived EVs (left blot), consistent with the constitutive activation of EGFR in PC9 cells. Total EGFR was present in EVs from both cell lines, confirming the incorporation of EGFR in EVs. Calnexin was absent in all EV samples, indicating the purity of the isolated EVs

Fig. 6

Effect of gefitinib treatment on EV size and secretion. A NTA showing the size distribution of EVs derived from A549 and PC9 cells, both with and without 20 nM gefitinib treatment for 24 h. No significant differences were observed in EV size across all conditions. B Gefitinib treatment significantly reduced the number of EVs secreted by PC9 cells (~ 47% decrease, p < 0.01), whereas it had no significant effect on A549-derived EVs. Error bars represent mean ± standard deviation (SD). Statistical significance: ns = not significant, ** p < 0.01

Fig. 7

Gefitinib inhibits p-EGFR expression in EVs. Western blot analysis of EVs isolated from A549 and PC9 cells, both treated with 200 μM gefitinib for 24 h. p-EGFR levels were significantly reduced in gefitinib-treated PC9-derived EVs, confirming the inhibition of EGFR signaling. Total EGFR was present in EVs from both cell lines, indicating its incorporation into EVs independent of phosphorylation. Calnexin was absent in all EV samples, confirming the purity of the isolated EVs and ruling out cellular contamination