Serum and exosome WNT5A levels as biomarkers in non-small cell lung cancer

Abstract

Background: Despite significant advances in the treatment of lung cancer (LC), there are no reliable biomarkers to effectively predict therapy response and overall survival (O/S) in non-small cell lung cancer (NSCLC) subtypes. While targeted therapies have improved survival rates in lung adenocarcinoma (LUAD), effective treatment options for lung squamous cell carcinoma (LUSC) are still limited. Recent evidence indicates that exosome-bound WNT5A may significantly contribute to disease progression. Our study assessed the WNT5A protein as a potential biomarker for diagnosing patients and predicting prognosis to assist in therapy selection.

Methods: Primary tumor tissue and serum samples were collected from a cohort of 60 patients with histologically confirmed NSCLC before therapy. Healthy serum donors served as controls. Exosomes were isolated, then exosome number and size were measured, and WNT5A protein levels were identified in tissue and in vesicle-free, vesicle-bound fractions of the serum by ELISA.

Results: Extensive statistical analysis (ROC, AUC, Cox, etc.) revealed that elevated WNT5A levels on the serum-exosome surface correlated with distant metastasis, advanced disease stage, and lymph node involvement in LUSC but not in LUAD patients. Moreover, a high WNT5A exosome surface expression was associated with a poor response to therapy and shorter O/S in LUSC patients. Additionally, serum-exosome surface + cargo WNT5A content distinguished LUAD and LUSC subtypes.

Conclusions: WNT5A, particularly its serum exosome-bound form, may serve as a valuable biomarker after further validation for differentiating NSCLC subtypes and predicting disease progression. Importantly, the information can become available from a simple serum sample at the time of diagnosis.

Keywords: Exosome; Lung adenocarcinoma; Lung squamous cell carcinoma; Non-small cell lung cancer; WNT5A.

Fig. 1

Patient selection and clinical parameters. (A) Flow chart of patient selection in our prospective study; (B) Clinicopathologic parameters of NSCLC patients

Fig. 2

Characterization of serum-derived exosomes (n = 5 each sample group). A-C: Size distribution of exosomes measured by NTA. D: Serum exosome concentration (NPs/mL) in HC, LUAD, and LUSC samples as detected with NTA. E: Exosomes isolated from NSCLC patients were observed under electron microscopy with 50–150 nm in diameter (bar = 200 nm). F: Representative Exo-Check Exosome Antibody Array for detecting exosome markers (CD81, CD63, ALIX, EpCam, ANXA5, and TSG101) and assessing cellular contamination (GM130)

Fig. 3

WNT5A levels in various fractions of the serum. A: The concentration of WNT5A protein in serum, supernatant, and exosomes. B: Distribution of WNT5A within exosomes in NSCLC and HC samples. Quantitative analysis of WNT5A levels in serum (C) and supernatant (D) among HC, LUAD and LUSC patients. E: WNT5A levels pg/particle as exosome surface F: WNT5A levels pg/particle as exosome surface + cargo

Fig. 4

WNT5A levels associated with overall survival, disease stage and therapy response. A: Kaplan–Meier O/S distributions stratified by supernatant WNT5A level (cut-off: 740 pg/mL) (p = 0.0042, from log-rank test). B: Kaplan–Meier O/S distributions stratified by exosome surface + cargo WNT5A level (cut-off: 2.5 × 10^− 9 pg WNT5A/ NP) (p = 0.028, from log-rank test). WNT5A on the surface of exosomes is elevated in the LUSC subtype and associated with distant metastasis (C), Univariate Cox regression analysis (D) Multivariate Cox regression analysis (E), positive lymph node status (F) and lack of response to therapy (G). D: ROC analysis was performed to calculate sensitivities and specificities for differentiation between LUAD and LUSC metastatic patients. AUC represents the diagnostic capacity. H: WNT5A transported on the exosome surface is a suitable marker in distinguishing LUAD patients with PD from LUSC patients with a lack of therapy response. (PR, partial response; PD, progressive disease; SD, stable disease)

Fig. 5

Schematic summary diagram of WNT5A exosome characteristics in sera of LUAD and LUSC patients

Fig. 6

Schematic diagram of the predictive, prognostic and diagnostic role of peripheral WNT5A in LUAD and LUSC patients. The cut-off values are summarized for each application. (Further validation is needed to confirm that the cut-off values are not data-set dependent)