uPAR+ extracellular vesicles: a robust biomarker of resistance to checkpoint inhibitor immunotherapy in metastatic melanoma patients

Abstract

Background: Emerging evidence has highlighted the importance of extracellular vesicle (EV)-based biomarkers of resistance to immunotherapy with checkpoint inhibitors in metastatic melanoma. Considering the tumor-promoting implications of urokinase-type plasminogen activator receptor (uPAR) signaling, this study aimed to assess uPAR expression in the plasma-derived EVs of patients with metastatic melanoma to determine its potential correlation with clinical outcomes.

Methods: Blood samples from 71 patients with metastatic melanoma were collected before initiating immunotherapy. Tumor-derived and immune cell-derived EVs were isolated and analyzed to assess the relative percentage of uPAR⁺ EVs. The associations between uPAR and clinical outcomes, sex, BRAF status, baseline lactate dehydrogenase levels and number of metastatic sites were assessed.

Results: Responders had a significantly lower percentage of tumor-derived, dendritic cell (DC)-derived and CD8⁺ T cell-derived uPAR +EVs at baseline than non-responders. The Kaplan-Meier survival curves for the uPAR⁺EV quartiles indicated that higher levels of melanoma-derived uPAR⁺ EVs were strongly correlated with poorer progression-free survival (p<0.0001) and overall survival (p<0.0001). We also found a statistically significant correlation between lower levels of uPAR⁺ EVs from both CD8⁺ T cells and DCs and better survival.

Conclusions: Our results indicate that higher levels of tumor-derived, DC-derived and CD8⁺ T cell-derived uPAR⁺ EVs in non-responders may represent a new biomarker of innate resistance to immunotherapy with checkpoint inhibitors. Moreover, uPAR⁺ EVs represent a new potential target for future therapeutic approaches.

Keywords: Biomarkers; CD8-Positive T-Lymphocytes; Dendritic Cells; Immunotherapy; Melanoma; Tumor.

Figure 1

EVs characterization by NTA and FCM analysis. (A) Representative NTA histograms reporting size and concentration of EVs purified from plasma samples at basal level of 3 MM patients. Scatter plots with median showing the percentage of (B) CD9⁺EVs, CD81⁺EVs and CD63⁺EVs and (C) double positive CD9⁺/CD81⁺ EVs, CD9⁺/CD63⁺ EVs, and CD81⁺/CD63⁺ EVs in 71 patients analyzed by FCM. (D) Scatter plot with median showing the percentage of uPAR⁺ EVs derived from melanoma cells (CD146⁺), T cells (CD8⁺) and B cells (CD19⁺), monocytes (CD14⁺) and DC (CD1a⁺) in responders (n=38) and non-responders (n=33). (E) Scatter plot of PDL-1⁺ EVs population in responders (n=38) and non-responders (n=33) and (F) of the sum of PDL-1⁺ EVs originating from melanoma cells, CD8⁺ T cells, B cells, monocytes and DCs in responders (n=190) and non-responders (n=165) (**p<0.01, ***p<0.001). DC, dendritic cell; EVs, extracellular vesicle; FCM, flow cytometry; NTA, nanoparticle tracking analysis.

Figure 2

PBMCs characterization by FCM analysis. Scatter plots with medians showing the percentage of immune cell populations (A), uPAR⁺ immune cells (B) and uPAR⁺ EVs derived from PBMC (C) in responders and non-responders obtained by FCM analysis (** p<0.01). EVs, extracellular vesicle; FCM, flow cytometry; uPAR, urokinase-type plasminogen activator receptor.

Figure 3

uPAR⁺ EVs derived from different cell types released in the plasma of patients clustered by response to therapy. (A) Scatter plot with median showing the percentage of uPAR⁺ EVs released in the plasma of responders (n=38) and non-responders (n=33). (B). Scatter plots with medians showing the FCM analysis of uPAR⁺ EVs (%) derived from melanoma cells and immune cells of non-responders (n=33) and responders (n=38) and (C) showing the sum uPAR⁺ EVs released from melanoma cells, CD8 +T cells and DCs of responders (n=114) and non-responders (n=99) (***p<0.001). DC, dendritic cell; EVs, extracellular vesicle; uPAR, urokinase-type plasminogen activator receptor.

Figure 4

Evaluation of PFS and OS in patients with uPAR⁺EVs derived from melanoma cells and immune cells. Kaplan-Meier survival curve analysis according to uPAR⁺ EVs quartiles, with uPAR⁺EVs from melanoma cells, CD8⁺ T cells and DCs as respect to PFS (A) and OS (B). For each analysis, a pairwise comparison of curves has been performed. P values are shown in tables below the graph. The distribution of the best responses stratifying patients by quartiles of uPAR⁺EVs from melanoma cells (C), CD8⁺ T cells (D) and DCs (E), respectively. (F) Multivariate Cox hazard regression analysis for OS. DC, dendritic cell; EVs, extracellular vesicles; OS, overall survival; PFS, progression-free survival; uPAR, urokinase-type plasminogen activator receptor.