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. 2021 May;9(5):e002372.
doi: 10.1136/jitc-2021-002372.

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

Letizia Porcelli #  1 , Michele Guida #  2 , Simona De Summa  3 , Roberta Di Fonte  1 , Ivana De Risi  2 , Marianna Garofoli  1 , Mariapia Caputo  3 , Antonio Negri  4 , Sabino Strippoli  2 , Simona Serratì #  5 , Amalia Azzariti #  6
Affiliations

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

Letizia Porcelli et al. J Immunother Cancer. 2021 May.

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.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
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
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
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
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.
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