Alix-normalized exosomal programmed death-ligand 1 analysis in urine enables precision monitoring of urothelial cancer

Abstract

Anti-programmed death-ligand 1 (PD-L1) Ab-based therapies have demonstrated potential for treating metastatic urothelial cancer with high PD-L1 expression. Urinary exosomes are promising biomarkers for liquid biopsy, but urine's high variability requires normalization for accurate analysis. This study proposes using the PD-L1/Alix ratio to normalize exosomal PD-L1 signal intensity with Alix, an internal exosomal protein less susceptible to heterogeneity concerns than surface protein markers. Extracellular vesicles were isolated using ExoDisc and characterized using various methods, including ExoView to analyze tetraspanins, PD-L1, and Alix on individual exosomes. On-disc ELISA was used to evaluate PD-L1 and Alix-normalized PD-L1 in 15 urothelial cancer patients during the initial treatment cycle with Tecentriq. Results showed that Alix signal range was relatively uniform, whereas tetraspanin marker intensity varied for individual exosome particles. On-disc ELISA was more reliable for detecting exosomal PD-L1 expression than standard plate ELISA-based measurement. Using exosomal Alix expression for normalization is a more reliable approach than conventional methods for monitoring patient status. Overall, the study provides a practical and reliable method for detecting exosomal PD-L1 in urine samples from patients with urothelial cancer.

Keywords: PD‐L1; exosome; normalization; urine; urothelial cancer.

FIGURE 1

Normalization method using exosomal Alix expression. (A) Exosomes (20–200 nm) are secreted from urothelial cancer (UC) and exist in urine in different numbers depending on the condition of the patient. (B) Exosomes were isolated and detected in scanning electron microscope (SEM) and transmission electron microscope (TEM) images. Scale bar, 50 nm. (C) Internal markers such as Alix may have less space for Ab binding compared to surface markers. (D) Intensity analysis of single exosomes for their surface tetraspanin markers (e.g. CD9, CD63, and CD81) and Alix. The intensity of tetraspanin markers (CD9, CD63, and CD81) of particles, isolated from T24 CCS using ExoDisc, attached on the surface coated with CD9, CD63, and CD81 showed varying intensity ranges, whereas Alix showed a uniform intensity range. One‐way AVOVA test was used for statistical analysis. *p < 0.05; **p < 0.005; ****p < 0.0001; A.U., arbitrary unit; N.S., not significant; PD‐L1, programmed death‐ligand 1.

FIGURE 2

Exosomal marker correlation with programmed death‐ligand 1 (PD‐L1) to identify a normalizing marker for PD‐L1. (A) Exosomes were isolated from cell culture supernatant (CCS) or urine by using ExoDisc. Following isolation, exosomes were labeled with individual Abs of tetraspanins (CD9, CD63, or CD81), PD‐L1, or Alix on the filter chamber of the disc, and their expression levels were quantified. (B) Exosomes isolated from different volumes of A549‐ and UMUC3‐CCS solutions were quantified for PD‐L1 expression using on‐disc ELISA. (C, D) On‐disc ELISA results show CD9, CD63, CD81, Alix, and PD‐L1 expression levels in exosomes isolated from (C) 2 mL of A549‐ and UMUC3‐CCS solution, as well as (D) 250 μL of urine from healthy donors (HD) and patients with urothelial cancer (UC). Error is denoted as SD. (E) PD‐L1 correlation tests for the clinical samples (HD, n = 5; UC, n = 5). There was a weak positive correlation between PD‐L1 and CD9, with Pearson's r value of 0.13. However, no significant correlation was observed between PD‐L1 and CD63 (Pearson's r = 0.014) or CD81 (Pearson's r = −0.17). In contrast, Alix showed a strong positive correlation with PD‐L1, with a Pearson's r value of 0.95. Pearson's r value for the correlation analysis were provided by Prism software. A.A., arbitrary amount; A.U., arbitrary unit; conc., concentration; OD, optical density.

FIGURE 3

Exosomal programmed death‐ligand 1 (PD‐L1) and Alix expression were quantified from extracellular vesicles isolated from clinical samples (healthy donors [HD], n = 12; urothelial cancer patients [UC], n = 26). (A) Exosomes in 1 mL urine were isolated using ExoDisc, and their exosomal PD‐L1 and Alix expression levels were analyzed with the corresponding particle number measured by nanoparticle tracking analysis (NTA). Relative quantification was calculated by standard curves and marked as an arbitrary amount (A.A.). (B) Correlation analysis between PD‐L1 and Alix expression shows a strong positive correlation with a Pearson's correlation coefficient of 0.96. (C) Pearson's correlation coefficient of 0.28 indicates a weak positive correlation between PD‐L1 and the number of particles measured by NTA. conc., concentration.

FIGURE 4

During the initial treatment cycle of Tecentriq in 15 patients with urothelial cancer, programmed death‐ligand 1 (PD‐L1), Alix, and the PD‐L1/Alix ratio of exosomes were quantified. Tumor size changes were observed for all patients. (A) Of the 15 patients, six showed stable disease from December 17, 2019 to the present (responder [R]1), from July 22, 2020 to the present (R2), from August 19, 2020 to the present (R3), from November 9, 2020 to the present (R4), from August 14, 2020 to the present (R5), and from July 16, 2020 to the present (R6). (B) Of the 15 patients, nine experienced recurrence (nonresponder [NR]1), progressive disease (NR4, NR5, NR6, NR8, and NR9), or cancer‐related death (NR2, NR3, and NR7 (computed tomography [CT] image not available due to cancer‐related death before obtaining follow‐up CT). During the first cycle of Tecentriq treatment, responders showed nonsignificant (N.S.) changes in the concentrations of PD‐L1 and Alix, while the PD‐L1/Alix ratio increased (*). Nonresponders showed an opposite trend of PD‐L1/Alix ratio (*) during the same period. (C) Representative CT image of responders (R2) showing the slightly reduced tumor size from 33.2 mm to 32.1 mm after 3 months. (D) Representative CT image of nonresponders (NR3) showing the increased tumor size from 47.9 to 73.5 mm after 1 month. Wilcoxon test was used for statistical analysis. *p < 0.05; A.A., arbitrary amount; N.S., not significant; conc., concentration; F/U, follow‐up.