Study design and patients
Paired tumor tissue samples and blood samples as well as clinicopathologic features were obtained from 51 various malignant tumor patients (ClinicalTrials.gov, NCT02890849). Repeated blood samples from forty other locally advanced/metastatic NSCLC patients treated with anti-PD-1/anti-PD-L1 antibody therapy were collected at baseline and at two months after the first intravenous transfusion (ClinicalTrials.gov, NCT03073902). In addition, blood samples from ten healthy donors (HDs) were collected. All patients and HDs provided informed consent. All tissue samples underwent overnight fixation in 10% phosphate-buffed formalin and then were processed and embedded in paraffin blocks for further analysis. All blood samples were centrifuged for 10 minutes at 2000 × g to obtain plasma and then stored at -80°C for further analysis. This study was approved by the ethics committee of the Xinqiao Hospital of Army Medical University (2016-No.054-01, 2017-No.011-01). The best objective response (bOR) to anti-PD-1/anti-PD-L1 antibody treatment was determined by iRECIST26 and included complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). PFS was defined as the time from the first dose of ICI treatment to PD. OS was defined as the time from the first dose of ICI treatment to death for any reason.
PD-L1 IHC staining and scoring
PD-L1 IHC staining was conducted on 3 μm thick sections of formalin-fixed paraffin embedded (FFPE) tumor blocks according to the VENTANA SP142 PD-L1 immunohistochemistry assay (Ventana, AZ, USA). The score of tPD-L1 expression on both TCs and tumor-infiltrating ICs was evaluated by digital image analysis software (Aperio membrane v9 and Aperio Genie Classifier, LEICA CAMERA AG,Wetzlar,Germany). The scoring criteria used were from a previous study27 (TC3, ≥50%; TC2, 5 to < 50%; TC1, ≥1 to < 5%; TC0, <1%; IC3, ≥10%; IC2, ≥5 to < 10%; IC1, ≥1 to < 5%; and IC0, < 1%). Additionally, all patients were divided into three groups according to tPD-L1 expression (TC0/IC0, TC1~2/IC1~2 and TC3/IC3).
Measurement of plasma PD-L1 mRNA
Total RNA was extracted using TRIzol Reagent (Invitrogen, Invitrogen, CA, USA), according to the manufacturer’s instructions. After the concentration and purity of the total RNA were determined, reverse transcription was performed using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). PLACON (Supplementary Fig. 1), a self-designed novel plasma external reference, was used for amplification and comparison to detect plasma PD-L1 mRNA. The relative expression level of plasma PD-L1 mRNA in tumor patients was calculated by referring to the average expression level of plasma PD-L1 mRNA in 10 HDs samples. The formula is y=2-(ΔCTx-ΔCT0). The following primer was used: PD-LI (Forward: 5’-GCTATGGTGGTGCCGACTAC-3’, Reverse: 5’-TTGGTGGTGGTGGTCTTACC-3’).
Measurement of soluble PD-L1
Soluble PD-L1 (sPD-L1) expression in plasma was determined using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, USA), according to the manufacturer’s instructions. The expression level of each sample was calculated according to standard curves. All samples were assayed in duplicate, and the mean value of each sample was reported.
Isolation of exosomes from plasma
Stored plasma samples were thawed in a water bath at 25°C. Exosomes were isolated from 200 μL of patient plasma using a Exosome Isolation Kit (Wayen Biotechnologies, Shanghai, China), according to the manufacturer's instructions. Then, isolated exosome samples were immediately stored at -80°C until further analysis.
Verification of isolated exosomes
We randomly selected one isolated exosome sample for verification. First, the size distribution of the isolated exosomes was determined through Nanosight Tracking Analysis (NTA) by utilizing ZetaView (Particle Metrix, Germany). Second, exosome morphology was analyzed by using transmission electron microscopy (TEM) (Tecnai G2 spirit BioTwin, FEI, USA). Third, exosomal proteins were subjected to SDS-PAGE followed by Western blotting (WB). The nitrocellulose membranes were blocked with 5% nonfat milk for 60 minutes at room temperature and incubated overnight at 4°C with the corresponding primary antibodies at dilutions recommended by the suppliers, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 hour. The blots were developed with enhanced chemiluminescence (ECL) PierceTM detection reagents (Thermo Scientific). CD63, CD9, and calnexin were used as exosome markers. Finally, immunoreactive proteins were visualized using a chemiluminescence detection system (FluorChem HD2, USA).
Measurement of exoPD-L1
Exosomal PD-L1 protein was measured with a SimoaTM NF-light Reagent Kit (Quanterix Corp, Lexington, MA). In short, all isolated exosome samples were loaded at a mass of 280 μg and then diluted with sample diluent to 130 μL for single-well detection. Standard samples were added to a 96-well plate. After the completion of the sample preparation, beads, detector, and SBG were loaded into the reagent holder, and RGP was loaded into the tube holder. Then, the sample was transferred to the Simoa Disc, using oil to seal the sample so that the signal was only in the well. Finally, pictures were taken, and the concentration was analyzed on a Simoa HD-1 platform (Quanterix Corp).
Statistical analysis
The difference in PD-L1 mRNA and sPD-L1 expression in different subgroups was calculated by using independent-samples t-test. The difference in tPD-L1 expression and bOR in different subgroups was calculated by using Pearson’s chi-square test or Fisher’s exact test. SPSS version 23.0 (IBM, Armonk, NY, USA) was used for performing these statistical analyses. The ‘survival’ and ‘survminer’ packages from R software (version 3.5.2) were used for calculating the best cutoff point of each biomarker, conducting statistical calculations, and drawing Kaplan–Meier curves. A two-sided P value < 0.05 was considered statistically significant.