ALPPL2 Is a Potential Diagnostic Biomarker for Pancreatic Cancer-Derived Extracellular Vesicles

Abstract

Pancreatic cancer is an aggressive malignancy that often goes undiagnosed in the early stages. Non-invasive, early, and accurate diagnosis is therefore undoubtedly the "holy grail" of pancreatic cancer research. However, despite extensive research efforts, there is no definitive biomarker for this cancer. Previously, we identified alkaline phosphatase placental-like 2 (ALPPL2) as a diagnostic biomarker for pancreatic ductal adenocarcinoma and developed a 2'-fluoro modified RNA aptamer toward it. In this study, we show that ALPPL2 is present in pancreatic cancer extracellular vesicles (EVs) and therefore has potential application in liquid biopsy-based diagnostic strategies. We also developed ALPPL2 direct and sandwich aptamer-linked immobilized sorbent assay (ALISA) for EVs, which could sensitively and specifically detect the protein. We believe that our ALISA format may have a potential diagnostic utility in screening pancreatic-cancer-derived EVs.

Keywords: ALISA; ALPPL2; aptamer; exosomes; extracellular vesicles; pancreatic cancer.

Figure 1

Characterization of PDAC Cell-Derived Extracellular Vesicles (A) SEM micrograph of PANC-1⁺-derived EVs acquired at 20k (left) and 100k (right) magnifications. (B) Size distribution of EVs by DLS assay. Commercial EV preparations from COLO-1 cells were used as a control. (C) NanoSight analysis (NTA) of EVs showing size distribution and (D) particle concentrations of cell culture supernatant (1 μg/mL) without ultracentrifugation. Data is represented as mean ± SD of three technical replicates.

Figure 2

ALPPL2 Is Present in PDAC Cell-Derived EVs Immunoblot analysis of EVs extracted from the serum-free conditioned media of PANC-1⁺ and Capan-1 showed significant ALPPL2 expression, whereas MIA PaCa-2 showed no expression of the protein. 15 μg of secretome (S), 15 μg of EV-depleted secretome (X), and 2.5 μg EVs (E) were used for immunoblotting. EV markers CD9, CD63, and TSG101 confirm the presence of EVs in samples S and E.

Figure 3

Aptamer SQ2-Based Direct ALISA for Quantitative Analysis of PDAC-Derived EVs (A) Schematic illustration of SQ2 aptamer-based direct ALISA for EV detection. (B) SQ2-based ALISA can detect recombinant ALPPL2 protein with a sensitivity of 1 ng (10 ng/mL). (C) Standard curve showing linearity in the broad range of 10 to 2,500 ng/mL of protein. (D) ALPPL2 estimation in the secretomes and EVs of (D) PANC-1⁺, (E) Capan-1, and (F) MIA PaCa-2 cells using SQ2-based ALISA. ALISA could detect ALPPL2 in EVs with much higher sensitivity than in the secretome. Results are mean ± SD of more than three independent experiments.

Figure 4

Quantitative Detection in ALPPL2 or CD9 Antibody/SQ2 Aptamer Sandwich ALISA (A) Scheme of sandwich ALISA. (B) ALPPL2 antibody/SQ2 aptamer-based sandwich ALISA for detecting recombinant ALPPL2 proteins with sensitivity of 3.5 ng (= 35 ng/mL) of ALPPL2. (C) The standard curve showed linearity in the range from 5 to 500 ng/mL. (D) ALPPL2 antibody/SQ2-based detection of ALPPL2 in PANC-1⁺ EVs (E) CD9 antibody/SQ2 aptamer-based sandwich ALISA for detecting PANC-1⁺ EVs. Sensitivity of detection for PANC-1⁺ EVs was as low as 0.1 ng (= 1 ng/mL). (F) CD9 antibody/SQ2 sandwich ALISA for detecting ALPPL2 in three PDAC-derived EVs (5 μg/mL) showed signals comparable to direct ALISA.

Figure 5

Proof-of-Principle Liquid Biopsy Test Using ALPPL2 Negative Human Serum Spiked with PANC-1⁺ EVs in CD9 Antibody/SQ2 Aptamer-Based Sandwich ALISA Serum was diluted in a ratio of 1:1 to 1:100 in PBS and then spiked with 0.5 μg of EVs. Samples were directly tested or EVs were isolated by Exoquick-based precipitation method and then tested with this sandwich ALISA.