Identification of Tumor-Specific Surface Proteins Enables Quantification of Extracellular Vesicle Subtypes for Early Detection of Pancreatic Ductal Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related mortality, largely due to late-stage diagnosis. Reliable early detection methods are critically needed. PDAC-derived extracellular vesicles (EVs) carry molecules that reflect their parental tumor cells and are detectable in early disease stages, offering a promising noninvasive diagnostic approach. Here, a streamlined PDAC EV Surface Protein Assay for quantifying PDAC EV subpopulations in 300-µL plasma through a two-step workflow is presented: i) click chemistry-mediated EV enrichment using EV Click Beads and trans-cyclooctene-grafted antibodies targeting three PDAC EV-specific surface proteins (MUC1, EGFR, and TROP2), and ii) quantification of enriched PDAC EVs through reverse transcription-quantitative polymerase chain reaction. The three PDAC EV-specific surface proteins are identified using a bioinformatics framework and validated on PDAC cell lines and tissue microarrays. The resultant PDAC EV Score, derived from signals of the three PDAC EV subpopulations, demonstrates robust differentiation of PDAC patients from noncancer controls, with area under the receiver operating characteristic curves of 0.94 in the training (n = 124) and 0.93 in the validation (n = 136) cohorts. This EV-based diagnostic approach successfully exploits PDAC EV subpopulations as novel biomarkers for PDAC early detection, translating PDAC surface proteins into an EV-based liquid biopsy platform.

Keywords: cancer diagnosis; extracellular vesicles; liquid biopsy; pancreatic ductal adenocarcinoma.

Figure 1

Pancreatic ductal adenocarcinoma (PDAC) Extracellular Vesicle (EV) Surface Protein Assay for noninvasive detection of early stage PDAC. A) PDAC EV Surface Protein Assay is conducted through a two‐step workflow—Step 1: Click chemistry‐mediated enrichment by EV Click Beads in the presence of one of the three trans‐cyclooctene (TCO)‐grafted PDAC EV‐specific antibodies targeting MUC1, EGFR, and TROP2; and Step 2: Quantification of the enriched PDAC EVs by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) detecting ACTB mRNA. B) A phase 2 (case‐control) biomarker study was developed to assess the diagnostic performance of PDAC EV Surface Protein Assay for distinguishing PDAC from noncancer controls). Plasma samples were collected from a training and a validation cohort. In the training cohort (University of California, Los Angeles [UCLA]), 124 plasma samples were collected from 61 PDAC patients, 63 noncancer controls. In the validation cohort (Suzhou Institute of Nano‐Tech and Nano‐Bionics [SINANO]), 136 plasma samples were collected from 49 PDAC patients, 87 noncancer controls. A logistic regression model was applied to generate PDAC EV Scores from the RT‐qPCR readouts for detecting PDAC. C) The resultant PDAC EV Scores can effectively distinguish PDAC from noncancer. EGFR, epidermal growth factor receptor; EV, extracellular vesicles; mTz, methyltetrazine; MUC1, mucin 1; PDAC, pancreatic ductal adenocarcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; SINANO, Suzhou Institute of Nano‐Tech and Nano‐Bionics; TCO, trans‐cyclooctene; TROP2, trophoblast cell‐surface antigen 2; UCLA, University of California, Los Angeles.

Figure 2

An integrated bioinformatic framework employed for identifying three PDAC EV‐specific surface proteins. The overall PDAC protein markers were compiled from two publicly available datasets: a) Clinical Proteomic Tumor Analysis Consortium (CPTAC) database, which identified 1559 upregulated differential expression proteins in PDAC tissues. b) Cancer Cell Line Encyclopedia (CCLE) protein database, which identified 12512 PDAC‐related proteins in PDAC cell lines. The framework employed sequential filtering steps: i) selection of PDAC‐specific proteins by including highly expressed markers in PDAC and excluding housekeeping and hematopoietic proteins, as well as those markers highly expressed in immune cell expression profiling (DMAP); ii) selection of PDAC EV‐specific surface proteins (TCSA GESP score >10), validating through Vesiclepedia (>10 supporting EV studies) and with high expression in mRNA level (CCLE); and iii) prioritization of three PDAC EV‐specific surface proteins that are potential PDAC treatment targets via Druggable Genome Database (Pharos) and ClinicalTrials.gov. ADC, antibody‐drug conjugate; CAR, chimeric antigen receptor; CCLE, Cancer Cell Line Encyclopedia; DMAP, Differentiation Map; EGFR, epidermal growth factor receptor; EV, extracellular vesicles; CPTAC, Clinical Proteomic Tumor Analysis Consortium; FC, fold change; FDR, false discovery rate; GESP, genes encoding surface protein; HR, hazard ratio; mAb, monoclonal antibody; MUC1, mucin 1; PDAC, pancreatic ductal adenocarcinoma; TCGA, The Cancer Genome Atlas; TCSA, The Cancer Surfacesome Atlas; TROP2, trophoblast cell‐surface antigen 2.

Figure 3

Validation of PDAC EV‐specific surface proteins using PDAC cell lines and PDAC tissue microarray (TMA). A) Representative immunofluorescence (IF) micrographs of the three PDAC EV‐specific surface proteins (MUC1, EGFR, and TROP2) on HPAF‐II and CFPAC1 PDAC cell lines. Blue: DAPI; green: FITC. Scale bar, 10 µm. B) Representative hematoxylin and eosin (H&E) and immunohistochemistry (IHC) images of the three PDAC EV‐specific surface proteins on PDAC TMA slides. Scale bar, 100 µm. C) IHC staining intensity and positive percentages for each marker across PDAC TMA. D) Quantitative IHC scoring is illustrated in pie and bar charts, summarizing the positive percentage of IHC scores for PDAC EV‐specific surface proteins and their combinations. EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; MUC1, mucin 1; TROP2, trophoblast cell‐surface antigen 2.

Figure 4

Preparation of EV Click Beads, characterization of PDAC cell‐derived EVs, and linearity study of PDAC EV Surface Protein Assay using synthetic plasma samples. A) Stepwise preparation of EV Click Beads. B) A representative transmission electron microscopy (TEM) image of HPAF‐II EVs. Scale bar, 100 nm. C) Size distribution of HPAF‐II measured by nanoparticle tracking analysis (NTA). D) A representative TEM image of HPAF‐II EVs enriched on an EV Click Bead in the presence of TCO‐anti‐MUC1, followed by immunogold staining with anti‐CD63‐grafted gold nanoparticles (gold arrows). Scale bar, 50 nm. E) A schematic illustration of the workflow developed for linearity study of PDAC EV Surface Protein Assay using synthetic plasma samples. Synthetic plasma samples were prepared by serially spiking HPAF‐II EVs into EV‐depleted HD plasma. Then the two‐step PDAC EV Surface Protein Assay was carried out: Step 1: The synthetic plasma samples were incubated with one of the three PDAC EV‐specific antibodies, followed by enrichment using EV Click Beads; Step 2: The enriched HPAF‐II EVs were quantified by detecting ACTB mRNA via RT‐qPCR. F–H) Dynamic linearity ranges of ACTB mRNA signals observed for the three subpopulations of PDAC EVs enriched by TCO‐grafted PDAC EV‐specific antibodies, i.e., TCO‐anti‐MUC1 F), TCO‐anti‐EGFR G), or TCO‐anti‐TROP2 H). Signal was defined as 40 − Ct value. APTES, 3‐aminopropyltriethoxysilane; EGFR, epidermal growth factor receptor; EV, extracellular vesicles; mPEG4‐NHS, methoxypolyethylene glycol succinate‐N‐hydroxysuccinimide; mTz, methyltetrazine; mTz‐PEG4‐NHS, methyltetrazine‐PEG4‐N‐hydroxysuccinimide; MUC1, mucin 1; NHS, N‐Hydroxysuccinimide; NP, nanoparticle; PDAC, pancreatic ductal adenocarcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; TCO, trans‐cyclooctene; TROP2, trophoblast cell‐surface antigen 2.

Figure 5

Diagnostic performance of PDAC EV Score for distinguishing PDAC patients from noncancer controls in the training cohort. A) A general workflow illustrating the application of the PDAC EV Surface Protein Assay using plasma samples from 61 PDAC patients and 63 noncancer controls in the training cohort. B) A heatmap summarizing ACTB mRNA signals in the three subpopulations of PDAC EVs, including MUC1⁺ PDAC EVs, EGFR⁺ PDAC EVs, and TROP2⁺ PDAC EVs. C–E) Boxplots illustrating significantly higher ACTB mRNA signals in the three subpopulations of PDAC EVs among PDAC patients compared to noncancer controls in the training cohort. The signal is represented as 40 – Ct value. F) Boxplots summarizing PDAC EV Scores in PDAC patients and noncancer controls. The dashed line indicates the optimal cutoff of −0.27. G) ROC curve of PDAC EV Score for distinguishing PDAC patients from noncancer controls in the training cohort. H) ROC curve of PDAC EV Score after leave‐one‐out cross‐validation (LOOCV) for distinguishing PDAC patients from noncancer controls in the training cohort. AUROC, area under the receiver operating characteristic curve; EGFR, epidermal growth factor receptor; EV, extracellular vesicles; MUC1, mucin 1; PDAC, pancreatic ductal adenocarcinoma; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; TCO, trans‐cyclooctene; TROP2, trophoblast cell‐surface antigen 2.

Figure 6

PDAC EV Score for distinguishing PDAC patients from noncancer controls in the validation cohort. A) Heatmaps summarizing ACTB mRNA signals in the three subpopulations of PDAC EVs, including MUC1⁺ PDAC EVs, EGFR⁺ PDAC EVs, and TROP2⁺ PDAC EVs. B–D) Boxplots illustrating significantly higher ACTB mRNA signals in the three subpopulations of PDAC EVs among PDAC patients compared to noncancer controls in the validation cohort. The signal is represented as 40 – Ct value. E) Boxplots showing PDAC EV Scores observed in PDAC patients compared to noncancer controls in the validation cohort. The dashed line indicates the fixed cutoff of −0.27, which is the same as the training cohort. F) ROC curve of PDAC EV Score for distinguishing PDAC patients from noncancer controls in the validation cohort. AUROC, area under the receiver operating characteristic curve; EGFR, epidermal growth factor receptor; EV, extracellular vesicles; MUC1, mucin 1; PDAC, pancreatic ductal adenocarcinoma; TROP2, trophoblast cell‐surface antigen 2.

Figure 7

The performance of the PDAC EV Score in all participants and subgroup analyses. A) Violin plots showing the PDAC EV Scores in different subgroups enrolled in this study. B) ROC curve of PDAC EV Score for detecting all stage PDAC (n = 110) from noncancer controls (n = 150). C) ROC curves of PDAC EV Score (blue line), serum CA19‐9 (purple line), and their combination (red line) for detecting serum CA19‐9 available all stage PDAC (n = 97) from noncancer controls (n = 59). D) ROC curve of PDAC EV Score for detecting early stage (stage I–II) PDAC (n = 92) from noncancer controls (n = 150). E) ROC curves of PDAC EV Score (blue line), serum CA19‐9 (purple line) and their combination (red line) for detecting serum CA19‐9 available early stage PDAC (n = 79) from noncancer controls (n = 59). F) ROC curve of PDAC EV Score for detecting early stage PDAC (n = 92) from high‐risk individuals (n = 41). G) ROC curve of PDAC EV Score for detecting early stage PDAC (n = 92) from benign pancreatic tumor (n = 22). H) ROC curve of PDAC EV Score for detecting early stage PDAC (n = 92) from HD (n = 87). ****p < 0.0001, ns: not significant. AUROC, area under the receiver operating characteristic curve; CA19‐9, carbohydrate antigen 19‐9; HD, healthy donor; PDAC, pancreatic ductal adenocarcinoma.