Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis

Abstract Detecting pancreatic duct adenocarcinoma (PDAC) in its early stages and predicting late‐stage patient prognosis undergoing chemotherapy is challenging. This work shows that the activation of specific oncogenes leads to elevated expression of mRNAs and their corresponding proteins in extracellular vesicles (EVs) circulating in blood. Utilizing an immune lipoplex nanoparticle (ILN) biochip assay, these findings demonstrate that glypican 1 (GPC1) mRNA expression in the exosomes‐rich (Exo) EV subpopulation and GPC1 membrane protein (mProtein) expression in the microvesicles‐rich (MV) EV subpopulation, particularly the tumor associated microvesicles (tMV), served as a viable biomarker for PDAC. A combined analysis effectively discriminated early‐stage PDAC patients from benign pancreatic diseases and healthy donors in sizable clinical from multiple hospitals. Furthermore, among late‐stage PDAC patients undergoing chemotherapy, lower GPC1 tMV‐mProtein and Exo‐mRNA expression before treatment correlated significantly with prolonged overall survival. These findings underscore the potential of vesicular GPC1 expression for early PDAC screenings and chemotherapy prognosis.


FigureFigure S3 .
Figure S2.ILN characterization Figure S3.Effect of capture antibody on the ILN assay Figure S4.Protein expression of GPC1, microvesicle markers, and exosome markers in MIA PaCa-2 and HPDE6c7 cells and cell derived EV subpopulations.

Figure S7 .
Figure S7.Dot charts, scatter plots, and ROC curves of GPC1 Exo-mRNA and tMV-mProtein expression as a dual biomarker for non-blinded validation PDAC patient samples from each hospital.

Figure S8 .
Figure S8.Dot charts, scatter plots, and ROC curves of GPC1 Exo-mRNA and tMV-mProtein expression as a dual biomarker for blinded validation samples from each hospital.

Figure S2 .
Figure S2.ILN biochip characterization.(A) Effect of neutravidin and neutravidin conjugated gold nanoparticle (Neutravidin-GNP) on TFI of GPC1 protein detection in HD and PDAC patients.(B) Non-specific binding of CD63 protein expression using the mIgG isotype as a control.Data were presented as means ± SD (n = 2 wells, each well with 100 images).(C) The TIRF images of minimal non-specific binding without (PBS) and with patient EVs on ILN biochips.(D) NanoSight nanoparticle tracking analysis of EVs from conditioned media of PANC-1 cells and different PDAC patients at low EV concentrations.(E) Effect of proteinase K (PK) on EV size, number, and protein contamination in PDAC patient serum (n = 20) from OSU.Data were presented as means (n = 3).

Figure S4 .
Figure S4.Protein expression of GPC1, microvesicle markers, and exosome markers in MIA PaCa-2 and HPDE6c7 cells and cell-derived EV subpopulations.The GPC-1 protein is highly expressed in MV and tMV fractions in EVs derived from PANC-1 and MIA PaCa-2 cell lines, but low in Exo fractions.Lower GPC-1 expression is found in non-cancerous cell line (HPDE6c7) and its EVs.

Figure S6 .
Figure S6.AUC/ROC curves for GPC1 Exo-mRNA and tMV-mProtein expression in Stage III/IV PDAC patients as single-or dual-biomarkers.PDAC patients from OSU compared to a control cohort consisted of HD and BPD samples.

Figure S7 .
Figure S7.Dot charts, scatter plots, and ROC curves of GPC1 Exo-mRNA and tMV-mProtein expression as a dual biomarker for non-blinded validation PDAC patient samples from each hospital.(A-E) MSKCC in the US.(F-J) TVGH in Taiwan.All data were presented as means (n = 2 wells, each well with 100 images).Pairwise comparison P values were determined by the Mann-Whitney U test.**P < 0.01, ***P < 0.001, n.s.not significant.

Table S1 .
Clinical characteristics of patients with BPD and IPMN for biomarker screening

Table S2 .
Clinical characteristics of CGMH PDAC patients undergoing chemotherapy

Table S3 .
Clinical characteristics of TVGH PDAC patients undergoing chemotherapy

Table S4 .
Clinical characteristics of NCKUH PDAC patients undergoing chemotherapy