Fig 1: sEV-MASP2 and serum CA 19–9 alterations and survival outcomes in APC cohorts. (A, C) Changes in serum sEV-MASP2 concentrations (pg/mL) of the R patient group (n = 25) tracked across (A) pre-t, BR, and TR, and (C) pre-t, month 2 (M2), and TR. (B,D) Histograms illustrating the groupwise mean alterations in MASP2 concentrations within the R group (n = 25) through (B) pre-t, BR, and TR, and (D) pre-t, M2, and TR timepoints. (E) Changes in serum sEV-MASP2 concentrations (pg/mL) of the NR patient group (n = 15) at pre-treatment (pre-t) and post-treatment (post-t) timepoints. (F) Histograms illustrating the groupwise mean alterations in MASP2 concentrations within the NR group (n = 15) at pre-t and post-t timepoints. (G, H) Waterfall plot showing percentage changes in (G) serum sEV-MASP2 levels, and (H) serum CA 19–9 levels in R and NR cohorts at month 2 compared to pre-t (baseline). For G&H, Patterned bars represent cases with a minimum 50 % reduction in serum CA 19–9 levels from baseline (baseline CA 19–9 reported as >10,000 U/mL); # denotes instances where CA 19–9 value at baseline or month 2 were unavailable. Chemotherapeutic drug (FOLFIRINOX or Gemcitabine/nab-paclitaxel) and PC stage (III or IV) are indicated for each patient. (I-K) Kaplan-Meier (K-M) plot for overall survival (OS) in PC patients. (I) Compares a > 20 % rise (blue) vs. a > 20 % drop (red) in serum sEV-MASP2 levels at month 2 vs. pre-t (n = 60). (J) Compares a >20 % rise (blue) vs. >20 % drop in serum CA 19–9 levels at month 2 compared to pre-t in CA19–9 eligible patients within the APC cohort evaluated for sEV-MASP2 (n = 42). (K) Compares a >20 % rise (blue) vs. >20 % drop in serum CA 19–9 levels at month 2 compared to pre-t in all qualified PC patients (n = 54) with known CA 19–9 changes. (L) ROC curve from a nominal logistic regression model assessing the prognostic efficacy of sEV-MASP2, using a ± 20 % change to predict 6 months OS in PC patients. Statistical significance was evaluated using the Chi-Square test (=14.58, p = 0.0001). (M) K-M plot analyzing survival in PC patients with high bilirubin (≥ 1.3) or low serum CA 19–9 (< 37 U/mL) at pre-t (n = 23), for >20 % rise (blue) vs. > 20 % drop (red) in serum sEV-MASP2 levels at 2 months compared to pre-t. For panels I-K and M, median survival times, hazard ratio (HR), and log-rank test significance are presented to highlight the differences in the survival outcomes between the groups compared.
Fig 2: Functional assays and IHC assessments of MASP2 in PC cells and tissues. (A) Western blot analysis for MASP2 across PC cell lines (Panc-10.05, Panc-1, Capan2, BxPC3, SW1990, HPAF1, MIA PaCa2, and CFPAC1), and pancreatic epithelial cell line HPDE. (B, C) Western blot for MASP2 in CFPAC1 cell line transfected with (B) control vector (CTRL) or MASP2 OE plasmid, and (C) scrambled control (siCT) or siMASP2. For panels A-C, relative protein levels were normalized to GAPDH. (D) Wound-healing assays were conducted using CTRL, MASP2 OE, siCT, or siMASP2 transfected CFPAC1 cells. Representative images for areas occupied by cells at 0 h and 48 h post-wounding. Scale bars = 1000 µm. (E) Quantitative analysis of wound closure, based on five independent experiments. (F) Representative images from the EdU proliferation assay showing the growth of CFPAC1 cells transfected with CTRL, MASP2 OE, siCT or siMASP248 h post-transfection. GFP staining (green) indicates proliferating cells in the S phase of mitosis, while nuclear DAPI staining (blue) represents all cells present. (G) Quantitative analysis of the percentage of EdU-positive cells in each group, demonstrating the proportion of cells undergoing DNA synthesis. Data presented as Mean ± SEM, n = 5. (H) Abundance and distribution of MASP2 measured by IHC staining in primary (n = 8) and liver metastatic (n = 8) PDAC biopsies, normal pancreas (n = 4) and normal liver (n = 4). IHC panels display representative images for H&E (top panel), negative control (Negative CTRL) (middle panel), and MASP2 (bottom panel) staining in the tissues. Black arrows indicate areas of positive staining in primary and liver metastatic PDAC tissues. Scale bars = 20 µm. (I) Quantitative evaluation of MASP2 expression in acinar and ductal cells from normal pancreas, and tumor cells from primary and liver metastatic PDAC tissues. (J) Quantitative assessment of MASP2 expression in stromal cells in normal pancreas, and in primary and liver metastatic PDAC tissues. Data presented as Mean ± SEM. (K) Box plot depicting MASP2 levels in PDAC tumors (n = 137) vs. paired normal pancreatic tissues (n = 74) from the CPTAC PDAC dataset. z-values represent standard deviations from the median across samples. Log2 spectral count ratios were first normalized within individual sample profiles and then normalized across all samples.
Fig 3: Mass-spectrometry workflow and identification of differentially expressed sEV proteins in discovery dataset. (A) Changes in CA 19–9 levels in patients (IDs: 33, 41, 47, 70 and 74) across pre-t, BR and treatment TR timepoints, with corresponding representative CT scans. (B) Schematic for experimental setup and replication sets for mass spectrometry. (C) Venn diagram showing overlapping and unique DE sEV-proteins across pre-t, BR and TR timepoints. (D) Volcano plot showing DE sEV-proteins between pre-t and BR, highlighting upregulated (red) and downregulated (blue) proteins (|fold change| ≥ 1.4; Tukey’s HSD p value ≤ 0.05). (E) Heatmap with hierarchical clustering depicting z-score normalized differential expressions of sEV-proteins between pre-t and BR for the five tested patients. (F) Volcano plot showing DE sEV-proteins between BR and TR, highlighting upregulated (red) and downregulated (blue) proteins (|fold change| ≥ 1.4; Tukey’s HSD p value ≤ 0.05). (G) Heatmap with hierarchical clustering depicting z-score normalized differential expressions of sEV-proteins between BR and TR for the five tested patients. (H) Normalized abundance with corresponding p values of the nine overlapping DE sEV-proteins (AOC1, IDE, PLPBP, MASP2, EPHA10, IGKJ4, MBL2, APOL1, and TF) across pre-t, BR and TR in the five APC patients in the dataset. (I) Heatmap of z-score normalized expression profiles for the nine overlapping DE sEV-proteins across the three timepoints in the discovery dataset.
Supplier Page from Abcam for Human MASP2 ELISA Kit