Fig. 1

uPAR knockout strategy and sequencing gene and uPAR re-expression A: Schematic representation of the uPAR knockout strategy using CRISPR/Cas9. B: List of gRNAs for uPAR and PCR primers used to detect gene knockout. C: Fluorescence-activated cell sorting (FACS) analysis of GFP/RFP double-positive cells. D: PCR screening for gRNA target sites and potential deletions. E: Sanger sequencing of mutations at the gRNA target sites. F: Sequences of siRNA oligonucleotide templates used in this study. G: Re-expression of uPAR protein levels measured by ELISA (n=3, ***: P<0.01, Student’s t-test, two-sided). H: Immunoblot analysis showing de novo expression of uPAR in clone 12 and rescued uPAR cells."

Fig. 2

CRISPR/Cas9-mediated uPAR knockout in AsPC-1 cells A: Immunoblot analysis showing uPAR protein levels in the pancreatic cell lines AsPC-1, CAPAN-1, CAPAN-2, MIA PaCa-2, PANC-1, and PaTu8988T. B: ELISA quantification of uPAR protein levels in 6 human pancreatic cell lines. C: Immunoblot analysis comparing uPAR levels between AsPC-1 uPAR knockout (KO) and wild-type (WT) cells. D: ELISA results showing uPAR levels in AsPC-1 uPAR KO cells compared to WT cells. At least three biological replicates were performed. GAPDH was used as a loading control. ***: P<0.001, multiple comparison tests were used to assess differences between groups."

Fig. 3

uPAR knockout inhibits pancreatic cancer cell growth and migration, and enhances resistance to gemcitabine A: Growth curves over 4 days showing significantly reduced proliferation rates in AsPC-1 uPAR KO clones compared to WT controls. B: Representative colony formation assay images in methylcellulose comparing uPAR KO and WT cells. C: Quantitative analysis showing a significant reduction in clonogenicity in uPAR KO cells compared to WT cells. D-E: Reduced migratory capacity of uPAR KO cells compared to WT cells. F: Immunofluorescence staining showing fewer stress fibers in uPAR KO cells compared to WT cells (×400, red: phalloidin, blue: DAPI). G: Immunoblot analysis of epithelial and mesenchymal markers indicating Mesenchymal-to-epithelial transition (MET) in uPAR KO cells. H: Increased resistance to gemcitabine in uPAR KO cells (0.1, 0.5, 1 µmol/L, 72 h). At least three biological replicates were performed. GAPDH or PARK7 was used as a loading control. ***: P<0.001, multiple comparison tests were used to assess differences between groups."

Fig. 4

uPAR regulates FAK and p38MAPK activity and enhaces gemCitabine resensitivity of AsPC-1 uPAR knockout cells A: Immunoblot analysis showing increased phosphorylation of FAK and p38, along with elevated levels of LC3B, while p62 levels remain unchanged in uPAR knockout (KO) cells. B-D: Restoration of gemCitabine sensitivity (B) and wild-type (WT) signaling phenotype (D) in AsPC-1 uPAR KO cells after (C) FAK siRNA knockdown. E-F: Gemcitabine response following p38MAPK knockdown (F) using two siRNAs in AsPC-1 uPAR KO cells. G: Re-establishment of the WT signaling phenotype in uPAR KO cells following p38MAPK siRNA knockdown. H: Combined treatment with gemcitabine (0.1 µmol/L) and p38 inhibitor JX401 (3 µmol/L) for 72 h in uPAR KO cells. GAPDH was used as a loading control. At least three biological replicates. **: P <0.01, ***: P<0.001, Student’s t test, two-sided."

Fig.5

Inhibition of autophagy or uPAR re-expression restores gemcitabine sensitivity in uPAR knockout cells A: Immunoblot analysis of autophagy markers p62 and LC3B following treatment with 3-MA (5 µmol/L) or chloroquine (CQ, 5 µmol/L). B: GemCitabine sensitivity after 72 hours of treatment with 3-MA (5 µmol/L) or CQ (5 µmol/L). C: Immunoblot analysis showing altered expression of glycolytic enzymes (GLUL, MTHFD2) and cell cycle regulators (FOXM1, CCNB1), suggesting dormancy in uPAR knockout (KO) cells. D: Re-expression of uPAR in clone 12 cells restored p-p38MAPK and p62 levels to wild-type (WT) levels. E: Re-expression of uPAR restored gemcitabine sensitivity in clone 12 cells (0.1 µmol/L). F: Re-expression of uPAR significantly enhanced migratory capacity in clone 12 cells. GAPDH or PARK7 was used as a loading control. At least three biological replicates were performed. ***: P<0.001, Student’s t-test, two-sided."

Fig. 6

uPAR as a predictive biomarker for gemcitabine sensitivity in PDAC-derived organoids A: Representative bright-field images of patient-derived PDAC organoids (PDOs) showing three distinct morphologies: organoids with varying degrees of budding (top), solid organoids (center), and hollow organoids (bottom). Scale bar: 200 µm. B: Dose-response curves for gemcitabine in PDOs cultures (n=8) treated with gemcitabine (0.01-10.00 µmol/L). C: IC50 values for PDOs cultures treated with gemcitabine. D: Immunoblot analysis of uPAR and LC3B in protein extracts from eight PDOs, with PARK7 as a loading control. E: Pearson correlation analysis showing a significant inverse correlation between uPAR levels and gemcitabine sensitivity (IC50) in PDOs (r2=0.66, P=0.026 2). At least three biological replicates were performed. *: P<0.05, compared with each other; **: P<0.01, compared with each other; Student’s t test, two-sided. GEM: Gemcitabine."