Fig 1: Reduced CD74 expression leads to reduced tumor growth and inflammatory conditions in orthotopic mouse models. (A) BALB/C nude mice were orthotopically injected with Capan-1 cells (3 × 106 cells). After two months, lentivirus particles carrying shControl or shCD74 were injected (2 × 106 particles). (B) Representative image of mice injected with control and shCD74 lentiviral particles, and image of tumors from the mice. The tumor weight was measured in grams. (C) Expressions of CD74, S100A8/A9, HIF-1a, and TRAF6, IL-6 in mice injected with control and shCD74 lentivirus were measured via RT-qPCR. (D) Representative images of immunofluorescence (IF) staining analyses of CD74-S100A8 and CD74-S100A9 double-stained tumors. (E) Representative images of IF staining analyses of HIF-1a, TNF-a, IL-6, and p-NF?B. * p < 0.05, ** p < 0.01, *** p < 0.0001. Scale bar = 20 µm.
Fig 2: S100A8 and S100A9 induce expressions of pro-inflammatory cytokines in fibroblasts. (A) The protein expression of a-SMA in normal and cancer-associated fibroblasts (NFs and CAFs, respectively) was measured via Western blot. (B) Heatmap of differential expressions of inflammatory response genes of NFs and CAFs. (C) Schematic of the indirect co-culture assay. (D) Expressions of S100A8 and S100A9 in control, siS100A8, siS100A9, and siS100A8/A9 double-knockdown Capan-1 cells were measured via RT-PCR. (E) Secretion of S100A8/A9 heterodimer in the conditioned medium of control, siS100A8, siS100A9, and siS100A8/A9 double-knockdown Capan-1 cells was measured via ELISA analysis. (F) Expressions of TNF-a, HIF-1a, IL-6, CXCL-1/6 in CAFs, NFs, NF grown in control Capan-1-conditioned medium, and NF grown in siCD74 Capan-1-conditioned medium were measured via RT-qPCR. (G) Expressions of indicated genes in CAFs, NFs, NFs grown in control Capan-1-conditioned medium, and NFs grown in siS100A8 and siS100A9 Capan-1 conditioned medium were measured via RT-qPCR. * p < 0.05, ** p < 0.01, *** p < 0.0001.
Fig 3: CD74 regulates expression and secretion of S100A8/A9 through the TRAF6-NF?B pathway. (A) Venn diagram of differentially expressed genes in the cell migration, inflammatory response, and secretion-related genes in control and siCD74 Capan-1 cells. S100A8 was the most downregulated gene among the categories. (B) The mRNA expressions of genes indicated in the TRAF6-NF?B pathway in control and siCD74 Capan-1 cells were estimated via PCR. (C) The protein expression of indicated genes was estimated via Western blot. (D) Secretion of S100A8/A9 heterodimer in the conditioned medium of control and siCD74 Capan-1 cells was measured via ELISA analysis. (E) Kaplan–Meier curves of overall and disease-specific survival of PDAC patients with high S100A8, low CD74-low S100A8, low CD74–high S100A8, high CD74-low S100A8, and high CD74-high S100A8 based on TCGA data. (F) S100A8 and S100A9 expressions in GEPIA2 database. The red represents expression level in the tumor tissues while the gray represents the normal tissues. (G,H) The mRNA and protein expression of S100A8/A9 in normal and PDAC tissues via qRT-PCR and Western blot. * p < 0.05, ** p < 0.01, *** p < 0.0001.
Supplier Page from Abcam for Human Calprotectin ELISA kit (S100A8/S100A9)