Fig 2: IRF7 expression in GEM-sensitive and -resistant patients(A) Immunohistochemistry (IHC) was used to localize IRF7 expression in patient tissues, with sensitivity and resistance groups compared. Representative images of IRF7 IHC from 25 GEM-sensitive (defined by stable disease/partial response with no progression for ≥6 months) and 25 GEM-resistant patients (defined by disease progression within 3 months of GEM treatment). Scale bars, 100 μm.(B) Western blot analysis quantified IRF7 protein levels in GEM-sensitive and -resistant patient samples. Representative images of IRF7 protein from 25 GEM-sensitive and 25 GEM-resistant patients.(C) qPCR assessed IRF7 mRNA expression, with results presented as mean ± standard deviation (SD). IRF7 mRNA expression was quantified in clinical specimens derived from 25 GEM-sensitive and 25 GEM-resistant patients with PDAC. Statistical significance was determined using an unpaired two-tailed Student’s t test. The data were presented as mean ± SD; ∗p < 0.05, ∗∗p < 0.01.

Fig 3: Synergistic effects of TMZ and GEM on pancreatic cancer cell proliferation and inducing apoptosis(A) A map generated from the Genomics of Drug Sensitivity in Cancer (GDSC) database shows drugs that modulate the expression of IRF7, highlighting potential therapeutic targets.(B) Dose-response curve of TMZ on SW1990 cells, determining the IC50, which is essential for establishing effective drug concentrations in subsequent experiments. Curve fitting was based on three independent biological replicates.(C) Proliferation assay using ZenCell technology assessing the combined effect of GEM and TMZ on SW1990 cells. The data demonstrate a synergistic inhibition of cell proliferation when both drugs are applied together compared to single-agent treatments. Assays were performed in three independent biological replicates.(D and E) Colony formation assays showing the impact of combined GEM and TMZ treatment on SW1990 cells. Reduced colony formation indicates enhanced antiproliferative effects with the combination therapy. Assays were repeated in three independent biological replicates.(F) In vivo, tumor growth inhibition in mice xenografted with SW1990 cells treated with GEM and TMZ alone or in combination. The combination treatment shows a significant reduction in tumor volume compared to single-agent treatments. Seven mice were included per group.(G) TUNEL staining of tumor sections from treated mice, indicating increased apoptosis in tumors treated with the combination of GEM and TMZ. Seven tumors per group. Data are presented as mean ± SD from three independent experiments. p < 0.01 vs. CV. Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test. The data were presented as mean ± SD; ∗p < 0.05, ∗∗p < 0.01.

Fig 4: CCL5 mediates resistance to GEM, a process facilitated by IRF7(A and B) Cells were subjected to knockdown or overexpression of IRF7 based on their expression profiles, and downstream CCL5 expression was simultaneously assessed. mRNA and protein levels of CCL5 were quantified by qPCR and western blot, respectively. Both qPCR and western blot experiments were performed in three independent biological replicates.(C) Western blot validation of IRF7 knockdown efficiency and CCL5 overexpression in the “shIRF7+CCL5” group, where CCL5 was ectopically overexpressed in IRF7-knockdown PDAC cells. Control groups include shNC (negative control for shRNA) and shIRF7 (IRF7 knockdown alone) to confirm the specificity of the manipulations. Western blots were repeated in three independent biological replicates.(D) The indicated cell lines were treated with or without GEM, and their viability was assessed using the ZENcell owl system. Viability assays were performed in three independent biological replicates.(E) Apoptosis rates were measured by flow cytometry 24 h after GEM administration. Flow cytometry experiments were performed in three independent biological replicates.(F) Colony formation assays were performed to evaluate cell survival post-GEM treatment. Assays were repeated in three independent biological replicates.(G and H) In vivo, validation was conducted by subcutaneously xenografting the indicated cells into nude mice, which were subsequently treated with or without GEM. Tumor volumes were measured every other day, and the weights of harvested tumors were recorded. Seven tumors were analyzed in each group.(I) Apoptosis within the tumors was assessed using TUNEL staining after tumor harvest and slicing. All scale bars represent 100 μm. TUNEL staining was validated in seven tumors. Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test. The data were presented as mean ± SD; ∗p < 0.05, ∗∗p < 0.01.

Fig 5: IRF7 enhances CCL5 secretion and attraction of M2-like TAMs(A) Multiplex immunofluorescence was used to assess the expression of IRF7, CCL5, and the M2 macrophage marker protein CD206. High-magnification insets (right) highlight CD206 membrane localization on M2-like macrophages. Scale bars, 100 μm. mIF staining was repeated in three independent clinical samples per group.(B) Spearman correlation revealed a borderline-significant association between IRF7 and CCL5 transcript levels in human clinical samples (R = 0.3332, p = 0.0181, n = 50 total samples: 25 GEM-sensitive, 25 GEM-resistant).(C) Western blot analyses were conducted to determine the expression levels of IRF7 and CCL5 in clinical samples. Blots were repeated in three independent biological replicates using distinct patient samples.(D) A cell chemotaxis assay was performed by co-incubating M2-like macrophages with BxPC3 cells in the presence of IgG or CCL5 antibody. Scale bars, 100 μm.(E) Quantitative analysis was performed across three independent experiments (p < 0.01; n = 3).(F) A cell chemotaxis assay was conducted by co-incubating M2-like macrophages with BxPC3 cells in the presence of DMSO or Maraviroc (MVC). Scale bar, 200 μm.(G) Quantitative analysis was performed across three independent experiments (p < 0.01; ns, not significant; n = 3). Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test. The data were presented as mean ± SD; ∗p < 0.05, ∗∗p < 0.01.