To investigate the changes in the tumor microenvironment of pancreatic cancer (PDAC) complicated with diabetes mellitus (DM) in a mouse model of hyperglycemia and orthotopic pancreatic cancer by analyzing transcriptome and single-cell transcriptome data in order to identify potential therapeutic targets.

By integrating single-cell transcriptome and bulk transcriptome data, bioinformatics analysis was conducted to compare the characteristics of tumor cells and tumor immune microenvironment between PDAC patients with DM (DM group) and those without DM (non-DM group). Twenty male C57BL/6 mice (6 weeks old, weighing 18~20 g) were randomly divided into a hyperglycemic group [STZ group, continuous intraperitoneal injection of 50 mg/kg streptozocin (STZ)(final concentration of1%) dissolved in citrate buffer], and a control group (Control group, an equivalent volume of citrate buffer without STZ at the same time points), with 10 mice in each group.Tail-tip blood glucose level was measured to monitor glycemic status. After orthotopic inoculation of pancreatic cancer cells in both Control and STZ groups, tumor-infiltrating immune cells were harvested. Flow cytometry was employed to determine the effects of hyperglycemia on: total CD8⁺ T cell and Treg cell populations; CD8⁺ T cell subsets expressing Ki67, TNF-α, granzyme B (GZMB) and IFN-γ; surface expression of PD-1, lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and mucin domain-3 (Tim-3) on CD8⁺ T cells; programmed death-ligand 1 (PD-L1) expression on tumor cells; and tumor-associated macrophage surface expression of major histocompatibility complex class Ⅰ (MHC-Ⅰ) and cluster of differentiation 206 (CD206).

Bioinformatics analysis revealed that, compared to the non-DM group, the genes significantly up-regulated in the DM group were associated with poor prognosis (P<0.001). The proportion of type 2 ductal cells was increased in the DM group, exhibiting higher levels of copy number variation (P<0.001). In the tumor immune microenvironment of the DM group, there was an increase in the proportion of Treg cells (P<0.05) and an elevated exhaustion score for CD8⁺ T cells (P<0.001), accompanied by down-regulated expression of effector molecules, up-regulated expression of inhibitory checkpoints, and a significant increase in the M2 score of M2-like macrophages (P<0.001). Animal experiments and flow cytometry found that, compared to the Control group, the STZ group had a shorter survival time (P<0.001), with decreased proportions of total CD8⁺ T cells (P<0.01) and CD8⁺ T cells expressing Ki67, TNF-α, GZMB and IFN-γ (P<0.01), increased proportion of Treg cells (P<0.001), up-regulated expression of PD-1, LAG-3 and Tim-3 on the surface of CD8⁺ T cells (P<0.001), and up-regulation of PD-L1 on tumor cell surface (P<0.001) and enhanced expression of CD206 on the surface of tumor-associated macrophages, while down-regulated expression of MHC-Ⅰ (P<0.001).

High glucose promotes the formation of an immunosuppressive microenvironment in PDAC, and targeting type 2 ductal cells and immunosuppressive cells in the tumor microenvironment, combined with dual immune checkpoint antibody therapy, may improve patient prognosis.

To explore the role of mitophagy in pancreatic cancer cachexia-induced muscle atrophy and its underlying mechanism.

Six male C57BL/6J mice (8 weeks old, weighing 20~30 g) were equally and randomly divided into a control group (intrapancreatic injection of normal saline) and a cachexia group (orthotopic pancreatic injection of KPC1199 cells). After successful model establishment, gastrocnemius muscles were harvested for transmission electron microscopy (TEM) to assess mitochondrial ultrastructure. Western blotting was performed to quantify mitochondrial respiratory chain complexes (Ⅰ ~ Ⅳ) and autophagy-related proteins, while immunofluorescence staining was conducted to evaluate mitochondrial-lysosomal colocalization. In in vitro experiments, C2C12 myoblasts were differentiated into myotubes, and then divided into a control group (standard culture) and a cachexia group (co-cultured with KPC1199 cells for 48 h using transwell chambers). Mitochondrial-lysosomal colocalization and autophagy-related protein expression were analyzed with immunofluorescence assay and Western blotting. The mitochondrial division inhibitor Mdivi-1 (20 μmol/L) was added to the co-culture system to assess its myotube diameter.

Compared to the control mice, the cachectic mice exhibited mitochondrial swelling, reduced cristae density, and significantly increased mitochondrial-lysosomal colocalization in gastrocnemius muscle (P<0.05). Western blotting revealed the expression levels of mitochondrial respiratory chain proteins complex Ⅰ (1.00±0.04 vs 0.51±0.04, P<0.05), complex Ⅱ (1.00±0.13 vs 0.73±0.15, P<0.05), complex Ⅲ (1.00±0.20 vs 0.64±0.01, P<0.05), complex Ⅳ (1.00±0.06 vs 0.65±0.02, P<0.05) and PGC1α (1.00±0.03 vs 0.62±0.06, P<0.05) were decreased, and the levels of mitophagy markers, LC3-Ⅰ/Ⅱ (1.00±0.14 vs 1.65±0.25, P<0.05), PINK1 (1.00±0.11 vs 1.51±0.05, P<0.05), and BNIP3 (1.00±0.22 vs 2.02±0.10, P<0.05) were elevated when compared to the control. In the C2C12 myotube model, tumor cell co-culture increased mitochondrial-lysosomal colocalization and upregulated mitophagy-related protein expression (P<0.05), consistent with the in vivo findings. Mdivi-1 treatment increased myotube diameter from 220.6±35.5 μm to 315.0±39.1 μm (R²=0.6665, P<0.05).

Mitophagy is activated in pancreatic cancer cachexia-induced muscle atrophy. Inhibiting mitophagy can effectively alleviate muscle atrophy induced by pancreatic cancer cachexia.

To observe the effect of bilirubin (Bil) on sorafenib (Sor) in the treatment of liver cancer and explore the involved molecular mechanisms.

In vitro, Huh7 cells were treated with different concentration of Sor (0, 2, 4, 8, 16, 32 μmol/L), of Bil (0, 2.5, 5, 10, 20, 40 μmol/L) and of Sor (0, 2, 4, 8, 16, 32 μmol/L) +Bil (20 μmol/L) for 24, 48 and 72 h. Cell viability was detected by CCK-8 assay. Huh7 cells were treated with Sor (4 μmol/L), Bil (20 μmol/L) and Sor (4 μmol/L)+Bil (20 μmol/L), cell cycle was detected by flow cytometry after 48 h, and the effects of drugs on cell proliferation were detected by clone formation assay after 14 d. In vivo, 16 mice bearing xenografts of Huh7 cells were divided into Con group, Bil group (25 mg·kg^-1·d^-1), Sor group (15 mg·kg^-1·d^-1) and Bil (25 mg·kg^-1·d^-1) +Sor (15 mg·kg^-1·d^-1) group (n=4), according to the S type sampling method. The expression levels of peroxisome proliferator-activated receptor (PPARα) and carnitine palmityl transferase 1A (CPT1A) were detected by Western blotting and real time PCR. After etomoxir (Eto), the inhibitor of CPT1A, was added, CCK-8 and clone formation assay were used to verify the mechanisms correlated with PPARα/CPT1A.

CCK-8 results showed that the inhibitory effect of Sor on Huh7 cells was in a dose and time-dependent manner (P<0.05), and Bil had no significant effect on Huh7 cells (P>0.05). Compared with Sor group, the cell proliferation in Bil+Sor group was increased (P<0.05), the relative cell colony formation rate was grown (P<0.05), and the number of cells in S phase was decreased (P<0.05). In vivo, results showed that the tumor volume and weight of Sor group were lower than that of Con group (P<0.05), and the tumor volume and weight of Bil+Sor group were higher than those of Sor group (P<0.05). Western blotting and RT-qPCR results showed that PPARα and CPT1A expression levels were increased in Bil+Sor group compared with Sor group (P<0.05). After Eto was added, the cell proliferation and the relative cell clone formation rate of Bil+Sor+Eto group were decreased compared with Bil+Sor group (P<0.05).

Bil impairs the antitumor effects of sorafenib treatment in hepatocellular carcinoma through fatty oxidation by up-regulating PPARα/CPT1A.