Journal: Cancer Research

Article Title: Spatial Multiomics Analyses Reveal That Diabetes Promotes Pancreatic Cancer Progression by Stimulating Cholesterol-Induced Neutrophil Extracellular Trap Formation

doi: 10.1158/0008-5472.CAN-25-2854

Figure Lengend Snippet: Spatial TME architecture unveils areas of neutrophil movement in PDAC with DM. A, Representative image of hematoxylin and eosin (H&E) staining from PDAC tissue showing tumor and stromal tissue regions. B, Volcano plot showing DEGs in tumor regions between DM and non-DM groups ( n = 5). C, GO analysis showing top 10 pathways (biological functions) enriched by upregulated genes in tumor region between DM and non-DM groups. D, Gene signatures enriched in neutrophil chemotaxis pathway between DM and non-DM groups. E, Spatially resolved gene expression images and expression levels of CXCL1 and CXCR2 between DM and non-DM patients. F, GO analysis indicating enriched top 10 pathways (biological functions) by upregulated genes between DM and non-DM groups in stromal region. G, KEGG analysis showing top 10 pathways enriched by upregulated genes between DM and non-DM groups in stromal region. H, Ligand–receptor interactions between tumor and stromal regions predicted by CellPhoneDB. I, The proportion of different clusters in stromal area between DM and non-DM groups. J, Bubble plots showing the overlaps between clusters and cell types. Red depth and black scales represent the score, indicating stronger association with the subtype. P values were determined by two-tailed ordinary one-way ANOVA with the Tukey post hoc test or Student t test. Data are represented as mean ± SEM.

Article Snippet: The primary antibodies used were CD66b (Abcam, cat. #ab300122, RRID: AB_3097803, 1:1,000), CXCL1 (Proteintech, cat. #12335-1-AP, RRID: AB_2087568, 1:100), CXCR2 (ABclonal, cat. #A3301, RRID: AB_2769086, 1:100), CXCR1 (Bio-Techne, MAB330-sp, 1:100), and Panck (Absin, 123684, 1:400).

Techniques: Staining, Chemotaxis Assay, Gene Expression, Expressing, Two Tailed Test

Journal: Cancer Research

Article Title: Spatial Multiomics Analyses Reveal That Diabetes Promotes Pancreatic Cancer Progression by Stimulating Cholesterol-Induced Neutrophil Extracellular Trap Formation

doi: 10.1158/0008-5472.CAN-25-2854

Figure Lengend Snippet: Inhibition of SREBP2-mediated cholesterol biosynthesis restrains CXCL1 expression and PDAC growth under DM. A, Experimental outline of murine PDAC orthotopic model. WT and db/db mice were implanted with Pan02 cells and sacrificed 3 weeks after tumor implantation. B, Representative images of tumors at the endpoint. The tumor volume and weight were compared. Scale bars, 5 mm; n = 6. C, The serum CXCL1 and cholesterol levels in the WT mice compared with the db/db mice. D, Representative immunofluorescence images and quantification of CD45 + Ly6g + and MPO + CitH3 + cells in the tumors. Scale bars, 50 µm; n = 6. Green, CD45- and CitH3-positive cells; red, Ly6g- and MPO-positive cells. E, Flow cytometric quantification of CXCR1 + and CXCR2 + neutrophils in the tumors between STZ-induced diabetic mice and control mice ( n = 5). F, Representative Western blot images and quantitative comparison of SREBP2 protein between WT and db/db mice ( n = 4). G, Representative Western blot images and quantitative comparison of SREBP2 protein between control and hyperglycemia samples ( n = 3). H, The relative mRNA expression of SREBP2 in the Panc-1 cells stimulated by high concentration of glucose versus normal glucose ( n = 3). I, The level of CXCL1 in cellular supernatant ( n = 3). J, A schematic diagram showing the experiment design. Three days after injection, mice were administered with betulin or simvastatin as indicated. K, The tumor volume and weight were compared. Scale bars, 5 mm; n = 6. L, The serum level of CXCL1 between control, betulin, and simvastatin groups. Representative immunofluorescence images and quantification of CD45 + Ly6g + and MPO + CitH3 + cells in the tumors. Scale bars, 50 µm; n = 5. M, Dotplot showing top 10 pathways enriched by upregulated genes in the DM group using KEGG analysis. N, Western blots of tumor samples showed the upregulation in phosphorylated AKT and NF-κB proteins and total AKT and NF-κB proteins. P values were determined by two-tailed ordinary one-way ANOVA with the Tukey post hoc test or Student t test. Data are represented as mean ± SEM.

Article Snippet: The primary antibodies used were CD66b (Abcam, cat. #ab300122, RRID: AB_3097803, 1:1,000), CXCL1 (Proteintech, cat. #12335-1-AP, RRID: AB_2087568, 1:100), CXCR2 (ABclonal, cat. #A3301, RRID: AB_2769086, 1:100), CXCR1 (Bio-Techne, MAB330-sp, 1:100), and Panck (Absin, 123684, 1:400).

Techniques: Inhibition, Expressing, Tumor Implantation, Immunofluorescence, Control, Western Blot, Comparison, Concentration Assay, Injection, Two Tailed Test

Journal: Cancer Research

Article Title: Spatial Multiomics Analyses Reveal That Diabetes Promotes Pancreatic Cancer Progression by Stimulating Cholesterol-Induced Neutrophil Extracellular Trap Formation

doi: 10.1158/0008-5472.CAN-25-2854

Figure Lengend Snippet: DM-mediated SREBP2 increases CXCL1 secretion by activating the PI3K/AKT and NF-κB signaling pathways. A, A schematic diagram showing the experiment design. B, WT and PAD4 −/− mice were implanted with shNC Pan02 cells or SREBP2 silencing Pan02 cells and sacrificed 3 weeks after tumor implantation. Scale bars, 5 mm; n = 5. The tumor volume and weight were compared. C, The serum level of CXCL1 between groups. D, The expression of CK5, Ly6g, CXCL1, CXCR2, and CXCR1 between control and SREBP2 knockdown tumors by mIHC. Scale bars, 100 μm ( n = 5). E, Representative immunofluorescence images and quantification of MPO + CitH3 + cells in the tumors. Scale bars, 50 µm; n = 5. F, Western blots showing the upregulation in phosphorylated AKT and NF-κB proteins and total AKT and NF-κB proteins in tumor tissues ( n = 4). G, Representative Western blot images and quantitative comparison of total and phosphorylation of NF-κB and AKT proteins in shNC and shSREBP2 tumor cells ( n = 3). H, A schematic diagram showing the experiment design. I, The tumor volume, weight, and CXCL1 level were compared between shNC and shSREBP2 with or without DHMEQ treatment. J, Representative immunofluorescence images and quantification of MPO + CitH3 + cells within tumors. Scale bars, 50 µm; n = 5. K, The tumor volume, weight, and CXCL1 level were compared between shNC and shSREBP2 with or without PI3K/AKT-IN-1 treatment. L, Representative immunofluorescence images and quantification of MPO + CitH3 + cells in the tumors. Scale bars, 50 µm; n = 5. P values were determined by two-tailed ordinary one-way ANOVA with the Tukey post hoc test or Student t test. Data are represented as mean ± SEM. ns, nonsignificant.

Article Snippet: The primary antibodies used were CD66b (Abcam, cat. #ab300122, RRID: AB_3097803, 1:1,000), CXCL1 (Proteintech, cat. #12335-1-AP, RRID: AB_2087568, 1:100), CXCR2 (ABclonal, cat. #A3301, RRID: AB_2769086, 1:100), CXCR1 (Bio-Techne, MAB330-sp, 1:100), and Panck (Absin, 123684, 1:400).

Techniques: Protein-Protein interactions, Tumor Implantation, Expressing, Control, Knockdown, Immunofluorescence, Western Blot, Comparison, Phospho-proteomics, Two Tailed Test

Journal: Cancer Research

Article Title: Spatial Multiomics Analyses Reveal That Diabetes Promotes Pancreatic Cancer Progression by Stimulating Cholesterol-Induced Neutrophil Extracellular Trap Formation

doi: 10.1158/0008-5472.CAN-25-2854

Figure Lengend Snippet: Cancer cell–derived cholesterol promotes NET formation through activating CXCL1/CXCR1/CXCR2 axis. A, Experimental outline of blockade of the CXCL1/CXCR1/CXCR2 axis in the orthotopic PDAC tumor model using neutralizing antibody reparixin. B, WT mice and STZ-induced diabetic mice were implanted with Pan02 cells and sacrificed 3 weeks after tumor implantation. Representative images (duplicates) of orthotopic PDAC tumors. Scale bar, 5 mm. The tumor volume and weight were compared ( n = 5). C, Representative images and quantification of Ki-67–positive cells in tumor tissues by IHC. Scale bars, 50 µm; n = 5. D, Representative immunofluorescence images and quantification of CD45 + Ly6g + and MPO + CitH3 + cells in the tumors. Scale bars, 100 µm; n = 5. Red, CD45-and MPO-positive cells; green, Ly6g- and CitH3-positive cells. E, Experimental outline of blockade of the CXCL1/CXCR1/CXCR2 axis in the orthotopic PDAC tumor model using neutralizing antibody SX-517. F, The tumor volume and weight were compared between WT, STZ, and STZ + SX517 groups. G, Representative immunofluorescence images and quantification of MPO + CitH3 + cells in the tumors. Scale bars, 50 µm. H, A schematic diagram showing the experiment design. I, The tumor volume and weight were compared between shNC and shCXCL1 groups. J, Representative immunofluorescence images and quantification of MPO + CitH3 + cells in the tumor tissues. Scale bars, 50 µm; n = 6. K, Flow cytometric quantification of CXCR1 + and CXCR2 + neutrophils in the tumors ( n = 6). L, Schematic representation of bicellular neutrophil-tumor cell coculture system. Representative immunofluorescence staining images of SYTOX to indicate NETs in the neutrophils. Scale bars, 50 µm. P values were determined by two-tailed ordinary one-way ANOVA with the Tukey post hoc test or Student t test. Data are represented as mean ± SEM.

Article Snippet: The primary antibodies used were CD66b (Abcam, cat. #ab300122, RRID: AB_3097803, 1:1,000), CXCL1 (Proteintech, cat. #12335-1-AP, RRID: AB_2087568, 1:100), CXCR2 (ABclonal, cat. #A3301, RRID: AB_2769086, 1:100), CXCR1 (Bio-Techne, MAB330-sp, 1:100), and Panck (Absin, 123684, 1:400).

Techniques: Derivative Assay, Tumor Implantation, Immunofluorescence, Staining, Two Tailed Test

Journal: Cancer Research

Article Title: Spatial Multiomics Analyses Reveal That Diabetes Promotes Pancreatic Cancer Progression by Stimulating Cholesterol-Induced Neutrophil Extracellular Trap Formation

doi: 10.1158/0008-5472.CAN-25-2854

Figure Lengend Snippet: DM promotes cholesterol biosynthesis and drives CXCL1-dependent NET formation in human PDAC. A, An UMAP plot displaying distinct clusters of 311,346 cells obtained from PDAC with DM ( n = 6) and without DM ( n = 13). B, Proportion of cell types. C, Enrichment score of cholesterol biosynthesis pathway in the cancer cells between DM and non-DM groups. D, The expression of SREBP2 in the cancer cells, ****, P < 0.0001. E, UMAP plot showing cell clusters of myeloid cells. Heatmap displayed the top DEGs between clusters. F, KEGG analysis indicating enriched pathways by upregulated genes in the neutrophils between PDAC with and without DM. G, Heatmap plotting different serum metabolites between DM and non-DM groups. H, Correlation between the levels of CXCL1 and cholesterol in the serum and overall survival was analyzed using Spearman correlation analysis. I, The staining of Panck, CD66b, CXCL1, CXCR2, and CXCR1 in PDAC tissues from patients with or without DM by mIHC. Scale bars, 100 μm ( n = 116). J, The percentages of CD66b + and CXCL1 + Panck + cells were calculated (PDCA patients, n = 96; PDAC with DM patients, n = 20). K, The staining of MPO, Panck, and CitH3 in PDAC tissues from patients with or without DM by mIHC. Scale bars, 100 μm ( n = 116). H&E, hematoxylin and eosin. L, Bar plots comparing mean distances from CXCR2 + neutrophils to tumor cells in PDAC with DM ( n = 96) vs. PDAC without DM ( n = 20). M, The number of MPO + CitH3 + cells in field were calculated.

Article Snippet: The primary antibodies used were CD66b (Abcam, cat. #ab300122, RRID: AB_3097803, 1:1,000), CXCL1 (Proteintech, cat. #12335-1-AP, RRID: AB_2087568, 1:100), CXCR2 (ABclonal, cat. #A3301, RRID: AB_2769086, 1:100), CXCR1 (Bio-Techne, MAB330-sp, 1:100), and Panck (Absin, 123684, 1:400).

Techniques: Expressing, Staining