Journal: iMeta

Article Title: Single‐cell sequencing reveals the role of IL‐33 + endothelial subsets in promoting early gastric cancer progression

doi: 10.1002/imt2.70050

Figure Lengend Snippet: Stromal cell remodeling in GC progression. (A) UMAP showing the CAF subcluster. (B) Representative marker genes of the CAF subcluster. (C) Proportions of each subset of CAFs. (D) Proportions of four representative CAF subpopulations (CAF mat ‐ POSTN , CAF infla ‐ PRSS35 , CAF infla ‐ MFAP5 , and CAF EMT ‐ KRT19 ) across tissue groups. (E) Expression specificity of PRSS35 and MFAP5 in CAFs. (F) Survival analysis of PRSS35 and MFAP5 in GC. (G) UMAP view of 7 endothelial cell clusters. (H) Proportions of each subset of endothelial cell clusters. (I) Proportions of representative IL‐33 + Venous‐1 subpopulations across tissue groups. (J) Heatmap of marker gene expression in endothelial cell subpopulations. Red signifies increased expression. (K) Survival analysis of IL‐33 in GC. (L) Expression specificity of IL‐33 in endothelial cells. (M) KEGG bubble map of DEGs between IL‐33 + Venous‐1 and other endothelial cell subpopulations. p values were calculated via one‐way Kruskal‒Wallis rank‒sum tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CAFs, cancer‐associated fibroblasts; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; UMAP, uniform manifold approximation and projection.

Article Snippet: Recombinant IL‐33 (#HY‐ P70475 , MCE, CA) was diluted with gastric cancer organoid medium to concentrations of 20, 30, 50, 80, and 100 ng/mL.

Techniques: Marker, Expressing, Gene Expression

Journal: iMeta

Article Title: Single‐cell sequencing reveals the role of IL‐33 + endothelial subsets in promoting early gastric cancer progression

doi: 10.1002/imt2.70050

Figure Lengend Snippet: IL‐33 drives endothelial angiogenesis, and IL‐33 + ECs promote EGC and AGC growth ex vitro. (A) IHC staining of IL‐33, CD31, ST2, and H&E. Representative images of NAG, EGC, and AGC tissues are shown. Scale bars, 100 and 200 µm. (B) Tube formation images and bar graphs of HUVECs with knockdown or overexpression of IL‐33. (C) Proliferation of HUVECs with IL‐33 knockdown or overexpression. (D) Adhesion ability of HUVECs with IL‐33 knockdown or overexpression. (E, F) Apoptosis of HUVECs with IL‐33 knockdown or overexpression. (G, H) Images and histograms of the effects of IL‐33 on the proliferative ability of organoids. EGC and AGC organoids were treated with the culture supernatant of HUVECs with knockdown or overexpression of IL‐33 and recombinant IL‐33, and the ATP of the organoids was detected. p values were calculated via Student's t ‐test. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. AGC, advanced gastric cancer; ATP, adenosine triphosphate; ECs, endothelial cells; EGC, early gastric cancer; H&E, hematoxylin‐eosin staining; HUVECs, human umbilical vein endothelial cells; IHC, immunohistochemistry.

Article Snippet: Recombinant IL‐33 (#HY‐ P70475 , MCE, CA) was diluted with gastric cancer organoid medium to concentrations of 20, 30, 50, 80, and 100 ng/mL.

Techniques: Immunohistochemistry, Knockdown, Over Expression, Recombinant, Staining

Journal: iMeta

Article Title: Single‐cell sequencing reveals the role of IL‐33 + endothelial subsets in promoting early gastric cancer progression

doi: 10.1002/imt2.70050

Figure Lengend Snippet: IL‐33 + ECs promote EGC and AGC angiogenesis and growth in vivo . (A) Diagram of a mouse subcutaneous transplanted tumor (EC cells and organoids). (B, C). Macroscopic images of the excised subcutaneous tumor mass upon sacrifice. No tumors were observed at the injection sites of the only EGC and AGC organoids (H&E‐confirmed inflammatory tissue). EC overexpression of IL‐33 promoted the growth of organoids (both EGC and AGC) subcutaneously (B). EC knockdown of IL‐33 inhibited the growth of subcutaneous organoids (both EGC and AGC) (both EGC and AGC) (C). (D, E) Tumor volume was monitored every four days, and tumor growth curves were drawn. EC‐OE‐NC had no significant effect on the growth of tumors compared with that of the only EGC organoid group (D). EC‐OE‐IL‐33 and EC‐OE‐NC had no significant effect on the growth of AGC organoids (E). (F, G) Tumor weights of the extracted subcutaneous tumors at the endpoint. ECs‐OE‐NC had no significant effect on the weight of tumors compared with the weight of tumors in the group with only EGC organoids. EC‐OE‐IL‐33 and EC‐OE‐NC had no significant effect on the weight of AGC organoids (F). The rate of subcutaneous tumor formation in the EC‐OE‐IL‐33 group of EGCs was greater than that in the EC‐OE‐NC group, and there was no difference in the other groups (G). (H) Ultrasound image of a mouse subcutaneous tumor. The EC‐sh‐IL‐33 group had more dark areas of fluid in the center of the subcutaneous tumors and a lower central blood supply. (I) Ultrasonic PSV detection of subcutaneous tumors in vivo at the endpoint. (J) Ultrasonic RI detection of subcutaneous tumors in vivo at the endpoint. The data represent the means ± SDs from six mice per group. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 versus empty vector control (two‐sided Wilcoxon rank‐sum test). AGC, advanced gastric cancer; B mode, brightness mode; CD mode, color Doppler mode; EGC, early gastric cancer; ECs, endothelial cells; H&E, hematoxylin‐eosin staining; NC, negative control; OE‐IL‐33, overexpression of IL‐33; PW mode, pulse‒wave Doppler mode; PSV, peak systolic velocity; RI, resistance index; sh‐IL‐33, knockdown of IL‐33.

Article Snippet: Recombinant IL‐33 (#HY‐ P70475 , MCE, CA) was diluted with gastric cancer organoid medium to concentrations of 20, 30, 50, 80, and 100 ng/mL.

Techniques: In Vivo, Injection, Over Expression, Knockdown, Plasmid Preparation, Control, Staining, Negative Control

Journal: iMeta

Article Title: Single‐cell sequencing reveals the role of IL‐33 + endothelial subsets in promoting early gastric cancer progression

doi: 10.1002/imt2.70050

Figure Lengend Snippet: IL‐33 upregulates the expression of related adhesion molecules in ECs and EGC. (A) KEGG bubble map of DEGs between IL‐33 ‐knockdown ECs and negative control ECs. (B) RT‐qPCR was used to verify the DEGs in ECs after knockdown or overexpression of IL‐33 . (C) WB was used to verify the gene expression of ECs after knockdown or overexpression of IL‐33. (D) ‌Tissue immunofluorescence showed that the expressions of IL‐33 ( p = 0.045) and CD34 ( p = 0.004) were higher in submucosal carcinoma than in intramucosal carcinoma. EGC with high expression of IL‐33 ( p = 0.048) and CD34 ( p = 0.011) was more malignant. (E) ‌DEG volcano plot of EGC organoids after the addition of IL‐33. (F) ‌GSEA revealed that DEGs were enriched in the cell adhesion and MAPK signaling pathways after ECs overexpressed IL‐33. (G) RT‒qPCR was used to verify the DEGs of EGC organoids after IL‐33 overexpression. (H) WB was used to verify the gene expression of organoids after IL‐33 overexpression. (I) Proposed a model of the mechanism in this study. IL‐33 activates the IL‐3/ST2 signaling pathway in an autocrine manner, enhances the expression of PECAM1 and CD34 in ECs, and promotes angiogenesis. High levels of IL‐33 also bind to ST2 in EGC organoids in a paracrine manner and activate the MAPK signaling pathway after the expression of KRT17 is upregulated, promoting the growth of EGC. IHC of IL‐33 at the boundary of normal gastric mucosa and EGC. Scale bar, 200 µm. The data are presented as the means ± SDs of three independent experiments. * p < 0.05; **** p < 0.0001 versus empty vector control (two‐sided Wilcoxon rank‐sum test). DEGs, differentially expressed genes; ECs, endothelial cells; EGC, early gastric cancer; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen‐activated protein kinase; NC, negative control; OE‒IL‒33, overexpressed IL‒33; RT‐qPCR, real‐time quantitative polymerase chain reaction; sh‒IL‒33, knocked down IL‒33; WB, western blot.

Article Snippet: Recombinant IL‐33 (#HY‐ P70475 , MCE, CA) was diluted with gastric cancer organoid medium to concentrations of 20, 30, 50, 80, and 100 ng/mL.

Techniques: Expressing, Knockdown, Negative Control, Quantitative RT-PCR, Over Expression, Gene Expression, Immunofluorescence, Protein-Protein interactions, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Western Blot