Journal: Advanced Science
Article Title: CYP4X1/sEH‐Dependent Endocannabinoid Metabolism Drives Fibroblast‐Mediated Immunosuppression to Limit Immunotherapy in Colon Cancer
doi: 10.1002/advs.202507695
Figure Lengend Snippet: CYP4X1/sEH‐derived 14,15‐EET‐EA triggers immunosuppression via GPR119‐Gs/β‐arrestin 2 signaling. MRC5 fibroblasts were treated with or without GPR119 knockdown, followed by stimulation with 14,15‐EET‐EA. A) αSMA, PD‐L1, CXCL12, and TGF‐β mRNA levels in the above‐treated MRC5 fibroblasts ( n = 5). B) Transwell migration analysis of Tregs cocultured with the above‐treated MRC5 fibroblasts ( n = 5). C) GZMB and IFNγ mRNA levels in CD8 + T cells cocultured with the above‐treated MRC5 fibroblasts were measured by qPCR ( n = 5). D) MC38‐luciferase cells were mixed with Gpr119 shRNA‐ or scramble shRNA‐treated L929 fibroblasts, and then co‐implanted into C57BL/6 mice. In vivo bioluminescent images and quantification of the indicated groups ( n = 8). E) Representative IF staining of αSMA (red) and vimentin (green) in MC38 tumor tissues ( n = 5). Scale bar, 50 µm. F) PD‐L1, CXCL12, and TGF‐β mRNA levels in the CAFs ( n = 5). G) The infiltration of CD8 + T cells in tumor tissues was determined by flow cytometry ( n = 5). H) Representative IF staining and frequency analysis of CD8 (red) and GZMB (green) or IFN‐γ (green) in tumor tissues ( n = 5). Scale bar, 50 µm. I) Treg accumulation in tumor tissues was analyzed and quantified by flow cytometry ( n = 5). J) IF analysis of tumor vessel normalization in the indicated groups ( n = 5). Scale bar, 50 µm. K) cAMP level in MRC5 and L929 fibroblasts incubated with the CM from CYP4X1 KD EPHX2 OE ‐expressed colon cancer cells with or without 14,15‐EET‐EA supplementation ( n = 5). L,M) β‐arrestin 2, p‐EGFR, and p‐Akt protein levels in MRC5 and L929 fibroblasts incubated with CM from CYP4X1 KD EPHX2 OE ‐expressed colon cancer cells with or without 14,15‐EET‐EA supplementation ( n = 5). N) β‐arrestin 2, p‐EGFR, and p‐Akt protein levels in L929 and MRC5 fibroblasts with or without GPR119 knockdown, followed by stimulation with 14,15‐EET‐EA. O,P) p‐STAT3 protein level, PD‐L1, CXCL12, and TGF‐β mRNA levels in MRC5 fibroblasts of the indicated groups ( n = 5). Data are presented as mean ± SEM. P values were determined using one‐way ANOVA (A‐C, K‐M, and P) or Student's t ‐tests (D, F‐J). * P < 0.05 and ** P < 0.01 vs. control or sh‐Scram; ^ P < 0.05 and ^^ P < 0.01 vs. 14,15‐EET‐EA+si‐Scram or 14,15‐EET‐EA; # P < 0.05 and ## P < 0.01 vs. CYP4X1 KD EPHX2 OE group.
Article Snippet: Next, slides were incubated with primary antibodies against CYP4X1 (1:100, Invitrogen, #PA5‐101319), sEH (1:200, Proteintech, #10833‐1‐AP), GPR119 (1:50, Affinity, #DF4892), α‐SMA (1:300, BOSTER, #BM0002), FOXP3 (1:400, Cell Signaling Technology, #12653), CD31 (1:300, Santa Cruz, #sc‐376764), CD8 (1:2, DAKO, #IR623), cytokeratin (CK, 1:2, DAKO, #IS05330‐2), PDPN (1:200, Huabio, #ET1703‐61), HLA‐DRA (1:1000, Huabio, #ET1610‐66), or CXCL12 (1:100, Huabio, #ER1902‐35).
Techniques: Derivative Assay, Knockdown, Migration, Luciferase, shRNA, In Vivo, Staining, Flow Cytometry, Incubation, Control