Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: Elevated ABHD16A triggered by ACh was associated with poor prognosis in patients with gastric cancer. A, Representative IHC images of ABHD16A expression in human normal gastric tissues and gastric cancer tissues from the TMA with magnifications of 40×, 100×, and 200×. Scale bar for 200×, 100 μm. B–D, Relationship of ABHD16A IHC score to clinical stage ( B ), pT stage ( C ), and lymph node metastasis ( n = 80; D ). E, Cumulative survival curves of patients with gastric cancer with high or low ABHD16A expression based on the TMA. F, IHC images and score of ABHD16A in S100 + or S100 − gastric cancer tissues. Scale bar, 100 μm. G, Procedure for coculture of DRG neurons and gastric cancer cells. H, Western blotting was used to analyze ABHD16A levels in MFC cells cocultured with DRG neurons or DRG neuron–derived CM. GAPDH served as the loading control. I, Expression of HIF1A and ABHD16A in neurotransmitter [HA, dopamine (DA), 5-HT, norepinephrine (NE), ACh]-treated MFC cells and ACh-treated MGC-803 cells. J, ACh concentration was detected in DRG CM by ELISA. K, Tumor volume of orthotopic gastric cancer tumors with or without vagotomy. L, Representative mIF staining images of FOXP3, CD163, CD11b, CD8, PD-L1, and Pan-CK in gastric cancer tissues with high or low expression of ABHD16A. Scale bar, 100 μm. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Expressing, Western Blot, Derivative Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: IL22 upregulates PD-L1 expression in gastric cancer cells through the UPR IRE1α–XBP1 axis. A, mIF images show the alterations of PD-L1 + tumor cells (purple), CD4 + (green), and CD8 + (red) T cells in orthotopic gastric cancer tumors from control and Abhd16a -knockdown mice following IL22 treatment. Scale bar, 50 μm. B, KEGG pathway enrichment analysis of RNA-seq data of gastric cancer tissues with or without IL22 treatment. C, RT-PCR was used to assess the mRNA expression of key downstream molecules of the UPR branches ( XBP1 , ATF4 , ATF6 ) in control and IL22RA1 -knockdown gastric cancer cells. D, Western blotting analysis of PD-L1 and XBP1s levels in control and IL22RA1 -knockdown MGC-803 cells treated with IL22 (100 μg/L). E, Western blotting detection of PD-L1 and XBP1s levels in XBP1- knockdown MGC-803 cells treated with IL22 and MGC-803 cells treated with IL22 or XBP1s inhibitor (STF083010, 30 μmol/L) in combination with IL22. F, The binding sequence of XBP1 on the CD274 promoter. G and H, ChIP ( G ) and luciferase reporter assay ( H ) showing the transcriptional regulation of CD274 by XBP1s under IL22 stimulation. I, Orthotopic gastric cancer mouse models ( n = 5 per group) were injected with anti-IL22 (200 μg per mouse), anti-CD90.2 antibody (150 μg per mouse), anti-CD90.2 antibody in combination with IL22 (500 ng per mouse), or anti-CD90.2 antibody in combination with XBP1s inhibitors (STF083010, 30 mg/kg) and IL22 for 2 weeks. IHC analysis was used to show IL22, XBP1s, and PD-L1 levels in gastric cancer tissues. Scale bar, 200 μm. J, Tumor volume of orthotopic gastric cancer models under treatments the same as in I . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Expressing, Control, Knockdown, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Sequencing, Luciferase, Reporter Assay, Injection

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: Combination therapy enhances the anti–PD-L1 immunotherapeutic effect in gastric cancer. A and B, After tumor formation, the orthotopic gastric cancer mice ( n = 5 per group) were treated with anti–PD-L1 (100 μg per mouse), GPR34 inhibitor (20 mg/kg), or XBP1s inhibitor (30 mg/kg) every 3 days or ACh inhibitor (2.5 mg/kg) daily. Combinations of anti–PD-L1 with each inhibitor followed the every 3-day dosing schedule for a total duration of 2 weeks via i.p. injection. Living images were used to monitor tumor progression at 5-day intervals from the time of drug administration ( A ); IHC and mIF were performed to detect PD-L1 and XBP1s levels and proportions of CD4 + (green) and CD8 + (red) T cells in gastric cancer tissues at the end of treatments ( B ). Scale bars, 1.000e+5 –∼ 5.000e + 5 p/s/cm 2 /sr for living images; 200 μm for IHC; 50 μm for immunofluorescence. C and D, Representative images ( C ) and tumor volume ( D ) of subcutaneous tumors. The administration protocol for the mice was consistent with the description provided in A and B . **, P < 0.01; ***, P < 0.001.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Injection, Immunofluorescence