Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Bar charts showing PD-L1 expression via flow cytometry. (B) Confocal microscopy images of MOC1, MOC2, and MOC2( PD-L1 ) cells merged from 3 channels (Supplemental Fig. 1A). Scale = 20 µm. MFI = median fluorescence intensity.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Expressing, Flow Cytometry, Confocal Microscopy, Fluorescence

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Saturation binding assay of 89 Zr-DFO-PD-L1 mAb on MOC2( PD-L1 ) cells. Curves for total bound (TB), specifically bound (SB), and nonspecifically bound (NSB) fractions fitted using given equation. (B) Specificity of binding assay of 89 Zr-DFO-PD-L1 mAb (2 nM; 1 h) on cancer cell lines pretreated with IFN-γ (20 ng/mL; 24 h) or PBS. Blocking with 100-fold molar excess of unlabeled PD-L1 mAb . Data were normalized to nontreated MOC2( PD-L1 ) cell–associated radioactivity. Significance determined using 2-way ANOVA with Bonferroni adjustment. C bound = bound ligand concentration; C ligand = ligand concentration; B max = maximum specific binding; K d = dissociation constant; ns = not significant.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Saturation Assay, Binding Assay, Blocking Assay, Radioactivity, Concentration Assay

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Coregistered PET/CT scans of MOC1, MOC2, and MOC2( PD-L1 ) tumor–bearing mice 48 h after 89 Zr-DFO-PD-L1 mAb injection (PET, coronal slice; CT, 3-dimensional maximum intensity projection). (B) 89 Zr-DFO-PD-L1 mAb biodistribution profile of MOC1, MOC2, and MOC2( PD-L1 ) tumor–bearing mice 48 h after injection (110 μg; adjusted specific activity = 0.018 MBq/μg). Data pooled from 3 studies. Inset graph shows correlation of PET quantification and biodistribution (BioD). Linear regression fit with 95% CI (dashed lines). (C) PD-L1 immunohistochemistry of tumor sections. BAT = brown adipose tissue; p.i. = postinjection.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Activity Assay, Immunohistochemistry

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: Experimental timeline of 89 Zr-DFO-PD-L1 mAb immuno-PET studies to monitor PD-L1 expression after single intratumoral dose of RP1 or PBS in MOC1 tumor–bearing mice. Figure created with BioRender.com. i.v. = intravenous; s.c. = subcutaneous.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Expressing

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Coregistered PET/CT scans of RP1 and PBS-treated mice on days 3 and 7 after treatment (PET, coronal slice; CT, 3-dimensional maximum intensity projection). Spleens of RP1-treated mice are highlighted (arrowheads). (B) Bar charts of 89 Zr-DFO-PD-L1 mAb uptake on day 3 vs. day 7 after RP1 or PBS. Significance determined using 2-way ANOVA with Bonferroni adjustment on log-transformed data. (C) Radiomics heatmaps of tumor and spleen VOIs from day 3 scans. x -axis shows feature numbers from Supplemental Tables 3 and 4. Feature values normalized to z score. Decision trees shown to left of each heatmap. TD Ln = tumor-draining lymph node.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Positron Emission Tomography-Computed Tomography, Transformation Assay

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) PD-L1 immunohistochemistry of MOC1 tumor and spleen sections from immuno-PET studies on day 3 and 7 after single intratumoral RP1 or PBS dose. (B) Concentrations of GM-CSF, IFN-α, IFN-β and IFN-γ in tumors on day 3 after RP1 or PBS. Significance determined using multiple unpaired t tests on log-transformed data with Benjamini-Hochberg FDR correction (FDR < 1%).

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Immunohistochemistry, Transformation Assay

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Representative fluorescent images of MOC1 and MOC2 cells after 48 h of incubation with increasing titers of GFP-expressing RP1-15. GFP shown in gray scale. (B) MOC1 and MOC2 cell viability after 48 h with increasing RP1 titers in vitro, assessed via CellTiter-Glo assay. Significance determined using 1-way ANOVA with Dunnett test. RLU = relative light units.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Incubation, Expressing, In Vitro, Glo Assay

Journal: Genes & Diseases

Article Title: Chloride channel accessory 4 suppresses stem cell-like properties of colorectal cancer and enhances anti-PD-1 immunotherapy

doi: 10.1016/j.gendis.2025.101859

Figure Lengend Snippet: CLCA4 overexpression enhanced the therapeutic effect of anti-PD-1. (A) In vivo tumorigenicity assay in nude mice was performed to detect the therapeutic effect of CLCA4 overexpression combined with anti-PD-1 antibody. (B) Tumor growth curve was monitored from nude mice with different treatment groups. (C) Weights of tumors from nude mice with different treatments were detected. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (D) Analysis of the survival times of mice in each group ( n = 8 per group), and the experiment was terminated 60 days after tumor inoculation. Unpaired two-tailed t -test (mean ± standard deviation). (E) Immunofluorescence or immunohistochemistry staining was performed to detect the infiltration levels of CD8 + T cells, GZMB, Perforin + cells, Ki67 + , Bmi-1 + , and Oct4 + cells in tumors from different treatment groups. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (F) Hematoxylin-eosin staining of liver metastases in each group. (G) Quantitative analysis of the liver weight in each group. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (H) Quantitative analysis of the liver metastasis area/total liver area in each group. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation).

Article Snippet: After 7 days, mice were intraperitoneally treated with either an in vivo blocking antibody against mouse PD-1 (Clone: 29F.1A2, BioXcell, Cat# BP0273) or a rat IgG2a isotype control antibody (Clone: 2A3, BioXcell, Cat# BP0089).

Techniques: Over Expression, In Vivo, Tumorigenicity Assay, Standard Deviation, Two Tailed Test, Immunofluorescence, Immunohistochemistry, 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: 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: ABHD16A promotes ILC3 aggregation and IL22 release in the immune microenvironment. A, A total of 5 × 10 6 MFC-luc or Abhd16a -knockdown MFC-luc cells were injected into the epidermis of the greater curvature of the stomach in the 615 mice. Tumor growth was monitored weekly through bioluminescence imaging from the first day of MFC cell inoculation. Scale bar, 2.000e+4 – 5.000e + 5 p/s/cm 2 /sr. B and C, Representative images of the excised stomachs ( B ) and the orthotopic tumor volume ( C ) on day 28 after gastric cancer cells were implanted orthotopically ( n = 9 per group). D, H&E images of the stomach, peritoneum, liver, intestine, and lung on day 42 after MFC or Abhd16a -knockdown MFC cells were implanted into 615 mice. Scale bar, 400 μm. E–G, Representative images ( E ), volume ( F ), and weight ( G ) of control and Abhd16a -knockdown subcutaneous tumors ( n = 5 per group). H, IHC images of PD-L1 in control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues. Scale bar, 500 μm. I, Detection of mouse cytokines in the TIF of orthotopic gastric cancer tissues by proteome profiler mouse XL cytokine array. J, Flow cytometry gating strategy and frequencies of ILC3s and Th17/Th22 cells out of CD45 + cells in control and Abhd16a -knockdown gastric cancer tissues in 615 mice. K, Flow cytometry gating strategy and frequencies of ILC3s out of ILCs in control and Abhd16a -knockdown gastric cancer tissues in Rag1 −/− mice. ***, 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: Knockdown, Injection, Imaging, Control, Flow Cytometry

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: High expression of ABHD16A mediates the cumulative release of LysoPS into the TME. A, mIF images of the orthotopic gastric cancer (GC) tissues stained for PD-L1, RORC, and CD3. Yellow, PD-L1 + tumor cells; red, RORC + ILC3s; green, CD3 + cells. Scale bars, 100 μm (left) and 25 μm (right). B and C, Flow cytometry gating strategy and frequencies of ILC3s ( B ) and proportions of ILC3s in total CD45 + cells ( C ) isolated from control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues ( n = 3 per group). D and E, Flow cytometry gating strategy and frequencies of ILC3s ( D ) and proportions of ILC3s in total CD45 + cells ( E ) derived from the peripheral blood of healthy controls (HC) and patients with gastric cancer ( n = 8 per group). F and G, LC-MS/MS analysis of 18:0 and 18:1 LysoPS in the control and Abhd16a -knockdown orthotopic ( F ) and subcutaneous ( G ) gastric cancer tissues ( n = 3 per group). H–J, ELISA was used to measure levels of LysoPS in gastric cancer cell supernatant ( H ), TIF ( I ), and in vitro tumor culture supernatant ( J ) of control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tumors ( n = 3 per group). K, LysoPS levels in the serum of healthy controls and patients with gastric cancer detected by ELISA. *, P < 0.05; **, 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: Expressing, Staining, Flow Cytometry, Isolation, Control, Knockdown, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, In Vitro

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

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: For immunotherapy, anti-mouse PD-L1 antibody (100 μg/mouse, TargetMol, T78270 ), GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg, MERYER, 907952), and XBP1s inhibitor (STF-083010, 30 mg/kg, MedChemExpress, HY-15845) were intraperitoneally injected once every 3 days, and ACh receptor inhibitor (darifenacin, 2.5 mg/kg, MedChemExpress, HY–A0033) was intraperitoneally injected every day after tumor formation.

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: ABHD16A promotes ILC3 aggregation and IL22 release in the immune microenvironment. A, A total of 5 × 10 6 MFC-luc or Abhd16a -knockdown MFC-luc cells were injected into the epidermis of the greater curvature of the stomach in the 615 mice. Tumor growth was monitored weekly through bioluminescence imaging from the first day of MFC cell inoculation. Scale bar, 2.000e+4 – 5.000e + 5 p/s/cm 2 /sr. B and C, Representative images of the excised stomachs ( B ) and the orthotopic tumor volume ( C ) on day 28 after gastric cancer cells were implanted orthotopically ( n = 9 per group). D, H&E images of the stomach, peritoneum, liver, intestine, and lung on day 42 after MFC or Abhd16a -knockdown MFC cells were implanted into 615 mice. Scale bar, 400 μm. E–G, Representative images ( E ), volume ( F ), and weight ( G ) of control and Abhd16a -knockdown subcutaneous tumors ( n = 5 per group). H, IHC images of PD-L1 in control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues. Scale bar, 500 μm. I, Detection of mouse cytokines in the TIF of orthotopic gastric cancer tissues by proteome profiler mouse XL cytokine array. J, Flow cytometry gating strategy and frequencies of ILC3s and Th17/Th22 cells out of CD45 + cells in control and Abhd16a -knockdown gastric cancer tissues in 615 mice. K, Flow cytometry gating strategy and frequencies of ILC3s out of ILCs in control and Abhd16a -knockdown gastric cancer tissues in Rag1 −/− mice. ***, P < 0.001.

Article Snippet: For immunotherapy, anti-mouse PD-L1 antibody (100 μg/mouse, TargetMol, T78270 ), GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg, MERYER, 907952), and XBP1s inhibitor (STF-083010, 30 mg/kg, MedChemExpress, HY-15845) were intraperitoneally injected once every 3 days, and ACh receptor inhibitor (darifenacin, 2.5 mg/kg, MedChemExpress, HY–A0033) was intraperitoneally injected every day after tumor formation.

Techniques: Knockdown, Injection, Imaging, Control, Flow Cytometry

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: High expression of ABHD16A mediates the cumulative release of LysoPS into the TME. A, mIF images of the orthotopic gastric cancer (GC) tissues stained for PD-L1, RORC, and CD3. Yellow, PD-L1 + tumor cells; red, RORC + ILC3s; green, CD3 + cells. Scale bars, 100 μm (left) and 25 μm (right). B and C, Flow cytometry gating strategy and frequencies of ILC3s ( B ) and proportions of ILC3s in total CD45 + cells ( C ) isolated from control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues ( n = 3 per group). D and E, Flow cytometry gating strategy and frequencies of ILC3s ( D ) and proportions of ILC3s in total CD45 + cells ( E ) derived from the peripheral blood of healthy controls (HC) and patients with gastric cancer ( n = 8 per group). F and G, LC-MS/MS analysis of 18:0 and 18:1 LysoPS in the control and Abhd16a -knockdown orthotopic ( F ) and subcutaneous ( G ) gastric cancer tissues ( n = 3 per group). H–J, ELISA was used to measure levels of LysoPS in gastric cancer cell supernatant ( H ), TIF ( I ), and in vitro tumor culture supernatant ( J ) of control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tumors ( n = 3 per group). K, LysoPS levels in the serum of healthy controls and patients with gastric cancer detected by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For immunotherapy, anti-mouse PD-L1 antibody (100 μg/mouse, TargetMol, T78270 ), GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg, MERYER, 907952), and XBP1s inhibitor (STF-083010, 30 mg/kg, MedChemExpress, HY-15845) were intraperitoneally injected once every 3 days, and ACh receptor inhibitor (darifenacin, 2.5 mg/kg, MedChemExpress, HY–A0033) was intraperitoneally injected every day after tumor formation.

Techniques: Expressing, Staining, Flow Cytometry, Isolation, Control, Knockdown, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, In Vitro

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: For immunotherapy, anti-mouse PD-L1 antibody (100 μg/mouse, TargetMol, T78270 ), GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg, MERYER, 907952), and XBP1s inhibitor (STF-083010, 30 mg/kg, MedChemExpress, HY-15845) were intraperitoneally injected once every 3 days, and ACh receptor inhibitor (darifenacin, 2.5 mg/kg, MedChemExpress, HY–A0033) was intraperitoneally injected every day after tumor formation.

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: For immunotherapy, anti-mouse PD-L1 antibody (100 μg/mouse, TargetMol, T78270 ), GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg, MERYER, 907952), and XBP1s inhibitor (STF-083010, 30 mg/kg, MedChemExpress, HY-15845) were intraperitoneally injected once every 3 days, and ACh receptor inhibitor (darifenacin, 2.5 mg/kg, MedChemExpress, HY–A0033) was intraperitoneally injected every day after tumor formation.

Techniques: Injection, Immunofluorescence