Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: Nectin-4 is significantly expressed in esophageal squamous cell carcinoma (A) Gene distribution showing m6A methylation sites identified through MeRIP-seq analysis. (B) Distribution pattern of m6A enrichment peaks. (C) Volcano plot analysis of differentially expressed membrane proteins from transcriptome sequencing data (|log2 FC| ≥ 2 and P ≤ 0.05). (D) Venn diagram depicting the overlap between differentially expressed membrane protein genes from transcriptome sequencing and genes with CDS region methylation identified by MeRIP-seq. (E) RT-qPCR validation comparing expression levels of Nectin-4, GJB3, and DSG2 between normal esophageal epithelium and esophageal squamous carcinoma cell lines. (F) Nectin-4 expression analysis in esophageal cancer using the GEPIA2 online database. (G) Western blot analysis comparing Nectin-4 protein levels between five ESCC cell lines (TE-1, KYSE-30, KYSE-150, KYSE-410, and KYSE-510) and normal esophageal epithelial cells (HET-1A). *P < 0.05; **P < 0.01.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Methylation, Membrane, Sequencing, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: Nectin-4 facilitates the progression of esophageal squamous carcinoma (A,B) Cell proliferation effects of Nectin-4 in KYSE-410 and KYSE-510 esophageal squamous carcinoma cell lines were validated by CCK8 and colony formation assays. (C,D) Impact of Nectin-4 on migration and invasion capabilities of KYSE-410 and KYSE-510 cells was assessed through wound healing and Transwell assays. (E) In vivo effects of Nectin-4 were evaluated using a subcutaneous xenograft model in nude mice with KYSE-410 cells (n = 8). (F,G) Immunohistochemical and H&E staining for Nectin-4, Ki-67, and vimentin were performed to evaluate the effects of control and Nectin-4 knockdown on tumor growth, proliferation, and metastatic potential (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Migration, In Vivo, Immunohistochemical staining, Staining, Control, Knockdown

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: METTL3 affects the progression of esophageal squamous cell carcinoma by facilitating the stabilization of Nectin-4 via m 6 A modification (A) Designated binding sites for Nectin-4 that exhibit m6A methylation according to MeRIP-seq data. (B) The m6A methylation target database M6A2Target was used to predict online that Nectin-4 undergoes m6A methylation via associated methyltransferases. (C) The online database TIMER 2.0 was used to analyze the expression of METTL3 in esophageal cancer. (D,E) RT-qPCR and western blot analyses confirmed the expression of METTL3 in five ESCC cell lines relative to that in normal esophageal epithelial cells. (F) The SRAMP online database was used to predict the m6A methylation sites of Nectin-4. (G) Dual-luciferase activity assay validation confirmed that METTL3 facilitates the m6A methylation alteration of Nectin-4. (H) MeRIP-qPCR confirmed that METTL3 facilitates alterations in the m6A methylation of Nectin-4. (I) Actinomycin D confirmed that METTL3 influences the stability of Nectin-4 through m6A modification. (J) RT-qPCR validation of the impact of METTL3 on Nectin-4 mRNA levels. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Modification, Binding Assay, Methylation, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Biomarker Discovery

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: METTL3 facilitates the progression of esophageal squamous cell carcinoma (A,B) Cell proliferation effects of METTL3 in KYSE-410 and KYSE-510 esophageal squamous carcinoma cell lines were evaluated by CCK8 and colony formation assays. (C,D) Impact of METTL3 on migration and invasion capabilities of KYSE-410 and KYSE-510 cells was assessed through wound healing and Transwell assays. (E) In vivo effects of METTL3 were evaluated using a subcutaneous xenograft model in nude mice with KYSE-410 cells (n = 8). (F, G) Immunohistochemical staining for METTL3, vimentin, Ki-67, and Nectin-4 was performed to evaluate the effects of control and METTL3 knockdown on tumor growth, proliferation, and metastatic potential, with H&E staining used to assess METTL3-mediated metastasis (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Migration, In Vivo, Immunohistochemical staining, Staining, Control, Knockdown

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: Nectin-4 overexpression mitigates the suppressive effect of METTL3 knockdown on the progression of esophageal squamous cell carcinoma (A, B) CCK8 and colony formation assays demonstrated that Nectin-4 overexpression attenuated the anti-proliferative effects of METTL3 knockdown in KYSE-410 and KYSE-510 esophageal squamous cell carcinoma cell lines. (C,D) Wound healing and Transwell assays revealed that Nectin-4 expression counteracted the inhibitory effects of METTL3 knockdown on migration and invasion in KYSE-410 and KYSE-510 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Over Expression, Knockdown, Expressing, Migration

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: VNN1 facilitates the progression of esophageal squamous carcinoma (A) Heatmap showing differential gene expression following Nectin-4 knockdown transcriptome sequencing. (B) Venn diagram illustrating overlap between downregulated genes in Nectin-4-knockdown cells (log2 FC ≤ –1.5 and P ≤ 0.01) and highly expressed genes in esophageal cancers from TCGA database. (C) RT-qPCR validation comparing expression levels of VNN1, CCL28, and PLEKHS1 between normal esophageal epithelial cells and esophageal squamous carcinoma cell lines. (D) RT-qPCR analysis of VNN1,CCL28, and PLEKHS1 expression changes following Nectin-4 knockdown. (E) VNN1 expression analysis in esophageal carcinoma using GEPIA2 database. (F) Western blot analysis of VNN1 protein levels following Nectin-4 knockdown. (G) Western blot analysis comparison of VNN1 protein expressions between normal esophageal epithelium and esophageal squamous carcinoma cell lines. (H) KEGG pathway enrichment analysis of downregulated genes from transcriptome sequencing. (I,J) Cell proliferation effects of METTL3 in KYSE-410 and KYSE-510 cells assessed by CCK8 and colony formation assays. (K,L) Impact of METTL3 on cell migration and invasion evaluated through wound healing and Transwell assays. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Gene Expression, Knockdown, Sequencing, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot, Comparison, Migration

Journal: Acta Biochimica et Biophysica Sinica

Article Title: METTL3-mediated m 6 A modification facilitates Nectin-4-induced VNN1 upregulation and promotion of ESCC progression

doi: 10.3724/abbs.2025108

Figure Lengend Snippet: Nectin-4, METTL3, and VNN1 in tissues from individuals with esophageal squamous carcinoma hold clinical importanceare clinically important (A) Immunohistochemical analysis of Nectin-4 expression in esophageal squamous carcinoma specimens and adjacent normal tissues (n = 50). (B–D) Analysis of correlations between Nectin-4 expression and tumor grade, stage, age, and sex in ESCC patients, with statistical comparison between tumor and non-tumor tissues. (E) Immunohistochemical analysis of METTL3 expression in ESCC specimens and adjacent normal tissues (n = 50). (F–H) Analysis of correlations between METTL3 expression and tumor stage, grade, age, and sex in ESCC patients, with statistical comparison between tumor and non-tumor tissues. (I) Immunohistochemical analysis of VNN1 expression in ESCC tissues and adjacent normal tissues (n = 50). (K–L) Analysis of correlations between VNN1 expression and tumor grade, stage, age, and sex in ESCC patients, with statistical comparison between tumor and non-tumor tissues. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies used were Nectin-4 and METTL3 polyclonal antibodies (21903-1-AP and 15073-1-AP, 1:1000 dilution; Proteintech, Wuhan, China), VNN1 (21745-1-AP, 1:500 dilution; Proteintech), Ki-67 (1:4000 dilution; Proteintech), and vimentin (1:2500 dilution; Proteintech).

Techniques: Immunohistochemical staining, Expressing, Comparison

Journal: Nature Communications

Article Title: Cancer cell plasticity defines response to immunotherapy in cutaneous squamous cell carcinoma

doi: 10.1038/s41467-024-49718-8

Figure Lengend Snippet: a Percentage of PD-L1 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 27), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 23), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 27). b Percentage of CD112 + cells within the indicated cancer cells ( n = 12 tumors per group). c Percentage of Gal9 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 27), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 17), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 21). d Percentage of CD80 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 24), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 18), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 21). e Percentage of CD155 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 10), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 9), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 12). f , h Representative immunofluorescence images of Ecad + (green), f CD80 + or h CD155 + (red), and DAPI nuclear (blue) staining in the indicated patient cSCCs. Scale bar, 100 µm. g Percentage of CD80 + cancer cells relative to total cancer cells in the indicated patient cSCCs ( n = 4 per group). i Percentage of CD155 + cancer cells relative to total cancer cells in epithelial ( n = 4), mixed ( n = 5), and mesenchymal ( n = 4) patient cSCCs. Each dot indicates the average quantification of at least five fields from different tumor regions. j Percentage of CD80 − Ecad + , CD80 + Ecad + , and CD80 + Ecad − cancer cells relative to total cancer cells in the indicated patient cSCCs ( n = 4 per group). k Percentage of CD155 − Ecad + , CD155 + Ecad + , and CD155 + Ecad − cancer cells relative to total cancer cells in epithelial ( n = 4), mixed ( n = 5), and mesenchymal ( n = 4) patient cSCCs. Data are represented as the mean ± SD ( a – e ) or ± SEM ( g , i , j , k ), and n values indicate independent tumors ( a – e , g , i ). P values are determined by one-way ANOVA with Dunnett’s ( a – e ) or Tukey’s ( g , i ) multiple comparison tests. See Supplementary Fig. for the gating strategy ( a – e ). Source data are provided as a Source Data file.

Article Snippet: For cell-surface staining, cells were blocked with 1 mg/ml IgG (Sigma, I5381) and stained with a cocktail of cell-surface antibodies in staining buffer (5% FBS in PBS) for 30 min at 4 °C: from Biolegend, CD11b-APC 1:250 (M1/70, 101211), CD11b-PE/Cy7 1:250 (M1/70, 101215), CD152 (CTLA-4)-PE/Cy7 1:250 (UC10-4B9, 106313), CD155-PE/Cy7 1:200 (TX56, 131511), CD223 (LAG-3)-PE/Cy7 1:250 (C9B7W, 125225), CD226 (DNAM-1)-PE/Cy7 1:250 (10E5, 128811), CD25-PE/Cy7 1:200 (PC61, 102015), CD274 (PD-L1)-PE/Cy7 1:200 (10F.9G2, 124313), CD279 (PD-1)-APC/Cy7 1:250 (29F.1A12, 135223), CD28-PE/Cy7 1:250 (37.51, 102125), CD3ε-APC 1:200 (145-2C11, 100311), CD366 (TIM-3)-PE/Cy7 1:250 (B8.2C12, 134009), CD4-PE/Cy7 1:200 (RM4-5, 100528), CD49f (α6-integrin)-FITC 1:10 (GoH3, 313605), CD69-PE/Cy7 1:200 (H1.2F3, 104511), CD8a-PE 1:200 (53-6.7, 100707), CD80-PE/Cy7 1:250 (16-10A1, 104733), F4/80-APC/Cy7 1:200 (BM8, 123118), Galectin9-PE/Cy7 1:250 (108A2, 137913), Ly-6G/Ly-6C (Gr-1)-PE/Cy7 1:250 (RB6-8C5, 108415), Ly-6C-PE/Cy7 1:250 (HK1.4, 128017), Ly-6G-APC 1:250 (1A8, 127613), NK-1.1-PE 1:200 (PK136, 108707), TIGIT (Vstm3)-PE/Cy7 1:250 (1G9, 142107); from BD Bioscience, CD11b-PE 1:250 (M1/70, 557397); from eBioscience, CD206-APC 1:200 (MR6F3, 17-2061-80), CD326 (EpCAM)-APC-eF780 1:400 (G8.8, 47-5791-82); from TONBO, CD45-PE 1:350 (30-F11, 50-0451); from R&D Systems, CD112 (Nectin-2)-APC 1:200 (829038, FAB3869A).

Techniques: Immunofluorescence, Staining, Comparison

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 1 High expression of Nectin4 and FAP in a variety of cancers. (A) Expression of Nectin4 and FAP in glioma, liposarcoma, and leiomyosarcoma was assessed by IHC. (B) Expression of Nectin4 on lung-metastasized esophageal cancer, lung-metastasized liver cancer, and bone-metastasized triple-negative breast cancer (TNBC). Also see Supplementary Figure 3. Nectin4 is mainly located in the membrane (strongly positive) and cytoplasm (weakly positive) of cancer cells; FAP is mainly located in the membrane (strongly positive) and cytoplasm (weakly positive) of stromal cells, shown in brown.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Expressing, Membrane

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 2 CAR structure and characterization of Nectin4-7.19 CAR-T cells. (A) Schematic illustration of Nectin4 CAR and Nectin4-7.19 CAR lentiviral vector. LTR: long terminal repeats; SP: CD8 signal peptide; TM: transmembrane region; P2A: 2A polypeptide element. (B) CAR expression in Nectin4 CAR-T and Nectin4-7.19 CAR-T cells was measured by flow cytometry. UTD indicates the untransduced T cells as a negative control. (C) Relative CAR expression in CD4+ and CD8+ T subsets. (D) Representative CAR-T cell phenotyping plot based on CD45RA and CCR7 in CD4+

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Plasmid Preparation, Expressing, Cytometry, Negative Control

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 3 Efficient cytotoxicity of Nectin4-7.19 CAR-T cells in vitro. (A) Expression of Nectin4 on a panel of cancer cell lines. (B) Cytotoxicity of Nectin4 CAR-T cells against ABC-1, HT1376, and MDA-MB-453 cells was detected by xCELLigence RTCA label-free technology. The left panel compares the cytotoxicity between Nectin4 CAR-T and CD19 CAR-T cells against target cells at an Effect/Target ratio of 10:1; the right panel shows the killing efficacy of Nectin4 CAR-T cells at different Effect/Target ratios. Arrows refer to the addition of CAR-T cells. The y-axis is the normalized cell index generated by the RTCA software and displayed in real time to reflect the vitality of tumor cells. The x-axis is the time of cell culture in hours. (C) Nectin4 and GFP expression in Luc. ABC-1 cells transfected with lentivirus encoding the Luciferase-T2A-GFP gene. (D) Quantified data on the specific lytic levels of Nectin4 CAR-T and Nectin4-7.19 CAR-T cells against Luc. ABC-1 cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. UTD served as a negative control. (E) Expression level of immune checkpoints was detected by flow cytometry after co-culture of Nectin4 CAR-T or Nectin4-7.19 CAR-T cells with ABC-1 cells at an Effect/Target ratio of 1:1 for 5 days. Data represent the mean ± SD of three independent experiments; ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, t-test.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: In Vitro, Expressing, Generated, Software, Cell Culture, Transfection, Luciferase, Negative Control, Cytometry, Co-Culture Assay

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 4 Therapeutic effect of Nectin4 mCAR-T cells on metastatic colorectal cancer in fully immune-competent mice. (A) The murine CAR construct was inserted upstream of an IRES-GFP marker in the MSCV retroviral plasmid pMIGR1. (B) mCAR expression of Nectin4 mCAR-T cells transfected with pMIGR1-mCAR-IRES-GFP retroviral particles. mUTD indicates the untransduced mouse T cells. (C) Nectin4 and GFP expression of Luc. MC38 cells and hNectin4-Luc. MC38 cells. (D) Quantified data on the specific lytic levels of Nectin4 mCAR-T cells against Luc. MC38 or hNectin4-Luc. MC38 cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. ***p < 0.001, t-test. (E) Secretion of IFN-g in CD4+ and CD8+ T subsets was assessed by flow cytometry after co-culture of Nectin4 mCAR-T cells or mUTD with hNectin4-Luc. MC38 cells for 12 h. ***p < 0.001, t-test. (F, G) C57BL/6 mice were s.c. inoculated with 1 × 106 hNectin4-Luc. MC38 cells on Day 0 and injected i.v. with 5.0 × 105 to 5.0 × 106 Nectin4 mCAR-T cells on Day 10. A total of 5.0 × 106 mUTD served as a negative control (N = 6 mice per group). Solid lines represent each individual mouse (F). Kaplan–Meier survival curve is shown in (G). p-values of log-rank tests were as follows: p = 0.35 (mUTD vs. 0.5 × 106 Nectin4 mCAR-T); p = 0.09 (mUTD vs. 1.5 × 106 Nectin4 mCAR-T); p = 0.0012 (mUTD vs. 5.0 × 106

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Construct, Marker, Retroviral, Plasmid Preparation, Expressing, Transfection, Luciferase, In Vitro, Cytometry, Co-Culture Assay, Injection, Negative Control

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 5 Significant anti-tumor effect of Nectin4-7.19 CAR-T therapy on metastatic lung cancer without on-target off-tumor toxicity. (A) NSG mice were i.v. inoculated with 1.0 × 106 Luc. ABC-1 cells on Day 0 and received an administration of 3 × 106 Nectin4-7.19 CAR-T cells on Day 7 (N = 3 mice per group). Mice treated with the same dosage of UTD cells served as a negative control. (B–D) Tumor xenografts were monitored via bioluminescence imaging. Representative bioluminescence images of three independent experiments are shown in (B); bioluminescence kinetics are shown in (C); solid lines represent each individual mouse. Kaplan–Meier survival curve is shown in (D), p = 0.0246 (Nectin4-7.19 CAR-T vs. UTD), N = 3, log-rank test. (E) Body weight of mice since the tumor inoculation.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Negative Control, Imaging

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 6 Synergistic effect of Nectin4-7.19 CAR-T with FAP-12 CAR-T therapy on metastatic lung cancer mouse model. (A) Schematic illustration of FAP CAR and FAP-12 CAR lentiviral vector. LS: leader signal. (B) CAR expression on FAP CAR-T and FAP-12 CAR-T cells. (C) Expression of CD45RA and CD45RO in CD4+ or CD8+ T subset to assess the subtypes of T cells. (D) 293T cells were transduced with lentivirus encoding the human FAP-Firefly-Luciferase-GFP gene or the murine FAP-Firefly-Luciferase-GFP gene to generate hFAP-Luc. 293T and mFAP-Luc. 293T cells, respectively. Expression of GFP was measured by flow cytometry. 293T cells served as negative controls. (E) Quantified data on the specific lytic levels of FAP CAR-T cells against hFAP-Luc. 293T and mFAP-Luc. 293T cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. UTD served as a negative control. (F) Cytotoxicity of FAP CAR-T and FAP-12 CAR-T cells was detected at an Effect/Target ratio of 10:1 by xCELLigence RTCA label-free technology. (G) Specific lysis of FAP CAR-T and FAP-12 CAR-T cells against hFAP- Luc. 293T was detected by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. (H) NSG mice were inoculated with 1.0 × 106 Luc. ABC-1 cells i.v. on Day 0 and received an administration of 2 × 106 FAP-12 CAR-T cells, 2 × 106 Nectin4-7.19 CAR-T cells, or an admixture of 1 × 106 Nectin4-7.19 CAR-T cells and 1 × 106 FAP-12 CAR-T cells on Day 3 (N = 3 mice per group). A total of 2.0 × 106 UTD served as a negative control. (I) Tumor xenografts were monitored via bioluminescence imaging. Representative bioluminescence images of three independent experiments in each group of mice were shown. (J) Bioluminescence kinetics of the tumor growth in the model. (K) Kaplan– Meier survival curve. p-values of log-rank tests were as follows: p = 0.0246 (Nectin4-7.19+FAP-12 vs. UTD); p = 0.0246 (Nectin4-7.19+FAP-12 vs. FAP-12); p = 0.1161 (Nectin4-7.19+FAP-12 vs. Nectin4-7.19), N = 3. (L) Body weight of mice since the tumor inoculation. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, t-test.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Plasmid Preparation, Expressing, Transduction, Luciferase, Cytometry, In Vitro, Negative Control, Lysis, Imaging

Journal: Virology

Article Title: Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor.

doi: 10.1016/j.virol.2012.10.033

Figure Lengend Snippet: Fig. 1. Infection of the parental, nectin4-expressing, and SLAM-expressing Vero cells with wild-type CDV strains. (A) Vero/hNectin4 (left panel) and Vero/dNectin4 (right panel) cells were stained with a goat anti-human nectin4 polyclonal antibody (gray empty profile) or a control goat IgG (filled black profile), followed by staining with Alexa Fluor 488-conjugated anti-goat IgG. (B) Vero, Vero/ hNectin4, Vero/dNectin4, Vero/hSLAM, and Vero.DogSLAMtag cells were infected with wild-type CDV strains (Ac96I-VDS, 82Con, 55L, M24Cr, and Th12) or mock- infected. At 48 h (Vero, Vero/hNectin4, Vero/dNectin4, and Vero/hSLAM) or 24 h (Vero.DogSLAMtag) post-infection, the cells were stained with the Giemsa solu- tion, and observed under a phase-contrast microscope. (C) Replication kinetics of Ac96I. Vero/hNectin4, Vero/dNectin4, and parental Vero cells were infected with Ac96I at a MOI of 0.01. At various time intervals post-infection, the virus titers were determined by plaque assays.

Article Snippet: The cell surface expression of nectin4 was analyzed using a goat anti-human nectin4 polyclonal antibody (R&D Systems, Minneapolis, MN) as the primary antibody and Alexa Fluor 488-conjugated anti-goat IgG (Molecular probes, Eugene, Oregon) as the secondary antibody.

Techniques: Infection, Expressing, Staining, Control, Microscopy, Virus

Journal: Virology

Article Title: Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor.

doi: 10.1016/j.virol.2012.10.033

Figure Lengend Snippet: Fig. 2. Amino acid sequence comparison of the V domains of human, dog, and mouse nectin4. Dots indicate identical residues to those of human nectin4.

Article Snippet: The cell surface expression of nectin4 was analyzed using a goat anti-human nectin4 polyclonal antibody (R&D Systems, Minneapolis, MN) as the primary antibody and Alexa Fluor 488-conjugated anti-goat IgG (Molecular probes, Eugene, Oregon) as the secondary antibody.

Techniques: Sequencing, Comparison

Journal: Virology

Article Title: Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor.

doi: 10.1016/j.virol.2012.10.033

Figure Lengend Snippet: Fig. 4. Replication kinetics in NCI-H358 cells. (A) NCI-H358 cells were stained with a goat anti-human nectin4 polyclonal antibody (gray empty profile) or a control goat IgG (filled black profile), followed by staining with Alexa Fluor 488-conjugated anti-goat IgG. (B) NCI-H358 cells were infected with the Ac96I-VDS, 82Con, 55 L, M24Cr, or Th12 CDV strains at a MOI of 0.01. At 5 days post-infection, the virus titers were determined by plaque assays. (C, D) NCI-H358 (C) and II-18 (D) cells were infected with Ac96I-VDS or Ac96I-H358 at a MOI of 0.01. At 1, 3, 5, and 7 days post-infection, the virus titers were determined by plaque assays.

Article Snippet: The cell surface expression of nectin4 was analyzed using a goat anti-human nectin4 polyclonal antibody (R&D Systems, Minneapolis, MN) as the primary antibody and Alexa Fluor 488-conjugated anti-goat IgG (Molecular probes, Eugene, Oregon) as the secondary antibody.

Techniques: Staining, Control, Infection, Virus