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

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: AML cells exhibit no, or heterogeneous, expression of DNAM-1 ligands. (A) The indicated AML cell lines were analyzed for expression of CD112, CD155 and CD33 by flow cytometry. Iso refers to immunoglobulin isotype matched control antibody while stain indicates specific antibody staining. (B) The percentage of cells expressing the indicated ligands within populations of the indicated cell type, as analyzed from (A). (C) CD155 expression on the indicated AML cell lines was visualized by confocal microscopy. Scale bar represents 20 µm.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Expressing, Flow Cytometry, Control, Staining, Confocal Microscopy

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: DNAM-1 ligands are required for NK cell activity against AML targets. (A) K562 cells were stained with anti-CD112 and anti-CD155 antibodies, then FACS sorted into high expressing (both CD112 and CD155) and low expressing (both CD112 and CD155) populations. (B) K562 cells were FACS sorted as in (A), then used as targets in an NK cell degranulation assay. (C) K562 cells were FACS sorted as in (A), then used as targets in an NK cell chromium release assay. (D) MV4-11 cells were stained with anti-CD112 and anti-CD155 antibodies, then FACS sorted into high expressing (both CD112 and CD155) and low expressing (both CD112 and CD155) populations. (E) MV4-11 cells were FACS sorted as in (D), then used as targets in an NK cell degranulation assay. (F) MV-411 cells were FACS sorted as in (D), then used as targets in an NK cell chromium release assay. (G) NK cell chromium release assay against the indicated AML targets (5:1 E:T ratio) in the presence or absence of anti-DNAM-1-neutralizing antibody (5 μg/mL). (H–I) NK cell chromium release assay at the indicated E:T ratios using FACS sorted high and low CD112/CD155 expressing K562 and MV-411 cells, in the presence or absence of anti-DNAM-1-neutralizing antibody (5 μg/mL). Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 3). *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Activity Assay, Staining, Expressing, Degranulation Assay, Release Assay

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: DNAM-1 ligands increase the frequency of ‘normal’ NK-target cell synapses. (A–B) FACS sorted (CD112/155 high and low) K562 and MV-411 cells were seeded in chamber slides using serum free media, then overlaid with NK cells 30 min later, followed by fixing. The percentage of targets that had conjugated with an NK cell was then quantitated by confocal microscopy. A minimum of 20 fields of view was analyzed and is representative of two independent experiments. (C) FACS sorted (CD112/155 high and low) MV4-11 cells were seeded in chamber slides using serum free media, then overlaid with NK cells 30 min later. After 1 h, cells were fixed, stained with the antibody combinations indicated, then analyzed by confocal microscopy. Representative images of NK-target cell synapses are presented. Scale bar represents 10 µm. (D) The percentage of NK cells that had polarized LFA-1 and perforin to the synapse was quantified from (C). A minimum of 20 NK-target cell synapses was analyzed and data from two independent experiments was pooled. Error bars represent the mean ± SEM *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Confocal Microscopy, Staining

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: Live imaging reveals that AML cells lacking DNAM-1 ligand expression drive NK cell failed killing. (A) FACS sorted (CD112/155 high and low) MV4-11 cells were seeded in chamber slides using serum free media, then overlaid with NK cells labeled with fluo-4 acetoxymethyl AM (green) to indicate calcium signaling, and analyzed by time-lapse microscopy. PtdIns (red) (100 μg/mL) was added to the medium to indicate perforin-induced target membrane puncture. Representative still images at the indicated time-points are depicted (hr:min). (B–C) Individual NK-MV-411 CD112/CD155 high and low contacts were monitored for events that did (successful kill) or did not (failed kill) result in target killing, as indicated by PI influx and apoptotic morphology. (D) The time interval between initial NK-target cell contact and target cell death (PtdIns influx) was analyzed. (E–G) K562 cells were used as targets in the assays described in (B–D) above. All quantification data is pooled from individual movies (n = 3). Error bars represent the mean ± SEM *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Imaging, Expressing, Labeling, Time-lapse Microscopy, Membrane

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: NK cells preferentially target DNAM-1 ligand-expressing cells and drive clonal selection of DNAM-1 ligand negativity. (A) FACS-sorted K562 CD112/CD155 high and low cells were labeled with CFSE and CTV, respectively, then exposed to NK cells at the indicated E:T ratios. After 4 h, cells were analyzed by flow cytometry and loss of dye was monitored from viable populations. (B) Extended E:T ratio titration for the assay described in (A), using FACS-sorted K562 and MV-411 CD112/CD155 high and low cells as targets. Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 2). *p < 0.05 by unpaired Student's t test. (C–D) K562 and MV-411 cells were either exposed to NK cells (1:1 E:T Ratio), or not, for 5 d. Viable AML cells (fixable yellow negative, CD33 positive) were then analyzed for CD112 and CD155 expression by flow cytometry, and compared to parental cells (no NK cell exposure). Bar charts represent the number of CD112/CD155 double positive cells after 5 d in the presence or absence of NK cell exposure. Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 3). *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Expressing, Selection, Labeling, Flow Cytometry, Titration

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) A series of PVRL4 deletion constructs were designed and their expression confirmed by Western blot. ( C ) PVRL4 mutants from ( A ) were tested for their ability to induce anchorage-independent colony formation in triplicate (error bars ± SD). ( D ) Cells with full-length PVRL4 or the cytoplasmic region deletion mutant were assayed for viability under conditions of anchorage deprivation by measuring total ATP content in cells cultured on ultra-low attachment plates for 72 hr. Values were normalized to an empty vector-transduced sample. Assays were performed in triplicate (error bars ± SD). ( E ) and ( F ) TL-HMECs expressing empty vector, full-length PVRL4 or cytoplasmic region deletion mutant containing cells were cultured on tissue culture-treated (adherent) or ultra-low attachment (suspension) dishes for 72 hr. RNA was isolated and mRNA levels for TGM1 ( E ) and KRT6A and IVL ( F ) were measured by RT-qPCR. Transcript levels were normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.004

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Construct, Expressing, Western Blot, Mutagenesis, Cell Culture, Plasmid Preparation, Suspension, Isolation, Quantitative RT-PCR

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) PVRL4 promotes cell clustering of TL-HMECs. Cells were dissociated off the tissue culture surface with trypsin-free cell dissociation buffer and kept in suspension for 1 hr. Small (3–5 cells) and large (>5 cells) cell clusters per field of view were counted, n = 3 (error bars ± SD). ( C ) GFP-labeled PVRL4-expressing TL-HMECs were allowed to aggregate with dsRed-labeled cells expressing either a PVRL1-targeting shRNA or a control shRNA. Representative phase-contrast and fluorescent images (red and green channels superimposed) are shown. ( D ) PVRL4 was co-expressed with the indicated shRNAs and anchorage-independent colony formation in TL-HMECs was assayed. Values were normalized to an empty vector-transduced sample. Assays were performed in triplicate (error bars ± SD). ( E ) PVRL4-expressing TL-HMECs were assayed for clustering in the presence of the indicated antibodies or isotype controls. Cell clusters were quantified as before. ( F ) Anchorage-independent growth induced by PVRL4 or an shRNA against PTEN was assayed in the presence of PVRL4-targeting antibody or control IgG. Colony numbers were normalized to the control sample. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.005

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Suspension, Labeling, Expressing, shRNA, Control, Plasmid Preparation

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4 expressing TL-HMECs were stably transduced with dsRed or GFP and mixed in equal proportions, followed by (i) immediate plating into semi-solid medium, or (ii) co-culturing on an adherent surface for 2 d, followed by plating into semi-solid medium. Resulting colonies were visualized under a fluorescent microscope and each colony was assessed for the presence of red and green fluorescence. Representative phase-contrast and fluorescent images (red and green channels) are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.006

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing, Stable Transfection, Transduction, Microscopy, Fluorescence

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: The colony formation efficiency of TL-HMECs transduced with empty vector or vector expressing PVRL4 was compared between unfiltered cell suspensions and cell suspensions that were filtered through a 35 μm nylon mesh strainer prior to plating into methylcellulose. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.007

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Transduction, Plasmid Preparation, Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4-expressing TL-HMECs were allowed to aggregate in the presence of the indicated antibodies or isotype controls. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.009

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4-expressing TL-HMECs were assayed for clustering in the presence of the indicated antibodies or isotype controls. Cell clusters were quantified as before. DOI: http://dx.doi.org/10.7554/eLife.00358.010

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Schematics of chimeric constructs containing extracellular domains of PVRL4 or an shRNA-resistant version of PVRL1 fused to the transmembrane domain of CD8 (blue). ( B ) and ( C ) TL-HMECs were stably transduced with the indicated combinations of expression constructs and assayed for anchorage-independent growth ( B ) and clustering ( C ). Colony numbers were normalized to the control sample. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). ( D ) Expression levels of endogenous and chimeric proteins were verified by Western blot. DOI: http://dx.doi.org/10.7554/eLife.00358.011

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Construct, shRNA, Stable Transfection, Transduction, Expressing, Control, Western Blot

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: TL-HMEC cells infected with PVRL4-CD8tm or empty vector were fixed with methanol and stained with goat polyclonal anti-PVRL4 antibody followed by anti-goat Alexa Fluor 488 secondary antibody. DOI: http://dx.doi.org/10.7554/eLife.00358.012

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Infection, Plasmid Preparation, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Clustering assays were performed with TL-HMECs expressing the following transgenes: (i) empty vector/control shRNA; (ii) PVRL4-CD8tm/control shRNA; (iii) PVRL4-CD8tm/anti-PVRL1 shRNA; and (iv) a 1:1 mixture of (iii) and (i). DOI: http://dx.doi.org/10.7554/eLife.00358.013

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing, Plasmid Preparation, Control, shRNA

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Cell surface-localized proteins interacting with HA/FLAG-tagged PVRL4, but not with HA/FLAG-tagged GFP, as determined by mass spectrometry. ( B ) TL-HMECs expressing HA/FLAG tagged PVRL4 or HA/FLAG-tagged GFP were detached from the adherent surface with the enzyme-free cell dissociation buffer and incubated in suspension for 1 hr. Immunoprecipitations were performed with HA beads, followed by Western blot with FLAG and integrin β4 antibody. ( C ) TL-HMECs expressing vector control, PVRL4, or myr-PI3K were stably transduced with the indicated shRNA constructs and integrin β4 levels were assayed by Western blot. ( D ) TL-HMECs from ( C ) were assayed for anchorage-independent colony formation. Colony numbers were normalized to the vector control sample. Assays were performed in triplicate (error bars ± SD). ( E ) TL-HMECs stably transduced with the indicated constructs were detached from the adherent surface with enzyme-free cell dissociation buffer and incubated in 0.5% methylcellulose in suspension for 6 hr or cultured on an adherent surface for 48 hr. Levels of pY416-SFK (Src family kinases), total SFK, and vinculin loading control were measured by Western blot. Band intensity was measured with ImageJ software. ( F ) PVRL4-expressing TL-HMEC cells transduced with control or anti-PVRL1 shRNA were incubated in suspension in the conditions indicated. Levels of pY416-SFK (Src family kinases) and tubulin loading control were measured by Western blot. Band intensity was measured with ImageJ software. ( G ) TL-HMECs stably expressing PVRL4 or control vector were assayed for anchorage-independent colony formation in the presence of PP2 or vehicle control. Colony numbers were normalized to the vector sample. Assays were performed in triplicate (error bars ± SD). ( H ) TL-HMECs expressing PVRL4 were stably transduced with the indicated shRNA constructs and assayed for anchorage-independent colony formation. Colony numbers were normalized to the vector control sample. Assays were performed in triplicate (error bars ± SD). ( I ) SHP-2 levels were assayed by Western blot in TL-HMEC lysates from ( H ). DOI: http://dx.doi.org/10.7554/eLife.00358.019

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Mass Spectrometry, Expressing, Incubation, Suspension, Western Blot, Plasmid Preparation, Control, Stable Transfection, Transduction, shRNA, Construct, Cell Culture, Software

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Anti-HA beads were used to immunoprecipitate HA/FLAG-PVRL4 from the indicated lysates. Immunoprecipitates were blotted with anti-integrin β4 and anti-FLAG antibodies. ( B ) Immunoprecipitations with anti-integrin β4 antibodies or with a control IgG were performed from TL-HMEC lysates expressing either full-length PVRL4 or its cytoplasmic deletion mutant. Immunoprecipitates and input lysates were blotted with anti-integrin β4 and anti-PVRL4 antibodies. Asterisks denote heavy chains of control and anti-integrin β4 antibodies, which cross-react with an anti-goat secondary antibody. DOI: http://dx.doi.org/10.7554/eLife.00358.020

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Expressing, Mutagenesis

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) A view from the integrated Genome Viewer program showing focal amplification of the PVRL4 locus in SUM190 cells. The degree of amplification is denoted by the intensity of the color. ( B ) PVRL4 mRNA was stably depleted from SUM190 cells by four independent shRNAs. Transcript levels were measured by RT-qPCR and normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). PVRL4-depleted and control cells were assayed for clonogenic survival and anchorage-independent colony formation. Assays were performed in triplicate (error bars ± SD). All values were normalized to the uninfected control sample. ECM: extracellular matrix. ( C ) The PVRL4-CD8 chimeric construct was used to rescue the defect in clonogenic survival observed with RNAi-mediated PVRL4 depletion. Assays were performed in triplicate (error bars ± SD). Colony numbers were normalized to the control shRNA sample. ( D ) Expression levels of endogenous and chimeric proteins were verified by Western blot. DOI: http://dx.doi.org/10.7554/eLife.00358.021

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Amplification, Stable Transfection, Quantitative RT-PCR, Control, Construct, shRNA, Expressing, Western Blot

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4 transcript levels were measured by RT-qPCR and normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.026

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: The clonogenic potential of the indicated cell lines in the presence of control or PVRL4 shRNA constructs was assessed. Assays were performed in triplicate (error bars ± SD). Colony numbers were normalized to the control shRNA sample. DOI: http://dx.doi.org/10.7554/eLife.00358.027

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, shRNA, Construct

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Cell line growth in methylcellulose-containing media on an ultra-low attachment surface was assessed in the presence of control or PVRL4 shRNA constructs. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.028

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, shRNA, Construct

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) Female nude mice were injected into their mammary fat pads with SUM190 cells expressing PVRL4-targeted or control shRNA (n = 10 per group, error bars ± SEM). The resulting tumors were excised, scaled ( A ), and photographed ( B ). ( C ) SUM185 cells were stably transduced with PVRL4-targeted or control shRNA and injected into the mammary fat pads of female nude mice (n = 10 per group, error bars ± SEM). Tumor volume was measured with calipers at the indicated time points. ( D ) Female nude mice with ∼50 mm 3 SUM190-eGFP xenografts were randomized into two cohorts (n = 7 per group) and injected with anti-PVRL4 monoclonal antibodies or control IgG on the indicated days. Tumor volume was measured with calipers (error bars ± SEM). ( E ) Levels of PVRL4 protein were measured in tumor lysates from anti-PVRL4 antibody or control-treated mice, 7 days after the last treatment. ( F ) Tumor sections from control IgG ( A – C ) or anti-PVRL4 antibody-treated ( D – F ) mice were stained with hematoxylin/eosin and photographed. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.029

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Injection, Expressing, Control, shRNA, Stable Transfection, Transduction, Bioprocessing, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Freshly explanted tumors from control IgG or anti-PVRL4 antibody-treated mice (N = 3 per group) were visualized using a two-photon confocal microscope. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.031

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Microscopy

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Europium-labeled SUM190 cells were incubated with fresh human NK cells in the presence of an isotype control or anti-PVRL4 antibody, and the degree of lysis was measured by the DELFIA europium assay. The maximum signal was determined by a complete lysis of labeled SUM190 cells in DELFIA lysis buffer. As a positive control, hMB humanized mouse lymphoma cells were mixed with effector cells in the presence of ADCC-competent anti-CD52 antibody. ADCC: antibody-dependent cytotoxicity. DOI: http://dx.doi.org/10.7554/eLife.00358.032

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Labeling, Incubation, Control, Lysis, Positive Control

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Paraffin-embedded sections of SUM190 xenografts from mice treated with either control IgG or anti-PVRL4 antibody were stained with anti-mouse F4/80 antibody. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.033

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Tumor lysates from control antibody- or anti-PVRL4 antibody-treated mice were blotted for E-cadherin and vimentin. ( B ) PVRL4 was stably depleted by two independent shRNAs in SUM190 cells and lysates were blotted for E-cadherin and vimentin. DOI: http://dx.doi.org/10.7554/eLife.00358.034

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Stable Transfection

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: 293T cells were transfected with empty pQCXIN (green line), pQCXIN-human PVRL4 (blue line), or pCMV-SPORT6-mouse PVRL4 (magenta line). Live-cell FACS was performed with mouse anti-human PVRL4 antibody followed by anti-mouse secondary antibody conjugated to Alexa Fluor 488 fluorophore. The FITC-A fluorescent signal for three labeled cell populations is shown. DOI: http://dx.doi.org/10.7554/eLife.00358.035

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Transfection, Labeling