Journal: bioRxiv

Article Title: A novel Gorilla-derived oncolytic Adenovirus with natural selective replication in cancer cells

doi: 10.64898/2026.02.26.708271

Figure Lengend Snippet: (A) Schematic representation of the genetic structure of the GRAd32Fk25 EV20 SC vector. (B) A549, NCI-H727 or NCI-H1975 were infected with GRAd32Fk25 EV20 SC expressing a single-chain antibody targeting HER3, based on the EV20 sequence. Infection was performed at MOI 1-10-50-100, and supernatant-containing antibodies were separated on SDS-PAGE and blotted with an anti-human antibody. (C) MRC5 cells were infected either with GRAd32Fk25 expressing the EV20 SC, or with GRAd25 expressing the same EV20 SC at an MOI of 10 or 100. The supernatant-containing antibody were separated on SDS-PAGE and blotted with an anti-human antibody. (D) Affinity for HER3 (ECD) of supernatants containing GRAd-produced EV20 SC (1:2 or 1:10 diluted) derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC or mock control. Affinity was evaluated through direct ELISA and absorbance values are expressed in OD (450nm) are reported. (E) Affinity for cell surface HER3 on A375m exposed to supernatants containing GRAd-produced EV20 SC derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC or mock control. Affinity was evaluated by FACS and levels are expressed in arbitrary units (A.U.) of mean fluorescence intensity (M.F.I.). (F) Western blotting images for the evaluation of p-HER3, total HER3, p-Akt, total Akt, and actin (loading control) protein levels in A375m exposed to supernatants containing GRAd-produced EV20 SC (1:2 or 1:10 diluted) derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC and treated or not with neuregulin-1β (NRG-1β). Equal amounts of protein samples were loaded per lane. All adenoviruses contained in the supernatants were heat-inactivated for 30 minutes at 56°C; EV20 is the positive control, EV20 heated is the positive control treated for 30 minutes at 56°C.

Article Snippet: For the evaluation of CD46 and CAR cell surface levels, 2-3 x 10 5 cells (MRC5, HUVEC, A549, NCI-H1299, NCI-H1975, NCI-H727) were collected and incubated with anti-human CD46 (1:50; mouse monoclonal; #12239-MM05, Sino Biological) or with anti-human CAR (1:50; rabbit monoclonal; #10799-R271, Sino Biological) for 30 min at 4°C.

Techniques: Plasmid Preparation, Infection, Expressing, Sequencing, SDS Page, Produced, Derivative Assay, Control, Direct ELISA, Fluorescence, Western Blot, Positive Control

Journal: Cell reports

Article Title: Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma.

doi: 10.1016/j.celrep.2023.112103

Figure Lengend Snippet: Figure 4. CXADR serves as the SFRP2 receptor in retinoblastoma (A) CXADR is highly expressed in retinoblastoma cell lines and tumors. CXADR RNA expression was analyzed by qRT-PCR and normalized to the levels in human ARPE-19 retinal pigment epithelial cells (n = 3). (B) SFRP2 co-immunoprecipitates with endogenous CXADR in 293T cells. The indicated FLAG-tagged proteins were transfected into 293T cells, and their interaction with endogenous CXADR was assessed by anti-FLAG immunoprecipitation followed by anti-CXADR immunoblotting. C-terminally FLAG-tagged SFRP2 specifically co-immunoprecipitated with endogenous CXADR. (C) CXADR serves as the SFRP2 receptor in retinoblastoma. C-terminally FLAG-tagged SFRP2 secreted from transfected 293T cells was incubated with Y79 cells that were transfected with CXADR siRNA or control siRNA as indicated. Cells were washed, and the whole-cell lysate was prepared to assess the binding of SFRP2 to the cells. SFRP2-FLAG bound to control siRNA-transfected cells, and the binding was abolished by CXADR silencing. The silencing of CXADR was verified by immunoblotting. (D) Microscale thermophoresis analysis of SFRP2-CXADR interaction. The interaction between recombinant SFRP2 (6838-FR-025, R&D Systems) and re- combinant CXADR (3336-CX-05, R&D Systems) was analyzed by microscale thermophoresis, which determined the KD of interaction to be 11.9 nM. (E) CXADR silencing rescues growth inhibition following SFRP2 silencing. RB383 and Y79 cells were transfected with SFRP2 siRNA, CXADR siRNA, and/or control siRNA as indicated. Cell proliferation was assessed by IncuCyte.

Article Snippet: Using microscale thermophoresis, we determined the KD of interaction between recombinant SFRP2 and recombinant CXADR (3336-CX-05, R&D Systems) to be 11.9 nM (Figure 4D).

Techniques: RNA Expression, Quantitative RT-PCR, Transfection, Immunoprecipitation, Western Blot, Incubation, Control, Binding Assay, Microscale Thermophoresis, Recombinant, Inhibition

Journal: Cell reports

Article Title: Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma.

doi: 10.1016/j.celrep.2023.112103

Figure Lengend Snippet: Figure 4. CXADR serves as the SFRP2 receptor in retinoblastoma (A) CXADR is highly expressed in retinoblastoma cell lines and tumors. CXADR RNA expression was analyzed by qRT-PCR and normalized to the levels in human ARPE-19 retinal pigment epithelial cells (n = 3). (B) SFRP2 co-immunoprecipitates with endogenous CXADR in 293T cells. The indicated FLAG-tagged proteins were transfected into 293T cells, and their interaction with endogenous CXADR was assessed by anti-FLAG immunoprecipitation followed by anti-CXADR immunoblotting. C-terminally FLAG-tagged SFRP2 specifically co-immunoprecipitated with endogenous CXADR. (C) CXADR serves as the SFRP2 receptor in retinoblastoma. C-terminally FLAG-tagged SFRP2 secreted from transfected 293T cells was incubated with Y79 cells that were transfected with CXADR siRNA or control siRNA as indicated. Cells were washed, and the whole-cell lysate was prepared to assess the binding of SFRP2 to the cells. SFRP2-FLAG bound to control siRNA-transfected cells, and the binding was abolished by CXADR silencing. The silencing of CXADR was verified by immunoblotting. (D) Microscale thermophoresis analysis of SFRP2-CXADR interaction. The interaction between recombinant SFRP2 (6838-FR-025, R&D Systems) and re- combinant CXADR (3336-CX-05, R&D Systems) was analyzed by microscale thermophoresis, which determined the KD of interaction to be 11.9 nM. (E) CXADR silencing rescues growth inhibition following SFRP2 silencing. RB383 and Y79 cells were transfected with SFRP2 siRNA, CXADR siRNA, and/or control siRNA as indicated. Cell proliferation was assessed by IncuCyte.

Article Snippet: Recombinant human CXADR protein (3336-CX-050, R&D Systems) was labeled using the protein labeling kit (RED-NHS second generation, MO-L011, Nano Temper Technologies) according to the manufacturer’s protocol.

Techniques: RNA Expression, Quantitative RT-PCR, Transfection, Immunoprecipitation, Western Blot, Incubation, Control, Binding Assay, Microscale Thermophoresis, Recombinant, Inhibition

Journal: Cell reports

Article Title: JAML immunotherapy targets recently activated tumor-infiltrating CD8 + T cells.

doi: 10.1016/j.celrep.2023.112040

Figure Lengend Snippet: Figure 6. Agonistic JAML antibody treatment impedes tumor growth (A and B) Tumor volume of C57BL/6J (A, n = 10 mice for isotype control group and n = 9 for anti-PD-1 and anti-JAML groups, p = 0.0141 for isotype control vs anti- JAML, and p = 0.0227 for anti-PD-1 vs anti-JAML) or CD8/ (B, n = 7 mice/group for isotype control and anti-JAML and n = 6 mice/group for anti-PD-1) mice subcutaneously inoculated with B16F10-OVA cells and treated with isotype control antibodies, anti-PD-1 antibodies or anti-JAML antibodies at indicated time points. (C and D) Tumor volume (C, p < 0.0001 for B16F10 vs. OT-Iwt, p = 0.0014 for B16F10 vs. OT-I JAML/, p = 0.033 for OT-Iwt vs. OT-I JAML/) (n = 13 mice/group for the control group, n = 8 mice/group for OT-Iwt, and n = 10 mice/group for OT-I JAML/), and frequencies of tumor-infiltrating OT-I T cells (D, n = 6 mice/group for OT-Iwt and n = 8 mice/group for OT-I JAML/) of mice subcutaneously inoculated with B16F10-OVA cells and treated with 1 3 106 adoptively transferred wild- type OT-I T cells or JAML/ OT-I T cells at day 6 after tumor inoculation. (E) Tumor volume of mice subcutaneously inoculated with CXADR+/+ or CXADR/ MC38-OVA cells and treated with either isotype control antibodies or anti- JAML antibodies at indicated time points (n = 8 mice/group for CXADR+/+ + isotype control and n = 7 mice/group for CXADR+/+ + anti-JAML, p = 0.61; n = 8 mice/ group for CXADR/ + isotype control and n = 7 mice/group for CXADR/ + anti-JAML, p = 0.041). All data are mean ± SEM and are representative of at least two independent experiments. Significance for comparisons was computed using two-tailed Mann-Whitney test; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies KLRG1 – BV421 (mouse) Biolegend Cat#138414; RRID: AB_2565613 CD3 – BV510 (mouse) Biolegend Cat#100353; RRID: AB_2565879 PD-1 – BV605 (mouse) Biolegend Cat#135220; RRID: AB_2562616 CD19 – BV650 (mouse) Biolegend Cat#115541; RRID: AB_11204087 CX3CR1 – BV711 (mouse) Biolegend Cat#149031; RRID: AB_2565939 FOXP3 – FITC (mouse) eBioscience Cat#11-5773-82; RRID: AB_465243 CD4 – PE-D594 (mouse) Biolegend Cat#100566; RRID: AB_2563685 CD8 – BB700 (mouse) BD Biosciences Cat#566409; RRID: AB_2744467 CD103 – BV421 (mouse) Biolegend Cat#121421; RRID: AB_2562901 JAML – APC (mouse) Miltenyi Cat#130-114-680; RRID: N/A CD25 – BB700 (mouse) BD Biosciences Cat#566498; RRID: AB_2744345 4-1BB – PE (mouse) Biolegend Cat#106106; RRID: AB_2287565 CD69 – BV786 (mouse) Biolegend Cat#104543; RRID: AB_2629640 CXADR – A647 (mouse) BEI Resources NR-9216; RRID: N/A ICOS – BV786 (human) Biolegend Cat#313534; RRID: AB_2629729 GITR – BV711 (human) Biolegend Cat#371212; RRID: AB_2687161 PD-1 – BV421 (human) BD Biosciences Cat#562584; RRID: AB_2737668 4-1BB – APC (human) Biolegend Cat#309810; RRID: A AB_830672 CD25 – PE (human) BD Biosciences Cat#555432; RRID: AB_395826 CD69 – BV605 (human) Biolegend Cat#310938; RRID: AB_2562307 Chemicals, peptides, and recombinant proteins CXADR Fc R&D Systems Cat#3336-CX-050 RRID: N/A Deposited data Raw and analyzed data This paper GEO: GSE185162 Experimental models: Cell lines MC38-OVA Peter MacCallum cancer center N/A

Techniques: Control, Two Tailed Test, MANN-WHITNEY