Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a Representative images of immunohistochemistry (IHC) for B7-H3 and CD31 in CRC tissues and matched normal tissues from the 125 clinical CRC patients. Scale bar, 100 μm. b , c B7-H3 ( b ) and CD31 ( c ) protein expression based on their staining index in CRC specimens and matched normal tissues. d CD31 protein expression is shown in patients stratified into B7-H3 low (median value) groups. e Correlation analysis of the staining index of the protein expression levels of B7-H3 and CD31 in human CRC specimens ( n = 125). The correlation coefficient (r) is shown. The data represent the means ± SEM. *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a Western blot analysis of B7-H3 in CRC stable cell lines with B7-H3 inhibition (shB7-H3) or their control cell lines (sh-NC) (left). Western blot analysis of B7-H3 in CRC stable cell lines overexpressing B7-H3 (B7-H3) or their control cell lines (EV) (right). β-actin served as a loading control. b , c Cell migration ( b ) and invasion ( c ) in HUVECs were examined by transwell assays after HUVECs were plated and treated with conditioned medium (CM) from sh-NC cells or shB7-H3 cells. Scale bar, 100 μm. One representative image from three reproducible experiments is shown. Migrated and invaded HUVEC numbers are shown in the bar graph. d Effect of CM from sh-NC cells or shB7-H3 cells on tube formation in HUVECs. Scale bar, 100 μm. The number of tubes counted is shown in the bar graph. One representative result from three reproducible experiments is shown. e , f Cell migration ( e ) and invasion ( f ) in HUVECs were examined by transwell assays after HUVECs were plated and treated with CM from EV cells or B7-H3 cells. Migrated and invaded HUVEC numbers are shown in the bar graph. g Effect of CM from EV cells or B7-H3 cells on tube formation in HUVECs. The number of tubes counted is shown in the bar graph. The data represent the means ± SEM. ** P < 0.01, *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Western Blot, Stable Transfection, Inhibition, Migration

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a The expression of angiogenesis-related genes was detected by RT-qPCR in shB7-H3 HCT116 and RKO cells. b Western blot analysis of B7-H3 and VEGFA in the sh-NC and shB7-H3 CRC cell lines. β-actin served as a loading control. c Representative images of IHC for VEGFA in CRC tissues and matched normal tissues from the 125 clinical CRC patients. Scale bar, 100 μm. d VEGFA protein expression based on the staining index of CRC specimens and matched normal tissues. e VEGFA protein expression is shown for patients stratified into B7-H3 low (median value) groups. f Correlation analysis of the staining index of the protein expression levels of B7-H3 and CD31 in human CRC specimens ( n = 125). The correlation coefficient (r) is shown. The data represent the means ± SEM. NS no significant difference; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a , b Cell migration ( a ) and invasion ( b ) in HUVECs were examined by transwell assays after HUVECs were co-treated with CM from sh-NC cells or shB7-H3 cells and IgG or recombinant VEGFA (rVEGFA). Migrated and invaded HUVEC numbers are shown in the bar graph. c The tube formation of HUVECs co-treated with CM from sh-NC cells or shB7-H3 cells and IgG or rVEGFA was examined. The number of tubes counted is shown in the bar graph. d , e Cell migration ( d ) and invasion ( e ) in HUVECs were examined by transwell assays after HUVECs were co-treated with CM from EV cells or B7-H3 cells and siRNA negative control (NC) or VEGFA siRNA. Migrated and invaded HUVEC numbers are shown in the bar graph. f The tube formation of HUVECs co-treated with CM from EV cells or B7-H3 cells and NC or VEGFA siRNA was examined. The number of tubes counted is shown in the bar graph. The data represent the means ± SEM. NS no significant difference; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Migration, Recombinant, Negative Control

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a The expression of p-STAT3, p-p65, p-AKT, p-JNK, and p-ERK1/2 in HCT116 (left) and RKO (right) stable cell lines with B7-H3 inhibition (shB7-H3) or their control cell lines (sh-NC) was analyzed by Western blot. β-actin was used as an internal control. b Relative mRNA level of VEGFA in B7-H3 CRC cells after treatment with perifosine, BAY11–7082 or cryptotanshinone were analyzed by RT-qPCR. c The protein expression of VEGFA in B7-H3 CRC cells after treatment with perifosine, BAY11–7082 or cryptotanshinone was analyzed by ELISA. d The protein expression of VEGFA in B7-H3 CRC cells after treatment with BAY11–7082 was detected by Western blot. e NF-κB activity in EV cells or B7-H3 cells was examined by luciferase reporter assay. f Schematic representation of the proposed B7-H3/NF-κB/VEGFA axis. g , h Cell migration ( g ) and invasion ( h ) in HUVECs were examined by transwell assays after HUVECs were co-treated with CM from EV cells or B7-H3 cells and BAY11–7082. Migrated and invaded HUVEC numbers are shown in the bar graph. i The tube formation of HUVECs co-treated with CM from EV cells or B7-H3 cells and BAY11–7082 was examined. The number of tubes counted is shown in the bar graph. The data represent the means ± SEM. NS no significant difference; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Expressing, Stable Transfection, Inhibition, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Luciferase, Reporter Assay, Migration

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a , b CD31 ( a ) and VEGFA ( b ) protein expression based on their IHC staining index results in subcutaneous tumors formed by sh-NC-HCT116 and shB7-H3-HCT116 cells. N = 5. c , d CD31 ( c ) and VEGFA ( d ) protein expression based on their IHC staining index results in subcutaneous tumors formed by EV-HCT116 and B7-H3-HCT116 cells. N = 5. e , f CD31 ( e ) and VEGFA ( f ) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with BAY11–7082 (6 mg/kg). g , h CD31 ( g ) and VEGFA ( h ) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with bevacizumab (1 mg/kg). i , j CD31 ( i ) and VEGFA ( j ) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors co-treated with 3E8 (5 mg/kg) and BAY11–7082 (6 mg/kg) or bevacizumab (1 mg/kg). N = 5. The data represent the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Expressing, Immunohistochemistry

Journal: Cancer Medicine

Article Title: Higher preoperative serum levels of PD ‐L1 and B7‐H4 are associated with invasive and metastatic potential and predictable for poor response to VEGF ‐targeted therapy and unfavorable prognosis of renal cell carcinoma

doi: 10.1002/cam4.754

Figure Lengend Snippet: Cox regression analysis for various potential prognostic factors in overall survival in all cases

Article Snippet: The serum levels of B7 family molecules, CD28, and VEGF were measured by using the following ELISA kits: human CD80 (CSB‐E15768h, Cusabio Biotech, Wuhan, China), human CD86 (CSB‐E08542h, Cusabio Biotech, Wuhan, China), human B7‐H4 (CSB‐E15013h, Cusabio Biotech, Wuhan, China), human PD‐1 (CSB‐E13643h, Cusabio Biotech, Wuhan, China), human PD‐L1 (CSB‐E13644h, Cusabio Biotech, Wuhan, China), human CD28 (CSB‐E09296h, Cusabio Biotech, Wuhan, China), human B7‐H3 (DB7H30, R&D Systems, Minneapolis, MN), human VEGF (DVE00, R&D Systems Minneapolis, MN), and human CTLA‐4 (KA0165, Abnova, Taipe, Taiwan), as described previously .

Techniques:

Journal: Cancer Medicine

Article Title: Higher preoperative serum levels of PD ‐L1 and B7‐H4 are associated with invasive and metastatic potential and predictable for poor response to VEGF ‐targeted therapy and unfavorable prognosis of renal cell carcinoma

doi: 10.1002/cam4.754

Figure Lengend Snippet: Cox regression analysis for various potential prognostic factors in recurrence‐free survival in 108 N0M0 cases at radical nephrectomy

Article Snippet: The serum levels of B7 family molecules, CD28, and VEGF were measured by using the following ELISA kits: human CD80 (CSB‐E15768h, Cusabio Biotech, Wuhan, China), human CD86 (CSB‐E08542h, Cusabio Biotech, Wuhan, China), human B7‐H4 (CSB‐E15013h, Cusabio Biotech, Wuhan, China), human PD‐1 (CSB‐E13643h, Cusabio Biotech, Wuhan, China), human PD‐L1 (CSB‐E13644h, Cusabio Biotech, Wuhan, China), human CD28 (CSB‐E09296h, Cusabio Biotech, Wuhan, China), human B7‐H3 (DB7H30, R&D Systems, Minneapolis, MN), human VEGF (DVE00, R&D Systems Minneapolis, MN), and human CTLA‐4 (KA0165, Abnova, Taipe, Taiwan), as described previously .

Techniques:

Journal: JACS Au

Article Title: Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

doi: 10.1021/jacsau.2c00328

Figure Lengend Snippet: (A,B) Structural and functional design of a LTzC . The construct comprises: (a) BMS-202-derived end (blue) to target PD-L1-overexpressing cells; (b) PEG-linker (black), as a solubility tag; (c) a tetrazine (magenta) for IEDDA reactions; and (d) dithiolane ring (green), as a redox agent. BMS-202 attachment strategy was identified using molecular modelling studies (PDB: 5J89 ). (C) Synthesis of LTzC . (a) Pd(OAc) 2 , Xphos, CsCO 3 , toluene, 80 °C, 24 h, 47%. (b) tert -Butyl (2-(ethylamino)ethyl)carbamate, sodium triacetoxyborohydride, DCE, room temperature, 24 h, 53%. (c) (i) 1 N HCl in dioxane, 2 h; (ii) Amberlyst A-21, DCM, 30 min; (iii) BocNH-PEG6-COOH, BOP, DIPEA, DMF, r.t., overnight, 88%. (d) (i) 1 N HCl in dioxane, 2 h; (ii) Amberlyst A-21, DCM, 30 min; (iii) Tz 1 , BOP, DIPEA, r.t., overnight, 62%. (e) (i) 1 N HCl in dioxane, 2 h; (ii) lipoic acid, BOP, DIPEA, DMF, r.t., overnight, LTzC = 47%. Overall yield = 6.4%. (D) Synthesis of Non-targeting LTzC . (f) (i) TFA/DCM (1:1), 30 min; (ii) lipoic acid, BOP, DIPEA, DMF, r.t., overnight, 59%. (g) Boc(NH)-PEG7-NH 2 , BOP, DIPEA, DMF, r.t., overnight, 42%. Overall yield = 24.7%.

Article Snippet: The membrane was blocked with a blocking buffer for 1 h at r.t., followed by incubation with the primary antibody (1:1000, Human PD-L1 Mab, R&D Systems Inc.) at 4 °C for 24 h. The membrane was washed twice with TBST, followed by incubation with the secondary antibody (1:1000, Goat Anti-Rabbit IgG HRP, R&D Systems Inc.) for 2 h. The membrane was washed thrice with TBST, followed by addition of Clarity ECL western blotting substrates (Bio-Rad) before imaging in the ChemiDoc XRS + Imaging System (Bio-Rad).

Techniques: Functional Assay, Construct, Derivative Assay, Solubility

Journal: JACS Au

Article Title: Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

doi: 10.1021/jacsau.2c00328

Figure Lengend Snippet: Schematic strategy and confocal imaging of LTzC / SRB probe bioconjugation method. (A) Structure of the fluorescent exo -norbornene-tagged sulforhodamine B probe ( SRB probe ) and ligation strategy with LTzC . (B) Western blot analysis of PD-L1 expression in MDA-MB-231 and MCF-7 in the presence and absence of IFN-γ (activator of PD-L1 expression). (C) Targeted bioorthogonal labeling of PD-L1 proteins in PD-L1-positive and -negative cell lines. Cells were first treated with LTzC (3 μM) for 4 h to enable targeted binding of PD-L1, followed by removal of unbound LTzC and addition of SRB Probe (100 nM) for 24 h. Unbound SRB probe was removed, and the cells were fixed for staining and imaging experiments. Nuclei were stained by Hoechst 33342; actin was stained using Phalloidin-Alexa Fluor 647. PD-L1 was labeled using antibody-labeling (Human PD-L1 Mab/Alexa Fluor 488 Goat anti-Rabbit IgG) post-fixation to study co-localization of its fluorescent signals with bioorthogonal-labeling ( LTzC / SRB Probe ) in MDA-MD-231 cells (merge).

Article Snippet: The membrane was blocked with a blocking buffer for 1 h at r.t., followed by incubation with the primary antibody (1:1000, Human PD-L1 Mab, R&D Systems Inc.) at 4 °C for 24 h. The membrane was washed twice with TBST, followed by incubation with the secondary antibody (1:1000, Goat Anti-Rabbit IgG HRP, R&D Systems Inc.) for 2 h. The membrane was washed thrice with TBST, followed by addition of Clarity ECL western blotting substrates (Bio-Rad) before imaging in the ChemiDoc XRS + Imaging System (Bio-Rad).

Techniques: Imaging, Ligation, Western Blot, Expressing, Labeling, Binding Assay, Staining, Antibody Labeling

Journal: JACS Au

Article Title: Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

doi: 10.1021/jacsau.2c00328

Figure Lengend Snippet: PD-L1 cell surface and total protein expression after IFN-gamma, BMS-202, or LTzC treatment in MDB-MB-231 cell line. (A) Workflow of flow cytometry measurement of surface PD-L1 using antibody-labeling. (B) Quantification of surface PD-L1 via flow cytometry. Data are represented as mean fluorescent intensity ratio of control cells vs treated cells ( n = 3). (C) Western Blot analysis of total PD-L1 expression ( n = 3). Representative western blots are provided for each data set.

Article Snippet: The membrane was blocked with a blocking buffer for 1 h at r.t., followed by incubation with the primary antibody (1:1000, Human PD-L1 Mab, R&D Systems Inc.) at 4 °C for 24 h. The membrane was washed twice with TBST, followed by incubation with the secondary antibody (1:1000, Goat Anti-Rabbit IgG HRP, R&D Systems Inc.) for 2 h. The membrane was washed thrice with TBST, followed by addition of Clarity ECL western blotting substrates (Bio-Rad) before imaging in the ChemiDoc XRS + Imaging System (Bio-Rad).

Techniques: Expressing, Flow Cytometry, Antibody Labeling, Western Blot

Journal: JACS Au

Article Title: Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

doi: 10.1021/jacsau.2c00328

Figure Lengend Snippet: (A) Structure and bioorthogonal activation of 7-oxa-norbornadiene-masked prodrugs Pro-INK128 and Pro-Dox . (B) Track-&-treat strategy: PD-L1 positive cells vs PD-L1 negative cells. (C) Dose response curves of INK128 and Pro-INK128 (left) and doxorubicin and Pro-Dox (right) in MDA-MB-231 cells. (D) Study of the LTzC -triggered activation of prodrugs in MDA-MB-231 and MCF-7 cancer cells. Cells were first treated with LTzC (3 μM) for 4 h, followed by removal of unbound LTzC and addition of Pro-Dox (0.3 μM) and/or Pro-INK128 (0.1 μM). Studies were performed with and without IFN-γ treatment to compare different levels of PD-L1 expression. Cell viability was measured at day 5 using PrestoBlue reagent. Negative controls: LTzC (3 μM)/ Pro-Dox (0.3 μM) or Pro-INK128 (0.1 μM); positive control: doxorubicin (0.3 μM) or INK128 (0.1 μM). The data are average of triplicates and the error bars indicate standard deviations.

Article Snippet: The membrane was blocked with a blocking buffer for 1 h at r.t., followed by incubation with the primary antibody (1:1000, Human PD-L1 Mab, R&D Systems Inc.) at 4 °C for 24 h. The membrane was washed twice with TBST, followed by incubation with the secondary antibody (1:1000, Goat Anti-Rabbit IgG HRP, R&D Systems Inc.) for 2 h. The membrane was washed thrice with TBST, followed by addition of Clarity ECL western blotting substrates (Bio-Rad) before imaging in the ChemiDoc XRS + Imaging System (Bio-Rad).

Techniques: Activation Assay, Expressing, Positive Control

Journal: Cancers

Article Title: Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs

doi: 10.3390/cancers13040785

Figure Lengend Snippet: Cross-binding of human ICIs with canine homologues. ( A ) Schematic workflow for the evaluation steps of ICIs in dogs. ( B , C ) Plate-based assay to determine cPD-1 binding by nivolumab, pembrolizumab and cemiplimab was performed by coating 10 μg/mL of cPD-1His (red) on a microwell plate and incubation with various concentrations of respective ICIs, with ipilimumab acting as a negative control. Subsequently ICI binding was detected with anti-human IgG-HRP. ( B ) Schematic representation of the experiment, ( C ) Duplicates with fitted curve (log agonist versus response). Representative plot from two independent experiments. ( D ) Flow cytometric histograms show binding profiles of nivolumab, pembrolizumab and cemiplimab to canine T cells after 48 h of ConA stimulation. Pregated on Live Single CD45+CD5+ cells. Representative plots from 2 independent experiments ( n = 3). ( E,F ) Flow cytometric evaluation of atezolizumab, avelumab and durvalumab binding cPD-L1 on canine SCC1 cell line stimulated with cIFN-γ. ( E ) Schematic representation of the experiment with upregulation of MHCII (blue) and cPD-L1 (purple) ( F ) Representative flow cytometric plots of cIFN-γ stimulated versus non-stimulated cells. Pregated on Live Single cells. ( G ) MFI binding curve comparison of atezolizumab, avelumab and durvalumab binding to cPD-L1. Triplicates with fitted curve (log agonist versus response). Representative plot from 3 independent experiments.

Article Snippet: Soluble canine PD-1 with a human fragment crystallized (Fc) IgG1 fusion tag (scPD-1Fc) (70109-D02H) or with fusion His-tag (scPD-1His) (70109-D08H), as well as soluble canine PD-L1 with a human IgG1 fusion tag (scPD-L1Fc) (70110-D02H) were all obtained from Sino Biological (Beijing, China).

Techniques: Binding Assay, Incubation, Negative Control

Journal: Cancers

Article Title: Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs

doi: 10.3390/cancers13040785

Figure Lengend Snippet: Blockade of interaction of PD-1 and PD-L1. ( A , B ) Outline of experimental setup blocking cPD-1/cPD-L1 binding by atezolizumab, avelumab and nivolumab. 20 μg/mL cPD-L1Fc (purple) was coated on a microwell plate and binding of 20 μg/mL cPD-1His (red) was detected on the plate, either ( A ) after blockade of cPD-L1 on the plate with atezolizumab and avelumab, or ( B ) preincubated with various concentrations of nivolumab ( C ) Percentage of cPD-1 binding to cPD-L1 after blockade with indicated ICIs. Triplicates with fitted curve (log agonist versus response). Representative plot from three independent experiments (Mean, SEM, Two-way ANOVA with Bonferroni post-test comparing all pairs of columns, ** p < 0.01, **** p < 0.0001). ( D , E ) Sequence analysis of canine and human ( D ) PD-1 and ( E ) PD-L1. Black arrows depict crucial residues for binding each other and for blockade of the interaction by ICIs .

Article Snippet: Soluble canine PD-1 with a human fragment crystallized (Fc) IgG1 fusion tag (scPD-1Fc) (70109-D02H) or with fusion His-tag (scPD-1His) (70109-D08H), as well as soluble canine PD-L1 with a human IgG1 fusion tag (scPD-L1Fc) (70110-D02H) were all obtained from Sino Biological (Beijing, China).

Techniques: Blocking Assay, Binding Assay, Sequencing

Journal: Cancers

Article Title: Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs

doi: 10.3390/cancers13040785

Figure Lengend Snippet: Functional effects of cPD-1/L1 blockade on cIFN-γ production by cPBMCs. ( A – C ) Flow cytometric analysis of GzmB production by canine NK and T cells for freshly isolated PBMCs, PBMCs isolated after overnight (O/N) storage of whole blood at 4 °C and freshly isolated, cryopreserved and thawed PBMCs. ( A ) Representative flow cytometric plots depicting gating for T cells (CD3+CD14−) and non-monocytes or T cell fraction, which contains canine NK cells (CD3−CD14−). ( B , C ) Bar graphs showing percentages of GzmB producing cells in ( B ) T cells ( C ) NK cell containing fraction (representative plots from three independent experiments, Mean, SEM, Paired One-way ANOVA, n = 10). ( D , F ) Dog peripheral blood mononuclear cells ( n = 12, ) were obtained from healthy donors and stimulated with 50 ng/mL of SEB in the presence of 10 μg/mL of atezolizumab or avelumab. Durvalumab was used as a non-binding isotype (hIgG1) control antibody. ( G – I ) SEB stimulation as in D–F with addition of 10 μg/mL of scPD-L1 ( D,G ) schematic outlines of the experiments for ( E , F ); ( H , I ), respectively. For evaluation of cIFN-γ production, the culture supernatant was harvested on day 3, and measured by ELISA. Pooled data from 2 independent experiments (Paired Student’s t test with p values indicated on the plots).

Article Snippet: Soluble canine PD-1 with a human fragment crystallized (Fc) IgG1 fusion tag (scPD-1Fc) (70109-D02H) or with fusion His-tag (scPD-1His) (70109-D08H), as well as soluble canine PD-L1 with a human IgG1 fusion tag (scPD-L1Fc) (70110-D02H) were all obtained from Sino Biological (Beijing, China).

Techniques: Functional Assay, Isolation, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs

doi: 10.3390/cancers13040785

Figure Lengend Snippet: Characteristics of healthy donors used in the study. Responders (R), non-responders (NR) to atezolizumab.

Article Snippet: Soluble canine PD-1 with a human fragment crystallized (Fc) IgG1 fusion tag (scPD-1Fc) (70109-D02H) or with fusion His-tag (scPD-1His) (70109-D08H), as well as soluble canine PD-L1 with a human IgG1 fusion tag (scPD-L1Fc) (70110-D02H) were all obtained from Sino Biological (Beijing, China).

Techniques:

Journal: Cancers

Article Title: Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs

doi: 10.3390/cancers13040785

Figure Lengend Snippet: Functional effects of cPD-1/L1 blockade on cIFN-γ production by cancer patient derived cPBMCs. ( A ) Representative staining for PD-L1 on CD5−CD14−MHCII+ APCs with atezolizumab, which was subsequently detected with PE labeled anti-human Fc antibody. ( B ) Pooled data from two independent experiments with each point representing a healthy donor or a lymphoma patient (Mean, SEM, Unpaired Student’s t test with p value indicated on the plot). ( C – F ) cPBMCs from cancer patients ( n = 27, ) were stimulated and evaluated as in D–F. ( C , D ) Lymphoma patients, ( E , F ) non-lymphoma cancer patients . Pooled data from three independent experiments (Paired Student’s t test with p values indicated on the plots). ( G , H ) Cancer patient PBMC samples classified as non-responders (NR, n = 6) or responders (R, n = 19) as well as healthy donor PBMC samples classified as responders (HD, n = 11) (1500 cells/sample) were flow cytometrically analyzed and concatenated; all lymphomas were excluded. ( G ) UMAP clustering analysis with different identified populations colored according to the cluster they were assigned to using the FlowSOM algorithm and manual annotation. ( H ) The heat map represents the normalized median fluorescence intensity (MFI) for respective markers within each cellular population for the nine clusters from concatenated data and was used to annotate the clusters in G. ( I ) Bar graph shows the frequency of CD8+ T cells in the PBMCs of NR vs. R, only T cell lymphomas were excluded. Each point represents a patient (Mean, SEM, Unpaired Student’s t test with p value indicated on the plot). ( J ) Schematic summary of the evaluation of the effect of human ICIs for dogs.

Article Snippet: Soluble canine PD-1 with a human fragment crystallized (Fc) IgG1 fusion tag (scPD-1Fc) (70109-D02H) or with fusion His-tag (scPD-1His) (70109-D08H), as well as soluble canine PD-L1 with a human IgG1 fusion tag (scPD-L1Fc) (70110-D02H) were all obtained from Sino Biological (Beijing, China).

Techniques: Functional Assay, Derivative Assay, Staining, Labeling, Fluorescence

Journal: Cancer Medicine

Article Title: Higher preoperative serum levels of PD ‐L1 and B7‐H4 are associated with invasive and metastatic potential and predictable for poor response to VEGF ‐targeted therapy and unfavorable prognosis of renal cell carcinoma

doi: 10.1002/cam4.754

Figure Lengend Snippet: Cox regression analysis for various potential prognostic factors in overall survival in all cases

Article Snippet: The serum levels of B7 family molecules, CD28, and VEGF were measured by using the following ELISA kits: human CD80 (CSB‐E15768h, Cusabio Biotech, Wuhan, China), human CD86 (CSB‐E08542h, Cusabio Biotech, Wuhan, China), human B7‐H4 (CSB‐E15013h, Cusabio Biotech, Wuhan, China), human PD‐1 (CSB‐E13643h, Cusabio Biotech, Wuhan, China), human PD‐L1 (CSB‐E13644h, Cusabio Biotech, Wuhan, China), human CD28 (CSB‐E09296h, Cusabio Biotech, Wuhan, China), human B7‐H3 (DB7H30, R&D Systems, Minneapolis, MN), human VEGF (DVE00, R&D Systems Minneapolis, MN), and human CTLA‐4 (KA0165, Abnova, Taipe, Taiwan), as described previously .

Techniques:

Journal: Cancer Medicine

Article Title: Higher preoperative serum levels of PD ‐L1 and B7‐H4 are associated with invasive and metastatic potential and predictable for poor response to VEGF ‐targeted therapy and unfavorable prognosis of renal cell carcinoma

doi: 10.1002/cam4.754

Figure Lengend Snippet: Cox regression analysis for various potential prognostic factors in recurrence‐free survival in 108 N0M0 cases at radical nephrectomy

Article Snippet: The serum levels of B7 family molecules, CD28, and VEGF were measured by using the following ELISA kits: human CD80 (CSB‐E15768h, Cusabio Biotech, Wuhan, China), human CD86 (CSB‐E08542h, Cusabio Biotech, Wuhan, China), human B7‐H4 (CSB‐E15013h, Cusabio Biotech, Wuhan, China), human PD‐1 (CSB‐E13643h, Cusabio Biotech, Wuhan, China), human PD‐L1 (CSB‐E13644h, Cusabio Biotech, Wuhan, China), human CD28 (CSB‐E09296h, Cusabio Biotech, Wuhan, China), human B7‐H3 (DB7H30, R&D Systems, Minneapolis, MN), human VEGF (DVE00, R&D Systems Minneapolis, MN), and human CTLA‐4 (KA0165, Abnova, Taipe, Taiwan), as described previously .

Techniques: