Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Strategy and materials in this study. (A) Diagram of the procedure in the study. The primary tumor cells and tumor organoids were generated from patients' tumor tissues and ROBO1-NK were constructed from patients' PBMCs. (B) IHC of ovarian tumor tissues. Negative control was used irrelevant IgG replacing primary antibody for incubating. Strong positive of ROBO1 expression on cell membrane was marked by red circles. (C) ROBO1 positively expressed in tumor tissues of the clinical ovarian cancer patient #01, #02, #03 and #04.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Generated, Construct, Negative Control, Expressing, Membrane

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Characters of ROBO1-NK cells. (A) Morphology comparison of PBMCs (upper panel), PBMC-NK cells (mid panel) and ROBO1-NK cells (bottom panel) under bright field. (B) Biomarkers determination of ROBO1-NK cells by flow cytometry. For the biomarkers CD34, CD45, CD3, CD56 and CD16, negative controls were used irrelevant IgG replacing primary antibody for incubating, presented as red peaks. For the ROBO1-CAR detection, negative control was used PBMC-NK incubating with anti-CAR antibody, presented as red peak. Samples in detection were presented as blue peaks.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Comparison, Flow Cytometry, Negative Control

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: ROBO1-NK lyse ovarian cancer cell line and primary cancer cells. (A) Positive expression of ROBO1 on SKOV-3 cells. (B) Rate of SKOV-3 cell lysis with different E-T ratio. E: number of effective cells; T: number of target cells. (C) Time curve of different NK cells, including Mock-CAR-NK (blue), PBMC-NK (green), ROBO1-NK (red) and control (medium, black), lysing SKOV-3 cells. (D) Lysis rate of primary ovarian tumor cells from patient #01, #02, #03 and #04 by PBMC-NK and ROBO1-NK after 24 h, detected with CCK-8 assay. (E) Photograph of PBMC-NK and ROBO1-NK lysing efficacy on primary ovarian tumor cells after 48 h co-culturing. The primary cells were presented as spindle-like form while NK cells were spherical form with clusters.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Expressing, Lysis, Control, CCK-8 Assay

Journal: International Journal of Nanomedicine

Article Title: Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma

doi: 10.2147/IJN.S455633

Figure Lengend Snippet: Effect of PEG-Liker (PEG-IALLIPF), Trp, or MPP-Trp on the production of NO and Pro-inflammatory cytokines. ( A ) In Control-PBMCs, Asthma-PBMCs, Asthma-PBMCs + PEG-Liker, Asthma-PBMCs + Trp and Asthma-PBMCs + MPP-Trp group, NO production were examined by NO assay kit. Pro-inflammatory cytokines ( B ) TNF-α, ( C ) IL-1β, and ( D ) IL-6 contents in all groups were examined by ELISA assay. Data were presented as mean ± SD of three independent experiments. * P <0.05, ** P <0.01, *** P <0.001 vs Asthma-PBMCs group.

Article Snippet: The total peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of control group and asthma group by mouse peripheral blood mononuclear cell isolation kit (P6340; Solarbio, Beijing, China) according to the manufacturer's protocol.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma

doi: 10.2147/IJN.S455633

Figure Lengend Snippet: MPP-Trp was closely correlated with the balanced Th1/Th2 level and Th1/Th2-type cytokine production. ( A ) In Control-PBMCs, Asthma-PBMCs, Asthma-PBMCs + PEG-Liker, Asthma-PBMCs + Trp and Asthma-PBMCs + MPP-Trp group, Th1 population (CD4+IFN-γ+) and Th2 population (CD4+IL-4+) were selected by flow cytometry assay. ( B ) Relative mRNA expressions of IFN-γ, IL-4, IL-13, and IL-5 in all groups were assessed by qRT-PCR. ( C ) The IFN-γ, IL-4, IL-13, and IL-5 contents in all groups were examined by ELISA assay. Data were presented as mean ± SD of three independent experiments. * P <0.05, ** P <0.01, *** P <0.001 vs Asthma-PBMCs group.

Article Snippet: The total peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of control group and asthma group by mouse peripheral blood mononuclear cell isolation kit (P6340; Solarbio, Beijing, China) according to the manufacturer's protocol.

Techniques: Control, Flow Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma

doi: 10.2147/IJN.S455633

Figure Lengend Snippet: MPP-Trp altered cytokine gene expression and production in a concentration-dependent way. In Control-PBMCs, Asthma-PBMCs, and Asthma-PBMCs + MPP-Trp (10, 50, 100 and 200 μg/mL) group, ( A ) Th1/Th2 cytokine gene expressions (IFN-γ, IL-4, IL-13, and IL-5) were determined by RT-qPCR. ( B ) The cytokines (IFN-γ, IL-4, IL-13, and IL-5) levels in all groups were detected by ELISA. Data were presented as mean ± SD of three independent experiments. ** P <0.01, *** P <0.001 vs Control-PBMCs group; # P <0.05, ## P <0.01, ### P <0.001 vs Asthma-PBMCs group.

Article Snippet: The total peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of control group and asthma group by mouse peripheral blood mononuclear cell isolation kit (P6340; Solarbio, Beijing, China) according to the manufacturer's protocol.

Techniques: Gene Expression, Concentration Assay, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma

doi: 10.2147/IJN.S455633

Figure Lengend Snippet: MPP-Trp altered cytokine gene expression and production in a time-dependent way. ( A ) In Control-PBMCs, Asthma-PBMCs, and 100 μg/mL Asthma-PBMCs + MPP-Trp (6, 12, 24, and 48 h) group, IFN-γ, IL-4, IL-13, and IL-5 mRNA levels were determined by RT-qPCR. ( B ) The Th1/Th2-type cytokines (IFN-γ, IL-4, IL-13, and IL-5) productions in all group were examined by ELISA. Data were presented as mean ± SD of three independent experiments. *** P <0.001 vs Control-PBMCs group; # P <0.05; ## P <0.01; ### P <0.001 vs Asthma-PBMCs group.

Article Snippet: The total peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of control group and asthma group by mouse peripheral blood mononuclear cell isolation kit (P6340; Solarbio, Beijing, China) according to the manufacturer's protocol.

Techniques: Gene Expression, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: B7H3 expression in cell lines and the binding of delivered B7H3 BiTE to T and target cell. ( A ) B7H3 expression on MGC-803, SH-SY5Y, U87, SGC-7901 and Raji cells were detected using flow cytometry. ( B ) Binding of delivered B7H3 BiTE to target cells. Several cell lines were incubated with supernatants of the OAd-B7H3-BiTE-infected HEK293 cells. B7H3 BiTE binding was detected via flow cytometry with anti–anti-CD3-scFv antibody. ( C ) CD3 expression on Jurkat cells was detected using flow cytometry. ( D ) Binding of delivered B7H3 BiTE to T cells. PBMCs were incubated with supernatants of the OAd-B7H3-BiTE-infected HEK293 cells. B7H3 BiTE binding was detected via flow cytometry with anti–anti-CD3-scFv antibody. ( E ) Competition between delivered B7H3 BiTE and OKT3. PBMCs were incubated with supernatants of OAd or OAd-B7H3-BiTE-infected HEK293 cells. Then, cells were stained with fluorescein-conjugated OKT3 and examined throughflow cytometry. ( F ) Cell-bridging assay. A mixture of MGC-803/U87 and PBMCs was incubated with the supernatants of the OAd or OAd-B7H3-BiTE-infected HEK293 cells. The percentage of T/target cell pairs was quantified using flow cytometry. Statistical analysis was performed by unpaired Student’s t-test (n=3). NS: not significant; **p<0.01 and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; OAd, oncolytic adenovirus; PBMC, peripheral blood mononuclear cell; scFv, single-chain variable fragment.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy adults using a PBMC Isolation Solution Kit (P8680; Solarbio).

Techniques: Expressing, Binding Assay, Flow Cytometry, Incubation, Infection, Staining, Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: T-cell activation mediated by oncolytic viruses-delivered B7H3 BiTE. ( A – D ) PBMCs were cultured alone or co-cultured with the indicated cell lines in the absence (Mock) or presence of the supernatants of OAd or OAd-B7H3-BiTE-infected cells for 48 hours. ( A ) Representative brightfield images are shown. ( B ) Representative flow cytometry histograms showing FSC-A of CD4 + and CD8 + T cells. ( C ) Cumulative data of flow cytometry showing CD69 or CD107a expression in CD4 + and CD8 + T cells. ( D ) The levels of IFN-γ and IL-2 in supernatants at 48 hours measured by ELISA. ( E ) T-cell proliferation assay. CFSE-stained PBMCs were co-cultured with MGC-803 cells and incubated with or without the supernatants of OAd or OAd-B7H3-BiTE-infected cells. Proliferation was determined after incubation for 4 days. Statistical analysis was performed by unpaired Student’s t-test (n=3). NS: not significant; **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester; IFN, interferon; IL, interleukin; OAd, oncolytic adenovirus; PBMC, peripheral blood mononuclear cell.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy adults using a PBMC Isolation Solution Kit (P8680; Solarbio).

Techniques: Activation Assay, Cell Culture, Infection, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Staining, Incubation

Journal: Journal for Immunotherapy of Cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: T-cell cytotoxicity mediated by OAd-B7H3-BiTE. ( A – C ) After labeling with CFSE, MGC-803 ( A ), U87 ( B ) or Raji ( C ) cells were infected with OAd or OAd-B7H3-BiTE; uninfected cells (Mock) were used as a control. After 24 hours, peripheral blood mononuclear cells were added and co-cultured for 48 hours. Representative flow cytometry plots (left) and cumulative data (right) are shown. Dead target cells are defined as CFSE + Zombie Dye + . The percentage of dead target cells is calculated as follows: CFSE + Zombie-Dye + /CFSE + . Statistical analysis was performed by unpaired Student’s t-test (n=3). **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester; OAd, oncolytic adenovirus.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy adults using a PBMC Isolation Solution Kit (P8680; Solarbio).

Techniques: Labeling, Infection, Control, Cell Culture, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: OAd-B7H3-BiTE treatment elicits potent therapeutic effects in vivo. ( A ) Schematic diagram of treatment schedule for MGC-803 bearing mice ( B – G ). ( B ) Representative flow cytometry plots showing human CD45 + CD3 + T cells in tumor and spleen of untreated mice. Flow cytometry plots gated on live-cell population. ( C ) Summary data for average tumor growth (left) and body weight changes (right) for all treatment groups. ( D ) Tumor pictures and weights for all treatment groups. ( E – F ) Primary tumors were collected and analyzed with reverse transcription-quantitative PCR to examine the expression of E1a ( E ) and B7H3 BiTE ( F ). ( G ) Representative H&E staining of the major organs for all treatment groups. Statistical analysis was performed by unpaired two-way analysis of variance ( C ) or unpaired Student’s t-test ( D ) (n=5). ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; mRNA, messenger RNA; OAd, oncolytic adenovirus; PBS, phosphate-buffered saline; PBMC, peripheral blood mononuclear cell; s.c., subcutaneous injection.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy adults using a PBMC Isolation Solution Kit (P8680; Solarbio).

Techniques: In Vivo, Flow Cytometry, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Staining, Saline, Injection