Journal: EMBO Molecular Medicine

Article Title: A therapeutic regimen using neoantigen-specific TCR-T cells for HLA-A*2402-positive solid tumors

doi: 10.1038/s44321-024-00184-1

Figure Lengend Snippet: ( A , B ) Tcr-T1 cells were generated as mentioned in the methods. Schematic for TCR-T cell generation, purification, and in vitro expansion. ( A ) Representative flow cytometric images indicated the electroporation efficiency and purification of Tcr-T1 cells ( B ). ( C ) HLA-A*2402-positive MKN45-Luci cells were treated with Tcr-T1 cells, with or without Pep-4 pulsing. 24 h later, cell viability was tested via CCK-8 assay. ( D ) Human synovial sarcoma cell line SW982 was modified with stable expression of the HLA-A*2402 molecule to generate target cells (SW982/2402). Then these target cells, SW982 and SW982/2402, were transfected with vectors expressing SYT-SSX fusion mutated peptide containing/deleting Pep-4 sequence (SYT-SSX ΔPep4, SYT-SSX, SYT-SSX ΔPep4/2402, and SYT-SSX/2402). Target cells were incubated with Tcr-T1 cells at ratio 0:1, 0.2:1, 1:1, and 5:1 (T-cell/SW982). Cell viability was determined by using CCK-8. ( E ) SW982 cells overexpressing HLA-A*2402 and SYT-SSX fusion mutated peptide containing Pep-4 sequence (SYT-SSX/2402) were incubated with Tcr-T1 cells ( E : T = 1:1). Cell viability was determined by CCK-8. IFN-γ secretion was examined using a CBA assay ( n = 3 independent replication). ( F ) Cytotoxicity of Tcr-T1 against HLA-A*2402-positive MKN45 was determined as indicated ( n = 3 independent replication). ( G – I ) Treatment scheme ( G , up). SYT-SSX/2402 and SYT-SSX ΔPep4/2402 cells (1 × 10 7 cells/mouse) were injected subcutaneously in nude mice ( n = 6). When tumor volume reached to ~100 mm 3 , Tcr-T1 cells, and UTD T cells (5 × 10 6 cells/mouse) were intravenously injected. Mice were sacrificed at the endpoint. Representative photographs of tumor tissues isolated from each group of mice ( G , bottom). Tumor volumes of each group were recorded ( H ). The overall survival of each group was recorded. ( I ) Data are presented as mean ± SEM. Statistical significance was calculated using the Student’s t test ( C , E ), one-way ANOVA ( C ), two-way ANOVA ( D – F , H ), and Kaplan–Meier survival analysis ( I ). ns not significant, * P < 0.05; ** P < 0.01; *** P < 0.001. .

Article Snippet: Human synovial sarcoma cell line SW982 (HLA-A24-, SYT/SSX fusion-negative), human gastric adenocarcinoma cell line MKN45 (HLA-A24 + , SYT/SSX fusion-negative), human breast cancer cell line MDA-MB-231 (HLA-A02 + , SYT/SSX fusion-negative) and human transporter-associated protein (TAP)-deficient cell line T2 was originally purchased from ATCC, China Center for Type Culture Collection (CCTCC) and the typical cell culture collection of the Committee of the Chinese Academy of Sciences Library.

Techniques: Generated, Purification, In Vitro, Electroporation, CCK-8 Assay, Modification, Expressing, Transfection, Sequencing, Incubation, Injection, Isolation

Journal: EMBO Molecular Medicine

Article Title: A therapeutic regimen using neoantigen-specific TCR-T cells for HLA-A*2402-positive solid tumors

doi: 10.1038/s44321-024-00184-1

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Human synovial sarcoma cell line SW982 (HLA-A24-, SYT/SSX fusion-negative), human gastric adenocarcinoma cell line MKN45 (HLA-A24 + , SYT/SSX fusion-negative), human breast cancer cell line MDA-MB-231 (HLA-A02 + , SYT/SSX fusion-negative) and human transporter-associated protein (TAP)-deficient cell line T2 was originally purchased from ATCC, China Center for Type Culture Collection (CCTCC) and the typical cell culture collection of the Committee of the Chinese Academy of Sciences Library.

Techniques: Cell Culture, Recombinant, Bicinchoninic Acid Protein Assay, Software

Journal: Marine Drugs

Article Title: Marine Antimicrobial Peptide TP4 Exerts Anticancer Effects on Human Synovial Sarcoma Cells via Calcium Overload, Reactive Oxygen Species Production and Mitochondrial Hyperpolarization

doi: 10.3390/md19020093

Figure Lengend Snippet: TP4 triggers cell death in synovial sarcoma cells. Dose-dependent cytotoxicity of TP4 in SW982 cells ( A , B ) and Aska-SS cells ( C ). Cells were incubated with various concentrations of TP4 for 5 h. Time-dependent cytotoxicity of TP4 in SW982 cells ( D , E ) and Aska-SS cells ( F ). Cells were treated with TP4 (20 μg/mL) for the indicated times. Cytotoxicity was determined by the MTS/PMS assay ( A , C , D , F ) and trypan blue exclusion assay ( B , E ). *: p < 0.05 (Compared to 0 min or 0 μg/mL). IC50 values were calculated with GraphPad Prism 8 using a non-linear regression fit (log(inhibitor) vs. normalized response—variable slope).

Article Snippet: The SW982 human synovial sarcoma cell line was purchased from ATCC (The Global Bioresource Center; Manassas, VA, USA).

Techniques: Incubation, Trypan Blue Exclusion Assay

Journal: Marine Drugs

Article Title: Marine Antimicrobial Peptide TP4 Exerts Anticancer Effects on Human Synovial Sarcoma Cells via Calcium Overload, Reactive Oxygen Species Production and Mitochondrial Hyperpolarization

doi: 10.3390/md19020093

Figure Lengend Snippet: Necrotic cell death is induced by TP4 in synovial sarcoma cells. Cells were treated with TP4 (20 μg/mL) or staurosporine (1 μM) for 5 h and 24 h, respectively. Vehicle control was 0.5% DMSO. Necrotic cell death was determined by the propidium iodide incorporation assay ( A ). ( B ) Cells (SW982 and Aska-SS) were treated as described above. Cell lysates and culture supernatants were collected and immunoblotted with indicated antibodies. Band intensities were analyzed with ImageJ software. *: p < 0.05 (Compared to vehicle). Cells (SW982 and Aska-SS) were preincubated with GSK’872 (5 μM) ( C , E ), Necrostatin-1 (10 μM) ( D , F ), or Z-VAD-FMK (100 μM) ( G , H ) for 1 h, followed by TP4 (20 μg/mL) for additional 5 h. Cell viability was analyzed using the trypan blue exclusion assay. *: p < 0.05 (Vehicle vs. TP4; TP4 vs. TP4 + inhibitor). Vehicle control: appropriate amount of DMSO.

Article Snippet: The SW982 human synovial sarcoma cell line was purchased from ATCC (The Global Bioresource Center; Manassas, VA, USA).

Techniques: Control, Software, Trypan Blue Exclusion Assay

Journal: Marine Drugs

Article Title: Marine Antimicrobial Peptide TP4 Exerts Anticancer Effects on Human Synovial Sarcoma Cells via Calcium Overload, Reactive Oxygen Species Production and Mitochondrial Hyperpolarization

doi: 10.3390/md19020093

Figure Lengend Snippet: TP4 triggers oxidative stress. Synovial sarcoma cells were treated with TP4 (20 μg/mL) for the indicated times, and intracellular ROS was evaluated by DCF-DA ( A , B , E , F ) and DHE ( C , D ) fluorescence using flow cytometry. *: p < 0.05 (compared to 0 min). SW982 ( G ) and AsKa-SS ( H ) cells were preincubated with TEMPO (150 μM) for 1 h, followed by TP4 (20 μg/mL) for additional 5 h. Cell viability was determined using the trypan exclusion assay. *: p < 0.05 (Vehicle vs. TP4; TP4 vs. TP4 + TEMPO). ( I ) SW982 cells were treated with TP4 (20 μg/mL) for the indicated times, and cell lysates were collected and immunoblotted with the indicated antibodies. UCP-2: uncoupling protein-2; GPx: glutathione peroxidase; SOD-1: superoxide dismutase-1; SOD-2: superoxide dismutase-2. ( J – N ) Band intensities were analyzed with ImageJ. *: p < 0.05 (Compared to 0 min).

Article Snippet: The SW982 human synovial sarcoma cell line was purchased from ATCC (The Global Bioresource Center; Manassas, VA, USA).

Techniques: Fluorescence, Flow Cytometry, Exclusion Assay

Journal: Marine Drugs

Article Title: Marine Antimicrobial Peptide TP4 Exerts Anticancer Effects on Human Synovial Sarcoma Cells via Calcium Overload, Reactive Oxygen Species Production and Mitochondrial Hyperpolarization

doi: 10.3390/md19020093

Figure Lengend Snippet: TP4 induces mitochondrial hyperpolarization and mitochondrial ROS generation. SW982 ( A , B ) and AsKa-SS ( C , D ) cells were treated with TP4 (20 μg/mL) for 0, 15, 30, 60, 180 and 300 min. TMRE intensity was analyzed by flow cytometry. SW982 ( E , F ) and AsKa-SS ( G , H ) cells were treated with TP4 (20 μg/mL), as described in ( A – D ), followed by MitoSOX Red (5 μM) for an additional 15 min. MitoSOX Red fluorescence intensity was determined by flow cytometry. *: p < 0.05 (Compared to 0 min).

Article Snippet: The SW982 human synovial sarcoma cell line was purchased from ATCC (The Global Bioresource Center; Manassas, VA, USA).

Techniques: Flow Cytometry, Fluorescence

Journal: Marine Drugs

Article Title: Marine Antimicrobial Peptide TP4 Exerts Anticancer Effects on Human Synovial Sarcoma Cells via Calcium Overload, Reactive Oxygen Species Production and Mitochondrial Hyperpolarization

doi: 10.3390/md19020093

Figure Lengend Snippet: TP4 triggers calcium overload. SW982 ( A , B ) and AsKa-SS ( C , D ) cells were preloaded with Fluo-4 (5 μM) for 15 min, followed by TP4 treatment. After 0, 15, 30, 60, 180 or 300 min, Fluo-4 intensity was analyzed by flow cytometry. *: p < 0.05 (compared to 0 min). SW982 ( E , F ) and Aska-SS ( G ) cells were preincubated with BAPTA (10 μM) for 1 h, followed by TP4 (20 μg/mL) for 5 h. Then, cells were incubated with DCF-DA for 15 min. DCF-DA intensity was analyzed by flow cytometry. *: p < 0.05 (Vehicle vs. TP4; TP4 vs. TP4 + BAPTA). SW982 ( H ) and AsKa-SS ( I ) cells were preincubated with BAPTA (10 μM) for 1 h, followed by TP4 (20 μg/mL) for an additional 5 h. Cell viability was determined using the trypan exclusion assay. *: p < 0.05 (Vehicle vs. TP4; TP4 vs. TP4 + BAPTA). SW982 ( J ) and AsKa-SS ( K , L ) cells were incubated with TP4 (20 μg/mL) for 300 min. TMRE intensity was analyzed by flow cytometry. *: p < 0.05 (Vehicle vs. TP4; TP4 vs. TP4 + BAPTA).

Article Snippet: The SW982 human synovial sarcoma cell line was purchased from ATCC (The Global Bioresource Center; Manassas, VA, USA).

Techniques: Flow Cytometry, Incubation, Exclusion Assay