Journal: Oncotarget

Article Title: Imaging the distribution of an antibody-drug conjugate constituent targeting mesothelin with 89 Zr and IRDye 800CW in mice bearing human pancreatic tumor xenografts

doi:

Figure Lengend Snippet: A. A representative brightfield image (400x) of a tumor with an IHC staining for mesothelin. B. Fluorescence microscopy at 800 nm for IRDye 800CW labelled to AMA; C. at 460 nm for DAPI staining, D. and overlay of fluorescent images.

Article Snippet: For analysis of receptor expression, cells were incubated with anti-human mesothelin phycoerythrin (catalog number: FAB32652P, R&D systems).

Techniques: Immunohistochemistry, Fluorescence, Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model

doi: 10.3389/fimmu.2024.1362904

Figure Lengend Snippet: Lentiviral vector production and titration. Schematic representation of three second-generation CAR constructs (A) . MSLN-CAR contains an anti-mesothelin (MSLN) scFv, human CD8α-derived hinge, 4–1BB-derived transmembrane (TM) domain, and two intracellular domains, including 4–1BB and CD3ζ. A2aR.KD and Tim3.KD.MSLN-CAR constructs include an anti-A2aR or Tim3 shRNA sequence after the CD3ζ sequence. ShRNA sequences were designed to target the A2aR and Tim3 genes. Fluorescence microscopy images and histogram plots show the percentage of HEK293T cells transfected with lentiviral vectors containing GFP (right) compared to untransfected HEK293T cells (left) (B) . Flow cytometry histograms show the titration of concentrated virus-containing media (VCM) on 1×10 6 Jurkat cells/well (C) . The percentage of CAR-expressing Jurkat cells (GFP%) three days after transduction with 0.01, 0.1, 1, 10, and 100 μL of VCM is shown.

Article Snippet: The expression of mesothelin was verified by flow cytometry using a PE-conjugated anti-human mesothelin antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Plasmid Preparation, Titration, Construct, Derivative Assay, shRNA, Sequencing, Fluorescence, Microscopy, Transfection, Flow Cytometry, Virus, Expressing, Transduction

Journal: Frontiers in Immunology

Article Title: Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model

doi: 10.3389/fimmu.2024.1362904

Figure Lengend Snippet: Generation of different types of MSLN-CAR T cells. Flow cytometry histogram plots show the percentage of CAR-positive T cells generated from one donor 4 days after transduction of anti-CD3/CD28-activated T cells with the indicated volume of lentiviral vectors at an MOI of ~5. Untransduced T cells refer to T cells that did not undergo transduction with lentiviral vectors. Mock T cells were transduced with a pCDH-CMV-MCS-EF1 vector without the MSLN-CAR sequence. MSLN-CAR T cells refer to T cells transduced with a lentivector containing the anti-mesothelin CAR. Tim3.KD.MSLN-CAR T cells refer to T cells containing both MSLN-CAR and Tim3 shRNA. A2aR.KD.MSLN-CAR T cells refer to T cells containing both MSLN-CAR and A2aR shRNA. Tim3/A2aR.KD.MSLN-CAR T cells were cotransduced with half of the indicated volume of lentivectors containing MSLN-CAR and Tim3 shRNA and MSLN-CAR and A2aR shRNA.

Article Snippet: The expression of mesothelin was verified by flow cytometry using a PE-conjugated anti-human mesothelin antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Flow Cytometry, Generated, Transduction, Plasmid Preparation, Sequencing, shRNA

Journal: Frontiers in Immunology

Article Title: Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model

doi: 10.3389/fimmu.2024.1362904

Figure Lengend Snippet: Antigen-dependent cytotoxicity of MSLN-CAR T cells. Line plots comparing the average percentage of dead HeLa target cells coincubated with MSLN-CAR T cells, Tim3.KD.MSLN-CAR T cells, A2aR.KD.MSLN-CAR T cells, Tim3/A2aR.KD.MSLN-CAR T cells and mock T cells at four different effector-to-target ratios (1:1, 5:1, 10:1, and 20:1) in the absence or presence of 1 μM NECA for 4 hours (A-D) . The cells were stained with propidium iodide (PI) dye. Data are representative of two independent experiments, each performed in duplicate. Two-way ANOVA followed by Tukey’s post hoc test was used for statistical analysis. P< 0.05 was considered statistically significant. (***P< 0.001). NECA: 5′-(N-ethylcarboxamido) adenosine; SD: standard deviation; MSLN-CAR T cells: fully human anti-mesothelin CAR T cells; Tim3.KD.MSLN-CAR T cells: MSLN-CAR T cells containing Tim3 shRNA; A2aR.KD.MSLN-CAR T cells: MSLN-CAR T cells containing A2aR shRNA; Tim3/A2aR.KD.MSLN-CAR T cells: MSLN-CAR T cells containing both Tim3 and A2aR shRNA; Mock T cells: T cells containing an empty vector.

Article Snippet: The expression of mesothelin was verified by flow cytometry using a PE-conjugated anti-human mesothelin antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Staining, Standard Deviation, shRNA, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model

doi: 10.3389/fimmu.2024.1362904

Figure Lengend Snippet: Proliferation potency of MSLN-CAR T cells in the presence of antigen and adenosine. 2 × 10 5 CAR T cells from four groups, including MSLN-CAR T cells, Tim3.KD.MSLN-CAR T cells, A2aR.KD.MSLN-CAR T cells, and Tim3/A2aR.KD.MSLN-CAR T cells were labeled with CFSE dye and cocultured with mitomycin C-treated HeLa cells at a 1:1 ratio for 72 hours in the absence or presence of 1 μM NECA. Mock T cells containing an empty vector were used as control cells. The bar graphs show the percentage of divided cells between MSLN-CAR T cells (in presence and absence of Neca) and mock T cells against media and mesothelin positive (HeLa) and negative (Panc-1) cancer cells (A) . Antigen specific proliferation of MSLN-CAR T cells and Tim3.KD.MSLN-CAR T cells in presence and absence of Neca (B) . Antigen specific proliferation of MSLN-CAR T cells and A2aR.KD.MSLN-CAR T cells in presence and absence of Neca (C) . Antigen specific proliferation of MSLN-CAR T cells and Tim3/A2aR.KD.MSLN-CAR T cells in presence and absence of Neca (D) , and among all groups (E) . The data are presented as the mean ± standard deviation (SD). Mean comparisons were performed using Brown-Forsythe and Welch ANOVA followed by Dunnett T3’s post hoc test. Statistical significance was set at P< 0.05. The results represent two independent experiments (*P< 0.05, **P< 0.01, and ***P< 0.001). NECA, 5′-(N-ethylcarboxamido) adenosine; SD, standard deviation; MSLN-CAR T cells, fully human anti-mesothelin CAR T cells; Tim3.KD.MSLN-CARs, T cells containing MSLN-CAR and Tim3 shRNA; A2aR.KD.MSLN-CARs, T cells containing MSLN-CAR and A2aR shRNA; Tim3/A2aR.KD.MSLN-CARs, T cells containing MSLN-CAR and both Tim3 and A2aR shRNA; Mock T, T cells containing an empty vector.

Article Snippet: The expression of mesothelin was verified by flow cytometry using a PE-conjugated anti-human mesothelin antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Labeling, Plasmid Preparation, Control, Standard Deviation, shRNA

Journal: Frontiers in Immunology

Article Title: Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model

doi: 10.3389/fimmu.2024.1362904

Figure Lengend Snippet: Cytokine production of different types of MSLN-CAR T cells. Fully human anti-mesothelin CAR T cells (MSLN-CAR T cells), Tim3 knockdown MSLN-CAR T cells (Tim3.KD.MSLN-CAR T cells), A2aR knockdown MSLN-CAR T cells (A2aR.KD.MSLN-CAR T cells), Tim3 and A2aR knockdown MSLN-CAR T cells (Tim3/A2aR.KD.MSLN-CAR T cells), and mock T cells containing empty vector were cocultured with HeLa target cells at a 1:1 ratio or media in the absence or presence of 1 μM NECA (5′-Nethylcarboxamido adenosine). After 48 hours, the supernatant was harvested, and the concentrations of IL2 (A) , TNFα (B) , and IFN-γ (C) cytokines were measured using ELISA. Data are presented as the mean ± standard deviation (SD) from two independent experiments. Mean comparisons were performed using one-way ANOVA followed by Tukey's post hoc test, with P< 0.05 considered statistically significant (**P< 0.01, ***P< 0.001, and ****P< 0.0001). MSLN-CAR T cells: fully human anti-mesothelin CAR T cells; Tim3.KD.MSLN-CARs: MSLN-CAR T cells containing Tim3 shRNA; A2aR.KD.MSLN-CARs: MSLN-CAR T cells containing A2aR shRNA; Tim3/A2aR.KD.MSLN-CARs: MSLN-CAR T cells containing both Tim3 and A2aR shRNA; Mock T cells: T cells containing empty vector.

Article Snippet: The expression of mesothelin was verified by flow cytometry using a PE-conjugated anti-human mesothelin antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Knockdown, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Standard Deviation, shRNA

Journal: Frontiers in Immunology

Article Title: Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model

doi: 10.3389/fimmu.2024.1362904

Figure Lengend Snippet: The effect of CAR T cells on tumor growth. The effect of CAR T cells on tumor growth was evaluated in six groups of C57BL/6-nude mice (n=5) treated with 1x10 7 different CAR T cells, including MSLN-CAR T cells, Tim3.KD.MSLN-CAR T cells, A2aR.KD.MSLN-CAR T cells, and Tim3/A2aR.KD.MSLN-CAR T cells. PBS-1X and Mock T cells were used as control groups. Tumor volume was measured on day 0 (the time of intervention) and every 24–48 hours (A) . Line plots depict the rate of tumor growth over time for each of the five mice in each group (B) . Each color represents the tumor growth trend curve for one mouse. The bar graph displays the tumor volume for each group (C) . Data are presented as the mean ± SD. Mean comparisons were performed using two-way ANOVA followed by Tukey's post hoc test. A P value less than 0.05 was considered statistically significant (*P< 0.05, **P< 0.01, ***P< 0.001). The results are representative of two independent experiments. The CAR T cells used in the study were fully human anti-mesothelin CAR T cells (MSLN-CAR T cells), T cells containing MSLN-CAR and Tim3 shRNA (Tim3.KD.MSLN-CAR T cells), T cells containing MSLN-CAR and A2aR shRNA (A2aR.KD.MSLN-CAR T cells), T cells containing MSLN-CAR and both Tim3 and A2aR shRNA (Tim3/A2aR.KD.MSLN-CAR T cells), and mock T cells containing an empty vector. The PBS-1x group received phosphate-buffered saline.

Article Snippet: The expression of mesothelin was verified by flow cytometry using a PE-conjugated anti-human mesothelin antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Control, shRNA, Plasmid Preparation, Saline

Journal: Biomedicines

Article Title: Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients' Sera.

doi: 10.3390/biomedicines10081942

Figure Lengend Snippet: Figure 1. Mesothelin expression in PaC and ovarian cancer cell lines by WB (a,b), and with or without PNGaseF digestion (c). (a) MSLN expression in cell lysates (top), stripping and reblotting with GADPH as loading control (bottom): 20 µg total protein loaded per lane; (b) MSLN expression in secretion media: 25 µg total protein loaded per lane. For (a,b) 25 ng rMSLN were used as a positive control; (c) MSLN from 10 µg protein lysates or 25 ng rMSLN detected with an anti-MSLN antibody (clone MN-1) under reducing conditions (left) and WB on 50 ng rMSLN detected with Aleuria aurantia lectin (fucose detection) under non-reducing conditions (right); ND: not digested; D: digested.

Article Snippet: A commercial recombinant human mesothelin standard (rMSLN, #3265-MS, R&D Systems, Minneapolis, MN, USA) which is produced in a murine myeloma cell line (NS0derived) was used throughout the study as a control, both for the MSLN expression and glycosylation.

Techniques: Expressing, Stripping Membranes, Control, Positive Control

Journal: Biomedicines

Article Title: Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients' Sera.

doi: 10.3390/biomedicines10081942

Figure Lengend Snippet: Figure 6. Mesothelin expression in pancreatic tissue lysates by WB under non-reducing conditions. Each number represents a different patient in each condition. A total of 20 µg protein was loaded per lane, and 25 ng rMSLN used as a positive control. All membranes were equally exposed to chemiluminescence for lane comparison. Control samples included two healthy pancreas (H) and pan- creatic non-tumor tissues adjacent to the cancer region from cholangiocarcinomas (C), from duodenum adenocarcinomas (D), from ampulloma (A) and from PaC (NT). Cancer tissues included thirty-one PaC of different stages and other tumors: one ampulloma (A) and one cholangiocarcinoma (C)).

Article Snippet: A commercial recombinant human mesothelin standard (rMSLN, #3265-MS, R&D Systems, Minneapolis, MN, USA) which is produced in a murine myeloma cell line (NS0derived) was used throughout the study as a control, both for the MSLN expression and glycosylation.

Techniques: Expressing, Positive Control, Comparison, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: Mesothelin and MUC16 are heterogeneously expressed in patient samples and human-derived cancer cell lines. A ) Dot plot of mesothelin (APC) and MUC16 (PE) expression in cells obtained from three patients diagnosed with ovarian cancer. Numbers in each quadrant represent percentages of the total “Alive” cell population as determined by DAPI staining via flow cytometry. ( B ) RNA expression of mesothelin and MUC16 in ovarian and pancreatic PDXs in Log2(TPM+1) units. Expression levels were obtained from the cBioPortal for Cancer Genomics, available through the Center for Patient Derived Models (CPDM) database at Dana-Farber, one-way ANOVA test was used to compare RNA expression levels of mesothelin and MUC16 between pancreatic and ovarian PDXs. ( C, D ) Tissue array of ovarian ( C ) and pancreatic ( D ) tumors together with adjacent normal tissue area, stained for mesothelin and MUC16 by IHC. Each magnified picture details the same tissue area for both stainings. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Adenoc., adenocarcinoma; ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; IHC, immunohistochemistry; meso, mesothelin; MUC16, Mucin16; PDX, patient-derived xenograft, TPM, transcripts per million.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: Derivative Assay, Expressing, Staining, Flow Cytometry, RNA Expression, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: Tandem scFv screening to target mesothelin and MUC16. ( A ) Schematic of the six tandem scFv designs. ( B ) MFI values of soluble FITC-labeled mesothelin (upper panel) and soluble His-tag-labeled MUC16 (lower panel) bound to each CAR construct expressed in Jurkat cells. MFI values were obtained from Jurkat-mCherry + cells. Bars represent the mean±SEM of three technical replicates. ( C, D ) MFI values of soluble mesothelin ( C ), and MUC16 ( D ) bound to each tandem CAR as single staining (empty dot) or sequential double staining (filled dot), where one antigen was added, the cells were washed, and the second antigen was added. For the sequential double staining we stained first with soluble mesothelin, washed, and then we added soluble MUC16. The MFI of the double staining for each antigen is relative to the MFI value of the corresponding single antigen staining. ( E ) Flow cytometry histograms showing mesothelin and MUC16ecto expression in ASPC-1 and OVCAR3 cancer cell lines that endogenously express (endo) or do not express (neg), are knocked out (KO) for, or are artificially transduced (TR) for mesothelin or MUC16ecto. (F) Heat map of GFP MFI values from CAR-Jurkat NFAP-GFP reporter cells exposed to tumor cells for 24 hours. MFI values were obtained from mCherry + CAR-Jurkat NFAT-eGFP cells. ( G and H) Multiparametric representation of the MFI of antigen bound to CAR-Jurkat cells (X and Y values) along with the MFI of the GFP (color scale) and the percentage of GFP + cells (circle size) in CAR-Jurkat NFAT-eGFP mCherry + cells in response to (G) ASPC-1 cells and (H) OVCAR3 cells. MFI values of GFP (color scale) and the percentage of GFP + cells (circle size) are the mean of each CAR-Jurkat NFAT-eGFP reporter cell co-cultures with the different ASPC-1 and OVCAR3 (E) tumor cells, excluding the double negative tumor cells. In B, significance between the different tandem scFv constructs was measured using one-way ANOVA test with Turkey’s multiple comparison test. In C and D, significance between the single and combinatorial staining for each tandem scFv was measured using two-way ANOVA test with Fisher LSD test. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; FITC, fluorescein Isothiocyanate; Fisher LSD test, Fisher least significant difference test; east Significance difference test; meso, mesothelin; MFI, mean fluorescence intensity; MUC16, Mucin16; MUC16ecto, Mucin16 ectodomain; ns, not significant; scFv, single-chain variable fragment, TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: Labeling, Construct, Staining, Double Staining, Flow Cytometry, Expressing, Comparison, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: TanCAR in vitro antitumor activity, cytokine production, and avidity . ( A, B ) Bar graph showing the percent cytotoxicity after 24 hours of ( A ) ASPC-1 or ( B ) OVCAR3 cancer cells co-culture with CAR-T cells (SS1, 4H11, TanCAR1 or TanCAR3) or UTD-T cells at an E:T ratio of 3:1, assessed by luciferase-based killing assay. Bars represent the mean±SEM of three healthy donors measured in triplicate or duplicate. Stars indicate significant differences between monospecific CAR-T cells and tandem CAR-T cells using a two-tailed Mann-Whitney test. ( C, D ) Heat maps showing the pg/mL concentration of IL-2, IFNγ, TNFα, or GM-CSF in supernatant collected from killing assays after 24 hours of co-culture of CAR-T cells with ( C ) ASPC-1 or ( D ) OVCAR3 cancer cells at an E:T ratio of 10:1. Data is the mean from two healthy donors. In A and B, significance between monospecific CAR-T cells and tandem CAR-T cells using a two-tailed Mann-Whitney test, only statistical significance is depicted on the graph. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. CAR, chimeric antigen receptor; ecto TR , transduced with the MUC16 ectodomain; endo , endogenous expression of the antigen; E:T, effector to target; IFNγ, interferon-gamma; IL, interleukin; KO , CRISPR KO of the endogenous antigen; meso, mesothelin; MUC16, Mucin16; MUC16ecto, Mucin16 ectodomain; neg , endogenously negative for the antigen; TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: In Vitro, Activity Assay, Co-Culture Assay, Luciferase, Two Tailed Test, MANN-WHITNEY, Concentration Assay, Transduction, Expressing, CRISPR

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: TanCAR-T cell binding avidity to tumor cells expressing one or both antigens. ( A ) Schematic of one antigen at a time (left) or two antigens at a time (right panel) hypotheses for the binding of tandem CAR to two antigens ( B ) Bar graph of mesothelin and MUC16ecto MFI values in different cell lines and isotype controls, where H/H stands for Meso H MUC16ecto H ; H/L, Meso H MUC16ecto L ; M/M, Meso M MUC16ecto M ; L/H, Meso L MUC16ecto H ; and L/L, Meso L MUC16ecto L . ( C–G ) Avidity curves showing the ratio of T cells bound relative to UTD to ASPC-1 tumor cells (C, Meso H MUC16ecto H ; D, Meso H MUC16ecto L ; E, Meso M MUC16ecto M ; F, Meso L MUC16ecto H ; G, Meso L MUC16ecto L ) per acoustic force unit applied in picoNewtons (pN). ( H ) Schematic of one antigen-driven (left) or two antigen-driven (right) tandem CAR avidity profile. ( I ) Multiparametric representation of the MFI of mesothelin and MUC16ecto expression in different ASPC-1 shown in 4B (X and Y values) along with the mean ratio of bound TanCAR1-T cells relative to UTD to each ASPC-1 cell type (data from 4C–G) (color scale). ( J ) Avidity curves showing the ratio of TanCAR1-T cells bound relative to UTD to each ASPC-1 tumor cell line, per pN of acoustic force applied. ( K ) Dot plot graph of the ratio of bound TanCAR-T cells at 1000 pN in each ASPC-1 cell line. Stars indicate significant differences as measured by a two-way ANOVA with Fisher’s LSD test in C–G, and as measured by one-way ANOVA with Fisher’s LSD test in K. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, non-significant. ANOVA, analysis of variance; CAR, chimeric antigen receptor; Fisher LSD test, Fisher least significat difference test; meso, mesothelin; MFI, mean fluorescence intensity; MUC16ecto, Mucin16 ectodomain; TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: Binding Assay, Expressing, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: Tandem CAR-T cells overcome tumor heterogeneity. ( A ) Schematic of a mixed tumor cell culture with ASPC-1 Meso endo MUC16 neg , Meso KO MUC16ecto TR , and Meso endo MUC16ecto TR cells. ( B ) Violin plot of the tumor area 96 hours after co-culture with CAR-T or UTD-T cells, relative to time 0, measured via real-time killing assays using an IncuCyte Live-Cell Analysis system. The dotted line at y=1 represents the threshold for tumor clearance (below 1). Each dot in the violin plot represents a replicate of the experiment, with four technical replicates of three healthy donor T cells. ( C ) Bar graph showing the mean absolute number of each tumor cell population at the end of the real-time killing assay in ( B ), per CAR-T cell treatment group. Each color of the stacked bars represents the tumor populations depicted in ( A ). D ) Dot plot of the absolute number of each tumor cell population shown in ( C ), per CAR-T cell treatment group. Each dot represents a replicate of the experiment, with two technical replicates of three healthy donor T cells. ( E, F ) Schematic of the mixture culture of ASPC-1 Meso endo MUC16 neg iRFP+ cells and Meso KO MUC16ecto TR GFP+cells and their meso and MUC16ecto MFI values. ( G–I ) Total tumor area relative to time 0 (start of the co-culture) ( G ), measured using an IncuCyte Live-Cell Analysis system of a real-time cytotoxicity assay with CAR-T cells or UTD-T cells co-cultured for 96 hours 1:1 with a mixture of ASPC-1 Meso endo MUC16 neg iRFP+ cells and Meso KO MUC16ecto TR GFP+cells shown in E; and blue ( H ) and green ( I ) cell area relative to time 0. Curves represent the mean±SEM of three technical replicates of two healthy donors for each treatment group. ( J ) Dot plot of blue (Meso endo MUC16 neg ) and green (Meso KO MUC16ecto TR ) tumor cell area at 96 hours of co-culture with TanCAR1 relative to time 0 from ( H ) and ( I ). ( K–M ) Mixed tumor spheroids were created with Meso endo MUC16 neg iRFP+ (blue) and Meso KO MUC16ecto TR GFP+ (green) cells and treated with CAR T cells. ( I ) Representative images from different time points of mixed-tumor spheroids treated with CAR-T cells. ( L–M ) Violin plots of the ( L ) blue and ( M ) green tumor cell area at 106 hours relative to time 0, measured using an IncuCyte Live-Cell Analysis system. Dots represent technical triplicates and two healthy donors for each treatment group. Differences measured by Mann-Whitney test for each comparison in ( B, J, L, M ), Kruskal-Wallis test for ( D ) and a two-way ANOVA with Fisher’s LSD test for (G–I). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; Fisher LSD test, Fisher least significat difference test; meso, mesothelin; MUC16, Mucin16; MUC16ecto, Mucin16 ectodomain; ns, non-significant; TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: Cell Culture, Co-Culture Assay, Cell Analysis, Cytotoxicity Assay, MANN-WHITNEY, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time

doi: 10.1136/jitc-2025-012822

Figure Lengend Snippet: In vivo antitumor activity of TanCAR1-T in double-positive tumor cells heterogeneous tumor models. ( A ) Schematic of the in vivo experiments in NSG mice engrafted intraperitoneally with OVCAR3 tumor cells and treated 14 days later with CAR-T or UTD-T cells or left untreated (tumor alone). ( B ) Quantification of the flux (photons/s 2 ) from mice treated as indicated. Curves represent the median+IQR of each treatment group, with 14 mice per group treated with T cells from two healthy donors. ( C ) Schematic of the in vivo experiments in NSG mice engrafted subcutaneously with mixed ASPC-1 tumor cells and treated 14 days later with CAR-T or UTD-T cells or left untreated (tumor alone). ( D ) Caliper measurements from mice treated as indicated. Curves represent the mean±SEM of each treatment group, with 14 mice per group, repeated with T cells from two healthy donors. ( E ) Percentages of each tumor cell population from mice in ( D ), at the time of injection compared with the time of tumor collection from the mice. Each dot represents the mean±SEM of each treatment group treated with T cells from one healthy donor: SS1 CAR group contains primary tumor and lung metastasis from one mouse, 4H11 CAR group contains primary tumors from three mice, and TanCAR1 group shows primary tumors from six mice. The statistical analysis from 4H11 and TanCAR1 groups is shown. ( F ) Diagram explaining the skewed killing of TanCAR1-T cells towards tumor cells expressing high levels of one of the cognate antigens. ( G ) Schematic of the in vivo experiment in NSG mice engrafted subcutaneously with mixed ASPC-1 tumor cells, treated 14 days later with CAR-T or UTD-T cells, and euthanized at day 21 after CAR/UTD-T cells injection. ( H ) Representative images of IHC slides of tumors from mice after 21 days of CAR/UTD-T cell administration. Lower panels are magnifications of highlighted area in the upper panels. ( I ) Dot plot graph showing CD3 + cells per mm 2 in each group of treated mice. Differences measured by a two-way ANOVA test with Fisher’s LSD test for B and D, a Mann-Whitney test for E and a one-way ANOVA with Holm-Šídák’s multiple comparisons test for I. Only comparisons between CAR-T cells (TanCAR1, SS1CAR, and 4H11CAR) are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; CAR, chimeric antigen receptor; IHC, immunohistochemistry; IP, intraperitoneal; IV, intravenous; Fisher LSD test, Fisher least significat difference test; meso, mesothelin; MUC16, Mucin16; MUC16ecto, Mucin16 ectodomain; ns, non-significant; TanCAR, tandem CAR configuration; UTD, untransduced.

Article Snippet: For direct staining of anti-meso scFv (SS1) expression, we used His-tag-FITC-Labeled Human Mesothelin (ACROBiosystems, MSN-HF223) and FITC-Labeled Human Mesothelin (Kactus, MSL-HM280F).

Techniques: In Vivo, Activity Assay, Injection, Expressing, MANN-WHITNEY, Immunohistochemistry

Journal: PLoS ONE

Article Title: Assessment of miR-103a-3p in leukocytes—No diagnostic benefit in combination with the blood-based biomarkers mesothelin and calretinin for malignant pleural mesothelioma diagnosis

doi: 10.1371/journal.pone.0275936

Figure Lengend Snippet: Distribution of medians of mesothelin (A), calretinin (B) and miR-103a-3p (C) in malignant pleural mesothelioma cases and population controls in a Mexican population. Black bars represent cases and gray bars represent controls.

Article Snippet: Mesothelin and calretinin were measured in plasma using commercial enzyme-linked immunosorbent assays (ELISA) for mesothelin (DY3265, R&D Systems, Minneapolis, MN) and calretinin (DLD Diagnostika GmbH, Hamburg, Germany) according to manufacturer’s instructions.

Techniques:

Journal: PLoS ONE

Article Title: Assessment of miR-103a-3p in leukocytes—No diagnostic benefit in combination with the blood-based biomarkers mesothelin and calretinin for malignant pleural mesothelioma diagnosis

doi: 10.1371/journal.pone.0275936

Figure Lengend Snippet: Sensitivity and specificity of biomarkers among malignant pleural mesothelioma cases and population controls according to sex groups in a Mexican population.

Article Snippet: Mesothelin and calretinin were measured in plasma using commercial enzyme-linked immunosorbent assays (ELISA) for mesothelin (DY3265, R&D Systems, Minneapolis, MN) and calretinin (DLD Diagnostika GmbH, Hamburg, Germany) according to manufacturer’s instructions.

Techniques: