Journal: Research

Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

doi: 10.34133/research.1105

Figure Lengend Snippet: Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression in AsPC-1 cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.

Article Snippet: The cells were again centrifuged and resuspended with 1 μl (10 μl/10 6 cells) Human Mesothelin Alexa Fluor 488-conjugated Antibody (R&D Systems, FAB32652G) and 0.5 μl of Invitrogen CellTrace Calcein Red-Orange, AM (Fisher Scientific, 50-113-7411) in 500 μl of flow cytometry buffer.

Techniques: Binding Assay, Electroporation, Membrane, Flow Cytometry, Control, Expressing, Imaging, Cell Characterization

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Ibrutinib and PD-1 blockade potentiate mesothelin-targeting CAR-T cell therapy in preclinical models of pancreatic cancer

doi: 10.1158/1078-0432.CCR-25-2907

Figure Lengend Snippet: A) Schematic of dual-targeting Meso-FAP CAR TEAM T cells engineered to co-target PDAC tumor cells, through a mesothelin-targeting CAR, and tumor-associated CAFs, through a secreted FAP-targeting TEAM molecule. The secreted FAP TEAM molecule can redirect the cytotoxicity of both meso-CAR-T cells as well as CAR-negative bystander T cells that are present in the tumor microenvironment 8 . Created in BioRender.com . Escobar, Giulia. https://app.biorender.com/illustrations/66eeece9f2d42f9534e90be7?slideId=be6d217b-9bf9–4f4e-8420-dc13e4db9d9bB ). Schematic of the experimental design. Mice are implanted with subcutaneous AsPC-1 tumors cells and then adoptively transferred with meso-CAR-T cells (3e6) or UTD T cells by intravenous injection at 14 days post tumor challenge. C) Tumor growth kinetic (tumor volume, mean ± SEM) in AsPC-1 tumor-bearing mice treated as shown in B (n=6 mice per group). Two-way ANOVA. HD53 was used to generate meso-CAR-T cells (transduction: 72%, viability at infusion: 74%). D) Survival curve of mice treated as shown in B. Mantel-Cox test. E) Schematic of the experimental design. Mice are injected intraperitoneally with AsPC-1 tumor cells and allowed to form peritoneal tumors. 7 days post tumor challenge, mice were adoptively transferred with meso-CAR-T cells (2e6) or UTD T cells by intravenous or intraperitoneal injections. F) Tumor growth kinetic (flux, photons/s, mean ± SEM) as measured by bioluminescence imaging (BLI) in mice treated as shown in E (n=6–7 mice per group). Two-way ANOVA. HD53 was used to generate meso-CAR-T cells (transduction: 72%, viability at infusion: 91%). G) Tumor burden in each individual mouse from F as measured by BLI. H) Absolute numbers (mean ± SEM) of meso-CAR-T cells in the blood of AsPC-1 tumor-bearing mice from F, treated as indicated and as shown in E. Two-way ANOVA. I) Proportion of CD4 and CD8 T cells within CAR-T cells in the blood of mice in F, at day15 post adoptive CAR-T cell transfer by intravenous or intraperitoneal injection. Unpaired Student’s t test. J) Phenotype of CD4-positive and CD8-positive CAR-T cells in the peripheral blood of tumor-bearing mice from F, at day 15 following CAR-T cell adoptive transfer by intraperitoneal or intravenous injection. T stem cell memory cells (TSCM, CD45RA+CCR7+CD95+), central memory (CM, CD45RA-CCR7+), effector memory (CD45RA-CCR7-) and terminally differentiated effector memory (TEMRA, CD45RA+CCR7-) T cells. Unpaired Student’s t test. K) Expression of PD-1, TIM3 and CD39 markers (frequency, mean ± SEM) on the surface of CD4-positive and CD8-positive CAR-T cells in the blood of mice from F, at day 15 post adoptive CAR-T cell transfer by intravenous or intraperitoneal injection. Unpaired Student’s t test. **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: To analyze soluble mesothelin in the culture supernatant, the Human Mesothelin DuoSet ELISA (R&D Systems, DY3265) kit was used according to manufacturer protocol.

Techniques: Injection, Transduction, Imaging, Adoptive Transfer Assay, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Ibrutinib and PD-1 blockade potentiate mesothelin-targeting CAR-T cell therapy in preclinical models of pancreatic cancer

doi: 10.1158/1078-0432.CCR-25-2907

Figure Lengend Snippet: A) Representative histograms of ADAM-10 and ADAM-17 expression in the indicated pancreatic cancer cell lines as measured by flow cytometry. B) Representative flow histograms of mesothelin expression in the indicated pancreatic cancer cell lines. C-E) Representative flow histograms (C) and quantification (C-E; mean ± SEM; frequency and mean fluorescence intensity, MFI) of mesothelin expression in AsPC-1, CAPAN-2 and BxPC-3 tumor cells either left untreated (DMSO) or treated with the indicated concentrations of aderbasib for 72 hours. Mesothelin knock-out (Meso-KO) AsPC-1 tumor cells are included as a negative control. Shown is one of two independent experiments per tumor cell line. One-way ANOVA with Dunnett’s multiple comparison test, vs DMSO-treated condition. F) Quantification (mean ± SEM) of soluble mesothelin by ELISA in the culture supernatant of the indicated pancreatic tumor cell lines treated for 72 hours with increasing concentrations of aderbasib. Meso-KO AsPC-1 cells are included as a negative control. One-way ANOVA with Dunnett’s multiple comparison test, vs DMSO-treated condition. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: To analyze soluble mesothelin in the culture supernatant, the Human Mesothelin DuoSet ELISA (R&D Systems, DY3265) kit was used according to manufacturer protocol.

Techniques: Expressing, In Vitro, Flow Cytometry, Fluorescence, Knock-Out, Negative Control, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Ibrutinib and PD-1 blockade potentiate mesothelin-targeting CAR-T cell therapy in preclinical models of pancreatic cancer

doi: 10.1158/1078-0432.CCR-25-2907

Figure Lengend Snippet: A) Fold change over timepoint 0 of the green area (mean ± SEM) of GFP-positive pancreatic tumor cells cultured alone or together with untransduced T cells (UTD) or meso-CAR-T cells in the absence (DMSO) or presence of 5uM aderbasib. Healthy donor (HD) 105 was used to manufacture meso-CAR-T cells. Two-way ANOVA is calculated between the meso-CAR + DMSO and the meso-CAR + aderbasib groups and between the UTD + DMSO and UTD + aderbasib groups. B) Schematic of the experimental design. AsPC-1-tumor bearing mice received aderbasib treatment by oral gavage (60mg/kg) starting 3 days prior and up to 14 days post adoptive transfer of 3e6 meso-CAR-T cells. C) Tumor growth kinetic (tumor volume, mean ± SEM) in AsPC-1 tumor-bearing mice treated with UTD T cells or 3e6 meso-CAR-T cells either as monotherapy or in combination with aderbasib (treatment window is indicated by the lilac square. n=6 mice per group). HD53 was used to manufacture meso-CAR-T cells (transduction: 66%, viability at infusion: >80%). Two-way ANOVA. D) Absolute numbers (mean ± SEM) of meso-CAR-T cells in the blood of AsPC-1 tumor-bearing mice treated as indicated and as shown in B. E) Schematic of the experimental design. AsPC-1 tumor-bearing mice received aderbasib treatment by oral gavage (60mg/kg) starting 4 days post CAR-T cell transfer (1.5e6 cells) and up to day 25. F) Tumor growth kinetic (tumor volume, mean ± SEM) in AsPC-1 tumor-bearing mice treated with UTD T cells or 1.5e6 meso-CAR-T cells either as monotherapy or in combination with aderbasib (treatment window is indicated by the lilac square; n=4–6 mice per group). Two-way ANOVA. HD207 was used to generate meso-CAR-T cells (transduction: 68%, viability at infusion: >80%). G) Representative flow histograms and quantification (frequency and MFI, mean ± SEM) of mesothelin expression in AsPC-1 (GFP+) tumors harvested at day 28 from mice treated with UTD T cells alone or in combination with aderbasib from F. Statistical significance was determined using unpaired Student’s t test. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: To analyze soluble mesothelin in the culture supernatant, the Human Mesothelin DuoSet ELISA (R&D Systems, DY3265) kit was used according to manufacturer protocol.

Techniques: In Vitro, In Vivo, Cell Culture, Adoptive Transfer Assay, Transduction, Expressing

Journal: bioRxiv

Article Title: Epicardial slices: a 3D organotypic model for the study of epicardial activation and differentiation

doi: 10.1101/2020.08.07.219931

Figure Lengend Snippet: Confocal microscopy analysis of formaldehyde-fixed OCT-embedded porcine epicardial/myocardial slices confirms the presence of an intact epicardial layer identified by the expression of WT1 (A) the sporadic expression of E-cadherin (B) and extensive positivity to mesothelin (MSLN) (C). The cardiomyocytes within the slice display intact α-sarcomeric actin structures (B) and gap junctions connexin 43 (D), indicative of well-preserved myocardial architecture. CD31 identifies vascular structures (E), including arterioles surrounded by smooth muscle cells expressing α-SMA (E) and capillaries associated with the occasional NG2 + pericytes (F). Nuclei are labelled with DAPI. Scale bar, 50µm.

Article Snippet: Primary antibody incubation was performed overnight at 4°C (WT1 1:50, E-cadherin 1:50, CD31 1:100, NG2 1:500 (water bath antigen retrieval performed) all from Abcam; Mesothelin 1:100 from Novus Biologicals; α-Actinin (Sarcomeric) 1:800, α-SMA 1:400, PCNA 1:100 all from Sigma-Aldrich; Connexin 43 1:300, Vimentin 1:100 all from Thermo Fisher Scientific), followed by the appropriate Alexa Fluor (Thermo Fisher Scientific) secondary antibody diluted 1:200 for 1 hour at 37°C, and nuclei staining with DAPI (4′,6-diamidino-2-phenylindole, Merck) for 10 minutes at RT.

Techniques: Confocal Microscopy, Expressing

Journal: International journal of cancer

Article Title: Difference in mesothelin-binding ability of serum CA125 between patients with endometriosis and epithelial ovarian cancer.

doi: 10.1002/ijc.29185

Figure Lengend Snippet: Figure 1. A sandwich ELISA involving a recombinant mesothelin and anti-CA125 mAb (CA125meso assay). (a) A schematic diagram of the CA125meso assay. (b) Linearity study of the CA125meso assay. Serum specimens from patients with endometriosis (n 5 2) and EOC (n 5 2), and OVCAR-3 cells-produced CA125 were diluted to give the indicated concentrations of CA125 and then analyzed by means of the CA125meso assay. (c and d) The serum levels of CA125meso (c) and CA125 (d) were examined in patients with endometriosis (n 5 59) and EOC (n 5 36). The horizontal dotted lines represent the arbitrary cutoff values; 15 U/ml in (c) and 35 U/ml in (d). (e) ROC curves for the CA125 and CA125meso assays for discriminating endometriosis from EOC.

Article Snippet: For measurement of serum CA125meso, the wells of Maxisorp Immunoplates (Nunc, Roskilde, Denmark) were coated with a recombinant human mesothelin (R&D Systems, Minneapolis, MN).

Techniques: Sandwich ELISA, Recombinant, Produced

Journal: International journal of cancer

Article Title: Difference in mesothelin-binding ability of serum CA125 between patients with endometriosis and epithelial ovarian cancer.

doi: 10.1002/ijc.29185

Figure Lengend Snippet: Figure 2. Evaluation of the mesothelin-binding ability of CA125 (CA125meso/CA125). (a) Comparison of the CA125meso/CA125 levels between patients with endometriosis and EOC. (b) Relationship between the serum CA125 and CA125meso/CA125 levels in patients with EOC. (c) Compar- ison of the CA125meso/CA125 levels between premenopausal and postmenopausal patients with EOC. (d) Comparison of the CA125meso/CA125 levels according to the clinical stage of EOC. (e) Comparison of the CA125meso/CA125 levels according to the histological type of EOC. In (a) and (c)–(e), the horizontal bars represent the mean values. In (a), (d) and (e), the horizontal dotted line represents the arbitrary cutoff value (1.08).

Article Snippet: For measurement of serum CA125meso, the wells of Maxisorp Immunoplates (Nunc, Roskilde, Denmark) were coated with a recombinant human mesothelin (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Comparison

Journal: International journal of cancer

Article Title: Difference in mesothelin-binding ability of serum CA125 between patients with endometriosis and epithelial ovarian cancer.

doi: 10.1002/ijc.29185

Figure Lengend Snippet: Figure 3. Effects of soluble mesothelin and MUC1 on the measurement of CA125meso. (a and d) The serum levels of mesothelin (a) and CA15- 3 antigen (d) were determined in patients with endometriosis (n 5 59 or 32) and EOC (n 5 36 or 30). (b and e) Relationship between the CA125meso/CA125 levels and the endogenous serum mesothelin (b) or CA15-3 (e) level. (c) OVCAR-3 cells-produced CA125 was diluted to 35 U/ml and then analyzed by means of the CA125meso assay in the presence of the indicated concentrations of the recombinant soluble human mesothelin (0–640 ng/ml). The data represent the means6 SE (n 5 4). In (a) and (d), the horizontal bars represent the mean values.

Article Snippet: For measurement of serum CA125meso, the wells of Maxisorp Immunoplates (Nunc, Roskilde, Denmark) were coated with a recombinant human mesothelin (R&D Systems, Minneapolis, MN).

Techniques: Produced, Recombinant