Journal: Cancers

Article Title: Minimally Invasive Preclinical Monitoring of the Peritoneal Cavity Tumor Microenvironment

doi: 10.3390/cancers14071775

Figure Lengend Snippet: Design of in vivo intraperitoneal lavage (IVIPL) and its usage in studying CAR-T therapy. ( A ) Schematic representation of IVIPL. Mice are injected i.p. with 500 uL PBS and the abdomen is gently kneaded. Peritoneal fluid is then aspirated from the mice by inserting a needle while the mouse is held supine to avoid organs and rotating the mouse to allow fluid buildup in the lower abdomen. ( B – J ) Studying the in vivo antitumor efficacy of mesothelin-targeting CAR-T cells in an OVCAR8-FG human ovarian cancer xenograft NSG mouse model. ( B ) Experimental design to monitor CAR T cells using IVIPL in an ovarian cancer model. ( C ) Tumor growth curve (n = 4 per group) and representative bioluminescence images ( D ). ( E ) FACS plots showing the change in CAR-T cell phenotype over time, summarized in ( F ). hCD3 + hCD45 + are gated on live IVIPL cells, CAR and PD1/TIM-3 plots gated on hCD45 + CD3 + cells. Black bars indicate pre-infusion in vitro MCAR-T cells. ( G ) FACS plots showing the change in tumor PD-L1 expression between treatment groups on day 20, summarized in ( H ). Gated on GFP + tumor cells. ( I ) FACS plots showing the change in tumor-associated macrophage accumulation and phenotype on day 20, summarized in ( J ). Left FACS plots gated on mCD45 + cells, right FACS plots gated on mCD45 + mCD11b + F4/80 + cells. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Fluorochrome-conjugated isotype and antibody specific for human mesothelin (Clone 420411) were purchased from R&D Systems.

Techniques: In Vivo, Injection, In Vitro, 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) 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: PLoS ONE

Article Title: Enhanced Binding of Poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 Promoter via an Extended Cytosine-Adenosine Repeat

doi: 10.1371/journal.pone.0008743

Figure Lengend Snippet: A. One mg of Dami nuclear extract protein was incubated with one of four biotinylated (CA) 12 oligonucleotide probes in binding buffer. Oligonucleotide/protein complexes were adsorbed to streptavidin-agarose beads, and the bound proteins were eluted, separated by SDS-PAGE and visualized using silver stain. The following protein samples are depicted: (lane1) Starting nuclear extract; (lane 2) Proteins bound to CA12 in the absence of calf thymus DNA; (lane 3) Proteins bound to CA12 in the presence of calf thymus DNA; (lane 4); Proteins bound to CA12 in the presence of calf thymus DNA+ 5-fold molar excess of control oligonucleotide; and (lane 5) Proteins bound to control oligonucleotide in the presence of calf thymus DNA. The two prominent protein bands with MWApp of 120 kDa and 80 kDa (positions indicated by arrows a and b, respectively, to the right of the panel) were excised and processed by MS/MS. Peptides recovered and sequenced by MS/MS are depicted in . The positions held by molecular weight standards (Amersham Biosciences, Pittsburgh, PA) are indicated to the left of the panel. B. Dami nuclear extract proteins were incubated in vitro with the biotin-conjugated oligonucleotide probes: CA12, CA11, CA10 or control oligonucleotide (C). The resultant oligonucleotide/protein complexes were pulled down with streptavidin agarose. Nuclear proteins present in the complexes were separated by SDS-PAGE and identified by western blotting using the specific antibodies indicated to the left of the figure. The presence of PARP-1, Ku80 and Ku70 was confirmed in this manner. Antibodies specific for Sp1 served as a negative control, since the oligonucleotides used in these assays does not contain the Sp1 binding site. To confirm comparable oligonucleotide loading, the same samples were electrophoresed in a 2% agarose gel and stained with ethidium bromide (probes; negative image).

Article Snippet: The human megakaryocytic cell line Dami was obtained as previously described , and the human cell lines HeLa and HEK293 were obtained from ATCC (Manassas, VA).

Techniques: Incubation, Binding Assay, SDS Page, Silver Staining, Control, Tandem Mass Spectroscopy, Molecular Weight, In Vitro, Western Blot, Negative Control, Agarose Gel Electrophoresis, Staining

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: A. Total cells from ascites (upper panels) or solid tumors (middle panels) from ovarian cancer patients gated on CD14 + CD45 + (left panels) after exclusion of cells stained with EpCam and 7-AAD. Gated cells were labeled with APC anti-CD206 and PE anti-mesothelin K1, as indicated. CD14 + CD45 + cells freshly purified from peripheral blood of healthy donors (lower panels) and isotypes IgG1 (APC and PE) were used as negative controls. B. Percentages of CD45 + CD14 + CD206 + cells in solid tumors ( grey bars , n = 8), ascites samples ( black bars , n = 6), and healthy donors ( white bars , n = 12) that bind (CD206 + K1 + ) or not (CD206 + K1 - ) to mesothelin. Statistical significance was determined by unpaired t-test analysis (***, P = 0.001). Error bars represent standard error of mean (SEM).

Article Snippet: Goat anti-human mesothelin was purchased from R&D Systems (cat# AF3265).

Techniques: Staining, Labeling, Purification

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: A–B. Short term incubations. In vitro differentiated CD206 + macrophages ( A ) and CD206 − monocytes ( B ) from healthy donors were incubated with conditioned media from OVCAR3, OVCAR5, 293 MESOIg, or ascites fluids from patients #1714 and #1647 after blocking with 10 µg/ml of human IgG. As negative controls, cells were incubated with conditioned media from 293WT cell line, RPMI+10% FBS medium. Isotype control IgG1 antibodies (APC and PE) on cells incubated with RPMI/FBS. C. Transwell co-cultures: Freshly isolated monocytes were cocultured with OVCAR5 or 293mesoIg. As negative controls, monocytes were incubated with 293WT or with RPMI+10% FBS medium only. Grey area s, PE isotype IgG1 control; open area , PE-conjugated anti-mesothelin mAb (K1). Results representative of 3 or more independent experiments.

Article Snippet: Goat anti-human mesothelin was purchased from R&D Systems (cat# AF3265).

Techniques: In Vitro, Incubation, Blocking Assay, Control, Isolation

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: Macrophages were labeled with PE anti-mesothelin antibody K1 ( lines ) or with PE isotype control Ab ( grey area ) after in vitro differentiation with IL4/IL10 and 72 hr co-culture with OVCAR3 cells ( bold lines ). Blocking conditions ( dashed lines ) included ( A ) mannan, or ( B–D ) anti-CRD4 MR scFvs #B2 ( B ), #G11 ( C ) or #H11 ( D ). As control, CD206 high macrophages were incubated in medium only ( dotted lines ). Results representative of 3 or more independent experiments.

Article Snippet: Goat anti-human mesothelin was purchased from R&D Systems (cat# AF3265).

Techniques: Labeling, Control, In Vitro, Co-Culture Assay, Blocking Assay, Incubation

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: Macrophages were double stained with PE anti-CD206 mAb and APC anti-mesothelin antibody (K1) after in vitro differentiation with IFN-γ/LPS and 72 hr co-culture with OVCAR3 cells ( B ). Blocking conditions included anti-CRD4 MR scFvs #G11 ( C ) and #B2 ( D ). As negative control, CD206 low macrophages were labeled with isotype control antibodies ( A ). Results representative of 3 or more independent experiments.

Article Snippet: Goat anti-human mesothelin was purchased from R&D Systems (cat# AF3265).

Techniques: Staining, In Vitro, Co-Culture Assay, Blocking Assay, Negative Control, Labeling, Control

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: ELISA assays of ascites fluids from ovarian cancer patients (#1714; #1686; #1647; #1753; #1756; #1773) and conditioning media derived either from ovarian cancer cell lines (A1847; OVCAR3; OVCAR5) or from 293 cell lines, wild type (293WT) or transfected to secrete a GPI anchor-truncated mesothelin fused to Ig (293 MESOIg). ( A ) Anti-mesothelin ELISA double determinant assay using anti-mesothelin mAbs as capture and detection antibodies (R&D Systems). Results representative of two independent experiments. ( B ) ELISA double determinant assay using anti-mesothelin mAb (K1) as capture antibody and biotinylated Endotoxin Alpha as detection reagent, followed by HRP-labeled streptavidin. Colorimetric signal was developed with TMB substrate solution, quenched with sulfuric acid and read at 450 nm on a Biotek ELISA reader.

Article Snippet: Goat anti-human mesothelin was purchased from R&D Systems (cat# AF3265).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Transfection, Labeling

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: ( A ) Tandem mass spectrometry (ESI_MS2) of choline-containing phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM). Lipids were extracted by the chloroform/methanol solvent Folch mixture from their association with the immunoprecipitated mesothelin from OVCAR3 cell culture supernatant. The high proportion of SM relatively to PC in the supernatant is revealed by the amplitude of peak m/z 703 (palmityl-SM) compared with peaks at m/z 732 (32∶1 PC), 760 (34∶1 PC) and 786 (36∶2 PC). Parent phospholipids of the phosphorylcholine ion (+184) are indicated as (total carbon number in acyl chains:double-bond number PC). Cholesterol assay by MS2 is obtained after acetylation of the non esterified sterol by recording of the transition 446 (acetyl-cholesterol + NH+4) to 369 (not shown). SM/cholesterol molar ratios are given as mole/mole after calibration. ( B ) Western Blot analysis of immunoprecipitated mesothelin from tumor conditioned media (right lane) and tumor cell lysates (left lane) as positive control. Membranes were probed with anti-Mesothelin, -TSG101, - Alix, or -β actin, as indicated. Signals were detected by ECL.

Article Snippet: Goat anti-human mesothelin was purchased from R&D Systems (cat# AF3265).

Techniques: Mass Spectrometry, Solvent, Immunoprecipitation, Cell Culture, Cholesterol Assay, Western Blot, Positive Control

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

Article Title: KTN0158, a humanized anti-KIT monoclonal antibody, demonstrates biologic activity against both normal and malignant canine mast cells

doi: 10.1158/1078-0432.CCR-16-2152

Figure Lengend Snippet: (A) KTN0158 inhibited KIT activation and downstream signaling in M-07e cells. Dose-dependent inhibition of KIT, ERK1/2 and AKT activation were observed flowing KTN0158 treatment. KTN0062C is an isotype control antibody. (B) KTN0158 was a more potent inhibitor of SCF-dependent proliferation in M-07e cells compared to imatinib and nilotinib. Dashed lines represent proliferation observed in controls grown in the presence (+SCF) or absence (-SCF) of SCF. Representative data are shown for six independent experiments. Analyses were performed in duplicate (KTN0158) or triplicate (all others) and data are plotted as means ± standard error of the mean (SEM). (C) KTN0158 was a more potent inhibitor of SCF-induced KIT phosphorylation in primary human mast cells in vitro than imatinib. Representative data are shown for four independent mast cell preparations and runs. Analyses were performed in duplicate and data are plotted as means ± SEM. (D) KTN0158 was a potent inhibitor of SCF enhancement of mast cell degranulation. In both (C) and (D), the isotype control is KTN0062C and analyses were performed in duplicate with data plotted as means ± SEM.

Article Snippet: The human acute megakaryoblastic leukemia cell line M-07e ( 19 ) was obtained from the Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH (Braunschweig, Germany) and used within 6 months of receipt.

Techniques: Activation Assay, Inhibition, Control, Phospho-proteomics, In Vitro

Journal: Blood Advances

Article Title: Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

doi: 10.1182/bloodadvances.2017010975

Figure Lengend Snippet: Schematic of the megakaryocyte/platelet studies. Schematic of studies beginning with CD34+ HPCs to in vitro megakaryocyte differentiation in the presence of inhibitors to then in vitro and in vivo studies of the resulting day 12 megakaryocytes and EV-PLPs. Megs, megakaryocytes.

Article Snippet: Human megakaryocytes, collected at day 12, spun at 1200 rpm for 3 minutes, and resuspended in 200-μL phosphate-buffered saline (Invitrogen), or human donor-derived platelets (hdPlts) isolated as previously described 16 from healthy volunteers were infused through the tail vein of 8- to 12-week-old NSG mice.

Techniques: In Vitro, In Vivo

Journal: Blood Advances

Article Title: Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

doi: 10.1182/bloodadvances.2017010975

Figure Lengend Snippet: Size and granularity of in vitro–grown megakaryocytes in the presence of inhibitors. (A) Representative data of FSC on day 12 of in vitro–grown, CD42bHigh megakaryocytes. Cells have a wide size distribution, but with most of them being small megakaryocytes. Horizontal lines in the graphs indicate size range of the megakaryocytes classified as “large” megakaryocytes with FSC >70% of FSC of the control sample. (B) Mean percentage ± 1 SEM of the day 12, in vitro–grown, CD42bHigh inhibitor-treated and control megakaryocytes. P values were determined using 1-way ANOVA in comparison with the control. (C) Representative data of SSC of day 12, in vitro–grown, CD42bHigh megakaryocytes. Cells have wide granularity distribution, with most of them being low-granular megakaryocytes. Horizontal lines in the graphs indicate granularity range of the megakaryocytes classified as high-granular megakaryocytes with SSC >70% of the SSC in the control sample. (D) Mean percentage ± 1 SEM of the day 12, in vitro–grown, CD42bHigh inhibitor-treated and control megakaryocytes that are high-granular (HG). In both panels B and D, N = ≥6 independent experiments. P values were determined using 1-way ANOVA in comparison with the control.

Article Snippet: Human megakaryocytes, collected at day 12, spun at 1200 rpm for 3 minutes, and resuspended in 200-μL phosphate-buffered saline (Invitrogen), or human donor-derived platelets (hdPlts) isolated as previously described 16 from healthy volunteers were infused through the tail vein of 8- to 12-week-old NSG mice.

Techniques: In Vitro, Control, Comparison

Journal: Blood Advances

Article Title: Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

doi: 10.1182/bloodadvances.2017010975

Figure Lengend Snippet: Inhibitors effect on yield and injury of in vitro–grown megakaryocytes. (A) Yield of viable day 12 CD41+ megakaryocytes per HPC plated with or without the addition of SU6656, Y27632, and AZD1152. Mean yield ± 1 SEM is shown for N = 6 independent experiments. P values were determined using 1-way ANOVA in comparison with the control. (B) Yield of viable day 12 CD41+CD42bHigh megakaryocytes per HPC plated with or without the addition of SU6656, Y27632, and AZD1152 as in panel A. Mean yield ± 1 SEM is shown for N = 6 independent experiments. P values were determined using 1-way ANOVA in comparison with the control. (C) Percentage of viable CD41+ megakaryocytes that were also CD42bHigh on day 12 of culture. (D) Percentage of Annexin V+ CD41+CD42bHigh day 12 megakaryocytes was done as in panel B, but for Annexin binding to the megakaryocytes at day 12. For panels C and D, mean percentage-positive cells ± 1 SEM is shown for N ≥ 7 independent experiments. P values were determined using 1-way ANOVA in comparison with the control.

Article Snippet: Human megakaryocytes, collected at day 12, spun at 1200 rpm for 3 minutes, and resuspended in 200-μL phosphate-buffered saline (Invitrogen), or human donor-derived platelets (hdPlts) isolated as previously described 16 from healthy volunteers were infused through the tail vein of 8- to 12-week-old NSG mice.

Techniques: In Vitro, Comparison, Control, Binding Assay

Journal: Blood Advances

Article Title: Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

doi: 10.1182/bloodadvances.2017010975

Figure Lengend Snippet: Ploidy of in vitro–grown megakaryocytes. (A) Example of ploidy distribution of day 12 CD42bHigh megakaryocytes after the indicated treatments. (B) Same as panel A, but showing mean ploidy ± 1 SEM. (C) Same as in panel B but mean percentage ± 1 SEM of megakaryocytes that were ≥8N. In both panels B and C, N = ≥5 independent experiments. P values were determined using 1-way ANOVA in comparison with the control. AZD, exposed to the aurora kinase inhibitor AZD1152; Ctl, control exposed to DMSO only; RI, exposed to the ROCK inhibitor Y27632; SU, exposed to the Src inhibitor SU6656.

Article Snippet: Human megakaryocytes, collected at day 12, spun at 1200 rpm for 3 minutes, and resuspended in 200-μL phosphate-buffered saline (Invitrogen), or human donor-derived platelets (hdPlts) isolated as previously described 16 from healthy volunteers were infused through the tail vein of 8- to 12-week-old NSG mice.

Techniques: In Vitro, Comparison, Control

Journal: Blood Advances

Article Title: Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

doi: 10.1182/bloodadvances.2017010975

Figure Lengend Snippet: Influence of inhibitors on in vitro proplatelet formation and released EV-PLPs. (A) Representative fields of megakaryocytes extending proplatelet shafts and branching extensions on fibronectin-coated plates. Original magnification ×200. (B) Proplatelet protrusions (PPP) and (C) proplatelet extensions (PPE) were quantitated on day 12 megakaryocytes that had or had not been exposed to the various inhibitors. Mean ± 1 SEM are shown with N = ≥20 cells counted per condition. P values were determined using 1-way ANOVA in comparison with the control. (D) Representative FSC vs SSC for hdPlts (Donor, left), and PLPs harvested from the media from day 12 plates of control and treated megakaryocytes (right 4 graphs). (E) Shows a difference in number of EV-PLPs (from the normal donor Plt FSC/SSC window) obtained from treated megakaryocytes in reference to control ones. (F) Representative FSC vs SSC for hdPlt (Donor, left), and EV-PLPs harvested from the media from day 12 plates of control and treated megakaryocytes (right 4 graphs). The blue indicates CD42b+ hdPlts and EV-PLPs among all others released particles. (G) Fold-difference in number of CD41+CD42b+AnnexinV− (uninjured) EV-PLPs obtained from treated megakaryocytes in reference to control ones. Mean ± 1 SEM are shown with N = 3 independent experiments. P values were determined using 1-way ANOVA in comparison with the control. (H) Percentage of healthy EV-PLPs (as in panel G) compared with the entire EV-PLP population. Mean ± 1 SEM are shown with N = 3 independent experiments. P values were determined using 1-way ANOVA in comparison with the control.

Article Snippet: Human megakaryocytes, collected at day 12, spun at 1200 rpm for 3 minutes, and resuspended in 200-μL phosphate-buffered saline (Invitrogen), or human donor-derived platelets (hdPlts) isolated as previously described 16 from healthy volunteers were infused through the tail vein of 8- to 12-week-old NSG mice.

Techniques: In Vitro, Comparison, Control

Journal: Blood Advances

Article Title: Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

doi: 10.1182/bloodadvances.2017010975

Figure Lengend Snippet: In vivo–released platelets from infused megakaryocytes. (A) Determined released or infused platelet half-lives. Mean ± 1 SEM are shown. N = 9 for hdPlt infusions. N ≥ 6 for megakaryocyte infusions. (B-C) Fold-difference in the number of human platelets released after infusing megakaryocytes expressed relative to control megakaryocyte infusions corrected for (B) the number of CD41+CD42bHigh megakaryocytes infused or (C) the number of HPCs initially plated. Mean ± 1 SEM are shown; N = ≥6 per arm. Horizontal lines are the control megakaryocytes value at 1. *P ≤ .05 compared with control by 1-way ANOVA analysis. Plt, hdPlt infusion.

Article Snippet: Human megakaryocytes, collected at day 12, spun at 1200 rpm for 3 minutes, and resuspended in 200-μL phosphate-buffered saline (Invitrogen), or human donor-derived platelets (hdPlts) isolated as previously described 16 from healthy volunteers were infused through the tail vein of 8- to 12-week-old NSG mice.

Techniques: In Vivo, Control

Journal: Blood Advances

Article Title: Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

doi: 10.1182/bloodadvances.2017010975

Figure Lengend Snippet: Number of healthy platelets obtained from infused megakaryocytes

Article Snippet: Human megakaryocytes, collected at day 12, spun at 1200 rpm for 3 minutes, and resuspended in 200-μL phosphate-buffered saline (Invitrogen), or human donor-derived platelets (hdPlts) isolated as previously described 16 from healthy volunteers were infused through the tail vein of 8- to 12-week-old NSG mice.

Techniques: Control

Journal: Blood Advances

Article Title: Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors

doi: 10.1182/bloodadvances.2017010975

Figure Lengend Snippet: Functionality of in vivo–released platelets. (A-B) Studies with whole murine blood isolated 4 hours after human megakaryocytes or hdPlts were infused. Mean ± 1 SEM of (A) PAC-1 binding as measured by MFI and (B) CD62P surface expression MFI pre- and post-high dose (10 U/mL) thrombin activation. N = 3 independent studies for hdPlt infusion, and N = ≥6 independent studies for infused megakaryocytes arms. *P < .05 compared with hdPlts by 1-way ANOVA analysis. (C) Representative confocal images of laser-induced cremaster arteriole thrombi formed in mice infused with either hdPlts or the various treated in vitro–grown megakaryocytes. Mouse platelets are labeled with CD41–Alexa 647 (dark blue). Human platelets derived from infused donor-derived platelets or megakaryocytes are calcein-AM labeled (green or cyan blue when overlapped with mouse platelets). Original magnification ×20. (D) Quantification of the incorporation into the mice clot standardized per the percentage of human platelets in the mice circulation and thrombus size after infused hdPlts or megakaryocytes. Individual study results are shown with N ≥ 16 independent studies per arm. Mean value is shown as a single horizontal line in each arm.

Article Snippet: Human megakaryocytes, collected at day 12, spun at 1200 rpm for 3 minutes, and resuspended in 200-μL phosphate-buffered saline (Invitrogen), or human donor-derived platelets (hdPlts) isolated as previously described 16 from healthy volunteers were infused through the tail vein of 8- to 12-week-old NSG mice.

Techniques: In Vivo, Isolation, Binding Assay, Expressing, Activation Assay, In Vitro, Labeling, Derivative Assay

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: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: A. Total cells from ascites (upper panels) or solid tumors (middle panels) from ovarian cancer patients gated on CD14 + CD45 + (left panels) after exclusion of cells stained with EpCam and 7-AAD. Gated cells were labeled with APC anti-CD206 and PE anti-mesothelin K1, as indicated. CD14 + CD45 + cells freshly purified from peripheral blood of healthy donors (lower panels) and isotypes IgG1 (APC and PE) were used as negative controls. B. Percentages of CD45 + CD14 + CD206 + cells in solid tumors ( grey bars , n = 8), ascites samples ( black bars , n = 6), and healthy donors ( white bars , n = 12) that bind (CD206 + K1 + ) or not (CD206 + K1 - ) to mesothelin. Statistical significance was determined by unpaired t-test analysis (***, P = 0.001). Error bars represent standard error of mean (SEM).

Article Snippet: Anti-human Mesothelin (K1), K1-PE, anti c-myc mAb and anti-mouse HRP labeled antibody were purchased from Santa Cruz.

Techniques: Staining, Labeling, Purification

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: A–B. Short term incubations. In vitro differentiated CD206 + macrophages ( A ) and CD206 − monocytes ( B ) from healthy donors were incubated with conditioned media from OVCAR3, OVCAR5, 293 MESOIg, or ascites fluids from patients #1714 and #1647 after blocking with 10 µg/ml of human IgG. As negative controls, cells were incubated with conditioned media from 293WT cell line, RPMI+10% FBS medium. Isotype control IgG1 antibodies (APC and PE) on cells incubated with RPMI/FBS. C. Transwell co-cultures: Freshly isolated monocytes were cocultured with OVCAR5 or 293mesoIg. As negative controls, monocytes were incubated with 293WT or with RPMI+10% FBS medium only. Grey area s, PE isotype IgG1 control; open area , PE-conjugated anti-mesothelin mAb (K1). Results representative of 3 or more independent experiments.

Article Snippet: Anti-human Mesothelin (K1), K1-PE, anti c-myc mAb and anti-mouse HRP labeled antibody were purchased from Santa Cruz.

Techniques: In Vitro, Incubation, Blocking Assay, Control, Isolation

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: Macrophages were labeled with PE anti-mesothelin antibody K1 ( lines ) or with PE isotype control Ab ( grey area ) after in vitro differentiation with IL4/IL10 and 72 hr co-culture with OVCAR3 cells ( bold lines ). Blocking conditions ( dashed lines ) included ( A ) mannan, or ( B–D ) anti-CRD4 MR scFvs #B2 ( B ), #G11 ( C ) or #H11 ( D ). As control, CD206 high macrophages were incubated in medium only ( dotted lines ). Results representative of 3 or more independent experiments.

Article Snippet: Anti-human Mesothelin (K1), K1-PE, anti c-myc mAb and anti-mouse HRP labeled antibody were purchased from Santa Cruz.

Techniques: Labeling, Control, In Vitro, Co-Culture Assay, Blocking Assay, Incubation

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: Macrophages were double stained with PE anti-CD206 mAb and APC anti-mesothelin antibody (K1) after in vitro differentiation with IFN-γ/LPS and 72 hr co-culture with OVCAR3 cells ( B ). Blocking conditions included anti-CRD4 MR scFvs #G11 ( C ) and #B2 ( D ). As negative control, CD206 low macrophages were labeled with isotype control antibodies ( A ). Results representative of 3 or more independent experiments.

Article Snippet: Anti-human Mesothelin (K1), K1-PE, anti c-myc mAb and anti-mouse HRP labeled antibody were purchased from Santa Cruz.

Techniques: Staining, In Vitro, Co-Culture Assay, Blocking Assay, Negative Control, Labeling, Control

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: ELISA assays of ascites fluids from ovarian cancer patients (#1714; #1686; #1647; #1753; #1756; #1773) and conditioning media derived either from ovarian cancer cell lines (A1847; OVCAR3; OVCAR5) or from 293 cell lines, wild type (293WT) or transfected to secrete a GPI anchor-truncated mesothelin fused to Ig (293 MESOIg). ( A ) Anti-mesothelin ELISA double determinant assay using anti-mesothelin mAbs as capture and detection antibodies (R&D Systems). Results representative of two independent experiments. ( B ) ELISA double determinant assay using anti-mesothelin mAb (K1) as capture antibody and biotinylated Endotoxin Alpha as detection reagent, followed by HRP-labeled streptavidin. Colorimetric signal was developed with TMB substrate solution, quenched with sulfuric acid and read at 450 nm on a Biotek ELISA reader.

Article Snippet: Anti-human Mesothelin (K1), K1-PE, anti c-myc mAb and anti-mouse HRP labeled antibody were purchased from Santa Cruz.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Transfection, Labeling

Journal: PLoS ONE

Article Title: Mannose Receptor (MR) Engagement by Mesothelin GPI Anchor Polarizes Tumor-Associated Macrophages and Is Blocked by Anti-MR Human Recombinant Antibody

doi: 10.1371/journal.pone.0028386

Figure Lengend Snippet: ( A ) Tandem mass spectrometry (ESI_MS2) of choline-containing phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM). Lipids were extracted by the chloroform/methanol solvent Folch mixture from their association with the immunoprecipitated mesothelin from OVCAR3 cell culture supernatant. The high proportion of SM relatively to PC in the supernatant is revealed by the amplitude of peak m/z 703 (palmityl-SM) compared with peaks at m/z 732 (32∶1 PC), 760 (34∶1 PC) and 786 (36∶2 PC). Parent phospholipids of the phosphorylcholine ion (+184) are indicated as (total carbon number in acyl chains:double-bond number PC). Cholesterol assay by MS2 is obtained after acetylation of the non esterified sterol by recording of the transition 446 (acetyl-cholesterol + NH+4) to 369 (not shown). SM/cholesterol molar ratios are given as mole/mole after calibration. ( B ) Western Blot analysis of immunoprecipitated mesothelin from tumor conditioned media (right lane) and tumor cell lysates (left lane) as positive control. Membranes were probed with anti-Mesothelin, -TSG101, - Alix, or -β actin, as indicated. Signals were detected by ECL.

Article Snippet: Anti-human Mesothelin (K1), K1-PE, anti c-myc mAb and anti-mouse HRP labeled antibody were purchased from Santa Cruz.

Techniques: Mass Spectrometry, Solvent, Immunoprecipitation, Cell Culture, Cholesterol Assay, Western Blot, Positive Control

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 1 Schematic diagram illustrating the preparation of A-MFS probe and its T2-weighted images. Abbreviations: IO, Fe3O4; A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody; APTES, (3-aminopropyl)triethoxysilane; MRI, magnetic resonance imaging; TEOS, tetraethylorthosilicate; h, hours.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: Modification, Magnetic Resonance Imaging

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 2 The electron microscopic image of A-MFS and its enlarged view (A), the distribution of hydrodynamic diameter (B), relaxation rate (r1, r2) of A-MFS versus Fe concentration (C), and XPS result (D). Notes: A-MFS has a spherical appearance and a regular shape, and most of its hydrodynamic diameters range between 110 and 130 nm, r1 =0.549 mM/s and r2 =59.435 mM/s. A-MFS contains Fe, O, C, N, and Si. Abbreviations: A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody; XPS, X-ray photoelectron spectroscopy.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: Concentration Assay, Modification, Spectroscopy

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 3 CCK-8 assay of SW1990 cell viability after incubation with different concentrations of A-MFS for 2, 6, and 24 hours. Note: With the increase in the concentration of A-MFS from 0 to 500 mg/L, the cell viability exceeded 86%. Abbreviations: A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody; CCK-8, Cell Counting Kit-8.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: CCK-8 Assay, Incubation, Concentration Assay, Modification, Cell Counting

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 4 In vitro targeting test. Note: The red fluorescent dots were found more frequently on the surface of SW1990 cells, after incubation with A-MFS. Abbreviations: A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody; QD, quantum dot.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: In Vitro, Incubation, Modification

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 5 Flow cytometry. Note: MSLN expressed on the surface of SW1990. Abbreviations: A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody; FS, Fe3O4@SiO2; MSLN, mesothelin; PE-A, Phycoerythrin-Conjugated Antibody.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: Flow Cytometry, Modification

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 6 (A) and (B) respectively represent T2-weighted images in MRI (3.0 T) of tumors in vivo before and after injection of saline solution and A-MFS. Notes: The images were obtained at the following time points: before injection, 30 minutes later, 1, 1.5, 2.5, and 3.5 hours after injection. The tumor signal decreased after injection of A-MFS. When injected for 2.5 hours, the T2-weighted signal values decreased to the lowest level. The signal remained almost constant after injection of saline solution. Abbreviations: A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody; MRI, magnetic resonance imaging; min, minutes; h, hours.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: In Vivo, Injection, Saline, Modification, Magnetic Resonance Imaging

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 7 The T2-weighted quantitative signals of tumor before and after injection of A-MFS (A); the T2-weighted quantitative signal before and after injection of saline solution (B). Notes: (A) Shows decreased tumor signal after injection of A-MFS via the mouse tail vein, and at ~2.5 hours, it decreased to the lowest level. (B) Illustrates the insignificant decrease in signal levels after injection of saline. Abbreviations: A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody; SE, standard error.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: Injection, Saline, Modification

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 8 Fe distribution (μg/g) in heart, liver, spleen, lung, kidney, and tumor, before and after injection of FS and A-MFS intravenously via the tail vein. Note: The Fe levels in the tumor differ significantly following injection of FS and A-MFS. Abbreviations: A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody; FS, Fe3O4@SiO2.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: Injection, Modification

Journal: International Journal of Nanomedicine

Article Title: In vitro and in vivo targeting imaging of pancreatic cancer using a Fe3O4@SiO2 nanoprobe modified with anti-mesothelin antibody

doi: 10.2147/ijn.s104501

Figure Lengend Snippet: Figure 9 Histology of heart, liver, spleen, lung, and kidney in the two groups, following injection of saline solution and A-MFS, respectively. Note: No obvious differences were seen in the control and experimental groups. Abbreviation: A-MFS, Fe3O4@SiO2 modified with anti-mesothelin antibody.

Article Snippet: Materials All initial reagents were obtained commercially and used as-received: sodium acetate, iron trichloride hexahydrate (FeCl 3 ⋅6H 2 O), ethylene glycol, ethanol, deionization water (DI water), ammonium hydroxide, tetraethylorthosilicate, (3-aminopropyl)triethoxysilane, polyethylene glycol (PEG) diacid solution (molecular weight, 6,000), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl), borate saline buffer (50 mM, pH 8.2), phosphatebuffered saline (PBS), bovine serum albumin, sodium azide, microplate reader, Cell Count ing Kit-8 (CCK-8), formazan dye, dehydrogenase, pancreatin, and mesothelin antibody (R&D Systems, Inc., Minneapolis, MN, USA). cell lines Human pancreatic cancer SW1990 cell line was supplied by the Department of Gastroenterology, the first hospital affiliated to the Second Military Medical University in Shanghai, People’s Republic of China.

Techniques: Injection, Saline, Control, Modification

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: Alternatively, a directly conjugated anti-mesothelin antibody from Miltenyi was used, clone REA1057 | 420411, Cat: 130–118-096; RRID:AB_2733436).

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: Alternatively, a directly conjugated anti-mesothelin antibody from Miltenyi was used, clone REA1057 | 420411, Cat: 130–118-096; RRID:AB_2733436).

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: Alternatively, a directly conjugated anti-mesothelin antibody from Miltenyi was used, clone REA1057 | 420411, Cat: 130–118-096; RRID:AB_2733436).

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

Journal: Oncotarget

Article Title: Mesothelin as a novel biomarker and immunotherapeutic target in human glioblastoma

doi: 10.18632/oncotarget.20303

Figure Lengend Snippet: Paraffin-embedded tissue sections were stained with rat anti-human mesothelin primary antibody. Detection was performed using a goat anti-rat IgG secondary antibody labelled with Alexa Fluor 488-conjugated polyclonal goat anti-rat secondary antibody. The primary antibody was omitted in the negative control while the pancreatic cancer tumour cell line PaTu was used as positive control . Shown are representative photographs for GBM tissue from one patient.

Article Snippet: The quantitative determination of human mesothelin concentration in plasma was performed using the human mesothelin immunoassay kit (R&D systems, Minneapolis, MN) according to the manufacturer's protocol.

Techniques: Staining, Negative Control, Positive Control

Journal: Oncotarget

Article Title: Mesothelin as a novel biomarker and immunotherapeutic target in human glioblastoma

doi: 10.18632/oncotarget.20303

Figure Lengend Snippet: Whole-blood obtained from patients with glioma (GBM, A ; astrocytoma, B ; OD, C ; metastasis, D ) were cultured with mesothelin or its derivatives in the absence of cytokine conditioning, with IL-2/IL-7 or IL-2/IL-15/IL-21 conditioning over seven days. Supernatants were then harvested for IFN-γ detection by ELISA. Shown are dot plots representing responses of individual patients. Mann Whitney test of medians was performed to gauge statistical significance. * p < 0.05; ** p < 0.001.

Article Snippet: The quantitative determination of human mesothelin concentration in plasma was performed using the human mesothelin immunoassay kit (R&D systems, Minneapolis, MN) according to the manufacturer's protocol.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Oncotarget

Article Title: Mesothelin as a novel biomarker and immunotherapeutic target in human glioblastoma

doi: 10.18632/oncotarget.20303

Figure Lengend Snippet: 42 non-overlapping 15-mer peptides spanning the entire mesothelin molecules were exposed to peripheral blood of GBM patients over a seven-day period. Supernatants were then harvested for IFN-γ detection by ELISA. Absolute IFN-γ concentrations (pg/ml) produced by each patient for a single peptide A. as well as the average value per peptide (total IFN-γ/no. of patients) normalised to the sum of IFN-γ production for the entire peptide pool in percentage B. are shown. Immune hotspots within the mesothelin component, defined by peptide-specific IFN-γ production, identify several peptides which may represent viable targets to expand T-cells in host-directed therapies.

Article Snippet: The quantitative determination of human mesothelin concentration in plasma was performed using the human mesothelin immunoassay kit (R&D systems, Minneapolis, MN) according to the manufacturer's protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Produced

Journal: Oncotarget

Article Title: Mesothelin as a novel biomarker and immunotherapeutic target in human glioblastoma

doi: 10.18632/oncotarget.20303

Figure Lengend Snippet: An indirect ELISA method was used for quantifying mesothelin-specific IgG titres in plasma, while a commercially available ELISA kit was used for measuring mesothelin protein. IgG titres and mesothelin levels are expressed as ng/ml of plasma. Mann Whitney test of medians was performed to gauge statistical significance. * p < 0.05; ** p < 0.001.

Article Snippet: The quantitative determination of human mesothelin concentration in plasma was performed using the human mesothelin immunoassay kit (R&D systems, Minneapolis, MN) according to the manufacturer's protocol.

Techniques: Indirect ELISA, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Oncotarget

Article Title: Mesothelin as a novel biomarker and immunotherapeutic target in human glioblastoma

doi: 10.18632/oncotarget.20303

Figure Lengend Snippet: TIL isolated from GBM tumor tissue were cultured in vitro with IL-2/IL-15/IL-21 and exposed to mesothelin peptides over a 6-hour period. Cells were then stained with CD3, CD4, CD8, IFN-γ and TNF-α to visualise intracellular cytokine production by mesothelin-specific T-cells. Shown are frequencies (%) of responding cells based on CD3+ TIL of one representative patient. Cell frequency of more than 0.2% is a considered a legitimate response.

Article Snippet: The quantitative determination of human mesothelin concentration in plasma was performed using the human mesothelin immunoassay kit (R&D systems, Minneapolis, MN) according to the manufacturer's protocol.

Techniques: Isolation, Cell Culture, In Vitro, Staining