Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Biopanning of a phage library to identify mesothelin-binding peptides. (A) Schematic of biopanning. A phage library was incubated with parental HEK 293T cells; unbound phage was then incubated with HEK 293T cells transfected with a green fluorescent protein (GFP)-tagged mesothelin (MSLN) expression vector (MSLN-HEK 293T). Cell-bound phage was eluted and amplified for the next round. Created with BioRender.com . (B) MSLN-HEK 293T cells 48 h after transfection (green) and parental HEK 293T cells stained with an anti-MSLN antibody (red) and diamidino-2-phenylindole (DAPI) (blue). (C) Phage titer enrichment across screening rounds. pfu, plaque-forming units. (D) Phage-cell-binding ELISA showing binding of individual phage clones to parental versus MSLN-expressing HEK 293T cells. OD, optical density.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Incubation, Transfection, Expressing, Plasmid Preparation, Amplification, Staining, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Binding of MSLNpep to MSLN-expressing cells. (A) Parental and GFP-tagged (green) MSLN-expressing HEK 293T cells were incubated with tetramethylrhodamine-5-maleimide (TAMRA)-labeled MSLNpep or a control peptide (red) at 4 °C for 1 h and DAPI (blue). (B) Immunoblotting of MSLN expression in AsPC-1 and MIA PaCa-2 pancreatic tumor cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) AsPC-1 and MIA PaCa-2 cells were incubated with fluorescein isothiocyanate (FITC)–MSLNpep (green) at 4 °C for 1 h and an anti-MSLN antibody (red). Nuclei were stained with DAPI (blue), and images were merged. (D and E) AsPC-1 cells were treated with small interfering RNA (siRNA) against MSLN (100 and 200 nM) to knock down MSLN expression or with control (Ctrl) siRNA (200 nM). Cells were incubated with an anti-MSLN antibody (D) or 25 μM FITC–MSLNpep (E) and analyzed by flow cytometry. *** P < 0.001 by one-way ANOVA. (F) AsPC-1 cells were treated with MSLN-siRNA or Ctrl-siRNA (200 nM). After silencing, cells were incubated with 25 μM FITC–MSLNpep (green) at 4 °C for 1 h and an anti-MSLN antibody (red) (1:1,000). Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Expressing, Incubation, Labeling, Control, Western Blot, Staining, Small Interfering RNA, Knockdown, Flow Cytometry

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Binding of MSLNpep to MSLN protein. (A) HEK 293T and AsPC-1 cell lysates (20, 200, and 400 μg) were incubated with biotin-labeled MSLNpep or control peptide (Ctrlpep) and pulled down with streptavidin-coated magnetic beads. Immunoblotting of the precipitates was performed using an anti-MSLN antibody. (B) Quantification of band intensity (%) for each pull-down relative to input in (A) using ImageJ software. Data are mean ± standard error from 3 independent experiments. *** P < 0.001 by one-way analysis of variance (ANOVA). (C) HEK 293 and AsPC-1 cells were incubated at 4 °C for 1 h with FITC-labeled MSLNpep together with either soluble or mature MSLN. Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (D) HEK 293 and AsPC-1 cells were pretreated with an anti-MSLN antibody at room temperature for 1 h and incubated with FITC–MSLNpep (green) at 4 °C for 1 h. Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (E) ELISA quantifying MSLNpep binding to soluble and mature MSLN. Data are mean ± standard error from 3 independent experiments. ** P < 0.01 by unpaired t test. (F) In silico modeling of MSLNpep binding to MSLN using the PEPFOLD program. MSLN, green; MSLNpep, blue.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Incubation, Labeling, Control, Magnetic Beads, Western Blot, Software, Staining, Enzyme-linked Immunosorbent Assay, In Silico

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Cytotoxicity of an MSLN-targeted mitochondrial-membrane-damaging peptide. (A) AsPC-1 cells were incubated at 37 °C for 2 h for 1 h with 25 μM FITC-labeled control peptide (Ctrlpep) or MSLNpep (green). Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (B) Diagram of MSLNpep-KLA (L-form) and MSLNpep-kla (D-form). (C to E) AsPC-1 (C), MIA PaCa-2 (D), and HEK 293T (E) cells were incubated with phosphate-buffered saline (PBS), Ctrlpep-KLA, Ctrlpep-kla, MSLNpep-KLA, or MSLNpep-kla for 24 h. After incubation, cell viability was measured using Cell Counting Kit reagents. The half-maximal inhibitory concentration (IC50) values are presented as mean ± standard error from 3 independent experiments. Created with BioRender.com .

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Membrane, Incubation, Labeling, Control, Staining, Saline, Cell Counting, Concentration Assay

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Inhibition of orthotopic pancreatic tumor growth by an MSLN-targeted mitochondrial-membrane-damaging peptide. (A) Treatment schema for mice bearing orthotopic AsPC-1-luc pancreatic tumors. Mice received PBS, Ctrlpep-kla, or MSLNpep-kla at 10 mg/kg body weight. (B) Whole-body bioluminescence imaging on days 0 and 16 after treatment. (C) Total bioluminescent flux after treatment. Data are mean ± standard error ( n = 5). *** P < 0.001; ns, not significant by 2-way ANOVA. (D to F) Total bioluminescent flux for each mouse after treatment with PBS (D), Ctrlpep-kla (E), or MSLNpep-kla (F). (G) Survival rates. (H) Body weights. (I) Serum aspartate aminotransferase (AST) levels. (J) Serum alanine aminotransferase (ALT) levels. (K) Serum alkaline phosphatase (ALP) levels. (L) Serum blood urea nitrogen (BUN) levels. (M) Serum creatinine (CRE) levels. Created with BioRender.com .

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Inhibition, Membrane, Imaging

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Cytotoxicity of an MSLN-targeted mitochondrial-membrane-damaging peptide in human pancreatic cancer patient-derived organoids (PDOs). (A) Immunoblot analysis of MSLN levels in human pancreatic cancer PDOs. Red, MSLN-high organoid; blue, MSLN-low organoid. AsPC-1 and MIA PaCa-2 cells served as controls. (B) Cytotoxicity of MSLNpep-kla in human pancreatic cancer PDOs. Red, MSLN-high organoid; blue, MSLN-low organoid. (C) Correlation between MSLN levels and IC50 values in PDOs ( R 2 = 0.7952). (D) Microscopic morphology of SPT#145 and SPT#144 organoids after treatment with Ctrlpep-kla (100 μM) or MSLNpep-kla (25, 50, and 100 μM). Scale bars, 200 μm.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Membrane, Derivative Assay, Western Blot

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Single-cell expression analysis of MSLN in human pancreatic cancer tissues. Violin plots showing the expression distribution of MSLN across various cell types identified within the pancreatic cancer tumor microenvironment (dataset from Ellerby et al. ). The y -axis represents log-normalized expression levels.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Single Cell, Expressing

Journal: bioRxiv

Article Title: Actionable spatial prostanoid barriers constrain BiTE-driven adoptive T cell immunity in intact human tumors

doi: 10.64898/2026.03.26.713601

Figure Lengend Snippet: A, Schematic of the tiered benchmarking assay comparing anti-MSLN CAR-T cells and anti-MSLN BiTE T cells in OVCAR-8 2D monolayers and matrix-free 3D spheroids after 24 hrs coculture at a 1:1 effector-to-target (E:T) ratio. B, Representative flow cytometry plots and quantification of CD8 + CD137 + cells among CD8 + T cells after coculture of MSLN-CAR T cells alone (T cell only), with 2D OVCAR-8 cells, or with 3D OVCAR-8 spheroids. C, Representative flow cytometry plots and quantification of CD8 + CD137 + cells among CD8+ T cells after coculture of MSLN-BiTE T cells alone (T cell only), with 2D OVCAR-8 cells, or with 3D OVCAR-8 spheroids. D, Supernatant concentrations of granzyme B, TNFα, and IFNγ after 24-hour coculture of MSLN-CAR (top) or MSLN-BiTE (bottom) T cells with 2D OVCAR-8 cells or 3D spheroids. E, Representative confocal immunofluorescence images of embedded 3D spheroids stained for nuclei (DAPI), CD8, and MSLN at baseline and at 5 and 24 hrs after exposure to MSLN-CAR or MSLN-BiTE T cells. Scale bars, 100 μm. F, Quantification of intratumoral CD8 density in 3D spheroids across conditions. Statistical analysis using one-way ANOVA (+Kruskal-Wallis) G, Quantification of intratumoral CD8 density in 3D spheroids across conditions. H, Interpatient heterogeneity of MSLN expression in PDTs assessed by flow cytometry, schematic of Matrigel-free PDT coculture with MSLN-BiTE T cells at a 1:1 E:T ratio, and correlation between epithelial target abundance (%CK + MSLN + of CK + cells) and CD8 activation (FC of CD8 + CD137 + over UT control). I , Multiparametric functional profiling of PDT responses to MSLN-BiTE T cells. Upper part, CD8 activation (%CD8⁺CD137⁺) under untreated versus +MSLN-BiTE conditions. Below, heatmap summarizing changes in secreted mediators (log2 fold-change over UT control) following co-culture across the indicated tumoroid lines. One-way repeated-measures ANOVA (+Tukey) was performed in GraphPad Prism v10.2 (n=3-4 technical replicates per patients). Exact P values and comparisons are indicated in the panels. In B and C , representative plots are shown and quantification reflects 3 to 4 technical replicates per condition. In D , n = 3 technical replicates per condition. In F , n = 12 to 22 regions of interest per condition. Exact P values and comparisons are indicated in the panels. Figure created with BioRender.

Article Snippet: When indicated, tumor cells were identified using anti EpCAM (BioLegend Cat# 324232, RRID:AB_2564301) and anti-MSLN (Santa Cruz Biotechnology Cat# sc-33672, RRID:AB_627930).

Techniques: Flow Cytometry, Immunofluorescence, Staining, Expressing, Activation Assay, Control, Functional Assay, Co-Culture Assay