Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Endogenous PAR1 expression in different human pancreatic ductal adenocarcinoma (PDAC) cells. Six human PDAC cell lines, namely MIA PaCa-2, HPAF-II, SU.8686, Capan-1, ASPC-1, and CFPAC-1, were cultured for PAR1 level screening. A RT-PCR analysis of endogenous PAR1 mRNA levels (*** p < 0.001). B Western blot analysis of PAR1 expression (~ 66 kDa) in whole-cell lysates among the six cell lines (*** p < 0.001). C The mean fluorescence intensity (MFI) of PAR1 surface expression by tumor cells was determined by a flow cytometric analysis using a phycoerythrin (PE)-anti-PAR1 antibody (Ab) versus an isotype control. Propidium iodide (PI) levels were used to examine apoptotic cells. D Immunocytofluorescence (IF) analysis of PAR1 expression patterns in PDAC cells using antihuman PAR1 with signal enhancement through m-IgGκ BP-FITC labeling (left panel). Quantified statistics of green fluorescent protein-positive (GFP + ) to DAPI. + cell ratio of IF results are also shown (right panel). Individual scale bars are shown. All data are presented as the mean ± SD. of three experiments. *** p < 0.001

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Fluorescence, Control, Labeling

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Suppression of PAR1-expressing MIA PaCa-2 and CFPAC-1 cells by PAR1CAR-T cells in vitro. A A standard 24-h MTT cytotoxicity assay using three replicates ( n > 3) with increasing effector/tumor (E/T; effector: PAR1CAR-T cells) ratios of 0, 0.1, 1, 5, 10, and 20 against pancreatic ductal adenocarcinoma (PDAC) cell lines of MIA PaCa-2, CFPAC-1, and HPAF-II. Cytotoxic activities were compared to those of non-transduced CD3 + T-cell-treated cells, and mock-transduced T-cell-treated cells served as the control PAR1CAR-T cells ( n > 3; * p < 0.05 and *** p < 0.001). B Real-time monitoring of cytotoxic activities used for comparison between non-transduced CD3 + T cell-treated and mock-transduced T cell-treated cells, and 1% Triton-X-100-treated cells served as a positive control. Real-time monitoring of PAR1CAR-T-cell-treated cells revealed specific growth inhibition of PAR1-expressing CFPAC-1 (low levels; n > 3; * p < 0.05 and ** p < 0.01) and MIA PaCa-2 cells (high levels; n > 3; * p < 0.05, ** p < 0.01, and *** p < 0.001) compared to PAR1 non-expressing HPAF-II cells, as observed using the x-CELLigence System. Data are presented as the mean ± SD of three independent experiments

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, In Vitro, Cytotoxicity Assay, Control, Comparison, Positive Control, Inhibition

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Transforming growth factor (TGF)-β-mediated PAR1 upregulation enhances pancreatic ductal adenocarcinoma (PDAC) cell responsiveness to PAR1CAR-T-cell-specific suppression. A Human PDAC cell lines (HPAF-II, CFPAC-1, and MIA PaCa-2) were exposed to TGF-β (18 ng/mL), and cells were collected at indicated times over 48 h. PAR1 expression was measured by flow cytometry. Results revealed original and enhanced levels of PAR1 by quantifying the mean fluorescent intensity (MFI) (left panel), expression fold-changes (right panel), and cell fold-changes (middle panel) over incubation times. B Standard 24-h cytotoxic activities of PAR1CAR-T cells toward tumor cells were measured using MTT assays with increasing effector/tumor (E/T) ratios of 0, 0.1, 1, 5, 10, and 20 against HPAF-II, CFPAC-1, and MIA PaCa-2 cells following 18 ng/mL TGF-β stimulation (18 ng/mL) for 48 h. Cytotoxic activities were compared to those of non-transduced CD3 + T-cell-treated cells, and mock-transduced T-cell-treated cells served as control PAR1CAR-T cells ( n > 3; * p < 0.05 and *** p < 0.001, respectively). Results are the mean ± SD of three independent experiments

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, Flow Cytometry, Incubation, Control

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Relationship between transforming growth factor (TGF)-β-modulated PAR1 and regulatory T cell (Treg) function and pancreatic ductal adenocarcinoma (PDAC) cell response to PAR1CAR-T cell targeting. A Western blotting results of 24-h stimulation with TGF-β on the MIAPaCa-2 and HPAF-II PDAC cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. B Analysis of tissue factor (TF) and thrombin expressions in individual cell lines treated for 24 h with the TGF-β growth factor. C Effect of adding cancer-associated fibroblasts (CAFs) and different phenotypes of cytokine-independent T cells on cell viability according to different treatments. Different co-culture combinations also resulted in various tumor-derived TGF-β levels. D A schematic diagram shows the role of immuno-mediated TGF-β affecting Treg function and transformation in PDAC treated with PAR1CART cells

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Western Blot, Control, Co-Culture Assay, Derivative Assay, Transformation Assay