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

Journal: Translational Oncology

Article Title: The pancreatitis-cancer transformation-related factor, human rhomboid family-1, promotes pancreatic cancer progression through the SRC/YAP signaling pathway

doi: 10.1016/j.tranon.2025.102346

Figure Lengend Snippet: Increased RHBDF1 levels in chronic pancreatitis linked to PDAC are associated with accelerated disease progression. (A) Transcriptomic analysis of tumor and surrounding tissues from FUSCC revealed significant differences in the level of RHBDF1 mRNA expression between CP-PDAC and adjacent normal tissues ( N = 11, P < 0.001). (B) Transcriptomic analysis of TGCA and GTEx databases revealed significant differences in the level of RHBDF1 mRNA expression between PDAC and normal pancreas tissues. (C) Kaplan–Meier plots illustrating the relationship between RHBDF1 expression and overall survival from TCGA data. (D) Western blot analysis of RHBDF1 in pancreatic ductal adenocarcinoma cell lines and CP, CP-PDAC, and adjacent tissues. (E, F) IHC staining analysis show the relative levels of RHBDF1 in CP, CP-PDAC, and adjacent tissue. (G, H) Immunohistochemical analysis of RHBDF1 expression in LSL-KrasG12D and Ptf1aCreER mice with chronic pancreatitis induced by cerulein. *P <0.05, **P <0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Human PDAC cells (Capan-1, MiaPaCa-2, SW1990, PANC-1, and BxPC-3), human pancreatic ductal epithelial cells (HPDE), human pancreatic stellate cells (HPSCs), and cancer-associated fibroblasts (CAFs) were obtained from the American Type Culture Collection.

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunohistochemistry, Immunohistochemical staining

Journal: Translational Oncology

Article Title: The pancreatitis-cancer transformation-related factor, human rhomboid family-1, promotes pancreatic cancer progression through the SRC/YAP signaling pathway

doi: 10.1016/j.tranon.2025.102346

Figure Lengend Snippet: RHBDF1 increases the proliferation, migration, and invasion of pancreatic tumor cells. (A-C) Three short hairpin RNAs (shRNAs) aimed at the RHBDF1 junction site were designed. Following stable transfection with sh-RHBDF1, the levels of RHBDF1 mRNA and protein in PDAC cells were measured by qRT-PCR and western blotting. (D–F) Cell proliferation was assessed by the CCK-8 and EdU incorporation assays. Scale bars, 100 μm. (G-I) The cell cycle was evaluated using flow cytometry. (J, K) Transwell assays were performed to assess the migration and invasion abilities of PDAC cells. Scale bars, 100 μm. * P <0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human PDAC cells (Capan-1, MiaPaCa-2, SW1990, PANC-1, and BxPC-3), human pancreatic ductal epithelial cells (HPDE), human pancreatic stellate cells (HPSCs), and cancer-associated fibroblasts (CAFs) were obtained from the American Type Culture Collection.

Techniques: Migration, Stable Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: The pancreatitis-cancer transformation-related factor, human rhomboid family-1, promotes pancreatic cancer progression through the SRC/YAP signaling pathway

doi: 10.1016/j.tranon.2025.102346

Figure Lengend Snippet: RHBDF1 activates SRC to exert carcinogenic effects. (A) Volcano plot illustrating the differentially expressed genes (DEGs) between the RHBDF1-high and RHBDF1-low groups analyzed using DESeq2. |Log2fold change| > 1, FDR < 0.05. (B) Protein-protein internation network of overexpressed mRNAs in the RHBDF1-high group, as constructed by Network Analyst. The size of each mRNA represents their topology level in the network. (C–D) RHBDF1 and SRC are co-expressed at the mRNA level in the CCLE (C) and TCGA datasets (D). (E–M) The expression levels of RHBDF1 and SRC mRNA and protein in PDAC cells were measured by qRT-PCR and western blotting. (N) Co-immunoprecipitation of RHBDF1 and SRC in PDAC cells. (O–Q) Cell growth was evaluated using the CCK-8 and EdU incorporation tests. Scale bars, 100 μm. (R, S) The migration and invasion potential of PDAC cells was assessed using the Transwell assay. Scale bars, 100 μm. * P <0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human PDAC cells (Capan-1, MiaPaCa-2, SW1990, PANC-1, and BxPC-3), human pancreatic ductal epithelial cells (HPDE), human pancreatic stellate cells (HPSCs), and cancer-associated fibroblasts (CAFs) were obtained from the American Type Culture Collection.

Techniques: Construct, Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation, CCK-8 Assay, Migration, Transwell Assay

Journal: Translational Oncology

Article Title: The pancreatitis-cancer transformation-related factor, human rhomboid family-1, promotes pancreatic cancer progression through the SRC/YAP signaling pathway

doi: 10.1016/j.tranon.2025.102346

Figure Lengend Snippet: RNHBDF1 activates the YAP signaling pathway through SRC. (A) Differences between the shRHBDF1 and shNC groups were analyzed by principal component analysis and volcano plot analysis. (B) The differentially expressed genes (DEGs) in the two sets of transcriptomics data (clinical samples and cell line RHBDF-1 knockdown) were performed. (C, D) Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. (E-H) Western blot analysis was employed to confirm the expression levels of SRC/YAP key proteins in ShNC, shRHBDF1, and shRHBDF1 combined with SRC inhibition groups. (I–K) Cell proliferation was assessed using the CCK-8 and EdU incorporation assays. Scale bars, 100 μm. (L, M) The migration and invasion abilities of PDAC cells were assessed using the Transwell assay. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human PDAC cells (Capan-1, MiaPaCa-2, SW1990, PANC-1, and BxPC-3), human pancreatic ductal epithelial cells (HPDE), human pancreatic stellate cells (HPSCs), and cancer-associated fibroblasts (CAFs) were obtained from the American Type Culture Collection.

Techniques: Knockdown, Functional Assay, Western Blot, Expressing, Inhibition, CCK-8 Assay, Migration, Transwell Assay

Journal: Theranostics

Article Title: Chimeric peptide supramolecular nanoparticles for plectin-1 targeted miRNA-9 delivery in pancreatic cancer.

doi: 10.7150/thno.38327

Figure Lengend Snippet: Figure 1. miR-9 enhances doxorubicin sensitivity in PDAC cells. (A) PDAC cells were incubated with indicated concentration (0, 0.125, 0.25, 0.5, 1, 2 µg/ml) of doxorubicin for 48 hr. Cell viability was assessed using Cell Counting Kit-8 assay. (B) Quantitative IC50 analysis of doxorubicin and quantitative RT-PCR analysis of miR-9 abundance in PDAC cells (n=3 independent experiments). (C) The correlation between miR-9 expression and IC50 value of PDAC cell lines for doxorubicin. (D) Quantitative RT-PCR analysis of miR-9 abundance in paired Adjacent and Tumor from PDAC patients (n=16). (E) PDAC cells were treated with 0.5 µg/ml doxorubicin for 48 hr. Shown are quantitative RT-PCR analysis of miR-9 abundance. (F-H) PDAC cells were treated with indicated concentration of doxorubicin for 48 hr after lipofectamine 2000 (Lipo) mediated miR-9 or control transfection in PANC-1 cells and CFPAC-1 cells. Quantitative RT-PCR analysis of miR-9 abundance (F). Cell viability was assessed using Cell Counting Kit-8 assay (G). Shown are quantitative IC50 analysis of doxorubicin (n=3 independent experiments) (H). (I) EdU analysis of proliferation in PDAC cells. Cells were treated with doxorubicin after lipofectamine mediated miR-9 or control transfection in PANC-1 cells and CFPAC-1 cells. Shown are representative EdU labeling images (left) and quantifications of EdU-positive cells in percentages (right), respectively. Scale bars, 50 µm. Data are presented as the mean ± SD, and analyzed with Student’s t-test or one-way ANOVA. *P < 0.05, **P < 0.01

Article Snippet: Cell lines and Cell culture Human PDAC cell lines (CFPAC-1, PANC-1 and CAPAN-1) and lung cancer cell lines (NCI-H1299 and A549) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Incubation, Concentration Assay, Cell Counting, Quantitative RT-PCR, Expressing, Control, Transfection, Labeling

Journal: Theranostics

Article Title: Chimeric peptide supramolecular nanoparticles for plectin-1 targeted miRNA-9 delivery in pancreatic cancer.

doi: 10.7150/thno.38327

Figure Lengend Snippet: Figure 2. miR-9 ameliorates doxorubicin sensitivity though inhibiting autophagy in PDAC cells. (A) PDAC cells were incubated with 0.5 µg/ml doxorubicin for 48 hr along with or without 10 µΜ Chloroquine. LC3 and P62 protein expression were assessed using Western blot. β-actin was used as the loading control. (B) PDAC cells with lipofectamine mediated miR-9 or control transfection, treated with or without 0.5 µg/ml doxorubicin for 48 hr. LC3 and P62 protein expression were assessed using Western blot. β-actin was used as the loading control. (C) mRFP-GFP-LC3 stable PANC-1 and CFPAC-1 cells with different treatment were visualized by confocal microscopy. The numbers of GFP+/mRFP+-LC3 (yellow) and GFP−/mRFP+-LC3 (red) dots were recorded at least in 50-100 cells. Scale bars, 10 µm. (D-G) PDAC cells were transfected with Lipo mediated miR-9 or control, then cells were incubated with indicated concentration of

Article Snippet: Cell lines and Cell culture Human PDAC cell lines (CFPAC-1, PANC-1 and CAPAN-1) and lung cancer cell lines (NCI-H1299 and A549) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Incubation, Expressing, Western Blot, Control, Transfection, Confocal Microscopy, Concentration Assay

Journal: Theranostics

Article Title: Chimeric peptide supramolecular nanoparticles for plectin-1 targeted miRNA-9 delivery in pancreatic cancer.

doi: 10.7150/thno.38327

Figure Lengend Snippet: Figure 4. PL-1 motif-functionalized nanoparticles are more stable and deliver miR-9 to PDAC cells with high specificity. (A) Self-assembly of PL-1 polypeptides with miR-9 payloads to form nanocomplexes. The PDAC-specific peptide PTP was fused to the N-terminus of nine D-arginine residues via a four-glycine linker. (B) Agarose gel (3%) retardation assay at different molar ratios of PL-1 to miR-9. (C) The average hydrodynamic diameter of the nanoparticles in ddH2O, as assessed by DLS, was 180 ± 17 nm. (D) Stability of naked miR-9 and miR-9 complexed with PL-1 at a molar ratio of 20:1, after incubation with RNase A (upper). Serum stability after incubation in 50% mouse-serum solution (lower). (E) Representative confocal fluorescence microscopy images of PANC-1 and CFPAC-1 cells treated with Cy3 (red)-labeled miR-9 (50 nM)/PL-1 peptide complex. Nuclei and endosomes/lysosomes were stained with Hoechst (blue) and FITC-labeled Dextran (green, endo/lysosome tracker). Scale bars, 10 µm. (F-G) PDAC cells were transfected with miR-9 using PL-1 or Lipo for 48 hr. Quantitative RT-PCR analysis of the mRNA abundance of miR-9 and eIF5A2 (F). eIF5A2 protein expression was assessed using Western blot (G). Experiments were repeated in three times. Data are presented as the mean ± SD, and analyzed with Student’s t-test or one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell lines and Cell culture Human PDAC cell lines (CFPAC-1, PANC-1 and CAPAN-1) and lung cancer cell lines (NCI-H1299 and A549) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Agarose Gel Electrophoresis, Incubation, Fluorescence, Microscopy, Labeling, Staining, Transfection, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Molecular Mechanisms Involved in the Synergistic Interaction of the EZH2 Inhibitor 3-Deazaneplanocin A with Gemcitabine in Pancreatic Cancer Cells

doi: 10.1158/1535-7163.mct-12-0037

Figure Lengend Snippet: Figure 3. Effects of DZNeP, gemcitabine, and their combination on PDAC cells migration. Results of wound-healing assay in PANC-1 (A), MIA-PaCa-2 (B), and LPC006 (representative picture at 48 hours; C) cells. Cells were exposed to 5 mmol/L DZNeP, gemcitabine at IC50, and their combination. Modulation of E-cadherin after 24 hours as determined by real-time RT-PCR (D) and immunocytochemistry (E). Columns, mean values obtained from 3 independent experiments; bars, SEM. , significantly different from controls.

Article Snippet: Eight PDAC cell lines (PANC-1, MIA-PaCa-2, BxPc3, Capan-1, PL45, HPAC, HPAF-II, and CFPAC-1), the human pancreatic duct epithelial-like cell line hTERT- HPNE, and skin fibroblasts Hs27 were obtained from the American Type Culture Collection, whereas 7 primary PDAC cultures (LPC006, LPC028, LPC033, LPC067, LPC111, LPC167, and PP437) were isolated from patients at theUniversityHospital of Pisa (Pisa, Italy), as described previously (18).

Techniques: Migration, Wound Healing Assay, Quantitative RT-PCR, Immunocytochemistry

Journal: Cancer research

Article Title: Post-transcriptional regulation of PARG mRNA by HuR facilitates DNA repair and resistance to PARP inhibitors

doi: 10.1158/0008-5472.CAN-16-2704

Figure Lengend Snippet: Cell survival of PDA cell lines (A), HuR-knockout CRIPSR cell lines, MIA PaCa-2 and Hs 766T [HuR(+/+) vs HuR(−/−)] (B) and HuR-silenced MiaPaCa-2 and Capan-1 cells (C) treated with increasing doses of olaparib for 7 days. (D) Representative images of MIA.HuR(+/+) vs MIA.HuR(−/−) and HST.HuR(+/+) vs HST.HuR(−/−) cells seeded and cultured in soft agar in the presence of respective IC50 doses of olaparib for 4 weeks. (E) HuR expression in MIA PaCa-2 cells treated with indicated IC50 doses of PARPi for 12hr, and fractionated as indicated. Lamin A/C and α-Tubulin used as controls to determine the integrity of nuclear and cytosolic lysates respectively. Mitomycin C used as positive control for cytoplasmic translocation of HuR. (F) Immunofluorescent images of HuR (green) in MIA PaCa-2 cells treated with PARPi for 12hr. Nuclei were stained with DAPI. Magnification 40X.

Article Snippet: PDA cell lines (MIA PaCa-2, PANC-1, Capan-1, Hs 766T, PL11) were purchased from ATCC (Manassas, VA, 2012).

Techniques: Knock-Out, Cell Culture, Expressing, Positive Control, Translocation Assay, Staining

Journal: Cancer research

Article Title: Post-transcriptional regulation of PARG mRNA by HuR facilitates DNA repair and resistance to PARP inhibitors

doi: 10.1158/0008-5472.CAN-16-2704

Figure Lengend Snippet: Models to evaluate the role of HuR in PARPi response Table indicates the IC 50 values of 1) (CRISPR)-generated PDA cell lines DDR- P MIA PaCa-2 and DDR- D Hs 766T with HuR genetically knocked out [HuR (+/+), HuR (−/−)]; 2) siRNA oligos against the HuR coding region as previously described ( 17 , 20 ); 3) a small molecule inhibitor, MS-444 ( 40 ).

Article Snippet: PDA cell lines (MIA PaCa-2, PANC-1, Capan-1, Hs 766T, PL11) were purchased from ATCC (Manassas, VA, 2012).

Techniques: CRISPR, Two Tailed Test, Knock-Out, Inhibition

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a , Meta-analysis of enriched pathways in regions accessible in CLA PDAC cell lines CAPAN1 and CAPAN2 (by ATAC-seq) and bound in CAPAN1 by JUNB (by ChIP-seq). Node color indicates significance of enrichment, link width the number of overlapping genes between gene sets. b - h , IHC analysis in 105 PDAC patients for epithelial JUNB expression. b , IHC for JUNB, GATA6 and ECAD in cores classified as JUNB low and JUNB high . Scale bar 200 μm. c - h , Quantification of b , for GATA6 ( c - e ) and ECAD ( f - h ) in JUNB low and JUNB high expression per patient ( c , f ), per TMA core across all patients ( d , g ) and in heterogeneous patients showing matched levels in JUNB low and JUNB high cores ( e , h ). i , Correlation of JUNB and GATA6 (left), as well as JUNB and CDH1 (right) in laser-capture microdissection-enriched human PDAC tumor epithelia and stroma. Top, RNA-seq expression (transcriptome, n=29 patients), bottom mass-spectrometry quantification (proteome, n=32 patients). Linear regression with 95% CI, as well as Spearman’s R and associated P value. j , IF for JUNB and GATA6 in resection tissue of PDAC patients at representative JUNB high and JUNB low regions, with overlayed cell classification by QuPath. Green: nuclear JUNB + , magenta: nuclear GATA6 + , white: JUNB-GATA6 double-positive. Scale bar 50 μm. k , Quantification of j for per-patient average nuclear GATA6 + cells and nuclear JUNB + cells relative to the total number of cells, plotted as in i . n=23. l , Epithelial-specific transcriptional RNA-seq profiles of resected PDAC patients were generated by fluorescence-activated cell sorting of EPCAM + /CD45 − /CD31 − cells. Correlation analysis for epithelial-specific JUNB and GATA6, plotted as in i . n=31. m , Gene set enrichment analysis for Chan-Seng-Yue PDAC subtypes in genes correlating with JUNB in epithelial compartment-sorted transcriptomes of l . Normalized enrichment score (NES) and FDR q value are indicated. n , Correlation analysis for JUNB and GATA6 in LCM-enriched epithelia of patients of the COMPASS trial (stage I-IV). Linear regression, as well as Spearman’s R and associated P value. n=486. o , JUNB expression in the dataset as in n , classified for the Chan-Seng-Yue PDAC subtypes. Kruskal-Wallis test. p , IF for JUNB, pan-cytokeratin (panCK), and GATA6, in tissue of treatment-naive, neo-adjuvant-treated, and chemo-radiation-treated PDAC patients. Overlayed cell classification by QuPath for panCK-JUNB-GATA6 triple-positive cells shown in white. Scale bar 50 μm. q , Quantification of p for average percentage of ductal (cytoplasmic panCK + cells) that are additionally double-positive for nuclear GATA6 and nuclear JUNB (blue) or not (grey). Naive, n=4; neo-adjuvant, n=4; chemo-radiation, n=3.

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: ChIP-sequencing, Expressing, Laser Capture Microdissection, RNA Sequencing, Mass Spectrometry, Generated, Fluorescence, FACS, Adjuvant

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a - c , Gene expression correlation of JUNB with GATA6 in the TCGA ( a ; n=177), Puleo ( b ; n=309), and QCMG ( c ; n=96) cohorts. Linear regression with 95% CI, as well as Spearman’s R and associated P value. d - f , IHC analysis in 105 PDAC patients for epithelial JUNB expression. d , IHC for cytokeratin 19 and JUNB in TMA cores classified as JUNB low and JUNB high , indicating tumor-specific JUNB + cells. Inserts show higher magnification of JUNB staining. Scale bar: overview 200 μm; insert 50 μm. e , Spatial heterogeneity of tumor-specific JUNB expression within different TMA cores of each patient. f , Correlation of JUNB and GATA6 IHC quantification per patient (left) or per TMA core across all patients (right), plotted as in a - c . l , Representative DAPI staining with QuPath overlay for cell detection of staining for JUNB, pan-cytokeratin (panCK), and GATA6, in tissue of treatment-naive, neo-adjuvant-treated, and chemo-radiation-treated PDAC patients (shown in ). Classifications for cytoplasmic panCK + cells that are additionally positive for nuclear GATA6 (yellow), nuclear JUNB (green), both nuclear GATA6 and JUNB (blue) or only panCK (“negative”; magenta), are indicated. Scale bar 50 μm. h , Quantification of g showing percentage of panCK + cells. Each patient is shown individually as stacked bar graphs. Naive, n=4; neo-adjuvant, n=4; chemo-radiation, n=3.

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: Gene Expression, Expressing, Staining, Adjuvant

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a - d , Coverage of previously published JUNB ChIP-seq data in CAPAN1, as well as publicly available H3K27ac data, for loci of JUNB ( a ), GATA6 ( b ), HNF1B ( c ), and FOXA1 ( d ). ChIP-qPCR validation regions are indicated. e , f , ChIP-qPCR for regions indicated in a - d , showing signal relative to input for JUNB ( e ) and H3K27ac ( f ) pulldown with mean ± s.d. and average IgG isotype control. n=3. g - i , Integration of RNA-seq data performed after JUNB silencing (siJUNB; n=3) or control siRNA (siCtrl; n=2) in CAPAN1, with ChIP-seq for JUNB. g , Violin plot of log 2 fold change (FC) in siJUNB RNA-seq data for all (n=36.740) or JUNB-bound (n=698) genes. Median and quartiles are indicated. Student’s t-test with Welch’s correction. h , As in g , showing the number of genes that display a significant upregulation (sigUP) or downregulation (sigDN), or no significant change (ns). i , Gene ontology analysis of significantly upregulated, JUNB-bound genes following JUNB silencing with –log 10 ( q -value) indicated. Hallmark (H) and curated (C2) signature collections of the Molecular Signature Database (MSigDB) are shown. j , k , Gene set enrichment analysis plots for “inflammatory response” ( j ) and “TNF-α signaling via NFκB” ( k ) Hallmark signatures of the MSigDB for siJUNB versus siCtrl in CAPAN1 cells. Normalized enrichment score (NES) and FDR q value are indicated. l , Heatmap of TCGA expression data for JUNB and macrophage markers as well as MCPcounter scores for the monocytic lineage. Cell color indicates z score. JUNB high/low annotation based on top/bottom half of patients for JUNB expression. n=177. m , n , Overall survival and hazard ratio in TCGA (n=177), Puleo (n=288), and QCMG (n=96) patients stratified by JUNB repression signature ( h ) score. m , Kaplan-Meier survival analysis for the lower/upper quartiles (n=140 each) and mid group (n=281) for JUNB repression signature scores. Median survival (ms) with 95% confidence interval (CI). Log-rank test. n , Cox proportional hazard groups as in j . Hazard ratio (to lower quartile) with 95% CI. P values are shown right. o , p , As in m , n , for progression-free survival in the TCGA cohort. q , r , JUNB repression signature scores in AJCC stages ( q ) and pathological grading ( r ) for TCGA and QCMG cohorts combined

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: ChIP-sequencing, ChIP-qPCR, Biomarker Discovery, Control, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a , PCA plot for RNA-seq data performed for JUNB silencing (siJUNB; n=3) or control siRNA (siCtrl; n=2) in CAPAN1. b - g , Transwell invasion assay for CAPAN2 ( b - d ) and GCDX62 ( e - g ) with siJUNB or siCtrl. b , e , Immunoblot for JUNB and b-actin after siJUNB or siCtrl in CAPAN2 ( b ) and GCDX62 ( e ), validating silencing for the invasion assay. n=3. c , f , DAPI staining of invaded CAPAN2 ( c ) or GCDX62 ( f ) cells. Scale bar 100 μm. d , g , Quantification of c , f , for number of invaded cells. Average counts per FOV with mean ± s.d. shown. d , n=6 inserts from n=3 independent experiments. g , n=7 inserts from n=4 independent experiments. h , i , Gene set enrichment analysis plots for ‘TGFβ signaling’ and ‘INFγ response’ Hallmark signatures of the Molecular Signature Database (MSigDB) for siJUNB versus siCtrl in CAPAN1 cells. Normalized enrichment score (NES) and FDR q value are indicated. j , k , Heatmap of Puleo ( j ; n=309) and QCMG ( k ; n=96) expression data for JUNB and macrophage markers as well as MCPcounter scores for the monocytic lineage. JUNB high/low annotation based on top/bottom half of patients for JUNB expression. Cell color indicates z score.

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: RNA Sequencing, Control, Transwell Invasion Assay, Western Blot, Invasion Assay, Staining, Expressing

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a , Heatmap showing expression of cytokines present in the core enrichment of the gene sets shown in , and , for JUNB silencing (siJUNB; n=3) versus control siRNA (siCtrl; n=2) in CAPAN1 cells. Cell color indicates z score. b , qRT-PCR analysis for indicated target genes in siJUNB conditions (red), normalized to siCtrl (grey), in CAPAN1. Relative mRNA expression with mean ± s.d. shown. n=3. Student’s t-test with Welch’s correction. c - e , Coverage of JUNB ChIP-seq data in CAPAN1 , as well as publicly available H3K27ac data, for loci of cJUN ( b ), IL1A/B ( c ), and CXCL9/10/11 ( d ). ChIP-qPCR validation regions are indicated. f , Gene set enrichment analysis for curated signatures (C2) of the Molecular Signature database (MSigDB) for siJUNB versus siCtrl in CAPAN1 cells. Normalized enrichment score (NES) and FDR q value are indicated. g , h , Immunoblot for JUNB, HDAC1, and β-actin after JUNB pulldown, IgG isotype control or input in CAPAN1 ( g ) and CAPAN2 ( h ). n=3. i , j , ChIP-qPCR for regions indicated in c - e , showing signal relative to input for JUNB ( i ) and HDAC1 ( j ) pulldown with mean ± s.d. and average IgG isotype control. n=3. k , l , Representative immunoblot for JUNB, cJUN, and β-actin in CAPAN2 ( k ) and GCDX62 ( l ) after siJUNB or siCtrl. n=3. m , Dual-luciferase reporter assay for cJUN promoter firefly luciferase constructs in CAPAN2 cells transfected with varying concentrations of JUNB overexpression plasmids (or EV controls), together with Renilla luciferase control and firefly luciferase (Luc) reporters. Relative Luc activity to control with mean ± s.d. shown. One-way ANOVA. n - p , Integration of RNA-seq data performed in three biological replicates for overexpression of cJUN (cJUN-OE) or empty vector (EV) control in GCDX62 with ChIP-seq for cJUN. n , Violin plot of log 2 fold change (FC) in cJUN-OE RNA-seq data for all (n=24.118) or cJUN-bound (n=224) genes. Median and quartiles are indicated. o , As in n , showing the number of genes that display a significant upregulation (sigUP) or downregulation (sigDN), or no significant change (ns). p , Gene ontology analysis of significantly upregulated (red) or downregulated (blue), cJUN-bound genes following cJUN-OE with –log 10 ( q -value) indicated. Hallmark (H) and curated (C2) signature collections of the MSigDB are shown. Not enriched pathways ( q -value>0.25) are indicated by striped bars. q , r , Immunoblot for JUNB, cJUN, and b-actin in CAPAN1 ( q ) and CAPAN2 ( r ) cells with overexpression of cJUN (cJUN-OE) or empty vector (EV) control. n=3.

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: Expressing, Control, Quantitative RT-PCR, ChIP-sequencing, ChIP-qPCR, Biomarker Discovery, Western Blot, Luciferase, Reporter Assay, Construct, Transfection, Over Expression, Activity Assay, RNA Sequencing, Plasmid Preparation

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a , Heatmap of CLA and BL PDAC identity, in previously published RNA-seq data of CAPAN1 cells treated with TNF-α or vehicle control (VC) for 18 h. Cell color indicates z score. n=3. b - f , Virtually microdissected RNA-seq data of orthotopically transplanted CAPAN1 tumors treated with TNF-α or VC for three weeks. n=3 tumors; one stroma-specific transcriptome was excluded from the analysis. b , Flash-frozen tumors from orthotopically transplanted CAPAN1 tumors in NMRI- Foxn1 nu/nu mice treated with TNF-α or VC were bulk RNA-sequenced and subsequently aligned to human and murine reference genomes, to generate tumor and stromal cell-specific transcriptomes (Methods). c , d , Tumor cell-specific transcriptome. Gene set enrichment analysis (GSEA) for Hallmark signatures of the Molecular signature database (MSigDB) ( c ) and PDAC subtype signatures ( d ), for TNF-α versus VC. Normalized enrichment score (NES) and FDR q -value are indicated. e , As in c , for stroma-specific transcriptome. f , MCPcounter analysis in stroma-specific transcriptome. Cell color indicates z score. g , Relative MCPcounter scores for the indicated lineages in 582 patients of the TCGA, QCMG and Puleo cohort, separated into quartiles based on the JUNB repression signature score (as in ). MCPcounter scores were min-max normalized and standardized to the mean of the lower JUNB repression signature score group for merging of the different cohorts. Mean ± s.d. shown. h , IF for JUNB in orthotopically transplanted CAPAN1 tumors treated with TNF-α or VC, with overlayed cell detection for nuclear JUNB + cells by QuPath. Scale bar 50 μm. i , Quantification of h , for per-animal average nuclear JUNB intensity with mean ± s.d. shown. n=5. j , As in h , for ECAD and GATA6 staining and overlayed cell detection for nuclear GATA6 + cells. k , Quantification of j for per-animal average ECAD intensity per FOV with mean ± s.d. shown. k , As in i , for nuclear GATA6 intensity of j . j , k , VC, n=7; TNF-α, n=8. g , i , k , l , Student’s t-test with Welch’s correction.

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: RNA Sequencing, Control, Staining

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a - j , IHC analysis in 105 PDAC patients or TNF-α expression. a , Spatial heterogeneity of TNF-α expression within different TMA cores of each patient. b , IHC for TNF-α and CD68 in cores classified as TNF-α low and TNF-α high . Scale bar 200 μm. c , d , Quantification of b , in TNF-α low , TNF-α intermediate (TNF-α int ), and TNF-α high expression per patient ( c ) and per TMA core across all patients ( d ). e , Lymphoid compartment distribution in TNF-α low/int/high patients. Line and percentages denote patients above a third of the maximum value. f , Representative IHC staining of TMA cores for TNF-α in deserted, intermediate, and reactive subTMEs. Scale bar 200 μm. g , h , Quantification for TNF-α per patient ( g ) or TMA core ( h ) classified as deserted, intermediate, and reactive.

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a - i , IHC analysis in 105 PDAC patients for TNF-α expression. a , IHC for TNF-α, CD3, CD4, and CD8 in cores classified as TNF-α low and TNF-α high . Sale bar 200 μm. b - g , Quantification of a , for CD3 ( b , c ), CD4 ( d , e ), and CD8 ( f , g ) in TNF-α low , TNF-α intermediate (TNF-α int ), and TNF-α high expression per patient ( b , d , f ) and per TMA core across all patients ( c , e , g ). h , i , KPC cells were orthotopically implanted into syngeneic C57BL6/J mice and treated with CCR2 inhibitor (CCR2i) RS504393 or vehicle control (VC). h , Kaplan-Meier survival analysis. Median survival is indicated. Groups are not significantly different as per log-rank test. i , H&E staining of CCR2i and VC tumors. Scale bar 100 µm.

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: Expressing, Control, Staining

Journal: bioRxiv

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

doi: 10.1101/2023.10.30.563552

Figure Lengend Snippet: a , KPC cells were orthotopically implanted into syngeneic C57BL6/J mice and treated with an anti-TNF-α antibody in combination with gemcitabine (GEM) chemotherapy, or vehicle control (VC). b , Kaplan-Meier survival analysis of a . Median survival indicated. Log-rank test. c , H&E staining of anti-TNF-α+GEM and VC tumors. Scale bar 100 μm. d , IF for CD45 with CD68, and CD45 with TNF-α, in anti-TNF-α+GEM and VC tumors. Scale bar 50 μm. e , f , Quantification of d for CD45/CD68 ( e ) and TNF-α/CD45 ( f ) double-positive cells. Per-animal average counts per FOV with mean ± s.d. shown. g , IHC for CD3 and CD8 in orthotopically transplanted anti-TNF-α+GEM and VC tumors. Scale bar: overview, 100 μm; insert, 30 μm. h , i , Quantification of g for CD3 + ( h ) and CD8 + ( i ) cells. Per-animal average percentage of positive cells with mean ± s.d. shown. e , f , h , i , Student’s t-test with Welch’s correction. i , Model of AP1 dichotomy in PDAC subtype identity and immune recruitment.

Article Snippet: Established human PDAC cell lines CAPAN1 and CAPAN2 were purchased from ATCC (Manassas, VA) and authenticated (CAPAN1, RRID:CVCL_0237), (CAPAN2, RRID:CVCL_0026) by the German Collection of Microorganisms and cell culture GmbH (DSMZ).

Techniques: Control, Staining