Journal: International journal of cancer. Journal international du cancer

Article Title: Induction of Osteopontin Expression by Nicotine and Cigarette Smoke in the Pancreas and Pancreatic Ductal Adenocarcinoma Cells

doi: 10.1002/ijc.24388

Figure Lengend Snippet: Nicotine induces OPN accumulation in cultured PDA cells. AsPC-1 (A), Hs766T (B), and PK9 (C) cells were treated with nicotine (3×10−9 – 3×10−7 mol/L) for 24 and 72 hr. Significant induction of OPN mRNA expression is seen with the maximum induction after 24 hr at 3×10−8 mol/L of nicotine.

Article Snippet: HS766T and PK9 cells were generously donated by Dr. Scott Kern, Johns Hopkins University School of Medicine, Baltimore, MD.

Techniques: Cell Culture, Expressing

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Endoglin is highly expressed on CAFs in human pancreatic tumors. ( A ) Representative images of human pancreatic cancer (representative from n = 25 PDAC patients) and normal pancreas stained for α-SMA, endoglin, cytokeratin, and vimentin. Endothelial cells (black arrow) and endoglin expressing CAFs (white arrow). ( B ) Immunofluorescent double staining for α-SMA and endoglin in human PDAC tumors. ( C ) Endoglin mRNA expression by human cells; ECRF endothelial cells, MIA PaCa-2, PANC-1 and BxPC-3 PDAC cells and 8 patient derived primary pancreatic CAFs. ( D ) Endoglin protein expression on human pancreatic fibroblasts. Basal and BMP9-induced downstream signaling (pSMAD1) was inhibited with TRC105 (full-length blot shown in Supplementary figure 4A – C ).

Article Snippet: The mouse PDAC cell line KPC-3 (Kras G12D/+ LSL-Trp53 R172H/+ Pdx-1-Cre), (kindly supplied by the department of Immunology, LUMC) with a targeted insertion of codon-optimized Luc-2 (pGL4.10) [luc2] (Promega Leiden, the Netherlands), mouse MC38 cells (kindly supplied by the department of Immunology, LUMC) and primary fibroblasts were all cultured in DMEM/F12 glutamax medium (Invitrogen, Landsmeer, the Netherlands), with 10% fetal bovine serum (FBS) (Gibco, Bleiswijk, the Netherlands), 0.01 M HEPES, 0.1 μg/mL Gentamycin, 40U/mL Penicillin and 40 μg/mL Streptomycin (all Invitrogen Landsmeer, the Netherlands) at 37°C and 5% CO2.

Techniques: Staining, Expressing, Double Staining, Derivative Assay

Journal: Pharmaceutics

Article Title: Nanoparticle-Based Therapeutic Strategies for Enhanced Pancreatic Ductal Adenocarcinoma Immunotherapy

doi: 10.3390/pharmaceutics14102033

Figure Lengend Snippet: Nanoparticle-based therapeutic strategies for enhanced PDAC immunotherapy.

Article Snippet: gemcitabine-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles + M2-like macrophages peptides (M2pep) +PD-L1 antibody , biomimetic dual-targeting nanomedicine , iv , elimination of PD-L1-positive macrophages and the downregulation of PD-L1 expression; reprogrammed macrophages, downregulated PD-L1 expression, and sustained T cell populations, , 2022 , [ ] .

Techniques: Modification, Molecular Weight, Clinical Proteomics, Injection, Polymer, Expressing, Recombinant, Liposomes, Irradiation, In Situ

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: PDA cells reprogram macrophages in TME through DNA methylation. a Tumor and macrophage co-culture experimental schema. b Nqo-1 and Aldh1a3 methylation was examined by methylation-specific PCR (MSP) in mouse BMDMs after co-culturing with KPC PDA cells. * P < 0.05 (paired t test). c The schema of the candidate gene selection process. d Expression of the key genes in glucose metabolism and OXPHOS pathway in mouse BMDMs after co-culturing with KPC cells. The mRNA expression of these genes was measured by RT-PCR and β-actin was used for normalization. e , f Nqo - 1 and Aldh1a3 methylation after pretreating BMDMs with DAC. * P < 0.05 ( d , e , f , Mann–Whitney U test) . g Methylation of Nqo - 1 and Aldh1a3 in TAMs, CD4 + and CD8 + T cells from primary PDA and BMDMs (consider as M0 macrophages) of the same KPC mice, and BMDMs after co-culturing with KPC cells. * P < 0.05 (ANOVA). Data are means ± SEM from technical duplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: DNA Methylation Assay, Co-Culture Assay, Methylation, Selection, Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: DNA methylation of the metabolism genes in macrophages is induced by direct interaction with PDA cells through GARP/TGF-βRII-integrin αV/β8. a , b Nqo-1 and Aldh1a3 methylation in mouse BMDMs in a transwell system separated from KPC cells by an 8-μm or 1-μm pore membrane that, respectively, allows or not allows tumor cells to migrate through and direct contract with macrophages and in BMDMs cultured with TCM. Nqo-1 and Aldh1a3 methylation quantified as described in Supplementary Methods. * P < 0.05 (Mann–Whitney U test). c Lucifer Yellow labeled-KPC cells were co-cultured with unlabeled BMDMs. Thick arrows indicate macrophages that contain Lucifer Yellow spread from KPC cells (thin arrow) around them. Scale bar: 20 μm. d GARP expression on M0, M1-like, and M2-like macrophages measured by immunofluorescent staining with FITC-conjugated anti-GARP antibody. Arrow indicates macrophages that have the highest fluorescence within each image. Scale bar: 20 μm. * P < 0.05 (ANOVA). Histogram (right panel) shows quantification of fluorescence intensity. e Multiplex immunohistochemistry (IHC) was performed on a single slide of human PDA tissues for GARP (in green), CD68 (in red) and CD163 (in purple). A representative among 20 human PDAs tested is shown. Arrows (both panels) indicate GARP-expressing CD68 + CD163 + (M2-like) macrophages ; and arrowheads (left panel) indicate GARP-expressing CD68 + CD163 - (M1-like) macrophages . Notched arrowheads (right panel) indicate CD68 + CD163 + (M2-like) macrophages with little GARP expression. Scale bar: 50 μm. f Multiplex IHC staining of GARP (in green) on F4/80 + (in red) macrophages in PDAs from KPC mice. Scale bar: 50 μm. g TGF-βRII and GARP on cell surface of M0, M1-like, and M2-like macrophages co-stained and analyzed by flow cytometry. h Quantification of the percentages of TGF-βRII on cell surface of M0, M1-like, and M2-like macrophages by flow cytometry. * P < 0.05 (ANOVA). i Integrin subunits ɑV and β8 cell-surface expression was measured by flow cytometry. j IHC staining of PDA and normal pancreas tissues from KPC mice with anti-integrin ɑV and β8 antibodies. Scale bar: 100 μm. Data were from technical triplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: DNA Methylation Assay, Methylation, Membrane, Cell Culture, MANN-WHITNEY, Labeling, Expressing, Staining, Fluorescence, Multiplex Assay, Immunohistochemistry, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: GARP mediates Nqo-1 methylation and M2-like phenotypical changes in M1-like macrophages after co-culturing with PDA cells. a Nqo-1 methylation in mouse WT M1-like macrophages compared to GARP KO M1-like macrophages. * P < 0.05 (paired t test). b RT-PCR of M2 marker genes in WT vs. GARP KO M1-like macrophages after co-cultured with KPC cells. Fold changes of these marker genes in co-cultured vs. monocultured M1-like macrophages were shown. Fold change >1: upregulation; fold change <1: downregulation. All results were first normalized by respective β-actin and then respective monocultured BMDMs. * P < 0.05 (Mann–Whitney U test). c Expression of the M2 cytokine IL-10 in WT vs. GARP KO M1-like macrophages after co-culturing with KPC cells, measured by flow cytometry analysis of percentages of IL-10-positive cells with intracellular staining of IL-10. * P < 0.05 (Mann–Whitney U test). d Fold changes of MSP results of the Nqo-1 gene in co - cultured vs. monocultured M1 - like macrophages treated with RGD or TGF-βRII blocking antibody. * P < 0.05 (ANOVA). e Fold changes of RT-PCR results of M2 marker genes in co-cultur e d vs. monocultured M1-like macrophages treated with RGD or TGF-βRII blocking antibody. Data were first normalized by respective β-actin and then respective monocultured M0 macrophages. * P < 0.05 (ANOVA). f Mitochondrial membrane potentials in mouse M0, M1-like and M2-like macrophages after co-culturing with KPC cells by measuring mean fluorescence intensity of TMRM signals on the PE channel of flow cytometry, comparing mono- vs. co-cultured macrophages. * P < 0.05 (ANOVA). g Glucose uptake activities in M0, M1-like, and M2-like macrophages by measuring mean fluorescence intensity of 2-NBDG signals, comparing mono- vs. co-cultured macrophages. * P < 0.05 (ANOVA). h KPC cells were co-cultured with mouse BMDMs or DAC pretreated BMDMs in upper chamber of a transwell system with 8-μm pore membrane that allows them migrating to the lower chamber. Migrated KPC cells were examined by immunofluorescent staining with FITC-conjugated anti-Pan-CK antibody and counted. Fold changes of migrated KPC cell number in co-cultured vs. monocultured group (normalized as 1) were shown. * P < 0.05 (Mann–Whitney U test). i KPC cells were co-cultured with BMDMs pretreated with DAC, glucose uptake inhibitor WZB-117, or DAC + WZB-117, respectively, in the transwell system. Numbers of migrated KPC cells were counted as described in ( h ) and shown. * P < 0.05 (ANOVA). j Il-10 expression per RT-PCR in untreated, DAC, or WZB-117 pretreated BMDMs before (normalized as 1) and after co-culturing with KPC cells. * P < 0.05 (Mann–Whitney U test). Data are means ± SEM from technical duplicates and representative of two experiments

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: Methylation, Reverse Transcription Polymerase Chain Reaction, Marker, Cell Culture, MANN-WHITNEY, Expressing, Flow Cytometry, Staining, Blocking Assay, Membrane, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism

doi: 10.1038/s41392-021-00769-z

Figure Lengend Snippet: Downregulation of genes in the metabolic pathway in TAMs from murine PDA. a mRNA expression of metabolism genes as indicated were measured by RT-PCR in TAMs, CD4 + , and CD8 + T cells from primary pancreatic tumors and BMDMs of the same KPC mice. Tumors were identified by ultrasound before sacrifice. β-actin used for normalization. Data are means ± SEM from triplicates and representative of two experiments. * P < 0.05 (ANOVA). b The schematic model of the GARP/integrin-mediated interaction between tumor cells and macrophages in the TME of PDAC and the mechanisms of metabolic, phenotypical, and functional reprogramming of macrophages from M1-like to M2-like macrophages in a DNA methylation-dependent manner

Article Snippet: The KPC PDA tumor cell line was developed from a KPC mouse as described previously., Human pancreatic cancer cell line Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Functional Assay, DNA Methylation Assay