Journal: JCI Insight

Article Title: Loss of TRPV4 reduces pancreatic cancer growth and metastasis

doi: 10.1172/jci.insight.196280

Figure Lengend Snippet: ( A ) Graphical illustration of experimental design. KPC cells (100,000 cells) were injected into the pancreas, followed by injection of KPCY cells (500,000 cells) into the spleen 10 days later. ( B ) Representative images of immunostaining of mouse liver for Ki67 and GFP (marking KCPY cells) at low (upper panels) and high magnification (lower panels). Scale bars: 100 μm (top) and 50 μm (bottom). ( C and D ) Quantitative analysis of Ki67 positively stained cells and liver lesions of WT and TRPV4-KO liver. The analysis was performed on large sections of pancreatic tissues using tiling and stitching of images obtained with a Zeiss 880 Airyscan confocal microscope. Lesions were defined as an aggregate of at least 10 KPCY cells. ( E and F ) Representative images of immunostaining of liver sections with CD68 and quantitative analysis of CD68 + cells in liver sections from WT or TRPV4-KO mice. Scale bars: 100 μm. Statistical analyses were performed using Student’s t test. Animal number, n = 4–5. Results were expressed as mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: KPC cells were purchased from Cancertools (catalog 153474).

Techniques: Injection, Immunostaining, Staining, Microscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer

doi: 10.1136/jitc-2021-003549

Figure Lengend Snippet: TERT expression in human and mouse PDAC tissues. (A) Representative H&E staining and TERT IHC analysis in human normal pancreas and PDAC tissues (G2/G3 tumor grade). PDAC tissues were scored as negative (-), positive (+), highly positive (++) according to hTERT antigen. (B) Patients with pancreatic cancer were stratified according to TERT expression. (C) Representative TERT IHC analysis (top panel) and H&E staining (bottom panel) in pancreas specimens isolated from KPC mice at different stages of neoplastic evolution, or in tumors isolated from C57BL/6 mice orthotopically injected with FC1242 cells (D) and sacrificed at different time points (10–30 days) from tumor challenge. Scale bar (A, C, D), 100 µm. (E) TERT activity and expression in normal pancreatic cells and KPC-derived cell lines by TRAP assay (top panel; untreated samples, black; heat-inactivated-negative control samples, white; TPG) and Western blot (bottom panel), respectively. Representative samples are shown. IHC, immunohistochemistry; PDAC, pancreatic ductal adenocarcinoma; TERT, telomerase; TPG, total product generated.

Article Snippet: KPC mice were kindly supplied by Dr D Tuveson (Cold Spring Harbor Laboratory, New York, USA).

Techniques: Expressing, Staining, Isolation, Injection, Activity Assay, Derivative Assay, TRAP Assay, Negative Control, Western Blot, Immunohistochemistry, Generated

Journal: Journal for Immunotherapy of Cancer

Article Title: Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer

doi: 10.1136/jitc-2021-003549

Figure Lengend Snippet: TERT-based ACT restrains PDAC progression. (A, B) C57Bl/6 mice were orthotopically challenged with mouse KPC-derived FC1242-Luc (A) or FC1199-Luc (B) cells. Mice were randomized in two groups which received mTERT-specific (treatment group, n=10) or OVA-specific (control group n=10) mouse CTLs ACT. Therapeutic efficacy was evaluated over time in terms of tumor progression (top panel) and survival (bottom panel). (C) Tracking of adoptively transferred T lymphocytes in PDAC specimens. Congenic immunocompetent CD45.1 mice were orthotopically challenged with mouse KPC-derived FC1242 or FC1199 cells and subjected to CD45.2 + mTERT-specific mouse CTLs ACT or left untreated. Tumor-infiltrating T lymphocytes (CD3 + CD8 + cells) were stained with both CD45.1 (endogeneous lymphocytes) and CD45.2 (transferred lymphocytes) antibodies. (D) KPC mice were enrolled in two groups receiving mTERT-specific (treatment group, n=20) or OVA-specific (control group n=20) mouse CTLs ACT. Therapeutic efficacy was evaluated over time by Kaplan-Meier curves for OS. (E) Immunodeficient NOG mice were orthotopically challenged with human PDAC HLA-A2 + HF2_Luc cell line. Mice were randomized in two groups which received, respectively, hTERT-specific (treatment group) or HCV-specific (control group) engineered human T lymphocytes. Tumor growth was monitored by in vivo imaging (left panel). Therapeutic efficacy was evaluated over time in terms of tumor progression (middle panel) and survival (right panel). Tumor growth was evaluated by in vivo imaging (IVIS-Perkin Elmer: A, B, E; Vevo ultrasound imaging: D): a representative image for each analyzed time point was reported (A, B, E). Data are reported as mean±SE of a representative experiment of two (A, B, E) and three (C) independent replicates. Statistical analysis was performed using one-wayANOVA (A, B, E), Mantel-Haenszel (long-rank) test (A, B, D, E), two-tailed Student’s t -test (C). ACT, adoptive cell therapy; ANOVA, analysis of variance; CTL, cytotoxic T lymphocyte; OS, overall survival; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: KPC mice were kindly supplied by Dr D Tuveson (Cold Spring Harbor Laboratory, New York, USA).

Techniques: Derivative Assay, Control, Drug discovery, Staining, In Vivo Imaging, Imaging, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer

doi: 10.1136/jitc-2021-003549

Figure Lengend Snippet: AT38 renders PDAC responsive to immunotherapy. (A) C57BL/6 mice were orthotopically challenged with FC1242 cells, randomized in four groups receiving, respectively, vehicle (CTRL, n=10), AT38 only (AT38, n=10), mTERT-specific mouse CTLs only (TERT-ACT, n=10), AT38+mTERT-specific mouse CTLs (n=10). Tumor growth was evaluated by in vivo imaging: a representative image for each analyzed time point was reported (left panel). Therapeutic efficacy was evaluated over time in terms of tumor progression (middle panels) and survival (right panel). Data are reported as mean±SD of a representative experiment of two independent replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for tumor growth and Mantel-Haenszel (long-rank) test for survival. (B) KPC mice were enrolled in four groups receiving, respectively, vehicle (CTRL), AT38 only (AT38), mTERT-specific mouse CTLs only (TERT-ACT), AT38+mTERT-specific mouse CTLs (AT38+TERT ACT). Tumor growth was evaluated by high-resolution ultrasound images of pancreatic tumor in KPC mice before (-, left panel) and 10 days after treatment (+, left panel) and shown as waterfall plots (right panel) of therapeutic response. (C) Kaplan-Meier curves for overall survival of KPC mice with invasive disease untreated (n=25) or treated with AT38 (n=20), TERT-ACT (n=15) or combined approach (n=20). Statistical analysis was performed using Mantel-Haenszel (long-rank) test. ACT, adoptive cell therapy; ANOVA, analysis of variance; CTL, cytotoxic T lymphocyte; PDAC, pancreatic ductal adenocarcinoma; TERT, telomerase.

Article Snippet: KPC mice were kindly supplied by Dr D Tuveson (Cold Spring Harbor Laboratory, New York, USA).

Techniques: In Vivo Imaging, Drug discovery, Clinical Proteomics

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: 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