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