Journal: eLife

Article Title: Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins

doi: 10.7554/eLife.86920

Figure Lengend Snippet:

Article Snippet: Cell line, human ( H. sapiens ) , Panc , American Type Culture Collection (ATCC) , ATCC CRL-2553 RRID: CVCL_0480 , Shh-expressing cancer cell line (pancreatic duct).

Techniques: Variant Assay, Expressing, Knock-Out, Control, Transfection, Construct, Plasmid Preparation, Recombinant, Functional Assay, Software, Western Blot, Derivative Assay, Binding Assay, Immunoprecipitation

Journal: Research

Article Title: Novel Combination of Irreversible Electroporation and Allogenic Chimeric Antigen Receptor T-Cell Therapy Synergizes Therapeutic Outcomes in a Preclinical Human Pancreatic Cancer Mouse Model

doi: 10.34133/research.1105

Figure Lengend Snippet: (A) Calculated irreversible electroporation (IRE) lethal electric field thresholds for Pan02 mouse pancreatic cancer using a 3-dimensional (3D) collagen hydrogel; mean ± SD; n = 5. (B) COMSOL Multiphysics finite element model (FEM) representing subcutaneous mouse tumors with a 5-mm spacing shown. (C) Simulated percent tumor coverage by the lethal electric field using the subcutaneous FEM with randomized tissue conductivities and tumor sizes (4 to 8 mm); mean ± SD; n = 30. (D) In Vivo Imaging System (IVIS) imaging of FLuc-eGFP + Pan02 cells in immunodeficient NOD/SCID/IL2gc-KO (NSG) mice (m) follows complete ablation and subsequent microtumor recurrence after IRE treatment at 2,500 V/cm; n = 6. (E) Tumor scabbing and flattening at day 3 post-treatment. (F) Total photon flux within the tumor region of interest from the IVIS images; mean and range presented; multiple 2-tailed t tests between the data compared to the initial day 0 total photon flux; n = 6. (G) Measured tumor size over time from tumor inoculation; mean and range presented; multiple 2-tailed t tests between the data compared to pre-treatment tumor size on day 30; n = 6. ** P < 0.01; **** P < 0.0001. FLuc-eGFP, firefly-luciferase-enhanced green fluorescent protein. SAT, subcutaneous fat.

Article Snippet: Pan02 mouse pancreatic cancer cells (Cytion, 300501), AsPC-1 human pancreatic cancer cells (American Type Culture Collection [ATCC], CRL-1682), and Jurkat immortalized human T lymphocytes (ATCC, TIB-152) were cultured in RPMI 1640 medium (Thermo Fisher, 11875093) supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific, FB12999102) and 1% (v/v) 10,000 U/ml penicillin–streptomycin (Gibco, 16140122).

Techniques: Electroporation, In Vivo Imaging, Imaging, Luciferase

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: NAM+GEM significantly improved clinical parameters, including survival, in mouse PDAC. Orthotopic Panc-02 model. Tumors and metastases were generated and treatments with NAM+GEM and controls were performed as outlined in . At the conclusion of the experiment tumor weight (A) and number of metastases (B) was measured, as well as the grade of ascites (T0–5) (C). Average of two experiments with n=10 mice per group. Orthotopic KPC model. Tumors and metastases were generated and treatments with NAM+GEM and controls were performed as outlined in . At the conclusion of the experiment tumor weight (D) and number of metastases (E) was measured, as well as the grade of ascites (T0–5) (F). Average of two experiments with n=10 mice per group. Significant differences were determined by Mann-Whitney U test. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars represent SE of the mean (SEM). Survival study orthotopic Panc-02 model. Tumors and metastases were generated and treatments with NAM+GEM and controls were performed as outlined in . At the end of treatments mice were monitored until death, and survival was assessed (G) when defined clinical endpoints were reached. The results of 2 experiments were averaged with n=10 mice per group. Significant differences were determined by the Mantel-Cox test. GEM, gemcitabine; NAM, nicotinamide; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Generated, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: CD4 T cells significantly reduced pancreatic cancer by NAM+GEM in immune competent mice, while little effect of NAM+GEM was observed in nude mice. Orthotopic Panc-02 tumors were generated in immune competent C57BL/6 mice and depleted for CD4 and CD8 T cells during NAM+GEM treatment as outlined in . Antibodies to CD4 T cells (300 µg/200 µL) or to CD8 T cells (300 µg/200 µL) or isotype control were administered IP every 3rd day for 2 weeks. At the conclusion of the experiment the average tumor weight (A) and number of metastases (B) was determined in the NAM+GEM-treated/T cell-depleted compared with NAM+GEM-alone treated mice, with n=5 mice per group. Mann-Whitney U test. *P<0.05, **p<0.01 is significant. depletion of CD4 and CD8 T cells was confirmed by flow cytometry in the spleen (C). Orthotopic Panc-02 tumors were generated in nude mice (Fox n1nu/J) and treated with NAM+GEM as outlined in . At the conclusion of the experiment the average tumor weight (D) and number of metastases (E) was determined in the NAM+GEM-treated compared with the saline mice with n=5 mice per group. Mann-Whitney U test. GEM, gemcitabine; NAM, nicotinamide; ns, non-significant.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Generated, MANN-WHITNEY, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: NAM+GEM decreased the production of collagen I and HABP, and increased αSMA and CD31, in pancreatic tumors of orthopic Panc-02 model. Tumors were analyzed for collagen I by trichrome staining (A) and quantified (B) and shown in more detail (C). n=5 mice per group and the results of 5 fields were averaged. HABP fibrils was analyzed by RT-PCR (D). αSMA protein was by analyzed by IHC (E), and quantified (F), and shown in more detail (G). For both trichrome and αSMA the percentage of positive areas were determined and the results of n=5 mice per group was averaged. The presence of blood vessels in the pancreatic tumors was analyzed by IHC using anti-CD31 antibodies (H) and quantified (I) and shown in more detail (J). n=5 mice per group and the results of 10 fields were averaged. Mann-Whitney U test. *P<0.05, **p<0.01 is significant. Error bars represent SEM. αSMA, α-smooth muscle actin; GEM, gemcitabine; HABP, hyaluronic acid binding protein; IHC, immunohistochemistry; SEM, SE of the mean.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Binding Assay, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: Peritumoral and intratumoral LNS in pancreatic tumors of orthotopic Panc-02 model. Detail of a peritumoral LNS (A). Peritumoral LNS, characterized by well-developed follicular structures with occasional germinal centers. CD4 + and CD8 + T cells are concentrated in the deep cortical zone/paracortex, where they were arranged in dense sheets, and are scattered within follicles and surrounding tissue. The peritumoral LNS is surrounded by a fibroblast layer (red arrows). For more detail about peritumoral and intratumoral LNS (see ). Immunostaining for CD31 highlights the presence of vessels within peritumoral and intratumoral LNS in pancreatic tumors (black arrows) (B), which may allow the T cells to migrate to the tumor areas. LNS, lymph node-like structures.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Immunostaining

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: NAM+GEM increased the expression of genes involved in T cell migration and activation, and reduced the expression of genes involved in invasion of tumor cells. Orthotopic Panc-02 tumors of the different treatment groups were analyzed by Nanostring through gene expression profiles involved in T cell migration, activation, epitope spreading and invasion of tumor cells. A heatmap of relevant genes is shown (A), and the function of each gene and p values (B). Gene expression levels in the NAM+GEM group were compared with the saline group and analyzed by ANOVA p<0.05 is significant. To confirm the Nanostring data, sections of tumors in the different treatment groups were analyzed for the expression of Ccl21a and Fcer by RNAscope (C D), and quantified (E F). Whole tissues were quantified using HALO Image Analysis; the number of cells with red dot(s) was determined. Images were acquired using ×40 magnification. Data are presented as SE of the mean (SEM); statistical analysis was performed using a one-way ANOVA. *p<0.05 is significant. ANOVA, analysis of variance; GEM, gemcitabine; NAM, nicotinamide.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Expressing, Migration, Activation Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Anti-Bv8 Antibody and Metronomic Gemcitabine Improve Pancreatic Adenocarcinoma Treatment Outcome Following Weekly Gemcitabine Therapy 1 2

doi: 10.1016/j.neo.2014.05.011

Figure Lengend Snippet: The combination of weekly and daily gemcitabine therapy improves therapeutic outcome in mice bearing pancreatic tumors. Eight-week-old C57Bl/6 mice were implanted orthotopically with 5 × 10 6 Panc-02 cells stably transfected with luciferase. After 7 days, they were either left untreated (Control) or treated with gemcitabine in the following regimens: maximum tolerated dose administered weekly (MTD-G), metronomic dose administered daily (MC-G), or the combination of the two regimens (COMB-G) as described in Figure W1. (A) Tumor growth was evaluated over time using IVIS following injection with luciferin as previously described . (B) Tumor growth was quantified by counting the number of photons collected at 8 minutes after luciferin injection when compared to their pre-treatment values. *, P < .05 when compared to pre-treated control. (C) Survival of mice from all groups was plotted using the Kaplan-Meier curve. (D) In a parallel experiment, 28 days following the different gemcitabine regimens, mice were imaged by MRI Spectra 1T. Non–tumor-bearing mice (NBT) or mice bearing tumors on day 5 pre-treatment (Tumor 5d) were also imaged for comparison. Representative images are shown.

Article Snippet: Cells (5 × 10 6 ) stably transfected with luciferase were mixed in Matrigel (BD Biosciences, San Jose, CA) and were injected transperitoneally into the head of the pancreas of 6-week-old female Severe Combined Immunodeficiency (SCID) (for Panc-1) or C57Bl/6 (for Panc-02) mice (Harlan, Jerusalem, Israel) after incision of the skin and fixation of the pancreas.

Techniques: Stable Transfection, Transfection, Luciferase, Control, Injection, Comparison

Journal: Neoplasia (New York, N.Y.)

Article Title: Anti-Bv8 Antibody and Metronomic Gemcitabine Improve Pancreatic Adenocarcinoma Treatment Outcome Following Weekly Gemcitabine Therapy 1 2

doi: 10.1016/j.neo.2014.05.011

Figure Lengend Snippet: Weekly gemcitabine induces host pro-tumorigenic effects that promote tumor cell invasion and increase the number of MDSCs. Non–tumor-bearing C57Bl/6 mice were either untreated (Control) or treated with gemcitabine administered weekly at the maximum tolerated dose (MTD-G), daily at a metronomic dose (MC-G), or in a combination of the two regimens (COMB-G). (A) Plasma was drawn after 7 days of therapy and used in a Boyden chamber assay for the assessment of Panc-1 and Panc-02 tumor cell invasion as described in the Materials and Methods section. Cells migrating to the bottom of the filter were counted and plotted. (B) In a parallel experiment, blood from mice treated with the different gemcitabine regimens was sampled on a daily basis for 10 sequential days. The number of MDSCs in the peripheral blood was evaluated by flow cytometry. (C) In a separate experiment, mice bearing orthotopic pancreatic tumors were treated with the different gemcitabine treatment regimens as indicated above. After 21 days, blood was drawn by cardiac puncture, bone marrow cells were flushed from the bone marrow, and tumors were removed. The percentage of MDSCs in the blood, bone marrow, and tumor samples was assessed by flow cytometry. The MDSC count in non–tumor-bearing mice is shown in the inset. *, P < .05; **, .01 > P > .001; ***, P < .001.

Article Snippet: Cells (5 × 10 6 ) stably transfected with luciferase were mixed in Matrigel (BD Biosciences, San Jose, CA) and were injected transperitoneally into the head of the pancreas of 6-week-old female Severe Combined Immunodeficiency (SCID) (for Panc-1) or C57Bl/6 (for Panc-02) mice (Harlan, Jerusalem, Israel) after incision of the skin and fixation of the pancreas.

Techniques: Control, Clinical Proteomics, Boyden Chamber Assay, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: Anti-Bv8 Antibody and Metronomic Gemcitabine Improve Pancreatic Adenocarcinoma Treatment Outcome Following Weekly Gemcitabine Therapy 1 2

doi: 10.1016/j.neo.2014.05.011

Figure Lengend Snippet: Weekly gemcitabine therapy increases Bv8 expression and tumor angiogenesis. (A) Non–tumor-bearing C57Bl/6 mice were either untreated (Control) or treated with gemcitabine administered weekly at the maximum tolerated dose (MTD-G), daily at a metronomic dose (MC-G), or in a combination of the two regimens (COMB-G). Seven days after treatment initiation, mice were sacrificed. The level of Bv8 mRNA was assessed by reverse transcription–polymerase chain reaction in the peripheral blood (PBL), bone marrow cells (BM), and MDSCs (Gr1+CD11b +) 7 days after treatment initiation. (B) Pancreatic tumor (Panc-02) bearing mice were treated with the different gemcitabine regimens as indicated above. After 21 days of treatment, blood was drawn by cardiac puncture, mice were sacrificed, and tumors were removed. The relative expression of Bv8 mRNA was assessed by reverse transcription–polymerase chain reaction on sorted MDSCs from both peripheral blood and tumor cells that were prepared as single cell suspensions. (C) Left panel: Tumor sections from all treatment groups were stained for CD31 representing endothelial cells (in red), hypoxia using hypoxic probe (in green), and Hoechst for assessment of perfusion (in blue). Right panel: To visualize MDSCs within the tumor, the sections were immunostained with anti-Gr1 (red) and anti-CD11b (green) antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue); scale bar, 200 μm. Microvessel density, the ratio of hypoxia over perfusion, and cells expressing both Gr1 and CD11b were quantified from the stained tumor sections from all groups ( n > 20 fields per group) and subsequently plotted. *, P < .05; **, .01 > P > .001; ***, P < .001.

Article Snippet: Cells (5 × 10 6 ) stably transfected with luciferase were mixed in Matrigel (BD Biosciences, San Jose, CA) and were injected transperitoneally into the head of the pancreas of 6-week-old female Severe Combined Immunodeficiency (SCID) (for Panc-1) or C57Bl/6 (for Panc-02) mice (Harlan, Jerusalem, Israel) after incision of the skin and fixation of the pancreas.

Techniques: Expressing, Control, Reverse Transcription, Polymerase Chain Reaction, Single Cell, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Anti-Bv8 Antibody and Metronomic Gemcitabine Improve Pancreatic Adenocarcinoma Treatment Outcome Following Weekly Gemcitabine Therapy 1 2

doi: 10.1016/j.neo.2014.05.011

Figure Lengend Snippet: Anti-Bv8 antibodies block MDSC mobilization and tumor homing. Eight-week-old C57Bl/6 mice were orthotopically implanted with Panc-02 tumor cells (5 × 10 6 cells/100 μl). After 7 days, treatment with weekly gemcitabine (MTD-G) with or without anti-Bv8 antibody (ABv8) administered twice weekly for 2 weeks was initiated. (A) Tumor growth was assessed by IVIS. Representative images are shown in the upper panel. Tumor size was quantified by detecting photon intensity (lower panel). (B) In a different experiment, non–tumor-bearing mice were treated with the drug combinations as indicated above, and blood was sampled daily (through retro-orbital sinus bleed) for the evaluation of MDSC number in peripheral blood by flow cytometry. (C) Tumor-bearing mice from (A) were treated as indicated above, and 14 days after treatment was initiated, mice were sacrificed and tumors were removed. Half of each tumor was prepared as a single cell suspension for the assessment of the percentage of MDSCs in the tumors by flow cytometry. (D) The other half of the tumor was sectioned and stained with anti-CD31 antibodies (red) to detect endothelial cells, hypoxic probe (green) to detect hypoxia, and Hoechst (blue) to assess perfusion (upper micrographs). To visualize MDSCs within the tumor, the sections were immunostained with anti-Gr1 (red) and anti-CD11b (green) antibodies. Nuclei were stained with DAPI (blue; lower micrographs); scale bar, 200 μm. Microvessel density, the ratio of hypoxia over perfusion in tumors, and cells expressing both Gr1 and CD11b were quantified in tumor sections from all groups ( n > 20 fields per group) and subsequently plotted. *, P < .05; **, .01 > P > .001; ***, P < .001.

Article Snippet: Cells (5 × 10 6 ) stably transfected with luciferase were mixed in Matrigel (BD Biosciences, San Jose, CA) and were injected transperitoneally into the head of the pancreas of 6-week-old female Severe Combined Immunodeficiency (SCID) (for Panc-1) or C57Bl/6 (for Panc-02) mice (Harlan, Jerusalem, Israel) after incision of the skin and fixation of the pancreas.

Techniques: Blocking Assay, Flow Cytometry, Single Cell, Suspension, Staining, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Anti-Bv8 Antibody and Metronomic Gemcitabine Improve Pancreatic Adenocarcinoma Treatment Outcome Following Weekly Gemcitabine Therapy 1 2

doi: 10.1016/j.neo.2014.05.011

Figure Lengend Snippet: Anti-Bv8 antibodies improve therapy outcome. (A) Eight-week-old non–tumor-bearing C57Bl/6 mice were treated with gemcitabine (500 mg/kg, MTD-G) with or without anti-Bv8 antibody (5 mg/kg, A-Bv8). After 24 hours, blood was drawn by cardiac puncture and plasma was separated and used in a Boyden chamber assay for the assessment of Panc-1 and Panc-02 tumor cell invasion as described in the Materials and Methods section. Cells migrating to the bottom of the filter were counted and plotted. (B) Eight-week-old C57Bl/6 mice were orthotopically implanted with Panc-02 tumor cells (5 × 10 6 cells/100 μl). After 7 days, treatment with weekly gemcitabine (MTD-G) with or without anti-Bv8 antibody (ABv8) administered twice weekly for 2 weeks was initiated. Mouse survival was assessed. A Kaplan-Meier curve is plotted. *, P < .05; **, .01 > P > .001.

Article Snippet: Cells (5 × 10 6 ) stably transfected with luciferase were mixed in Matrigel (BD Biosciences, San Jose, CA) and were injected transperitoneally into the head of the pancreas of 6-week-old female Severe Combined Immunodeficiency (SCID) (for Panc-1) or C57Bl/6 (for Panc-02) mice (Harlan, Jerusalem, Israel) after incision of the skin and fixation of the pancreas.

Techniques: Clinical Proteomics, Boyden Chamber Assay