Journal: Mediators of Inflammation

Article Title: The Vitamin D3 Analog Calcipotriol Attenuates Pancreatic Cancer Malignancy via Downregulating Thrombospondin 1 in Pancreatic Stellate Cells

doi: 10.1155/mi/2632235

Figure Lengend Snippet: Effects of Cal on aPSC activation. (A) (Left) VDR mRNA expression in PDAC cell lines (AsPC‐1, MIA PaCa‐2, and PANC‐1) and aPSCs was determined by qRT‐PCR ( n = 3). (Right) CYP24A1 mRNA expression in PDAC or aPSCs treated with DMSO or Cal (100 nM and 48 h) was examined by qRT‐PCR ( n = 3). (B) VDR protein expression in PDAC cell lines (AsPC‐1, MIA PaCa‐2, and PANC‐1) and aPSCs was determined by western blot ( n = 3). (C) Correlation analysis between α‐SMA and VDR mRNA expression in aPSCs, with GAPDH normalization ( n = 9). (D) VDR and α‐SMA gene expression in aPSCs treated with DMSO or Cal (100 nM and 48 h) was evaluated by qRT‐PCR ( n = 3). (E) VDR and α‐SMA protein expression in aPSCs treated with DMSO or Cal (100 nM and 48 h) ( n = 4). (F) Immunocytochemistry showing α‐SMA expression in aPSCs treated with DMSO or Cal (100 nM and 48 hr) ( n = 3). (G) EZ4U assay indicating the impacts of Cal on the proliferation of aPSCs ( n = 3). (H) Transwell migration assay and (I) wound healing showing the effects of Cal on aPSCs’ migration ability ( n = 3). caPSCs, PSCs derived from pancreatic cancer; cpPSCs, PSCs derived from chronic pancreatitis; cuPSCs, culture‐activated PSCs derived from normal tissue; aPSCs, activated PSCs; HPF, high‐power field; Ctr, control group treated with DMSO. All experiments were conducted in triplicate. ns, not significant. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Human PDAC cell lines PANC‐1 (male, American Type Culture Collection [ATCC] CRL‐1469, RRID: CVCL_0480), MIA PaCa‐2 (male, ATCC CRL‐1420, RRID: CVCL_0428), and AsPC‐1 (female, ATCC CRL‐1682, RRID: CVCL_0152) were purchased directly from the ATCC (Manassas, VA, USA) in 2015.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Gene Expression, Immunocytochemistry, Transwell Migration Assay, Migration, Derivative Assay, Control

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: Chimeric antigen receptor (CAR) target binding analysis following irreversible electroporation. (A) Viable and intact cells following electroporation are still available for cell membrane mesothelin (MSLN) binding, while necrotic cells experience a decrease in binding. (B) Flow cytometry gating to isolate single cells and plots of calcein AM versus mesothelin at 3 h following IRE delivery using 0 V/cm (control), 1,000 V/cm, and 2,000 V/cm. (C) Control using mesothelin-negative Jurkats. (D) Mesothelin expression in AsPC-1 cells compared to that in Jurkats. (E) Cell viability 3 h after electroporation at different electric field strengths; one-way analysis of variance (ANOVA) with Tukey’s posttest and correction; mean ± SD; n = 4. (F) Percent mesothelin (Mes) expression of high-viability and low-viability cell populations 3 h after electroporation; multiple 2-tailed t test; mean ± SD; n = 4. (G) Live (green) and dead (red) imaging at 3 h and 7 d after treatment; the scale bar is 1 mm. (H) Viable cell count at different electric fields after IRE and following recovery; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. (I) Mesothelin binding for recovered cells at day 7; one-way ANOVA with Tukey’s posttest and correction within each timepoint; mean ± SD; n = 4. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IL-2, interleukin-2; IL-15, interleukin-15; IFNγ, interferon-γ; FSC-H, forward scatter height; FSC-A, forward scatter area.

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: Binding Assay, Electroporation, Membrane, Flow Cytometry, Control, Expressing, Imaging, Cell Characterization

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: In vitro assay for longitudinal combinatorial treatment evaluation. (A) In vitro multicellular tumor spheroid (MCTS) assay to assess the treatment response to electroporation and CAR T-cell therapy. (1) MCTSs were formed within a low-adherent U-bottom 96-well plate and (2) then moved to a 4-well rectangular plate with low-conductivity buffer to (3) deliver electroporation via parallel-plate electrodes. (4) The MCTSs were immediately moved back into the original U-bottom well, where (5) adjuvant CAR T-cell therapy or sham was delivered. (B) Live (green) and dead (red) imaging of AsPC-1 MCTSs at 3 and 72 h after treatment at different electric field magnitudes; the scale bar is 1 mm. Normalized absorbance for the XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay at (C) 3 and (D) 72 h post-treatment at different electric fields; one-way ANOVA with Tukey’s post hoc and correction; mean ± SD; n = 3. (E) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs over time and across different electric field intensities. (F) Normalized green fluorescent protein (GFP) intensity and (G) MCTS area over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); multiple one-sample Wilcoxon signed-ranked tests between that timepoint and the initial zero timepoint ( # P < 0.05); n = 3.

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: In Vitro, Electroporation, Adjuvant, Imaging

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: In vitro evaluation of anti-tumor efficacy and infiltration following IRE and CAR T-cell therapy. (A) Green fluorescent intensity of FLuc-eGFP + AsPC-1 MCTSs, (B) deep-red intensity of CellTracker-stained CAR T cells, and (C) merged images over time for the CAR-T-cell-only and combinatorial treatments (both). (D) Measured MCTS area and (E) normalized eGFP intensity over time; one-way ANOVAs with Tukey’s post hoc between groups on the last timepoints (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001); n ≥ 3. (F) Comparison of deep-red intensity within the tumor spheroid over time; 2-tailed t tests between groups at each timepoint ( # P < 0.05; ## P < 0.01); n ≥ 3.

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: In Vitro, Staining, Comparison

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: Subcutaneous mouse model of human pancreatic cancer for comparing tumor response and survival following combinatorial IRE and CAR T-cell treatment. (A) Schematic of the treatment timeline: NSG mice were inoculated with MSLN + AsPC-1 cells on day 0. Mice received IRE followed by peritumoral CAR T-cell injection on day 25 and were tracked for 35 d until day 60. (B) Pre- and post-treatment IVIS imaging verifies successful peritumoral FLuc + αMLSN CAR T-cell injection. (C) Relative rodent weight over time post-treatment; mean ± standard error of the mean (SEM); n = 24. (D) Representative images of rodents over the post-treatment tracking period (blanks for mice sacrificed). (E) Tumor measurements from inoculation for all mice. (F) The average measured tumor volume from inoculation; mean ± SEM; n = 6 (the sample size drops as mice reach the tumor size endpoint). (G) Progression-free survival and (H) overall survival for each group; Kaplan–Meier with Bonferroni multiple comparisons; n = 6 (* P < 0.05; ** P < 0.01).

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: Injection, Imaging

Journal: Cancers

Article Title: Externally Applied Electromagnetic Fields and Hyperthermia Irreversibly Damage Cancer Cells

doi: 10.3390/cancers15133413

Figure Lengend Snippet: Effect of EMFs and HT on cancer cell viability. ( A ) Effect of EMFs. Cancer cells were seeded and 24 h later exposed to EMFs (100–500 kHz × 1–5 h). Control values (0 kHz) were 1.14 ± 0.03 A2058, 1.13 ± 0.04 AsPC1, and 1.20 ± 0.05 MDA-MB-231 (×10 6 ) viable cells ( n = 5 in all cases). * p < 0.05 comparing all conditions versus controls (0 kHz) ( n = 5 t -test). ( B ) Effect of HT. Cancer cells were seeded and 24 h later exposed to HT (42–52 °C × 20–40 min). * p < 0.05 ** p < 0.01 comparing all conditions versus controls (37 °C) ++ p < 0.01 comparing 40 min versus 20 min ( n = 5 t test). ( C ) Effect of EMFS and HT. Cancer cells were seeded and 24 h later exposed to EMFs (100 kHz × 4 h) and HT (52 °C × 40 min from min 120 to min 160 of the 4 h period where cells were constantly exposed to the EMFs). The surviving cells were cultured for 24 additional hours without further exposure to EMFs and HT. A two-way analysis of variance (ANOVA) was used to make comparisons among the different groups after 4 h of treatment with EMFs + HT and 24 h after. Different letters indicate differences p < 0.05 ( n = 5).

Article Snippet: Human AsPC1/Luciferase Stable Cells were obtained from GenTarget Inc. (San Diego, CA, USA).

Techniques: Control, Cell Culture

Journal: Cancers

Article Title: Externally Applied Electromagnetic Fields and Hyperthermia Irreversibly Damage Cancer Cells

doi: 10.3390/cancers15133413

Figure Lengend Snippet: Effect of EMFs and HT on ROS generation and the molecular mechanisms of apoptosis.

Article Snippet: Human AsPC1/Luciferase Stable Cells were obtained from GenTarget Inc. (San Diego, CA, USA).

Techniques:

Journal: Cancers

Article Title: Externally Applied Electromagnetic Fields and Hyperthermia Irreversibly Damage Cancer Cells

doi: 10.3390/cancers15133413

Figure Lengend Snippet: Effect of EMFs + HIFU-induced HT, gemcitabine and/or PT on the growth of AsPC1 pancreas carcinoma. Cancer cells were inoculated subcutaneously on day 0, and mice were treated with EMFs and HIFU as described under Materials and Methods. ( A ) EMFs and HIFU were applied once per day per three consecutive days (Monday to Wednesday) for two consecutive weeks starting on day 14 after tumor inoculation. Gemcitabine (50 mg/kg) was administered twice on days 14 and 21. A one-way analysis of variance (ANOVA) was used to make comparisons among the different experimental groups at each time point. Different letters indicate statistical differences p < 0.05 ( n = 15 mice per experimental group). ( B ) A disodium salt of PT phosphate (Chromadex Inc. Los Angeles CA) (100 mg of PT/kg) was administered i.p. (one dose 30 min before starting each irradiation session with EMFs and HT). A one-way analysis of variance (ANOVA) was used to make comparisons among the different experimental groups. Different letters indicate statistical differences p < 0.05 ( n = 12 mice per experimental group). ( C ) Representative images of mice inoculated with AsPC1/Luciferase Stable Cells and treated with EMFs HT and gemcitabine (GEM) or EMFs HT gemcitabine and PT.

Article Snippet: Human AsPC1/Luciferase Stable Cells were obtained from GenTarget Inc. (San Diego, CA, USA).

Techniques: Irradiation, Luciferase

Journal: International Journal of Surgery (London, England)

Article Title: Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study

doi: 10.1097/JS9.0000000000001226

Figure Lengend Snippet: Antitumor activity of BiTP combinatorial chemotherapy in orthotopic PDAC models. (A) Treatment plan and schedules. (B) Schematic diagram of the KPC model. The approximate time frame of mouse tumor progression to borderline resectable (BR), locally advanced (LA), and terminal (TERM) are days 7, 14, and 21, respectively. (C) Representative tumor images of orthotopic PDAC mice receiving different therapies. (D) Tumor weights and volumes on day 30. (E) The table included the survival rate (%), tumor proliferation (T/C, %), tumor growth inhibition (TGI, %), and resectability status of orthotopic PDAC mice receiving different therapies on day 30, and the median survival time (MST) in survival assays. (F) Kaplan–Meier plot survival curve and pairwise comparison results of orthotopic PDAC mice receiving different therapies in survival assays. Data presented in the graphs represent mean±standard deviation (SD) (**** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns P >0.05). BiTP, anti-PD-L1/TGF-β antibody; CTH, chemotherapy; PDAC, pancreatic ductal adenocarcinoma; PD-L1, programmed cell death 1 ligand 1.

Article Snippet: Human PDAC cell lines ASPC-1 (with Kras G12D mutation) and CFPAC-1 (with Kras G12V mutation) and human pancreatic ductal epithelial cell line HPDE6-C7 were obtained from China Center for the Conservation of Typical Cultures (CCTCC).

Techniques: Activity Assay, Inhibition, Comparison, Standard Deviation

Journal: International Journal of Surgery (London, England)

Article Title: Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study

doi: 10.1097/JS9.0000000000001226

Figure Lengend Snippet: Decisions about resectability status in PDAC murine models.

Article Snippet: Human PDAC cell lines ASPC-1 (with Kras G12D mutation) and CFPAC-1 (with Kras G12V mutation) and human pancreatic ductal epithelial cell line HPDE6-C7 were obtained from China Center for the Conservation of Typical Cultures (CCTCC).

Techniques: Membrane

Journal: International Journal of Surgery (London, England)

Article Title: Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study

doi: 10.1097/JS9.0000000000001226

Figure Lengend Snippet: Preclinical assessment of neoadjuvant BiTP combinatorial chemotherapy for the treatment of PDAC. (A) Treatment plan and schematic diagram of treatment schedules. (B) Schematic diagram of distal pancreatectomy in orthotopic PDAC mice model. (C) Representative images of tumors. (D) Tumor weights and volumes of direct surgery (day 7) and post-neoadjuvant therapy resections (day 24). (E) Table of resectability status (day 24), operative mortality (OM), and median survival time (MST) of orthotopic PDAC mice receiving different therapies. (F–H) Kaplan–Meier plot survival curve and pairwise comparison results of orthotopic PDAC mice receiving different therapies in survival assays. Data presented in the graphs represent mean±standard deviation (SD). (**** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns P >0.05). AC, adjuvant chemotherapy; ACI, adjuvant chemo-immunotherapy; BiTP, anti-PD-L1/TGF-β antibody; CTH, chemotherapy; NAC, neo-adjuvant chemotherapy; NACI, neo-adjuvant chemo-immunotherapy; NAT, neo-adjuvant therapy; SRG, surgery.

Article Snippet: Human PDAC cell lines ASPC-1 (with Kras G12D mutation) and CFPAC-1 (with Kras G12V mutation) and human pancreatic ductal epithelial cell line HPDE6-C7 were obtained from China Center for the Conservation of Typical Cultures (CCTCC).

Techniques: Comparison, Standard Deviation, Adjuvant

Journal: International Journal of Surgery (London, England)

Article Title: Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study

doi: 10.1097/JS9.0000000000001226

Figure Lengend Snippet: BiTP reverses TGF-β-induced EMT (epithelial–mesenchymal transition) of PDAC (pancreatic ductal adenocarcinoma) cells. (A) CCK-8 (Cell Counting Kit-8) assay to measure the effect of BiTP on TGF-β-mediated chemoresistance in PDAC cells. After 10 ng/ml TGF-β1, 10 5 pM BiTP or hIgG treatment for 24 h. HPDE6-C7, CFPAC-1, ASPC-1, and KPC cells growing in 96-well plates were exposed to serial dilutions of gemcitabine and nab-paclitaxel for 72 h and CCK-8 assays was performed. (B) The representative images of IHC (immunohistochemistry) staining of Ki67 in orthotopic PDAC mice model and statistical graph of the percentage of Ki67-positive cells. (C) The representative images of TUNEL staining for apoptosis (green) and nuclei (DAPI, blue) in orthotopic PDAC mice model and statistical graph of the percentage of apoptosis cells. (D) Transwell assays to measure the effect of BiTP on TGF-β-mediated migration and in PDAC cells. After 10 ng/ml TGF-β1, 10 5 pM BiTP or hIgG treatment for 96 h. 1×10 4 CFPAC-1 and KPC cells were seeded in the upper chambers and Transwell assays were performed. (E) Western blotting assays exploring the chemotherapy-induced EMT and the blocking effect of BiTP. (F) The representative images of IHC staining of E-cadherin in orthotopic PDAC mice model. Statistical graph of the AOD (average optical density) of E-cadherin. (G) The representative images of IF staining of Vimentin (green) and α-SMA (rose red) in orthotopic PDAC mice model. Statistical graph of the arbitrary units of Vimentin and α-SMA. (H) The representative images of Masson staining in orthotopic PDAC mice model. Statistical graph of the percentage of collagen volume fraction (%). Data presented in the graphs represent mean±standard deviation (SD). (**** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns P >0.05). BiTP, anti-PD-L1/TGF-β antibody; CTH, chemotherapy; KPC, LSL-Kras(+/G12D);LSL-Trp53(+/R172H);Pdx1-Cre; PDAC, pancreatic ductal adenocarcinoma; PD-L1, programmed cell death 1 ligand 1; TGF-β, transforming growth factor-β.

Article Snippet: Human PDAC cell lines ASPC-1 (with Kras G12D mutation) and CFPAC-1 (with Kras G12V mutation) and human pancreatic ductal epithelial cell line HPDE6-C7 were obtained from China Center for the Conservation of Typical Cultures (CCTCC).

Techniques: CCK-8 Assay, Cell Counting, Immunohistochemistry, Staining, TUNEL Assay, Migration, Western Blot, Blocking Assay, Standard Deviation