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