Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 1. Influence of effector cell preparation, choice of effector cell, and E/T ratio on the measure- ment of trastuzumab-mediated ADCC using LDH ADCC assay. a–d. Influence of cryopreservation and the recovery cultivation time (4 h to overnight in RPMI-1640 with 10% FBS) on the recovery rate (a), viability of recovered PBMCs (b), the rate of NK cell isolation from the PBMCs (c), and the viability of isolated NK cells (d). Data were analyzed with GraphPad Prism using one-way ANOVA mixed-effects statistical analysis based on matched rows and recovery time as repeated measure with Tukey’s post hoc test. Overall p values for Figure 1a–d are <0.0001, 0.0126, 0.2553 (no statistical significance among three groups), and 0.0185, respectively; * p < 0.05 and **** p < 0.0001 (e) ADCC activity of trastuzumab with NK cells from immediately thawed PBMCs or overnight recovered PBMCs as effector cells with E/T ratio 20:1 measured after treatment for 5 h, 24 h, or 48 h. (f) ADCC activity of trastuzumab with immediately thawed or overnight recovered PBMCs as effector cells with E/T ratio 20:1 after treatment for 24 h. Data were analyzed with GraphPad Prism using two-way ANOVA with Šídák’s multiple comparisons test. Overall p value < 0.0001 for unrecovered compared to overnight recovered PBMCs. For (e,f), SKBR-3 cells were treated with trastuzumab (0–1 µg/mL) and NK cells, and killing of SKBR-3 was measured using the LDH ADCC assay. NC stands for the no-cell or media-only control, E stands for the effector-cell-only control, and T stands for the target-cell-only control. Data were analyzed with GraphPad Prism using two-way ANOVA, and p values are <0.0001. (g) Comparing Emax and EC50 values of LDH ADCC activity between different

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Cell Isolation, Isolation, Activity Assay, Control

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 2. Influence of the recovery culturing time and method on LDH ADCC assay and cellular expression of granulation proteins. (a) Comparison of ADCC activity of trastuzumab with NK cells isolated from PBMCs recovered for different times 0 h (NR), 4 h, or 24 h. Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001; (b) Comparison of trastuzumab induced ADCC activity using NK cells that underwent overnight recovery after isolation from thawed cryopreserved PBMCs (R-iso for “recovered after isolation”), and NK cells isolated from the overnight recovered PBMCs (R-PB). Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001. SKBR-3 cells were treated with trastuzumab (0–0.01 µg/mL) and NK cells with E/T ratio 5:1 for 24 h, and killing of SKBR-3 was measured with LDH ADCC assay; (c) Representative antiperforin, antigranzyme B, and Anti GAPDH immunoblot in NK cells isolated from unrecovered (NR) PBMCs or from PBMCs after recovery cultivation for different time points (4 h, 24 h) with E/T ratio 5:1. Lanes 1–3, 4–6, and 7–9 show expression levels for NK cells after incubation for 24 h hours with media only, with SKBR-3 only, and with SKBR-3 and trastuzumab, respectively. All lanes were run in the same gel, and images were

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Expressing, Comparison, Activity Assay, Isolation, Western Blot, Incubation

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 3. Influence of the recovery culturing time and method on the extracellular release of gran- ulation proteins and cytokines secretion. (a–d) Comparison of the secretion levels of perforin (a), granzyme B (b), TNFα (c), and IFNγ (d) using ELISA in the 24 h cell culture media of the target cell SKBR-3 (T), the effector cells NK (E), coculture of SKBR-3 and NK (E+T), and trastuzumab-treated coculture of SKBR-3 and NK (Tras) with E/T ratio 5:1. Data in this figure were analyzed with Graph- Pad Prism using one-way ANOVA analysis with Tukey’s post hoc test (**** p < 0.0001). * Fresh NK cells were prepared from a different donor; thus, they were not included in statistical analysis.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 4. The recovery culturing downregulates expression of CD16 and CD56 on NK cells. (a) Represen- tative immunoblot of CD16A (FcγRIIIa) in the whole cell lysates of NK cells isolated from unrecovered

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Expressing, Western Blot, Isolation

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 5. Phenotyping by flow cytometry indicates activation of NK cells by upregulation of CD69 during the recovery cultivation. (a) Flow cytometry analysis of surface CD69 expression in NK cells

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Flow Cytometry, Activation Assay, Expressing

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 6. Choice of target cell lines in the LDH ADCC assay and ADCC reporter assay. (a) Represen- tative immunoblots using lysates from HER2-positive cell lines; (b) Quantitation of the immunoblot; (c) Dose-dependent curves of trastuzumab-mediated LDH ADCC assay using SKBR-3, BT-474, and NCI-N87 cells. Target cells were treated with trastuzumab (0–1 µg/mL) and NK cells with E/T ratio 5:1 for 24 h and killing of SKBR-3 was measured with LDH ADCC assay; (c) Dose-dependent curves of trastuzumab-mediated ADCC activity using ADCC reporter assay in SKBR-3, BT-474 and NCI-N87 cells. Target cells were treated with trastuzumab (0–5 µg/mL) and Promega ADCC reporter cells with E/T ratio 6:1 and luminescence was measured after 6 h.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Reporter Assay, Western Blot, Quantitation Assay, Activity Assay

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 7. Influence of assay buffer, target cell plating method, and treatment time on the measurement of ADCC of trastuzumab. (a) Luminescence fold changes from the LDH ADCC assay using RPMI-1640 with 1% BSA or 1% FBS. Data were analyzed with GraphPad Prism using two-way ANOVA with Šídák’s multiple comparisons test. p value < 0.0001 for 1% BSA vs. 1% FBS; (b) Luminescence fold changes using the LDH ADCC assay performed with either overnight or same-day culture of SKBR-3 target cells plated with 1% BSA or overnight culture of SKBR-3 in 10% FBS. Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001. SKBR-3 cells were treated with trastuzumab (0–1 µg/mL) and NK cells with E/T ratio 5:1, and the killing of SKBR-3 was measured with the LDH ADCC assay, where “E” stands for effector-cell-only control.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Control