Computational protocol: Trogocytosis-mediated expression of HER2 on immune cells may be associated with a pathological complete response to trastuzumab-based primary systemic therapy in HER2-overexpressing breast cancer patients

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Protocol publication

[…] Isolated PBMCs, monocytes, and NK cells (effector cells) were co-cultured with different ratios (1:1, 10:1, 100:1, or 1000:1) of human breast cancer cell lines or breast cancer patient tumor cells (target cells) in RPMI 1640 alone or in RPMI 1640 containing different concentrations of normal human plasma. Cells were co-cultured in Eppendorf 500 Tubes® (C153008O, Eppendorf AG, Hamburg, Germany) for 60 min at 37°C, 5% CO2, in the presence of different concentrations of trastuzumab (0, 0.1, and 1 μg/mL; provided by Chugai Pharmaceutical Co., Ltd.). The optimal time and effector:target (E:T) cell ratios were determined in preliminary studies (data not shown). After co-culture, the cells were transferred to fluorescence-activated cell sorter tubes (Corning, Cat. No. 352235), washed with 0.5% BSA-PBS, and centrifuged at 300 × g for 5 min. The supernatant was discarded and the cells were re-suspended in 0.5% BSA-PBS and analyzed by flow cytometry using a FACSCalibur (BD Biosciences). The expression of HER2 (stained with FITC, PE, or APC-conjugated anti-HER2 antibodies; BD Biosciences) was determined on target breast cancer cells and on CD14+ (stained with FITC-conjugated anti-CD14 antibodies; BD Biosciences, Cat. No. 555397), CD56+ (stained with PE-conjugated anti-CD56 antibodies; BD Biosciences, Cat. No. 555516), CD19+ (stained with FITC-conjugated anti-CD19 antibodies; BD Biosciences, Cat. No. 557398), and CD3+ (stained with PE-conjugated anti-CD3 antibodies; BD Biosciences, Cat. No. 555340) cells. Antibodies were diluted 1:20 with Flow Cytometry Staining Buffer (BD Biosciences, Cat. No. 00-4222-26) prior to staining. Positive cell populations were gated with reference to negative isotype control matched antibody staining reactions (Mouse IgG1 PE and APC, Cat. No. 559320 and 554681, respectively; Mouse IgG2a FITC, Cat. No. 553456; BD Biosciences) and baseline HER2 expression on CD14+, CD56+, CD19+, and CD3+ cells from normal healthy volunteer PBMCs (without cancer cell co-culture). Data were stored electronically for reanalysis (FlowJo Version 7.6.5 software; TreeStar). [...] Statistical analyses, including the Student’s t test, Wilcoxon signed-rank test, and Chi-square test were performed using JMP Pro 11. […]

Pipeline specifications

Software tools FlowJo, JMP Pro
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms