Type

Journal Article

Authors

John Crown
Norma O'Donovan
Kenneth J O'Byrne
Stephen F Madden
Anthony Davies
Connla Edwards
Clare Hughes
Kathy Gately
Denis M Collins

Subjects

Biochemistry

Topics
breast cancer antibody dependent cell cytotoxicity tyrosine kinase inhibitor flow cytometry cancer cell lines high content analysis cell line tyrosine kinase

Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines. (2017)

Abstract Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high cytotoxic capacity, TKIs have the ability to augment the passive immunotherapeutic potential of trastuzumab in SKBR3, a model of HER2+ breast cancer. ADCC levels detected by LDH release assays are extremely low in MCF-7; the flow cytometry-based CFSE/7-AAD method is more sensitive and consistent for the determination of ADCC in HER2-low models.
Collections Ireland -> Dublin City University -> PubMed

Full list of authors on original publication

John Crown, Norma O'Donovan, Kenneth J O'Byrne, Stephen F Madden, Anthony Davies, Connla Edwards, Clare Hughes, Kathy Gately, Denis M Collins

Experts in our system

1
John Crown
Dublin City University
Total Publications: 104
 
2
Norma O'Donovan
Dublin City University
Total Publications: 59
 
3
Kenneth J O'Byrne
Trinity College Dublin
Total Publications: 37
 
4
Stephen F Madden
Dublin City University
Total Publications: 45
 
5
Anthony Davies
Trinity College Dublin
Total Publications: 28
 
6
Connla Edwards
Dublin City University
Total Publications: 3
 
7
Kathy Gately
Trinity College Dublin
Total Publications: 22
 
8
Denis M Collins
Dublin City University
Total Publications: 26