Flow Cytometry Analysis to Detect Lapatinib-Induced Modulation of Constitutive and IFN-γ-Induced HLA Class I Expression in HER2-Positive Breast Cancer Cells.

Drug-induced modulation of HLA molecules on cancer cell lines can easily be detected using flow cytometry and HLA-specific antibodies to ascertain the number of positive cells and their expression levels. Loss or downregulation of HLA-I molecules on cancer cells, a well-documented immune escape mechanism, may occur via activation and integration of numerous signalling pathways that are operative in cancer. Whereas IFN-γ, produced during an adaptive anti-tumor immune response upregulates HLA expression, activation of the human epidermal growth factor receptor 2 (HER2) pathway and its downstream signalling pathways are reported to decrease HLA-I. Here we describe the flow cytometry procedure used to determine whether lapatinib, known to negate HER2 signalling, increased HLA-I expression on HER2+ cell lines, in the presence and absence of IFN-γ. Contrary to our prediction, the flow cytometry data clearly show lapatinib-mediated downregulation of both constitutive and IFN-γ-induced HLA class I expression. These results, for which we do not yet have an explanation, may have important implications for our understanding of lapatinib resistance in metastatic HER2+ cancer.