Mechanisms of immune regulation by CD8+ T cells in triple-negative breast cancer Irina Perlitch1, Bruce Huang1, Dr. Tina Gruosso1, Dr. Morag Park1 1McGill University Introduction: Breast cancer, which affects 1 in 9 Canadian women, consists of multiple subtypes associated with different prognosis and standard of care. The triple-negative subtype (TNBC) consists of ~15% of cases, is negative for common biomarkers, lacks a targeted therapeutic approach, and has the worst outcome. Several cancer immunotherapy approaches target regulatory interactions required to negatively balance the level of the immune response, referred to as “immune checkpoints”. Uncoupling these checkpoints boosts host anti-tumour response and the cytotoxic activity of CD8+ T cells, whose high infiltration levels within TNBC tumours correlate with good prognosis. By performing laser capture microdissection we have identified distinct TNBC tumour microenvironments. Tumours with an immune-cold microenvironment display restriction of CD8+ T cells to tumour margins, elevated expression of the PD-L1 family member B7-H4, and poor outcome. I hypothesize that B7-H4, when expressed in tumour epithelia, promotes immunosuppression and is a potential therapeutic target for a subset of TNBC, but little is known about its regulation.
Methods/Results:I have developed a high throughput, functional genetic screen using a genome-wide CRISPR/Cas9 library to identify regulators of B7-H4. B7-H4 cell surface expression measured by flow cytometry was used as a read-out. After CRISPR/Cas9 library infection, the breast cancer cell line, MDA-468, sorts into two subpopulations with high and low B7-H4 levels of expression which can be separated using a fluorescence-activated cell sorting (FACS) technique. CRISPR targets in each population (inducing up or downregulation of B7-H4 expression) were then identified by next-generation sequencing. Using Model-based Analysis of Genome-Wide CRISPR-Cas9 Knockout (MAGeCK), target genes were ranked based on their effect on B7-H4 expression levels. The top-ranked genes cause the strongest shift of cells towards high or low B7-H4 expression. Validation of potential B7-H4 regulators is currently in progress. Conclusion:Combination of a genome-wide CRISPR library and FACS is a robust tool to identify regulators of cell surface expression of B7-H4. Identification of novel regulators of B7-H4 can lead to potential clinically relevant targets and companion diagnostics in several cancers.
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