L Boquete Vilarino1; L Escudero-Ibarz2; V Quarantotti2; D Ross-Thriepland2; P Kozik1;
1 MRC Laboratory of Molecular Biology, UK; 2 Discovery Sciences, R&D, AstraZeneca, Cambridge, UK, UK
AbstractTumour immunogenicity is defined as the ability of the
tumour to induce an immune response. Immunogenicity is determined by two main components: antigenicity (presence of neoantigens) and adjuvanticity (presence of signals that will activate antigen presenting cells). While genomic studies have facilitated identification of neoantigens, identification of signals that activate the immune system and facilitate initiation of immune responses remains challenging. To identify pathways that contribute to tumour cell adjuvanticity, we have developed a high-throughput co-culture assay and used it in a whole genome arrayed CRISPR/Cas9 screen.
Our co-culture assay provides an in vitro model of tumour
cell - antigen presenting cell communication. We use the dendritic cell (DC) line MutuDC as our antigen presenting model as they can be expanded in an immature state and activated by pathogen-derived and endogenous signals. We selected the lung adenocarcinoma cell line A549, which did not activate MutuDC in our co-culture assay, as our cancer model.
We developed a high throughput pipeline where Cas9 inducible
A549 cells are transfected with an arrayed sgRNA library, and edited cells are co-cultured with MutuDC. To monitor DC activation, we stain for membrane CD83 and CD86, and quantify the staining signal in DCs using high throughput confocal microscopy and image analysis.
Using the co-culture activation assay we have performed the
first to our knowledge whole genome arrayed CRISPR/Cas9 screen for cancer cell-derived activators of dendritic cells. We have identified more than 300 candidate genes with a robust Z-score for dendritic cell activation higher than 2.5. Pathway and gene set enrichment analysis of these candidate genes reveal a potential role for arachidonic acid metabolism and eukaryotic translation initiation factor 4f complex in the regulation of tumour cell adjuvanticity.
We are currently validating the candidate genes with the aim to discover novel targets to modulate cancer cell adjuvanticity. Our
co-culture assay can be easily adapted to other cancer cell models and screening technologies. Better understanding of the mechanisms involved in tumour cell – antigen presenting cell communication may facilitate design of new combination treatments with other immunotherapies, as well as provide tools for patient stratification or prognosis.