Anais Elewaut

Pooled genetic screening with CRISPR–Cas9 has enabled genome-wide, high-resolution mapping of genes to phenotypes, but assessing the effect of a given genetic perturbation requires evaluation of each single guide RNA (sgRNA) in hundreds of cells to counter stochastic genetic drift and obtain robust results. However, resolution is limited in complex, heterogeneous models, such as organoids or tumors transplanted into mice, because achieving sufficient representation requires impractical scaling. This is due to bottleneck effects and biological heterogeneity of cell populations. Here we introduce CRISPR-StAR, a screening method that uses internal controls generated by activating sgRNAs in only half the progeny of each cell subsequent to re-expansion of the cell clone. Our method overcomes both intrinsic and extrinsic heterogeneity as well as genetic drift in bottlenecks by generating clonal, single-cell-derived intrinsic controls. We use CRISPR-StAR to identify in-vivo-specific genetic dependencies in a genome-wide screen in mouse melanoma. Benchmarking against conventional screening demonstrates the improved data quality provided by this technology.

The tumour microenvironment is programmed by cancer cells and substantially influences anti-tumour immune responses^1,2. Within the tumour microenvironment, CD8⁺ T cells undergo full effector differentiation and acquire cytotoxic anti-tumour functions in specialized niches^3–7. Although interactions with type 1 conventional dendritic cells have been implicated in this process^3–5,8–10, the underlying cellular players and molecular mechanisms remain incompletely understood. Here we show that inflammatory monocytes can adopt a pivotal role in intratumoral T cell stimulation. These cells express Cxcl9, Cxcl10 and Il15, but in contrast to type 1 conventional dendritic cells, which cross-present antigens, inflammatory monocytes obtain and present peptide–major histocompatibility complex class I complexes from tumour cells through ‘cross-dressing’. Hyperactivation of MAPK signalling in cancer cells hampers this process by coordinately blunting the production of type I interferon (IFN-I) cytokines and inducing the secretion of prostaglandin E₂ (PGE₂), which impairs the inflammatory monocyte state and intratumoral T cell stimulation. Enhancing IFN-I cytokine production and blocking PGE₂ secretion restores this process and re-sensitizes tumours to T cell-mediated immunity. Together, our work uncovers a central role of inflammatory monocytes in intratumoral T cell stimulation, elucidates how oncogenic signalling disrupts T cell responses through counter-regulation of PGE₂ and IFN-I, and proposes rational combination therapies to enhance immunotherapies.