Although immunotherapy has shown major success in the clinic, most lung cancer patients fail to respond long-term to the current treatment options. Immune cell composition and spatial organisation of the tumour microenvironment (TME) determine patient response to immunotherapy. However, how cancer cells modulate cellular arrangements within the TME remains poorly understood. Here, we combine a novel in vivo spatial CRISPR screen with spatial omic technologies to identify genes regulating the TME in non-small cell lung cancer and decipher mechanisms of resistance to immunotherapy.
We curated a candidate gene library based on differentially expressed genes in two RNA-seq datasets of lung adenocarcinoma patient biopsies: 1) comparing tumours sensitive or resistant to anti-PD-L1 therapy and 2) comparing tumours with high or low immune infiltration. Correlating these two transcriptomic datasets identified genes associated with both, response to immune checkpoint blockade and immune infiltration, suggesting these gene products might be involved in immunotherapy resistance via modulation of the tumour immune microenvironment.
Using a combinatorial protein barcoding system, called Perturb-map, this project aims to functionally validate these potential TME regulators and evaluate their function in an in vivo pooled CRISPR screen. Perturb-map uses protein-barcoded sgRNA vectors that can be resolved by imaging, hence allowing the identification of extracellular gene functions. Contrary to conventional in vivo CRISPR screens that can only assess how depletion of individual target genes impact tumour cell fitness, the protein barcodes add spatial resolution and additionally enable the assessment of potential effects on the tumour architecture. Finally, combining Perturb-map with multiplex-ion beam imaging and spatial transcriptomics enables us to investigate how individual gene perturbations influence the TME and immune cell composition, providing detailed mechanistic insights into these important determinants of immunotherapy efficacy.
Defining the spatial relationships within the tumour microenvironment is necessary to fully understand cell-cell interactions and mechanisms controlling immune infiltration. Our findings may help identifying patients responsive to immunotherapy, enabling more targeted and personalised therapy. Further, regulators of the TME may provide potential targets for future cancer treatments. Genes identified in our study may suggest novel drug targets and improve patient outcome in combination with conventional immunotherapies.