Divide and conquer: using patient-derived tumour organoids to dissect intra-tumour immune heterogeneity of non-small cell lung cancer

Immune checkpoint blockade (ICB) has revolutionised treatment of patients with non-small cell lung cancer (NSCLC), but is effective in only ~20% of patients.

Anti-tumour immunity is highly heterogeneous within tumours, and intra-tumour heterogeneity (ITH) is a major driver for treatment resistance.

However, the mechanistic basis of intra-tumour immune heterogeneity (ITIH) is unclear, largely due to the absence of appropriate functional models.

Here, I aim to identify the genetic or transcriptomic drivers of ITIH, and their impact on immune surveillance and control.

I have recently developed an organoid – T-cell co-culture system that I will use to generate personalised models of ITIH.

Leveraging the TRACERx lung cancer evolution study, I will establish multiple clonal organoid lines from the same tumour from NSCLC multi-region biopsies.

Each organoid subline will be co-cultured with autologous T-cells to evaluate how they differ in sensitivity to T-cells.

For each patient, the 6 most sensitive and 6 most resistant sublines will be used for DNA and RNA sequencing to identify mutations (including neo-antigens), copy number alterations and differentially expressed genes associated with resistance to T-cells. Candidate subclonal immune evasion factors will be validated in organoids by CRISPR-Cas9.

I will prioritise genes by cross-referencing with genes associated with immune-cold regions in the TRACERx cohort.

I will also perform pooled enrichment screens as a less biased approach.The integration of novel organoid technology with ‘big data’ from TRACERx allows moving beyond merely descriptive studies of ITIH. This will result in the most fine-grained mechanistic study of ITIH to date.