Multi-omic and spatial dissection of immunotherapy response groups in non-small cell lung cancer

The composition and activation status of the cellular milieu contained within the tumour microenvironment (TME) is becoming increasingly recognized as a driving factor for immunotherapy response. Here, we employed multiplex immunohistochemistry (mIHC), and digital spatial profiling (DSP) to capture the targeted immune proteome and transcriptome of tumour and TME compartments from an immune checkpoint inhibitor (ICI)-treated (n = 41) non-small cell lung cancer (NSCLC) patient cohort. We demonstrate by mIHC that the interaction of CD68⁺ macrophages with PD1⁺ , FoxP3⁺ cells is enriched in ICI refractory tumours (p = 0.012). Patients responsive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p = 0.028) within their tumour compartments, which corresponded with increased IL2 mRNA (p = 0.001) within their stroma. In addition, stromal IL2 mRNA levels positively correlated with the expression of pro-apoptotic markers cleaved caspase 9 (p = 2e^-5 ) and BAD (p = 5.5e^-4 ) and negatively with levels of memory marker, CD45RO (p = 7e^-4 ). Immuno-inhibitory markers CTLA-4 (p = 0.021) and IDO-1 (p = 0.023) were suppressed in ICI-responsive patients. Tumour expression of CD44 was depleted in the responsive patients (p = 0.02), while higher stromal expression of one of its ligands, SPP1 (p = 0.008), was observed. Cox survival analysis also indicated tumour CD44 expression was associated with poorer prognosis (hazard ratio [HR] = 1.61, p = 0.01), consistent with its depletion in ICI-responsive patients. Through multi-modal approaches, we have dissected the characteristics of NSCLC immunotherapy treatment groups and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy.