Prognostic role of macrophage phenotypes, spatial distribution and differential iNOS expression in NSCLC

The immune microenvironment has been shown to significantly impact the prognosis of various tumor entities. Spatial immunophenotyping has emerged as a promising technique to characterize the tumor microenvironment and explore new biomarkers aiding in prognostic evaluation. In this study, we aimed to elaborate further on the prognostic role of macrophages in the microenvironment of non-small cell lung cancer (NSCLC). For that purpose, we performed multiplex immunofluorescence on a large binational cohort, dividing the macrophage phenotype spectrum into CD68 + CD163 - iNOS - macrophage-like, M1-like, M1M2-like, and M2-like phenotype. Quantitatively, we observed the CD68 + CD163 - iNOS - population to be the most abundant subset. High M1-like and CD68 + CD163 - iNOS - densities seemed to confer a better prognosis, whereas M1M2-like seemed unfavorable in this context. In line with this, the combination of low CD68 + CD163 - iNOS - and low M1-like cell counts showed a significantly worse outcome than other combinations. Interestingly, semi-quantitative expression analysis revealed that iNOS expression in intratumoral macrophages, which in our study characterized M1-like polarization, correlated inversely with prognosis. In multivariate nearest neighbor analysis, we demonstrated that short tumor-to-M1-like distances, as well as short tumor-to-CD68 + CD163 - iNOS - distances, seemed to convey a generally better prognosis independent of age, sex and stage. Again, the combination of these markers seemed to strengthen this effect. Various immune-to-immune cell distances also showed a strong impact on prognosis. On a molecular level, we did not appreciate any impact of KRAS and EGFR status on macrophage density or spatial configuration between different macrophage phenotypes. We did, however, observe a varied effect on tumor cell-immune cell proximity in KRAS and EGFR mutated tumors. Hence, our data put forward a complex, yet prognostically important role for macrophages in NSCLC microenvironment. Our data further validate the important role of macrophages in the microenvironment of NSCLC and suggest candidate biomarkers for prognostic estimation.