Dissecting the spatial heterogeneity of different immune cell subsets in non-small cell lung cancer

To date, most studies investigating the immune tumor microenvironment of non-small cell lung cancers (NSCLC) only consider a small number of immune cell subsets or do not reflect the distribution of these cells between different tumor compartments as they were performed on tissue microarrays (TMA). To address this, we analyzed the immune infiltrate in surgically resected NSCLCs, focusing on potential spatial heterogeneity. We evaluated 45 NSCLCs based on whole-slide sections using immunohistochemistry with eleven different antibodies (CD3, CD4, CD8, CD20, CD68, Gata3, FOXP3, T-bet, kappa, lambda, PD-L1). While most markers were relatively evenly distributed among different tumor compartments as well as within the same tumor compartment, some immune cell subsets showed a considerable variance. Notably, the immune infiltrate at the tumor invasion front was dominated by B cells. Concerning markers for T cell differentiation, FoxP3 (Th2) was predominantly expressed in stromal lymphocytes, while T-bet (Th1) was most commonly expressed in intraepithelial immune cells. Although most immune cell subtypes showed a heterogenous distribution within in the intraepithelial compartment, the results from a simulated TMA and core biopsy were mostly in line with the results from whole slide evaluation. Regarding disease specific survival, there were no clear correlations. Interestingly, patients with intraepithelial T-bet positive lymphocytes had a significantly better outcome (p = 0.039), however, this difference was not preserved in multivariate analysis. In conclusion, our study shows that the immune tumor microenvironment of NSCLCs is complex and partially heterogenous, especially concerning markers for T cell differentiation.