Gene expression analysis of sarcoma-infiltrating NK cells identifies signatures correlating with favorable prognosis and potential new targets for augmenting NK function

Abstract

Abstract Success of current T-cell based immunotherapies in soft tissue sarcomas (STS) is limited while pre-clinical and clinical studies have shown evidence of NK activity in STS. To investigate the effects of the tumor microenvironment (TME) on tumor-infiltrating NK cell gene expression, we isolated circulating and tumor-infiltrating NK and CD3 T cells from sarcoma patients undergoing surgery (N=4) and performed RNA sequencing analysis. Comparing Differential Gene Expression (DGE) of flow-sorted intra-tumoral NK cells to circulating NK cells, we identified upregulation of genes involved in mitogen signaling inhibition (DUSP4) and metabolic function (SMPD3, SLC7A5), but not of genes typically associated with cytotoxic function (e.g. IFNG, GZMB). In contrast, intra-tumoral T cells showed significant upregulation of both activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. We then queried the TCGA STS dataset and stratified cases into “NK Activating TME” and “NK Suppressing TME” based on the CIBERSORT NK signature analysis program (based on high or low proportion of activated NK cells, respectively). Patients with NK Activating TME had significantly improved survival compared to NK Suppressive TME (log-rank p = 0.03). DGE identified unique tumor-associated gene ontology between NK activated versus suppressive subsets with upregulation of the retinoic acid pathway. Unlike T cells which demonstrate significant DGE between circulating and tumor-infiltrating subsets, NK cells appear to have similar gene expression between blood and tumor. Therefore, although NK cells appear to be prognostic in STS, their anti-tumor effects may be dictated by systemic rather than local factors.