부산대학교 도서관

The epithelial to mesenchymal transition (EMT) is an important pathway in renal cell carcinoma (RCC) progression in which tumor cells lose epithelial features and gain more aggressive mesenchymal features that promote invasion and metastasis. Sarcomatoid RCC (sRCC) is a de-differentiated tumor state which occurs by EMT. sRCC tumors are highly responsive to immunotherapy, although the reasons behind this remain unknown. Here, we demonstrate that EMT modulates tumor cells and their immune environment to confer sensitivity to PD-1/PD-L1 directed immunotherapy. Multiplex immunofluorescent staining (Vectra Polaris) was performed on 29 human RCC specimens for epithelial marker E-cadherin, mesenchymal marker N-cadherin, PD-L1, CD8 (CD8 T-cells), CD4 (CD4 T-cells), and CD163 (macrophages). Spatial correlation of markers was assessed. One specimen underwent single cell spatial transcriptomics for spatially resolved cell type and gene expression using the NanoString CosMx platform. In vitro, expression of EMT markers and PD-L1 were measured using Western Blot in two ccRCC lines and five sRCC lines. EMT was induced in cells through treatment with cytokines TGFB or IFNg, treatment with recombinant HGF or SPP1 (proteins of interest from prior data), and by overexpression of EMT transcription factors Snail or Zeb1. To evaluate the role of PD-1/PD-L1 signaling on EMT, cells were treated with a siRNA against PD-L1, the PD-L1 inhibitor durvalumab, or recombinant PD-1 protein. Effects on EMT-related protein expression were measured using Western Blot. In 29 RCC specimens, N-cadherin (mesenchymal marker) was strongly associated with PD-L1 expression (Figure1A). Macrophages in N-cadherin high areas also exhibited high PD-L1 expression and were closely spatially associated with CD8 T-cells (Figure1B-E). Single cell transcriptomic data revealed these CD8 T-cells expressed high levels of PD-1 but otherwise cytotoxic traits (Figure1F). In vitro, PD-L1 expression positively correlated with increasing EMT state in all 7 RCC lines. Induction of EMT in RCC cells by all methods tested led to a concurrent strong upregulation of PD-L1 expression. Co-culture of EMT high RCC cells with macrophages led to upregulation of PD-L1 expression in the macrophages. PD-L1 knock down with siRNA or inhibition with durvalumab, both led to a decreased EMT state in the cells whereas activation of PD-L1 by treatment with recombinant PD-1 led to an enhanced EMT state. These findings demonstrate that EMT alters tumor cells and their immune microenvironment to enhance PD-L1 expression and promote co-localization of PD-1 expressing CD8 T-cells. Our data suggests that EMT high RCC tumors may be dependent on PD-1/PD-L1 signaling to maintain or enhance the EMT status of tumor cells which can be blocked with PD-1/PD-L1 inhibition. This provides biologic insight to sRCC's high responsiveness to PD-1/PD-L1 directed immunotherapy, and provides important rationale to explore EMT related factors as biomarkers for immunotherapy response in RCC. [ABSTRACT FROM AUTHOR]