12: Epithelial to mesenchymal transition (EMT) and PD-1/PD-L1 signaling provide reciprocal feedback in renal cell carcinoma progression with implications for immunotherapy response

Background: 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 EMT modulates tumor cells and their immune environment to confer sensitivity to PD-1/PD-L1 directed immunotherapy.

Methods: Multiplex immunofluorescent staining (mIF) was performed on 29 human RCC specimens for EMT and immune markers. One specimen underwent single cell spatial transcriptomics via NanoString CosMx. Spatial correlation of cell types and gene expression was assessed. In vitro experiments were performed using 7 RCC cell lines. EMT markers and PDL1 were measured by Western Blot.

Results: mIF staining of 29 RCC specimens demonstrated strong association of PD-L1 with N-cadherin (mesenchymal marker) expression on tumor cells as well as nearby macrophages. PD-L1 expressing cells were spatially associated with CD8 T-cells. Single cell transcriptomic data revealed these were cytotoxic CD8 T-cells with high PD-1 expression. In vitro, induction of EMT led to concurrent strong upregulation of PDL1 in RCC cells and in co-cultured macrophages. PD-L1 knock down with siRNA or inhibition with durvalumab, led to a decreased EMT state whereas activation of PD-L1 by treatment with recombinant PD-1 led to an enhanced EMT state.

Conclusions: These findings demonstrate 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 EMT high RCC tumors may be dependent on PD-1/PD-L1 signaling to maintain or enhance their EMT state. These findings provide mechanistic insight to sRCC’s high responsiveness to PD-1/PD-L1 directed immunotherapy, and provide rationale to explore EMT related factors as biomarkers for immunotherapy response in RCC.