Abstract PR014: Macrophage induced epithelial to mesenchymal transition in sarcomatoid renal cell carcinoma
Authors: May Allison , Williams Claire , The Stephanie , McGue Jake , Kim Sean , Schurman Nathan , Shelley Greg , Robinson Tyler , Salami Simpa , Mehra Rohit , Frankel Timothy , Udager Aaron , Keller Evan 
Published: August 2023

Abstract
Intro: Sarcomatoid de-differentiation in renal cell carcinoma (sRCC) leads to aggressive tumors that may be uniquely responsive to adjuvant immunotherapy. sRCC is thought to arise by epithelial to mesenchymal transition (EMT) of the parental tumor, most commonly clear cell RCC (ccRCC). However, factors that drive sRCC are unknown and no biomarkers exist to predict the transition. Furthermore, the immune microenvironment in sRCC is not well understood and may provide additional therapeutic targets. Here, we use spatial biology and in vitro studies to explore EMT/immune cell crosstalk in sRCC. Methods: Single cell spatial transcriptomics was performed on a human sRCC specimen with Nanostring CosMx platform. Semi-supervised clustering referenced to scRNAseq data was used to define cell types and map them spatially. Differential gene expression and cell distance analysis were performed. Multiplex immunofluorescent staining (mIF) was performed by Vectra Polaris. In vitro work used ccRCC cell line, 786-O, and sRCC cell line, UOK-127. M0, M1, or M2 macrophages were derived from THP-1 cells. Analysis of macrophage markers and EMT markers were performed by western blot and RT-qPCR. Results: Single cell spatial transcriptomic data revealed a ccRCC population, 2 sRCC cell types, and a novel transitional cell type along the EMT continuum and spatially between ccRCC and sRCC. Importantly, matched H&E shows the transitional cell type present in areas histologically defined as ccRCC, demonstrating molecular evidence of transition towards sRCC prior to histologic changes. Cell to cell distance revealed strong correlation of M2 macrophages with transitional and sRCC cells. The ccRCC population expressed CCL20, a factor known to assist in macrophage recruitment, and CCL20 was upregulated in the transitional cell type. FZD4 was the highest fold changed gene lost from the ccRCC to transitional cells. In vitro culture of RCC cells with M2 macrophages led to CCL20 upregulation and loss of FZD4. Overexpression of CCL20 and knockdown of FZD4 in RCC cells both led to EMT. Exposure of macrophages to CCL20 led to M2 polarization. To validate the role of these genes, analysis of the human protein atlas (877 RCC specimens) revealed high expression of CCL20 and low expression of FZD4 are both associated with poor survival. mIF of 20 human sRCC specimens demonstrated CD163 (M2 macrophage marker) was associated with sRCC or ccRCC areas with high expression of mesenchymal marker N-cadherin. Conclusion: We report the first detection of a transitional cell type along the de-differentiation pathway from ccRCC to sRCC. This cell type is detectable in areas histologically defined as ccRCC, demonstrating strong potential as a transcriptional biomarker to inform adjuvant immunotherapy. We show that CCL20 on tumor cells leads to macrophage recruitment and polarization, resulting in FZD4 loss and CCL20 upregulation, which induce EMT and propagates a vicious cycle of EMT/macrophage crosstalk in sRCC. This suggests potential for therapeutic targeting of macrophages or CCL20 in RCC.
Citation Format: Allison May, Claire Williams, Stephanie The, Jake McGue, Sean Kim, Nathan Schurman, Greg Shelley, Tyler Robinson, Simpa Salami, Rohit Mehra, Timothy Frankel, Aaron Udager, Evan Keller. Macrophage induced epithelial to mesenchymal transition in sarcomatoid renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR014.