Abstract B92: VHL inactivated kidney epithelial cells reprogram macrophage behavior through IL-6 and CXCL-1 secretion
Authors: Nguyen Thi-Ngoc , Hsu Tien 
Published: March 2020

Abstract
Introduction: There is a very close causal correlation between clear-cell renal cell carcinoma (ccRCC) formation and inactivation of the tumor suppressor gene von Hippel-Lindau (VHL). Up to 80% of sporadic ccRCC and nearly 100% familial ccRCC (in VHL disease) carry VHL mutations. To study this correlation, we generated a novel mouse with Vhlh gene inactivation in the kidney tubules. This model suggested a pre-ccRCC condition that produced highly penetrant cystic and inflammatory phenotypes. Interestingly, abundance of infiltrating macrophages also was detected in the Vhlhdel/del tissue in connection with inflammation and cancer formation. Macrophage infiltration in the inflammatory microenvironment has been observed in previous studies of ccRCC models containing VHL mutations. However, the mechanism by which VHL loss of function cells attract macrophage during ccRCC formation has remained unclear.
Methods: Transwell coculture was used to mimic the extravasation process in vitro, containing three types of cell lines including human kidney cells (HK2 with or without VHL knockdown), human microvascular endothelium (HMEC-1), and macrophage precursor THP-1 cells. Invasive macrophages were quantified using ICC/IF staining; their polarization was analyzed by RT-qPCR and flow cytometry. Cytokine composition from conditioned media of HK2 cells was detected by cytokine bead array. The secreted cytokines program was tested for its action on endothelial dysfunction and macrophage differentiation and extravasation. Vhlh knockout mouse and ccRCC tissues were used to validate the in vitro results.
Results: First, VHL loss of function kidney cells secreted specific cytokines such as IL-6 and CXCL1 to induce endothelial dysfunction with upregulation of ICAM1, JAM1, JAM2 and downregulation of PECAM1 and VE-cadherin. Second, under a long-term IL-6 and CXCL1 stimulation, recruited macrophages tend to polarize toward tumor-associated macrophage (TAM) with upregulation of CD206, CD163, and Arg-1. Third, antibody blockade of IL-6 and CXCL1 reduced macrophage extravasation, and macrophage phenotype shifts from M2/TAM to M1 subtype with high expression of CD86, IL-12, and TNFα. Fourth, the upregulation of IL-6 and CXCL1 in VHL inactivated cell was mediated through STAT3 and NF-kB pathways. Finally, double knockdown of VHL and HIF-1α significantly reduced secreted IL-6 and CXCL1 levels, and inhibited macrophage trafficking and differentiation.
Conclusions: We found that VHL knockdown cells recruit macrophages across endothelium and induce their polarization toward M2/TAM phenotype through secreting cytokines such as IL-6 and CXCL1. Blockade of IL6 and CXCL1 or double knockdown of VHL and HIF-1α significantly reduces macrophage trafficking and differentiation. These cytokines may be potential targets for early detection or therapeutics for ccRCC.
Citation Format: Thi-Ngoc Nguyen, Tien Hsu. VHL inactivated kidney epithelial cells reprogram macrophage behavior through IL-6 and CXCL-1 secretion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B92.