DEVELOPMENT OF EX VIVO PATIENT-DERIVED MODELS TO UNCOVER THE TUMOR-IMMUNE MICROENVIRONMENT IN RENAL CELL CARCINOMA

Abstract

Introduction

With the rise of immune checkpoint inhibitors (ICIs) as the primary treatment option for metastatic renal cell carcinoma (RCC), investigating the role of T cells within the tumor microenvironment (TME) is a critical component of understanding both treatment response and resistance. Prior efforts, including single-cell transcriptomic approaches, have provided an important landscape of T cell transcriptional phenotypes. However, these immuno-profiling efforts require validation through functional interrogation of the TME to facilitate the development of novel immunomodulatory therapies. Thus, we established a patient-derived tumor model (PDTM) system to directly assess the effect of inhibitory immune interactions on T cell function and anti-tumor activity in the RCC TME. In this initial proof-of-concept study, we evaluated T cell activation in the RCC TME using the PDTM system.

Methods

Fresh tumor samples were obtained from surgical resections of RCC at Yale-New Haven Hospital. The tumor was minced to ∼1-3 mm³ pieces and suspended in an air-liquid interface system, consisting of tumor fragments embedded in a collagen matrix on an insert with a semi-permeable membrane, exposed to culture media. The tumor fragment and matrix suspension were carefully pipetted onto the Millicell insert, which served as the top layer. The PDTM setup includes an inner dish containing the bottom gel layer and the tissue-containing top layer. To complete the assembly, a low-dose T cell stimulant, 1.5 ml of DMEM media with or without 500 nM of anti-PD-1 monoclonal antibody (aPD1mAb) was added to the outer dish surrounding the insert.

Results

We successfully optimized PDTM experimental workflows for culture, dissociation, and analysis using immunohistochemistry (IHC), flow cytometry (FCM), and enzyme-linked immunosorbent assays (ELISA). Hematoxylin and eosin (H&E) staining and IHC showed that the TME cellular architecture and immune cell composition was broadly preserved during the three-day experimental period. Using FCM to analyze the dissociated tumor samples, we identified well-preserved CA9+ tumor cells, CD4+ and CD8+ T cell populations, CD4+CD25+ regulatory T cells, CD56+ natural killer cells, CD20+ B cells, and CD14+CD11b+ myeloid subsets including monocytes and CD163-/+macrophages. Among the T cells, we detected PD1+, LAG3+, TIM3+, and TIGIT+ cells. We tested the effect of the anti-PD-1 antibody on PDTM, and importantly, found that treatment of the PDTM with aPD1mAb resulted in more activated CD8 T cells and higher IFN-γ production than the control samples.

Conclusions

Through optimization of assays evaluating T cell cytokine production, we were able to assess multiple axes of T cell function in the RCC TME. This study revealed that our PDTM system preserves the RCC TME for functional interrogation. Furthermore, our system for assessing T cell phenotype and cytokine production successfully demonstrated the activity of PD-1 blockade ex vivo. Taken together, this novel ex vivo PDTM system has extensive applications in the study of RCC, including assessing the impact of ICIs and new combination therapies on T cell function.