Defining candidate mRNA and protein EV biomarkers to discriminate ccRCC and pRCC from non-malignant renal cells in vitro

Richard C Zieren,Sarah R Amend,Louis Vermeulen,Leandro Ferreira Moreno,Liang Dong,Michael Schnaubelt,Tung-Shing M Lih,Theo M De Reijke,Morgan D Kuczler,Kengo Horie,Hui Zhang,David J Clark,Kenneth J Pienta

doi:10.1007/s12032-021-01554-2

Abstract

Renal cell carcinoma (RCC) accounts for over 400,000 new cases and 175,000 deaths annually. Diagnostic RCC biomarkers may prevent overtreatment in patients with early disease. Extracellular vesicles (EVs) are a promising source of RCC biomarkers because EVs carry proteins and messenger RNA (mRNA) among other biomolecules. We aimed to identify biomarkers and assess biological functions of EV cargo from clear cell RCC (ccRCC), papillary RCC (pRCC), and benign kidney cell lines. EVs were enriched from conditioned cell media by size exclusion chromatography. The EV proteome was assessed using Tandem Mass Tag mass spectrometry (TMT-MS) and NanoString nCounter technology was used to profile 770 cancer-related mRNA present in EVs. The heterogeneity of protein and mRNA abundance and identification highlighted the heterogeneity of EV cargo, even between cell lines of a similar pathological group (e.g., ccRCC or pRCC). Overall, 1726 proteins were quantified across all EV samples, including 181 proteins that were detected in all samples. In the targeted profiling of mRNA by NanoString, 461 mRNAs were detected in EVs from at least one cell line, including 159 that were present in EVs from all cell lines. In addition to a shared EV cargo signature, pRCC, ccRCC, and/or benign renal cell lines also showed unique signatures. Using this multi-omics approach, we identified 34 protein candidate pRCC EV biomarkers and 20 protein and 8 mRNA candidate ccRCC EV biomarkers for clinical validation.

Highlights

Renal cell carcinoma (RCC) is a lethal disease which accounts for more than 400,000 new cases worldwide and over 175,000 deaths annually [1]
Extracellular vesicles (EVs) were isolated from cell-conditioned media (CCM) of clear cell RCC (ccRCC) cells (786-O, 769-P, and Caki1), papillary RCC (pRCC) cells (ACHN and Caki2), and immortalized benign epithelial kidney cells (HK2 and RPTEC/ TERT1)
Only few studies focus on the identity of cargo from EVs derived directly from RCC cells

Summary

Introduction

Renal cell carcinoma (RCC) is a lethal disease which accounts for more than 400,000 new cases worldwide and over 175,000 deaths annually [1]. The incidence of localized RCC increased > 2.3-fold between the years 2000 and 2017, compared with 1.6-fold for regional metastatic and 1.3-fold for distant metastatic RCC [2] This shift is attributed to the discovery of incidental renal masses through the increased use of imaging modalities for other symptoms [3]. The most common (~ 75%) histopathological subtype of RCC is clear cell RCC (ccRCC) followed by papillary RCC (pRCC, ~ 15%). The subtypes and their genetic subtypes (e.g., papillary type 1 and 2) vary in lethality and biological tumorigenesis, resulting in various response to targeted therapies [6, 7].

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