Diagnostic potential of serum extracellular vesicles expressing prostate-specific membrane antigen in urologic malignancies

Kyojiro Kawakami,Ikuroh Ohsawa,Takuya Koie,Yasunori Fujita,Masafumi Ito,Yuri Miura,Yasuo Katagiri,Taku Kato,Kengo Horie,Hiroki Tsumoto,Keitaro Umezawa,Tatsuhiko Miyazaki,Kosuke Mizutani

doi:10.1038/s41598-021-94603-9

Abstract

We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies. First, we optimized the conditions for sandwich ELISA measuring the PSMA level on EVs captured from serum by Tim4 and found that the use of highly-purified EVs released from Tim4 that had captured EVs in serum reduced the background. Second, we confirmed its validity by studying mouse xenograft model for prostate cancer (PC). Lastly, we measured PSMA-EVs in serum of patients with urologic malignancies. The PSMA-EV levels were significantly higher in metastatic PC and castration-resistant PC (CRPC) patients than in therapy-naïve PC patients. In renal cell carcinoma (RCC) patients, PSMA-EVs were elevated in those with metastasis compared with those without metastasis, which may reflect the development of the neovasculature positive for PSMA in tumors. In conclusion, we developed a sandwich ELISA for detection of PSMA-EVs using highly-purified EVs isolated from serum by Tim4. Our results suggest that PSMA-EVs may be useful to diagnose and monitor not only PC but also RCC and possibly other hypervascular solid tumors.

Highlights

We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies
Our preliminary experiments suggested that the development of PSMA-EV sandwich ELISA was difficult when EVs were captured from serum by antibody
We demonstrated that the PSMA-EV sandwich ELISA we developed is useful to diagnose and monitor metastatic prostate cancer (PC) and castration-resistant PC (CRPC) patients as well as metastatic renal cell carcinoma (RCC) patients

Summary

Introduction

We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies. Abbreviations ABs Apoptotic bodies BPH Benign prostatic hyperplasia CRPC Castration-resistant prostate cancer EVs Extracellular vesicles EXs Exosomes MPs Microparticles PC Prostate cancer PSMA Prostate-specific membrane antigen PSMA-EVs PSMA-expressing EVs PSMA-PET/CT PSMA-positron emission tomography/computed tomography RCC Renal cell carcinoma Tim[4] T-cell immunoglobulin domain and mucin domain-containing protein 4. The contamination is problematic to develop an immunoassay such as ELISA, especially when dealing with targets that are of low abundance in blood such as cancer-derived EVs. The non-specific binding of serum or plasma constituents to the vessels such as microplates and beads as well as to target proteins causes high background and low sensitivity, which is referred to as the “matrix effect”[7]

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Conclusion