Diagnostic liquid biopsy biomarkers in renal cell cancer.

The clinical presentation of renal cell cancer (RCC) is shifting towards incidental and early detection, creating new challenges in RCC diagnosis. Overtreatment might be reduced with the development of new diagnostic biomarkers to distinguish benign from malignant small renal masses (SRMs). Differently from tissue biopsies, liquid biopsies are obtained from a patient's blood or urine and, therefore, are minimally invasive and suitable for longitudinal monitoring. The most promising types of liquid biopsy biomarkers for RCC diagnosis are circulating tumour cells, extracellular vesicles (EVs) and cell-free DNA. Circulating tumour cell assays have the highest specificity, with low processing time and costs. However, the biological characteristics and low sensitivity limit the use of these markers in SRM diagnostics. Cell-free DNA might complement the diagnosis of high-volume RCC, but the potential for clinical application in SRMs is limited. EVs have the highest biological abundance and the highest sensitivity in identifying low-volume disease; moreover, the molecular characteristics of these markers make EVs suitable for multiple analytical applications. Thus, currently, EV assays have the greatest potential for diagnostic application in RCC (including identification of SRMs). All these liquid biomarkers have potential in clinical practice, pending validation studies. Biomarker implementation will be needed to also improve characterization of RCC subtypes. Last, diagnostic biomarkers might be extended to prognostic or predictive applications.

Advancements in the identification of EV derived mRNA biomarkers for liquid biopsy of clear cell renal cell carcinomas.

OBJECTIVE: To propose EV-derived mRNA as a potential diagnostic biomarker detecting the presence of clear cell renal cell carcinoma (ccRCC). There is currently no kidney cancer specific screening or diagnostic technology. Therefore, one-third of kidney cancer diagnoses occur after the cancer has metastasized and is past curative measures MATERIALS AND METHODS: Urine, plasma, normal tumor adjacent tissue, and tumor tissue was collected from a limited population of ccRCC patients. Extracellular vesicle (EV) isolation was performed on each sample, followed by mRNA extraction from isolated EVs. NanoString nCounter technology was utilized to count the mRNA transcripts present in matched plasma, urine, tumor tissue, and normal tumor adjacent tissue samples. RESULTS: 770 mRNA transcripts related to gene's affecting cancer's progression and metastasis processes were evaluated. Four EV derived mRNA transcripts (ALOX5, RBL2, VEGFA, TLK2) were found specific to urine and tumor tissue samples. CONCLUSION: Four candidate RCC-specific urine EV biomarkers were identified. However, due to the lack of a true negative control and urine collection techniques, further re-examination is necessary for validation. This study demonstrates the promise of defining disease-specific EV biomarkers in liquid biopsy patient samples.

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

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.

High-Throughput Simultaneous mRNA Profiling Using nCounter Technology Demonstrates That Extracellular Vesicles Contain Different mRNA Transcripts Than Their Parental Prostate Cancer Cells.

Extracellular vesicles (EVs) are nano-sized lipid bilayer encapsulated particles with a molecular cargo that appears to play important roles within the human body, such as in cell-to-cell communication. Unraveling the composition of EV cargos remains one of the most fundamental steps toward understanding the role of EVs in intercellular communication and the discovery of new biomarkers. One of the unmet needs in this field is the lack of a robust, sensitive, and multiplexed method for EV mRNA profiling. We established a new protocol using the NanoString low RNA input nCounter assay by which the targeted mRNA transcripts in EVs can be efficiently and specifically amplified and then assayed for 770 mRNAs in one reaction. Prostate cancer cells with epithelial (PC3-Epi) or mesenchymal (PC3-EMT) phenotypes and their progeny EVs were analyzed by the same panel. Among these mRNAs, 157 were detected in PC3-Epi EVs and 564 were detected in PC3-EMT EVs. NOTCH1 was the most significantly abundant mRNA transcripts in PC3-EMT EVs compared to PC3-Epi EVs. Our results demonstrated that when cells undergo epithelial-to-mesenchymal transition (EMT), a more active loading of cancer progression-related mRNA transcripts may occur. The mRNA cargos of EVs derived from mesenchymal prostate cancer cells may contribute to the pro-EMT function. We found that mRNA transcripts are different in progeny EVs compared to parental cells. EV cargos are not completely reflective of their cell origin, and the underlying mechanism of cargo sorting is complicated and needs to be further elucidated.

Extracellular vesicle isolation from human renal cancer tissue.

Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive biomarkers are needed to aid diagnosis and treatment. Extracellular vesicles (EVs), membranous vesicles secreted by all cells, are a promising potential source for cancer biomarkers, but new methods are required that are both sensitive and specific for cancer identification. We have developed an EV isolation protocol optimized for kidney tumor and normal kidney tissue that yields a high vesicle concentration, confirmed by nanoparticle tracking analysis (NanoSight) and by nanoscale flow cytometry (NanoFCM). Using Western blot, we confirmed presence of EV markers CD81, CD63, flotillin-1, and absence of cellular debris, calnexin. Transmission electron microscopy images demonstrate intact membranous EVs. This new method improves existing protocols with additional steps to reduce contaminants in the EV product. Characterization of our isolation product confirms successful isolation of EVs with minimal contamination. The particle yields of our protocol are consistent and high as assessed by both standard and novel methods. This optimized protocol will contribute to biomarker discovery and biological studies of EVs in renal cancer.

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.

One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co-separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density-gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co-separate contaminants when the non-EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non-EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

Metastatic prostate cancer remains incurable, why?

Metastatic prostate cancer patients present in two ways-with already disseminated disease at the time of presentation or with disease recurrence after definitive local therapy. Androgen deprivation therapy is given as the most effective initial treatment to patients. However, after the initial response, almost all patients will eventually progress despite the low levels of testosterone. Disease at this stage is termed castration resistant prostate cancer (CRPC). Before 2010, the taxane docetaxel was the first and only life prolonging agent for metastatic CRPC (mCRPC). The last decade has witnessed robust progress in CRPC therapeutics development. Abiraterone, enzalutamide, apalutamide and sipuleucel-T have been evaluated as first- and second-line agents in mCRPC patients, while cabazitaxel was approved as a second-line treatment. Radium-223 dichloride was approved in symptomatic patients with bone metastases and no known visceral metastases pre- and post-docetaxel. However, despite significant advances, mCRPC remains a lethal disease. Both primary and acquired resistance have been observed in CRPC patients treated by these new agents. It could be solely cell intrinsic or it is possible that the clonal heterogeneity in treated tumors may result from the adaptive responses to the selective pressures within the tumor microenvironment. The aim of this review is to list current treatment agents of CRPC and summarize recent findings in therapeutic resistance mechanisms.

Recent advances in extracellular vesicle research for urological cancers: From technology to application.

Urological malignancies, including prostate cancer, bladder cancer and kidney cancer, are major causes of morbidity and mortality worldwide. Because of the high incidence, diversity in biology, and especially direct interaction with urine, urological cancers are an important resource for both scientists and clinicians for novel diagnostic and therapeutic discovery. Extracellular vesicles (EVs) are lipid bilayer encapsulated particles released by cells into the extracellular space. Since EVs work as a safe way to transport important biological information through the whole body, they are now recognized as an important mechanism of cell-cell communication and have opened a new window for us to gain a better understanding of cancer biology, novel diagnostics, and therapeutic options. In recent years, numerous evolutions in EV technologies and novel biological and clinical findings continue to be reported in the research field of urological cancers. This comprehensive review aims to give an update of recent advances in EV technologies and summarize the state-of-the-art knowledge of EVs related to prostate cancer, bladder cancer and kidney cancer, particularly focusing on the potential of EV as biomarkers and their biological roles in promoting cancer and metastasis.