Chasing Circulating Biomarkers in Renal Cell Carcinoma: the Case of Extracellular Vesicles and their miRNA Cargos

1. Renal Cell Carcinoma: clinical diagnosis and staging

Renal cell carcinoma (RCC) is the second most frequent cancer in the urological system [1] and represents about 3% of all tumors affecting adults in western world [2]. Every year 64,000 new RCC cases are diagnosed and 14,000 mortality cases due to this malignancy are registered in the United States (US), projecting to increase in burden worldwide [3]. RCC early diagnosis is critical to reduce mortality rate, however the disease is characterized by an asymptomatic clinical course with most cases accidentally diagnosed during clinical evaluations for other concomitant pathologies [2]. Nevertheless, some clinical signs could be related with RCC, such as polycythemia, hypercalcemia, hypertension and Cushing’s syndrome, respectively correlated to erythropoietin, parathyroid-related hormone, renin and ACTH over-production by cancer. Furthermore, leukocytosis, fever and weight loss are common symptoms, while only about 10% of cases show the “classic triad” of RCC symptomatology, consisting of flank pain, hematuria, and palpable masses [4].

The incidence of the disease varies widely throughout the world and appears to be highest in more developed countries. White people have a lower risk of RCC than Native, African and Hispanic Americans probably due to the disparities in diets, lifestyles, socioeconomic conditions, education, and economical possibility for healthcare. Risk factors connected to RCC could be distinguished in (a) unmodifiable such as age, race and gender and (b) modifiable that include diet, alcohol, occupational exposure and drugs [4]. Peak incidence is mostly around sixty and eighty, when the average is 64 years in the US [3].

Unmodifiable and modifiable risk factors are often related: RCC is doubly diagnosed in men where some modifiable risks such as hypertension, tobacco smoking and obesity are more frequently observed.

RCC originates from kidney cortex, in particular from the epithelial cells of renal tubular [3,4] and is commonly classified into three main histopathological subtypes: (1) clear cell renal cell carcinoma (ccRCC) represents the 75% of all RCC cases and the 90% of all RCC deaths due to tendency to metastasize [2]; it derives from renal cancer progenitors, namely RCC cancer stem cells (CSCs) and it is often associated with distant disease to bones, liver and lung through hematologic way [4]; (2) around 10% of diagnosis consists in papillary renal cell carcinoma (pRCC) characterized by histological sub-classification in type I and II, where the first has a tropism for lymphatic recurrence; (3) chromophobe renal cell carcinoma (chRCC) is the less aggressive subtype and represents the 5% of all RCC [4]. The RCC stage at diagnosis is an important prognostic factor. Indeed, for organ-confined tumors (stage I) over the 90% of individuals survive at 5 years from onset, while the survival percentage is reduced to 72,5% for regional disease (stage II/III). Unfortunately, about 30% of RCC are diagnosed at metastatic disease (stage IV) where the prognosis is poor with a 5-year survival rate of 12% [4].

2. RCC treatments: advances and challenges in the lack of biomarkers

RCC is a chemo- and radio-resistant neoplasia. The current therapeutic strategies are based on the surgical approach of radical and/or partial nephrectomy that remain the only effective therapy for clinically localized RCC [5]. Traditionally, these tumors have been treated aggressively, most often with radical nephrectomy, however it predisposes patients to chronic kidney disease with attendant cardiovascular risks and increased mortality [6]. Nephron-sparing approaches such as partial nephrectomy, thermal ablation and active surveillance have also emerged as viable options for the management of these patients [7]. Treatment options are thus individualized and related to disease features (above all tumor stage, size and grade) [2,4,8], weighing the risks and benefits of partial versus radical nephrectomy. Nevertheless, RCC is characterized by a high rate of relapse and unfortunately about 20-50% of those treated with curative intent will progress to stage IV. Features related to a worse prognosis are the tumor size (>7cm), the tumor necrosis, a fat invasion, the performance status (KPS < 80) together with nodal metastases and age [4]. The lack of sensitivity to chemotherapy and radiation therapy prompted efforts into novel treatment options. In the last few years, the therapeutic approach for metastatic RCC (mRCC) in the first-line setting is radically changed due to novel combinatory approaches impacting on both immunity and intracellular molecular pathways inhibition.

Those approaches are targeting the peculiar hyper-vascularity of RCC and trigger the immune cell infiltration by immune checkpoint inhibitors, with or without anti-angiogenic tyrosine kinase inhibitor (TKI)-based combinations [9]. Thus, the current standard of care for frontline advanced ccRCC includes an “immuno-oncology” (IO) combination that may include a vascular endothelial growth factor (VEGF) inhibitor with a checkpoint inhibitor (VEGF-IO) or of two checkpoint inhibitors together (IO-IO) that showed efficacy and safety [10,11]. Interestingly, the anti-angiogenic TKI in RCC are given without actionable mutations to screen. They are oral drugs able to block angiogenesis pathway by binding VEGF receptor, c-MET, RET. For RCC, TKIs can be distinguished in three generations drugs, according to spectrum of action and side effects: sunitinib, pazopanib and sorafenib (first-generation); axitinib and tivozanib (second-generation) and cabozantinib (third-generation) [12].

ICIs can stimulate the host immune response against cancer, through PD1 inhibitors (Nivolumab and Pembrolizumab) or anti-CTLA4 inhibitors (Ipilimumab). The first group can block the interaction PD1/PD-L1 able to downregulate the host T cells and therefore the immune response [11]. Many studies investigated the role of PD-L1 expression as a biomarker, such as Iacovelli et al [13] who performed a meta-analysis of six studies on 1323 patients, showing a high level of PD-L1 expression associated with a higher risk of death. Indeed, PD-L1 expression may be considered as a negative prognostic factor in RCC [14].

However, the response and tolerance to these combination treatments are not always satisfying, probably due to the lack of recognized molecular targets, an intra-tumoral heterogeneity and different RCC molecular subtypes.

The therapeutic strategy is based on the clinical prognostic score (IMDC, MSKK criteria), drug toxicities, patients’ comorbidities, and preference. In fact, nowadays the International mRCC Database Consortium risk model remains the only prospectively known predictive biomarker in mRCC [15].

Although targeted therapies represent the standard treatment options for RCC, nearly all patients treated with targeted drugs will eventually experience disease progression. Unfortunately, a significant number of RCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilization nor clinical benefits, probably due to the intra-tumoral heterogeneity and molecular subtypes [15,16].

Therefore, the identification of new promising biomarkers in RCC is an urgent clinical need that leads clinicians and researchers to investigate the role of cell components as microRNAs secreted by tumor cells and packaged into extracellular vesicles (EVs) released in biological fluids [17].

3. Liquid biopsy in RCC

For decades, surgical tissue-biopsy has firmly been considered the gold standard for solid tumor diagnosis, however it may become a diagnostic obstacle for its invasiveness, unrepeatability and especially for its peculiarity to immortalize that moment without taking into account the tumor heterogeneity and dynamism [18]. The molecular tumor pattern can dynamically evolve over time, driven by microenvironmental stimuli and clonal selection under treatment pressure, rendering future therapeutic decisions based on tissue biopsy suboptimal [19,20].

Over the past decade, liquid biopsy (LB) revolutionized the field of clinical oncology, providing the ease in tumor sampling and continuous tumor monitoring [21].

Thus, LB is gaining relevance in the clinical scenario in cancer for early detection and treatment stratification, as well as residual disease and recurrence monitoring [20].

In particular, overall survival in mRCC patients remains low despite the development of novel targeted therapeutic approaches. LBs could provide an attractive and non-invasive method to determine the risk of recurrence or metastatic dissemination during follow-up and thus improve clinical outcomes and quality of life of RCC patients [22]. Several biological body fluids, such as blood, urine, saliva, breast milk and other patient samples, can be collected and used for clinical investigations, as they contain numerous biomarkers. Peripheral blood, as well as other biological fluids, can be a source of cancer material, such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and tumor-derived EVs [23] (Figure 1).

The identification of biomarkers in LB could enable continuous and real-time collection of RCC patients’ information for diagnosis, prognosis assessment, treatment monitoring and response evaluation [24].

Many studies have attempted to use LB as a routine method for the clinical diagnosis of RCC and for the prediction of patients’ grades, stages, and survival to identify high-risk patients for metastasis and recurrence [25,26,27,28,29]. In particular, in recent decades most efforts have been spent in analyzing CTCs and ctDNA [30,31], however their low level in the blood makes their capture and analysis very difficult [24].

EVs and miRNAs are emerging as a platform with potentially broader and complementary applications in LB [32].

4. Extracellular vesicles: biogenesis and role in RCC

EVs are small lipid bilayer-enclosed vesicles, released continuously by all viable cells, in physiological and pathological conditions [33]. According to ISEV guidelines, the term EVs includes three different subtypes of vesicles based on their biogenesis and size [34]: (a) exosomes have a diameter of 30-150 nm and originate by the fusion between intracytoplasmic multi-vesicular bodies (MVB) and cellular plasma membrane; (b) microvesicles with a size of 100-1000 nm generate directly from the plasma membrane by budding process; (c) apoptotic bodies are larger than 1000 nm and are released by dying cells [35] (Figure 2). A list of minimal information for studies of EVs was provided from MISEV-2014 [36] and updated in MISEV-2018 [35] pointing out EVs separation/isolation and characterization techniques and functional studies.

EVs can transport tumor-derived material, including lipids, a wide variety of RNA (microRNAs, mRNAs and long non coding-RNAs), DNA, proteins, enzymes and metabolites. They play a key role in cell-to-cell communication [37] by transferring their information to nearby or long distance recipient cells, influencing their phenotype and functions [38].

These small messengers are involved in tumorigenesis and tumor metastasis due to their widespread distribution throughout the blood and lymphatic circulation. Several studies indicate that tumor released EVs may orchestrate tumor progression stimulating proliferation, angiogenesis, chemo-resistance and immune-escape [19,39,40]. In addition, EVs are highly stable in biological fluids and protect their content from RNase and protease activity thanks to their lipid bilayer structure [41].

Recently, EVs showed an increasing interest in the field of urological malignancies [42]. A growing number of studies are focusing on EV cargoes, particularly miRNAs, in relation to diagnostic accuracy, treatment prognosis, treatment response as well as numerous biological processes [43,44].

The recent knowledge is related to the biological role of EVs shed by RCC tumor cells in RCC progression, such as angiogenesis, immune escape and tumor growth [45].

Horie K et al analyzed the hypoxic conditions in RCC that stimulate the release of tumor cells-derived EVs. Their results suggested the possibility that carbonic anhydrase 9 (CA9) enriched in exosomes and released from hypoxic RCC may enhance angiogenesis in microenvironment, thereby contributing to cancer progression [46]. In addition, RCC-EVs were studied for their modulation of vascular permeability affecting metastatization, thanks to their enrichment in the azurocidin protein (AZU1) significantly higher in serum EVs from ccRCC patients compared to those from healthy donors [47].

More, specific pro-tumorigenic roles have been attributed to EVs released by CD105+ renal CSCs, due to their cargo enriched in several pro-angiogenic mRNAs and miRNAs [48]. Those cargos can also impact other tumor microenvironmental cells, as demonstrated by Lindoso et al, considering the participation of renal CSC-derived EVs in the interaction between tumor and stroma. They found that CSC-derived EVs promoted phenotypical changes in stromal cells characterized by an increased expression of genes associated with cell migration, matrix remodeling, angiogenesis and tumor growth [49]. Grange et al. additionally demonstrated the impact of CSC-derived EVs isolated from RCC as main mediators for additional tumor microenvironment element, impacting on monocyte differentiation into dendritic cells [50].

A specific role for renal CSC-derived EVs has been additionally described for the formation of the pre-metastatic niche, which consist of a complex network of information exchanges. These EVs may sustain an unfavorable outcome of the tumor by enhancing tumor vascularization and by contributing to the establishment of a pre-metastatic niche [48]. More recently, it has been demonstrated that renal CSC EVs were enriched by miRNAs influencing cell growth, tumor invasion and metastases, thus giving to circulating miRNAs a promising role of biomarkers in RCC [45].

5. Conclusions

RCC is considering one of the most unfavorable tumor disease, due to the late diagnosis and the poor prognosis. Nowadays, specific biomarkers validated for RCC early detection are not available and the actual treatments are often unable to avoid recurrence of disease. LB could provide an attractive and non-invasive tool to assist the research for biomarkers in RCC tumors to capture a larger amount of the molecular heterogeneity compared to tissue biopsy and give prompt information on the risk of recurrence/relapse during follow-up.

Many efforts are directed towards the research for circulating miRNAs that play a role in almost all aspects of cancer biology and development. In particular, miRNAs packaged in EVs and released in blood flow could allow to expand the spectrum of potential biomarkers for future using in diagnosis and prognosis of RCC and in prediction of therapeutic response.

However, nowadays there are no miRNAs widely applied as biomarker in the clinical setting, partly due to the lack of isolation and quantification standardized protocols, the heterogeneity of the study cohorts and the variety of body fluids under investigation. Several studies analyzed plasma and serum-derived miRNAs, without focusing on the vesicle compartment. EV-derived miRNAs appear to be more stable than free miRNAs, as EVs seem to protect them from degradation by macrophages. The EVs double-layered membrane and nanoscale size allow the miRNA stability, thus prolonging their circulation half-life and enhancing their biological activity [84]. After release, EVs are taken up by neighboring or distant cells, and the miRNAs encapsulated modulate several processes, as interfering with tumor immunity and the microenvironment, possibly facilitating tumor growth, invasion, metastasis, angiogenesis and drug resistance [85]. Therefore, EV-derived miRNAs have a significant function in regulating cancer progression. More investigations on this matter are warranted. Nonetheless, the most exciting but challenging application will be to utilize EVs and their cargo as a clinical tool to diagnose and monitor disease. Thus, EV-derived miRNAs could provide a novel therapeutic tool in RCC clinical practice, where the lack of adequate biomarkers makes functional investigations urgently needed.