Extracellular vesicles as a novel source of biomarkers in liquid biopsies for monitoring cancer progression and drug resistance
Introduction
Resistance of cancer cells to chemotherapeutic agents, molecular targeted therapies or immunotherapy, may be either intrinsic or acquired (Gottesman, 2002; Gottesman et al., 2016; Kalbasi and Ribas, 2019; Kelderman et al., 2014; Assaraf et al., 2019) and is often ultimately responsible for cancer patient’s reduced survival (Holohan et al., 2013). Some tumors present multidrug resistance (MDR), having cross-resistance to various anticancer drugs (Gottesman, 2002). Causes of drug resistance (Housman et al., 2014) may be categorized as host factors, tumor factors and tumor-host interactions (Alaoui-Jamali et al., 2004; Alfarouk et al., 2015; Assaraf et al., 2019). Host factors include genetic variants and drug-drug interactions (Mukerjee et al., 2018; Riechelmann and Del Giglio, 2009; Riechelmann et al., 2007). Tumor factors include mostly alterations in drug targets (Yaghmaie and Yeung, 2019), activation of prosurvival pathways (Indran et al., 2011), decreased intracellular drug concentration (caused by decreased drug influx, increased efflux, or drug sequestration in intracellular vesicles and compartments) (Goler-Baron and Assaraf, 2011; Gonen and Assaraf, 2012; Gong et al., 2013; Peetla et al., 2013; Robey et al., 2018), enhanced DNA damage repair (Buys et al., 2017; Sakthivel and Hariharan, 2017), epigenetic alterations (Brown et al., 2014; Kagohara et al., 2018) and/or deregulation of microRNAs (An et al., 2017), intratumor heterogeneity and dynamics (Burrell and Swanton, 2014; Turajlic et al., 2019), as well as the presence of cancer stem cells (Doherty et al., 2016; Mansoori et al., 2017; Najafi et al., 2019; Turdo et al., 2019). Tumor-host interactions refer to interactions of the tumor cells with the tumor microenvironment (TME) and the intracellular transfer of traits mediated by extracellular vesicles (EVs) (Junttila and de Sauvage, 2013; Namee and O’Driscoll, 2018; Samuel et al., 2017; Wu and Dai, 2017).
The ability to frequently monitor cancer progression and early diagnose tumor drug resistance could contribute to informed clinical decisions and personalized treatment of cancer. This may be possible with liquid biopsies, given that liquid biopsies might allow to survey tumor heterogeneity and clonal evolution (Babayan and Pantel, 2018; Liebs et al., 2019). The term “liquid biopsy” refers to a test performed on a sample of biofluid including blood or urine aiming to detect cancer cells or cancer-derived molecules (Babayan and Pantel, 2018). Liquid biopsies are obtained by non-invasive or minimally invasive means that allow serial sampling, therefore they have a potential utility for the detection of minimal residual disease (MRD) or recurrence, tracking tumor evolution and predicting the emergence of chemoresistance in solid tumors and hematological malignancies. Currently, the most widely studied analytes of liquid biopsies are circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and circulating tumor RNA (ctRNA) (Babayan and Pantel, 2018; Kawaguchi et al., 2016). Some of the CTC and ctDNA tests, such as CTC enumeration by CellSearch® technology or Cobas EGFR Mutation test have been approved by the regulatory authorities and are available for clinical use. However, there are many scientific, technological and regulatory challenges that still need to be overcome in order to introduce liquid biopsies into the standard clinical workflows (Ossandon et al., 2018; Reimers and Pantel, 2019).
The EVs have recently emerged as novel analytes for liquid biopsies. The term “EVs” refers to all kinds of particles naturally released from cells that are delimited by a lipid bilayer and cannot replicate (Thery et al., 2018). Various subtypes of EVs differ in their biogenesis, size and physical properties, molecular composition and functions in the body. In live cells, the main pathways for EV biogenesis are the release of the intraluminal vesicles of multivesicular endosomes in the extracellular space and direct budding from the cell surface. EVs of endosomal origin are called exosomes and the majority of them range between 30–150 nm in diameter, while vesicles derived by budding of plasma membrane may reach 1000 nm in diameter and have been referred to as microvesicles, ectosomes, shedding vesicles or microparticles (Colombo et al., 2014; Vestad et al., 2017). Apoptotic cells can also release a variety of EVs by blebbing of apoptotic membrane, formation of membrane protrusions such as microtubule spikes, apoptopodia, and beaded-apoptopodia. Apoptotic cell-derived EVs are commonly referred to as apoptotic bodies, and the majority of them are in size range from 1 to 5 μm in diameter, though the formation of smaller vesicles during the progression of apoptosis has also been reported (Caruso and Poon, 2018). Although the mean size of various EV subtypes is different, accurate separation of EV subtypes based on the size, biochemical properties or surface markers is currently not feasible. Therefore, the International Society for Extracellular Vesicles (ISEV) recommends using operational terms for EV subtypes such as size, density, marker profile etc., instead of using terms like exosomes or microvesicles, unless their origin is clearly established (Thery et al., 2018).
EVs are released by virtually all cell types in the body and they have been found in various biofluids, including blood, urine, saliva, bile and cerebrospinal fluid (Murillo et al., 2019; Yanez-Mo et al., 2015). EVs contain various lipids (Skotland et al., 2017), proteins (Vagner et al., 2019), metabolites (Royo et al., 2019), mRNA fragments and non-coding RNAs (Turchinovich et al., 2019) and even DNA fragments (Vagner et al., 2018). Importantly, EVs contain molecular signatures reminiscent of their cell of origin (Broggi et al., 2019; Platko et al., 2019). EVs isolated from cancer patients’ biofluids have been shown to contain cancer-associated molecules such as amplified oncogenes, oncoproteins, specific miRNA signatures, and mutated mRNA or DNA fragments (Al-Nedawi et al., 2008; Broggi et al., 2019; Garcia-Silva et al., 2019; Lazaro-Ibanez et al., 2014). Moreover, it is increasingly recognized that cancer-derived EVs contribute to cancer progression via transferring phenotypic traits among cancer cells and mediating crosstalk with the TME, pre-metastatic niche and immune system (Becker et al., 2016; Whiteside, 2017b). These findings have raised the idea that the analysis of molecular content of EVs could inform about the presence, molecular profile and behavior of cancer and therefore they could serve as liquid biopsies. In this review, we summarize studies investigating EVs or their cargo as biomarkers for tracking cancer dynamics and predicting drug resistance and discuss the advantages, limitations and challenges of exploiting EVs as liquid biopsies of cancer.
Section snippets
EV levels in biofluids – a marker on its own?
There is conflicting evidence regarding the levels of EVs in cancer patients as opposed to healthy controls (Cappello et al., 2017; Xu et al., 2018). Some studies show that cancer patients have increased EV levels in their blood (Alegre et al., 2016; Caivano et al., 2015; Duijvesz et al., 2015; Logozzi et al., 2009; Matsumoto et al., 2016; Nawaz et al., 2014; Ogorevc et al., 2013; Rodriguez Zorrilla et al., 2019). Moreover, several studies have shown that levels of plasma EVs may be associated
Sorting of molecular cargo into EVs
One of the main features that makes EVs particularly attractive as analytes for liquid biopsies is that tumor-derived EV’s cargo bares a strong pathological resemblance to the intracellular status of the cancer cell of origin (Baran et al., 2010; Rabinowits et al., 2009; Szajnik et al., 2013; Tanaka et al., 2013). However, several recent studies suggest that the sorting of molecular cargo into EVs is a regulated process leading to the enrichment or depletion of EVs in specific nucleic acids,
EVs as analytes of liquid biopsies
A growing body of evidence suggests that cancer-derived EVs propagate drug-resistant phenotypes and facilitate cancer progression by transferring aggressive phenotypic traits to other cancer cells, modulating the anti-tumor immune response, remodeling the TME and promoting the formation of pre-metastatic niches (Fig. 1). These effects can be triggered by internalization of EVs and release of their cargo into recipient cells, where they initiate various intracellular signaling events, or by
Clinical relevance of EVs for monitoring cancer progression
Despite overwhelming evidence on the contribution of EVs to cancer progression, so far only a limited number of clinical studies have reported EV-associated biomarkers for monitoring cancer progression and tracking cancer evolution. As preclinical studies demonstrating the role of EVs in cancer progression have been recently reviewed in several excellent recent articles (Kikuchi et al., 2019; Milman et al., 2019; Zhang and Yu, 2019), here we focused on longitudinal clinical studies reporting
Pre-clinical evidence on the relevance of EVs for the prediction of drug resistance
The role of EVs shed by drug-resistant tumor cells on the protection of recipient cancer cells upon drug treatment has been reported in different cancer cell types (Table 2). For example, in CRC, EVs derived from cetuximab-resistant cell lines induced resistance in sensitive cell lines, by downregulating PTEN expression and increasing the phosphorylation of Akt levels (Zhang et al., 2017). Moreover, EVs released by mesenchymal NSCLC cell lines transferred their MDR phenotype to the parental
Clinical evidence for the relevance of EVs in the prediction of drug resistance
An overwhelming amount of evidence, corroborated by multiple research groups, suggests that EVs derived from drug-resistant cancer cells and/or cells present in the TME, are causally involved in disseminating a drug-resistance phenotype in clinical context (Chen et al., 2018; Corcoran et al., 2012; Martinez et al., 2017; Mikamori et al., 2017; Ning et al., 2017; Soldevilla et al., 2014; Wang et al., 2014a; Wei et al., 2017a). Importantly, these alterations in circulating EVs cargo, responsible
EVs in diagnostics and therapeutics: from the bench to bedside
With a plethora of EV-based technologies just over the horizon, it is of utmost importance to fulfil their potential into clinically useful enabling tools for diagnosis and treatment of cancer patients. Indeed, EVs-derived products have the potential to fulfil two major unmet clinical needs in a new era of precision medicine: their use as (i) biomarkers for diagnosis, prognosis, prediction of drug response and monitoring tumor evolution and as (ii) naturally engineered drug-delivery magic
Perspectives and future challenges
Collectively, these studies have provided proof of concept that EVs can be used for real-time monitoring of tumor burden, tracking cancer evolution, predicting response to treatment and monitoring the emergence or progression of drug resistance. However, most of the clinical studies were based on very small sample size, therefore the identified biomarkers must be validated in large longitudinal studies before they can be translated into clinically applicable tools. Moreover, the future of
Declaration of Competing Interest
AL, AĀ and CX have no conflict of interest to disclose. MHV and HC are members of the research team of a project financed by Celgene and MHV is member of the team of a grant co-financed by AMGEN. These companies had no role in the decision to publish nor were they involved in the writing of this manuscript.
Acknowledgements
This article is based upon work from COST Action 17104 STRATAGEM, supported by COST (European Cooperation in Science and Technology).
The Vasconcelos group is supported by FEDER – Fundo Europeu de Desenvolvimento Regional through COMPETE 2020 and by FCT - Foundation for Science and Technology, in the framework of project POCI-01-0145-FEDER-030457.
The Linē group is supported by the Latvian Council of Science Project No. lzp-2018/0269 and ERA-NET TRANSCAN-2 project PROSCANEXO.
Artūrs Ābols is
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