**Extracellular Vesicles: Biology and Potentials in Cancer Therapeutics**

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**William C. S. Cho**

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## **Contents**

Tiny Actors in the Big Cellular World: Extracellular Vesicles Playing Critical Roles in Cancer Reprinted from: *Int. J. Mol. Sci.* **2020**, *21*, 7688, doi:10.3390/ijms21207688 .............. **165**

## **About the Editor**

**William C. S. Cho**'s main research interests focus on cancer studies to discover biomarkers for cancer diagnosis, treatment prediction, and prognostication. As a seasoned researcher, Dr Cho has conducted cancer research using molecular biology, proteomics, genomics, immunology, as well as bioinformatics; his current H-index is 60. Dr Cho has published over 400 peer-reviewed papers (e.g., *Lancet Oncology, Annals of Oncology*, *Journal of Extracellular Vesicles*, *Advanced Science*, *Nature Communications*, *PNAS*, *Journal of the National Cancer Institute*, *Clinical Cancer Research*, *Clinical Chemistry*, *Molecular Cancer*, *Theranostics*, etc.) covering cancer biomarkers, proteomics, non-coding RNAs, and Chinese medicine, and these papers have received over 18,000 citations. Dr Cho has also composed 30 book chapters and edited 20 academic books, including "MicroRNAs in Cancer Translational Research", "An Omics Perspective on Cancer Research", "Supportive Cancer Care with Chinese Medicine", "Drug Repurposing in Cancer Therapy: Approaches and Applications", etc.

## *Editorial* **Extracellular Vesicles: Biology and Potentials in Cancer Therapeutics**

**William C. S. Cho**

Department of Clinical Oncology, Queen Elizabeth Hospital, Hong Kong, China<EMAIL_ADDRESS>
#### **Background**

Extracellular vesicles (EVs) are particles wrapped in a lipid bilayer membrane and are naturally released from cells. This kind of cargo vessel is a nanostructure, which mainly transfers lipids, proteins, various nucleic acid fragments, and metabolic components to neighboring cells or distant parts of the body through the circulatory system. EV is of great significance to the communication mechanism between cells. This Special Issue aims to collect articles to enhance our understanding of the biological characteristics of EVs and their potential applications. It features a set of high-impact articles from leading experts in the field. Through a rigorous peer-review process, a total of nineteen articles were accepted, including eight original research articles, ten review articles, and one communication article.

#### **Biology of EVs**

Menck et al. [1] summarized the current knowledge about the biology and composition of microvesicles (MV), as well as their role in the tumor microenvironment (TME). Increasing evidence shows that although MVs are biologically different, they have the tumor-promoting properties of endosomal-derived small exosomes in TME. Due to their larger size, they can be easily collected from the patient's blood and characterized by conventional flow cytometry using excess molecules expressed on their surface.

Zmigrodzka et al. [ ˙ 2] reviewed the biogenesis and cargo molecules of platelet extracellular vesicles (PEV), as well as their effects on cancer progression. During platelet activation or apoptosis, PEV is formed, which presents a highly heterogeneous EV group and is the most abundant EV group in the circulatory system. Since the role of platelets in cancer development is well known, and PEV is the most abundant EV in the blood, its possible impact on cancer growth has been strongly discussed. The crosstalk of PEV can promote proliferation, change TME, and promote the formation of metastasis. In many cases, these functions are related to specific cargo molecules transferred from PEV to recipient cells.

Today, in multiple studies exploring TME, the interaction between tumors and macro phages is certainly interesting. Among them, tumor-associated macrophages include a subgroup that has a variety of tumor-promoting effects (including general immunosuppression) in tumor development, which can be identified based on the high expression of mannose receptor/CD206 [3].

Radiation affects not only target cells, but also neighboring cells that have not been exposed to radiation. This response is described as radiation-induced bystander effects (RIBE). Molecular communication between irradiated and unirradiated adjacent cells can trigger RIBE and out-of-field (distance) effects. Cagatay et al. [4] evaluated the changes in the spectrum of exosomes and the role of exosomes as possible molecular signaling mediators for radiation damage. After 24 hours and 15 days of irradiation, exosomes derived from the whole body or partial body of irradiated mouse organs were transferred to recipient mouse embryonic fibroblast (MEF) cells. The changes in cell viability, DNA damage and calcium, reactive oxygen species, and nitric oxide signals were compared with exosome-treated MEF cells from unirradiated mice. Their results showed that whole and

**Citation:** Cho, W.C.S. Extracellular Vesicles: Biology and Potentials in Cancer Therapeutics. *Int. J. Mol. Sci.* **2021**, *22*, 9586. https://doi.org/ 10.3390/ijms22179586

Received: 24 August 2021 Accepted: 1 September 2021 Published: 3 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

local irradiation would increase the number of exosomes and induce changes in MEF cells treated by exosomes.

#### **EV Isolation and Characterization**

Many isolation techniques typically discard large EVs in the early stage of sEV or exosome isolation protocols. Johnson et al. [5] described the stepwise separation and characterization of large EV subsets in a medulloblastoma cell line using fluorescent light microscopy, transmission electron microscopy, and tunable resistance pulse sensing. They developed a labeling and strict gating strategy to explore the expression of EV markers (CD63, CD9, and LAMP 1) on a single EV in a wide heterogeneous population. Their data strongly support the exploration of large EVs in clinical samples to obtain potential biomarkers, which are very useful in diagnostic screening and disease monitoring.

EV concentrations often result in low final yields or severe contamination of vesicle samples, which greatly limits further applications and data reproducibility, and contamination greatly affects a wide range of functional studies. Simon et al. [6] described a new combination of three well-known methods (size exclusion chromatography (SEC), Western blotting, and transmission electron microscopy) to obtain medium to high yields of EVs while reducing protein contamination. They believe that this method may have great benefits for in vitro and in vivo functional studies.

Efforts have also been made to standardize the separation and characterization techniques of sEV. Current protocols often result in the co-separation of soluble proteins or lipid complexes with other EVs. Bordas et al. [7] reported an optimized protocol for the isolation of sEV from human and murine lymphoid tissues. To separate sEV from freshly resected human lymph nodes and mouse spleen, two different methods were compared: (1) ultracentrifugation on a sucrose density pad and (2) a combination of ultracentrifugation and SEC. The purity of sEV preparations was analyzed using Western blotting, nanoparticle tracking analysis, and electron microscopy. Their results clearly demonstrated the superiority of SEC in improving the yield and purity of sEV.

On the other hand, it is questionable whether clinical laboratories can conduct indepth research through flow cytometry to evaluate EV surface cargo in various diseases. Lucchetti et al. [8] reported the difficulty of evaluating small and medium-sized EVs through traditional flow cytometry. Running a sample of medium EVs stained with equal amounts of Calcein-green and Calcein-violet, they found that the cluster detection produced false double positive events. This phenomenon was significantly reduced by sample dilution, but it was not completely eliminated. In addition, running highly diluted samples required a longer cytometer time. Their findings question the routine applicability of traditional flow cytometry in EV analysis.

#### **Therapeutic Potential of EVs**

It is well known that the potential use of EVs as therapeutic agents lies not only in their cell membrane-bound components but also in their cargo. Jurj et al. [9] highlighted the characteristics of EV involvement in cancer cells, paying special attention to those molecular processes that were affected by EV cargo. In addition, they explored the role of RNA types and proteins carried by EVs in triggering the drug resistance phenotype. Interestingly, in various in vivo and in vitro studies and multiple clinical trials, engineered EVs have been proposed as therapeutic agents.

The composition of exosomes and the possibility of interacting with cells make exosomes a multifaceted regulator of cancer development. Lorenc et al. [10] discussed the role of tumor-derived exosome (TEX) in the progression of prostate cancer and the potential use of exosomes in the management of prostate cancer. The biophysical properties of EVs (such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity) are the key to the successful development of innovative drug delivery systems. EV has enhanced circulation stability and biological barrier permeability, so it can be used as an effective chemotherapy carrier to improve the regulation of target tissues and organs. Exosomes can deliver different types of cargo and target specific cells. They may help deliver chemotherapy drugs, natural products, and RNA-based cancer gene therapy [11].

The combination of ultrasound and microbubbles has been shown to trigger cancer cells to release EVs. Yuana et al. [12] used microbubbles-assisted ultrasound (USMB) to load the model drug CellTracker green fluorescent dye (CTG) or bovine serum albumin coupled with fluorescein isothiocyanate (BSA FITC) into primary human endothelial cells in vitro. They found that USMB loaded CTG and BSA FITC into human endothelial cells and triggered the release of EVs containing these compounds in the cell supernatant within two hours after treatment. The amount of EV released seems to be related to the increase in ultrasound acoustic pressure. They concluded that USMB can load model drugs into endothelial cells and trigger the release of EV-carrying model drugs, highlighting the potential of EV as a drug nanocarrier for future cancer drug delivery.

Sun et al. [13] elaborated about the role of EV in the development of cancer therapeutics. They emphasized that some EVs (such as tumor exosomes) have a tumor-homing tendency, which has led to the use of EV as a drug carrier to effectively provide cancer treatment. The results of preclinical research and early clinical trials were mainly reviewed. For example, a phase I clinical trial designed to evaluate the ability of plant sEVs to prevent oral mucositis during chemotherapy and radiotherapy of head and neck cancer revealed the potential of using plant sEVs to reduce side effects during cancer treatment. It was also reported that isolating fibroblast-like mesenchymal cell-derived sEV and loading siRNA or shRNA targeting *KRAS* mutation (*KrasG12D*) could more effectively inhibit the progression of pancreatic ductal adenocarcinoma in vitro and in vivo compared with other drug carriers.

Interestingly, human breast milk (HBM) is an irreplaceable source of nutrition for early infant growth and development. Kim et al. [14] reviewed the various components of HBM (especially exosomes and miRNA) and their therapeutic potential for cancer. Milk-derived exosomes play a variety of physiological and therapeutic functions in cell proliferation, inflammation, and immune regulation, mainly due to their cargo molecules (such as proteins and miRNA). Exosomal miRNA is not affected by enzymatic digestion and acidic conditions, and it plays a key role in immune regulation and cancer. In addition, milk-derived exosomes have been developed as drug carriers for the delivery of small molecules and siRNA to tumor sites.

Although cancer treatments encounter physiological obstacles in TME, they must be delivered to their target to improve efficacy and reduce toxicity. Choi et al. [15] summarized the biological function and therapeutic potential of exosomes as diagnostic biomarkers and drug delivery vehicles for cancer treatment. They also explored whether exosomes could be used as effective nanocommunicators to promote drug design for personalized cancer immunotherapy.

Despite the tremendous technological advances in the treatment of head and neck squamous cell carcinoma (HNSCC), the overall survival rate is usually low. Ebnoether and Muller [16] summarized the diagnostic and therapeutic applications of exosomes in HNSCC. They also reviewed the impressive preliminary results obtained in recent studies.

Since TEX is involved in the crosstalk between cancer and immune cells, it plays a key role in suppressing the anti-tumor immune response to promote tumor progression. Most of the available information about the molecular composition and function of TEX was obtained using small EV (sEV) isolated from the supernatant of cancer cell lines. However, new data linking plasma TEX levels to cancer progression focuses on TEX in the peripheral circulation of patients as a potential biomarker for cancer diagnosis, development, activity, and response to treatment. Zebrowska et al. [17] published an article on the signaling of melanoma cell-derived sEV, with particular emphasis on exosome-mediated signal transduction between melanoma cells and the host immune system. They argue that the signaling of TEX may impact melanoma progression.

Dobra et al. [18] initiated the first proteomic study comparing whole-serum and serumderived sEV. They aimed to determine the characteristic protein fingerprints associated with

central nervous system (CNS) tumors. A total of 96 human serum samples were obtained from four patient groups (glioblastoma multiforme, brain metastases from non-small-cell lung cancer, meningioma, and lumbar disc herniation). Among the 311 identified proteins, 10 whole serum proteins and 17 serum-derived sEV proteins showed the highest difference between each group. A total of 65 proteins were significantly enriched in serum-derived sEV samples, while 129 proteins were significantly reduced compared with whole-serum sEV samples. Based on principal component analysis, serum-derived sEV was more suitable for distinguishing patient groups and has a greater potential for monitoring CNS tumors than whole-serum sEV.

Glioblastoma is a devastating disease, and there is an urgent need for biomarkers that can predict prognosis. Tzaridis et al. [19] analyzed a set of serum microRNAs (miRNAs) in the EV from glioblastoma patients and examined their relevance to the prognosis of these patients. Using RT-qPCR and CD44 immunoprecipitation (sinusoidal endothelial cell + CD44), the levels of 15 miRNAs in EVs separated by SEC were compared with those of glioblastoma patients and healthy volunteers. Combining miR-15b-3p in serum or miR-106a-5p in CD44 immunoprecipitation EVs with any of the other three miRNAs in CD44 immunoprecipitation EV could stratify the prognosis of glioblastoma patients.

Transporting the biologically active cargo of MVs to target cells can affect their fate and behavior and change their microenvironment. Szyposzynska et al. [20] evaluated the biological activity of MV derived from human immortalized mesenchymal stem cells of adipose tissue-origin (HATMSC2-MVs) against two ovarian cancer cell lines ES-2 and OAW-42. They prove that HATMSC2-MVs can effectively metastasize to ovarian cancer cells and inhibit cell proliferation, apoptosis, and/or necrosis through different pathways, which may be related to the presence of different anti-tumor factors secreted by ES-2 and OAW-42 cells.

#### **Conclusions**

We hope that all these efforts will improve our understanding of the EV world and provide some insights into its potential clinical applications. With its multiple encapsulated macromolecules and powerful extraction platform, we envision a more comprehensive understanding of EV biology through multi-omics approaches.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

## *Review* **Microvesicles in Cancer: Small Size, Large Potential**

### **Kerstin Menck 1,**†**, Suganja Sivaloganathan 1,**†**, Annalen Bleckmann 1,2 and Claudia Binder 2,\***

Received: 30 June 2020; Accepted: 27 July 2020; Published: 28 July 2020

**Abstract:** Extracellular vesicles (EV) are secreted by all cell types in a tumor and its microenvironment (TME), playing an essential role in intercellular communication and the establishment of a TME favorable for tumor invasion and metastasis. They encompass a variety of vesicle populations, among them the well-known endosomal-derived small exosomes (Exo), but also larger vesicles (diameter > 100 nm) that are shed directly from the plasma membrane, the so-called microvesicles (MV). Increasing evidence suggests that MV, although biologically different, share the tumor-promoting features of Exo in the TME. Due to their larger size, they can be readily harvested from patients' blood and characterized by routine methods such as conventional flow cytometry, exploiting the plethora of molecules expressed on their surface. In this review, we summarize the current knowledge about the biology and the composition of MV, as well as their role within the TME. We highlight not only the challenges and potential of MV as novel biomarkers for cancer, but also discuss their possible use for therapeutic intervention.

**Keywords:** microvesicles; biomarker; cancer; tumor microenvironment; therapy

#### **1. Introduction**

Tumor development is a multistep process that is accompanied by various cellular reprogramming events in which cells acquire the hallmarks of cancer cells to gain and sustain abnormal growth and invasive capacity [1]. The complex process of tumor formation and spreading not only depends on the tumor cells themselves but also requires rewiring of the surrounding stromal cells. This can be induced by cell intrinsic events (genetic or epigenetic aberrations) or by external factors originating from direct cell–cell contact or from indirect cell communication. While soluble factors, such as chemokines, cytokines, and growth factors, have long been known for their role during tumor development, extracellular vesicles (EV) have recently attracted increasing attention as mediators of intercellular communication. Conserved from prokaryotes to eukaryotes, the secretion of EV is observed in all cell types [2]. The term EV encompasses all types of secreted membrane-enclosed vesicles, which are highly heterogeneous. On the basis of various characteristics, ranging from size, biogenesis, cell of origin, morphology, and content, EV are categorized into four main classes: endosomal-derived small exosomes (Exo) (50–150 nm), plasma membrane-derived middle-sized microvesicles (MV) (100–1000 nm), and large oncosomes (LO) (1000–10,000 nm), as well as apoptotic bodies (500–4000 nm) that are released from dying cells [3,4]. Although commonly used to categorize EV, this classification has been challenged by recent evidence demonstrating that, for instance, the Exo contain further subtypes with different biological and biochemical properties [5]. In order to avoid terminological ambiguities, the term "small EV" was coined for EV harvested at > 100,000 g. However, since most authors still use the term Exo, we will adhere to this nomination throughout the review.

In cancer, EV have been shown to be essential for various steps during tumor initiation and progression. By horizontal transfer of bioactive molecules between cancer cells and the neighboring stroma, EV interfere

with signaling and regulation of gene expression in the recipient cell. Thus, the malignant cells can convert the phenotype of surrounding benign cells into a tumor-supporting one and create a favorable environment for cancer progression and metastatic spread. While much attention has been paid to the role of Exo in cancer, the function of the larger MV is still poorly defined. This seems surprising, since MV, in contrast to Exo, are easily accessible in patients' blood and are characterizable by routine methods that should make them ideal candidates for "liquid biopsies". Additionally, MV have long been known for their involvement in metastasis formation. This was initially attributed to their procoagulant activity, favoring the formation of microthrombi and facilitating the extravasation of the thus captured circulating tumor cells (reviewed in [6]). However, more recently, accumulating evidence points to a plethora of different ways in which MV are involved in the various steps of tumor progression.

In this review, we discuss the current knowledge about the role of MV during cancer development and shed light on how these insights can be exploited clinically for cancer diagnostics. Moreover, we highlight the divergent features of MV compared to Exo as cancer therapeutics and illustrate the various ways in which MV can either promote or counteract cancer therapy. For this purpose, we specifically included studies in which EV were harvested at 10,000–20,000 g and collectively refer to them as MV.

#### **2. Preparation of MV**

In order to decipher the role of MV in cancer development and progression, effective methods are required that allow for the stringent isolation of pure MV populations from different cell types and biological fluids. A major caveat in MV research is that the currently available isolation methods potentially co-isolate LO or Exo, yielding a mixed population of EV. This may explain many of the apparently conflicting results in the field of EV research. To address this major challenge, new technologies are under development, but they are not yet suitable for laboratory use. In an endeavor to standardize the experimental procedures and limit experimental variability in the field, scientists of the International Society of Extracellular Vesicles (ISEV) published a position paper indicating the appropriate methods for isolation of EV from cells or biological fluids and highlighting the current knowledge and major caveats of these procedures [7]. A variety of methods are available on the basis of different principles for enriching the various EV subpopulations, including density gradient centrifugation, size-exclusion chromatography, precipitation via volume-excluding polymers, immunoaffinity capture methods, high-pressure liquid chromatography, field flow fractionation, and flow cytometry [7,8].

To date, differential centrifugation is still the method of choice for isolating MV since it yields a reasonably good separation of MV from Exo with regard to protein and RNA content as well as function [9–11]. In principle, samples are spun down at 2000 g to initially pellet large EV such as LO, followed by centrifugation at 10,000–20,000 g to sediment the middle-sizedMV, while ultracentrifugation at ≥100,000 g leads to a harvest of small Exo [8]. Although ultracentrifugation has been criticized for inducing vesicle aggregation and thus affecting downstream applications [12–14], these studies were conducted on the 100,000 g Exo pellet. It is unclear to what extent these problems also apply to the 10,000 g MV pellet as well. Another popular method to separate EV from contaminations with non-vesicular proteins or RNA aggregates is ultracentrifugation of EV samples on density gradients. However, the typically used sucrose gradients lack sufficient resolution to separate EV that have slightly different densities and are released by different mechanisms [15]. Since the density of MV and Exo is comparable, they cannot be separated on sucrose gradients easily [11]. Likewise, precipitation methods such as protein organic solvent precipitation (PROSPR) have been demonstrated to co-isolate MV, but the preparations were mainly enriched in smaller Exo, with MV being only a minor side population [16]. Immunoaffinity capture methods rely on antibodies for specific EV surface markers that are coupled to beads and used to isolate specific vesicle subpopulations. Due to the current lack of specific markers for MV, this method has been scarcely used for the preparation of MV. Currently, novel micro-/nano-based devices for MV isolation from clinical samples are being developed

(reviewed in [17]). However, one major problem is that the thus isolated vesicle preparations have often been poorly characterized and it is unclear whether they yield MV with a purity comparable to sequential centrifugation protocols. The same applies to methods originally established for Exo isolation such as precipitation or size-exclusion chromatography. While none of the currently available isolation procedures for MV and Exo yield pure vesicle populations, but rather MV- or Exo-enriched fractions, more effective techniques that separate pure MV from Exo might help to shed light on their distinct cell biological features such as biogenesis, uptake, and cargo trafficking routes. Then again, from a clinical viewpoint, the isolation of pure MV populations might not be absolutely necessary for their use as biomarkers, since recent studies have demonstrated the potential of MV for cancer diagnostics or therapy monitoring, even when the preparations contain some smaller Exo.

No matter which method is chosen for EV isolation, the researchers of the ISEV community highly recommend validating the obtained EV by different techniques [7]. This includes the analysis of marker protein expression comprising (i) transmembrane, (GPI)-anchored, as well as cytosolic EV marker proteins (e.g., CD63, CD9, Alix, Syntenin, Rgap1, along with negative controls consisting of (ii) non-EV co-isolated proteins (e.g., ApoB, albumin), (iii) proteins typically present in non-EV subcellular structures such as the Golgi or endoplasmatic reticulum (e.g., GM130, Calreticulin, histones), and (iv) secreted proteins (e.g., collagen, epidermal growth factor (EGF), interleukins). Moreover, EV should be characterized by at least two distinct techniques including their visualization by, for instance, electron or atomic force microscopy, as well as the analysis of their biophysical properties via, for example, nanoparticle tracking analysis (NTA) or Raman spectroscopy. Nanoparticle tracking analysis (NTA) is the most suitable method for analyzing the isolated MV in terms of quantification and sizing. NTA tracks the Brownian movement of laser-illuminated particles and calculates the diameter based on the Stokes–Einstein equation [18]. While NTA remains the most commonly used method for quantitative MV analysis, other methods such as tunable resistive pulse sensing or dynamic light scattering are also available [19]. However, a major limitation of these methods is that they cannot efficiently analyze larger vesicles and that they do not yield any information on the molecular composition of the MV. They are, therefore, combined with other methods such as tunable resistive pulse sensing and Raman spectroscopy or NTA with fluorescence labelling to obtain more information about the isolated MV [19].

A major caveat in MV research is that the currently available isolation methods potentially co-isolate LO or Exo, yielding a mixed population of EV. This may explain many of the apparently conflicting results in the field of EV research. To address this major challenge, new technologies are under development, but are not yet suitable for laboratory use. In an endeavor to standardize the experimental procedures and limit experimental variability in the field, scientists of the International Society of Extracellular Vesicles (ISEV) published a position paper that indicated the appropriate methods for isolation of EV from cells or biological fluids and highlighted the current knowledge and major caveats of these procedures [7]. Furthermore, the researchers of the ISEV community highly recommend validating different techniques for various cell types and biological fluids. A crowdsourcing knowledge base was established to create further transparency with regards to experimental and methodological parameters of EV isolation (http://evtrack.org) [20]. This platform encourages researchers to upload published and unpublished experiments, thereby creating an informed dialogue among researchers about relevant experimental parameters. This represents a major step in facilitating standardization in EV, as well as MV, research.

#### **3. Biogenesis of MV**

MV directly bud off from the outer cell membrane. The shedding process comprises molecular rearrangements of the plasma membrane regarding lipid and protein composition as well as Ca2<sup>+</sup> levels. Ca2<sup>+</sup>-dependent aminophospholipid translocases, flippases, floppases, scramblases, and calpain drive the translocation of phosphatidylserine from the inner to the outer membrane leaflet, which is considered a typical characteristic of MV (comprehensively reviewed in [21]). Apoptotic bodies, which are larger

in size, also externalize phosphatidylserine on their surface [22,23]. Therefore, the isolation of MV should be conducted solely from healthy and viable cells to avoid contamination with apoptotic bodies, which otherwise can be difficult to discriminate from MV. Ca2<sup>+</sup> levels regulate membrane rigidity and curvature and maintain physical bending of the membrane, which leads to restructuring and contraction of the underlying actin cytoskeleton enabling MV formation and pinching (reviewed in [24]). MV formation and release are also affected by the lipids ceramide and cholesterol [25]. Neutral sphingomyelinase activity, which hydrolyses lipid sphingomyelin into phosphorylcholine and ceramide, was shown to be involved in Exo and MV release. Inhibition of the enzyme led to a reduction in Exo release, while simultaneously increasing MV budding [26], which suggests that the release of both EV subpopulations is interconnected, albeit on the basis of distinct biogenetic mechanisms.

In addition to lipids, enzyme machineries involved in cytoskeletal regulation play a key role in MV formation and budding. One example is the small GTPase protein ADP-ribosylation factor 6 (ARF6), which stimulates phospholipase D (PLD) that subsequently associates with extracellular signal-regulated kinase (ERK) at the plasma membrane. ERK activates a signaling cascade downstream of the myosin light chain kinase (MLCK) that results in contraction of actinomyosin and enables MV release [27]. Similar to MV, LO are thought to derive from the plasma membrane. However, in contrast to MV, the shedding of LO has been exclusively attributed to aggressive cancer cells that have acquired an amoeboid phenotype to facilitate motility and invasiveness ([4]). Other Rho family small GTPases such as RhoA and RHO-associated protein kinases are equally important regulators of actin dynamics relevant for MV formation [28]. In addition, the endosomal sorting complex required for transport (ESCRT), which is mainly known for its role in the biogenesis of Exo [29], is also involved in MV formation and the last phase of their release. Interaction of arrestin domain-containing protein 1 (ARRDC1) with the late endosomal protein tumor susceptibility gene 101 (TSG101) results in relocation of TSG101 from the endosomal to the plasma membrane, which then induces the release of MV [30].

#### **4. Membrane Composition of MV**

EV-mediated cell–cell communication requires targeting and uptake into the recipient cell to deliver the bioactive cargo, which then induces functional and phenotypical changes. These events depend on the composition of the EV membrane, as surface molecules on EV are responsible for binding and docking to recipient cells [31,32]. The molecular composition of the MV membrane closely resembles that of the parental cell [33]. It is enriched in phospholipid lysophosphatidylcholine, sphingolipid sphingomyelins, acylcarnitine, and fatty acyl esters of l-carnitine [34]. ARF6, a key regulator of MV biogenesis, was shown to mediate MV surface molecule selection by recruiting proteins such as ß1 integrin receptor, major histocompatibility complex (MHC) class I and II molecules, membrane type 1-matrix metalloproteinase (MT1-MMP), vesicular SNARE (v-SNARE), and vesicle-associated membrane protein 3 (VAMP3) to tumor MV [27]. Moreover, the bioactive cargo of MV depends on the conditions the parental cells are subjected to, such as inflammation or other stressors. An additional example is hypoxia, which induces recruitment of the RAS-related protein Rab22a to the site of MV budding in breast cancer cells, thus influencing MV formation and loading [35].

Since both LO and MV are derived from the plasma membrane, it is not surprising that some transmembrane proteins are present on either of these EV. Analysis of protein marker expression on LO and MV revealed a common signature, underlining the fact that the definition of MV-specific markers remains challenging. Of note, some of the markers initially thought to be specific for Exo, including tetraspanins (CD9, CD63, CD81, HSP60, HSP70, HSP90), membrane transporters and fusion proteins (annexin, flotillin), and multivesicular body (MVB) synthesis proteins (Alix, TSG101) were also found in varying amounts on MV and LO [9]. The fact that, despite their different routes of origin, EV share some common surface molecules and MV-specific markers are still lacking represents major challenges in EV research. It further emphasizes that to correctly characterize EV populations it is indispensable to combine a variety of parameters in addition to marker expression, such as size, sedimentation coefficient, and others.

#### **5. Role of Cancer-Associated MV and Their Protein Cargo in Tumor Progression**

EV-mediated cell–cell communication within the tumor microenvironment (TME) is highly complex and has recently been comprehensively reviewed in [36,37]. The role of EV in promoting tumor progression has mostly been elucidated in studies on mixed populations of EV without focusing on a specific subpopulation. Therefore, the specific contribution of MV in this complex process remained enigmatic for a long time. However, with increasing awareness of the presence of large MV, accumulating evidence has begun to unravel the tumor-promoting role of MV in TME communication, as described below by selected examples. The function of MV largely depends on their bioactive cargo, in particular the shuttling of tumor-specific proteins to the surrounding cells. While researchers initially concentrated on the role of nucleic acids transported via EV (e.g., DNA, mRNA, or miRNA), the focus has more recently shifted towards the analysis of the EV proteome. The protein content of MV has been found to be strikingly different from that of the Exo proteome and is enriched in proteins involved in microtubule/cortical actin and cytoskeleton networks, ARF6, its effector phospholipase D2, and parts of the ESCRT family (ESCRT-I) [27,38]. Carrying these bioactive cargo molecules, MV are able to influence either the adjacent tumor cells in an autologous way or the neighboring stromal cells in a heterologous kind of cell–cell communication.

MV-mediated autologous communication transfers oncogenic traits between tumor cells, resulting in enhanced tumor growth and progression. One example is multiple myeloma in which MV shed by the cancer cells were shown to enhance tumor cell proliferation and thereby stimulate tumor growth [39]. Interestingly, this effect was related to the enrichment of the extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) on the tumor MV. This protein is known to be frequently overexpressed in solid tumors as well as in some lymphomas and leukemias [40]. In line with this finding, another study in breast cancer cells showed that the highly glycosylated isoform of EMMPRIN in particular is present in high levels on breast cancer cell-derived MV and stimulates tumor cell invasion via activation of p38/MAPK signaling [11]. Strikingly, a similar, high-EMMPRIN expression was also found on blood MV from patients with metastatic breast cancer where it was co-expressed with the tumor marker Mucin-1 (MUC1/CA 15-3) [11]. Another tumor-specific factor implicated in pro-tumorigenic tumor–tumor crosstalk via MV is the truncated oncogenic form of the epidermal growth factor receptor (EGFR), EGFRvIII, which is commonly expressed in aggressive brain tumor cells. It was shown that MV secreted by U373 glioma cells contained EGFRvIII, enabling them to transfer malignant characteristics from highly aggressive tumor cells to EGFRvIII-negative, more benign tumor cells, thereby promoting their oncogenic transformation [41]. Hence, MV are convenient communicators within the TME that can either mediate the horizontal transfer of oncogenic material or activate oncogenic signaling pathways in neighboring cancer cells, stimulating their proliferative, survival, mitogenic, and angiogenic potential and shifting them to a highly invasive phenotype.

In addition to tumor–tumor communication, MV were also found to mediate reciprocal crosstalk between the tumor and the surrounding stroma cells. Such heterologous interactions were observed, for instance, between tumor cells and surrounding immune cells, being seemingly essential for cancer immune evasion. As shown for breast cancer cells, the secretion of both tumor MV as well as Exo induced the expression of Wnt5a in tumor-associated macrophages. Macrophage Wnt5a was then, in turn, delivered to breast cancer cells via macrophage-derived MV and Exo, where it activated ß-catenin-independent Wnt signaling, leading to increased tumor invasion [42]. This example indicates that EV-based cell–cell communication can occur bidirectionally in a reciprocal loop and reprogram tumor-associated immune cells towards a tumor-supporting phenotype. The finding that MV-enriched preparations isolated at 50,000 g induced the differentiation of monocytes producing the anti-inflammatory cytokine IL-10 further supports this notion [43,44]. In line with this, early stimulation with tumor MV triggered macrophage polarization towards an anti-inflammatory phenotype with decreased anti-tumor cytotoxic potential [45].

Apart from macrophages, other immune cells can be affected by MV, such as the T cells. As T cells represent the first line of the immune defense, tumor cells seem to have established strategies to suppress T cell activity and dampen antitumoral immune response by exploiting MV-mediated cell–cell communication. For instance, leukemia-derived MV deliver miRNAs to T cells, which then interact with their targets, resulting in a T cell exhaustion phenotype [46]. Moreover, MV released by irradiated breast cancer cells were shown to carry an increased amount of immune-modulating proteins, such as programmed cell death ligand 1 (PD-L1). Via transfer of this immunosuppressive protein, tumor MV inhibited cytotoxic T cell activity and enabled tumor growth [47].

Tumor cell-derived MV were also shown to modulate fibroblasts in the tumor stroma. Stimulation of fibroblasts with prostate cancer MV converted them to an activated phenotype and triggered the release of tumor-promoting fibroblast MV [48]. Similar observations were made in oral squamous carcinoma when normal human fibroblasts were treated with tumor-derived MV [49]. In this study, the switch to cancer-associated fibroblasts (CAF) was mainly mediated via metabolic reprogramming of the fibroblasts to aerobic glycolysis, with an increase in glucose uptake and lactate secretion. Co-culturing the generated CAF with oral squamous cell carcinoma cells again led to enhanced cancer cell invasion and migration. Interestingly, MV-induced fibroblast activation and spreading seems to occur in particular in the stiff matrix environment that is typically found in the tumor periphery [50]. Tumor MV are also able to modulate angiogenesis within the primary tumor. In a study on a mixed population of MV and Exo, normal endothelial cells were found to endocytose tumor EV, which activated PI3K/Akt signaling and promoted the motility as well as the tube formation activity of the endothelial cells [51]. As the pro-angiogenic factor vascular endothelial growth factor (VEGF) has been found on MV and Exo, this might be one important factor contributing to the stimulatory effect of tumor MV on endothelial cells [52]. Of note, MV transport a unique 90 kDa form of VEGF that has only a weak affinity for bevacizumab, the clinically used monoclonal anti-VEGF antibody, which might thus be ineffective in blocking MV-mediated activation of the VEGF receptor (VEGFR) [53]. Similarly, MV derived from multiple myeloma cells were shown to transfer CD138, a myeloma cell marker, to endothelial cells. This stimulated the endothelial cells to proliferate, invade, and secrete the angiogenic factors IL-6 and VEGF to promote tube formation [54]. Taken together, these observations emphasize the important role of MV in tumor–stroma crosstalk during tumor progression.

Using the same mechanisms as discussed above, MV were found to modulate the surrounding environment beyond the primary tumor by shaping the formation of pre-metastatic niches over long distances. In pancreatic cancer, tumor MV were able to enter the liver microcirculation and extravasate through the vessel wall in a CD36-dependent manner where they were taken up by perivascular macrophages and primed the liver metastatic niche [55]. Furthermore, the highly metastatic melanoma cell line B16F10 releases large amounts of tumor MV into its surroundings. These MV were found to be able to induce metastasis formation in BALB/c mice, which are normally resistant to the B1610 tumor cell line [56]. Metastatic niche formation might be essentially influenced by tumor MV uptake into organ-specific macrophage populations, and this uptake seems to be further influenced by systemic changes such as inflammation [57]. However, further in vivo studies are required to characterize the uptake kinetics and to clarify which cells are specifically targeted by tumor MV during systemic spreading. In summary, an increasing number of studies confirms that MV play a critical role in cell–cell communication within the TME and support local tumor growth and progression as well as tumor spreading to distant sites through the priming of metastatic niches (Figure 1).

**Figure 1.** Microvesicles (MV) shaping the local tumor microenvironment (TME) and distant metastatic niches to promote tumor growth. On the one hand, MV shed from the plasma membrane of tumor cells act in an autologous way on other tumor cells by transferring oncogenic cargo and stimulating pro-tumorigenic signaling. On the other hand, MV are able to mediate heterologous cell–cell communication by acting on cells present within the TME, such as macrophages, T cells, fibroblasts, or endothelial cells. As a result, these cells become activated and change their phenotype to enhance tumor growth. Once the tumor has gained access to the circulation, tumor MV can be distributed throughout the body and mediate cargo transfer and cell–cell communication at distant sites. This induces the formation of pre-metastatic niches that offer a favorable environment for subsequent seeding of tumor cells in secondary organs.

#### **6. Analysis of MV in Peripheral Blood**

MV have been successfully detected in almost all body fluids, including saliva [58], urine [59], cerebrospinal fluid [60], breast milk [61], ejaculate [62], synovial fluid [63], bronchoalveolar lavages [64], and blood, wherein they were described as early as 1967 by Peter Wolf as "platelet dust" [65]. The advantage of body fluids apart from blood is that they contain MV specifically enriched for the cancers drained by them. Nevertheless, some of these liquids are either difficult to obtain or provide additional methodological problems. For instance, MV in urine are of special interest as biomarkers for urothelial cancers. However, their isolation poses two additional challenges: (1) the most abundant protein in urine, the Tamm–Horsfall protein, associates and co-precipitates with EV, and (2) standard protocols pre-clear urine samples at 17,000 g to deplete the urinary sediment, and thus also deplete the MV contained within. Therefore, the number of studies on MV in urine meeting the criteria mentioned in the introduction is very limited. Since blood is the most thoroughly studied body fluid, as well as the most likely application route for using MV with regard to diagnostic and therapeutic interventions, we chose to focus this review on MV in blood.

When isolating MV from blood, several studies have shown that the choice of anticoagulant used for blood collection has a critical influence on the obtained MV, although the results seem to be contradictory. While the use of protease inhibitors, either sodium heparin or a mix of hirudin supplemented with a factor Xa inhibitor, was initially recommended because of the high number of preserved MV [66], other reports have argued for the use of sodium citrate since the lower numbers of MV might point to less artificial MV shedding [67]. In contrast, Jamaly et al. recently used various anticoagulants to isolate MV and measure their total plasma concentrations by NTA without finding any significant differences in numbers [68]. Apart from total MV numbers, the isolation of MV from serum drawn into lithium heparin tubes seems to result in the aggregation of platelet-derived MV, with sticky vesicle pellets and a reduction in platelet MV numbers, thereby specifically affecting certain MV subpopulations [69,70]. In addition to the anticoagulant, other preanalytical factors such as centrifugation, temperature, freeze–thaw cycles, or agitation can influence MV in blood samples, which should be taken into account when setting up isolation protocols for clinical studies [66,67,71]. However, long-term storage of plasma samples without repeated freeze–thaw cycles seems feasible without significant MV loss [67,69,72]. Another important factor for the analysis of large MV in blood is the initial removal of platelets and blood cells to obtain platelet-poor plasma (PPP). Since PPP is often prepared by centrifugation at 2500–3000 × *g*, this step might result in a significant loss of larger MV, which already pellet at this force [9]. It has been proposed that two centrifugation steps at 1500× *g* might be preferable for MV isolation [70]; however, more elaborate sorting methods might be required to specifically isolate larger EV from blood.

A current study by Brennan et al. compared different EV isolation methods, including ultracentrifugation, density gradient centrifugation, and size exclusion chromatography, as well as different combinations thereof, for their potential to isolate EV from plasma [73]. While none were found to be clearly superior, the authors showed that the isolation method significantly affected the yield, size, and degree of contamination with serum (lipo)proteins [73]. This must therefore be considered carefully on the basis of the planned downstream application. Unfortunately, the study was conducted on whole EV preparations, and thus it is again unclear whether the results are also applicable for larger MV. A novel approach to specifically reduce the contamination of plasma EV preparations with lipoproteins is to use anti-apolipoprotein B (ApoB) antibody-coated magnetic beads prior to EV isolation [74]. However, the method might lead to a high loss of vesicles.

The preferred method for analyzing MV from clinical samples is flow cytometry because of its wide availability in most labs and clinical centers, its capability for rapid preparation and acquisition of a large number of samples, as well as its potential for standardization. Since the size threshold of standard flow cytometers is ~300 nm (see [75] for a current instrument comparison), this allows the quick measurement and quantification of MV protein expression in contrast to Exo, which have to be coupled prior to analysis to larger latex beads following a time-consuming protocol. Protocols exist for staining of single or multiple markers on plasma-derived MV for flow cytometry [76,77]. Even though flow cytometry might not allow the detection of very small MV <300 nm and thus not reproduce the whole spectrum of MV in blood, several studies have successfully demonstrated the potential of flow cytometry for measuring MV-associated cancer biomarkers, as discussed in detail below. However, analysis of MV samples by flow cytometry bears several pitfalls, and the settings should be chosen with care to avoid misinterpretation of the results [78]. When measuring MV by flow cytometry, their presence should be confirmed by additional methods such as NTA or electron microscopy as the correct sizing of vesicles by flow cytometry can be challenging [79]. When discriminating MV from background noise using annexin V staining, the use of saline was recommended since the routinely used PBS seemed to create artificial nano-sized vesicles and could thus lead to false positive results [80]. Another pitfall is that the insoluble fraction of immune complexes and MV have overlapping size profiles, which might interfere with their flow cytometric analysis. To confirm that MV and not immune complexes are measured, samples can be treated with low concentrations of detergents (e.g., 0.05% Triton X-100), which lyse MV, thus resulting in a loss of signal in contrast to immune complexes that keep their fluorescent signal [63]. Recently, another label-free approach called flow cytometry scatter

ratio (Flow-SR) has been introduced to discriminate MV <500 nm from lipoprotein particles, which are highly abundant in plasma samples [67].

Apart from flow cytometry, there are protocols for surface profiling of plasma MV by antibody microarrays. However, this is a semiquantitative method and thus more useful for initial screening of patient samples for the expression of potential novel biomarkers [81]. In summary, the current studies have shown that isolation and analysis of MV from human biofluids, such as plasma, is feasible using standard laboratory equipment. However, it is clear that transparent reporting and standardization of protocols are highly important for implementing and translating MV as novel biomarkers and therapeutics into the clinic [82,83].

#### **7. MV Levels in Cancer Patients**

Most studies have focused on measuring total MV levels in the plasma of cancer patients. In this context, it must be kept in mind that tumors can secrete large numbers of apoptotic bodies under therapy due to the massive induction of apoptosis in the tumor tissue. Apoptotic bodies can have a similar size as MV and are equally characterized by an externalization of phosphatidylserine, a marker that is often used to identify MV by flow cytometry. Hence, care should be taken to include only patients prior to treatment into these studies. While the observed results are very contradictory, the majority of reports have documented increased MV levels in cancer patients compared with healthy controls. Augmented MV levels have been detected by flow cytometric quantification, mostly using TruCount Beads, in the plasma of patients suffering from gastric cancer [84], lung cancer [85], breast and pancreatic cancer [86,87], and colorectal cancer [88], as well as in most hematologic malignancies including chronic lymphocytic leukemia (CLL) and multiple myeloma [89–91]. The same trend has been confirmed for hepatocellular carcinoma by measuring MV protein levels by bicinchoninic acid assay (BCA) [92]. In contrast, two studies investigating heterogeneous cohorts of patients with different types of advanced cancers did not detect significant differences in total plasma MV numbers by flow cytometric quantification or analysis of MV protein content [69,93]. Similar observations were made by a novel capture/imaging approach in glioblastoma patients [94]. Another report quantified the total number of annexin V+ MV by flow cytometry and found a significant decrease of MV levels in patients with colorectal cancer and benign colorectal diseases compared with healthy controls [95]. The discrepancy between the observations might be due to the methods used for MV quantification, the composition of the patient and control cohorts, or diverging effects in different cancer subtypes. Patient selection criteria and the clinical setting of sample acquisition (before/during/after treatment) are not always conclusively reported in the studies, which could be another reason for the heterogeneous results.

While the increased levels of MV in hematologic malignancies could be explained by additional vesicle shedding from the large numbers of cancer cells in blood, the source of the elevated MV counts in solid tumor patients remains undefined. In these patients, plasma levels of tumor MV might depend on the access of the tumor to the circulation or might originate from other blood cell populations as a reaction to the growing tumor. Other limitations are that all the studies are either single center studies; include a very small number of patients; or have compared MV levels between cancer patients and younger, healthy controls. It was recently found that total MV levels were higher in healthy, untreated elderly than in younger individuals, which might have biased the results [96].

An argument for a specific increase in MV levels in cancer patients is that MV levels seem to increase with advanced tumor stages and have been found to be elevated in late stage metastatic breast cancer [86,87], colorectal cancer [88], hepatocellular carcinoma [92], or CLL [90] compared with early stage patients. Of note, a significant increase was already observed in stage I colorectal cancer patients compared with healthy controls [88]. In line with this, total MV levels decreased 1 month after surgery in hepatocellular carcinoma patients and have thus been suggested as a potential diagnostic biomarker [92]. Moreover, higher MV levels in blood might additionally serve as a prognostic biomarker since they were found to correlate with a poor clinical outcome in glioblastoma

or CLL [90,97]. In contrast, in lung cancer, an increase in MV in blood was associated with better progression-free survival [85]. An interesting finding was the detection of very large vesicles (1–14 μm), measurable with the established CellSearch system used for the analysis of circulating tumor cells (CTCs) [98]. These epithelial cell adhesion molecule (EpCAM)+/cytokeratin CK+/CD45- large vesicles without nuclei were present at frequencies one order of magnitude higher than CTCs and were elevated in the plasma of prostate, breast, and colorectal, but not lung cancer patients, with high numbers correlating with shorter overall survival [99]. While these vesicles have not yet been validated to be "true EV" using the MISEV guidelines, their discovery might represent one additional opportunity of translating EV as biomarkers into the clinic using an instrument already applied in diagnostics.

#### **8. MV as Cancer Biomarkers**

The growing number of in vitro studies demonstrating that cancer cells shed large numbers of EV with tumor-specific material has fueled interest in using EV as biomarkers in the clinic. Most investigations thus far have focused on the detection of tumor-derived vesicles within the complex mixture of plasma EV. This seems to be a promising strategy for gaining information about the genetic makeup of the tumor and identifying targetable characteristics, in particular for solid tumors, which are not directly accessible without invasive procedures (e.g., pancreatic cancer, glioblastoma). In contrast to the classical soluble "tumor markers" in the serum, MV also allow for detection of non-secreted, membrane-bound, or even intracellular molecules. Thus, the MV-based liquid biopsy enlarges the spectrum of detectable tumor-associated factors that could help not only with regard to diagnosis but also with accurate patient risk stratification and therapy monitoring. Since tumors seem to gain access to the circulation quite early [100], tumor MV in blood are believed to be of potential interest as early diagnostic biomarkers. In contrast to the extremely sparse CTCs, they are present in significantly greater numbers, which might improve detection sensitivity. Recently, tumor EV, consisting of a mixture of small and large vesicles, were shown to be able to pass the intact blood–brain barrier in an orthotopic glioblastoma xenotransplant model of human cancer stem cells as well as in glioma patients [101]. In line with this finding, EV carrying tumor-specific alterations were detected in the blood of patients with low-grade glioma, most of whom still had an intact blood–brain barrier [101], thus further supporting the concept of using EV as early cancer biomarkers. Another important point is that tumors are known to be highly heterogeneous [102], a circumstance that currently gains more and more attention with the novel single cell analysis technologies. While a biopsy taken from a limited area within the tumor only partly mirrors the molecular composition of the whole tumor, EV are believed to give a more comprehensive picture. This might apply also to patients with advanced, metastatic cancer, considering that metastases often differ from the primary tumor, as well as among each other, and might thus express different markers or potential therapeutic targets [103,104]. Interestingly, an early study in glioblastoma described the detection of oncogenic EGFRvIII transcripts in plasma-derived EV in two patients who had been diagnosed as EGFRvIII-negative on the basis of a tumor tissue biopsy [105]. Thus, it may be speculated that EV might mirror tumor heterogeneity more accurately than limited biopsies.

When aiming at the specific detection of tumor-derived MV in blood, the first step is to identify cancer-specific factors that are not expressed on MV shed by other cell populations. Therefore, a thorough characterization of tumor MV is necessary, which usually has been performed on cancer cell line-derived vesicles for later translation to patient samples. A recent report has suggested that MV reflect the cellular transcriptomic landscape better than Exo and can thus give valuable information about mutation status or gene amplifications that can be used for cancer diagnostics [106]. In line with this, hallmark oncogenic fusion transcripts (e.g., BCR-ABL1, TEL-AML1, MLL-AF6) as well as other specific tumorigenic transcripts (e.g., HOXA9, MEIS1) were found in MV of leukemia cell lines [107,108]. Similarly, in a glioblastoma model, genomic DNA (gDNA) sequences of tumor-associated genes were identified with a distinct distribution in either apoptotic bodies, MV or Exo isolated from human cancer stem cells, or from the blood of xenotransplanted mice, including specific detection of PIK3CA, EGFR, AKT1, and MDM2

sequences in MV [101]. However, the question remains as to whether analysis of vesicle-associated transcripts is superior to mutation detection based on cell-free DNA. Encouraging results have been obtained in this context by the group of Krug et al., who demonstrated increased sensitivity in detecting EGFR mutations in the plasma of lung cancer patients with a combined analysis of circulating tumor and EV DNA compared to circulating tumor DNA alone [109].

MV and Exo have been shown to differ in their RNA composition [10]. While several populationspecific microRNAs have been identified for both MV and Exo, there seems to be a trend that miRNAs are relatively more enriched in Exo and mRNAs in MV [106,110,111]. In addition, an enrichment compared with the whole cell has been observed as several miRNAs that were almost undetectable in colorectal cancer cell lysates (<5 transcript per million (TPM)) were found at high levels (>1000 TPM) in the respective MV and Exo from the cells [111]. However, it is currently unclear whether sufficient amounts of RNA molecules are shuttled to EV in order to functionally influence recipient cells, as several critical reports have revealed that most RNA species are present at frequencies of much less than 1 copy per EV [106,110,112]. Therefore, the focus of the rest of this review is on MV-associated proteins that have the additional benefit that they could be used not only as biomarkers, but also for specific drug delivery and targeting. The protein content of EV has been evaluated largely by Western blotting, flow cytometry, as well as different proteomic approaches. While shot-gun proteomics was initially used to thoroughly characterize the whole EV proteome, novel targeted proteomic acquisition strategies such as selected (or multiple) reaction monitoring (SRM/MRM) allow the detection of a predetermined selection of specific target peptides with high sensitivity and quantitative accuracy [113]. These methods thus facilitate the discovery and large-scale validation of biomarker pipelines in complex biological samples (e.g., blood) [114]. In a recent study, SRM/MRM was successfully employed to identify novel potential biomarkers for prostate cancer on Exo in urine [115], demonstrating its potential for future EV-related biomarker studies.

The MV proteome has been proposed to be more similar to the cellular proteome than that of Exo [35]. However, not all proteins from the cellular plasma membrane are found on MV, and some are highly enriched, which suggests specific sorting mechanisms [116]. An overview of MV-associated tumor antigens that have been successfully detected in the blood of cancer patients and used as biomarkers is presented in Table 1. In hematologic malignancies, MV carrying the respective malignancy-associated antigen (e.g., CD38 for multiple myeloma, CD30 for Hodgkin lymphoma) were detected in cancer patients but were almost undetectable in healthy controls [91]. Similar observations have been made for solid tumors. In one of the earliest studies, the oncogenic receptor human epidermal growth factor receptor 2 (Her2/Neu) was identified and significantly elevated on blood-derived MV from gastric cancer patients [84]. Meanwhile this finding has been confirmed for colorectal cancer [117]. In the largest study to date of a cohort of 330 cancer patients with advanced solid tumors and 103 healthy, non-cancer controls, the number of MV carrying the tumor antigen EMMPRIN were found to be increased with advanced tumor stage [69]. Expanding the analyses on MV co-expressing EMMPRIN together with other tumor-associated molecules, such as epithelial cell adhesion molecule (EpCAM/CD326), MUC1/CA 15-3, or EGFR1, the numbers were not only significantly elevated in cancer patients versus controls but also associated with poor overall survival, thus suggesting that MV might indeed represent valuable prognostic cancer biomarkers. EpCAM is among the most studied antigens as it is known to be overexpressed on tumors of epithelial origin and is thus a bona fide tumor marker. The combination of EpCAM with other tissue-specific markers such as, for instance, the asialoglycoprotein receptor 1 (ASGPR1), exclusively expressed in liver cells, allowed the separation of patients with liver disorders into patients with or without liver tumors (including hepatocellular or cholangiocellular carcinoma) [118]. In line with this, the EpCAM+/EMMPRIN+ MV levels correlated with tumor size in colorectal cancer patients and decreased after surgical removal of the tumor [118,119]. In yet another study performed on plasma samples from metastatic prostate cancer patients, caveolin-1 (CAV1) was found on 5–10% of large EV (1–10 μm) isolated by filtration, but with a size profile similar to the 10,000 g pellet, whereas it was barely detectable in healthy controls [120].

**Table 1.** Common tumor antigens detected on patient-derived MV.

From the thus far published MV biomarker studies, one can deduce that the population of tumor MV carrying the respective tumor antigens constitutes up to 5–10% of the total MV in blood of advanced cancer patients [69,94,120]. A recent study has profiled MV at the single vesicle level and proposed that there is a huge heterogeneity among MV, and that not all MV, even from the same cell line, carry the respective tumor markers [94], which further hampers the detection of tumor MV in blood. Moreover, especially low-abundance tumor MV might be concealed in the heterogeneous mixture of blood MV, which might necessitate the development of novel protocols for specific enrichment of tumor MV populations. One suggestion is to remove the platelet-derived MV prior to analysis [70] since they represent the largest MV population in blood. A protocol was recently published for isolating different subpopulations of small and large EV from solid tumor tissue, in this example from melanoma [122,123]. This is an important step that will now allow for isolation of MV from primary tumor tissues and metastases to define not only tumor-specific but even patient-specific biomarkers. The comparison of MV profiles from the tumor tissues (primary tumor/metastases) with MV present in body fluids might then provide information on clonal evolution and serve as the basis for therapy selection and monitoring.

#### **9. MV Biomarker Signatures**

The studies conducted thus far have shown that while single MV-associated biomarkers are often elevated in the blood of cancer patients, each of the antigens alone failed to reliably distinguish cancer patients from healthy controls, mainly due to low sensitivity. In contrast, combinations of several markers have proved to be of superior diagnostic value, such as the combination of EMMPRIN/EpCAM/MUC1/EGFR, which reliably separated cancer patients from healthy controls with a high AUC value of 0.85 [69]. It seems likely that more comprehensive signatures comprising cancer subtype-specific markers can even further increase sensitivity and specificity. Importantly, it has to be considered that the overexpression of certain oncogenes might impact MV protein profiles as was shown for breast cancer for the oncogene Src for Exo [124] or Her2/Neu, which was shuttled along with its associated proteins onto MV and Exo [125]. The resulting specific signatures have already been successfully used for the correct determination of breast cancer subtypes from serum EV [126]. Compared to soluble cancer biomarkers, which often suffer from low specificity and a high rate of false positive results, the strength of MV is their potential as biomarker platforms that simultaneously carry multiple markers, allowing for the recognition and detection of specific patterns.

#### **10. The Microvesicle Reactome in Blood**

Several conditions, such as hypoxia, inflammation, exercise, or nutrition, influence levels of MV subpopulations in blood (reviewed in [127]). It might thus be worth considering the fact that not only the analysis of tumor MV in blood but also changes in blood MV composition or the markers on distinct blood MV subpopulations might give information about cellular transformation (Figure 2). Although, for example, endothelial cell-derived MV were detected at elevated levels in some hematologic malignancies [91], a slight increase was observed in platelet- as well as leukocyte-derived MV in solid tumor patients [69,88,128]. An analysis of the phospho-proteome of MV and Exo isolated from plasma samples of breast cancer patients by ultracentrifugation described 156 unique phosphosites, which differed significantly between the vesiculome of healthy individuals and patients [129].  The cell type of origin, however, remained unclear [84].

Among the EV subpopulations, MV are considered the main source of tissue factor (TF) activity [130]. Higher amounts of MV with TF activity were detected in the plasma of metastatic cancer patients, suggesting their use as novel markers for venous thromboembolism, a common complication in advanced cancer patients [131]. In addition to TF, phosphatidylserine, whose exposure on the outer membrane is a typical feature of all MV, can further induce thrombin generation [132]. This potential side effect should therefore be carefully taken into account when considering the application of MV as therapeutics, although first studies in mice did not show an effect of the injected MV on coagulation [133].

Taken together, analysis of the whole mixture of plasma vesicles does not only detect the presence or absence of tumor EV but yields more comprehensive information since it additionally mirrors the reaction of immune and other cells against the tumor EV.

**Figure 2.** The microvesiculome in blood. The growth of a tumor is accompanied by secretion of circulating tumor cells (CTCs) as well as of tumor-derived soluble factors and MV. When encountering any of these components, benign blood cells can become activated and react by shedding their own, altered MV. Taken together, this results in changes in the composition as well as expression pattern of MV in the blood of cancer patients. Liquid biopsy-based MV sampling should therefore not only be focused on the detection of tumor MV, but also on alterations in non-tumor blood MV.

#### **11. MV for Therapy Monitoring**

First studies have revealed the potential of MV for monitoring therapeutic responses in cancer patients. Krishnan et al. reported that plasma levels of CD138+ MV corresponded with therapeutic response in individual multiple myeloma patients [89]. In general, total levels of MV were found to decrease after surgical removal of the tumor, which was generally associated with a better outcome [92,97]. In line with this, the number of EpCAM+ MV decreased 10 days after surgery in colorectal cancer patients, although the effect was not seen for EpCAM+/EMMPRIN+ MV [119]. This might be explained by the additional presence of EMMPRIN on platelet- and immune cell-derived MV [11], which are directly influenced by the surgery. Prolonged high levels of total EV or endothelial cell-derived CD144+ MV after chemotherapy seem to predict a poor clinical response and were found to be associated with shorter progression-free and overall survival [134,135]. The same trend was observed in glioblastoma patients showing tumor progression after radiochemotherapy who generally exhibited higher MV levels compared with patients with pseudoprogression or stable disease [136]. Moreover, an increased concentration of large and small EV prior to therapy was associated with failure of chemotherapy in breast cancer patients [134]. Taken together, these results demonstrate that MV levels not only mirror the presence of tumor, but that MV might also have a protective role against chemotherapeutic agents.

#### **12. MV and Cancer Therapy**

As discussed above, MV collected by liquid biopsies can give valuable information about targetable characteristics in the growing tumor and thus be used as an innovative read-out for targeted therapy decisions and patient stratification for personalized medicine. However, it has also become clear that MV can interfere with cancer treatment and limit therapeutic success. On the one hand, MV have been identified as transporters for P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1), two plasma membrane multidrug efflux transporters, and have thus been recognized as vehicles for spreading multidrug resistance from resistant to sensitive tumor clones [137,138]. On the other hand, cancer cells export cytotoxic agents via their MV during chemotherapy as has been shown, for instance, for doxorubicin, cisplatin, gemcitabine, or docetaxel [133,139,140]. Cancer cells seem to shed higher numbers of MV during treatment, and it has been suggested that the potential of a cell to shed MV correlates with its resistance to treatment [140]. Therefore, it seemed to be a promising strategy to inhibit MV release in order to increase treatment efficiency. Indeed, the combination of chloramine and bisindolylmaleimide-I, two inhibitors blocking the release of both MV and Exo, highly increased apoptosis in cancer cell lines treated with chemotherapeutic agents [141]. In another study, reducing MV shedding by inhibition of calpain via siRNA or the specific inhibitor calpeptin significantly elevated intracellular levels of docetaxel. This increased the susceptibility of the cell to chemotherapy, allowing for treatment with lower doses of chemotherapeutic agent at higher efficiency in vitro as well as in a xenograft mouse model in vivo [139]. Considering the toxic effects of general inhibition of EV secretion, it still remains elusive as to whether this strategy is feasible for human patients or whether it will be possible to specifically target such inhibitors to the tumor tissue.

Another mechanism as to how EV can impede cancer therapy is that cancer cells have been shown to export therapeutic targets such as CD20, Her2/Neu, or PD-L1 onto Exo [142–144] that capture the administered therapeutic antibodies and shield the tumor cells from attack, resulting in therapy failure. Moreover, Exo have been recently shown to transfer resistance-mediating cargo from stromal to cancer cells [145]. Whether the same mechanisms indeed also apply for large MV is currently unknown.

Considering the evidence for a tumor-supporting role of MV, they should also be regarded as novel targets for therapeutic approaches. This notion is supported by a study by Keklikoglou et al. showing in a mixed population of MV and Exo that chemotherapy induces the release of Annexin A6 (ANXA6+) EV from murine breast cancer cells. When these chemotherapy-induced ANXA6+ EV were injected into the tail vein of mice, they activated endothelial cells to produce the chemokine Ccl2, which led to the expansion of Ly6C+CCR2+ lung monocytes and thus pre-conditioned the lungs for the seeding of breast cancer metastases [146]. Similar observations of EV-mediated pre-metastatic niche formation have been made for melanoma [147]. In line with these findings, MV isolated from cancer patients induced a tumor-supporting phenotype in human macrophages ex vivo and highly increased the invasive potential of benign breast cancer cells, while MV isolated from healthy controls had no such effect [69].

#### **13. MV as Cancer Vaccines**

Although the studies presented thus far argue for an unfavorable role of MV in hampering cancer therapy, the final two chapters discuss how MV could also be exploited positively with regard to therapeutic interventions. In a first study, Zhang et al. reported that while the immunization of mice with PBS or tumor cell lysates failed to prevent the growth of subsequently injected tumor cells, mice treated with tumor Exo or MV developed tumors in only 87.5% or 50% of the animals, respectively [148]. These initial results suggested that tumor MV, which are enriched in a multitude of tumor-specific proteins (see Table 1), could serve as a novel, cell-free source of tumor antigens that are more immunogenic than other parts of the tumor cells, including tumor Exo. A possible explanation for their superior efficacy compared with Exo might be the presence of DNA as well as mitochondrial fragments inside MV that discriminate them from Exo and might provide an additional immunostimulatory signal for innate immune cells. However, more studies are required to confirm the differential immunogenic effect of MV and Exo and explain the potential difference.

In general, the anti-tumor immune response is believed to occur largely via cytotoxic CD8+ T cells activated by tumor antigen-presenting dendritic cells. Since immunization with tumor MV prior to tumor growth is hardly possible in human patients, strategies will have to be developed to treat already existing tumors. Indeed, in the above-mentioned study, the injection of tumor MV into mice with already growing tumors failed to induce a sufficient activation of T cells [148]. Instead, the injection of dendritic cells primed with tumor MV overcame the problem of inadequate T cell activation, enhancing T cell infiltration in the tumor tissue as well as tumor cell killing [148]. Mechanistically, incubation of dendritic cells with tumor MV induced dendritic cell maturation with concordant upregulation of the co-stimulatory molecules CD80 and CD86, increasing their homing to the tumor-draining lymph nodes [148,149]. Further evidence that the use of MV as tumor vaccines might indeed lead to successful anti-tumor immune responses has been provided by a recent study by Pineda et al. who harvested MV from C6 rat glioma cells and subsequently administered them to rats, which had been inoculated with a subcutaneous C6 glioma. MV vaccination increased the number of infiltrating T cells in the tumor tissue, induced tumor cell apoptosis, and led to a reduction of tumor growth [150]. Interestingly, it might even be possible to deliver future MV vaccines orally, as it was discovered that tumor MV injected intragastrically into mice are taken up by intestinal epithelial cells in the ileum and transferred by transcytosis to dendritic cells at the basolateral side that can subsequently induce T cell activation and anti-tumor immune responses [151]. However, this treatment was only successful with concordant anti-acidic agents, as MV are sensitive to degradation by gastric acid.

Thus far, clinical studies testing the efficiency of tumor cell- or Exo-based vaccines have shown only limited efficiency and it remains to be seen whether this trend is different for MV. The efficiency of tumor MV vaccines will critically depend on whether it will be possible, on the one hand, to find ways of boosting their efficacy in eliciting an anti-tumor immune response, while, on the other hand, limiting their immunosuppressive functions.

#### **14. MV as Vehicles for Drug Delivery**

The finding that EV transport bioactive molecules (e.g., proteins, metabolites, or nucleic acids) raised interest early on in terms of their use as a novel drug delivery system. Compared with cell-based therapeutics, EV have the advantages that they do not possess the potential for transformation or unlimited growth, they are known to cross biological barriers such as the blood–brain barrier, and as non-living material they can be stored or transported more easily and be modified with more aggressive manipulation techniques. Another positive aspect is their natural origin, which makes them more biocompatible than artificial liposomal drug carriers, with enhanced stability, less immunogenicity, and less liver toxicity [152]. In general, EV as drug carriers offer protection for their content, such as from enzymatic degradation, which enhances cargo stability in biofluids. EV can be modified with several approaches to increase their bioactive potential, including either manipulation strategies for the secreting cell, such as genetic manipulation, cell stress, or hypoxia, or post-isolation loading methods, such as electroporation, sonication, heat-shock, or EV transfection. A current overview of the techniques, their advantages, and their limitations is presented in [153,154]. Although most methods have been solely tested for Exo, the majority should be equally applicable to MV, considering the similarities in their biophysical makeup.

The first report on MV as a drug delivery system was published by Tang et al., who successfully used chemotherapy-loaded MV to inhibit tumor growth in a murine hepatocarcinoma ascites model, as well as in severe combined immunodeficient (SCID) mice injected with ovarian cancer cells [133]. Delivery was even successful to solid tumors when MV were injected intravenously [133]. In a first in vivo approach, the same group tested the therapeutic potential of intrathoracic injections of cisplatin-loaded tumor MV in six end-stage, cisplatin-resistant lung cancer patients. The injections greatly reduced the number of tumor cells (>95%) in the metastatic malignant pleural effusions in three of the six patients, suggesting an efficacy of the MV injections and also their ability to reverse drug resistance [155]. However, the treatment did not show any benefit in the other three patients, raising the question about the underlying reasons for the treatment failure. Two clinical trials are currently registered to further explore the potential of MV as therapeutic tool in cancer: a phase I/II

study investigating the use of red blood cell-derived MV loaded with methotrexate in the treatment of malignant ascites (clinicaltrials.gov identifier: NCT03230708), and a phase II study aimed at generating methotrexate-loaded autologous tumor MV from malignant effusion and testing their effect on tumor growth and immune regulation (clinicaltrials.gov identifier: NCT02657460).

The majority of the conducted studies thus far has used EV from mesenchymal stem cells as native vehicles for therapeutic applications, and none of them has observed significant side effects [156]. Alternatively, red blood cell-derived EV have been suggested especially for the delivery of RNA drugs since they are easily available, do not contain DNA, and can be easily modified by electroporation [157]. However, in mouse models, their systemic administration as drug carrier was found to only be feasible for the therapy of leukemias, while successful delivery to solid tumors required intratumoral injections due to the otherwise low target organ specificity [157]. In general, EV biodistribution seems to depend on the kind of cell used for EV production, the EV delivery route, the dosage, and the uptake efficiency [158]. After systemic injection, a major part of the injected EV is trapped in the liver, spleen, or lung [158,159], which on the one hand might limit EV distribution to other organs, but on the other hand might be used for specific cargo delivery to these organs. Several engineering strategies are currently being tested to improve EV circulation kinetics and increase targeting to specific cell populations [160]. A hallmark of MV is that phosphatidylserine is exposed on their outer membrane surface, a feature which distinguishes them from Exo. Since externalized phosphatidylserine is a recognition signal for macrophages and triggers phagocytosis [161], MV might be superior for drug delivery to this specific cell population.

Interestingly, a recent report suggested that the choice of MV or Exo as drug carrier influences the intracellular delivery route of the transported cargo. While MV-loaded paclitaxel was mostly delivered by membrane fusion and endocytosis, Exo-loaded paclitaxel was predominantly taken up by endocytosis into prostate cancer cells [162]. Moreover, MV seem to promote drug entry into the nucleus, even into highly resistant tumor-repopulating cells [155]. Since the mechanism of internalization and intracellular trafficking significantly affects the functional efficiency of the delivered drug, further research aiming at elucidating the different fates of MV- and Exo-delivered cargo will help to choose the most accurate EV delivery system for a given drug.

While MV are larger and therefore allow for packaging larger amounts of drug molecules, it could be more difficult for them to penetrate into the tissue due to their size. One solution that has been presented suggests isolating MV from tumor cells grown in and adapted to soft fibrin gels, which resulted in the shedding of less stiff MV [163]. These softer MV with a higher capacity for deformation showed enhanced extravasation into the tumor with deep tissue penetration and, as a result, enhanced treatment efficacy when loaded with the chemotherapeutic agent doxorubicin [163]. Moreover, a weaker extravasation was observed in non-tumor tissue compared with MV isolated from cells cultured in 2D [163], suggesting less toxicity. A problem for both MV and Exo seems to be their limited lifespan in circulation. For fluorescently labeled pancreatic tumor MV injected into healthy mice, the signal was cleared after 60 min, while the majority of MV were already lost within 15 min after injection [164].

Taken together, while both Exo and large MV have been shown to possess the potential for drug delivery, more comparative studies are required to evaluate common as well as diverging features of both EV populations as therapeutic vehicles. Both still suffer from the lack of scalable, specific, cost-effective production and isolation methods as well as poor drug loading efficiency, problems that need to be solved for their standardized use in the clinic. Moreover, heterogeneity and batch-to-batch variability are known to strongly influence their therapeutic efficacy [165].

#### **15. Concluding Remarks and Future Perspectives**

Over the past decade, an increasing number of reports has indicated that plasma membrane-derived MV are a distinct vesicle population with some common but also several diverging features compared with the smaller Exo. While most researchers have focused on the analysis of Exo, MV offer several advantages as diagnostic and therapeutic tools. In particular, their isolation is easier and less time-consuming and does not necessarily require ultracentrifugation with its associated problems of vesicle aggregation, damage and loss, and instrument availability. Additionally, protocols are available for their rapid and thorough characterization by standard flow cytometry, a technique that is already well-established in routine clinical diagnostics. Interesting findings also include the possibility for delivery of larger amounts of drugs or distinct classes of biomolecules (e.g., mRNA) by MV compared to Exo, as well as more successful targeting of MV cargo to the nuclear or cytosolic compartment, aspects that surely require further exploration.

It has become clear that MV have a multifaceted influence on cancer therapy, as summarized in Figure 3. They can not only serve as innovative tools for targeted drug delivery but should also be considered as important therapeutic targets when developing novel treatment strategies. Therefore, it will be highly advantageous to further elucidate the molecular mechanisms underlying MV formation and secretion in order to learn how MV biogenesis, especially in cancer cells, can be specifically inhibited or modulated therapeutically. However, at present, the lack of standardization, multicenter studies, and technical challenges, such as storage, production, quality control, and targeted delivery, still hamper the use of MV as diagnostic and therapeutic tools in cancer and must be overcome for them to be exploitable in the clinic.

**Figure 3.** MV in cancer therapy. The figure summarizes the roles of MV in cancer therapy. Once a tumor has gained access to the circulation, circulating tumor MV in blood can be used as biomarkers in liquid biopsies. Moreover, they can mediate therapy resistance by capturing or exporting the administered anti-cancer drugs. Due to their role in therapy resistance and tumor microenvironment crosstalk resulting in pre-metastatic niche formation, they should be regarded as a novel therapeutic target. However, MV can also have a beneficial role in cancer therapy as they represent promising novel drug delivery systems with the benefits of high biocompatibility as well as opportunities for specific targeting and crossing of biological barriers. Vesicular functions, which have only been reported for endosomal-derived small exosomes (Exo) and lack experimental proof for MV, are highlighted in grey.

**Author Contributions:** Conceptualization, C.B., K.M., and A.B.; writing—original draft preparation K.M. and S.S. writing—review and editing, C.B., A.B., K.M., and S.S.; supervision, C.B., A.B., and K.M. All authors have read and agreed to the published version of the manuscript.

**Funding:**This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation-project 424252458), Gerdes-Stiftung, Innovative Medizinische Forschung (IMF, project ME 1 2 19 14), and the German Ministry of Education and Research (BMBF) e:Med project MyPathSem (031L0024).

**Acknowledgments:** Suganja Sivaloganathan is a member of CiM-IMPRS, the joint graduate school of the Cells-in-Motion Interfaculty Centre, University of Münster, Germany, and the International Max Planck Research School-Molecular Biomedicine, Münster, Germany.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Platelets Extracellular Vesicles as Regulators of Cancer Progression—An Updated Perspective**

#### **Magdalena Zmigrodzka \*, Olga Witkowska-Piłaszewicz and Anna Winnicka ˙**

Department of Pathology and Veterinary Diagnostics, Institute of Veterinary Medicine, Warsaw University of Life Sciences (WULS-SGGW), Nowoursynowska 159c, 02-787 Warsaw, Poland<EMAIL_ADDRESS>(O.W.-P<EMAIL_ADDRESS>(A.W.) **\*** Correspondence<EMAIL_ADDRESS>
Received: 26 June 2020; Accepted: 20 July 2020; Published: 22 July 2020

**Abstract:** Extracellular vesicles (EVs) are a diverse group of membrane-bound structures secreted in physiological and pathological conditions by prokaryotic and eukaryotic cells. Their role in cell-to-cell communications has been discussed for more than two decades. More attention is paid to assess the impact of EVs in cancer. Numerous papers showed EVs as tumorigenesis regulators, by transferring their cargo molecules (miRNA, DNA, protein, cytokines, receptors, etc.) among cancer cells and cells in the tumor microenvironment. During platelet activation or apoptosis, platelet extracellular vesicles (PEVs) are formed. PEVs present a highly heterogeneous EVs population and are the most abundant EVs group in the circulatory system. The reason for the PEVs heterogeneity are their maternal activators, which is reflected on PEVs size and cargo. As PLTs role in cancer development is well-known, and PEVs are the most numerous EVs in blood, their feasible impact on cancer growth is strongly discussed. PEVs crosstalk could promote proliferation, change tumor microenvironment, favor metastasis formation. In many cases these functions were linked to the transfer into recipient cells specific cargo molecules from PEVs. The article reviews the PEVs biogenesis, cargo molecules, and their impact on the cancer progression.

**Keywords:** extracellular vesicles; exosomes; ectosomes; neoplasia

#### **1. Introduction**

The number of research work and scientific papers that discuss the involvement of cell-derived extracellular vesicles (EVs) in multiple physiological and pathological processes has increased rapidly during the last two decades. EVs might have an influence on target cells by delivering ligands and signaling complexes, and transferring mRNA and transcription factors that cause the epigenetic reprograming of recipient cells. EVs are submicron spherical membrane bound structures, that are generated by different prokaryotic (termed as membrane vesicles) and eukaryotic cells [1–3]. EVs nomenclature take into account their cellular origin and size. Their size ranges between 10 nm to 5 μm and comprises three heterogeneous populations of vesicles—exosomes (EXSMs), ectosomes (ECTSMs) also named microparticles (MPs), and apoptotic bodies (ABs) [4,5]. EVs actively secreted form parental cells with a diameter of 10 to 100 nm are named EXSMs, and those with a diameter ranging between 100 nm to 1 μm are ECTSMs. Lipid bilayer membrane protects their cargo from enzymes like proteases and ribonucleases [6]. The largest of EVs are ABs (with diameter 1–5 μm) represented by clumps of material generated during the late stage of cell apoptosis [5–7].

During activation, maturation, proliferation, stress, aging, or apoptosis, cells shed EVs into the extracellular space [8]. Their presence in a number of body fluids including—urine, synovial fluid, bronchoalveolar lavage fluid, saliva, and bile was confirmed [7,9–11]. In the bloodstream, EVs are released by—erythrocytes, leukocytes, platelets (PEVs), megakaryocytes, and endothelial cells [10,12]. In addition, EVs are also secreted by cancer cells known as tumor-derived extracellular vesicles (TEVs) [4,12]. In both healthy subjects and those with a variety of pathologies, peripheral blood is a rich source of EVs, where the most abundant population are PEVs. Their percentage ranges between 70 to 90% of all EVs in the plasma of healthy individuals [13–15].

In 1967, Peter Wolf described "platelet dust"—a subcellular material derived from thrombocytes in the plasma and serum of healthy individuals [16,17]. This was a milestone in medicine research, allowing further examinations evaluating PEVs involvement in physiological and pathological processes. PEVs share many functional features with PLTs. These tiny fragments smaller than platelets (PLTs) were secreted during PLT activation and were known to be crucial in coagulation and clot formation [16,18]. Despite the fact that PLTs play a crucial role in hemostasis, PEVs coagulation capacity is several dozen higher than PLTs [19]. Platelets microparticles (PMPs) are enriched in tissue factor (TF), coagulation factors, and dozens of them expose about 3-fold higher phosphatidylserine (PS) concentration on the outer membrane than PLTs [20]. The coagulation process initiated by TF connection with coagulation factor VII, activates coagulation cascade. Activated PLTs, PMPs PS + offer a catalytic surface for the coagulation and binding of consecutive clotting factors. Moreover, in healthy individuals, the presence of integrin αIIbβ3 (CD41/CD61) on PMPs supports fibrin clot formation [21]. In various bleeding disorders, abnormalities in PMPs functions and their reduced number in blood were reported [22]. On the other hand, their increased amount was presented in thrombotic state and other pathologies [23]. PLTs of patients described by Castaman are unable to shed PMPs, conversely to patients with Scott syndrome in which the PMPs number is adequate, but the incorrect translocation of PS impairs prothrombinase activity, and causes hemorrhagic diathesis [22]. Patients with immune thrombocytopenia have higher PEVs level than healthy individuals, which might be an evolutionary way to prevent blood loss and maintain tissue integrity [24]. Additionally, contemporary papers showed that PEVs might be a potential biomarker or prognostic factor in other pathologies—inflammatory, cardiovascular, and autoimmune diseases, solid tumors and hematological malignancies [14,25].

In this review, the role of PEVs in the cancerogenesis, tumor growth, and metastasis formation in distant organs is reported. Furthermore, the possible evaluation of PEVs as markers for cancer detection, and effectiveness of anticancer treatment is discussed.

#### **2. EVs Biogenesis and Elimination**

Based on the current knowledge, the mechanism of EVs formation and secretion to the extracellular space vary, depending on the EXSMs or ECTSMs descent. The EXSM definition was originally used for microparticles secreted from variety of cultured cells, thereafter, Johnstone and colleagues in 1987 explained the mechanism of transferrin receptor loss during reticulocytes maturation via secretion of nanosize vesicles; for this term EXSMs is used [26]. The latest research confirmed that the pathways of EVs biogenesis might differ between the parental cells types and EVs secretion, which does not seem to be accidental [1,27].

#### *2.1. ECTSMs Formation*

The blebbing of the plasma was documented in apoptosis during ABs formation, but it was confirmed as well in ECTSMs biogenesis. Changes in lipid components affect the rearrangement within plasma membrane. This process is initiated by an increased level of intracellular calcium ions. It causes activation of floppase and scramblase enzymes and inhibition of flippase (Figure 1) [1,8]. The membrane phospholipids—PS and phosphatidyl-ethanolamine, are vertically translocated from the inner leaflet to the outer cell membrane surface. The rearrangement breaks the bonds between cytoskeleton and cell membrane phospholipids. Partial degradation of actin filaments leads to restructuring of the cytoskeleton filaments, which favor formation of ECTSMs [1,8,10].

**Figure 1.** Extracellular vesicle biogenesis and secretion. The exosomes (EXSMs) generation begins with the membrane bulging into the lumen of the ESE. Part of them form a part of the plasma membrane (recycling endosome), others are converted into multi vesicular body (MVB). Members of the Rab family, Rab27a and Rab27b, are involved in MVB transport and fusion with cell membrane. Transmembrane protein complex SNARE enables the MVB to dock with the cell membrane that leads to release of EXSMs to extracellular space. Ectosome (ECTSM) are formed directly by cell membrane blebbing. This process is initiated with an increase in intracellular calcium that causes the activation of enzymes floppase and scramblase and the inhibition of flippase. This causes the rearrangement of phospholipids in the cell membrane, as well as results in breaking bonds between cytoskeleton and partial degradation of actin filaments. During formation of EXSMs and ECTSMs, mRNA and miRNA that are located in cytoplasm are randomly entered.

The fast phospholipid membrane remodeling and PS exposure are relevant for PLTs physiological procoagulant response in hemostasis. PMPs formation in the circulation could result from PLTs activation via multiple agonists, high shear stress or apoptosis [20,28]. In the high shear rate, the loss of membrane integrity is initiated through the dislocated connection between the membrane glycoprotein Ib receptor (CD42b) and PLTs cytoskeleton, which began PMPs formation [20]. Natural PLTs activators, such as thrombin or collagen, induce PMPs formation via transmembrane integrin receptor gpIIb/IIIa (CD41/CD61) or tetraspanin 29 [29]. Altogether, these observations become the starting point for subsequent works assessing, how different types of PLTs activators induce PMPs formation, and how they affect the heterogeneity of PEVs population. Noticeably, a research conducted in 2017 confirmed that PS negative tubular PMPs population with structural similarities to filopodia could be formed during PLTs activation. Lack of PS expression on their surface implied that during their formation, there is no PS translocation [30].

#### *2.2. EXSMs Formation*

EXSMs generation begins with the inward bulging of the plasma membrane by endocytosis into the cytoplasm lumen. It leads to forming early sorting endosomes (ESEs) (Figure 1) [1].

Part of ESEs is returned into plasma membrane, other under the Rab5 control are changed into late endosomes or multivesicular bodies (MVBs) [1,10]. During this process, proteins and antigens are packaged into intraluminal vesicles (ILVs) and the budding of the ESEs membrane transform into MVBs [31]. Four protein subunits of the endosomal sorting complex required for transport (ESCRT) machinery are involved in this process. ESCTR-III is essential for the scission of ILVs into MVBs lumen. Cargo clustering and membrane budding can occur by ESCRT-dependent or -independent machinery [1]. ESCRT-0 recognizes ubiquitinated proteins (cargo) by the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), in association with clathrin. This complex helps ESCRT I and II to connect with ESCRT 0 and ubiquitinated cargo, on the part of the endosomal membrane, where it will finally pullulate. ESCRT III connects with the complex and ultimately bud ILVs into the endosome [32]. The MVBs fuse with the plasma membrane to secrete the ILVs as exosomes or absorb with lysosomes for their degradation [1]. Members of Rab family, Rab27a and Rab27b, are essential mediators in transport of MVBs and its fusion with cell membrane. Transmembrane protein complex SANRE enables dock EXSMs with cell membrane that leads to the release of EXSMs to extracellular space (exocytosis). Increased concentration of calcium ions is one of the EXSMs secretion regulators [33]. Targeting selected Rabs via specific inhibitors modulates their structure or secretory function and becomes a new promising strategy of limiting EXSMs formation, both by PLTs and cancer cells. Wang et al. showed in a pre-clinical study, that elevated number of PMPs in patients with sepsis after intravenous administration of small GTPase inhibitor NSC23766, reduced PMPs secretion for about 87% [34,35].

Aatonen et al. showed that PMPs and platelet derived EXSMs (PdEXSMs) biogenesis is also observed by non-activated PLTs [36]. Examination potency of various agonists on EVs formation confirmed that Ca2<sup>+</sup> ionophore is the strongest agonist, these include—thrombin, collagen, LPS, TRAP-6, and the weakest one is ADP [36]. Moreover, authors considered that Ca2<sup>+</sup> ionophore causes vesiculation in unselective way or fragmentation and ABs formation, and should be advisedly used as agonist. The strongest PdEXSMs activators are thrombin and collagen or collagen-related peptide XL. Interestingly, the proteins cargo in PdEXSMs derived from stimulated PLTs was richer than from resting PLTs [36]. Nowadays, the utility of EXSMs as a new diagnostic cancer marker is extensively studied. Recent work performed by Lea et al. showed an increased number of EXSMs with PS expression in peritoneal fluid and plasma of patients with ovarian carcinoma [37]. It confirmed that, when PS is routinely used as a PMPs marker, it is also present on cancer derived EXSMs and causes a possibility to exploit these results in early diagnostic tests of women with ovarian malignancies [37].

#### *2.3. EVs Elimination and Impact of Storage Conditions on PEVs Number*

The PEVs rapid clearance from circulation varied depending on their molecular content, and the induction signal in different species [38,39]. As they have pro-coagulant and pro-inflammatory nanosize structures, their rapid turnover is essential for prevention of thrombotic diseases. PMPs turnover in rabbits is less than ten minutes, compared to people where PMPs were shown in circulation for more than 3 h [20,38,39]. Flumenhaft found that mice PMPs are eliminated from bloodstream within 30 min [40]. PEVs could be phagocytized after their opsonization with thrombospondin or complement components C3b [40]. The PS on the PMPs outer leaflet of the plasma membrane is recognized by macrophages and it originates a signal to remove them. Moreover, the role of lactadherin (LA) in clearance of EVs from circulation is discussed [41]. LA secreted by macrophages and adipocytes is also detected on the circulating PMPs. An increased PEVs level was observed in lactadherin–deficient mice, which could suggest the role of LA as a one of "eat-me" signals for phagocytosis [41]. Dasgupta et al. showed that developmental endothelial locus–1 in endothelial cells mediates PS-positive PMPs elimination via endocytosis [17,42]. Shorter half-life of ECTSMs, compared to EXSMs in blood, might arise from the higher concentration of membrane lipids in ECTSMs and activity of phospholipase A2 in serum [43]. Furthermore, EXSMs elimination via IgM immunoglobulins binding to lipid lysophosphatidylcholine was reported and liver macrophages were shown to be crucial elements of EXSMs clearance [43,44].

In EVs analysis, preanalytical steps standardization is crucial for the minimization of false results of PEVs number and their quality tests. Different anticoagulants could activate PLTs during blood collection and storage. Wisgrill et al. confirmed that the EVs number and their functionality is stable in sodium citrate for 8 h in room temperature (RT), after blood samples collection [45]. In EDTA, routinely used in clinical practice, PMPs and erythrocytes derived EVs count is stable for 48 h in RT [45]. Thus, it could be an alternative when the collected samples are stored before analysis [45].

#### **3. Content of Platelet Extracellular Vesicles**

Physiological or pathological processes in parental cells define their EVs cargo and biological properties. As described above, the PEVs formation, membrane composition and specific markers expression on the outer membrane leaflet depends on the PLTs activators (Ca2<sup>+</sup> ionophore, adenosine diphosphate, thrombin, collagen, epinephrine) [20]. Most of the EVs circulating in plasma are classified as PEVs based on their surface receptors. Nevertheless, heterogeneity of PEVs surface receptors starts discussion about EVs derived from megakaryocytes (Mk-EVs), as a part of PEVs subpopulation [46]. The EVs phenotyping conception to distinguish PEVs from Mk-EVs involves the usefulness of cluster of differentiation (CD) CD41/CD61 as a constitutive marker for both PLTs and Mks, while CD62P and CD107a act as a PLTs activation markers [47]. Flaumenhaft et al. showed that mouse and human Mk-EVs are PS/CD41/CD61 positive and CD62/CD107a negative [46]. In support of this finding, after irradiation of bone marrow, the CD61 positive EVs population largely disappears from mice circulation, whereas CD62P remains unchanged [48]. A study by Brisson et al. showed that small PMPs population—PS negative and CD41 positive, is a result of cell membrane shedding without PS redistribution. Moreover, PMPs could contain organelles like mitochondria and dense granules [30]. EVs are identified based on their size and expression of characteristic surface markers. PS expression is an a ECTSMs marker, when the presence of tetraspanin CD63 is used for EXSMs identification. During PLTs activation, both ECTSMs and EXSMs are CD63 positive but the CD63 expression is higher on EXSMs. It could be useful for determining the purity of the EXSMs population [30,49]. A characteristic of PEVs is the diversity of their surface markers and cargo. PEVs display a wide array of bioactive molecules like adhesion molecules, chemo- and cytokines, apoptosis regulators, miRNAs. They also harbor a broad spectrum of coagulation factors, enzymes, complement proteins, and bioactive lipids (Table 1). PEVs express glycoprotein (gp) IIb/IIIa, Ib, IIa, as well as P-selectin and a lysosome-associated glycoprotein-1 (LAMP-1). C-type lectin domain family 1-member B (CLEC-2) and gp VI expression was documented on Mk-EVs [17]. PdEXSMs are substantial with proteins from α granules, whereas ECTSMs are substantial with lipid mediators and mitochondrial proteins [17,50].

Molecules presented on PEVs were involved in triggering receptors on the target cells or regulating them via bioactive molecules, signaling molecules or a plethora of genetic material including miRNAs [51]. PEVs can interact with donor cells in multiple ways—(i) stimulation via signaling complex, using specific PEVs surface receptors and lipids; (ii) transfer membrane receptors and adhesion molecules; (iii) horizontal transfer of heterogeneous proteins, miRNAs, bioactive lipids, and other factors including infectious particles (prions) or even organelles (mitochondria) [3].

*Int. J. Mol. Sci.* **2020**, *21*, 5195

PEVs are able to transfer receptors expressed on their surface (i.e., CD41, CD61, CD184, CD62P, PAR-1) to recipient cells (monocytes, myeloblasts, hematopoietic stem cells) and induce their adhesion or proliferation [3,27]. PEVs functional gpIIb/IIIa (CD41/CD61) transferred to neutrophils, activated NF-κB, in response to GM-CSF and enhanced inflammation [77]. Tang and colleagues showed that PEVs transfer arachidonate 12-lipoxygenase to mast cells, which increased synthesis of one of the negative regulator of inflammation lipoxin A4 (LXA4) [27,55]. Thus, PEVs play both positive and negative role in inflammation response, depending on the target cell.

PEVs are rich in sphingosine 1-phosphate, metalloproteinases, heparyanase, PDI, and arachidonic acid (AA) [3]. Transfer of AA by PMPs to monocytes and endothelial cells induced by prostanoids and cyclooxygenase 2 synthesis enhances these cells interactions [3]. Treatment of human umbilical vein endothelial cells (HUVECs) with PEVs showed intensification of angiogenesis and cell proliferation versus activated charcoal treated PEVs (removed nonpolar lipids), where a reduction of these effects was observed. This experiment showed that PEVs lipid components were involved in HUVECs stimulation [27,78]. The horizontal transfer of non-coding RNAs via EVs regulates gene expression by post-transcriptional repression. miRNA from parental cells encapsulated in EVs was protected from ribonuclease activity in circulation [51]. In human's, several PMPs miRNAs were detected, e.g., miR-19, miR-21, miR-22, miR-126, miR-133, miR-146, miR-185, miR-223, and miR-320b [3]. Moreover, it was confirmed that PEVs miRNA was transferred to macrophages, endothelial, and cancer cells. In macrophages, MiR-126–3p transferred from PEVs led to decreased ATF3 and ATP1B1 expression and protein synthesis [27]. Recently presented data support the notion of PEVs tumor microenvironment infiltration and interaction with cancer cells via the mechanisms described above.

#### **4. PEVs in Cancer Progression**

PEVs are highly interesting group of EVs because of their percentage participation in bloodstream, as well as their increased number in patients with cancer, such as glioblastoma, gastric, lung and skin cancer, and other diseases. This makes them potentially useful as a diagnostic marker [79]. It is known that PLTs facilitate cancer metastasis. Moreover, the number of papers that discuss PEVs contribution in cancerogenesis increased recently [51,80]. EVs as cell-to-cell messenger molecules can start phenotypic and functional changes in donor cells, by reaching the recipient cells and delivering EVs content. PEVs are also discussed as potentially early markers of disease progression.

#### *4.1. PEVs in Tumor Angiogenesis*

The cancer cells without blood circulation can grow up to 2 mm3 in diameter, forming a tumor and then stop and undergo apoptosis or necrosis [81]. Growth of the vascular network is pivotal for the cancer cells survival, proliferation, as well as metastatic spread of cancer [81]. Angiogenesis is essential for formation of a new vascular network that supplies nutrients, oxygen, and immune cells, and also removes waste products of cellular metabolism. Therefore, angiogenesis is a critical factor in the progression of cancer. The tumor microenvironment (TME) consists of diverse cellular populations, including tumor cells, endothelial cells, fibroblasts, infiltrating immune cells (monocytes, macrophages, neutrophils, mast cells, T cells), extracellular matrix, and newly formed blood vessels [79]. The PEVs interaction with TME components could reveal their functions in cancer progression. Newly stirring blood vessels permit tumor growth, which is critical in cancer progression. Interestingly, Happonen et al. demonstrated a mechanism of PEVs transfer to human aortic endothelial cells (HAECs) and HUVECs [82]. PS-positive PEVs are taken up by phagocytosis via tyrosine kinase receptor Axl, and its ligand protein Gas6 on endothelial cells [82]. Janowska-Wieczorek et al. used lung cancer cell lines to elucidate PEVs importance in cancer angiogenesis [83]. After PEVs stimulation of IL-8 (about 35-fold), vascular endothelial growth factor (VEGF) (3-fold) and scatter factor (4-fold) mRNA expression increased in the A549 cell line [83].

PEVs delivery of bioactive molecules like cytokines or microRNA to recipient cells could regulate tumor growth [84]. miRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally. Anene et al. demonstrated regulatory angiogenesis miRNAs transfer from PEVs to HUVECs cells during co-culturing on extracellular matrix gel [85]. A robust capillary-like structure formation and simultaneously decreased synthesis of anti-angiogenic thrombospondin-1 (THBS-1) was observed. miRNA Let-7 a from PEVs was delivered to HUVECs and targeted THBS-1 mRNA to induce angiogenic responses of HUVECs [85]. Blood vessel formation is controlled by a balance between localized production of pro- and anti-angiogenic molecules and changes in THBS-1 concentration is the key determinant of this "angiogenic switch" [85]. PEVs ability to bind TF and the platelet-activating factor potentiates their pro- angiogenic competence even more [86].

Pan et al. demonstrated that after incubation, PEVs with HUVECs cells miR-223 level in endothelial cells increased, which promoted glycation end-product-induced vascular endothelial cell apoptosis via targeting insulin-like growth factor 1 receptor [87]. Another work showed that HUVECs cells preferentially uptake miR-223 from PEVs generated by thrombin-activated PLTs [4,88]. This leads to the formation of functional Argonaute 2 (Ago2) miR-223 complexes. These complexes are able to regulate gene expression and protein level for ephrin A1 and F-box/WD repeat-containing protein 7 in HUVECs cells and conduces apoptosis [4].

Increased angiogenesis in TME could be a result of metalloproteinase-1 (MMP-1) transfer, as well as increased MMP-9, VEGF, and IL-8 mRNA expression in lung cancer cells lines, after co-incubation with PEVs [83]. Moreover, PEVs molecules from α granules like VEGF, platelet-derived growth factor and fibroblast growth factor are a component of their cargo with pro-angiogenic properties.

#### *4.2. PEVs in Migration, Invasion, and Metastasis*

A key for distant metastases formation is cancer cells passage through the newly formed vascular walls in primary tumor, surviving in the circulation, and finally proliferation at the distant tissue. In solid tumors, vasculature is highly permeable, allowing the possibility to PEVs infiltration to TEM and contact with cells. A great number of studies indicate the PEVs involvement in cancer progression and some discuss their anti-cancer properties. Michael et al. showed that PEVs have the ability to infiltrate murine and human tumors [84]. This ability creates conditions for the horizontal transfer of miRNA-24, which targets mitochondrial mt-Nd2, and Snora75. This entails mitochondrial dysfunction and results in an increased cancer cell apoptosis [84].

Bakewell and colleagues showed that platelets gpIIb/IIIa antagonists minimize formation of distant metastasis from B16 melanoma cells in bones, due to the inhibition of the interaction between cancer cells and PLTs [89]. Lung cancer cell line A549 increases adhesiveness to the fibrinogen and HUVECs, after receiving CD41 from PEVs. PEVs chemoattract lung cell lines from 2.5 to 7-fold more than the control [83]. Moreover, evaluation of PEVs interaction with lung cancer cell lines confirmed the activation of mitogen-activated protein kinase (MAPK) MAPK p42/44 and AKT, signaling pathways participating in proliferative responses [83]. Murine lung cells covered by PEVs injected intravenously into mince resulted in significant increase metastasis formation in lungs and bone marrow [83]. Transfer onto the surface of donor cells CD184-, a chemokine receptor type 4 from PEVs and respond to stromal cell-derived factor 1, which is rich niche in bone marrow in the murine model, confirmed their high metastatic potential [83]. Moreover, activation of cyclin D2 by PEVs in lung cancer cell lines could change the phenotype of cancer cells into a more invasive phenotype. Similar observations were made in human squamous carcinoma or breast cancer cell lines in murine in vivo model [90].

Interestingly, Gasperi and colleagues confirmed the modulatory influence of polyunsaturated fatty acids (PUFAs) diet, especially the ω3 and ω6 on cellular processes in carcinogenesis [62]. The PUFAs ω3 cancer preventive activity is well known, in contrast to high concentration of ω6 in diet, which correlates with higher risk of breast and prostate cancer [62,91]. Their role in cancerogenesis is related to changes in fatty acids compositions of membrane rafts in cells membranes. PEVs contains miR-126 and miRNA-223, which are important players in tumorigenesis. VEGF-dependent proliferation of endothelial cells is stimulated by miRNA-126, while miRNA-223 inhibit formation of new blood vessels by targeting endothelial β1 integrin [92]. Gasperi et al. examined the influence of increased level of PUFAs ω6

on both PEVs formation and their cargo [62]. The newly formed PEVs had an increased amounts of miRNA-123 and miRNA233. Breast cell line BT549 blocked its cell cycle and decreased cell migration after internalizing PEVs [62].

A Tang et al. study revealed an important PEVs role in the epithelial-to-mesenchymal transition of ovarian epithelial cancer cell line (SKOV3). miR-939 transfer leads to enhanced invasion and cancer progression [93,94]. Tropomyosin 3 (TPM3) contributes cancerogenesis in thyroid papillary carcinoma and esophageal squamous cell carcinoma by fusing neurotrophic receptor tyrosine kinase 1 and PDGF receptors [95]. Yao et al. demonstrated increased TPM3 mRNA in PLTs and revealed their transfer by PEVs into breast cancer cells and promotion of an invasion [94]. Moreover, in patients with distant metastases, compared to subjects without metastases TPM3 mRNA in PLTs was significantly increased [94].

Another interesting issue is the ability of cancer cells to educate PLTs. Zarà et al. demonstrated that breast cancer cell lines—highly aggressive MDM-MB-231 and MCF7 could educate PLTs to produce PEVs in an amount similar to that after thrombin activation [96]. Next, those PEVs were co-cultured with cancer cells to investigate if the newly formed PEVs impact cells. Only in the MDM-MB-231 cell line, authors observed cells activation and phosphorylation of p38MAPK and myosin light chain. Moreover, increased migration and invasion was noted. This experiment showed that PEVs can novel paracrine-positive feedback mechanism initiated by MDA-MB-231 to escalate their invasive phenotype [96].

PEVs formed by PLTs during apoptosis-like process show surface gpIIb/IIIa, and PS and stimulate their own phagocytic removal by monocytes, moreover, they are able to change macrophages into M2 macrophages [97,98]. In contrast to effect on endothelial cells, after PEVs miR-223 transfer into gastric cell line SGC7901, increased proliferation and invasion in vitro, as well as decreased apoptosis, was observed. This showed that horizontal miRNA transfer via PEVs could have diverse effect contingent on donor cells [4,99]. Another noteworthy experimental work showed that peripheral blood mononuclear cells (PBMCs) isolated from patients with B-precursor acute lymphoblastic leukemia had increased apoptotic markers CD95, active caspse-3, and an increased number of apoptotic cells, after two days of co-culturing with PEVs [100].

Cancer cells transmigration from circulation into the tissues is mediated likewise by tissue-specific enzymes, the majority of which belongs to the MMP family. Dashevsky et al. confirmed transfer of MMP-2 and its increased secretion from Cl-1 cells after co-culturing with PEVs. Interesting observation was made when Cl-1 cells were incubated with PEVs lysate. Values of MMP-2 concentration and secretion were similar to that after cells co-culturing with PEVs. It suggests that the transfer of MMP-2 is not dependent only on PEVs internalization. The other possible candidates for increased MMP-2 value might be free miRNA from PEVs lysate or lysophosphatidic acid (LPA) as an MMP-2 activator presented on PLTs and in prostate cancer cells [101].

Natural killer (NK) cells efficiently recognize and kill circulating tumor cells of almost any origin, but their effectiveness in TME is discussed. PEVs miR-183 transfer into NK cells suppressed activator adapter DAP12 and suppressed their cytolytic functions in tumor-associated NK cells [102]. PEVs could also horizontally transfer functional miR-126–3p into primary human macrophages. The PEVs dose-dependent down regulation of miR-126–3p targets CCL4, CSF1, and TNF was observed. Decreased secretion of cytokines/chemokines was correlated with reprogramming into phagocytic macrophages [88,103]. The role of TF in angiogenesis and metastasis formation is well documented, therefore, the role of TF-positive PEVs in tumor growth seems clear. Another interesting aspect of PEVs as a potentially important immune checkpoint in cancer biology is a presence of PS on PEVs surface. PEVs as an abundant source of PS might be a possible ligand for PS receptor (PSR) on the immune cells. Activation of PS–PSR pathway leads to the inhibition of innate and adaptive immune response in TME, as well as in circulation [104]. The new oncotherapy strategies examined the PSR inhibitors as a new anticancer target, but only a highly selective inhibition strategy could be applied in

the cancer treatment. Table 2 summarize PEVs pivotal role in crosstalk between PLTs and other cells, particularly with cancer cells (Table 2).

**Table 2.** The role of PEVs in cell-to-cell communication. PEVs secreted from activated PLTs transfer to target cells and their cargo promotes phenotypic changes and novel functions in donor cells.

Abbreviations: CCL-cancer cell line; \* murine cell line; (+) increase; (−) decrease.

#### **5. The Potential of PEVs as Diagnostics Cancer Biomarkers**

PEVs number in blood was raised about twice in myeloproliferative neoplasms, compared to healthy controls, up to four times in oral cancer and colorectal subjects and more than ten times in breast cancer patients [86,106]. The highest concentration of PEVs, more than 30-fold, was noticed in patients with IV stage of gastric cancer. In each group, the highest PEVs concertation were demonstrated in advanced cancer stages and in patients with distal metastases [86,106–108].

Investigation in patients with non-small cell lung cancer (NSCLC) categorized based on disease progression, showed the significantly higher number of circulating EVs from activated or apoptotic PLTs and from endothelial apoptotic cells, compared to healthy subjects. Changes in EVs levels in different stages of NSCLC showed that serial measurements of circulating PEVs are valuable prognostic biomarkers, mainly in the advanced stages of NSCLC [109].

PEVs as source of anionic phospholipids and TF on their surface are one of the important factors of procoagulant activity. Data demonstrated by Ren et al. showed the significantly increased number of EVs and PEVs in patients with oral squamous cell carcinoma (OSCC) in peripheral blood. PEVs level was also positively correlated with clinical stage and with fibrinogen concentration and patients hypercoagulable state [107]. Mege and colleagues showed correlations between increased PEVs number and the stage of the disease in patients with pancreatic cancer and colorectal cancer. They suggested that PEVs concentration in blood could be a useful marker for evaluation of the disease progression in these types of neoplasia [110].

Yenigürbüz et al. described another aspect of increased PEVs number in patients with neoplasia. Thromboembolism is one of the complications during induction of therapy in pediatric acute lymphoblastic leukemia (ALL) patients [111]. Children with ALL have increased levels of ABs, PEVs, endothelial-derived, and tissue factor-positive microvesicles during induction therapy. Further studies are needed to confirm the PEVs contribution in thromboembolism during the induction therapy period in children with ALL [111]. Similar observations were made in adult patients with myeloproliferative neoplasia, where the number of TF positive PMPs and endothelial derived EVs was significantly increased, which might also play a role in thrombotic complications in that group of subjects [112]. Tjon-Kon-Fat et al. demonstrated that tumor educated PLTs are a source of prostate cancer biomarkers [113,114]. In this context it seems to be interesting to evaluate the presence and role of EVs generated from tumor-educated PLTs.

#### **6. The Potential of PEVs in Cancer Therapy**

The paradigm of using nanoparticulate pharmaceutics as delivery vectors was established over the past decade [56]. To use EVs as drug transporters, their pharmacokinetics should be analyzed. Mice models of EXSMs distribution showed that the route of administration, EXSMs origin, and concentration critically influenced their biodistribution [115]. In the mice model, after intraperitoneal and subcutaneous administration of EXSMs, they preferentially localized in the pancreas and gastrointestinal tract. Whereas, intravenous administrated EXSMs were detected in the spleen and the liver [116]. In addition, EXSMs loaded with therapeutic anti-miRNA could be transferred locally into tumor or systemically. Other therapeutic strategies in cancer therapy were elimination of EXSMs from blood or prevention of EXSMs fusion with target cell [117,118]. Various strategies of using EXSMs in anticancer therapy are characterized in the literature, but more research is still needed.