Extracellular vesicles in leukemia

Highlights

• Comprehensive Review of Leukemia extracellular vesicles.

• Leukemia extracellular vesicle characterization.

• Leukemic extracellular vesicles alter their microenvironment.

• Cancer extracellular vesicles act as immunomodulators.

• Extracellular vesicle clinical, diagnostic, and therapeutic potential.

Abstract

Extracellular vesicles (EV) are nano-sized membrane enclosed vehicles that are involved in cell-to-cell communication and carry cargo that is representative of the parent cell. Recent studies have highlighted the significant roles leukemia EVs play in tumor progression, and ways in which they can lead to treatment evasion, thus meriting further investigation. Leukemia EVs are involved in crosstalk between the leukemia cell and its surroundings, transforming it into a cancer favorable microenvironment. Due to the diverse biological content found in leukemia EVs, they have an assortment of effects on the cells they interact with and can be harnessed as candidates for diagnostic and therapeutic treatments. This review focuses on EVs in the context of leukemia and the means by which they modulate their microenvironment, hematopoiesis, and the immune system to facilitate malignancy. We will also address current and prospective EV-based therapeutics.

Introduction

EVs are small, membrane-enclosed heterogeneous spheres of varying size that are directly secreted by all cells and carry a select cargo of cellular RNA, bioactive lipids, DNA and proteins that is usually representative of the parent cell [1], [2]. EVs are present in physiological and pathological circumstances, and their numbers, cellular origin, composition, and function are cell type and disease dependent [3]. They are known for their coagulant phenotype but can also affect inflammation, angiogenesis, and intercellular signaling [4]. There is increasing evidence suggesting that these vesicles have an important role in the regulation of immune stimulation or suppression that can drive inflammatory, autoimmune, and infectious disease pathology [5]. Furthermore, EVs have the potential to alter the fate of their target cells by regulating gene expression, partially through epigenetic changes in the recipient cells [6].

The first line of evidence that tumor cells shed membrane-vesicles came in 1978 from studies performed by Friend and colleagues on samples obtained from patients with Hodgkin’s disease [7]. In 1979, an independent study identified plasma-derived vesicles released by murine leukemia cells [8]. In the early 1980s, studies reported that EVs from pig hepatocellular carcinoma and mouse breast carcinoma cells were carriers of procoagulant activity. However, it was not until twenty years later that vesicles were acknowledged not to be artifacts but an active component of tumor microenvironment [9]. As in solid tumor malignancies, patients with hematologic malignancies have high levels of EVs in their biological fluids compared to normal controls that include cases of acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) [10], [11]. Moreover, EV cargo also increases in patients with hematological malignancies [12], [13].

EVs can be categorized based on their size. Microvesicles are 200–1000 nm in size and are formed by outward budding from the plasma membrane. Apoptotic bodies are 800–5000 nm in diameter and are released by cells undergoing programmed cell death [14]. More recently another category of EVs has been identified known as oncosomes that are much larger (1–10 μm) and are released as pinched membrane blebs from amoeboid cancer cells [15]. Exosomes are a subset of EV’s reported to vary between 30 and 100 nm, although the exact size varies with isolation approach and cellular conditions. Whereas microvesicles form by budding from the plasma membrane followed by fission of their connecting membrane stalks, exosomes are formed by a combination of processes starting with the inward invagination of clathrin-coated microdomains on the plasma membrane, suggesting a more active sorting mechanism. The Endosomal Sorting Complex Required for Transport (ESCRT) System facilitates the development of the invaginated vacuoles carrying ubiquitinated cargos into early endosomes. A second invagination of vesicles termed intraluminal vesicles (ILVs) occurs into the endosomes where they accumulate into large multivesicular bodies [16]. From this point they may be trafficked to lysosomes or fused with the plasma membrane for the release of ILVs at which point they are referred to as exosomes [17]. Sorting of exosomes by the ESCRT involves several proteins that are subsequently used to identify them as exosomes including ALG-2-interacting protein X (ALIX) and tumor susceptibility gene 101 protein (TSG101) [18]. Other proteins most frequently associated with identification of exosomes are tetraspanins (e.g., CD9, CD63, and CD81) membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), heat shock proteins, lipid-related proteins, and phospholipids [19]. Exosomes are involved in numerous biological functions such as intercellular communication, antigen presentation, protein secretion, and RNA shuttling [20]. Exosomes can play a key role in physiological and pathological processes depending on the cell of origin that induce proliferation, differentiation, inhibition, quiescence or cellular death.

To isolate EV populations various methodologies have been implemented including ultracentrifugation, size exclusion chromatography, microfluidics, immunoaffinity capture based techniques, size-based techniques, precipitation, and more [21]. Each methodology exploits a particular trait of EVs including size, density or immunophenotype [21]. Some techniques combine several different methodologies in an effort to isolate purer populations [22]. Due to the specific advantages and disadvantages each method provides, there is no standardized method for EV isolation [23]. For the purpose of this review, exosomes and microvesicles are referred to as EVs because of the potential heterogeneity that results from the different isolation procedures [23].

Due to EV ability to contain a subset of specific molecules and antigens from the parent cell, there is increasing evidence suggesting that EVs can serve as biomarkers [24]. Tumor EVs contain unique genetic information about the tumor related to its state of malignancy, cellular type, and susceptibility to therapeutic treatment. As such, they may be used for early diagnosis and monitoring of cancer. Importantly, EVs are used by tumors to evade or influence their environment to favor tumor progression. These tumor derived EVs are implicated in modulating the tumor microenvironment [25], acting as immunomodulators [26], and contributing to inhibition of anti-tumor activity [27]. The complexity of the biology of EVs is just being discovered (Fig. 1). This review focuses on the role of EVs in the pathogenesis of hematologic malignancies and their potential utilization in therapies (Table 1).