Leukaemia: The Purinergic System and Small Extracellular Vesicles

Abstract

Haematopoiesis is a tightly regulated process, by intrinsic and extrinsic factors, to produce lifelong blood cell lineages within the bone marrow. In the bone marrow microenvironment, mesenchymal stem cells and haematopoietic stem cells play important roles to ensure that haematopoiesis is maintained. These cells contain purines and pyrimidines that control intercellular process such as energy transport. However, in some cases, this process may be misregulated thus leading to the production of various diseases, including leukaemia. As a result, bone marrow cells may be stimulated via stress or induced hypoxia, and this leads to the release of purine and pyrimidine nucleotides and nucleosides into the extracellular space, and activation of autocrine/paracrine feedback loops. These extracellular nucleotides and nucleosides, and their respective cell surface receptors are involved in purinergic signaling that control different physiologic functions in cells including proliferation, differentiation, and cell death. These extracellular nucleotides and nucleosides include ATP, UTP, adenosine diphosphate (ADP), UDP and adenosine however the most important players are ATP and its metabolite adenosine. ATP is degraded via a sequential activity of ectonucleotidases. ATP, adenosine and these ectonucleotidases play very important roles in the tumour microenvironment crucial to disease development, progression, and aggressiveness by modulating immune response to leukaemia treatment and increasing homing of leukaemic cells.

1. Introduction

Leukaemia is a malignant disorder involving early blood-forming cells of the bone marrow in which there is lack of control in the haematopoietic process. As a result, abnormal or immature blood cells accumulate in the bone marrow, bloodstream or lymphatic system thus causing debilitating effects in the patient [1, 2]. Leukaemia is a heterogenous disease hence its diagnosis is based on a complete blood workup (full blood count) and bone marrow studies (aspiration and biopsy) incorporating clinical, morphological, immunophenotypical, cytogenetic and genetic data.

Leukaemia is classified into different types depending on the blood cell type that the cancer originates. Leukaemia could be myeloid (myelogenous) if it involves the myeloid lineage such as red blood cells (RBC), platelets and white blood cells (WBCs) or lymphoid (lymphocytic/lymphoblastic) if it involves cells originating from the lymphoid lineage such as lymphocytes [1, 3]. Leukaemia is also classified into acute and chronic based on the progression of the disease. Acute leukaemia is often abrupt and fast-growing due to the accumulation of immature cells in the bone marrow whilst chronic leukaemia is rather slow growing [1, 3]. Therefore, leukaemia is categorized into four main groups: acute myeloid leukemia (AML), acute lymphocytic leukaemia (ALL), chronic myeloid leukaemia (CML) and chronic lymphocytic leukaemia (CLL).

2. Epidemiology and aetiology of leukaemia

Leukaemia is one of the most common cancers in the world and accounts for about 2.6% of all cancer cases worldwide [4]. In 2020, leukaemia was reported to be the 13th most diagnosed cancer case and 10th leading cause of cancer death [5]. Caucasians especially the men are more predisposed to the disease compared to other ethnic groups. The disease progresses with age hence adults are more susceptible than children. However, the outlook for patients is much better than three decades ago, with better cure rates and longer-term disease-free survival. Chemotherapy and radiotherapy are the mainstay treatment for leukaemia however the overall survival rate has improved remarkably in recent years with almost half of the population diagnosed with leukaemia surviving for at least five years or more [3, 6]. This is due to the advent of different therapies including stem cell transplantation, targeted therapy, combined therapy, immune cell engineering such as chimeric antigen receptor (CAR)-T cell therapy, innovative clinical trials and immunotherapies, and patient’s improved access to these therapies [6, 7].

Though an increase in age is a well-known factor, the aetiology of leukaemia remains unclear. Exposure to certain viruses (for example, human T-cell leukaemia virus; HTLV-1), ionizing radiation, chemicals such as benzene or formaldehyde, smoking and other environmental cues have all been reported to be risk factors for leukaemia [8, 9, 10]. These environmental cues induce genetic and chromosomal aberrations such as chromosomal deletions or translocations, point mutations and epigenetic factors, that cause maturation arrest of the normal haematopoietic stem cells (HSCs). This leads to development of leukaemia due to the proliferation and clonal expansion of leukaemic stem cells (LSC). Leukaemia could also arise following exposure to chemotherapy, radiotherapy and/or stem cell transplantation [6, 7].

3. Leukaemia and the purinergic system

Haematopoiesis is a tightly regulated process that leads to lifelong production of a sustainable pool of functional blood cells within a compartmentalized bone marrow. The bone marrow consists of osteocytes, osteoblasts, osteoclasts, the bone matrix, perivascular cells, immune cells, sinusoidal endothelium as well as regenerative cells; mesenchymal stem cells (MSC) and HSC that inhabit a unique hypoxic microenvironment [11, 12]. These cells are responsible for the provision of cell signals required for the support and regulation of haematopoiesis as well as maintenance of homeostasis within the bone marrow microenvironment [12, 13].

Additionally, there is prevalence of purines and pyrimidines in these bone marrow cells thus modulating intracellular processes such as energy transport [14, 15]. These cells can also release the purine and pyrimidine nucleotides and nucleosides into the extracellular space under normal circumstance in the absence of any stimulus. However, the stimulation of these bone marrow cells via stress or induced hypoxia leads to the release of purine and pyrimidine nucleotides and nucleosides, and activation of autocrine/paracrine feedback loops [12, 13, 14, 15]. These extracellular nucleotides and nucleosides, and their respective cell surface receptors control a form of cell-to-cell communication called purinergic signaling. This complex network controls various physiologic functions in the body, including cell proliferation, differentiation, and cell death. As a result, any aberration to this network will lead to the development of diseases such as leukaemia [11, 16, 17, 18, 19].

These extracellular nucleotides and nucleosides include ATP, UTP, adenosine diphosphate (ADP), UDP and adenosine however the most important players are ATP and its metabolite adenosine [11, 19, 20]. ATP is degraded via a sequential activity of four ectonucleotide enzyme subtypes (ectonucleotide pyrophosphate, ectonucleotide triphosphate diphosphohydrolase, alkaline phosphatase and ecto5-nucleotidase) leading to adenosine, which binds P1 receptors, as end-product [2, 11, 17, 18, 21, 22, 23, 24]. Thus, extracellular ATP is the primary source of adenosine, and adenosine is degraded by adenosine deaminase (ADA). HSCs express several receptors for nucleotides and nucleosides, which belong to two different purinergic receptor families, P1 and P2. The P2 family includes eight receptors that have been identified so far (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14), which are G protein-coupled receptors that respond to stimulation by ATP, ADP, UTP or UDP, depending on the receptor subtype and seven ionotropic receptors (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7), which are sensitive to ATP [15, 18, 25]. The P1 receptor family consists of four G protein-coupled receptor subtypes, A₁, A_2A, A_2B, and A_3, which are activated by adenosine.

Extracellular ATP is a crucial component of the tumour microenvironment. The balance between extracellular ATP and adenosine is pivotal to cancer progression, promoting proliferation of HSCs and thus, cancer cell proliferation [19]. Extracellular ATP also mediate proinflammatory activity on specific P2Y (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y12) and P2X (P2X4 and P2X7) receptors. In addition, extracellular ATP exerts angiogenic effects on P2Y1, P2Y2 and P2X7 receptors and immunosuppressive effects on P2Y11 receptors thereby affecting interaction with the immune system via trafficking of granulocytes and monocytes and inhibiting proliferation and migration of leukemic cells [2, 16, 18, 23, 24, 26, 27]. Cellular stress or damage to stromal host cells promotes and controls inflammatory response resulting in ATP release and subsequent accumulation of ATP in extracellular space, which can be beneficial or harmful to the host depending on its concentration.

Notably, the concentration gradient of ATP is maintained by two important enzymes, CD39 (ectonucleotide triphosphate diphosphohydrolase-1) and CD73 (ecto5-nucleotidase) that are found in abundance in immune cells, endothelial cells, and tumour cells [18, 19, 28]. This enzymatic chain is also responsible for adenosine production, which can accumulate in the tumour environment and stroma where it acts as a potent immunosuppressant that exerts its effects mainly at A2A receptors. In addition, adenosine also modulates cell growth when acting on A3 receptors. Multiple mechanisms have been implicated in adenosine effects and these include deregulation of mononuclear phagocyte cell differentiation and maturation, suppression of effector T cells, inhibition of T helper 1 cell cytokine production, and generation of an angiogenic and matrix remodeling environment [17, 21, 23]. Extracellular adenosine also modulates and protects cells and tissues from excessive inflammatory and immune responses, which favor angiogenesis and thus, carcinogenesis [2, 18, 19, 29]. Adenosine acts on the A2A, A2B and A3 receptors to induce macrophages to promote the release of anti-inflammatory cytokine such as tumour necrosis factor (TNF), nitric oxide (NO), macrophage inflammatory protein (MIP), interleukin (IL)-6, IL-10, and IL-12. Therefore, ATP exerts dual mechanism in cancer, facilitating pro- and anti-tumoral immune response whilst adenosine is a known immunosuppressive mediator facilitating tumour immune evasion [11, 18, 19, 23]. An immunosuppressed microenvironment and inflammation enhance the development and metastasis of cancer via release of a wide variety of cytokines and other proinflammatory markers.

In various cancers, including leukaemia, it is widely acknowledged that purinergic receptors of the P2 family (P2Rs) are required for anti-cancer immune response induced by chemotherapy. In leukaemia, chemotherapeutic agents such as doxorubicin and daunorubicin induce intracellular ATP release to create an immunosuppressed tumour microenvironment, which leads to cell death [2, 19, 20]. Once released, the extracellular ATP can bind to P2 receptors to regulate cell invasion and migration. Treatment with ATP inhibitors or ATP analogues has a strong cytotoxic effect on several tumours, including leukaemia [23, 24]. This leads to a decrease in the intracellular concentration of ATP until it falls to undetectable levels when cells enter secondary necrosis. Low ATP doses have a growth-promoting effect and depending on the P2 receptor subtypes expressed, tumour cells may be more sensitive to the death inducing or the trophic effect of ATP [21].

Recent developments have shown that the purinergic system is potentially beneficial in leukaemia [2, 16, 29]. ATP/P2X7 axis is very vital in regulating the proliferation and homing of leukaemic initiating cells. Purinergic receptors, P2XRs, particularly P2X7R, are elevated in patients with acute and chronic forms of leukaemia [2, 11, 15, 16]. Studies show that patients with acute and chronic forms of leukaemia express higher levels of P2X7 than the healthy bone marrow controls, with the P2X7-loss-of-function polymorphism linked to increased capacity to evade apoptosis and therefore, to progression of chronic leukaemia [2, 11, 15, 16]. Knockdown studies have shown that ATP in the leukaemia microenvironment decreases upon knockdown of P2X7 thus leading to the lysis of leukaemic cells [16, 18, 19, 29]. P2X7 and P2Y11 were also identified in leukaemic cell lines, HL-60 and NB-4 cell lines [11, 29]. Leukaemic cell lines also express elevated levels of ecto-enzymes CD39 and CD73 [2, 19, 22]. In another study, the levels of these enzymes that are concerned with purine degradation, CD73, ADA and purine nucleoside phosphorylase were varied in patients with different forms of chronic leukaemia [19, 21, 22]. This infers that these enzymes may be beneficial to the survival of leukemic cells and promote metastatic spread.

Since promotion of inflammatory response is a hallmark of cancer, and ATP and adenosine exert pro-inflammatory and anti-inflammatory activities, the development of innovative agonists and antagonists that target these specific receptors will be a relevant therapeutic mechanism in leukaemia. Adenosine analogues have been proposed as a possible differentiation-inducing agent against AML. Adenosine exerts cytotoxic effects on leukaemic cells after ectoenzymic breakdown of ATP, with ATP increasing the cation permeability of acute and chronic leukaemia lymphocytes and ADP increasing the calcium mobilization of myeloid leukaemia cells [23, 24]. A voltage-gated potassium channel blocker, 4-aminopyridine, also induced apoptosis in human AML cells via increasing the calcium ions through P2X7 receptor pathways. Activation of the P2X7 receptor induces the formation of reactive oxygen species in erythroleukemia cells whilst P2X7 receptor agonists mediate cation uptake into human myeloid leukaemic cells [2, 23, 25, 29]. Evidence suggest that P2XR expression and activity may be relative to disease severity and depends on the level of activation as shown in lymphocytes of patients with varying forms of B-cell CLL [16, 23, 25]. Remission in patients with CML has also been associated with a frameshift polymorphism of the P2X5 receptor that elicits an allogeneic cytotoxic T lymphocyte response. Low levels of ATP triggered anti-proliferative effects in AML cells and except for P2X2, P2X3 and P2X6, AML cells are known to express all subtypes of P2Rs thereby suggesting targeting ATP is a promising alternative therapy in AML with favourable outcome in patients [2, 19, 21, 22]. Taken together, ATP/P2R axis demonstrate sustainable leukaemia-initiating cell signaling activities thus suggesting targeting and inhibiting ATP/P2XR signaling may completely hinder leukemogenesis.

4. The role of ectonucleotidases in leukaemia

Metabolic stress due to hypoxia, ischemia, and pro-inflammatory signals lead to abundant release of ATP in the extracellular space within the tumour microenvironment. CD39 and CD73 are ectonucleotidases that catabolize high extracellular levels of ATP to produce molecules that modulate intracellular calcium levels and activate the purinergic receptors [22, 28]. Under normal conditions, these ectonucleotidases potentially stimulate immune cells to fight against the tumour. However, these ectonucleotidases are also capable of modulating immune response to favour tumour growth.

CD39 converts extracellular ATP or ADP to adenosine monophosphate (AMP) whilst CD73 converts AMP to adenosine [22, 28]. Adenosine then accumulates in the tumour microenvironment thereby supporting tumour growth by skewing immune cells towards tolerance. Therefore, these enzymes have significant effects in pathogenesis and progression of leukaemia by enhancing chemoresistance and homing of leukaemic cells [22]. In support of this, high expression of ectonucleotidases increase homing of leukaemic cells to protected niches, survival, and proliferation of leukaemic cells and modulation of immune cells towards tolerance.

Since these enzymes are deeply involved in the pathogenesis of leukaemia, their expression in leukaemia may be of prognostic value in leukaemia, marking disease progression and aggressiveness, and immunosuppression. They may act as reliable markers to monitor and distinguish specific cellular populations or subset of patients characterized by a different prognosis. CD39 and CD73 are expressed in leukaemia of lymphoid and myeloid lineage such as AML, B-ALL and CLL [2, 19, 22]. In these subtypes of leukaemia, there’s a significant crosstalk between these leukaemic cells and the bystander non-leukaemic cells. Therefore, the levels of these ectonucleotidases, both on the leukaemic and bystander non-leukaemic cells, may be used as disease markers or prognostic factors.

In ALL, CD73 was identified as a differentially expressed molecule in a genome-wide analysis, which compared 270 newly diagnosed ALL patients to normal B cell progenitors [30]. Following chemotherapy, flow cytometry validation revealed that these patients had upregulated expression of these enzymes thereby suggesting that CD73 could serve as a useful marker for minimal residual disease (MRD) and predicting ALL relapse following leukaemia treatment.

In AML, a more permissive immune environment is associated to high expression of CD39 and CD73, which favour leukaemia progression and aggressiveness [2, 19, 22]. AML cells release ATP following exposure to chemotherapy and upregulate CD39 expression on immune cells, and through this they modulate immune cells and skew them towards tolerance. In contrast, increased expression of CD73 on immune cells elicits a reawakening of the immune response. However, CD73 can also influence leukaemic cell proliferation and aggressiveness via CAAT enhancer-binding protein alpha (CEBPA) gene [19, 22].

In CLL, an increase in expression level of CD39 in patients is linked to an increase in circulating leukaemic cells as this contributes to disease progression and aggressiveness [2, 19, 22]. Thus, expression of CD39 on these immune cells can be used as a marker for advanced disease stage and a predictive factor of treatment requirement. An increased expression of CD73 is significant in CLL cells and is associated with a higher cellular turnover, an increased recirculation to and from the lymphoid niche, a more aggressive clinical behavior and associated with time to disease progression after chemotherapy [2, 22]. Therefore, CLL cells are well equipped with CD39/CD73 that protect these cells from drug-induced toxicity thereby causing MRD, a reservoir that fuels disease progression. In addition, adenosine, which is produced by these enzymes, further acts on immune cells and polarize them towards tolerance thus sustaining leukaemic cell expansion. Upregulation of CD73 and adenosine is mediated by hypoxia thus influencing the metabolic adaptation of immune cells surrounding the tumour. This induces a switch towards glycolysis, with upregulation of glucose and lactate transporters and of lactate dehydrogenase and pyruvate kinase [18]. This further links hypoxia and CD73/adenosine in a common axis in which the tumour microenvironment is reshaped with tumour-supportive and immunosuppressive features.

5. Leukaemia and small extracellular vesicles

Extracellular vesicles (EVs) are nanosized lipid bilayered membrane vesicles that are virtually released by all cells and secreted at higher numbers in cancer cells. They can also be found in varied body fluids such as semen, blood, follicular fluid, and urine. Based on their size, intercellular origin and release mechanisms, these vesicles are grouped into microvesicles (≥200 nm and ≤1000 nm), exosomes (≤200 nm) and apoptotic bodies [31, 32]. These vesicles originate from plasma membrane (microvesicles) and endosomes (exosomes) from inward budding of the plasma membrane into the cytoplasm to form multivesicular bodies (MVBs). These vesicles are then released into the extracellular milieu to be transported to neighbouring or distant cells to induce phenotypic and functional changes in the recipient cells [31, 33]. There are also other types of small EV that have been mentioned in recent literature such as exomeres, large oncosomes and enveloped viruses [34, 35]. Nevertheless, the detection and classification of these vesicles remain an uphill task due to this heterogeneity.

The recipient cells internalize their vesicles via different routes in different ways such as clathrin-dependent and clathrin-independent pathways like phagocytosis, macropinocytosis, caveolin-mediated uptake or lipid raft-mediated internalization [36, 37]. However, the mechanism of vesicular uptake and internalization remains a conundrum, but it is postulated that it depends on some factors found on the surface membrane of the recipient cells and the vesicles such as surface proteins, glycoproteins and glycolipoproteins. Therefore, it can be inferred that these surface membrane factors promote adhesion of the vesicles to the recipient cells thus facilitating their internalization.

Upon uptake, the function and fate of these vesicles differ depending on the physiological or pathological state of origin cells releasing and receiving these vesicles [36, 37]. These vesicles often carry a cargo of bioactive molecules, including proteins, lipids, and nucleic acids, which they can transfer to the recipient cells to modulate and reprogram the bone marrow microenvironment to promote their survival and induce biological effects in these cells [33, 38]. These vesicles play important roles in the regulation of immune stimulation or suppression that can drive inflammatory, autoimmune, and infectious disease pathology. EV could also alter the fate of their target cells by regulating gene expression through epigenetic changes in the recipient cells [36, 37, 38]. EV produced by cancer cells are in abundance in the tumour microenvironment and can enhance malignancy by transferring regulatory factors to normal cells within the tumour microenvironment. They also enhance anti-tumour immune response by inducing immunity to antigens that are carried by tumour EV.

In leukaemia, EV have a key role in the early stages of leukemogenesis as predominant EV population changes during leukaemia progression. Leukaemic cells utilize EV to transfer functional information to either MSC, HSC or myeloid progenitor cells in the BM microenvironment in amounts sufficient to induce phenotypic and functional changes in these cells, which are necessary for the development of leukaemia [33, 39]. These vesicles bridge the gap between leukaemic cells and the stromal cells that reside in the BM microenvironment thus initiating a crosstalk between these cells. This crosstalk between the leukaemic cells and stromal cells is crucial in remodelling and transforming the BM microenvironment into a leukaemia-permissive space, where leukaemic cells could proliferate, grow, and survive [29, 40, 41]. Leukaemia-derived EV transform and potentiate the phenotypic change of healthy MSC into cancer associated fibroblasts, which then proliferate, release inflammatory cytokines, and increase angiogenesis. As a result, leukemic cells survive and protect against apoptotic stimuli, including cytotoxic chemotherapy. AML-derived EV also alter the differentiation of stromal cells upon uptake thus leading to a decrease in the development of osteoblasts [42, 43].

The main mechanism through which EV promote the development and progression of leukaemia is not yet fully elucidated but much evidence supports through the delivery of microRNAs (miRNAs) into HSC within the BM microenvironment to change its characterization for developing into leukaemic cells [43, 44, 45]. These miRNAs are short non-coding RNAs that can mediate RNA silencing and regulate genes at the post-transcriptional level thus influencing the translocation of genes and signaling cascades. As thus, these small RNAs play vital roles in different cellular processes, including cell cycle, proliferation, angiogenesis, inflammation, immune reaction, and cell death [45, 46]. These small RNAs are either oncogenic or tumour suppressive, and once transferred from leukaemic cells to bone marrow microenvironment induce epigenetic changes that will support leukemogenesis. For example, in ALL, leukaemic cells release EV that carries miR-43a-5p to the bone marrow microenvironment and upon internalization, targets Wnt signaling pathway [45, 47]. This signaling pathway is vital for regulating haematopoiesis and induces the inhibition of osteogenesis in the bone marrow. Once suppressed, malignant HSC can then transform to leukaemic cells. MSC in the bone marrow microenvironment also secrete EV containing miR-21, which are then delivered into HSCs to enhance the development of B-ALL cells [40, 43]. These vesicle-derived miR-21 also interacts with transforming growth beta (TGF-β) to suppress the anti-tumour immune responses in the BM microenvironment.

In AML, the levels of serum-derived EV containing miR-10b, which is crucial in abrogating granulocytic/monocytic differentiation in HSCs, are elevated in patients compared to healthy individuals [12, 40, 43]. This suggests that miR-10b may play a vital role in inducing AML by enhancing the proliferative capacity of immature myeloid progenitors thus leading to the development of AML. Another miRNA, miR-4532 that targets the signal transducer and activator of transcription (STAT-3) signaling pathway, could also be transferred to HSCs from AML-derived EV to suppress the expression of leucine zipper downregulated in cancer 1 (LDOC), a well-known inhibitor of the STAT-3 signaling pathway [47]. This leads to manipulation of the proliferative capacity of the cells in the BM microenvironment via this pathway. AML-derived EV also induce early leukemogenesis in myeloid progenitors through the transfer of miR-155 that inhibits c-Myb [31, 40, 47, 48]. c-Myb is a differentiating transcription factor in myeloid cells that induce differentiation arrest, which is critical in the development of AML. EV containing miR-155 and miR3-375 could also be transferred from MSC to AML cells to confer drug resistant phenotype against tyrosine kinase inhibitors [40]. Other miRNAs, such as miR-17-92 family, also induce chemoresistance in AML cells through activation of TGF-β and PI3K/Akt signalling axis. In CLL, EV released by leukaemic cells express a cell differentiating miRNA, miR-202-3p, which is taken up by stromal cells to influence the stroma cell transcriptome thereby resulting in altered growth characteristics [40].

However, miRNAs are not the only RNAs that have been implicated in the development and progression of leukaemia. Oncogenic messenger RNA (mRNA) derived from EV can be transferred to myeloid progenitors to encode nucleophosmin (NPM1) and FMS-like tyrosine kinase 3 (FLT3) gene with internal tandem duplication (ITD) (FLT3-ITD) thereby leading to the development of leukaemia [40]. Insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) mRNA enriched in AML vesicles are also transferred to stromal cells, leading to alterations in cell proliferation, secretion of growth factors and induction of downstream gene expression [31, 40, 47, 48]. AML-derived EV could also silence the expression of haematopoiesis-related growth factors such as IGF-1, C-X-C motif chemokine ligand 12 (CXCL12), KIT ligand and IL-7, and osteogenesis (osteocalcin; OCN, collagen type 1 alpha 1 chain; Col1A1, IGF1) whilst increasing the expression of genes that support AML growth such as Dickkop wnt signaling pathway inhibitor 1 (DKK1), IL-6 and C-C motif chemokine ligand 3 (CCL3) [31, 40, 47, 48].

As thus, these enforce HSC committed to the myeloid progenitors. Anti-apoptotic proteins such as myeloid leukaemia 1 (MCL-1), B-cell lymphoma 2 (BCL-2) and B-cell lymphoma extra-large (BCL-XL) could also be transferred to the immature myeloid blasts to guarantee their survival in the BM microenvironment [46, 48]. By modulating the promoting loss of apoptosis or cell death, EV play a vital role in drug resistance. Drug-resistant AML cells also transfer p-glycoprotein to induce chemoresistance in drug-sensitive leukaemia cells via their nucleic acid and multidrug resistance protein 1 (MRP1) cargo [40].

In ALL, leukemic vesicles reduce mitochondrial respiration and cause metabolic switch, from oxidative phosphorylation to anaerobic glycolysis, in MSC thereby changing their ability to respond to metabolic changes [46]. This could provide the desired energy for ALL development in the BM microenvironment. In CLL, these vesicles also mediate the activation of AKT signaling pathway, which in turn induces the production of vascular endothelial growth factor (VEGF) to enhance progression of CLL [40]. Furthermore, EV from MSC of CLL patients could rescue leukaemic cells from drug-induced apoptosis and enhance their migratory capacity [31, 40, 49]. Lastly, CML-EV also promote the growth and maintenance of leukemic cells via internalization of their own EV or activation of EGFR signaling following delivery of amphiregulin protein [40, 49, 50]. Amphiregulin is a ligand of EGFR and stimulates cell growth, survival, and migration via juxtacrine, autocrine and paracrine signaling. EV from CML also contain BCR-ABL transcripts that induce increased proliferation in MSC upon uptake [31, 40].

Since leukaemic EV are enriched in tumour signature molecules and cargo antigens and immunological molecules associated with leukaemia cells, the role of EV within the leukaemic microenvironment may provide insight for therapeutic advances. Drug resistance is a substantial impediment to successful treatment in leukaemia. Leukaemia EV could act as circulating biomarkers for diagnosis and detection of leukaemia [31, 40, 46]. Their evaluation in body fluids especially peripheral blood and urine could provide relevant information for early and highly sensitive method for detection and monitoring of leukaemia progression in patients. EV could also be harnessed for gene delivery and personalized therapy in leukaemia [31, 50]. EV offer beneficial characteristics that synthetic vectors cannot such as high physiochemical stability, long distance communication, inherent cell signaling, cell-to-cell communication and bioactive delivery whilst protecting the interior cargo from stress-induced necrosis and the environment. EV can cross the blood brain barrier and target neuronal cells; this may be useful in treating central nervous system (CNS)-associated leukaemia, which has poor prognosis [46]. EV can also be synthetically modified by attaching cell-specific targeting ligands to the EV surface to cargo chemotherapeutic drugs directly to leukaemia cells thereby enhancing their functionality, specificity of cell targeting and decrease adverse immune response. Chemotherapeutic drugs such as imatinib and paclitaxel have been incorporated into EV and delivered to target IL-3 receptor on the CML blasts [40]. However, it is important to mention that the study of EV as biomarkers in clinical medicine is still a new field. No standard methods have been established yet for proper enrichment and isolation of these circulating vesicles. Varied methods such as differential ultracentrifugation, density gradient centrifugation, polymer-facilitated precipitation, immune-affinity isolation, and size exclusion chromatography are currently employed for isolation of EV. This heterogeneity in isolation techniques causes variability in the results thus affecting the purity of EV and subsequent precise molecular characterization of EV.