Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio- and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellular vesicles (EV). In this review, the evidence for transfer of a hypoxia tolerance phenotype between tumour cells via EV is discussed. In particular, proteins, mRNA and microRNA enriched in EV, derived from hypoxic cells, that impact HIF-1α-, UPR-, angiogenesis- and autophagy signalling cascades are listed.


Introduction
In cancer cells, genetic and epigenetic changes allow uncontrolled growth and proliferation. In addition to these genomic alterations, the tumour microenvironment (TME) is increasingly recognized as an important contributor to cancer progression and therapy resistance [1]. The majority of solid tumours contain regions with microenvironments that are uncommon in healthy tissues. Low pH, nutrient depletion, high interstitial pressure, necrosis and hypoxia are frequently observed [2][3][4]. The high proliferative capacity and alterations in metabolism of cancer cells, as well as the highly irregular vasculature in tumours, further contribute to the existence of these features [3,5]. This results in the continuous selection of cells that have acquired resistance mechanisms to withstand these harsh conditions and contribute to increased tumour malignancy.
One of the main contributing TME features to tumour progression and malignancy is hypoxia. Tumour hypoxia is observed in the majority of solid tumours and is a very heterogeneous and dynamic feature [6]. The classical perception of tumour hypoxia is caused by a limitation of oxygen diffusion (chronic hypoxia). However, in tumours, there are additional regions displaying periodic cycling in oxygenation (acute hypoxia) [7], which can account for a large proportion of the hypoxic cells at any given time [8]. Hypoxia is therefore a very heterogeneous and dynamic feature of the TME [6]. From a clinical point of view, low oxygenation of tumours is associated with poor outcome in multiple cancer types [9], independent of treatment modality [10]. The adverse effect of tumour EV-associated miR-199a derived from bone marrow mesenchymal stromal cells (MSC) protected against I/R damage, potentially by suppression of UPR activation during reperfusion by targeting binding immunoglobulin protein (BiP/GR78) [40]. These studies implicate EV as important mediators for the transfer of hypoxia tolerance to remote organs. However, the underlying mechanism of remote preconditioning and how EVs mediate this remains poorly understood.
Although the phenomenon of remote preconditioning was described for normal cells, it is unclear to which extent cancer cells are capable of preconditioning/transferring hypoxia resistance to hypoxia-naïve cells. However, instances of horizontal transfer and phenocopying have been described [41][42][43]. One example is the EV-mediated transfer of drug resistance. Drug-resistant cells can incorporate functional drug-efflux pumps, such as P-glycoprotein 1 (P-gp) and ATP-binding cassette (ABC)-transporters, into EV. Upon uptake of these EV, previously drug-sensitive cells started expressing these pumps on their cell surface and were subsequently rendered resistant (reviewed in Reference [44]). Besides this direct transfer of resistance, EV also contain several miRNAs and lncRNAs that can alter cell signaling pathways, steering sensitive cells toward resistance (reviewed in References [44,45]). Sunitinib showed that renal cell carcinoma cells not only transferred resistance to other cancer cells, but also to normal endothelial cells [46], possibly contributing to therapy resistance in the entire TME. Besides therapy resistance, EV-mediated phenocopying is involved in the propagation of tumour growth and invasiveness, all contributing to increased malignancy. For instance, glioblastoma multifome (GBM) cells expressing epithelial growth factor receptor variant III (EGFRvIII), a constitutive active mutant form of EGFR, incorporate this receptor into EV. These EV can subsequently transfer it to GBM cells that did not possess the genetic mutation for vIII themselves [43], leading to increased oncogenic signaling. Similarly, in murine hepatocarcinoma, transfer of C-X-C motif chemokine receptor type 4 (CXCR4) via EV resulted in stimulation of migratory capacity [47]. In another study, Zomer et al. found that coculturing nonmigratory 4T1 cells with EV from highly migratory MDA-MB-231 cells increased the invasiveness of 4T1 cells [42]. By use of a Cre/LoxP reporter system, they were able to show that the increase in migratory capacity was only observed in 4T1 cells which had taken up EV [42]. Preliminary data of our lab and others indicate that EV derived from hypoxic cells are capable of changing the hypoxic response of cells and increase survival when exposed to low oxygen concentrations (unpublished observation, [48]).
In light of these findings, it is conceivable that hypoxic tumour cells are able to precondition surrounding cells towards a more hypoxia-tolerant phenotype through EV, leading to overall increased tumour survival and malignancy. Here, we will review the available evidence on the role of tumour hypoxia on EV cargo and its subsequent effects on the induction of the main hypoxia tolerance mechanisms in target cells, i.e., angiogenesis, HIF-1α signaling, UPR execution and autophagy.

Extracellular Vesicles & HIF1 Signaling and Angiogenesis
The most studied and best understood response to hypoxia is mediated by the HIF family of transcription factors (HIF1, HIF2, HIF3). These transcription factors consist of an instable, oxygen sensitive, α-subunit and a stable β-subunit. During normal oxygen concentrations, the instable α-subunit is rapidly hydroxylated at two proline residues in the oxygen dependent regions by one of three prolyl hydroxylases (PHDs). The α-subunit is subsequently ubiquitinated by the Von Hippel Lindau protein (VHL) and targeted for proteasomal degradation [49,50]. In the absence of oxygen, the α-subunits are not degraded and are available to dimerize with β-subunits resulting in nuclear translocation triggering a powerful transcriptional response.
HIF transcription factors regulate the expression of many genes that promote hypoxia tolerance by decreasing cellular oxygen consumption, increasing oxygen supply and regulating pH [11,51]. In general, this includes shifting the energy metabolism toward the glycolytic pathway and the expression of proteins for pH-regulation [52][53][54][55]. In addition, HIFs influence tumour oxygenation directly by promoting angiogenesis, endothelial cell survival and vasculogenesis through genes, such as vascular endothelial growth factor (VEGF), CXCR4 and SDF1 [11,52,[54][55][56][57]. Hence, failure to stabilize HIF results in sensitization of cells to hypoxia and reduces the hypoxic fraction within tumours [58,59]. In addition, targeting HIF-inhibitory molecules (PHD2 and factor inhibiting HIF (FIH) through miR-182 expression) increases the degree of HIF-stabilisation during hypoxia and increases the angiogenic potential of cells [60,61].
One of the main reasons that the level of hypoxia within tumours correlates with poor treatment outcome is due to increased efficiency in inducing angiogenesis in support of tumour growth. Interestingly, hypoxic tumour cells induce angiogenesis through the secretion of EV ( [62], unpublished observation). EV derived from hypoxic GBM cells are enriched in mRNA from HIF-1α target genes, including Bcl-2 interacting protein 3 (BNIP3), adrenamedullin (ADM), lysyl oxidase (LOX), N-myc downstream regulated 1 (NDRG1), procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) and serpin family E member 1 (SERPINE1) [62]. In addition to mRNA, levels of several HIF-1α-inducible proteins were enriched in these EV, including interleukin 8 (IL-8), insulin-like growth factor binding protein 1 (IGFBP1), IGFBP3 and carbonic anhydrase 9 (CAIX) [62,63]. These mRNAs and proteins are instrumental in hypoxia tolerance and their incorporation into EV suggests that an active HIF-1α phenotype can be transmitted to surrounding cells. This is further supported by findings that MSC overexpression of HIF-1α incorporated more JAGGED-1 into EV, a Notch ligand which increases angiogenesis [64]. Aga et al. even found functional HIF-1α in EV of nasopharyngeal carcinoma cells [65]. However, it is difficult to determine what the functional consequences of EV-associated HIF-1α would be, as recipient cells which experience hypoxia already express HIF-1α, while nonhypoxic cells would likely rapidly ubiquitinate and degrade the protein. Additional evidence for increased angiogenic potential of hypoxia-derived EV shows that exposure of tissue factor (TF)/VIIa on the outer membrane leaflet of EV can bind proteinase-activated receptor 2 (PAR-2) on endothelial cells leading to pro-angiogenic ERK1/2 signaling [66]. Likewise, EV-associated CAIX significantly increased angiogenesis and endothelial migration [63]. Hypoxic colorectal cell-derived EV stimulated angiogenesis through the transfer of WNT4 mRNA. This led to an upregulation of Wnt4 protein and induced β-catenin signaling in endothelial cells [67].
In addition to protein and mRNA, several miRNAs under the transcriptional control of HIF-1α are enriched in EV derived from hypoxic cells. Amongst these are miR-21, miR-23a, miR-126, miR-135b, miR-210. The incorporation of these hypoxia-activated miRNAs reflect the hypoxic status of the producing cells and may result in hypoxia-phenocopying in neighbouring and distant cells. In Table 1 we have summarized which miRNAs are enriched in EV derived from hypoxic cells and their effects on HIF-1α signaling and angiogenesis [68][69][70][71]. For instance, miR-21 is dependent on HIF-1α for transcription and is associated with increased resistance to apoptosis, angiogenesis and migration through the activation of Akt and ERK [72][73][74]. Its incorporation into EV supports the hypothesis that cells are able to communicate a hypoxia tolerance phenotype to other surrounding cells possibly contributing to survival of the entire tumour. Likewise, miR-23a was increased in EV from hypoxic lung cancer cells and targets PHD1 and PHD2 in acceptor cells, leading to increased HIF-1α stabilization [75]. Additionally, miR-23a targets several components necessary for apoptosis, such as BH3 interacting domain death agonist (BID), caspase-7 and NIX/BNIP3L [76], possibly heightening the threshold for hypoxia-induced cell death. Hypoxia-dependent enrichment of EV-associated miR-210 is transcriptionally regulated by HIF-1α, although many of its (predicted) target genes are not directly involved in the hypoxia response. Gene ontology analysis revealed roles for miR-210 in regulation of differentiation, membrane trafficking and amino acid catabolism, indicating that miR-210 might downregulate cellular processes which are not necessary during hypoxia, and can thus be an important contributor to tumour survival [77,78]. Other HIF-1α regulated miRNAs found in EV include miR-494, miR-127 and miR-135a [79][80][81]. Delivery of miR-494 to vascular endothelial cells targets regulators of PTEN and subsequently activates of Akt/eNOS pathway [79]. In addition, miR-494 upregulates HIF-1α expression via activation of the PI3K/Akt pathway and increases hypoxia tolerance [82]. Treatment with antagomir-494 inhibits angiogenesis and reduces tumour growth in vivo [79] Likewise, miR-4530 increases angiogenesis by targeting vasohibin 1 (VASH1) [83,84], while miR-127 was protective for I/R related tissue damage [80]. On the other hand, miR-135a targets a negative regulator of HIF-1α, estrogen-related receptor alpha (ERRα) [81]. These findings indicate that the net effect of EV will depend on the balance in the recipient cell.

Extracellular Vesicles & the Unfolded Protein Response and Autophagy
The functionality of many proteins depends on correct folding and post-translational modification. The disulfide bonds introduced during post-translational folding or isomerization are oxygen-dependent [85]. Hypoxia, therefore, results in the accumulation of misfolded proteins, leads to ER stress and rapid activation of the unfolded protein response (UPR) [86], allowing cells to survive hypoxia exposure [12,58,87,88]. This is an evolutionarily conserved pathway that responds to endoplasmic reticulum (ER) stress by the coordinate action of three ER stress sensors present within the ER membrane, protein kinase-like ER kinase (PERK/EIF2AK3), inositol-required enzyme 1 (IRE1/ERN1) and ATF6 [89]. In the absence of ER stress, these sensors are associated with BiP/GRP78, preventing their activation. BiP is a chaperone protein involved in proper protein assembly. During ER stress, it is sequestered by misfolded proteins, leading to activation of the three transmembrane proteins and their respective UPR branches in order to restore ER homeostasis. The balance between BiP expression and UPR-executors (i.e., the quantity of "free" BiP) determines the threshold and extent of UPR-activation. Despite several reports which show that ER stress and UPR can be transported to other surrounding cells [75,90], EV as vehicles of this transport have mostly been ignored. However, recently, tumour EV were shown to induce the IRE1 branch of the UPR in nonmalignant target cells [22]. Although this was not studied in the context of hypoxia, it suggests that an increased hypoxia-tolerant phenotype could be transmitted by EV through activation of UPR. However, indirect evidence for the involvement of EV exists, as some miRNAs that have been shown to be associated to EV from hypoxic tumour cells in other studies are known to influence UPR signaling (Table 1). For example, miR-204 which targets PERK [91] or miR-181a. MiR-181a is associated with decreased BiP/GRP78 protein levels, but increased levels of mRNA, suggesting translational arrest [92]. By reducing or blocking miR-181a the brain was protected from stroke [93]. MiR-433 reduces glutathione biosynthesis, in turn leading to more oxidative stress and UPR activation [94].
In addition to HIF-1α and UPR, hypoxia activates autophagy, the lysosomal degradation pathway that mediates both selective and bulk degradation of proteins, cytoplasmic content and organelles [12,[95][96][97][98][99][100]. Nonselective autophagy is referred to as macro-autophagy (autophagy in this review), whereas micro-autophagy, mitophagy and chaperone-assisted autophagy refer to more selective forms that are directed by specific proteins. Autophagy begins with the formation of a double-membrane structure, the autophagosome, to engulf cellular content. Once formed, the outer membrane fuses with a lysosome, releasing its contents to the degradative enzymes. Autophagy plays an important role during conditions of starvation or metabolic stress by 'recycling' amino acids and nutrients to maintain energy levels, protein synthesis and essential metabolic processes [98]. Failure to execute autophagy in response to hypoxia results in cell death and sensitization of tumours to therapy [12,96]. Furthermore, autophagy is essential for removal of mitochondria that might become cytotoxic and is used during cycling hypoxia to rapidly decrease mitochondrial mass and reduce ROS production [100,101]. As a consequence, maintaining high levels of autophagy during hypoxia is essential for survival and inhibiting autophagy exposes the cells to increased ROS and cell death [12,102,103]. Hypoxic cells can secrete various signaling factors including proteins and/or microRNAs that are capable of influencing the autophagic potential of distant cells and potentially prime the cells to withstand periods of low oxygenation through elevated autophagy induction.

The Importance of EV Isolation Methods for the Interpretation of Results
Isolation methods for EV are known to impact findings with regard to composition and function and are, subsequently, important to note for the interpretation of results. For instance, it was shown that the vast majority of extracellular RNA is, in fact, not EV-associated, but rather coupled to RNA carrier proteins, such as Argonaut-2 [144][145][146] or high-density lipoproteins [147]. However, this extracellular RNA, as well as lipoprotein particles, soluble protein, protein aggregates, and the recently discovered exomeres are co-isolated by ultracentrifugation at 100,000 g, one of the most commonly applied isolation methods for EV [33,[148][149][150]. The same holds true for polyethylene glycol (PEG) precipitation-based isolation methods, such as exoquick [146]. Although it is becoming more recognized that further purification steps, such as density gradient separation or size exclusion chromatography, are necessary to be able to distinguish between true EV effects or effects resulting from other biologically relevant sources, this has yet to become common practice [151][152][153][154]. However, these purification steps are relatively labour-intensive, require specialized equipment or result in significant loss of EV particles, hampering widespread implementation. For purposes of this review, we therefore did not discriminate based on EV isolation methods. However, as we do believe that these isolation methods are key to fully understanding published results, we summarized the EV isolation methods and biological sources used by the studies we discussed in Table 2. Where available, we also included the EV-TRACK scores of the papers discussed, as EV-TRACK aims to concisely report important methodological parameters for EV-related publications, creating more transparency and facilitating interpretation of results [153]. However, it should be taken into account that many publications are not yet recorded in the EV-TRACK database due to its recent development and the relevance of these publications cannot be evaluated based on these scores alone. Table 2. Isolation methods and EV-METRIC scores used by the studies discussed. EV-TRACK ID as referenced in the platform for Transparant Reporting And Centralizing Knowledge in Extracellular Vesicles research (EV-TRACK [153]). EV-METRIC score represents the completeness of reporting of generic and method-specific information necessary to interpret and reproduce the experiment. N/A: Not available.

Conclusions
Overall, there is a great deal of evidence pointing toward a key role for EV in tumour hypoxia tolerance induction (proposed model in Figure 1). Besides an ability to transfer ready-to-use molecules to other cells, miRNA's are able to finetune pathways necessary for cell survival during hypoxia. In addition, EV-mediated communication can alleviate long-term hypoxia by interacting with endothelial cells in the TME leading to angiogenesis. However, it should be noted that reports on hypoxia-derived EV that have been performed with EV purified from protein and RNA contaminants are currently lacking, and caution should be taken when interpreting these results as EV-mediated. As most extracellular RNA and protein is, in fact, not EV-associated [146], it is currently difficult to determine what the true hypoxia modulating capacity of EV is. This makes additional fundamental research, with protocols that include current knowledge on isolation procedures, into the effects of hypoxia on EV cargo and effects necessary. on hypoxia-derived EV that have been performed with EV purified from protein and RNA contaminants are currently lacking, and caution should be taken when interpreting these results as EV-mediated. As most extracellular RNA and protein is, in fact, not EV-associated [146], it is currently difficult to determine what the true hypoxia modulating capacity of EV is. This makes additional fundamental research, with protocols that include current knowledge on isolation procedures, into the effects of hypoxia on EV cargo and effects necessary.

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

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