Background

Hypoxia, a condition of insufficient oxygen, is a common feature of numerous solid tumors, related to tumor development, therapy resistance, and mortality [1]. Tumor cells in the microenvironment encounter low oxygen levels because of insufficient oxygen flow and physiological anomalies in tumor vessels, resulting in normoxic, hypoxic, and also necrotic regions [2]. In most solid tumors, the average concentration of oxygen is near 10 mmHg, whereas in the other tissues it reaches 40 and 60 mmHg [3]. Tumor cells juxtaposed to the normal vessels are functional, whereas cells situated around 150 μm from the vasculature bed may undergo necrosis and atresia [4], however, cells are located between these two cell populations habituate to an insufficient oxygen environment, hypoxia. Generally, cells located in a diameter of approximately 1 mm undergo genetic, molecular, and metabolic changes. These cells could reorganize the microenvironment appropriate for growth, metastasis, and therapy resistance [5]. In this situation, by the production of pro-angiogenic factors, tumor cells can induce angiogenesis for supplying nutrients and oxygen and removing waste. Besides, increased angiogenesis in tumor mass provides a way for tumor cells to migrate and metastasize [6].

In response to a hypoxic condition, the transcription profile of tumor cells such as hypoxia-inducible factors (HIFs), the key factor involved in regulating hypoxic condition, are altered [7]. HIFs, the dimeric proteins, are composed of a constitutive subunit named HIF-1β and an oxygen-regulated α-subunit (HIF-1, 2, 3α) [8]. The activity of HIF-1α is dynamic and related to the availability of oxygen. In the presence of oxygen, HIF-1 α is degraded by the proteasome system; so that, HIF-1α is hydroxylated by prolyl hydroxylases (PHD), in kee**, von Hippel-Lindau (VHL) recognizes the hydroxylated HIF-1α. This event subsequently ubiquitinates HIF-1α for degradation by the 26S proteasome [9, 10]. PHDs are inactive in a hypoxic condition and allow HIF-1α to link to the HIF-1β subunit.

According to previous studies, HIFs are active in almost solid tumors. HIFs regulate different signaling pathways, which are implicated in the cell viability, proliferation, epithelial-to-mesenchymal transition (EMT), angiogenesis, metastasis, and therapy resistance [11]. However, other pathways including mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinases (PI3K)-Akt, Wnt/β-catenin, nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPK), and NADPH oxidase (NOX) facilitate the adaption of tumor cells to hypoxia [1, 12, 13]. Besides, HIFs have been shown to facilitate exosomes biogenesis and secretion [14]. Exosomes a subfamily of extracellular vesicles (EVs) mediate intercellular communication by carrying different biological molecules among cells. These vesicles contain various biological molecules like proteins, RNAs (coding and non-coding RNAs), DNAs, and lipids, regulating activation of different signaling pathways in recipient cells located nearby or distant tissues [15, 16]. It has been shown that exosomes derived from tumor cells participate in modulating the tumor microenvironment and promoting tumorigenesis [17, 18]. Confirmed that, hypoxia induces exosomes biogenesis and secretion and thereby promotes tumor intercellular communication, representing the key role of exosomes in hypoxic tumors [14, 19]. The majority of experiments discussed in this review used 1% O2 as a hypoxic model that generally called the hypoxic condition, if not, we explained the condition. In the present review, we discuss exosomes biogenesis and loading and also possible underlying mechanisms under hypoxic. Also, we describe the key roles of hypoxic in tumorigenesis and tumor-therapy.

Tumor microenvironment

The tumor microenvironment is a dynamic environment around a tumor, containing the blood vessels, fibroblasts, immune cells, the extracellular matrix (ECM), and signaling molecules that support proliferation, growth, metastasis, and therapy resistance of tumor cells. Tumor cell proliferation, death, invasion, migration, angiogenesis, metabolic reprogramming, immune evasion, are all regulated by the complex interaction inside the tumor microenvironment. In this regard, autocrine, paracrine, and juxtacrine communication network orchestrate these biological functions. Paracrine-mediated communication plays pivotal roles in signal transduction between neighboring and distant cells [20,21,22].

Non-tumor cells including fibroblasts, endothelial cells (ECs), and immune cells contribute to tumor microenvironment interaction and are affected by tumor soluble factors, and their fate goes through tumor-like modifications, persistently accommodate to the tumor microenvironment and support tumor growth. In the tumor microenvironment, fibroblasts are motivated into cancer-associated fibroblasts (CAFs), these cells are the most resident stromal cells in the tumor microenvironment, producing an ECM that vary common ECM in inflexibility and arrangement properties that facilities migration and invasion of tumor cells [23]. In the tumor microenvironment, hypoxia induces tumor cells to produce angiogenic factors, which in turn affect ECs and up-regulate angiogenesis [24, 25]. In the tumor microenvironment, the resident immune cells demonstrate multiplicity and could suppress the immune responses. Also, anti-inflammatory molecules can inhibit the immune system, which is involved in the suppression of cancer cells [99]. More recently, Mo et al. [100] found that exosomes derived from hypoxic A549 lung cancer cells contain angiopoietin-like 4 protein that induces angiogenesis in HUVECs. Also, exosomal miR-210 released from hypoxic leukemia cells induced tubulogenesis in ECs [101]. These data show exosomes from hypoxic tumor cells can promote angiogenesis, thus these exosomes may serve as a novel target for cancer treatment.

Diagnostic application of hypoxic exosomes

Early diagnosis of cancer is the hallmark of cancer therapy that improves the survival rate and quality of a patient’s life [102]. As exosomes released from tumor cells can be distributed to several bio-fluids, thus, a simple liquid-biopsy from plasma, serum, urine, and CSF is a non-invasive way for acquiring detailed information about tumor environment/status [15]. As exosomes originate directly from tumor cells, they may serve as a diagnostic tool for predicting the extent of the physiological and pathological status of tumor cells. Exosomes cargo like proteins and nucleic acids are altered upon the change in the dynamic of parental cells, suggesting a prognostic and diagnostic tool for the treatment of cancer [103]. Analyzing exosomal cargoes (miRs and proteins) gives a chance for scientists to predict the status of a pathological condition like tumor progression. Therefore, as under hypoxia condition tumor cells release more exosomes with distinct cargoes; they may be potentially used as a biomarker for hypoxia tumors. Exosomal biomarker represents superiority against other approaches evaluating hypoxia. Currently, some of the approaches have clinical challenges in estimating hypoxia. For instance, pimonidazole and immunohistochemistry techniques are invasive and necessitate surgical elimination of tumors. Consequently, the application of tumor-derived exosomes from bio-fluids to obtain evidence of hypoxia standing in various cancers could be noteworthy. In this regard, exosomal miRs and protein obtained from bio-fluids have biomarker potential for the diagnosis of different cancers [104, 105]. For example, Matsumura et al. [106] reported that expression of miR-19a in exosomes isolated from the serum of CRC patients was up-regulated, which could be considered as a relapse biomarker of CRC. In the case of hypoxic tumors, confirmed that HIF-1 mRNA molecules are enriched within tumor-derived exosomes that are commonly considered as a typical biomarker for diagnosing cancer development as well as therapy consequences [79]. A study conducted by Kucharzewska et al. [107] demonstrated that exosomes obtained from both Glioblastoma multiform (GBM) cells culture medium and isolated from the GBM patients plasma abundantly contain hypoxia-regulated proteins and mRNAs including PDGFs, Caveolin 1, IL- 8, MMPs, and LOX. The authors conclude that the mRNA and proteome content of these exosomes reflect the hypoxic status of cancer and have biomarker potential for GBM. Also, miRs and metabolites cargo of hypoxic exosome would be useful as a biomarker for diagnosis and prognosis of different cancers such as prostate, colorectal, and pancreatic cancers [108,109,110]. For example, the expression pattern of exosomal miR-210 from the serum of CRC patients may function as a promising non-invasive biomarker for the diagnosis and prognosis of CRC [108]. During the hypoxic condition, miR-210 is the most extensively and consistently up-regulated miR that commonly shows tumorigenesis properties in different tumors [111]. Similar to hypoxic exosomes from prostate tumor cells (PCa and LNCaP), exosomes obtained from the serum of PCa patients contain a high level of miR-885 and miR-521 [109]. Besides, proteins cargo (VLA-4, TYRP2, HSP90, and HSP70) of exosomes derived from the plasma of melanoma patients are significantly increased in comparison with healthy persons [112]. VLA-4 and TYRP2 are up-regulated under the hypoxic condition and their high expression in exosomes correlates with stage 3 melanoma [113, 114]. Previous studies have shown that HSP90 and HSP70 are hypoxic related proteins and play roles in hypoxic condition [115, 116]. In this regard, exosomal proteins have diagnostic and prognostic value for the melanoma tumor development and hypoxic status. Therefore, hypoxic exosomes may be a useful tool for predicting the hypoxic status of solid tumors, however, it seems that this evidence is not sufficient and further scrutiny is essential to examine and confirm the potential application of hypoxic exosomes to quantify the degree of hypoxia to detect stages of tumor development.

Possible therapeutic application of exosomes

Exosomes can reach target cells and alter the function, fate, and morphology through different signaling pathways. As mentioned, tumor cells under hypoxic conditions produce more exosomes, promoting tumorigenesis. Thus, it seems that targeting exosomes formation and secretion particularly from the hypoxic tumor may provide us with a tool that reduces tumorigenesis. Recent findings have shown that it is possible to inhibit the exosomes biogenesis and secretion from different cells. For example, Manumycin A and GW4869 have been shown to inhibit exosome biogenesis and release from cells [117]. Datta et al. [118] reported that Manumycin A inhibited exosomes biogenesis and secretion from aggressive prostate cells mainly by suppression of Ras/Raf/ERK1/2 signaling and hnRNP H1. They concluded that Manumycin A is a potential drug candidate to inhibit exosome biogenesis and secretion. Suppression of Rab27a, a protein involved in exosomes secretion, has been shown to inhibit exosome-dependent and -independent tumor cells growth [119]. Inhibition of Rab proteins involved in intracellular trafficking of exosomes/MVB may inhibit exosomes biogenesis and release, thus they may be a target to inhibit exosome biogenesis. For example, Rab5a has been involved in the early step of exosomes biogenesis, while Rab11, Rab27a, and Rab35 regulate the MVBs-plasma membrane fusion and exosomes secretion. Moreover, inhibition of sphingomyelinase, an enzyme catalyzes the formation of ceramide from sphingomyelin, may lessen exosomes biogenesis and loading, thus prevents tumor growth [120]. In human prostate cancer (PC3) cells, it was shown that Imipramine profoundly inhibited the biogenesis of both microvesicles and exosomes [121]. A fascinating approach has been proposed by Marleau and colleagues based on the effective elimination of blood exosomes of breast cancer patients by extracorporeal hemofiltration associated with affinity agents like exosome-trap** antibodies and lectins. This approach was proposed to trap particles < 200 nm from the whole circulatory system [122]. Considering the existence of numerous experiments on exosomes inhibition, there are challenges regarding analyzing and conclusions of findings, because as various methods are used to isolate and characterize exosomes. Moreover, some researchers did not include ISEV guidelines regarding the exosomes confirmation and validation, as exosome-based studies had been performed before the 2014 and 2018 declaration of ISEV guidelines about exosome-based studies [30, 123]. Besides, it is vital to discover the non-toxic doses of the drugs for target cells to confirm that any decrease in exosomes secretion resulting from exosomes inhibition not from cell death. The majority of these experiments were pre-clinical performed, thus, clinical trials are essential for the approval. Furthermore, the non-targeting effects of these drugs on exosomes biogenesis from normal cells remain an important concern. At least, in the field of cancer, key studies must still be necessary to investigate their effects on exosomes production from both healthy and tumor cells and to progress methods to specifically deliver drugs into tumor cells.

Another approach that exosomes can be used as a therapeutic agent is the drug delivery potential of them. According to previous studies, exosomes can be used as a drug delivery system in two ways: (I); direct loading by which therapeutic agents directly sorted into exosomes; and (II); indirect loading where source cells co-cultured with therapeutic agents or manipulated genetically to produce optional exosomes. In this regard, different approaches for producing optional exosomes have been examined, which comprise: incubating exosomes with the agents, electroporation, sonication, sensitive fusogenic peptide, and cationic lipid, liposome, and exosome-coated metal–organic nanoparticle [124, 125]. An example of the direct method, Zhuang et al. [131]. Collectively, exosomes may serve as a new avenue to overcome cancer, however, translation of pre-clinical results into the clinic needs more experiments regarding exosomes biology and bio-applications in disease models.

Conclusion

Hypoxia increases exosome biogenesis and secretion in tumor cells. Moreover, it can alter exosomes cargo. Exosomes released from tumor cells play a pivotal role in promoting growth, metastasis, and resistance of hypoxic tumors. Furthermore, exosomes from hypoxic tumors have been suggested to be a promising non-invasive biomarker for cancer diagnosis through analyzing their components such as proteins and miRs. Inhibition of exosomes biogenesis and secretion may help to reduce tumorigenesis. Exosomes can be used as a drug delivery system for the treatment of cancer. However, despite many experiments, translation of the preclinical findings into the clinic requires additional examinations in this field. Therefore, further scrutiny is essential for a better understanding of the mechanisms behind exosome loading and production under hypoxic conditions, which could be useful in targeting exosomes biogenesis and prevent tumorigenesis.