Bone malignancies are the third leading cause of cancer-related deaths in children and adolescents (Jemal et al., 2007). Osteosarcoma is the primary bone malignancy that mainly affects this age group, accounting for about 60% of bone malignancies (Geller and Gorlick, 2010). Although localized osteosarcoma has an overall survival of >70% in the clinic, metastatic, refractory, and recurrent osteosarcoma have poorer survival rates. Thus, in this article, we mainly discuss the osteosarcoma with poor survival. The most common complication of osteosarcoma is the development of metastatic diseases (Marina et al., 2004). The lung is the most common site of metastasis accounting for >85% of metastatic disease, followed by the bone being the second most common site of distant metastasis (Isakoff et al., 2015). Lung metastases are the leading cause of death in over 30% of patients with osteosarcoma (Botter et al., 2014). The overall survival rate of metastatic osteosarcoma is 20% after 5 years and the prognosis is poor (Allison et al., 2012; Zhu et al., 2013; Friebele et al., 2015; Saraf et al., 2018). Therefore, early diagnosis is of great importance for increasing the long-term survival of patients, and it is urgent to find biomarkers with high sensitivity and specificity suitable for timely detection. For metastatic osteosarcoma, chemotherapy is the primary treatment, and drug resistance is common; thus, there is also a need for new approaches to improve the efficacy of chemotherapeutic agents and minimize their toxic side effects.

Nanodrug delivery systems have the potential to enhance and maintain the clinical efficacy of chemotherapeutic drugs with less side effects by improving the absorption, penetration, and distribution of chemotherapeutic drugs, and has been widely studied in tumor targeted drug therapy in recent years (Li et al., 2018; Rodríguez-Nogales et al., 2018; Tu et al., 2018). The secretion mode of exosomes, their good biocompatibility, and their nanoscale size makes them excellent potential nano-drug delivery carriers, which will be described in detail below.

Exosomes are widely accepted generic terms for naturally secreted lipid bilayer particles in cells, such as endogenous exosomes and plasma membrane derived exosomes (Théry et al., 2018). Exosomes contain transmembrane proteins and enveloped cytoplasmic nucleic acids, lipids, and proteins, which ultimately form a lipid bilayer that supports exosomes structure (Fu et al., 2019). Exosomes are very small, 30–150 nm vesicles, which have been proven to exert important communication between cells and are easily released by a variety of cells in vivo and in vitro, including cancer cells and normal cells (Keller et al., 2006). Exosomes can also be found in many body fluids, including blood, urine, semen, breast milk, saliva, amniotic fluid, and ascites (Xu et al., 2018). In addition, exosomes have been described as a source of biomarkers and are involved in various physiological processes including blood coagulation, angiogenesis, wound healing, tissue regeneration, pregnancy, autophagy, immune regulation, stem cell differentiation, and cancer progression (Wolf-Dennen and Kleinerman, 2020). Further, exosomes are important agents in metastasis, tumor growth, tumorigenesis, and angiogenesis (Peinado et al., 2012). Exosomes have been shown to contain a variety of cargoes, such as proteins, mRNAs, miRNAs, and single- and double-stranded DNA (Vlassov et al., 2012), which can reflect the identity of the cell of origin (Fu et al., 2019). Once released, exosomes can transmit multimolecular information to neighboring cells and distant targets through the extracellular fluid, thereby regulating their roles (Cappariello and Rucci, 2019).

The purpose of this review is to discuss the relationship between exosomes and osteosarcoma, focusing on the functions, mechanisms of action, and potential clinical applications of exosomes and their cargoes as biomarkers and therapeutic targets for osteosarcoma. But overall, the applicability of osteosarcoma derived exosomes can be used for all osteosarcoma subsets. Although there have been two recent reviews summarizing the relationship between exosomes and osteosarcoma, the first described only the role and application prospects of exosome derived non-coding RNAs for osteosarcoma, while the other focused more on exosome derived cargoes in osteosarcoma patients (Li and Wang, 2021; Zhang X. B. et al., 2021). This review provides a more comprehensive description of the function and application prospects of osteosarcoma exosomes, including not only as exosome cargoes in osteosarcoma, but also as carriers in osteosarcoma. In addition, all studies describing the relationship between exosomes and osteosarcoma were included.

The production of exosomes involves the double invagination of the plasma membrane and the formation of intracellular multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs) (Kalluri and LeBleu, 2020). Early endosomes are formed when cell-surface proteins and soluble proteins are endocytosed via inward budding of the plasma membrane, which indicates exosome formation has initiated (Merchant et al., 2017; Kalluri and LeBleu, 2020). Early endosomes also fuse with the endoplasmic reticulum (ER), trans-Golgi network (TGN), and mitochondria (Kalluri, 2016; Hessvik and Llorente, 2018; van Niel et al., 2018; Willms et al., 2018; Mathieu et al., 2019; McAndrews and Kalluri, 2019). Early endosomes mature into late endosomes and eventually generate MVBs containing numerous ILVs formed by the emboly of the endosomal membrane (Figure 1; Merchant et al., 2017). During emboly, ILVs incorporate cytosolic and membrane proteins, lipids, and RNAs (Raiborg and Stenmark, 2009; van Niel et al., 2018). The MVBs can be degraded or fuse with the plasma membrane and by fusing with lysosomes or autophagosomes they release the contained ILVs as exosomes (Raiborg and Stenmark, 2009; Kahlert and Kalluri, 2013; Lo Cicero et al., 2015; van Niel et al., 2018). Furthermore, exosomes could also be formed through plasma membrane (PM) directly. The main difference between the MVB-dependent secretion of exosomes and PM derived exosomes is the different molecular mechanisms involved in exosomes formation. The former is associated with the endosomal sorting complex required for the transport complex (ESCRT) family, lipids, tetraspanins, and the Ras-related proteins in the brain (RAB) family (including RAB11, RAB35, RAB7, and RAB27), and in turn the RAB act on different MVBs. The MVB pathway is associated with proteins of ADP-ribosylation factor 6 (ARF6), and part of the ESCRT family [including ALG2-interacting protein X (ALIX) and tumor susceptibility gene 101 (TSG101)] (Colombo et al., 2014). Nonetheless, the different extracellular fate of these two types of exosomes is currently unknown and may be related to the cargoes they derive.

When exosomes arrive at the surface of receptor cells then may induce intracellular signals by binding to surface receptors (Denzer et al., 2000; Miyanishi et al., 2007). Exosome uptake has been linked to multiple mechanisms, including macropinocytosis, phagocytosis, clathrin-dependent endocytosis, and clathrin-independent endocytosis (Mulcahy et al., 2014; van Dongen et al., 2016). The different types of endocytosis are the primary methods of exosome uptake. However, which type of endocytic mechanism is involved in osteosarcoma and is the most important has little consensus in research and differences in exosome uptake reveal the heterogeneity of exosomes and the original cells (Mulcahy et al., 2014). Thus, it is important to explore this problem in future studies. Furthermore, exosomes, by fusing with the plasma membrane, directly release exosome cargoes into the cytoplasm of the recipient cell where they play subsequent biological roles (Parolini et al., 2009).

Recent evidence has shown that exosomes play a vital role in the tumorigenesis, proliferation, metastasis, anti-apoptosis, immune evasion and chemoresistance of osteosarcoma. Garimella et al. first reported the function of exosomes in osteosarcoma. They detected factors such as MMPs, RANKL, TGFβ, and CD-9 in the exosomes of osteosarcoma cells, as well as changes in intracellular calcium concentration that can affect exosome biogenesis. They suggested a novel function of exosomes in driving bone resorption of osteoclastic by disrupting bone remodeling homeostasis in the osteosarcoma bone microenvironment (BME) (Garimella et al., 2014). This study opened a novel area in osteosarcoma research, linking emerging nanocarrier exosomes to the progression, treatment, prognosis, and even pathogenesis of osteosarcoma.

In addition to exosomes, exosomal cargoes can also have direct or indirect effects on the biological function of osteosarcoma cells or other cells. Exosomes-delivered bioactive molecules result in the intercellular exchange of genetic information and recipient cell reprogramming (Fu et al., 2019). Exosome derived cargoes can be used to clarify biogenesis processes, recognize parent cells, and regulate biological functions (Tang and Wong, 2015). Below, we will discuss exosomal cargoes carried by exosomes derived from osteosarcoma cells and other cells in the TME, including exosomal RNAs [such as Micro RNAs (miRNAs), Long non-coding RNAs (lncRNAs), and Circular RNAs (circRNAs)] and proteins (Table 1).

Exosomes could be secreted by different types of cells including osteosarcoma, BMSCs, CAFs, ADSCs, macrophages, and the secreted exosomes may contain a series of cargoes such as miRNAs, lncRNAs, circRNAs, and proteins. The exosomes and exosomal cargoes derived from these cells could exert biological activity and modify the functional activity of recipient cells, including proliferation, invasion, migration, angiogenesis, apoptosis, metastasis and chemoresistance. Moreover, several important branches are being explored to better define the clinical applications for exosomes and exosomal cargoes, including biomarkers, therapeutic targets and drug delivery vehicles (Figure 5).

Recent studies on exosomes have changed our understanding of the role of exosomes in osteosarcoma and provided new targets for the diagnosis and treatment of osteosarcoma. The role of exosomes in changing osteosarcoma behavior, remodeling TME and producing osteosarcoma drug resistance has attracted extensive attention. The existing studies of exosomes and their cargoes have partially unraveled the mechanism of action of osteosarcoma-associated exosomes. For example, there is molecular communication between osteosarcoma cells and MSCs that help establish a premetastatic niche in the TME. In addition, exosomes carrying specific cargoes in the circulation have potential diagnostic value, which reflects the progression and metastasis stages of osteosarcoma and predict the prognosis of osteosarcoma. Exosomes-based therapy has shown great promise. Blocking exosome-based cargo and its target or developing exosome-based drug delivery provides a novel strategy for the treatment of osteosarcoma.

Circulating exosomes derived cargoes have a great potential for early cancer detection, therapy, and prognosis. Nonetheless, using exosomes as biomarkers in diagnosis and prognosis, may result in false positive and negative findings due to the quality and heterogeneity of exosomes. Although the quality of exosomes has improved recently, and cancer therapy based on exosomes is supported by several early clinical trials. In pancreatic ductal adenocarcinoma (PDAC), engineered MSC derived exosomes carrying a commonly mutated gene KRAS siRNA is under research in early-phase clinical trial to treat metastatic pancreas cancer patients with the KRAS mutation (NCT03608631) (Kamerkar et al., 2017). The safety of dendritic cell (DC) derived exosomes for patients and the feasibility of producing DC derived exosomes are supported by early-phase clinical trials in cancer patients (Escudier et al., 2005; Morse et al., 2005). In colorectal cancer (CRC), tumor derived exosomes containing melanoma-related antigen recognized by T cells (Mart1) in ascites (Aex) was used to deliver Mart1 to DCs and Aex-containing exosomes plus granulocyte-macrophage colony-stimulating factor (GM-CSF) induced tumor-specific antitumor CTL response in early-phase clinical trials (Dai et al., 2008). In these three clinical trials, it is not difficult to appreciate that in addition to natural exosomes, engineered exosomes have been developed to meet the needs of clinical applications. There are two strategies used to produce engineered exosomes: direct extracellular modification and indirect intracellular modification. Extracellular modification often refers to the direct load of cargoes after exosomes isolation and purification, and intracellular modification means the parental cells are manipulated before exosomes release by physical or genetic approaches (Ma et al., 2020). However, the therapeutic potential of exosomes, the method of drug loading, the safety of exosomes as carrier, and the choice of exosomes doner cells are variables awaiting further investigation. A few exosome isolation techniques have been developed, including filtration, size-exclusion chromatography, ultracentrifugation, immunoaffinity capture, and microchip-based techniques (Zhang X. B. et al., 2021). However, the effects of different exosome isolation methods on the size, integrity, RNA, and protein cargoes of exosomes are unknown, which could lead to different sizes and concentrations of exosomes. Therefore, a standardized and stable isolation method is needed to produce exosomes for clinical application. Furthermore, the different storage temperatures of exosomes suitable for maintaining the vitality of exosomes in the clinical setting and in laboratory is also a significant challenge for clinicians (Park et al., 2018).

Although increasing attention has been paid to the application of exosomes in osteosarcoma, there are still several problems to be addressed before they can be used in the clinical treatment of osteosarcoma, including the specific mechanism of action of exosomes in osteosarcoma, isolation techniques, quantification, and the analysis of exosomes in body fluids of patients with osteosarcoma, as well as methods for detecting circulating osteosarcoma-specific exosomal cargoes (DNA, RNA, protein), and the preparation, storage and drug loading methods of engineered exosomes for treating osteosarcoma. In addition, there are many exosomes and exosomal cargoes awaiting to be discovered, providing more biological support for their role in the progression, treatment, and prognosis of osteosarcoma. Therefore, further efforts are needed to understand the role and mechanism of exosomes in osteosarcoma and to develop exosomes-based diagnosis, prognosis prediction and treatment options for osteosarcoma patients.

LY and XH: conceptualization, investigation, methodology, and writing-original draft. ZS: funding acquisition, writing-review and editing. HG, LW, WW, DJ, and WY: software, supervision, and writing-review and editing. All authors have given final approval to this version of the manuscript to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.