Functions and therapeutic potentials of exosomes in osteosarcoma

Abstract

Osteosarcoma, a primary malignant tumor of the skeleton, has a morbidity of 2.5 per 1 million people. The epiphysis of extremities is typically affected. Osteosarcoma has a high likelihood of early metastasis, rapid progression, and poor prognosis. The survival rate of patients with metastatic or recurrent osteosarcoma remains low; therefore, novel diagnostic and therapeutic methods are urgently needed. Exosomes, extracellular vesicles 30–150 nm in diameter, are secreted by various cells and are widely present in various body fluids. Exosomes are abundant in biologically active components, such as proteins, nucleic acids, and lipids. Exosomes participate in numerous physiological and pathological processes via intercellular substance exchange and signaling. This review presents the novel findings regarding exosomes in osteosarcoma diagnosis, prognosis, and therapeutics.

Main article text

1. INTRODUCTION

Osteosarcoma (OS) is a primary malignant bone tumor originating from primitive osteogenic mesenchymal cells in adolescents and young adults under the age of 20 years [1]. Although the quality of life of patients with OS has significantly improved over the past few decades, its etiology remains unclear. Studies aimed at determining causes of OS have typically focused on multiple factors, including genetics, epidemiology, and the environment [2]. Research has identified associations with secondary OS in patients with Paget disease, electrical burns, trauma, exposure to beryllium, exposure to alkylating agents, FBJ virus, osteochondromatosis, enchondromatosis, fibrous dysplasia, orthopedic prosthetics, or bone infarction and infection. Additionally, OS has been reported to correlate with exposure to ionizing radiation, radium, and archaic contrast agents, such as thorotrast [3]. Furthermore, several genetic aberrations have been identified in cases of primary OS, including hereditary retinoblastoma, Li-Fraumeni syndrome, Rothmund-Thompson syndrome, Bloom syndrome, and Werner syndrome [4]. Radiographs of OS present osteogenic, osteolytic, or mixed bone destruction at the lesion. “Codman’s triangle” and sun-exposed periosteal reaction [5] are typical radiographic features. MRI accurately depicts OS on the basis of tumor cell differentiation and proliferation [6]. Radionuclide scans can determine whether bone metastases occur in OS [7]. Frozen biopsies are used for rapid intraoperative diagnosis, and paraffin sections are used for obtaining accurate histological findings postoperatively [8]. High levels of serum alkaline phosphatase and lactate dehydrogenase are predictive of poor prognosis [9]. Treatments for OS include neoadjuvant chemotherapy, surgical resection, chemotherapy, and interventional therapy [10]. In addition, cellular immunotherapy, gene therapy, and stem cell therapy have made some progress in recent years [11]. However, these methods remain in experimental stages. Approximately 18% of patients present micrometastases at the time of diagnosis, and the 5-year survival rate remains poor for patients with metastasis and recurrence [12]. Treatment outcomes remain suboptimal, owing to the tendency of OS to remain asymptomatic, and to show early onset metastasis, and high malignancy. The 5-year survival rate of patients with OS without chemotherapy is below 30%. The leading cause of death is lung metastasis [13]. The 2-year survival rate of patients with OS with pulmonary metastasis is less than 25%, and the survival period after treatment shows a plateau, thus making breakthrough efficacy with traditional treatment regimens challenging [14]. Therefore, the underlying mechanisms of OS development and metastasis must be determined to enable the discovery of novel markers for clinical detection and effective therapeutic targets.

Exosomes have been reported to be involved in regulating cellular behavior by transferring cargoes (proteins, DNA, RNA, and lipids) intercellularly. Increasing evidence indicates that exosomes have high potential to promote OS progression and development; moreover, the therapeutic potential of exosomes in OS is receiving increasing attention. Exosomes are membranous vesicles 30–100 nm in diameter originating from late endosomes, which are formed by inward budding of the limited multivesicular body (MVB) membrane [15]. Exosomes were first identified as double-layered lipid structures containing no organelles in blood erythrocytes [16]. Exosomes contain various nucleic acids and evolutionarily conserved proteins [17], which transmit biological information through cellular communication for biological processes and disease progression [18]. In lung adenocarcinoma (LUAD), LINC00273 is induced by M2 macrophages and exosomal LINC00273 was transferred into LUAD cells to recruit NEDD4, thereby promoting LATS2 ubiquitination, inhibiting the Hippo pathway and YAP-induced RBMX transcription, and ultimately resulting in LUAD malignancy [19]. Anlotinib-resistant non-small-cell lung cancer (NSCLC) cells promote the proliferation of parental NSCLC cells by transferring functional miR-136-5p from anlotinib-resistant NSCLC cells to parental NSCLC cells via exosomes. Exosomal miR-136-5p can lead to anlotinib resistance in NSCLC cells by targeting PPP2R2A and promoting activation of the AKT pathway [20]. Exosomes secreted by various cells in OS enable intercellular communication of ncRNAs and protein components, thus effectively regulating the tumor microenvironment, and promoting proliferation and metastasis. In addition, exosomes’ stability in the circulatory system supports their diagnostic and therapeutic potential. This article reviews the biological properties of exosomes and their roles in the diagnosis and treatment of OS.

2. EXOSOME FORMATION AND BIOLOGICAL CHARACTERISTICS

Extracellular vesicles (EVs) are universally found in cells, and carry proteins, genetic material, and metabolites [21]. On the basis of their size and release mechanism, EVs are classified into exosomes (30–150 nm in diameter), microvesicles/extranuclear granulosomes (100–1000 nm in diameter), and apoptotic vesicles (50–1500 nm in diameter) [22]. Exosome formation involves dual invagination of the protoplasmic membrane and the formation of intracellular multivesicular bodies (MVBs), which contain intraluminal vesicles (ILVs) [23]. The endoplasmic reticulum also contributes to early endonucleosome formation [24]. Invagination of late endosomal membranes results in the formation of ILVs within large MVBs, which fuse with lysosomes or autophagosomes, thereby leading to cargo degradation, or fuse with the plasma membrane and release the contained ILVs as exosomes [25]. Exosomes are present in almost all body fluids, including plasma, urine, ascites, and breast milk [26].

2.1 Exosome formation

Exosome formation is activated by endosomal endocytosis, wherein the endosomal limiting membrane undergoes multiple deformations and invaginates, thus generating ILVs. The ILVs transform into MVBs with dynamic subcellular structures. MVBs are generated at the endosomal limiting membrane through either an endosomal sorting complex required for transport (ESCRT) mechanism or a non-ESCRT mechanism [27]. The ESCRT mechanism involves recognition of cytoplasmic protein complexes with ubiquitinated modified membrane proteins. The ESCRT-0 complex plays a vital role in the generation of multivesicular bodies by binding and clustering ubiquitinated proteins. The ESCRT-I complex recognizes and passes ESCRT-0 to ESCRT II. TSG101 in ESCRT I identifies disulfide bonds and consequently induces endosomal membrane invagination; shearing of the bud neck via ESCRT III then leads to the formation of MVBs [28]. In the absence of ESCRT, MVB formation is initiated by the accessory protein ALG-2 interacting protein X (AIix), which directly binds the intracellular bridging protein syntenin and participates in exosome formation [29]. The abundant tetratransmembrane protein can facilitate the production of these ESCRT-nondependent MVBs [30]. MVB fusion with lysosomes induces the degradation and recirculation of their cargo. Cholesterol abundance in MVBs plays an essential role in regulating their sorting: cholesterol-rich MVBs are targeted to the cell membrane for release as exosomes, whereas low-cholesterol MVBs are targeted for transport to lysosomes [31].

2.2 Exosome mechanisms in biological function

Exosome-mediated intercellular transmission relies on membrane receptors. Exosomes activate receptors on recipient cells, thereby activating the uptake of exosomes through cytokinesis [32]. Studies have focused on exploring the functions of cell-derived exosomes and the use of exosomes for disease treatment [33]. Target cell specificity may be determined by specific interactions between proteins enriched on the surfaces of exosomes and receptors on the membranes of recipient cells [34]. Known mediators include transmembrane tetraspanins, integrins, and extracellular matrix components [35].

2.3 Exosomes’ potential in tumor diagnosis and treatment

Exosomes primarily regulate the exclusion of redundant and nonfunctional cellular components [36]. Exosomes are intercellular linkers that transport proteins, lipids, and nucleic acids to target cells in various biological processes, such as angiogenesis, antigen presentation, apoptosis, and inflammation [37]. The specific cell components in the exosomes reflect cellular origin and physiological state, and show significant disease specificity, thus making them ideal biomarkers. Exosomes are involved in various cancer-associated processes, including proliferation, apoptosis, angiogenesis, and metastasis; consequently, they may serve as noninvasive biomarkers for cancer diagnosis [38, 39]. For example, miR-21, miR-222, and miR-124-3p in serum exosomes are detectable in early tumor progression after surgical treatment of patients with high-grade glioma [40]. Moreover, miR-21, miR-451, and miR-636 in urinary exosomes of patients with prostate cancer closely correlate with preoperative prostate-specific antigen levels; thus, urinary exosomal microRNAs (miRNAs) may potentially serve as noninvasive markers to predict prostate cancer metastasis and prognosis [41]. Plasma exosomal miR-363-5p has shown high diagnostic performance in discriminating patients with LN (+) versus LN (-) breast cancer. Elevated miR-363-5p expression levels have been found to indicate lower overall survival [42]. The therapeutic potential of exosomes is associated primarily with targeted drug delivery and biomedical regeneration. Exosomes have great potential in treating diseases, owing to their nontumorigenic, bactericidal, and low immunogenicity characteristics [43]. Ligand enrichment on engineered exosomes can induce or inhibit signaling in receptor cells, or can target exosomes to specific cells [44]. Exosomes loaded with chemotherapeutic agents have shown promise for antineoplastic drugs delivery with low toxicity and high tolerance [45].

3. EXOSOMES IN OS PROGRESSION

Exosomes can transmit intercellular signals that regulate proliferation and metastasis. Exosomes promote tumor proliferation and metastasis by inducing epithelial-mesenchymal transition, and accelerating tumor neovascularization and immunosuppression through regulating the microenvironment and transformation of cancer-associated fibroblasts [46, 47]. Exosomes have major roles in regulating proliferation, invasion metastasis, and OS angiogenesis, by participating in intercellular contacts and modulating cellular signaling.

3.1 Exosomes in OS proliferation

The potential to proliferate indefinitely is the fundamental feature of cancer cells [48]. OS cells express growth factor receptors and rarely show negative feedback regulation, thus resulting in continuous activation of signal stimulation, and unlimited cell division and proliferation [49]. Exosomes participate in various processes in the proliferation of OS ( Table 1 ). For example, miR-208a from bone marrow-derived mesenchymal stem cell (BMSC)-derived exosomes has been found to promote OS cell proliferation and inhibit apoptosis by suppressing PDCD4 expression, and activating the ERK1/2 and Hippo pathways. BMSC-derived exosomal miR-206 inhibits cell proliferation by targeting TRA2B [50]. In addition, BMSC-derived exosomes encapsulate PVT1 and translocate it to OS cells. PVT1 promotes tumor growth and metastasis by binding miR-183-5p, thus increasing ERG expression [51]. The MALAT1/miR-143/NRSN2/Wnt/β-catenin axis is another crucial target through which BMSE-EVs promote proliferation [52]. ADSC exosomes deliver COLGALT2 to OS cells, thus leading to OS malignancy [53]. BMSC-derived exosomes promote OS proliferation and metastasis via the LCP1/JAK2/STAT3 pathway. Moreover, targeting the miR-135a-5p/LCP1 axis inhibits OS progression [54]. MG-63-cell-derived exosomes promote the proliferation of OS and inhibit apoptosis. Hic-5 from MG-63-cell-derived exosomes interacts with Smad4 and regulates Wnt/β-catenin signaling by decreasing TCF/LEF activity [55]. OS-cell-derived exosomal miR-1307 promotes OS cell proliferation by inhibiting AGAP1 expression; consequently, the miR-1307-AGAP1 axis may serve as a potential therapeutic target for OS [56]. In patients with OS, exosomal miR-15a expression is diminished in plasma exosomes. Moreover, exosomal miR-15a has been found to inhibit the GATA2/MDM2 axis via the p53 signaling pathway, thereby inhibiting the proliferation and invasion of OS cells in vitro [57].

Table 1 |

Biological functions of exosomes in the proliferation and metastasis of osteosarcoma.

Exosomal target	Parent cell	Target cell	Mechanism	Biological function	Ref.
Proliferation and metastasis
miR-208	BMSCs	Osteosarcoma cells	PDCD4/ERK1/2	Increase the viability, migration, and clonogenicity of OS	[95]
miR-206	BMSCs	Osteosarcoma cells	TRA2B	Promote OS cell proliferation, migration, and invasion, and induce apoptosis	[50]
MALAT1	BMSCs	Osteosarcoma cells	MALAT1/miR-143/NRSN2/Wnt/β-catenin	Promote OS cell proliferation, migration, and invasion	[52]
PVT1	BMSCs	Osteosarcoma cells	PVT1/miR-183-5p/ERG	Promote OS growth and metastasis	[51]
ATG5	BMSCs	Osteosarcoma cells	/	Promote OS cell proliferation, migration, and invasion	[96]
COLGALT2	ADSCs	Osteosarcoma cells	/	Promote OS cell proliferation, migration, and invasion	[97]
Linc00852	Osteosarcoma cells with high AXL expression	Osteosarcoma cells with low AXL expression	Linc00852/miR-7-5p/AXL	Promote cell proliferation, migration, and invasion	[98]
LCP1	BMSCs	Osteosarcoma cells	miR-135a-5p/LCP1/JAK2/STAT3	Induce the proliferation and metastasis of OS cells	[54]
Hic-5	MG-63	MG-63 and HOS cells	Hic-5/smad4-TCF/LEF -Wnt/β-catenin	Promote cell proliferation and inhibit cell apoptosis	[55]
miR-1307	Osteosarcoma cells	Osteosarcoma cells	AGAP1	Promote OS cell proliferation, migration, and invasion	[56]
miR-15a	Serum-derived exosomes	Osteosarcoma cells	miR-15a/p5/GATA2/MDM2	Promote OS cell proliferation and invasion	[99]
miR-769-5p	BMSCs	Clinical specimens	DUSP16/JNK/p38 MAPK	Promote OS proliferation and metastasis	[100]
SHNG17	CAFs\NFs	HOS cells	miR-2861	Promote OS proliferation and metastasis	[101]
miR-143	/	Osteosarcoma cells	/	Inhibit cell invasion	[59]
miR-675	Osteosarcoma cells	hFOB1.19	CALN1	Promote cell migration and invasion	[25]
Rab22a-NeoF1/PYK2	PYK2-positive osteosarcoma cells	Macrophages	RhoA	Facilitate pre-metastatic niche formation	[62]
miR-1307	Osteosarcoma cells	Osteosarcoma cells	AGAP1	Promote cell proliferation, migration, and invasion	[62]
LCP1	BMSCs	Osteosarcoma cells	miR-135a-5p/Nrdp1/JAK2/STAT3	Promote OS proliferation and metastasis	[54]
LIFR-AS1	Macrophages	Osteosarcoma cells	miR-29a/NFIA	Promote cell proliferation and invasion, and restrain cell apoptosis	[102]
Angiogenesis
miR-25-3p	/	Osteosarcoma cells	DKK3	Promote capillary formation and the invasion of vascular endothelial cells	[42]
EWSAT1	/	Osteosarcoma cells	/	Increase sensitivity/reactivity of vascular endothelial cells	[103]
OIP5-AS1	Osteosarcoma cells	Osteosarcoma cells	miR-153/ATG5	Increase angiogenesis	[66]
miR-199a-5p	Osteosarcoma cells	HUVECs	VEGFA	Inhibit the growth and angiogenesis of osteosarcoma	[104]
miR-148a-3p and miR-21-5p	Osteosarcoma cells	Raw264.7 and HUVECs	/	Influence osteoclastogenesis, bone resorption, and tumor angiogenesis	[69]
Immunosuppressive
miR-148a-3p and miR-21-5p	Osteosarcoma cells	Raw264.7 and HUVECs	/	Influence osteoclastogenesis, bone resorption, and tumor angiogenesis	[69]
Tim-3	MG63	Macrophages	/	Induce M2 type differentiation of macrophages	[72]

3.2 Exosomes in OS metastasis

Epithelial-mesenchymal transition is a biological phenomenon in which epithelial cells lose their epithelial properties and acquire a mesenchymal phenotype. In this process, epithelial features are diminished. Cells change from polygonal to spindle-shaped fibroblast-like morphology; show loss of cell polarity and decreased adhesion; and acquire invasion and metastasis ability [58]. Exosomes are essential in the invasive metastasis of OS ( Table 1 ). For example, miR-143 is transferred to OS cells via exosomes and significantly inhibits tumor invasiveness [59]. Highly invasive OS cells secrete exosomal miR-675, which suppresses CALN1 expression in recipient cells. The expression of exosomal miR-675 in the serum in patients with OS is strongly correlated with prognosis [60]. Mazumdar et al. have found that both highly metastatic 143-B cells and weakly metastatic SAOS-2-cell-derived EVs induce the recruitment of bone marrow cells to the lungs, and that components in exosomes may inhibit remote metastasis of OS [61]. In OS, the Rab22a-NeoF1 fusion protein is assimilated into exosomes. The exosomal Rab22a-NeoF1 fusion protein promotes formation of the premetastatic lung niche by recruiting bone-marrow-derived macrophages [62]. OS-cell-derived exosomal miR-1307 promotes proliferation, migration, and invasion by regulating AGAP1 expression, thus indicating the inhibitory roles of miR-1307 in the malignant progression of OS [56].

3.3 Exosomes in OS angiogenesis

Proangiogenic and antiangiogenic factors have major roles in the formation of blood vessels [63]. Tumor cells require nutrient supply and metabolite excretion for survival and development [64]. Tumor-derived exosomes are critical mechanisms that promote angiogenesis ( Table 1 ). In OS tissues, an increase in miR-25-3p promotes tumor proliferation, metastasis, and drug resistance by inhibiting DKK3. EWSAT1 promotes OS angiogenesis by transfering it into the exosome-driven vascular endothelial cell, thus increasing the secretion and the sensitivity/responsiveness to angiogenic factors [65]. OS cells with high exosome abundance regulate OS tumor angiogenesis and autophagy through miR-153 and ATG5, by secreting exosomal lnc-OIP5-AS1, which is taken up by adjacent OS cells [66].

3.4 Exosomes in OS the immune response

Exosomes participate in the immune response and regulate immunocompetence [67]. Tumor-cell-derived exosomes carry tumor-associated antigens and stimulate immune cells to generate antitumor immune responses. However, they can interfere with immune recognition, and inhibit T cells and immune-associated cells, thereby accelerating tumor cell immune escape and metastasis [17]. Immune cells derived from the tumor microenvironment regulate proliferation and metastasis through exosomes [68]. Exosomes also have critical roles in the tumor immune microenvironment of OS ( Table 1 ). Exosomal miR-1228 secreted by cancer-associated fibroblasts (CAFs) promotes OS invasion and migration by targeting SCAI. This miRNA may serve as a potential therapeutic target for OS [42]. Exosomes enhance tube formation in endothelial cells and increase the expression of angiogenic markers. Next-generation sequencing has revealed that specific miRNAs, such as miR-148a and miR-21-5p, have essential roles in the tumor microenvironment [69]. The exosomes of metastatic OS cells secrete TGFβ2, which is taken up by tumor-associated macrophages, thus promoting the M2 phenotype and contributing to immunosuppression and tumorigenesis [70]. OS-cell-derived EVs promote myofibroblast/cancer-associated fibroblast differentiation, smooth muscle actin expression, and fibronectin production. In addition, they significantly promote the invasiveness of human lung fibroblasts [71]. OS-derived exosomes induce M2 polarization of macrophages via Tim-3, thereby promoting OS invasion and metastasis [72]. Exosomal Col6a1 converts normal fibroblasts into CAFs through the secretion of proinflammatory cytokines. Activated CAFs promote OS cell invasion and migration by mediating the TGF-β/COL6A1 signaling pathway [73]. The macrophage-derived exosomal long noncoding RNA LIFR-AS1 promotes the malignant progression of OS by binding miR-29a and consequently increasing NFIA expression [74].

4. APPLICATION POTENTIAL OF EXOSOMES IN OS

Exosomes contain various biologically active molecules in circulation and mediate remote intercellular interaction [75]. Tumor-derived exosomes contain multiple proteins, genetic material, lipids, and other molecules that reflect tumor physiological and pathological status [76]. The specific lipid bilayer structure of exosomes protects RNA molecules from degradation [77]. Therefore, detecting tumor exosomes provides major advantages in liquid biopsy. Exosomes have good application potential for early diagnosis, assessment of treatment efficacy, and monitoring of prognosis in various diseases. They have become new and ideal biomarkers, and may possibly serve as targeted drug carriers in clinical diagnosis and treatment.

4.1 Potential of exosomes in OS diagnosis

Exosomes are essential in the early diagnosis and prognostic assessment of OS. Eight novel miRNAs have been identified by next-generation sequencing in three distinct OS cell lines, five of which are present in the circulating exosomes of patients with OS [57]. EV-miR-101 expression levels are significantly lower in OS patients. In plasma from patients with OS metastasis, EV-miR-101 is lower than that in patients without metastasis and thus may serve as a potential diagnostic marker for OS [78]. Ye et al. have revealed that the expression levels of miR-92a-3p, miR-130a-3p, miR-195-3p, miR-335-5p, and let-7i-3p are significantly upregulated in the exosomes of patients with OS and therefore may serve as potential diagnostic markers [79]. HSATI, HSATII, LINE1-P1, and Charlie 3 are overexpressed at the RNA level in serum exosomes from patients with OS and thus may be potential OS biomarkers [80]. Exosome-derived SENP1 in patients with OS is closely correlated with tumor size, location, necrosis rate, lung metastasis, and surgical staging. Higher plasma exosome-derived SENP1 levels indicate poorer disease-free survival and overall survival [81]. Seven exosomal proteins have been identified as potential biomarkers of OS lung metastasis [82]. In addition, SERS and MALDI-TOF MS exosomes have shown great potential in the rapid diagnosis of OS [83].

4.2 Potential of exosomes in OS treatment

Exosomes have great potential in the treatment of OS. Multidrug-resistant OS cells secrete exosomes containing MDR-1 messenger RNA and P-glycoprotein, thus promoting doxorubicin resistance in sensitive cells. Exosomes targeting drug-resistant OS cells may inhibit the malignant progression of OS [84]. Compared with exosomes from normal osteoblasts, OS-derived exosomes contain immunomodulatory substances that significantly decrease T cell proliferation rates and promote T regulatory phenotypes, thereby facilitating OS progression [10, 85]. The miRNAs miR-135b, miR-148a, miR-27a, and miR-9 are highly expressed in the serum exosomes in patients with OS and may potentially be reliable biomarkers of chemotherapy sensitivity [16, 86]. Exosome-loaded doxorubicin has been found to enhance cellular uptake efficiency and antitumor effects in the OS MG63 cell line, while showing low cytotoxicity, thus potentially providing a good targeting regimen for OS [87]. OS cells promote OS lung metastasis by releasing exosomes containing PD-L1 and N-calcineurin. In addition, the expression levels of exosomal PD-L1 and N-calcineurin in the serum in patients with OS have been found to predict pulmonary metastasis progression [88]. Exosomes from cisplatin-resistant OS cells decrease the expression of multidrug resistance-associated protein 1 and P-glycoprotein in MG63 and U2OS cells; increase chemosensitivity to cisplatin; and inhibit apoptosis through exosomal-hsa_circ_103801 [89]. Moreover, exosomes from drug-resistant HMPOS-2.5R cell lines have been found to transfer drug resistance to drug-sensitive HMPOS cells, thereby decreasing the therapeutic sensitivity of OS [90].

5. CONCLUSIONS

Early diagnosis is critical for promoting good prognosis and survival in tumor patients [91]. Exosomes are stable and widespread in all tissues, organs, and body fluids, and are released by all types of cells ( Figure 1 ) [92]. Tumor exosomes regulate tumor progression, angiogenesis, metastasis, and immune escape by interacting with other cells in the tumor microenvironment [93]. Standard methods for liquid biopsy are needed to isolate exosomes quickly, easily, and specifically. Exosomes are a promising biomarker for the diagnosis of OS, predicting prognosis, and monitoring treatment response in real time. Therefore, large multicenter studies are needed to develop the validity of liquid biopsies. For study of biological functions, whether exosomes have similar regulatory functions in vivo and in vitro is impossible to determine. For therapeutic purposes, exosome-derived cells should be carefully selected to ensure the safety of the treatment. Erythrocytes are the most promising exosome-producing cells, because they are readily available in blood banks, do not contain nuclei, and lack genetic material. Beyond their potential as biomarkers, exosomes may support new research directions in the precision treatment of tumors [87]. To improve the effectiveness of antitumor drug therapy, development of drug-loading systems remains a key challenges. As natural therapeutic carriers, exosomes contain bioactive molecules and can avoid immune rejection [94]; in addition they enable exogenous drugs to maintain stability in vivo. These advantages make exosomes an ideal loading system providing a new paradigm for drug delivery, and are expected to be an important tool for the development of precision medicine for tumors. Han et al. have constructed fusion gene iRGD-Lamp2b-modified mesenchymal stem cells to isolate and purify exosomes and loaded anti-miRNA-221 oligonucleotides into exosomes. AMO-loaded exosomes have been found to effectively inhibit the proliferation and clonal formation of colon cancer cells in vitro [51].

Figure 1 |

The interaction of OS and related cells through exosomes.

Mesenchymal stem cells, CAFs, and CSCs secrete exosomes containing specific proteins and genetic material, which promote the proliferation, metastasis, and invasion of OS. OS cells generate exosomes targeting specific cells that promote angiogenesis, osteoclastogenesis, and immunomodulation of the target cells. OS promotes drug resistance, proliferation, and metastasis through exosome secretion. (Created in Biorender.com.)

This review discussed the biological functions of exosomes in the progression of OS and clinical applications. Exosomes from OS promote malignant progression by regulating tumor metastasis, angiogenesis, tumor immunity, and drug resistance. Exosomes therefore provide new potential therapeutic targets.

ABBREVIATIONS

Osteosarcoma, OS; microRNAs, miRNAs; non-small-cell lung cancer, NSCLC; extracellular vesicles, EVs; multivesicular bodies, MVBs; luminal vesicles, ILVs; endosomal sorting complex required for transport, ESCRT; bone marrow-derived mesenchymal stem cells, BMSCs; cancer-associated fibroblasts, CAFs.

ACKNOWLEDGEMENTS

This work was supported by the Liaoning Cancer Hospital & Institute (Shenyang), St. John’s University and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine. This work was supported by NCI grant R01 CA230916 and funds from the Sylvester Comprehensive Cancer Center/University of Miami. Zhe-Sheng Chen is supported by St. John’s University Department of Pharmaceutical Sciences.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

REFERENCES

1. Fujiwara T, Uotani K, Yoshida A, Morita T, Nezu Y, Kobayashi E, et al.. Clinical Significance of Circulating miR-25-3p as a Novel diagnostic and Prognostic Biomarker in Osteosarcoma. Oncotarget. 2017. Vol. 8:33375–33392

2. Zheng C, Tang F, Min L, Hornicek F, Duan Z, Tu C. PTEN in Osteosarcoma: Recent Advances and the Therapeutic Potential. Biochim Biophys Acta Rev Cancer. 2020. Vol. 1874:188405

3. Jerez S, Araya H, Thaler R, Charlesworth MC, López-Solís R, Kalergis AM, et al.. Proteomic Analysis of Exosomes and Exosome-Free Conditioned Media from Human Osteosarcoma Cell Lines Reveals Secretion of Proteins Related to Tumor Progression. Journal of Cellular Biochemistry. 2017. Vol. 118:351–360

4. Hameed M, Mandelker D. Tumor Syndromes Predisposing to Osteosarcoma. Advances in Anatomic Pathology. 2018. Vol. 25:217–222

5. Permata TBM, Sato H, Gu W, Kakoti S, Uchihara Y, Yoshimatsu Y, et al.. High Linear Energy Transfer Carbon-Ion Irradiation Upregulates PD-L1 Expression More Significantly than X-rays in Human Osteosarcoma U2OS Cells. Journal of Radiation Research. 2021. Vol. 62:773–781

6. Zheng Y, Duan X, Wang H, Zhao S. Pulmonary Artery Osteosarcoma Masquerading as Pulmonary Thromboembolism: The Role of Multimodality Imaging. ESC Heart Failure. 2021. Vol. 8:5565–5567

7. Qi J, Zhou Y, Jiao Z, Wang X, Zhao Y, Li Y, et al.. Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway. Cellular Physiology and Biochemistry. 2017. Vol. 42:2242–2254

8. Laskar S, Kakoti S, Khanna N, Manjali JJ, Mangaj A, Puri A, et al.. Outcomes of Osteosarcoma, Chondrosarcoma and Chordoma Treated with Image Guided-Intensity Modulated Radiation Therapy. Radiotherapy and Oncology. 2021. Vol. 164:216–222

9. Mahyudin F, Prawiragara FA, Edward M, Utomo DN, Basuki MH, Bari YA, et al.. The Escalation of Osteosarcoma Stem Cells Apoptosis after the Co-Cultivation of Peripheral Blood Mononuclear Cells Sensitized with Mesenchymal Stem Cells Secretome and Colony Stimulating Factor-2 In Vitro. Journal of Blood Medicine. 2021. Vol. 12:601–611

10. Troyer RM, Ruby CE, Goodall CP, Yang L, Maier CS, Albarqi HA, et al.. Exosomes from Osteosarcoma and Normal Osteoblast Differ in Proteomic Cargo and Immunomodulatory Effects on T Cells. Experimental Cell Research. 2017. Vol. 358:369–376

11. Huang Q, Liang X, Ren T, Huang Y, Zhang H, Yu Y, et al.. The Role of Tumor-Associated Macrophages in Osteosarcoma Progression - Therapeutic Implications. Cellular Oncology (Dordrecht). 2021. Vol. 44:525–539

12. Prudowsky ZD, Yustein JT. Recent Insights into Therapy Resistance in Osteosarcoma. Cancers (Basel). 2020. Vol. 13:83

13. Christie JD, Appel N, Canter H, Achi JG, Elliott NM, de Matos AL, et al.. Systemic Delivery of TNF-Armed Myxoma Virus Plus Immune Checkpoint Inhibitor Eliminates Lung Metastatic Mouse Osteosarcoma. Molecular Therapy Oncolytics. 2021. Vol. 22:539–554

14. Chen C, Xie L, Ren T, Huang Y, Xu J, Guo W. Immunotherapy for Osteosaarcoma: Fundamental Mechanism, Rationale, and Recent Breakthroughs. Cancer Letters. 2021. Vol. 500:1–10

15. Psaraki A, Ntari L, Karakostas C, Korrou-Karava D, Roubelakis MG. Extracellular Vesicles Derived from Mesenchymal Stem/Stromal Cells: The Regenerative Impact in Liver Diseases. Hepatology (Baltimore, Md.). 2022. Vol. 75:1590–1603

16. Xu JF, Wang YP, Zhang SJ, Chen Y, Gu HF, Dou XF, et al.. Exosomes Containing Differential Expression of microRNA and mRNA in Osteosarcoma that can Predict Response to Chemotherapy. Oncotarget. 2017. Vol. 8:75968–75978

17. Chen H, Chengalvala V, Hu H, Sun D. Tumor-Derived Exosomes: Nanovesicles Made by Cancer Cells to Promote Cancer Metastasis. Acta Pharmaceutica Sinica. B. 2021. Vol. 11:2136–2149

18. Isaac R, Reis FCG, Ying W, Olefsky JM. Exosomes as Mediators of Intercellular Crosstalk in Metabolism. Cell Metabolism. 2021. Vol. 33:1744–1762

19. Bayer A, Lennemann NJ, Ouyang Y, Sadovsky E, Sheridan MA, Roberts RM, et al.. Chromosome 19 microRNAs Exert Antiviral Activity Independent from Type III Interferon Signaling. Placenta. 2018. Vol. 61:33–38

20. Gu G, Hu C, Hui K, Zhang H, Chen T, Zhang X, et al.. Exosomal miR-136-5p Derived from Anlotinib-Resistant NSCLC Cells Confers Anlotinib Resistance in Non-Small Cell Lung Cancer through Targeting PPP2R2A. International Journal of Nanomedicine. 2021. Vol. 16:6329–6343

21. Hur JY, Lee KY. Characteristics and Clinical Application of Extracellular Vesicle-Derived DNA. Cancers (Basel). 2021. Vol. 13:3827

22. Brady JV, Troyer RM, Ramsey SA, Leeper H, Yang L, Maier CS, et al.. A Preliminary Proteomic Investigation of Circulating Exosomes and Discovery of Biomarkers Associated with the Progression of Osteosarcoma in a Clinical Model of Spontaneous Disease. Translational Oncology. 2018. Vol. 11:1137–1146

23. Chen J, Zhang Q, Liu D, Liu Z. Exosomes: Advances, Development and Potential Therapeutic Strategies in Diabetic Nephropathy. Metabolism. 2021. Vol. 122:154834

24. Ruan S, Greenberg Z, Pan X, Zhuang P, Erwin N, He M. Extracellular Vesicles as an Advanced Delivery Biomaterial for Precision Cancer Immunotherapy. Advanced Healthcare Materials. 2021. Vol. 11:e2100650

25. Gong L, Bao Q, Hu C, Wang J, Zhou Q, Wei L, et al.. Exosomal miR-675 from Metastatic Osteosarcoma Promotes Cell Migration and Invasion by Targeting CALN1. Biochemical and Biophysical Research Communications. 2018. Vol. 500:170–176

26. Tang XH, Guo T, Gao XY, Wu XL, Xing XF, Ji JF, et al.. Exosome-Derived Noncoding RNAs in Gastric Cancer: Functions and Clinical Applications. Molecular Cancer. 2021. Vol. 20:99

27. Chivero ET, Dagur RS, Peeples ES, Sil S, Liao K, Ma R, et al.. Biogenesis, Physiological Functions and Potential Applications of Extracellular Vesicles in Substance Use Disorders. Cellular and Molecular Life Sciences. 2021. Vol. 78:4849–4865

28. Lagerweij T, Perez-Lanzon M, Baglio SR. A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-Mediated Communication between Tumor and Mesenchymal Stem Cells. Journal of Visualized Experiments. 2018. 56932

29. Chen P, Wang L, Fan X, Ning X, Yu B, Ou C, et al.. Targeted Delivery of Extracellular Vesicles in Heart Injury. Theranostics. 2021. Vol. 11:2263–2277

30. Shehzad A, Islam SU, Shahzad R, Khan S, Lee YS. Extracellular Vesicles in Cancer Diagnostics and Therapeutics. Pharmacology & Therapeutics. 2021. Vol. 223:107806

31. Schubert A, Boutros M. Extracellular Vesicles and Oncogenic Signaling. Molecular Oncology. 2021. Vol. 15:3–26

32. Mohammadi M, Zargartalebi H, Salahandish R, Aburashed R, Wey Yong K, Sanati-Nezhad A. Emerging Technologies and Commercial Products in Exosome-Based Cancer Diagnosis and Prognosis. Biosensors & Bioelectronics. 2021. Vol. 183:113176

33. Kumar A, Deep G. Hypoxia in Tumor Microenvironment Regulates Exosome Biogenesis: Molecular Mechanisms and Translational Opportunities. Cancer Letters. 2020. Vol. 479:23–30

34. Nakamura K, Sawada K, Kobayashi M, Miyamoto M, Shimizu A, Yamamoto M, et al.. Role of the Exosome in Ovarian Cancer Progression and its Potential as a Therapeutic Target. Cancers (Basel). 2019. Vol. 11:1147

35. Li C, Hou X, Zhang P, Li J, Liu X, Wang Y, et al.. Exosome-Based Tumor Therapy: Opportunities and Challenges. Current Drug Metabolism. 2020. Vol. 21:339–351

36. Shao J, Zaro J, Shen Y. Advances in Exosome-Based Drug Delivery and Tumor Targeting: From Tissue Distribution to Intracellular Fate. International Journal of Nanomedicine. 2020. Vol. 15:9355–9371

37. Xu Z, Zeng S, Gong Z, Yan Y. Exosome-Based Immunotherapy: A Promising Approach for Cancer Treatment. Molecular Cancer. 2020. Vol. 19:160

38. Meng W, Hao Y, He C, Li L, Zhu G. Exosome-Orchestrated Hypoxic Tumor Microenvironment. Molecular Cancer. 2019. Vol. 18:57

39. Namee NM, O’Driscoll L. Extracellular Vesicles and Anti-Cancer Drug Resistance. Biochimica et Biophysica Acta - Reviews on Cancer. 2018. Vol. 1870:123–136

40. Olioso D, Caccese M, Santangelo A, Lippi G, Zagonel V, Cabrini G, et al.. Serum Exosomal microRNA-21, 222 and 124-3p as Noninvasive Predictive Biomarkers in Newly Diagnosed High-Grade Gliomas: A Prospective Study. Cancers (Basel). 2021. Vol. 13:3006

41. Shin S, Park YH, Jung SH, Jang SH, Kim MY, Lee JY, et al.. Urinary Exosome microRNA Signatures as a Noninvasive Prognostic Biomarker for Prostate Cancer. NPJ Genomic Medicine. 2021. Vol. 6:45

42. Yoshida A, Fujiwara T, Uotani K, Morita T, Kiyono M, Yokoo S, et al.. Clinical and Functional Significance of Intracellular and Extracellular microRNA-25-3p in Osteosarcoma. Acta Medica Okayama. 2018. Vol. 72:165–174

43. Yang E, Wang X, Gong Z, Yu M, Wu H, Zhang D. Exosome-Mediated Metabolic Reprogramming: The Emerging Role in Tumor Microenvironment Remodeling and its Influence on Cancer Progression. Signal Transduction and Targeted Therapy. 2020. Vol. 5:242

44. Santos P, Almeida F. Exosome-Based Vaccines: History, Current State, and Clinical Trials. Frontiers in Immunology. 2021. Vol. 12:711565

45. Gonzalez MJ, Kweh MF, Biava PM, Olalde J, Toro AP, Goldschmidt-Clermont PJ, et al.. Evaluation of Exosome Derivatives as Bio-Informational Reprogramming Therapy for Cancer. Journal of Translational Medicine. 2021. Vol. 19:103

46. Mashouri L, Yousefi H, Aref AR, Ahadi AM, Molaei F, Alahari SK, et al.. Exosomes: Composition, Biogenesis, and Mechanisms in Cancer Metastasis and Drug Resistance. Molecular Cancer. 2019. Vol. 18:75

47. Zhang P, Samuel G, Crow J, Godwin AK, Zeng Y. Molecular Assessment of Circulating Exosomes toward Liquid Biopsy Diagnosis of Ewing Sarcoma Family of Tumors. Translational Research. 2018. Vol. 201:136–153

48. Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011. Vol. 144:646–674

49. Huang T, Song X, Yang Y, Wan X, Alvarez AA, Sastry N, et al.. Autophagy and Hallmarks of Cancer. Critical Reviews in Oncogenesis. 2018. Vol. 23:247–267

50. Zhang H, Wang J, Ren T, Huang Y, Liang X, Yu Y, et al.. Bone Marrow Mesenchymal Stem Cell-Derived Exosomal miR-206 Inhibits Osteosarcoma Progression by Targeting TRA2B. Cancer Letters. 2020. Vol. 490:54–65

51. Zhao W, Qin P, Zhang D, Cui X, Gao J, Yu Z, et al.. Long Non-Coding RNA PVT1 Encapsulated in Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Promotes Osteosarcoma Growth and Metastasis by Stabilizing ERG and Sponging miR-183-5p. Aging (Albany NY). 2019. Vol. 11:9581–9596

52. Li F, Chen X, Shang C, Ying Q, Zhou X, Zhu R, et al.. Bone Marrow Mesenchymal Stem Cells-Derived Extracellular Vesicles Promote Proliferation, Invasion and Migration of Osteosarcoma Cells via the lncRNA MALAT1/miR-143/NRSN2/Wnt/β-Catenin Axis. Onco Targets and Therapy. 2021. Vol. 14:737–749

53. Abello J, Nguyen TDT, Marasini R, Aryal S, Weiss ML. Biodistribution of Gadolinium- and Near Infrared-Labeled Human Umbilical Cord Mesenchymal Stromal Cell-Derived Exosomes in Tumor Bearing Mice. Theranostics. 2019. Vol. 9:2325–2345

54. Ge X, Liu W, Zhao W, Feng S, Duan A, Ji C, et al.. Exosomal Transfer of LCP1 Promotes Osteosarcoma Cell Tumorigenesis and Metastasis by Activating the JAK2/STAT3 Signaling Pathway. Molecular Therapy Nucleic Acids. 2020. Vol. 21:900–915

55. Sha L, Ma D, Chen C. Exosome-Mediated Hic-5 Regulates Proliferation and Apoptosis of Osteosarcoma via Wnt/β-Catenin Signal Pathway. Aging (Albany NY). 2020. Vol. 12:23598–23608

56. Han F, Pu P, Wang C, Ding X, Zhu Z, Xiang W, et al.. Osteosarcoma Cell-Derived Exosomal miR-1307 Promotes Tumorgenesis via Targeting AGAP1. BioMed Research International. 2021. Vol. 2021:7358153

57. Cuscino N, Raimondi L, De Luca A, Carcione C, Russelli G, Conti L, et al.. Gathering Novel Circulating Exosomal microRNA in Osteosarcoma Cell Lines and Possible Implications for the Disease. Cancers (Basel). 2019. Vol. 11:1924

58. Pal A, Barrett TF, Paolini R, Parikh A, Puram SV. Partial EMT in Head and Neck Cancer Biology: A Spectrum Instead of a Switch. Oncogene. 2021. Vol. 40:5049–5065

59. Shimbo K, Miyaki S, Ishitobi H, Kato Y, Kubo T, Shimose S, et al.. Exosome-Formed Synthetic microRNA-143 is Transferred to Osteosarcoma Cells and Inhibits their Migration. Biochemical and Biophysical Research Communications. 2014. Vol. 445:381–387

60. De Feo A, Sciandra M, Ferracin M, Felicetti F, Astolfi A, Pignochino Y, et al.. Exosomes from CD99-Deprived Ewing Sarcoma Cells Reverse Tumor Malignancy by Inhibiting Cell Migration and Promoting Neural Differentiation. Cell Death & Disease. 2019. Vol. 10:471

61. Mazumdar A, Urdinez J, Boro A, Arlt MJE, Egli FE, Niederöst B, et al.. Exploring the Role of Osteosarcoma-Derived Extracellular Vesicles in Pre-Metastatic Niche Formation and Metastasis in the 143-B Xenograft Mouse Osteosarcoma Model. Cancers (Basel). 2020. Vol. 12:3457

62. Zhong L, Liao D, Li J, Liu W, Wang J, Zeng C, et al.. Rab22a-NeoF1 Fusion Protein Promotes Osteosarcoma Lung Metastasis through its Secretion Into Exosomes. Signal Transduction and Targeted Therapy. 2021. Vol. 6:59

63. Martin P, Gurevich DB. Macrophage Regulation of Angiogenesis in Health and Disease. Seminars in Cell & Development Biology. 2021. Vol. 119:101–110

64. Zhu L, Yu X, Wang L, Liu J, Qu Z, Zhang H, et al.. Angiogenesis and Immune Checkpoint Dual Blockade in Combination with Radiotherapy for Treatment of Solid Cancers: Opportunities and Challenges. Oncogenesis. 2021. Vol. 10:47

65. Fu CY, Chen MC, Tseng YS, Chen MC, Zhou Z, Yang JJ, et al.. Fisetin Activates Hippo Pathway and JNK/ERK/AP-1 Signaling to Inhibit Proliferation and Induce Apoptosis of Human Osteosarcoma Cells via ZAK Overexpression. Environmental Toxicology. 2019. Vol. 34:902–911

66. Li Y, Lin S, Xie X, Zhu H, Fan T, Wang S. Highly Enriched Exosomal lncRNA OIP5-AS1 Regulates Osteosarcoma Tumor Angiogenesis and Autophagy Through miR-153 and ATG5. American Journal of Translational Research. 2021. Vol. 13:4211–4223

67. Liu J, Ren L, Li S, Li W, Zheng X, Yang Y, et al.. The Biology, Function, and Applications of Exosomes in Cancer. Acta Pharmaceutica Sinica. B. 2021. Vol. 11:2783–2797

68. Zheng H, Siddharth S, Parida S, Wu X, Sharma D. Tumor Microenvironment: Key Players in Triple Negative Breast Cancer Immunomodulation. Cancers (Basel). 2021. Vol. 13:3357

69. Raimondi L, De Luca A, Gallo A, Costa V, Russelli G, Cuscino N, et al.. Osteosarcoma Cell-Derived Exosomes Affect Tumor Microenvironment by Specific Packaging of microRNAs. Carcinogenesis. 2020. Vol. 41:666–677

70. Wolf-Dennen K, Gordon N, Kleinerman ES. Exosomal Communication by Metastatic Osteosarcoma Cells Modulates Alveolar Macrophages to an M2 Tumor-Promoting Phenotype and Inhibits Tumoricidal Functions. Oncoimmunology. 2020. Vol. 9:1747677

71. Mazumdar A, Urdinez J, Boro A, Migliavacca J, Arlt MJE, Muff R, et al.. Osteosarcoma-Derived Extracellular Vesicles Induce Lung Fibroblast Reprogramming. International Journal of Molecular Sciences. 2020. Vol. 21:5451

72. Cheng Z, Wang L, Wu C, Huang L, Ruan Y, Xue W. Tumor-Derived Exosomes Induced M2 Macrophage Polarization and Promoted the Metastasis of Osteosarcoma Cells through Tim-3. Archives of Medical Research. 2021. Vol. 52:200–210

73. Zhang Y, Liu Z, Yang X, Lu W, Chen Y, Lin Y, et al.. H3K27 Acetylation Activated-COL6A1 Promotes Osteosarcoma Lung Metastasis by Repressing STAT1 and Activating Pulmonary Cancer-Associated Fibroblasts. Theranostics. 2021. Vol. 11:1473–1492

74. Galardi A, Colletti M, Di Paolo V, Vitullo P, Antonetti L, Russo I, et al.. Exosomal MiRNAs in Pediatric Cancers. International Journal of Molecular Sciences. 2019. Vol. 20:4600

75. Hwang S, Yang YM. Exosomal microRNAs as Diagnostic and Therapeutic Biomarkers in Non-Malignant Liver Diseases. Archives of Pharmacal Research. 2021. Vol. 44:574–587

76. Hosseini R, Asef-Kabiri L, Yousefi H, Sarvnaz H, Salehi M, Akbari ME, et al.. The Roles of Tumor-Derived Exosomes in Altered Differentiation, Maturation and Function of Dendritic Cells. Molecular Cancer. 2021. Vol. 20:83

77. Bai K, Li X, Zhong J, Ng EHY, Yeung WSB, Lee CL, et al.. Placenta-Derived Exosomes as a Modulator in Maternal Immune Tolerance during Pregnancy. Frontiers in Immunology. 2021. Vol. 12:671093

78. Zhang K, Dong C, Chen M, Yang T, Wang X, Gao Y, et al.. Extracellular Vesicle-Mediated Delivery of miR-101 Inhibits Lung Metastasis in Osteosarcoma. Theranostics. 2020. Vol. 10:411–425

79. Jerez S, Araya H, Hevia D, Irarrázaval CE, Thaler R, van Wijnen AJ, et al.. Extracellular Vesicles from Osteosarcoma Cell Lines Contain miRNAs associated with Cell Adhesion and Apoptosis. Gene. 2019. Vol. 710:246–257

80. Cambier L, Stachelek K, Triska M, Jubran R, Huang M, Li W, et al.. Extracellular Vesicle-Associated Repetitive Element DNAs as Candidate Osteosarcoma Biomarkers. Scientific Reports. 2021. Vol. 11:94

81. Wang L, Wu J, Song S, Chen H, Hu Y, Xu B, et al.. Plasma Exosome-Derived Sentrin SUMO-Specific Protease 1: A Prognostic Biomarker in Patients With Osteosarcoma. Frontiers in Oncology. 2021. Vol. 11:625109

82. Han Z, Peng C, Yi J, Wang Y, Liu Q, Yang Y, et al.. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Profiling of Plasma Exosomes Evaluates Osteosarcoma Metastasis. iScience. 2021. Vol. 24:102906

83. Han Z, Yi J, Yang Y, Li D, Peng C, Long S, et al.. SERS and MALDI-TOF MS Based Plasma Exosome Profiling for Rapid Detection of Osteosarcoma. Analyst. 2021. Vol. 146:6496–6505

84. Torreggiani E, Roncuzzi L, Perut F, Zini N, Baldini N. Multimodal Transfer of MDR by Exosomes in Human Osteosarcoma. International Journal of Oncology. 2016. Vol. 49:189–196

85. Ryskalin L, Busceti CL, Biagioni F, Limanaqi F, Familiari P, Frati A, et al.. Prion Protein in Glioblastoma Multiforme. International Journal of Molecular Sciences. 2019. Vol. 20:5107

86. Tu C, He J, Chen R, Li Z. The Emerging Role of Exosomal Non-coding RNAs in Musculoskeletal Diseases. Current Pharmaceutical Design. 2019. Vol. 25:4523–4535

87. Wei H, Chen J, Wang S, Fu F, Zhu X, Wu C, et al.. A Nanodrug Consisting Of Doxorubicin And Exosome Derived From Mesenchymal Stem Cells For Osteosarcoma Treatment In Vitro. International Journal of Nanomedicine. 2019. Vol. 14:8603–8610

88. Wang J, Zhang H, Sun X, Wang X, Ren T, Huang Y, et al.. Exosomal PD-L1 and N-cadherin Predict Pulmonary Metastasis Progression for Osteosarcoma Patients. Journal of Nanobiotechnology. 2020. Vol. 18:151

89. Pan Y, Lin Y, Mi C. Cisplatin-Resistant Osteosarcoma Cell-Derived Exosomes Confer Cisplatin Resistance to Recipient Cells in an Exosomal circ_103801-Dependent Manner. Cell Biology International. 2021. Vol. 45:858–868

90. Weinman MA, Ramsey SA, Leeper HJ, Brady JV, Schlueter A, Stanisheuski S, et al.. Exosomal Proteomic Signatures Correlate with Drug Resistance and Carboplatin Treatment Outcome in a Spontaneous Model of Canine Osteosarcoma. Cancer Cell International. 2021. Vol. 21:245

91. Wang M, Zhang B. The Immunomodulation Potential of Exosomes in Tumor Microenvironment. Journal of Immunology Research. 2021. Vol. 2021:3710372

92. Clark RA, Garman ZG, Price RJ, Sheybani ND. Functional Intersections between Extracellular Vesicles and Oncolytic Therapies. Trends in Pharmacological Sciences. 2021. Vol. 42:883–896

93. Wang JW, Wu XF, Gu XJ, Jiang XH. Exosomal miR-1228 From Cancer-Associated Fibroblasts Promotes Cell Migration and Invasion of Osteosarcoma by Directly Targeting SCAI. Oncology Research. 2019. Vol. 27:979–986

94. Mkhobongo B, Chandran R, Abrahamse H. The Role of Melanoma Cell-Derived Exosomes (MTEX) and Photodynamic Therapy (PDT) within a Tumor Microenvironment. International Journal of Molecular Sciences. 2021. Vol. 22:9726

95. Qin F, Tang H, Zhang Y, Zhang Z, Huang P, Zhu J. Bone Marrow-Derived Mesenchymal Stem Cell-Derived Exosomal microRNA-208a Promotes Osteosarcoma Cell Proliferation, Migration, and Invasion. Journal of Cellular Physiology. 2020. Vol. 235:4734–4745

96. Huang Y, Liu W, He B, Wang L, Zhang F, Shu H, et al.. Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Promote Osteosarcoma Development by Activating Oncogenic Autophagy. Journal of Bone Oncology. 2020. Vol. 21:100280

97. Wang Y, Chu Y, Li K, Zhang G, Guo Z, Wu X, et al.. Exosomes Secreted by Adipose-Derived Mesenchymal Stem Cells Foster Metastasis and Osteosarcoma Proliferation by Increasing COLGALT2 Expression. Frontiers in Cell and Developmental Biology. 2020. Vol. 8:353

98. Li Q, Wang X, Jiang N, Xie X, Liu N, Liu J, et al.. Exosome-Transmitted linc00852 Associated with Receptor Tyrosine Kinase AXL Dysregulates the Proliferation and Invasion of Osteosarcoma. Cancer Medicine. 2020. Vol. 9:6354–6366

99. Wu C, Li Z, Feng G, Wang L, Xie J, Jin Y, et al.. Tumor Suppressing Role of Serum-Derived Exosomal microRNA-15a in Osteosarcoma Cells through the GATA binding Protein 2/Murine Double Minute 2 Axis and the p53 Signaling Pathway. Bioengineered. 2021. Vol. 12:8378–8395

100. Liu W, Wang B, Duan A, Shen K, Zhang Q, Tang X, et al.. Exosomal Transfer of miR-769-5p Promotes Osteosarcoma Proliferation and Metastasis by Targeting DUSP16. Cancer Cell International. 2021. Vol. 21:541

101. Zhu B, Zheng S, Fan W, Zhang M, Xia Z, Chen X, et al.. Carcinoma-Associated Fibroblasts Promote the Proliferation and Metastasis of Osteosarcoma by Transferring Exosomal LncRNA SNHG17. American Journal of Translational Research. 2021. Vol. 13:10094–10111

102. Zhang H, Yu Y, Wang J, Han Y, Ren T, Huang Y, et al.. Macrophages-derived Exosomal lncRNA LIFR-AS1 Promotes Osteosarcoma Cell Progression via miR-29a/NFIA Axis. Cancer Cell International. 2021. Vol. 21:192

103. Tao SC, Huang JY, Wei ZY, Li ZX, Guo SC. EWSAT1 Acts in Concert with Exosomes in Osteosarcoma Progression and Tumor-Induced Angiogenesis: The “Double Stacking Effect”. Advanced Biosystems. 2020. Vol. 4:e2000152

104. Zhang L, Lu C, Zeng M, Li Y, Wang J. Exosomal MiR-199a-5p Inhibits Tumorigenesis and Angiogenesis by Targeting VEGFA in Osteosarcoma. Frontiers in Oncology. 2022. Vol. 12:884559