Through the transfer of cellular content between cells, exosomes are involved in the cellular communication across different cell types and different locations. Although great interest has been focused on exosomes under pathological conditions, especially cancer, these small extracellular vesicles have been identified in all cell types and in different biological states
[5][1]. Therefore, it is not surprising that exosomes are essential mediators of cellular behaviour under normal physiological conditions. For instance, they play important roles in immune response and surveillance
[34][30], neurotransmitter release by neurons
[35][31], or tissue repair
[36][32]. Similarly, cells use exosomes under pathological conditions, such as virus infections and spread
[37][33] or cancer
[7][3]. Furthermore, from a clinical point of view, exosomes and their cargo could be exploited as circulating biomarkers due to the ability to correlate their profile to the cell of origin
[38][34].
Different mechanisms associated with tumorigenesis have been linked to exosome release and uptake. Amongst these, we can find extracellular remodelling, transfer of oncogenes and oncoproteins, release of pro-inflammatory factors, or even education of stroma cells into forming a pre-metastatic niche by changing their phenotype
[7][3].
2. The Role of Exosomes in Bone Sarcoma Metastasis
Bone sarcoma metastasis represents the most adverse clinical factor and is associated with poor survival rates
[1][35]. This is partly due to the lack of understanding on the molecular mechanisms behind tumour dissemination and metastatic disease
[1,74,75][35][36][37]. In both OS and ES, the most common metastatic site is the lung, followed by bone and bone marrow. Interestingly, lung metastasis is associated with better prognosis than non-lung metastasis
[1,76][35][38].
Due to the importance of metastasis in these bone sarcomas, many studies have focused on deciphering the pathways behind disease progression. Amongst the different genes identified in OS, we find overexpression of
CD155 [77][39], loss of
TP53,
RB1, and
PTEN [78][40], and upregulation of Notch genes
[79][41] to be important in metastatic OS. Moreover, different novel treatment strategies are evaluating their efficacy in OS metastatic patients through clinical trials
[75][37], with the aim of finding novel treatment strategies for these patients. In ES, similar studies have resulted in several genes linked to metastasis, such as
ROR1 [80][42],
MSH2,
MSH6,
RPA2, and
RFC2 genes from the mismatch repair pathway
[81][43], PPP1R1A
[82][44] and TWIST1 proteins
[83][45], the Cad11 adhesion molecule
[84][46], or ERBB4 via activation of the PI3K-Akt-FAK cascade
[85][47]. Moreover, similar to other cancers, hypoxia has been associated with induction of metastasis in OS and ES via regulation of HIF1α through HIF1α
[86][48] or overexpression of CXCR4 in ES
[87][49], amongst others. Similar to OS, different clinical trials are evaluating the response of metastatic ES patients to different treatment approaches in order to improve survival rates
[76][38]. In contrast to OS and ES, CS is usually a non-metastatic disease with locally aggressive tumours
[88][50]. However, some evidence suggests integrins are involved in the metastatic organotropism of CS to the lungs
[89,90][51][52].
2.1. Exosomes in OS
OS is the most common primary bone tumour, with an incidence of 0.3 per 100.000 people per year
[91][53]. The peak incidence follows a bimodal distribution, with most cases between 0–24 years of age and 60–85 years of age
[92][54]. It is an intraosseous neoplasm with origin in bone regions with active cellular growth in which the balance between osteoclasts and osteoblasts is disrupted
[93][55]. Contrary to other sarcomas with a clear genetic driving event, OS is characterised by high genomic instability. This results in complex karyotypes involving copy number alterations
[94][56] and frequency of chromotripsis (high incidence of chromosomal rearrangements in a delimited genomic region)
[95][57]. All these factors, adding to the heterogeneity and rarity of OS, have made difficult the successful identification of better treatment strategies for patients.
Despite improvements in diagnosis and treatment over the last decades, survival rates are still poor for an important fraction of patients. Between 10% and 20% of patients have metastasis at diagnosis, with 5-year overall survival rates (OSR) lower than 20%
[75][37]. For localised disease at diagnosis, survival rates are higher (5-year OSR: 60–80%), although evidence suggests 80% of patients have micrometastasis at the time of diagnosis which will be refractory to chemotherapy. This results in 30–40% of non-metastatic OS patients developing metastasis and recurrent disease
[96,97][58][59]. Therefore, one of the focal points of OS research is to better understand the process of metastasis and how different factors modulate the TME to favour metastatic spread. This will help in identifying treatment strategies against metastatic and refractory disease, leading to improved survival rates.
Due to the role exosomes play in tumorigenesis in many cancers and the implications these small extracellular vesicles have in metastasis, it is not strange that researchers in the OS field are investigating the implications of exosomes in OS (). As exosomes are involved in cellular cross-talk via transfer of their cargo, one of the focal points has been to characterise the cargo of OS-derived exosomes. An analysis of OS-derived exosomes and conditioned media (exosome-free) revealed a different protein profile between both
[98][60]. Among the 250 most enriched proteins in exosomes, pathway analysis revealed an association with migration, adhesion, and angiogenesis, all important processes for cancer dissemination and modulation of the pre-metastatic niche. Following these data, the same group investigated the exosomal cargo of several OS cell lines
[99][61]. A difference in the miRNA content was seen between metastatic and non-metastatic OS-derived exosomes. Amongst the predicted target genes for the differential expressed miRNA, there was an enrichment of genes associated with tumour progression and metastasis. A deeper analysis of miRNAs enriched in exosomes from the most metastatic OS cell line (SAOS2) revealed that 4 miRNAs targeted 31 target genes from the same network associated with cellular adhesion and apoptosis
[99][61]. Therefore, data from these studies suggest that OS-derived exosomes could drive a pro-metastatic phenotype by transferring specific proteins and miRNA to other OS cells, resulting in induction of changes in migration, adhesion, and angiogenesis.
Table 1. Summary of exosome studies in OS.
Origin Cell |
Recipient Cell |
Cargo |
Change |
Ref. |
OS cells and conditioned media |
- |
Profiling of proteome and secretome |
Exosome proteins involved in migration, adhesion, and angiogenesis |
[98][60] |
Metastatic and non-metastatic OS cell lines |
- |
Profiling of miRNAs and target genes |
miRNA of metastatic OS exosomes target metastasis-associated genes, cell adhesion, and apoptosis |
[99][61] |
Metastatic and non-metastatic OS cell lines |
Osteoblasts |
ES cell linesmiR-675 (miRNA profiling) |
Osteoblasts, osteoclasts in 3D scaffoldMetastasis-associated exosomes induce migration and invasion in osteoblasts via miR-675/CALN1 axis |
[100][62] |
EZH2 mRNA |
| (target of interest) |
Transfer of EZH2 mRNA to MSC (increase expression), osteoblasts (no change), and osteoclasts (reduction expression) |
[118][82] |
Metastatic OS cell lines |
Macrophages, osteoclasts, endothelial cells |
miR-148a, miR-21-5p (RNA profiling) |
Induction of osteoclast-like gene expression (macrophage), increase in bone resorption (osteoclasts) and angiogenesis (endothelial cells) via miRNA transfer |
[101][63] |
ESCD99neg cell line model |
ES cell lines (normal CD99) |
Increased miR-34a |
Regulation of NFκB via miR-34a through reduction of Notch. Increase in neural differentiation (similar to direct CD99 silencing) |
[119][83] |
Metastatic and non-metastatic OS cell lines |
ESCD99neg cell line model | MSC |
TGFβ (induction of IL6) |
Internalization of TGFβ induces IL6 production, cell growth, and lung metastasis in vivo |
[102][64] |
Doxorubicin-resistant OS cell lines |
Sensitive OS cell lines |
MRP1, Pgp(multidrug resistant proteins) |
Increase in doxorubicin resistance in recipient cells; increase in MRP1 and Pgp mRNA levels. |
[103][65] |
Bone marrow (conditioned media) |
Metastatic and non-metastatic OS cells |
uPA (secreted, paracrine loop) |
Increase in migration on recipient cells, induction of OS metastasis in vivo |
[104][66] |
CAF |
OS cell lines |
miR-1228 (miRNA profiling) |
Increase in migration and invasion via miR-1228 transfer |
[105][67] |
MSC |
OS cell lines |
miR-143 (synthetic introduction) |
Reduction of migration via exosome transfer (better than transfection) |
[106][68] |
OS cell lines |
- |
Profiling of miRNA as OS biomarkers |
Better biomarker than ALP or patient stratification according to chemotherapy response |
[107,108][69][70], |