Extracellular Vesicles in Bone Tumors: How to Seed in the Surroundings Molecular Information for Malignant Transformation and Progression

Bone is a very dynamic tissue hosting different cell types whose functions are regulated by a plethora of membrane-bound and soluble molecules. Intercellular communication was recently demonstrated to be also sustained by the exchange of extracellular vesicles (EVs). These are cell-derived nanosized structures shuttling biologically active molecules, such as nucleic acids and proteins. The bone microenvironment is a preferential site of primary and metastatic tumors, in which cancer cells find a fertile soil to “seed and blossom”. Nowadays, many oncogenic processes are recognized to be sustained by EVs. For example, EVs can directly fuel the vicious cycle in the bone/bone marrow microenvironment. EVs create a favourable environment for tumor growth by affecting osteoblasts, osteoclasts, osteocytes, adipocytes, leukocytes, and endothelial cells. At the same time other crucial tumor-mediated events, such as the premetastatic niche formation, tumor cell dormancy, as well as drug resistance, have been described to be fostered by tumor-derived EVs. In this review, we will discuss the main body of literature describing how the cancer cells use the EVs for their growth into the bone and for educating the bone microenvironment to host metastases.

vesicles sorted in a multivesicular body (MVB) via a complex molecular machinery involving endosomal sorting complex required for transport (ESCRT) components, ceramide/ sphingomyelinase pathway members, and Ras-related proteins in brain (RAB) proteins (4). This MVB then fuses with the plasma membrane and releases the small EVs in the extracellular space. Medium/large EVs arise directly from the plasma membrane via ADP-ribosylation factor (ARF) 6-mediated activation of phospholipase D (PLD), resulting in the recruitment of extracellular signal-regulated kinase (ERK) and phosphorylation of the myosin light-chain kinase (MLCK). This cascade triggers the budding of the cellular membrane, culminating in the formation and release of EVs from the cell (4). During their biogenesis, EVs entrap different macromolecules, such as nucleic acids (DNA, mRNA, miRNAs, long non-coding RNAs), lipids, and proteins (cytosolic factors, receptors, and ligands), which are then transferred to target cells where they induce metabolic changes (1). osteocalcin (OCN), osteopontin (OPN), bone morphogenetic proteins (BMPs), insulin-like growth factor (IGF)-1 and -2, platelet-derived growth factor (PDGF), and calcium ions (18,19). Beside these molecules, many others concur to regulate this cellular crosstalk, such as stromal derived factor (SDF)-1, interleukin (IL)-1 and -6, fibroblast growth factor (FGF), parathyroid and parathyroid hormone related peptide (PTH/ PTHrP), Lipocalin-2 (LCN2), NOTCH family members, Ephrin ligands and receptors, Semaphorins, and vascular endothelial growth factors (VEGFs), that establish the so called "virtuous cycle of the bone" (20)(21)(22)(23). In addition, the BRU establishes a further crosstalk with the bone/bone marrow-resident cells thorough juxtacrine and paracrine communications (24)(25)(26). Indeed, EV involvement in bone physiology has been elucidated in many aspects. The very first observation for a contribution of EVs in the process of bone mineralization was reported by two different groups in late '60s (27,28). Later on, EVs from osteoblasts were demonstrated to bind, by means of annexin 2, calcium phosphate, and other ions, thus triggering the formation of nuclei of mineralization in the bone matrix (29). Moreover, EVs have been described to be active coupling factors in osteoblastosteoclasts crosstalk. In fact, it is known that osteoblasts secrete EVs shuttling RANKL, which in turn sustains in vitro osteoclast formation (30). In line with this data, we found that osteoblastderived RANKL-positive EVs injected in osteoclast-poor RANKL knock out mice induced the osteoclastic commitment and the appearance of tartrate-resistant acid phosphatase (TRAP) positive cells in these mice (31). These results demonstrate another way exploited by osteoblasts to regulate osteoclast formation besides the juxtacrine and paracrine signaling. Vice versa, osteoblast differentiation and function can be also regulated by osteoclasts, as shown by Ma et al., who found that apoptotic bodies released by mature resorbing osteoclasts trigger osteoblast differentiation by activating the RANKL reverse signaling in these cells (32,33). The pro-osteoblast differentiating effect could be also accomplished by an autocrine mechanism, since it has been demonstrated that EVs from mineralizing osteoblasts promote their own differentiation by activating Wnt-b-catenin pathway, while reducing Axin1 expression in bone marrow stromal cells (34). Recent reports show that osteocytes, the most abundant cells in bone having not only a mechanosensing role but also a regulatory role on osteoclast and osteoblast differentiation, accomplish these functions by releasing EVs. As a matter of fact, Morrel et al. found that under mechanical stress osteocytes release EVs, which in turn enhance in vivo bone formation (35). At the same time, osteocyte homeostasis is regulated by EVs, as demonstrated by Ren et al., who found that exosomes from adipose tissue MSCs counteracted the hypoxiaand serum deprivation-induced apoptosis of the osteocyte-like cell line MLO-Y4, eventually leading to a lower production of RANKL by these cells and, consequently, to an inhibition of osteoclast formation (36). Consistently, Lu and colleagues found that exosomes isolated from adipose tissue-derived MSCs promoted in vitro proliferation and osteogenic differentiation of primary human osteoblasts. Moreover, pre-conditioning of the donor cells with tumor necrosis factor (TNF)-a increased the pro-osteoblastogenic effect by a Wnt signaling dependent mechanism (37).
Finally, endothelial cells also contribute to bone homeostasis by releasing EVs, which induce osteogenic differentiation of MSCs by shuttling Galectin-3 (38).

EXTRACELLULAR VESICLES CONTRIBUTION TO TUMOR COLONIZATION OF BONE
Neoplastic bone diseases are fueled by EVs (39). As a matter of fact, the onset and progression of both primary and metastatic bone cancers are promoted by a massive release of EVs, characterized by an abnormal molecular cargo (40). This event is not surprising considering that many key mechanisms involved in cancer progression, such as microenvironment acidification or aberrant pathways activation, also induce EVs production (41). In the bone context, tumor cells exploit the molecular pathways involved in bone remodeling to their own advantage, thus converting this "virtuous cycle" in a "vicious" one (42).
Different types of tumors can thrive in the bone milieu: primary tumors such as osteosarcoma (OS), Ewing's sarcoma, chondrosarcoma or fibrosarcoma and, very frequently, metastatic tumors from breast, prostate, lung, renal, colon, and bladder cancers (43,44). All these tumors can hijack physiological stimuli to their advantage. Key examples are SDF-1, RANKL, and OPN, which can be exploited by cancer cells due to their chemoattractant and promitogenic activities, that summoning and promoting the engraftment of cancer cells into the bone microenvironment (45). The interaction between tumor cells and the bone induces a deregulation of bone giving rise to bone lesions can be classified as osteosclerotic, characterized by osteoblastic overactivation, osteolytic, due to an exacerbated osteoclast function, and mixed, when both features coexist in the same site (46,47). Indeed, OS can generate all the three types of lesions, due to a wide histologic variety: the majority of OS cases (>85%) has osteosclerotic features, telangiectatic OS (~7%) presents osteolytic lesions, while small cell OS (~2%) produces mixed lesions (48,49). In contrast, breast and lung cancers usually give rise to osteolytic bone metastases, while osteosclerotic lesions characterize most of prostate cancer-induced bone metastases (47)(48)(49)(50).

Extracellular Vesicles in Bone Tumors Growth
OS is the most common primary tumor of bone, predominantly occurring in adolescents, with a second peak in elderly adults (51). MSCs and osteoblast precursors undergo malignant transformation, eventually leading to the deposition of an aberrant, immature bone (52). As for other types of tumors ( Figure 1 and Table 1), growing evidence demonstrates a crucial impact of EVs on OS development. An interesting work by Macklin and colleagues demonstrated that the OS aggressive behavior can be transferred from the highly metastatic KHOS OS cell line to a non-metastatic KHOS subtype by means of EVs (67). This effect was due to an enrichment, inside the EVs, of molecules involved in G-protein coupled receptor signaling. Moreover, the highly metastatic OS cells secrete 3-fold more vesicles than the lower metastatic ones, while multiphoton microscopy with fluorescence lifetime in vivo imaging demonstrated a preferential seeding of lungs by EVs derived from the highly metastatic OS clonal variants (67). Interestingly, Qi et al. showed that proliferation of the MG63 OS cell line is supported by EVs derived from bone marrow stromal cells, eventually leading to the activation of the Hedgehog pathway in the recipient cells (68), while other studies found that aggressiveness and metastatic  potential of OS are directly related to the release of soluble and EVbound urokinase plasminogen activator (uPA) as well as EV-bound to PD-L1/N-cadherin (69, 70). OS derived-EVs are also able to influence bone resident cells to promote OS grown. As an example, OS derived EVs stimulate endothelial cells to increase the secretion of IL-6,-8 and VEGF, by shuttling miR-148a and miR-21-5p (53). Osteoclasts are important players in OS, whose number seems to be correlated with tumor aggressiveness and metastatization (71). Interestingly, Garimella et al. found that EVs isolated from the 143B OS cell line contain a proosteoclastogenic cargo including matrix metalloproteinases-1 and -13 (MMP-1, -13), TGF-b, CD-9, and RANKL, thus suggesting a role for these EVs in mediating bone degradation (72). Similarly, our recent work shows that EVs isolated from the MNNG/HOS human OS cell line significantly impair osteoblast differentiation while increasing osteoblast secretion of pro-osteoclastogenic/ inflammatory cytokines (i.e., IL-6, Lcn2, RankL, CCL2,5,6,12, and CXCL1,2,5) and of MMP3. Moreover, OS-derived EVs have a proangiogenic effect, evaluated both in vitro and in vivo (73).
Ewing's sarcoma is the second most common bone cancer in children and adolescents. It presents with small round cells derived from the neural crest and is usually associated with the chimeric fusion gene Ewing sarcoma breakpoint region 1/Friend leukemia integration 1 transcription factor (EWSR1/FLI1) (74). Miller et al. firstly described that Ewing's sarcoma-released EVs are enriched in the mRNA of the chimeric fusion gene, along with Six transmembrane epithelial antigen of the prostate 1 (STEAP1) and Lipase, member 1 (LIP1) mRNA (75). Interestingly, the specificity of this EV cargo could make circulating EVs suitable biomarker candidates for patients.
Chondrosarcoma accounts for approximately 20% of bone tumors and is due to a malignant transformation of chondroblasts (76). From an histological point of view, it is classified in three stages according to cytonuclear atypia, number of multinucleated cells, degeneration of chondroid matrix, and absence of mitosis. Also for this tumor, a contribution of EVs in its progression has been hypothesized. As an example, the SW1353 chondrosarcoma cellderived EVs shuttle the long non-coding RNA (lncRNA) RAMP2-AS1, which acts as a molecular decoy for miR-2355-5p to regulate VEGFR2 expression thus increasing the angiogenic ability of HUVEC endothelial cells (77).
Finally, also the aggressiveness of fibrosarcoma seems to be supported by EVs release. This is a very rare bone tumor (0.5 cases in a million per year) caused by malignant transformation of fibroblasts (78). Malignant cells are strongly positive for vimentin and have an altered production of collagen, which is inversely correlated to the histological grade (78). Hakulinen et al. found that EVs isolated from the human fibrosarcoma cells HT-1080 contain both the inactive cleaved and the active full-length forms of MT1-MMP, which in turn activate pro-MMP2, allowing type I collagen and gelatin degradation (79).

Extracellular Vesicles in Metastasis Development
OS frequently metastasizes to lungs and its aggressiveness is related to osteoclastogenesis and neo-angiogenesis (80,81). Likewise, it has been demonstrated that OS derived EVs increase both osteoclasts and neo-angiogenesis, by shuttling miR-148a and miR-21-5p to target cells (53). Jerez et al. investigated whether EV cargo can be predictive for metastatic potential of OS by investigating the miRNAs profile of EVs from metastatic SAOS2 and nonmetastatic MG63 osteosarcoma cell lines (82). They found that SAOS2-EVs were particularly enriched in miR-21-5p, miR-143-3p, miR-148a-3p, and 181a-5p compared to MG63-EVs. Consistently, bioinformatic analysis revealed, among the miRNA targets, some genes involved in ECM remodeling, such as the collagencrosslinking enzyme lysyl oxidase (LOX), and the tissue inhibitor metalloproteinase 3 (TIMP3), suggesting that SAOS2 related miRNAs may influence metastatic potential of OS at least in part by modulating ECM remodeling (82). Interestingly, another study highlights the protumoral role of cancer associated fibroblasts (CAFs) on OS by releasing EVs enriched in miR-1228. Once taken up by the OS cell lines MG-63 and HOS, miR-1228 suppressed SCAI (suppressor of cancer cell invasion), resulting in the promotion of migration and invasion of OS cells (83). Of note, MSCs have also been described to support osteosarcoma progression by releasing EVs (84). In particular, MSC-EVs enriched in miR-208a are taken up by SAOS2 and MG63 cells, which inhibited programmed cell death 4 (PDCD4) eventually stimulating tumor cells proliferation, migration and invasion (84).
Once reached the metastatic site, which is usually in the lungs, OS cells regulate the function of resident cells by means of EVs. Indeed, it has been reported that the LM7 metastatic osteosarcoma cell line releases EVs able to induce the production of IL-8, TGF-b2 and CCL22 from alveolar macrophages and the impairment of the macrophagic immune-surveillance function by promoting a switch of the M1-to-M2 phenotype (85). Interestingly, Mazumadar and colleagues found that EVs from the human OS cell lines 143B and SAOS2 (highly metastatic and non-metastatic, respectively) when injected intraperitoneally into SCID mice promote the recruitment of CD11+ Gr+ immune cells into lungs. Moreover, OS-derived EVs alone can recapitulate myeloid cell infiltration in the lungs of naïve mice but are insufficient to promote the development of OS metastasis, thus indicating that the establishment of the PMN in the lungs may require a combination of tumor-secreted factors along with EVs (86).
As already mentioned, to engraft and colonize a distant organ, cancer cells are able to influence the future host tissue by preparing the resident cells to receive tumor cells and support their growth (87). This process, generating a microenvironment suitable for cancer dissemination before the dissemination itself occurs, is called "premetastatic niche" (88). Many mechanisms and secreted factors inducing the premetastatic niche (PMN) have been discovered (89) and undoubtedly EVs are important players in this process. This is also true for bone metastases which, as already stated, represent the preferential secondary site of growth for several tumors. The accumulation of EVs in a metastatic site is accompanied by several modifications in the microenvironment, such as resident cells activation, matrix deposition and vascular proliferation ( Figure 2 and Table 1).
Prostate cancer (PrCa) is the tumor with the highest propensity to metastasize to bone. In PrCa-induced bone metastases, several reports have emerged, showing the crucial role of tumor-derived EVs cargo in the preparation of the "soil". Hashimoto et al. found that miR-940 is highly expressed in EVs from highly osteotropic prostate cancer cells, and promotes osteoblastic/osteosclerotic bone lesions by targeting Rho GTPase Activating Protein 1 (ARHGAP1) and family with sequence similarity 134 member A (FAM134A) (90). Consistently, Karlsson et al. found that EVs from the TRAMP-C1 prostate tumor cell line impair osteoclast formation, while enhancing abnormal bone formation at the metastatic site (91). On the same line, other authors showed that PC3 PrCa cells decrease osteoclasts formation by means of EVs, which reduce the expression of miR-214 and the phosphorylation of p65 in osteoclast cells, finally suppressing the NF-kB signaling and osteoclast differentiation (92).These events enhance angiogenesis and finally tumor proliferation and invasiveness. PrCa cells also affect bone marrow MSCs behavior by means of EVs, by promoting a shift from an adipogenic toward a myofibroblastic differentiation. The latter cells in turn secrete high levels of proangiogenic (VEGF-A) and pro-invasive factors like MMP-1,-3 and -13 (93). Consistently PrCa cell (i.e., C4-2B, PC-3 and RWPE-1)derived EVs contribute to tumorigenic reprogramming of MSCs by delivering oncogenic transcripts of H-Ras, K-Ras and oncomiRNAs miR-125b, miR-130b, and miR-155 as well as the Ras superfamily of GTPases Rab1a, Rab1b, and Rab11a (94). Finally, an autocrine effect of tumor cell-derived EVs was also demonstrated by Dai and Gao, who found that miR-183 shuttled by PC3 derived EVs promotes cancer cell proliferation, migration and invasion through the inhibition of Tropomyosin 1 (TPM1) (95).
With regards to breast cancer (BrCa), it also very frequently metastasizes to bone, inducing osteolytic lesions. EVs released by the SCP28 BrCa cells are enriched in miR-21 which is internalized by the osteoclasts (96). Inside these cells miR-21 represses PDCD4, a negative regulator of osteoclastogenesis, leading to an exacerbation of the osteoclast function. In agreement with these observations, breast cancer patients with bone metastasis showed higher expression of miR-21 in serum exosomes than those without metastasis or with non-bone metastasis (97).
Hypoxic BrCa cells 4T1 release miR-210-containing EVs in the tumor microenvironment resulting in inhibition of Ephrin-A3 and Protein tyrosine phosphatase 1B (PTP1B), as well as in the increase of angiogenesis and tumor proliferation (98). BrCaassociated fibroblasts secrete EVs that activate the Wnt-planar cell polarity (Wnt-PCP) and finally promote dissemination of cancer (99). Finally, we recently demonstrated that MDA-MB-231 derived-EVs affect bone cells in vitro, since they increase osteoclast formation while dramatically impairing osteoblast differentiation and activity. Moreover both in vitro (tube formation assay) and in vivo (Matrigel plug assay) experiments demonstrated the ability of these EVs to promote angiogenesis (100).
Melanoma is a very aggressive tumor, frequently generating visceral metastasis (101) while bone metastases occurr in around 15% of patients, with a dramatic worsening of quality of life and survival (102). An in vitro study from Mannavola and colleagues showed that the osteotropic melanoma LCP cells release EVs able to switch the non osteotropic WM-266 and SK-Mel28 cell lines to an osteotropic one, by increasing the expression of CXCR7 (103). In contrast, the expression of a 6 b 4 or a v b 5 integrins favors a specific affinity for the lung and the liver, respectively (54). In line with these observations, integrins b 4 and a v expression in EVs isolated from the plasma of melanoma patients predict metastasis to lung and liver (54).
Lung cancer can also metastasize to the bone, and EVs facilitate this event (55). Indeed, EVs from the CRL-2868 non-small cell lung cancer (NSCLC) cell line and from lung cancer patients contain Amphiregulin (AREG) which in turn promotes osteoclast differentiation and exacerbated bone resorption by an EGFRdependent mechanism (56). In another study, metastatization to bone of lung adenocarcinoma A549 cells is reported to be inversely correlated miR-192 expression (57). In particular, the authors found that EVs from the highly metastatic A549-M1 subclone presented with a lower content of miR-192 compared to A549 parental EVs. Interestingly, intravenous administration of miR-192-enriched EVs the day before the intracardiac injection of the A549-M1 subclone, drastically reduced the bone metastatic burden of these cells in the tibia, as well as the number of CD31 positive cells in comparison to naïve EVs (57).

EXTRACELLULAR VESICLES CONTRIBUTION TO TUMOR CELL DORMANCY IN THE BONE
Once cancer cells reach a distant organ, they can encounter a dormancy-permissive environment (58). In particular, the resident stem cell niche compartment can favors the survival of a distant cancer stem cell, by the same mechanisms usually employed to regulate the maintenance of the stem cell pool and the recruitment of uncommitted cells into the tissue (59,104,105). On the other side, the dormant tumor cells can acquire stem cell-like properties overexpressing specific genes to interact with the niche (46). As an example, BrCa cells can shift toward a dormant phenotype in the endosteal niche, activating a hematopoietic stem cell profile via the Notch2 pathway (106). Cancer dormancy accounts for a considerable clinical problem, since it is responsible for tumor recurrence; in fact the tumor awakening leads to the generation of secondary tumor lesions, which in many cases constitute the cause of the death of patients (107).
Several studies revealed that EVs can also trigger the dormant phenotype of tumor cells (Figure 1 and Table 2). Kling et al. found that under hypoxic conditions, the Ewing's sarcoma cells A673 and SK-ES-1 upregulate and load miR-210 in their EVs, which suppresses Caspase 8 Associated Protein 2 (CASP8AP2) expression in parental cells, eventually inhibiting OS cells apoptosis and inducing a stem-like phenotype (60). Furthermore, OS modulate its aggressiveness and dormant phenotype by the EVshuttled miR-101 (61). In fact, in vivo injection of SAOS2 and SOSP-9607 cells overexpressing miR-101 resulted in less lung metastases in mice, through the silencing of B cell lymphoma protein, BCL6. Consistently, when miR-101 was overexpressed in MSCs, the MSC-EVs were enriched in this miRNA and effectively reduced in vitro migration and invasion of SAOS2 and 143B cells. These effects have been confirmed in immunocompromised mice, in which the treatment with MSC-EVs-miR-101 reduced the lung metastatic foci after 2 weeks of intratibial injection of 143B. In addition, other studies found that plasma EV-miR-101 is able to distinguish between OS patients with metastasis from those without. Insulin like growth factor (IGF)2, was reported to be shuttled by EVs and was isolated from biological fluids of healthy subjects as well as of cancer patients (62). Moreover, OS survival is sustained by IGF2, that induced dormancy by triggering autophagic pathways and inducing chemoresistance to adriamycin in syngenic animal models injected with the OS murine ATX cells (117). Notably, in biopsies from OS patients with favorable prognosis after chemotherapy, IGF2 levels are decreased.
With regards to bone metastases ( Figure 2 and Table 2), in an in vitro study Ono et al. found that EVs from MSCs decrease the proliferation, invasion, and sensitivity to chemotherapeutics of the bone metastatic human BrCa BM2 cells (63). These effects were due to the shuttling of miR-23b from MSC-EVs to cancer cells,

EXTRACELLULAR VESICLES IN BONE CANCER DRUG RESISTANCE
Overcoming chemoresistance is a major clinical unmet need, since what is usually the first line treatment is not an option for patient with chemoresistant cancer. Many mechanisms have been elucidated in the establishment of chemoresistance, and EVs have been shown to shuttle the necessary molecular machinery (Figures 1, 2 and Table 3) to promote drug resistance (131).

Drug Resistance in Bone Tumors: Role of EVs
Primary OS MG63 cells treated with doxorubicin (DXR) increased their expression of P-glycoprotein (P-gp)1, a membrane transporter pumping xenobiotics outside the cell. Consistently, EVs from DXR treated MG63 presented with higher levels of both ABCB1 transcript and P-gp encoded pretein expression, that can be transferred to untreated MG63 cells, conferring them drug resistance (132). In another study, upregulation of miR-25-3p, which silences the Dickkopf WNT signaling pathway inhibitor 3 (DKK3) gene, was detected in human OS samples, where it is negatively correlated with clinical outcome (133). The same authors demonstrated that miR-25-3p upregulation supported tumor growth and drug resistance and that the same effects were observed after DKK3 silencing. Interestingly, miR-25-3p was found in cell-derived EVs. In another study by Weinman et al. the correlation between drug resistance and EVs has been investigated in a spontaneous canine model of OS (134). The authors clustered the animals in two cohorts based on the responsiveness to amputation and adjuvant carboplatin chemotherapy (good, disease-free interval > 300 days; poor, diseasefree interval < 100 days) and analyzed the protein profile of circulating EVs by mass spectrometry. The proteomic profile revealed differences in EV cargo, identifying tetranectin, which was decreased in the poor prognosis group, as the most reliable biomarker.
The clinical relevance of EV-mediated drug resistance in OS patients has been investigated by Pan et al. (135). The analysis of circulating EVs from 43 OS patients compared to healthy subjects revealed overexpression of the circular RNA circRNA103801 in the former. The level of this circRNA presented a prognostic value, being inversely correlated with the overall survival of patients. The authors further investigated the biological function of the circRNA103801 in an in vitro model of OS. The overexpression of circRNA103801 in MG63 cells increased the sensitivity to cisplatin, the OS cells released also EV enriched in the circRNA. When these EVs were incubated to naïve MG63 and U2OS cells, both the OS cells increased the sensitivity to cisplatin, upregulating the expression of P-gp and Multidrug Resistance Protein 1, MRP1. These experiments demonstrated that circRNA103801 is responsible for conferring chemoresistance in OS patients.

Drug Resistance in Bone Metastases: Role of EVs
Like in OS, the expression P-gp was increased in MCF7 BrCa cells after exposure to docetaxel (DOC) Moreover, EVs from DOC-treated MCF-7 expressed higher levels of P-gp compared to EVs from naïve MCF-7, and the incubation with DOC-MCF-7 EVs reduced cell apoptosis of naïve MCF-7 (108). It is known that ABCB1 can be activated by the ubiquitin carboxy-terminal hydrolase (UCH) L1 through MAPK/ERK pathway. Consistently, MCF7 treated with adriamycin (ADR) show high level of UCH-L1 and phospho-ERK compared to control cells and when naïve MCF7 cells were cultured with EVs from ADR-MCF7 they acquired a reduced sensitivity to ADR and increased p-ERK and P-gp levels (109). Interestingly, EVs from blood of BrCa patients were positive for UCH-L1 and show an inverse correlation with chemosensitivity. Chemotherapeutics resistance can also occur via EV-mediated miRNAs transfer. In fact, ADR/DOC-resistant BrCa cells release EVs enriched in miR-222, suppressing phosphatase and tensin homolog (PTEN) gene, a tumor suppressor which negatively regulates intracellular levels of phosphatidylinositol trisphosphate and the Akt signaling (136). These EVs are also taken up by M2 macrophages, eventually promoting their activation and polarization to support cancer cells. Accordingly, miR-222 has been also found in EVs from plasma and tissue of chemoresistant patients (110). Exosomal miR-222 is also responsible for the resistance to tamoxifen in MCF7 cells, suppressing p27 and Estrogen Receptor (ER) alpha expression (110).
Cancer cells can counteract chemotherapeutics like ADR by overexpressing the glutathione-S-transferase P1 (GSTP1), a phase II-metabolizing enzyme that detoxifies chemicals by conjugating with glutathione. Yang et al. found that this enzyme is present in EVs from ADR/MCF7 and sera of chemoresistant patients (111). Another study described that, when exposed to paclitaxel (PTX), MDA-MB-231 specifically released EVs enriched in Survivin, an inhibitor of apoptosis (112). The protective effects of Survivinenriched EVs were effective on drug-sensitive fibroblasts and SKBR3 cells when exposed to PTX. Furthermore, tumor cells can directly counteract chemotherapeutics by EV release, via a decoy-like system. Indeed, Ciravolo et al. described that the human epidermal growth factor receptor 2 (HER2)-positive BrCa cells SKBR3 and BT474 release HER2-positive EVs. These EVs are able to bind and neutralize the biological drug trastuzumab, an anti-HER2 antibody, while EVs from triple negative BrCa cells MDA-MD-231 do not (113). In fact, SKBR3 cells treated with autologous EVs were less sensitive to the antiproliferative effect of trastuzumab. Interestingly, the authors found that EVs purified from sera of HER2-positive BrCa patients showed lower binding to trastuzumab compared to EVs circulating in sera from patients with advanced disease.
Notably, cancer cells can also take advantage of CAFs derived EVs to acquire chemoresistance. Indeed, it has been shown that EVs from CAFs sustain radiation-and chemo-resistance of MDA-MB-231 BrCa cells by activating retinoic acid-inducible gene I (RIG-I), signal transducer and activator of transcription (STAT) 1, and NOTCH3 pathways (114). Similarly, Shan et al. dissected the role of CAF-EVs in taxane resistance-acquisition by PrCa cells, finding an enrichment of miR-423-5p in these EVs which, once internalized in PrCa cells (LNCAP, 22RV-1 and C4 cells) suppressed GREM2 (encoding for gremlin2 protein inhibitor of bone morphogenetic proteins family members, BMPs) and increased TGF-b eventually leading to a reduced sensitivity to taxane (115).

POTENTIAL APPLICATIONS OF EVS IN CLINICAL MANAGEMENT
Cancer-derived EVs can be also used for diagnosis and to monitor cancer progression, exploiting them in the field of liquid biopsy. Of note, fibronectin shuttled by EVs isolated from BrCa patients is becoming a reliable diagnostic marker for detecting tumor early stages (116). On the same line, developmental endothelial locus-1 protein (Del-1) has been proposed as an exosomal biomarker to discriminate between benign and malignant BrCa (119). Periostin is another potential EV-related biomarker, since it has been found enriched in circulating exosomes from BrCa patients, and allowing the stratification of patients with localized disease versus those with lymph node metastasis (120). HER2 expression in circulating EVs has been found to be consistent with the positivity assessed by immunohistochemistry on tumor biopsies (121). Similarly, PrCa diagnosis can be addressed via EVs, since the classical marker prostate specific antigen (PSA) can be measured in EVs from patients, distinguishing benign hyperplasia from malignant transformation (137). EV-bound survivin has been proposed as a biomarker and clinical tool for diagnosing or monitoring PrCa as well as PTEN, both being detectable only in patients (138). Melanoma can be also monitored by circulating EVs profile of caveolin 1, and lung cancers can be histologically discriminated through a multimarker model based on the expression on circulating EVS of tetraspanin 8/CD151/CD171 (122,139). Not only protein cargoes but also miRNA profiles of circulating EVs in patients suffering from cancers are pursued for prognostic and diagnostic aims (123)(124)(125)(126).
Another useful approach in the EVs field is their employment as pharmacological delivery system for the treatment of different cancers (127). Indeed, EVs present with attractive potentialities due to a relatively long half-life, high biocompatibility, and minimal or no adverse effects. As an example, Melzer et al. showed the efficacy of taxol-loaded MSC-derived exosomes in targeting MDA-MB-231 growth and metastases in NOD-SCID mice (128). Similarly, zoledronate and dasatinib were efficaciously delivered by osteoblast-EVs and suppressed osteoclast formation and function in vivo, thus potentially being efficacious in reducing tumor-induced osteolysis (31). Consistently, EVs from the monocyte/macrophage cell line RAW264.7 have been reported to be efficient vehicles for paclitaxel and doxorubicin shuttling and treatment of mice orthotopically injected with MDA-MB-231 cells (129).
Finally, EVs are promising tools for immunotherapy (130). As an example, EVs derived from overexpressing IL-12 transgenic renal and bladder cancer cells efficiently trigger a strong antitumoral activity of cytotoxic T cells through the FasL/ Fas signaling pathway, and the same strategy could be applied to bone primary tumors (140). Xu's group demonstrated that EVs from macrophages carry phagocytosed antigens to dendritic cells and strengthen T-cell responses, conferring anti-tumor immunity (141). Dendritic cell-derived EVs induced specific cytotoxic T cell activation suppressing the growth of lung adenocarcinoma in a xenograft mouse model (142), while Zhu et al. found that NK cell primed by IL-15 shuttled-EVs show a higher cytolytic effect against BrCa cells MDA-MB-231 injected in nude mice (143). Many other studies and ongoing clinical trials are endorsing these approaches.

OUTLOOK AND PERSPECTIVES
The whole body of evidence from the literature strongly indicates that EVs exert a critical role in the progression of both primary and secondary bone tumors. Bone is an attractive microenvironment for tumor cells growth and metastasis, through the establishment of a crosstalk among many different resident cell types, occurring via juxtacrine, paracrine, and EV-mediated mechanisms. The latter are an additional piece of the complex system by which tumor fuels this "vicious cycle". The translational significance of this aspect is endorsed by the growing interest of the application of EVs in diagnostic and therapeutic fields, as well as the patent applications/grants and clinical trials based on the use of EVs. However, many challenges are still present in the field. For the basic research side the main issues are: i) to isolate with very high purity the different EV subpopulations, ii) to identify specific mechanisms of selective EV uptake into target cells, iii) to track the kinetics and distribution of EVs towards distant tissues, and iv) to distinguish the real contribution of the molecular legacy of EVs into an complex microenvironment, such as the tumor milieu. The isolation and quantification methods are still a crucial issue. Depending of the technique used for the isolation, the composition and the effect of the EVs from the same source can be different, and the same goes for the quantification. Of course, there are consensus methods in the field, but every method presents pitfalls to take into account. Protein titration, one of the most widely used methods in laboratory practice, can be affected by "exogenous" contaminants and does not consider the size distribution. Methods of quantification based on physical features, such as dynamic light scattering and resistive pulse sensing, cannot estimate the entire EV population, since generally a size cut-off must be applied on the instruments, and cannot distinguish between intact and damaged EVs.
The possible experimental approaches to dissect biological aspects of EVs have pros and cons: the in vitro studies can help to quantify and estimate the contribution of a specific pathway in the cellular context. However, the in vitro cell system cannot take in consideration the paracrine and systemic contribution of all cell types on cancer metabolism and related EVs. Furthermore, the EV cargo is a balanced cocktail of molecules that can exert opposite effects (i.e., EVs can shuttle both RANKL and OPG), and generally the investigators focus only on one member of them, possibly overestimating the importance of the single factor using targeted rather than system/pathway-oriented overexpression or knockdown techniques. On the other side, the in vivo studies in animal models can help to assess the real contribution of a molecular player on a biological system, but many confounding factors are present due to the complexity of the system. The in vivo studies help to investigate the kinetics of release and integration of EVs, frequently using intravital microscopy approaches. These studies provide impactful data, but are limited by technical constraints, such as resolution, signal/noise ratio and cellular recycling of the tags used for tracking the EVs.
Finally, a real clinical setting of EVs is hindered by many limitations. The main technical issues are shared with basic research. Indeed, a lack of standardization methods for isolation, characterization, quality control, large scale production and storage conditions reduce the translational value of EVs (144). Moreover, the EVs and their molecular profile can change for several patient-related variables, including the circadian rhythm and lifestyle habits as well as comorbidities, affecting their prognostic reliability (145,146).
In conclusion, EVs are an active field of study for a better understanding of the biological bases of tumorigenesis, but also offer a promising translational tool for diagnosis, monitoring, and treatment of cancer patients.

AUTHOR CONTRIBUTIONS
AC and NR contributed to original draft conceptualization, preparation, and revision. Both authors have read and agreed to the final version of the manuscript. All authors contributed to the article and approved the submitted version.