Proteomic Profiling of Retinoblastoma-Derived Exosomes Reveals Potential Biomarkers of Vitreous Seeding

Retinoblastoma (RB) is the most common tumor of the eye in early childhood. Although recent advances in conservative treatment have greatly improved the visual outcome, local tumor control remains difficult in the presence of massive vitreous seeding. Traditional biopsy has long been considered unsafe in RB, due to the risk of extraocular spread. Thus, the identification of new biomarkers is crucial to design safer diagnostic and more effective therapeutic approaches. Exosomes, membrane-derived nanovesicles that are secreted abundantly by aggressive tumor cells and that can be isolated from several biological fluids, represent an interesting alternative for the detection of tumor-associated biomarkers. In this study, we defined the protein signature of exosomes released by RB tumors (RBT) and vitreous seeding (RBVS) primary cell lines by high resolution mass spectrometry. A total of 5666 proteins were identified. Among these, 5223 and 3637 were expressed in exosomes RBT and one RBVS group, respectively. Gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified in RBVS exosomes upregulated proteins specifically related to invasion and metastasis, such as proteins involved in extracellular matrix (ECM) remodeling and interaction, resistance to anoikis and the metabolism/catabolism of glucose and amino acids.


Introduction
Retinoblastoma (RB) is the most common intraocular malignancy of childhood and accounts for 3% of all childhood cancers [1]. About 40% of cases are hereditary and usually bilateral: 10% are due to a germline mutation of RB1 and 30% to a de novo germline mutation. The remaining 60% of cases are sporadic and non-hereditary, usually monolateral, with a somatic biallelic RB1 inactivation arising locally within the developing retina [2,3]. In both cases, the loss of RB1 protein function, which is a tumor suppressor located on chromosome 13q14, promotes uncontrolled cell division in retinal cells determining tumor formation [4,5]. The tumor can be endophytic in the vitreous, exophytic in the subretinal space, or have a mixed presentation. Vitreous seeding may occur when the tumor penetrates the inner limiting membrane of the retina, either spontaneously or by iatrogenic mechanisms (e.g., during focal ocular treatment). When the vitreous seeding is present at diagnosis, it is defined as "primary seeding", whereas if the disease course complicates independently of the initial growth pattern, it is termed "secondary seeding" [6]. The vitreous seeds remain a challenge in the management of intraocular RB and the enucleation of the affected eye may represent the only treatment option when the tumor is too advanced [7]. In this context, the identification of early prognostic biomarkers, which are predictive for vitreous seeding and are a reliable indicator of response to treatment, is urgently needed. Compared with other cancers, RB cannot be biopsied, due to the risk of extraocular dissemination, and much is known about the RB genetics derived from studies of tumors in enucleated eyes. Liquid biopsy is a minimally invasive alternative to surgical biopsies of solid tumors, based on the analysis of tumor-derived material in blood sample or other body fluids. Exosomes represent a novel source of biomarkers in liquid biopsies for monitoring tumor progression and drug resistance. Exosomes are cell membrane-derived nanovesicles (30-100 nM in diameter), containing RNA, microRNA, lipids and proteins. Secreted abundantly by aggressive tumor cells, those microvesicles can be isolated from several biological fluids [8]. In recent years, numerous efforts are being made to characterize the content of exosomes, both at the microRNA and protein levels [9,10]. Proteins and peptides are promising biomarkers, since they are functionally involved in biological processes. Thus, there is a correlation between their expression levels and various disease pathologies [11]. Moreover, proteomic technology platforms have developed rapidly, enhancing the precision and expedience of proteome analyses [12]. In particular, mass spectrometry has emerged as a promising approach for protein biomarker discovery, by exploring the protein content of body fluids, both in patients and controls [13].
The present study aimed to identify an exosome signature specifically associated with vitreous seeding. Here, we characterized the proteomic cargo of exosomes isolated from RB cell lines established from solid tumor tissue in the retina (retinoblastoma tumors (RBT)) and from tumor seeding in the vitreous humor (RBVS). We identified, in RBVS exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ECM) remodeling and interaction, resistance to anoikis and metabolism/catabolism of glucose and amino acids.

Characterization of Exosomes Derived from Primary RB Cell Lines
Exosomes were isolated from the cell culture conditioned media of primary RB cell lines generated from primitive mass (RBT1, RBT2, RBT5, RBT14) and vitreous seeding (RBVS1, RBVS3, RBVS10) by serial ultracentrifugations, as reported in Material and Methods. Scanning electron microscopy (SEM) analysis showed single and aggregate round-shaped nano-vesicles, the majority of which ranged from 50 to 70 nM ( Figure 1A). A NanoSight tracking system analysis revealed a relatively uniform size distribution of peaks from 100 to 150 nM, which is consistent with exosomes size ( Figure 1B). Exosome protein concentration is reported in Figure 1C. Western blot (WB) analysis showed an enrichment of exosomal-specific proteins, such as tumor susceptibility gene 101 protein (TSG101) and the tetraspanin CD9 ( Figure 1D). Altogether, these results confirmed that isolated microvesicles corresponded to exosomes.

Exclusive RBT-and RBVS-Exosomal Proteins
The comparison among all proteins highlighted 99 and 35 proteins exclusively present and statistically significant in RBT (Table 1) or RBVS-derived exosomes (Table 2), respectively. In RBT-derived exosomes were exclusively identified as the most significant, as present in all the samples analyzed, the proteins abl interactor 2 (ABI2), glutathione s-transferase Mu (GSTM), neurocan (NCAN), V-type proton ATPase subunit C 1 (ATP6V1C1), synaptosome associated protein 25 (SNAP25), serine/threonine-protein phosphatase 2A (PPP2R2A) and UBX domain protein 4 (UBXN4). In RBVS-derived exosomes were specifically found as the most significant as present in all the samples analyzed, the protein osteoglycin (OGN) and the low-density lipoprotein (LDL) receptor-related protein (LRP-1). The ABI family of adaptor proteins has been linked to signaling pathways involving the Abl tyrosine kinases and the Rac GTPase. ABI proteins localize to sites of actin polymerization in protrusive membrane structures and regulate actin dynamics in vitro, and cell morphogenesis and migration in vivo [15]. The homozygous deletion of murine abi2 results in cell migration defects in the neocortex and hippocampus in vivo and produces abnormal phenotype in the eye. In particular, ABI2 loss leads to the incorrect orientation and migration of secondary lens fibers, probably due to defect in actin polymerization [15]. There is no clear evidence of a role of ABI2 in tumors, but previous reports suggest that ABI2 functions as a tumor suppressor since ABI2 suppresses cell growth [16] and its truncated form accelerates the tumorigenesis [17].   GSTM protein belongs to the superfamily of enzymes that protect normal cells, by catalyzing conjugation reactions of electrophilic compounds, including carcinogens, to glutathione [18]. In several cancers, the association between GSTM1 polymorphisms and cancer risk has been suggested through meta-analysis studies. These studies showed that the GSTM1 null phenotype increases the risk of liver, gastric, breast, and prostate cancer [19,20]. Moreover, a role for this enzyme in the cisplatin-resistance of cancer cells has been reported [21]. NCAN is expressed abundantly in developing rat retina [22], and in Müller cells correlate with the invasive phenotype in low-grade astrocytoma [23]. Müller cells are the radial glial cells of retina, spanning the entire thickness of the retina and interacting with all retinal cell types. Under pathological conditions, Müller cells are involved in retinal angiogenesis. In response to hypoxia, high glucose, and inflammation conditions, multiple signaling pathways are activated in these cells, which are involved in retinal remodeling following retinal damage [24]. Furthermore, high NCAN expression has been associated with unfavorable outcome in neuroblastoma, as it induces an undifferentiated phenotype that promotes the malignancy [25]. ATP6V1C1 belongs to the vacuolar (H+)-ATPases (or V-ATPases) family that is responsible for the acidification of intracellular compartments in eukaryotic cells [26]. It is known that V-ATPases at the cell surface play a role in maintaining an alkaline intracellular environment favorable for growth, while maintaining an acidic extracellular environment favorable for invasion [27,28]. ATP6V1C1 is overexpressed cancer such as metastatic oral squamous cell carcinoma [29], oral cancer patients [30] and breast cancer [31], and has a role in tumor progression and metastasis. Synaptosomal-associated protein-25 (SNAP-25) is a component of the trans soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs)-complex, a key protein that mediates several cellular processes, including synaptic vesicles fusion, transmitter release, cell growth, cytokinesis and protein transport [32]. An increase in SNAP-25 expression was reported in neuroblastoma SH-SY5Y cells during neuritogenesis [33], and is overexpressed in tumor cells of prolactinomas [34]. On the other hand, a low expression level of SNAP-25 has been found in medulloblastoma tumors associated with defects in dendrite formation and in the impairment of targeted chemotherapy [35].
PPP2R2A is a major heterotrimeric serine/threonine phosphatase, counteracting the actions of Ser/Thr kinases, such as the components of mitogen activated protein kinase (MAPK) and AKT pathways, tumor suppressor pRB and p53, and cyclin-dependent kinase 1 (CDK1) substrates, which are often defective or deregulated in cancer [36]. Finally, UBXN2A protein is a positive regulator of p53, facilitating the translocation of WT-p53 to the nucleus, where p53 regulates its target genes, particularly those involved in apoptosis. For those characteristics, UBXN2A represents an important anticancer factor [37].
OGN, detected exclusively in all samples of RBVS, is a matrix molecule, belonging to the small leucine-rich proteoglycan (SLRP), which functions as an important component of the extracellular matrix. This protein serves as cell scaffold and is involved in collagen fibrillogenesis and cell adhesion [38]. Decreased OGN expression has been observed in a variety of different cancers, including gastric cancer [39], colorectal adenoma [40], squamous cervical and vaginal cancer [41], invasive ductal breast carcinoma [42], and laryngeal carcinoma [43]. In colorectal cancer, OGN expression reduces cell proliferation, inhibits invasion and limits cancer progression [44]. LRP-1 modulates retinal neovascularization by regulating proteolytic activity [45]. In mouse retina, LRP-1 has a role in endothelial cell proliferation and retinal neovascularization. LRP-1 knockdown results in increased PARP-1 activity and subsequent phosphorylation of both RB protein and cyclin-dependent kinase 2 (CDK2), with cell cycle progression and angiogenesis [46]. LRP-1 function depends on the tumor cell type [47]. Indeed, if low LRP-1 expression has been associated with advanced tumor stages and poor survival in several cancers (hepatocellular carcinoma [48], lung adenocarcinoma [49], melanoma [50], and Wilms tumors [51]), high LRP-1 expression has been related to advanced tumor stages (in endometrial carcinoma [52], breast cancer [53], prostate carcinomas [54], and colon cancer [55]).
We further identified molecular function ( Figure 4A), biological processes ( Figure 4B) and biological pathway ( Figure 4C) for proteins exclusively present in RTB-or RBVS-exosomes. Among molecular function, proteins with catalytic activity were abundant in both groups (6.9% RBT; 15.6% RBVS), while extracellular matrix structural constituents were only enriched in RBVS-exosomes (15.6%). The biological processes of proteins exclusively present in RBT-derived exosomes were essentially related to the regulation of energy pathway (13.9%), metabolism (13.9%) and transport (14.9%). There was an enrichment in energy pathway (21.9%), metabolism (25%), and cell growth and/or maintenance (15.6%), for proteins exclusively present in RBVS-exosomes. Intriguingly, the analysis of biological pathway highlighted an involvement in energy-related pathways and neurotransmitter release for proteins exclusively transported by RBT-exosomes, while RBVS-exosomes were enriched with proteins involved in integrins-mediated signaling and platelet aggregation ( Figure 4C). Integrins participate in cell-cell and cell-matrix interaction and have been involved in metastatic processes in several cancers [56][57][58]. In particular, αIIbβ3 integrin was first identified in platelets and later in cells derived from solid tumors, where its expression correlates with the invasive phenotype [59][60][61]. Moreover, it has been described that, integrin αIIbβ3 specifically activates c-Src to induce platelet spreading and thrombus formation [62], mediating tumor cell attachment to the vessel wall under flow condition [63]. Another interesting pathway that emerged links p130 Cas to integrins through the MAPK. p130 Cas is a multifunctional signaling adaptor protein which integrates signals generated from a variety of extracellular stimuli and regulates several cellular processes including cell death. p130 Cas has a particular role in anoikis, which is a programmed cell death induced upon cell detachment from extracellular matrix. Anoikis represents a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding the colonizing of distant organs. p130 Cas is specifically cleaved during anoikis; in anoikis-sensitive cells, but not in anoikis-resistant tumor cells [64]. Another biological pathway involves Netrins, secreted or membrane bound proteins originally proposed to play an important role in development of the central nervous system and in axon guidance, but recently shown to have a pivotal role in cancer [65,66]. The expression of Netrin-1 is increased in many cancers and the binding with its receptor, uncoordinated-5 homolog (UNC5B), results in the inhibition of p53-related apoptosis [66,67].
Exosomes derived from RBT cells showed upregulated proteins belonging to all the nodes listed above, while in exosomes isolated from RBVS cell lines, we found a significant representation of proteins involved in transmission across chemical synapses, platelet degranulation, response to elevated platelet cytosolic Ca 2+ , cardiac conduction, endoplasmatic reticulum unfolded protein response, cellular response to hypoxia, purine and ribonucleoside triphosphate metabolic process. In particular, 12 proteins were the most represented in the network for RBVS-exosomes: enolase 3 (ENO3), galactokinase-1 (GALK1), synaptotagmin like 2 (SYTL2), CREB binding protein (CREBBP), proteasome 26S subunit, non-ATPase (PSMD), Eps15 homology (EH) domain-containing protein 3 (EHD), filamin A (FLNA), talin-1 (TLN-1), asparagine synthetase (ASNS), saccharopine dehydrogenase (SCCPDH), tubulin alpha 4a (TUBA4A). Among those, ENO3, GALK1, ASNS and SCCPDH are all enzymes involved in metabolic pathways. ENO3, also known as phosphopyruvate hydratase, is a metalloenzyme responsible for the catalysis of the conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP), the ninth and penultimate step of glycolysis. GALK1 is an enzyme of the Leloir pathway, a metabolic pathway found in most organisms for the catabolism of β-D-galactose to glucose 1-phosphate. ASNS catalyzes the synthesis of asparagine and glutamate from aspartate and glutamine in an ATP-dependent amidotransferase reactin [68], and SCCPDH is an enzyme involved in the metabolism of the amino acid lysine, via an intermediate substance called saccharopine [69]. FLNA and TLN are important proteins for the connection of the cytoskeleton with ECM. In particular, FLNA is a well-known actin cross-linking protein that serves as a scaffold for over 90 binding partners and is involved in multiple cell functions, such as cell migration and adhesion [70]. The role of FLNA in cancer development is controversial and although its overexpression has been observed in several tumors [71][72][73][74][75][76][77][78][79][80], in some cases opposite roles have been described. For example, in breast cancer cells, the interaction of FLNA with cyclin D1 promotes migration and invasion [81], while on the other hand, the regulation of focal adhesion disassembly by FLNA leads to the suppression of breast cancer cell migration and invasion [82]. In prostate cancer, FLNA binding promoted androgen receptor (AR) localization to the nucleus [83], promoting prostate cancer progression, but once in the nucleus, it inhibited the transcriptional activity of the same transcription factor [84]. Moreover, being a scaffold protein, the function of FLNA depends on the binding partners available for its interaction.
TLN is a focal adhesion protein that links intracellular networks with the ECM, via its connection with the actin cytoskeleton and membrane integrins. TLN dysregulation can lead aberrant integrin activation and mechanotransduction, causing changes in cell spreading, migration and survival [85].

Comparison of Proteins Differentially Expressed between Exosomes Derived from RBT and RBVS of the Same Patient
We performed a comparison of exosomal proteins derived from RBT and RBVS cells of the same patients (RBT1 and RBVS1). This analysis showed a total of 1582 proteins for RBT1 and 1472 proteins for RBVS1, 1395 proteins were in common. Among all proteins, 187 proteins were exclusive for RBT1 and 77 for RBVS1 ( Figure 7A). A total of 92 proteins were differentially expressed: 66 were up-regulated in RBT and 26 in RBVS-derived exosomes ( Figure 7B). Gene enrichment analysis was performed by FunRich [14] software, to characterize common and exclusive exosomal proteins derived from RBT1 and RBVS1. Common proteins between RBT1 and RBVS1 were functionally involved in RNA binding (7.5%), were structural constituents of ribosome (5.2%), and presented a GTPase (3.7%), chaperone (3%) or translation regulator (2.2%) activity. Moreover, they were involved in biological processes such as regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism (20.7%), protein metabolism (17.8%), energy pathways (14.5%) and cell growth and/or maintenance (10.3%) ( Figure 7C). Instead proteins exclusively present in RBT1-derived exosomes were involved in molecular function such as RNA binding (6.4%) or protease activity ubiquitin-specific (6.4%), and in biological processes such as transport (10.1%), hormone metabolism (0.5%) and osmoregulation (0.5%) ( Figure 7D,E). Proteins exclusively detected in RBVS1-derived exosomes were principally involved in enzymatic (5%) and transporter activity (6.5%), and in nucleic acid metabolism (20.4%) ( Figure 7D,E).

Discussion
Although intraocular RB has achieved a high cure rate through multimodal therapies, there is still an important need to develop new treatment in order to improve globe retention, in the most advanced intraocular cases, and vision preservation, as well as to avoid the late effects. Indeed, even though the management of advanced intraocular RB is improving rapidly, with enucleation progressively being supplanted by new therapeutic approaches with local delivery of standard chemotherapy [86], refractory or recurrent diffuse vitreous seeding is associated, in most cases, with the failure of eye salvage in patients with RB [7]. For these reasons, novel prognostic biomarkers, predictive for vitreous seeding dissemination and response to therapy, and new therapeutic targets are needed. Due to the risk of extraocular spread, traditional biopsy is contraindicated in RB, thus, the application of liquid biopsy as a non-invasive way represents a promising surrogate marker to perform molecular studies in RB and to define the disease more in depth. Exosomes are emerging as new circulating biomarkers in the liquid biopsy field. Cell membrane-derived nanovesicles (30-100 nM in diameter), containing RNA, microRNA, lipids and proteins, exosomes are released by several cell types and mainly by tumor cells [9,87,88]. The possibility to isolate exosomes from different sources, as well as to monitor their content in a relatively simple way, allows one to follow tumor evolution and evaluate the response to therapy over time. In this study, we characterized the proteomic cargo of exosomes isolated from RBT with those obtained from RBVS. We analyzed proteins exclusively present in all samples of RBVS and RBT-derived exosomes and proteins enriched in RBVS samples with respect to RBT. Moreover, having the primary cell lines of both primary tumor and vitreous seeding available to the same patient, we compared also these two samples. Our aim was to identify new putative biomarkers and therapeutic target associated to vitreous seeding in RB. Vitreous seeding results from a clonal selection of RB cells that are able to proliferate in the avascular vitreous environment and survive in such hypoxic conditions [89]. The ability of these cells to survive and grow under hypoxic condition depends on metabolic reprogramming and/or resistance to anoikis. The anoikis is a form of programmed cell death occurring in anchorage-dependent cells, following the detachment from the surrounding ECM. From our analysis, RBVS exosomal proteins are involved in processes of remodeling and interaction with ECM, in pathways linked to integrins signaling and in resistance to anoikis. These observations reflect the ability of RB cells that seed in the vitreous to detach from the primary retinal tumor, migrate and survive under inappropriate conditions. Alteration in integrin expression have been observed in several cancers and is thought to play a central role in cancer spread and metastasis [56]. Integrins bind to multi-adhesive ECM components, organizing the cytoskeleton and activating intracellular signaling pathways. They have been shown to affect cell shape, polarization, cytoskeletal organization, cell motility, proliferation, survival and differentiation [90]. Moreover, integrins have been identified in exosomes, and their transfer through these vesicles is implicated in the promotion of cell adhesion, migration and the determination of organotropism in metastatic dissemination [58,91]. During retinal development, ECM constituents play versatile roles, which include cellular proliferation, differentiation, migration, adhesion and maturation, as well as axonal growth and guidance [92][93][94][95]. ECM components and their remodeling have a crucial role in several diseases of the retina, such as retinal injury and cancer, where changes in the interaction of cells and ECM components occur with a consequent disruption of the retinal homeostasis [96,97]. In particular RBVS cells secrete exosomes enriched with TLN, a key cytoplasmic protein that mediates integrin adhesion to the ECM. TLN is an adaptor protein forming the core of integrin adhesion complexes by linking integrins directly to actin; TLN has been demonstrated to have an important role in cancerogenesis, promoting the invasion and anoikis' resistance of human prostate cancer cells [98]. TLN-1 is upregulated in primary and metastatic prostate cancer compared to the normal prostate gland [98] and its downregulation led to a reduction of metastatic ability in vivo. Furthermore, the expression of TLN-1 has been reported as being significantly high in poorly differentiated prostate tumors and in cells with highly metastatic potential [99]. Intriguingly, the knockout of exosomal TLN-2 in murine lymphocytes TK1 leads to a reduced binding to the integrin ligand ICAM-1 and MAdCAM-1. Furthermore, TLN-2-deficient T-cell-exosomes were less efficiently internalized by murine bEnd.3 endothelial cells, compared with control exosomes, suggesting a critical role of TLN-2 in integrin functions in exosomes [100]. An interesting evidence is the involvement of TLN and integrin in invadosomes [101], microdomains including podosomes and invadopodia formed at the ventral surface of the cells capable of interacting with ECM and degrading it [102,103]. Invadosomes are formed by an F-actin core surrounded by a ring of regulatory and adhesive proteins such as integrins, TLN, vinculin and paxillin [101]. Furthermore, the ß3 integrin (ITGB3) founded exclusively in exosomes isolated from RBVS cells exerts several crucial roles in malignant tumor progression, such as metabolic reprogramming, maintenance of a stem-like phenotype and drug resistant acquisition [104]. Interestingly, vitreous seeds result from the clonal selection of RB cells that are able to proliferate in tumor-spheres likewise stem cells, and are prone to develop chemo-resistance properties. Additionally, GSTM1, exclusively detected in RBVS exosomes, has been implicated in cancer chemoresistance [105,106].
FLNA is another adaptor protein susceptible to proteolysis implicated in cytoplasm remodeling and interaction with the ECM. In the cytoplasm, full-length FLNA promotes the development of metastasis [107], whereas when nuclear, it was shown to be necessary for the inhibition of transcription and susceptibility to therapeutic interventions [84]. FLNA interacts with many proteins related to cancer metastasis and has a role in cancer progression [108][109][110]. Intriguingly, a role in podosomes formation and stabilization has been reported for FLNA in macrophages [111]. Through strategies of knock-out, knockdown and rescue it has been demonstrated that FLNA is involved in podosomes stability and their organization, regulates the proteolysis of the matrix and is required for podosomes formation in macrophages. A link between invadopodia formation and exosomes production has been described in the tongue squamous cell carcinoma SCC61 cell line [112]. In particular, it has been observed that the inhibition of invadopodia formation reduced exosomes secretion in culture media. On the other hand, the addition of purified exosomes or the inhibition of exosomes secretion affected the invadopodia formation, stabilization and exocytosis of proteinases indicating a key role for exosomes cargoes in the promotion of invasive activity. The fact that exosomes released by RBVS cells are enriched in proteins involved in ECM remodeling and invadosomes formation suggests their potential role in tumor dissemination during invasion and metastasis. OGN, detected exclusively in all samples of RBVS, is a small leucine rich proteoglycans of the ECM, where both structural and non-structural functions are exerted; it has been implicated in wound healing and inflammation of the eye [113]. Decreased OGN expression has been described in several malignancies, and for this reason, even if its precise role remains undefined, it is thought to be a tumor suppressor. The exclusive presence of this protein in the exosomes of RBVS cells suggests that it is eliminated by the metastatic cells to limit its tumor suppressor action or for its involvement in wound healing mechanisms related to tumor mass expansion and dissemination.
Netrins are a family of extracellular proteins that control axonal and cellular migration in embryogenesis. In particular, it has been suggested that Netrin acts through an adhesive process called haptotaxis [114], providing traction for a growth cone to navigate and generating a gradient to direct neurite growth.
In cancer, Netrin-1 has a double role: it is a survival factor and a promoter of cell invasion [115]. In particular, netrin-1 up-regulation has been observed to be the way to gain survival advantage for several cancers, such as colorectal cancer, neuroblastoma, glioblastoma and metastatic breast cancer [116][117][118][119]. Furthermore, higher Netrin-1 expression has been correlated with invasion and metastasis in distant sites in colon carcinoma, adenoma, pancreatic ductal adenocarcinoma and breast cancer [118,120,121].
In RBVS exosomes proteins involved in metabolic pathways or in resistance to anoikis were also found. Anoikis is a programmed cell death induced upon cell detachment from the extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding the colonizing of distant organs. If non neoplastic cells undergo anoikis in response to ECM detachment, cancer cells rapidly develop several mechanisms to resist anoikis. Cancer cells can achieve resistance to anoikis through several mechanisms, such as a specific switch in integrins, which leads them to adapt to the metastatic site or change their metabolism [122]. Cells' detachment from ECM strongly influences the metabolism of normal cells, reducing glucose uptake, glycolytic flux, mitochondrial respiration and the pentose phosphate pathway. The consequences of detachment from ECM are the reduction of both intracellular ATP and NADPH concentration, the reduction of fatty acid oxidation, the increase of ROS production and the induction of apoptosis. In recent years, it has been highlighted that the modulation of metabolic pathways in cancer cells contributes to the increase of anoikis resistance. Cancer cells metabolize high glucose levels through glycolysis, but most of the pyruvate obtained is transformed into lactate instead of being oxidized in mitochondria, a phenomenon described as the Warburg effect. Intriguingly, RBVS, which are metastatic cells able to survive in a hypoxic environment, release exosomes carrying up-regulated proteins involved in glycolysis, in glucose catabolism or in amino-acid synthesis, such as ENO3, GALK1, ASNS and SCCPDH. It is known that cells that manage to survive in the hypoxic environment of the eye and that are resistant to anoikis are able to escape from the primitive tumor site and establish a metastasis in the vitreous [89]. Based on our evidence, we hypothesize that these cells are able to produce exosomes loaded with proteins that facilitate ECM remodeling, resistance to anoikis and changes in metabolism ( Figure 8). The fate and function of these exosomes depend on which cell they interact with. Indeed, exosomes released in the vitreous by vitreous seeding cells can be both incorporated by metastatic cells in an autocrine way, strengthening the aggressive phenotype, or reach sites as far away as the cells of the primary tumor. In target cells, RBVS-derived exosomes may transfer proteins involved in invadosomes formation inducing an invasive phenotype that, together with the increased resistance to anoikis and changes in metabolism, makes the cells able to escape from the primitive mass, digest the matrix and survive in a hypoxic environment in the vitreous. Moreover, Netrin-enriched exosomes secreted by vitreous seeding cells could generate a gradient that drives nerve growth and facilitates tumor innervation, which is an emerging feature of tumor progression [123]. Human PDGF-BB (10 ng/mL, Peprotech, Cranbury, NJ, USA) and Heparin Solution 0.2% (2 µg/mL, Sigma Aldrich, St. Louis, MO, USA), as previously described [125]. Cell lines were maintained at 37 • C in a 5% (v/v) CO 2 humidified incubator.

Exosomes Purification and Characterization
Exosomes were purified from culture media after 3-4 days culture by sequential centrifugation. Supernatants were centrifuged at 500× g for 10 min to remove cell contamination, then at 12,000× g for 20 min to eliminate possible apoptotic bodies and large cell debris. Then, exosomes were collected by spinning at 100,000× g for 70 min, twice, using a 70Ti rotor (Beckman Coulter, Fullerton, CA, USA). The final pellet was resuspended in PBS or specific lysis buffer for proteomic analysis. Three different biological replicates were prepared for each cell line, and analyzed both for characterization and mass spectrometry analysis. Protein concentration was measured by bicinchoninic acid assay (BCA, Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Exosomes preparations were verified by SEM. Several drops of vesicles suspension (20 µL each) were placed on Parafilm. A formvar-coated nickel grid (Electron Microscopy Sciences, Hatfield, PA, USA) was placed on the top of each drop for 1 h in a humidified chamber. Grids were then fixed with 2.5% glutaraldehyde (Fluka, St. Louis, MO, USA) in phosphate buffer (PB), for 5 min at room temperature. The grids were attached on metal stubs, coated with chrome to a thickness of 10 nM and examined with a ZEISS-LEO 1525 (Laboratorio Universitario di Nanomateriali-University of Perugia). The NS500 nanoparticle characterization system (NanoSight, Salisbury, UK), equipped with a blue laser (405 nM), was used to characterize exosome size and particle number (CNIO, Madrid, Spain).

Sample Preparation for Mass Spectrometry, NanoLC and Mass Spectrometer Setup
The exosome pellets were re-suspended in 50 µL of lysis buffer (6M GdmCl, 10 mM TCEP, 40 mM CAA, 100 mM Tris pH 8.5). In eppendorf cells were lysed, reduced and alkylated; lastly, 5% ProteaseMAX surfactant (Promega, Madison, WI, USA) was added to enhance protein digestion by providing a denaturing environment prior to protease addition. To the samples were added 0.3 µg LysC and 0.7 µg Trypsin in 250 µL of a dilution buffer (10% ACN, 25 mM Tris HCl pH 8.5), to dilute the ProteaseMAX to 0.1%. After overnight digestion at 37 • C, the peptides were acidified with 0.1% TFA and loaded into StageTip. [126].
The tryptic peptides, after a speed vacuum concentration, were loaded directly into a 75 µm ID × 50 cm 2 µm, 100 Å C18 column, thermostated at 55 • C, and the peptides were separated with an organic solvent at a flow rate of 250 nL/min, using a non-linear gradient of 5-45% solution B (80% CAN and 20% H 2 O, 0.1% FA) in 140 min, and analyzed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific Instruments, Bremen, Germany). Orbitrap detection was used for both MS1 and MS2 measurements at resolving powers of 120 K and 30 K (at m/z 200), respectively. Data dependent MS/MS analysis was performed in top speed mode with a 2 sec cycle-time, during which precursors detected within the range of m/z 375−1500 were selected for activation in order of charge state, using CHarge Ordered Parallel Ion aNalysis (CHOPIN) [127].
Briefly, if precursor charge state is 2, then follow with CID fragmentation and by scan in the ion trap with an isolation window 1.6 m/z, AGC 3e4, maximum injection time 250 ms and normalized collision energy of 35%. If precursor charge state is 3-7 and precursor intensity is greater than 500,000, then follow with HCD fragmentation and by scan in the Orbitrap with a resolution 15,000, AgC 1e4 and maximum injection time 40 ms. If precursor charge state is 3-7 and precursor intensity is below 500,000, then follow with CID fragmentation and scan in the ion trap.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [128] partner repository with the dataset identifier PXD016488". (Reviewer account details: Username: reviewer66723@ebi.ac.uk; Password: 6oxpairt)

Data Analysis and Statistical Methods
MaxQuant software was used to process the raw data, setting a false discovery rate (FDR) of 0.01 for the identification of proteins, peptides and PSM (peptide-spectrum match); moreover, a minimum length of six amino acids for peptide identification was required. Andromeda engine, incorporated into MaxQuant software, was used to search MS/MS spectra against the Uniprot human database. For protein digestion, allowing for cleavage N-terminal to proline, trypsin was chosen as enzyme. Cysteine carbamidomethylation was selected as fixed modification, whereas acetylation protein N-terminal, oxidation (M) and deamidation (N, Q) have been selected as variable modifications. A tolerance of 7 ppm was set for the mass deviation of the precursor ions, while the maximum mass deviation for MS2 events was 0.5 Da. Algorithm MaxLFQ [129] was chosen for protein quantification, with the activated option 'match between runs' to reduce the number of the missing proteins. All bioinformatics analyses were done with the Perseus software of the MaxQuant computational platform [130]. Protein groups were filtered to require 70% valid values in at least one experimental group. The label-free intensities were expressed as base log2, and empty values were imputed with random numbers from a normal distribution for each column, to best simulate low abundance values close to noise level. For each group, a t-test with permutation-based FDR of 0.05 and a s0 of 0.1 was used. The Venn diagram of identified proteins was calculated using an online tool [131]. To visualize the profile of the experiment, a hierarchical clustering of resulting proteins was performed on log2 intensities after z-score normalization of the data for each exosome line, using Euclidean distances. Lastly, the network was built on significant proteins obtained by the t-test between two groups (RBT and RBVS) and plot the GO Enrichment using Cytoscape 3.7 and the ClueGo app.

Conclusions
In conclusion, in this study, we isolated exosomes from RB primary tumor and vitreous seeding cell lines and characterized their content with a proteomic approach. To the best of our knowledge, this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in RB. This characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in RB, as well as therapeutic targets.

Conflicts of Interest:
The authors declare that they have no conflict of interest.