Exosomes have been implicated in a wide range of pathological and nonpathological processes. Research on tumor-derived exosomes uncovered their role on major processes associated with disease progression. Uncontrolled cellular proliferation resulting in tumor growth, metastatic dissemination and modulation of the immune response, are only a few of the central pathological processes in which tumor-derived exosomes have been implicated. These in vivo studies rely on the administration of purified labeled exosomes from cell culture supernatants into circulation of animals or injections of genetically engineered cells that produce labeled exosomes. However, it is not clear that current available techniques actually translate the in vivo implications of exosomes in several biological processes. The variations seen when using different exosomes cell sources, the total amount of exosomes injected in mice and their route of administration as well as the fact that most studies are performed in immunodeficient animals, shows the difficulty to achieve conclusions which are biologically significant. Genetically engineered mouse models (GEMM) could be a promising approach to address the current technical limitations allowing tracing tumor-derived exosomes in a living organism. These models could enhance greatly our knowledge about exosomes in different fields of research, namely cancer.
- in vivo imaging
- tumor progression
During the last decades, extensive research on exosomes has contributed to the increasing knowledge on their composition, biogenesis and biological function . Exosomes intrinsic ability of horizontal cargo transfer, and their high stability in circulation, allows them to interact with neighbor and distant cells and phenotypically reprogram them, being important mediators of cell-to-cell communication . Numerous
Exosomes biodistribution research is based on two main approaches: administration of purified labeled exosomes from cell culture supernatants into circulation of animals or injections of genetically engineered cells that produce labeled exosomes (Figure 1). According to the literature, most studies performed so far used exogenously produced exosomes isolated from cell culture medium, following treatment using different routes of administration, including intraperitoneal (i.p.), intravenous (i.v.), subcutaneous (s.c.), retro-orbital, intranasal and
2. Bioluminescence reporter system
Genetically engineered bioluminescent proteins such as Gaussia luciferase, combined with transmembrane domains like lactadherin- or platelet-derived growth factor receptor (PDGFR), could reveal the spatiotemporal distribution of EVs in a quantitative manner in small animals [14, 16]. This approach overcomes the limitation of background auto-fluorescence when working with fluorescent proteins. Nevertheless, this system presents the disadvantage of attenuated signal when located in a deep organ. In 2013, Takahashi et al. designed a new reporter system based on bioluminescence that enables tissue biodistribution and pharmacokinetic studies . This system is based on a fusion protein comprising Gaussia luciferase (gLuc) and a N-terminal secretion signal of lactadherin and C1C2 domains of lactadherin. The gLuc is a reporter protein that emits a very strong chemiluminescent signal when its substrate, coelenterazine (CTZ), is present, while lactadherin is a membrane-associated protein mainly found in exosomes [17, 18]. N-terminal secretion signal of lactadherin was found to be necessary for the protein to be transported to exosomal compartments and C1C2 domains necessary for its retention on exosomes membrane . Exosomes derived from B16-BL6 murine melanoma cells transfected with GLuc lactadherine (GL exosomes) were collected and then used to intravenously inject mice. GL exosomes were administered on Balb/c mice
Lai et al. also developed an additional multimodal reporter for EVs imaging based on bioluminescence . A recombinant protein composed of a transmembrane domain of PDGFR fused to a biotin acceptor domain that is fused to the humanized Gaussia luciferase was expressed in the membranes of EVs. In the presence of coelenterazine (CTZ), the purified vesicles exhibited a strong bioluminescent signal. When conjugated to streptavidin-Alexa 680, the EVs can be imaged
3. Radiolabeling of exosomes
In 2014, Morishita et al. developed a method to quantitatively assess the biodistribution of B16BL6-derived exosomes using iodine-125 (125I) labeling on a streptavidin (SAV)-biotin system . B16BL6 cells were transfected with a plasmid vector encoding the fusion protein SAV-lacadherin, and the resulting exosomes were purified and incubated with (3-125I-iodobenzoyl) norbiotinamide (125I-IBB) to obtain 125I labeled exosomes. Balb/c mice were i.v. injected with 125I labeled B16BL6 exosomes or control conditions. 125I labeled B16BL6 exosomes first underwent a distribution step with a half-life of 1.5 min and then entered a clearance phase with a half-life of 346 min, indicating that exogenously administered exosomes have short half-lives in circulation. Furthermore, 125I labeled B16BL6 exosomes were found to distribute to the liver, spleen and lung after systemic administration. High levels of radioactivity signal were found in the liver at 1 min, reaching a peak at 30 min and following a decrease at 4 h. Spleen distribution pattern was the same as the liver though at lower levels. Liver and spleen make part of the mononuclear phagocyte system, which is rich in macrophages and potentially responsible for the clearance of exosomes. Notably, at 1 min, a considerable amount of radioactivity was detected in the lungs, which had its peak at 1 h and decreased at 4 h. This can be due to exosomes aggregation possibly through interaction with blood components. Authors concluded that radiolabeling of exosomes with iodine-125 using the SAV-biotin system is a better choice when quantitatively determining exosomes tissue biodistribution than approaches based on fluorescence or chemioluminescence.
Additional methods have been developed to radiolabel exosomes. Hwang et al. produced exosomes-mimetic nanovesicles (ENVs) from extrusion of macrophage cells and radiolabeled them with 99mTc-hexamethylpropyleneamineo (HMPAO) under physiologic conditions . The conversion of 99mTc-HMPAO in the hydrophilic form that is confined inside cells is accomplished by intracellular glutathione . Further monitoring of ENVs
4. Labeling exosomes with magnetic resonance contrast agents
Studies performed by Hood group in 2014 described a new electroporation method to load mouse B16-F10 melanoma-derived exosomes with super-magnetic iron oxide nanoparticles (SPION5) . This study was in agreement with their previous findings demonstrating that melanoma exosomes appear to home to the subcapsular sinus in lymph nodes (LN) . Since a right and left pair of popliteal (PO) and inguinal (IN) lymph nodes drains mouse feet, they serve as sentinel LN for footpad tumors. Authors found that animals treated with SPION5 loaded exosomes exhibited a growth in the cross-sectional area of ipsilateral peripheral LN when compared to pre-treatment with free SPION5, probably due to the activation of inflammatory signaling pathways. Moreover, at the 48-h time point, the accumulation of SPION5 in the ipsilateral node was higher for SPION5 loaded exosomes compared to free SPION5. Furthermore, nodes treated with SPION5 loaded exosomes did not display significant differences in the MRI signal when comparing pre-injection and 1-h post-injection conditions. Altogether, these findings demonstrate that a greater amount of SPION5 accumulates in the ipsilateral LN when distributed by exosomes, and that exosomes need more time to deliver SPION5 to the LN than the trafficking time of free SPION5. These observations suggest that exosomes home and stay trapped in sentinel LN. Contrarily, free SPION5 and liposomes follow an unspecific diffusion throughout the LN system. The predominant subcapsular distribution of exosomes carrying SPION5 was further validated by histological analysis with fluorescence microscopy and transmission electron microscopy (TEM).
In 2016, Busato et al. established a new protocol to label exosomes with ultrasmall super-magnetic iron oxide nanoparticles (USPIO) . USPIO range in size from 5 to 7 nm and are stable and biocompatible . The authors described a new methodology in which adipose stem cells (ASCs) were directly labeled with USPIO rather than exosomes. ASCs are known to incorporate USPIO as part of the endocytic pathway . Furthermore, other studies reported nanoparticles accumulation inside multivesicular bodies, being consequently incorporated into exosomes . This protocol allows the preservation of the integrity of exosomes membrane, since no electroporation is required. The resulting exosomes were isolated, purified and injected in mice intramuscularly. Histological examination of gastrocnemius confirmed the presence of iron and
5. Fluorescent labeling of exosomes
When administering exogenous preparations of exosomes to assess their biodistribution
5.1. Nucleic acids labeling
The use of fluorescent dyes or fluorescent reporters has been one of the gold standard approaches to label exosomes. In addition to proteins, exosomes have been shown to carry RNA and DNA . Therefore, exosomes can be fluorescently labeled using selective dyes for those nucleic acids. The SYTO 13 dye is cell permeable and has a high fluorescent yield when bound to DNA or RNA . Other DNA binding dyes include H33342 and Thiazole Orange . Alvarez-Erviti et al. research is one good example of this approach since they detect fluorescent signals in the central nervous system after i.v. injection of exosomes derived from dendritic cells (DC) genetically engineered to express RGV peptide on the membrane and loaded with siRNA fluorescently labeled with Cy-3 dye .
5.2. Membrane-intercalating fluorescent dyes
Exosomes labeling can also be achieved by using fluorescent lipid membrane dyes, including the commonly used PKH (PKH67, PKH26), which label cell membranes through the insertion of their aliphatic chains into the lipid bilayer [8, 11, 12]. Rhodamine B also known as R18, DiI, DiO and DiD, in addition to PKH, are other examples of lipophilic fluorescent membrane dyes [35–37]. The carbocyanine dyes, DiI (yellow/red fluorescent) and DiO (green fluorescent), are weakly fluorescent in aqueous solutions but become highly fluorescent and reasonably photo-stable when incorporated into cell membranes particularly, DiR (carbocyanine DiOC18(7)) . A limitation for
5.3. Membrane permeable fluorescent dyes
Exosomes labeling can be achieved using membrane permeable chemical compounds. These dyes become confined to the cytosolic lumen and fluoresce as a consequence of esterification and include carboxyfluorescein succinimidyl ester (CFSE), 5(6)-carboxyfluorescein diacetate (CFDA) . Another example is the use of calcein AM (an acetoxymethyl derivate of the fluorescent molecule calcein) that is a very good cytoplasmic fluorescent dye, since it attains high fluorescence intensities and exhibits an acceptable persistent labeling, given that it does not covalently link to intracellular molecules . Calcein-labeling strategy is based on a membrane-permeant molecule that is nonfluorescent until it is activated by intra-vesicular enzymes . Upon hydrolysis of the acetoxymethyl ester moieties by esterases, calcein becomes highly membrane impermeable . The detection of calcein-labeled exosomes through flow cytometry has been already described and its use in some experiments has also been reported [43, 44].
Most of the studies performed to date include the use of membrane-labeled exosomes as described in the following examples. Sun et al., in 2010 administered i.p. fluorescent-labeled exosomes carrying curcumin (an anti-inflammatory agent), collected from EL-4 mouse lymphoma cells . The exosomes accumulated in greater amounts in the liver, lungs, kidneys and spleen 1 h post-treatment. Interestingly, when exosomes were administered through the intranasal route, the distribution pattern was re-directed to the brain and intestines.
In 2011, Hood et al. demonstrated for the first time that exosomes isolated from melanoma cells supernatants induced LN conditioning
In 2012, Peinado et al. proposed a new role for melanoma-derived exosomes in which they educate bone marrow (BM) progenitor cells to acquire a pro-metastatic phenotype through MET signaling . First, fluorescently labeled B16-F10 exosomes (using PKH67 dye) to analyze exosomes biodistribution were i.v. administered into naive mice. Exosomes were detected in the blood vessels and organs within 5 min after injection. Twenty-four hours post-treatment, exosomes were no longer found in blood circulation. Instead, exosomes were found in the major organotropic sites for B16-F10 metastasis including interstitium lungs, BM, liver and spleen. Next, B16-F10-derived exosomes were injected 3 times a week for 3 weeks, 7 days after orthotopically injection of B16-F10mCherry cells to assess melanoma-derived exosomes role in primary tumor growth and metastasis. Mice showed lung micro-metastasis at day 19 after tumor cell injection in contrast with the control (synthetic unilamellar liposomes). To evaluate the role of the metastatic potential of the cells of origin, equal amounts of exosomes from highly (B16-F10) or poorly (B16-F1) metastatic melanomas were i.v. injected into mice 3 times a week over 28 days, and then subcutaneously implanted B16-F10 cells expressing luciferase. Injection with B16-F10 exosomes resulted in a higher metastatic burden in the lungs and greater tissue distribution, including bone and brain when compared to mice injected with control particles or with B16-F1 exosomes. Notably, these observations indicate that exosomes content can mediate metastatic potential and organotropism. To further investigate this hypothesis and taking into consideration the central role of bone marrow-derived cells (BMDCs) in metastatic progression, they postulated whether tumor-derived exosomes could educate BMDCs and affect metastatic development. In a process, they termed bone marrow education, GFP-expressing mice were treated with B16-F10 exosomes 3 times a week for 28 days. Next, lethally irradiated mice were transplanted with the educated bone marrow and the mice were also subcutaneously injected with B16-F10 cells expressing mCherry. By pre-educating BMDCs with exosomes from a highly metastatic cancer cell line, an increase in the metastatic tumor burden and distribution in target tissues was observed, even for tumors with a low metastatic capacity. Overall, this work shows that by educating BMDCs tumor exosomes can regulate tumor metastasis. Further proteomics studies revealed MET as a potential candidate implicated in BM education given its previous described role in migration, invasion, angiogenesis and BM cells mobilization. Indeed, additional studies demonstrated that B16-F10-derived exosomes could transfer MET to BM progenitor cells, this way mediating pro-vasculogenic and metastatic effects (enhanced cell mobilization).
In 2015, Costa-Silva et al. proposed a mechanism in which pancreatic ductal adenocarcinoma (PDAC)-derived exosomes induce liver pre-metastatic niche formation in naive mice . Authors demonstrated that Kupffer cells, macrophages present in the liver, uptake PDAC-derived exosomes, which activates the secretion of transforming growth factor β that in turn stimulates hepatic stellate cells to produce fibronectin. The resulting fibrotic microenvironment was showed to enhance the recruitment of BM-derived macrophages. Furthermore, macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its inhibition resulted in abrogation of liver pre-metastatic niche formation and metastasis. Collectively, these data suggest that PDAC-derived exosomal MIF primes the liver for metastasis.
In addition, Hoshino et al. took a step further in investigating pre-metastatic niche formation and unraveling exosomes organotropism . The authors seek to demonstrate tumor-derived exosomes contribution to the establishment of a permissive microenvironment at future metastatic sites, describing their nonrandom biodistribution patterns. In all experiments performed, the authors use prepared pools of exosomes labeled with PKH dyes. They show that exosomes from mouse and human lung-, liver- and brain-tropic tumor cells fuse preferentially with resident cells at their predicted destination sites to prepare the pre-metastatic niche. Surprisingly, treatment with exosomes from lung-tropic tumor cells was sufficient to redirect the metastasis of bone-tropic tumor cells. Further exosomes proteomic studies revealed distinct integrin expression patterns that differed from tumor cells. They found that exosomes expressing integrin αvβ5 specifically bind to Kupffer cells, mediating liver tropism, whereas exosomal integrins α6β4 and α6β1 mediated lung metastasis through binding with fibroblasts and epithelial cells. Moreover, when these integrins were blocked a decrease of exosomes uptake as well as metastasis formation was observed. Additionally, exosomes uptake by resident cells at metastatic sites mediated by the previously mentioned integrins was found to induce Src phosphorylation and activate the expression of pro-inflammatory S100 response. Altogether, these findings suggest that exosomal integrins are responsible for the adhesion of exosomes to target cells. Furthermore, this interaction activates, in the recipient cells, signaling pathways involved in inflammatory responses contributing to the formation of a microenvironment that supports the growth of metastatic cells.
Wen et al. associated exosomes derived from highly metastatic breast cancer cell lines with an increase in the metastatic potential partly due to an immune suppression of the tumor microenvironment . Exosomes isolated from murine breast cancer cell lines (metastatic EO771 and 4T1, nonmetastatic 67NR) were labeled with DiD, a lipid-associating fluorescent dye and i.v. injected into mice. Exosomes biodistribution was evaluated in several organs 24 h post-injection using
In 2014, Smyth et al. evaluated the tissue distribution of exosomes derived from breast and prostate cancer cell lines when i.v. administered into healthy or tumor-bearing mice . Exosomes were isolated from cell culture supernatants of 4T1, PC3 and MCF-7 cells. Mice were inoculated with 4T1 cells in the mammary fat pad (MFP) and 15 days after inoculation they were i.v. injected
Recently, Wiklander et al. established a set of experiments demonstrating that EVs biodistribution is dependent on many factors, including cell source, exosomes concentration and route of administration . They injected 1 × 1010 particles/gram body weight (p/g)
Another method consists on the genetic engineering of cells to direct the expression of fluorescent markers to the exosomal membrane resulting in labeled exosomes production. Exosomes can be isolated from these cells culture supernatants or cells can be directly injected into mice. One commonly used example is the GFP-CD63 construct. The CD63 is a tetraspanin, a membrane-associated protein and is known as a general marker of exosomes . Suetsugu and collaborators were one of the first ones to orthotopically inject stable expressing GFP-tagged CD63 cells . They demonstrated that tumor-derived exosomes serve as a central mediators for communication not only between cancer cells but also with their microenvironment components. They produced mouse breast cancer cells (MMT) and human breast cancer cells (MDA-MD-231) RFP labeled stably expressing GFP-tagged CD63 (MMT-RFP/GFP-Exo and MDA-MD-231-RFP/GFP-Exo, respectively). Hence, cells were red and producing green exosomes. To generate orthotopic mouse models of breast cancer metastasis to the lung, they orthotopically injected MMT-RFP/GFP-Exo or MDA-MD-231-RFP/GFP-Exo cells into the MFP of nude mice. Overall, using confocal laser scan microscopy (CLSM), they observed that both in primary tumors and in lung metastasis breast cancer cells secreted exosomes into the tumor microenvironment. To confirm GFP exosomes integration in mice host cells, RFP nude mice were orthotopically injected with the cells previously mentioned into the MFP. GFP-labeled exosomes were taken up by stromal cells namely fibroblasts. Finally, blood samples analysis by CLSM confirmed the presence of GFP exosomes in circulation of mice-bearing lung metastasis.
Nonetheless, when designing these fusion plasmids, one should consider the influence they may have in the protein normal functions. Interestingly, it has been shown that GFP fusion to the N- or C-terminus of CD63 influences protein distribution in rat basophilic leukemia (RBL) cells . When GFP was linked to the C-terminus of CD63 (CD63-GFP), the fused proteins were expressed on both the granule membranes and plasma membranes of RBL cells as native CD63 proteins. Contrarily, when the GFP was conjugated to the N-terminus of CD63 (GFP-CD63), it was homogeneously distributed in the cytoplasm, not being present on granules or the plasma membrane . These results suggested the possibility that the N-terminus of CD63 might play an important role in the establishment of protein localization.
Other approaches developed by Lai et al. in 2014 included a multiplex reporter system consisting of enhanced (EGFP) and tandem dimer tomato (tdTomato) fluorescent proteins fused at NH2-termini with specific palmitoylation signals, enabling EV membrane labeling . By treating cells with EVs carrying fluorescently labeled siRNA, they observed EVs uptake in donor cells. Notably, by combining fluorescent and bioluminescent EVs membrane reporters, they elegantly demonstrated EVs uptake and translation of nascent EV-derived cargo mRNAs in cancer cells
Recently, a new approach was put forward that allows the study of the function of transfer of EVs
6. Concluding remarks
Collectively, exosomes spatiotemporal distribution is still elusive, mostly because
SAM laboratory is supported by the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FEDER funds through the COMPETE Program (POCI-01/0145-FEDER-016618), national funds from FCT – Foundation for Science and Technology PTDC/BIM-ONC/2754/2014 and Maratonas da Saúde. SAM is supported by FCT – Foundation for Science and Technology (IF/00543/2013). We thank Dr. Nuno Barros for the help with the design of the figures included in this chapter.
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