Extracellular vesicle‐associated organotropic metastasis

Abstract Metastasis refers to the progressive dissemination of primary tumour cells and their colonization of other tissues and is associated with most cancer‐related mortalities. The disproportional and systematic distribution pattern of distant metastasis in different cancers has been well documented, as is termed metastatic organotropism, a process orchestrated by a combination of anatomical, pathophysiological, genetic and biochemical factors. Extracellular vesicles (EVs), nanosized cell‐derived membrane‐bound particles known to mediate intercellular communication, are now considered crucial in organ‐specific metastasis. Here, we review and summarize recent findings regarding EV‐associated organotropic metastasis as well as some of the general mechanisms by which EVs contribute to this important process in cancer and provide a future perspective on this emerging topic. We highlight studies that demonstrate a role of tumour‐derived EVs in organotropic metastasis via pre‐metastatic niche modulation. The bioactive cargo carried by EVs is of diagnostic and prognostic values, and counteracting the functions of such EVs may be a novel therapeutic strategy targeting metastasis. Further investigations are warranted to better understand the functions and mechanisms of EVs in organotropic metastasis and accelerate the relevant clinical translation.


| BACKG ROU N D
Metastasis is a multistep process in which tumour cells progressively spread from the primary sites of oncogenesis and colonize other tissues or organs. 1 It is considered one of the most catastrophic hallmarks of cancer. 2 Most cancer-related deaths are thought to link to metastasis. 3 Moreover, clinical observations have documented the disproportional distribution pattern of distant metastasis in various cancers, which is well recognized as metastatic organotropism. 4 Recently, extracellular vesicles (EVs) and their cargoes are reported to be crucial in organ-specific metastasis. Here, we concentrate on and summarize the recent findings regarding EV-associated organotropic metastasis as well as some general mechanisms involved and review the novel translational studies targeting metastasis with EVbased diagnostic, prognostic and therapeutic strategies before providing a future perspective on this topic.

| Multistep mechanism of metastasis
Tumour cells at primary sites of cancer undergo a series of complex but highly regulated processes to spread to and grow in distant organs and tissues. At first, abnormally proliferating tumour cells start to invade surrounding tissues, such as basement membrane, which is frequently accompanied by epithelial to mesenchymal transition and marked by decreased E-cadherin expression and enhanced N-cadherin expression. 5 Next, tumour cells intravasate into blood vessels and begin their journey in the circulation. Inflammatory responses are known to play a crucial role in intravasation by increasing vascular permeability. 6 Circulating tumour cells in migration are then arrested by adhesive interactions, or neutrophil aggregation or trapped by physical occlusion in vasculature and extravasate. 7 Upon landing at future metastatic sites, tumour cells resume secondary growth, requiring multiple factors for adaptation to the new environment. 8 The spatial concept inherent in metastasis, known as the tumour microenvironment, is of rapidly rising interest and relevance to research in cancer biology. 9

| Metastatic routes
Metastasis can happen via various routes, including the lymphatic system, haematogenous routes, transcoelomic spreads, and iatrogenic transplantation. The site-specific metastases of transcoelomic spreads and iatrogenic transplantation are largely associated with anatomical features. Transcoelomic metastasis occurs when tumour cells disseminate across body cavities such as pericardial, pleural, peritoneal, or subarachnoid spaces. 10 For instance, the transcoelomic route is the most frequent metastatic pattern in epithelial ovarian malignancy and is responsible for most mortalities. 11 Similarly, iatrogenic transplantation is caused by carryover of malignant cells during invasive diagnostic or surgical procedures. For this reason, routine biopsy is often not recommended for cases of hepatocellular carcinoma due to the risk of needle-track seeding. 12,13 Compared to transcoelomic spreads and iatrogenic transplantation, organ-specific tropism involved in lymphatic and haematogenous metastasis is more complex and regarded as the results from numerous factors including anatomical and functional characteristics, and molecular and cellular interactions. 14

| Extracellular vesicles
In recent years, EVs have been increasingly recognized as a key player in the tumour microenvironment, with diverse EV cargoes thought to contribute to metastasis and organotropism. Abundant in a variety of human biofluids, from blood 15 and urine 16 to cerebrospinal fluid 17 and semen, 18 EVs are a class of cell-derived, membrane-enclosed nanoparticles secreted by various types of cells, 19 including cancer cells, 20 immune cells, 21 and red blood cells. 22 Protected by a lipidbilayer membrane structure, EVs carry a variety of bio-functional molecules, both on the membrane and within the vesicles, including proteins, nucleic acids, and lipids. 23 EV function is largely dependent on the various imparted cargo it shuttles, which can be reflective of the cells of origin. 24 Based on distinct biogenesis pathways, EVs are typically categorized into three major groups: exosomes, microvesicles and apoptotic bodies.
Exosomes are endosomal in origin and formed by the fusion of multivesicular bodies (MVBs) with plasma membrane. MVBs contain intraluminal vesicles and they are subsequently released into the extracellular lumen when these MVBs fuse with the plasma membrane. 25 In comparison, microvesicles are generally larger, and they bud directly from the plasma membrane as the molecular cargo is transported to the cell surface. For this reason, they are often referred to as shedding vesicles or shedding bodies. This shedding of microvesicles can be stimulated by activation of the plasma membrane involving increased intracellular calcium levels. 26 Microvesicles, like exosomes, have unique lipid compositions, and are enriched in phosphatidylserine. 27 Many protein markers are known to express on exosomes and microvesicles, including tetraspanins such as CD9, CD62 and CD81, membrane transporters and fusion proteins such as annexins, EV synthesis proteins such as TSG101, and other EV-associated proteins. 25,28 Apoptotic bodies are predominantly secreted by cells experiencing apoptosis. 29 They are, in general, observed to be larger than exosomes and microvesicles, and contain fragments of DNA and cell organelles from the apoptotic cells. These apoptotic bodies are formed during blebbing of the plasma membrane. 30

| EV signalling
EV signalling is an important mode of intercellular communication, given the myriad of bioactive molecules, including DNA, RNA, and proteins, which are carried and transferred by EVs. Tumour metastasis and progression are not an exception to this. EVs play a pivotal role in communicating between tumour cells and other cells in the tumour microenvironment; via EVs, highly metastatic tumour cells are able to transfer biomolecules to less malignant cells. 31 Consequently, the less malignant cells receiving the EVs may start to display enhanced migratory and metastatic behaviour. 31 An example is the avß3 integrin, which is upregulated during cancer progression and acknowledged to account for the migration of cancer cells, hence leading to metastasis of tumours. EVs are able to transfer avß3 integrins from tumorigenic cells to other cells, causing recipient cells to express malignant traits, increasing avß3-dependent adhesion and migration. 32 EV signalling is also capable of inducing the changes of tumour cell characteristics and behaviours, such as drug resistance.
Challagundla and colleagues 33 found that human monocyte-derived EVs were able to transfer miR-155 to neuroblastoma cells; upon EV-mediated delivery, the miR-155 directly targeted TERF1, which is a component of the shelterin complex and also a telomerase inhibitor. Increased cisplatin resistance was hence conferred to the neuroblastoma cells. Similarly, Chen et al showed that adriamycin and docetaxel resistance can be conferred by EVmediated microRNA transfer. 34 Cell surface proteins of EVs contribute to drug resistance as well. Ko et al posited that small EVs (sEVs) promote angiogenesis via their EV-surface-specific isoform of vascular endothelial growth factor (VEGF), which cannot be neutralized by bevacizumab, 35 the therapeutic monoclonal antibody against VEGF reported to be effective in many solid cancer treatments. [36][37][38] The combination treatment of bevacizumab with a Hsp90 inhibitor, 17-N-allyamino-17-demothoxygeldanamycin (17AAG), can restore the bevacizumab sensitivity of EV surface VEGF and inhibit tumour growth in a breast cancer patient-derived xenograft model through 17AAG localizing to microvesicles (MVs), binding VEGF 90K and releasing this VEGF, thus increasing the efficacy of bevacizumab. 39 EVs may also play a role in immunosuppression allowing unchecked proliferation of tumours and subsequently, their metastasis.  EVs are also responsible for reprogramming cellular metabolism, which is essential in providing energy for dissemination and proliferation of cancer cells, especially at distant sites where vasculature and nutrients are lacking. 43 Cancer-associated fibroblast (CAF)-derived EVs inhibit mitochondrial oxidative phosphorylation, and shuttle de novo 'off the shelf' metabolites for cancer cells. 44 These respective findings demonstrate that EV signalling plays an important role in cancer initiation and progression. Further discoveries pertaining to organ-specific metastasis will be detailed in coming sections.

| E X TR ACELLUL AR VE S I CLE S IN ORG ANOTROPIC ME TA S TA S IS
In the context of cancer metastasis and organotropism, EVs are best known to function by modulating the pre-metastatic niche formation. Paget's 'seed and soil' hypothesis has established the conceptual architecture for organotropic metastasis studies. 45 In brief, tumour cells are considered as 'seeds' while metastatic sites as 'soil'.
In order for the 'seeds' to grow in the new 'soil', the tumour cells and the microenvironment at the metastatic sites need to be compatible: the 'seeds' require intrinsic properties to expand and the 'soil' has to be congenial. 45 Recent reports have shown that tumour cells at primary sites are able to signal resident cells at potential metastatic sites via EVs. 46 Primary tumour cells secrete EVs into circulation as messengers even before their arrival at distant sites. 47 Carrying bioactive molecules, EVs travel from primary tumour sites, arrive at the pre-metastatic niches, and are internalized by resident cells there. 47 After internalization, molecules transported by EVs participate in biological interactions in the recipient cells. For instance, the cargo de- Thus, EVs are responsible for tuning the distant microenvironment into a piece of favourable 'soil' for the 'seeds' tumour cells to grow. 47 A deeper understanding of EVs in site-specific metastasis may unravel novel uses of EVs for diagnostic and therapeutic purposes. We next review recent studies investigating EV-associated metastatic organotropism by organs where distant metastasis frequently happens-in the lungs, brain, bone and liver ( Figure 1; Table 1).

| Lung-tropic metastasis
Lung is a frequent metastatic site for breast, colorectal, and pancreas cancers as well as melanoma. Hoshino et al 51 demonstrated that tumour-derived, lung-tropic EVs bear integrins α6β1 and α6β4 and are favourably taken up by lung fibroblasts and surfactant protein C-expressing lung epithelial cells. Incorporation of such EVs by lung resident cells increase the expression of pro-inflammatory genes S100 and promote lung metastasis while blocking lung-tropic EVs surface integrins using HYD-1 peptides can inhibit the uptake by lung resident cells and reduce lung metastasis. This is consistent with our findings that tumour-derived, microRNA-125b-containing EVs are preferentially taken up by pulmonary resident fibroblasts and transform them into activated phenotypes, and thus promote lung metastasis. 52 Moreover, Ortiz et al 53 reported the protective effect of cholesterol 25-hydroxylase (CH25H) in healthy cells against pro-metastatic education by tumour-derived EVs. Interferon-inducible CH25H hampers tumour-derived EVs uptake by generating 25-hydroxycholesterol. In vivo assays showed that inability to downregulate IFN receptor and Ch25h protects the lung from pre-metastatic niche formation via ablating tumour-derived EVs education, thus reducing melanoma lung metastasis. Surprisingly, reserpine, the classic anti-hypertensive beta blocker, reduced lung metastasis by refusing tumour-derived EVs education, which appears to be a potential novel therapeutic target against lung metastasis.
One of the most characteristic features of the lung is its abundance in capillaries, providing the physiological territories for tumours cells to arrest, adhere, extravasate, and grow. 54 EVs are actively involved in influencing the lung vascular permeability as well. According to Zhou et al, 55 microRNA-105 can be transferred from metastatic breast cancer cells to endothelial cells via EV-mediated cell communication, which hampers the barrier functions of endothelial monolayers, increases vascular permeability, and promotes metastasis in lung via targeting the tight junction protein zonula occludens-1 (ZO-1). Besides, microRNA-105 in circulation has been proved to correlate with metastasis in patients with early-stage breast cancer. Similarly, F I G U R E 1 Extracellular vesicle (EV)-mediated organotropic metastasis via pre-metastatic niche modulation. Investigations showed that pre-metastatic niche modulation via EVs plays a major role. Primary tumour cells disseminate EVs which can travel to distant metastatic sites and modulate resident or stroma cells in the pre-metastatic microenvironment through various pathways, such as promotion of proinflammatory gene expression and cytokine secretion, direction of phenotype-specific induction/differentiation, and recruitment of specific cell types. The EV-mediated modulation prepares a favourable pre-metastatic niche at distance for metastatic tumour cells to reside in and grow

| Brain-tropic metastasis
Brain metastasis is mostly observed in breast, kidney, lung cancers, and melanoma. The distinctive biophysiological property that separates brain from other metastatic sites is the blood-brain barrier (BBB), which is a highly selective border controlling substance permeability. The BBB consists of continuous capillary wells of endothelium cells joint by tight junction with astrocyte end feet ensheathing and pericytes embedded in the basement membrane. 59 Intact BBB needs to be disrupted or compromised for tumour cells to metastasize to the brain. 60 Recently, Morad and colleagues 61 expression in brain-tropic EVs in comparison to lung-tropic or bone-tropic EVs and demonstrated that ablation of CEMIP in tumour cells hinders brain metastasis. CEMIP-expressing, braintropic EVs prompts remodelling and inflammation in brain vasculature, upon the uptake by brain endothelial and microglial cells via increasing secretion of pro-inflammatory cytokines. Elevated levels of CEMIP are also predictive of brain metastasis and poor prognosis in patients. In another study by Xing et al, 64

| Bone-tropic metastasis
Tumour cells from melanoma, prostate, lung, and breast cancers can metastasize to bone. One unique feature of the bone microenvironment is the distinct composition and dynamics of bone-resorbing osteoclasts, bone-forming osteoblasts, and the bone-residing osteocytes. 66

| Liver-tropic metastasis
Liver metastasis is primarily described in lung, breast, and gastrointestinal cancers. One of the most studied liver resident cell types

| E VS A S D IAG NOS TI C AND PROG NOS TI C B I OMARK ER S IN C AN CER M E TA S TA S I S
The transport and delivery of the bioactive cargo is fundamental to

EV-mediated intercellular communication and has motivated a num-
ber of translational studies examining and exploiting EVs for metastasis-associated diagnostic, prognostic, and potentially therapeutic applications.
From the perspective of diagnostic and prognostic studies, the secretion of EVs by donor cells and uptake by recipient cells have been widely observed in a broad spectrum of human diseases, including cancers, 20 cardiovascular conditions, 83 metabolic dysfunctions, 84 autoimmune disorders, 85 and neurological diseases. 86 Profiling EVs using sequencing and mass spectrometry empowers us to decode the molecular messages that the diseased cells try to send. Since EVs carry molecular features from parental cells that are highly biorelevant, their molecular profiles may be of unparalleled diagnostic and prognostic values as compared to other clinical benchmarks. 87 The abundance of EVs in biofluids provides the possibility to readily evaluate EVs in a minimally invasive manner. 88 EVs can serve as dynamic snapshots of the diseased parental cells to reflect real-time functional changes, which likely occur far before possible detection by observation of morphological changes or by imaging.
In terms of biomedical and translational research, a better understanding of EVs and their cargo can potentially reveal the molecular mechanisms involved in these diseases ( Table 2).
The overall EV count can provide prognostic value for cancer pa- is raised in serum EVs from CRC patients-this rise is detectable earlier than current biomarkers used for CRC diagnosis such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). The authors also showed that the ExoScreen platform

F I G U R E 3 Applications of organotropic metastasis-associated
EVs. These metastatic and organotropic aspects of tumour-derived EVs can be of great diagnostic, prognostic, and therapeutic potentials and of emerging interests in biomedical and translational research uses as little as one microlitre of EV-containing culture medium for detection and has superior results to conventional immunoblotting and ELISA.
Another method was devised by Dong and colleagues 90 to detect EVs by using the surface-enhanced Raman scattering (SERS) technique to monitor the protein phosphorylation process in EVs.
A 3D gold-coated TiO2 macroporous inversion opal (MIO) structure was developed that can coordinate the interaction of the laser used, sample, and the SERS substrate. 1087cm -1 SERS peaks of the EVs from prostate, lung, liver and colonic cancer cell lines were double that in normal cells. These changes were apparent from EVs taken from prostate cancer patients' plasma as well, showing promise in cancer diagnostics via EV monitoring.
Specific EV proteins associated with certain cancers have been identified as possible biomarkers for cancer diagnosis. Ji et al showed that integrin beta-like 1 (ITGBL1) was highly expressed in primary CRC and metastatic sites in comparison to normal tissues, and its high levels were also detectable within plasma EVs of CRC patients. 91 Similarly, levels of TGM2, an extracellular matrix protein, U2AF1, U2AF2, and HNRHPU, which are intracellular spliceosomal proteins, were raised in EVs from ascitic fluid of ovarian cancer patients compared to normal ascitic fluid. 92 In some cases, the level of these biomarkers can be used for prognostic purposes-in stage IV melanoma patients, the melanoma biomarkers MIA and S100B are raised and detectable in EVs from their sera; raised levels of these biomarkers are also associated with shorter median survival. 93 In  EVs derived from macrophages of the M1 phenotype stimulated pro-inflammatory cytokine production, in turn allowing the cancer vaccine to induce a stronger cytotoxic T-cell response. Accumulation of immune cells in tumours increased, and tumour growth was significantly inhibited.

| NOVEL E V-A SSO CIATED THER APEUTI C S TR ATEG IE S AG AIN S T C AN CER M E TA S TA S I S
In brief, researchers are utilizing EVs as promising therapeutic agents against cancer-either taking advantage of their unique properties, or actively countering their role in tumour progression and metastasis. The above section represents a recent and novel classification of the various studies done in this field. Further research will uncover more potential therapeutic applications of EVs in cancer therapy.

| SUMMARIE S AND FUTURE PER S PEC TIVE S
To summarize, organotropic metastasis involves the convergence of many complex biological interactions and processes, orchestrated action, and internalization mechanism and signalling initiation upon uptake. We believe that emerging novel techniques in EV research, such as label-free EV visualization 122 and blood oncogenic EV elimination, 118 will surely deepen our understanding and accelerate the clinical translation of EVs in organotropic metastasis.

ACK N OWLED G EM ENTS
The figures, including the graphical abstract and the cover image, were generated using BioRender with a paid licence to publish. We would like to thank our funders for support. They do not play a role in the design of the study and collection, analysis or interpretation of data or in writing the manuscript.

CO N FLI C T O F I NTE R E S T
MTNL is a cofounder, advisor and shareholder of Carmine

DATA AVA I L A B I L I T Y S TAT E M E N T
Original data can be found in the corresponding references. No new datasets were generated for this review.