Tumor-derived microvesicles: The metastasomes

Medical Hypotheses 80 (2013) 75–82

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Tumor-derived microvesicles: The metastasomes Reza Ghasemi a,b, Antonino Grassadonia a, Nicola Tinari a, Enza Piccolo a, Clara Natoli a, Federica Tomao c Stefano Iacobelli a,⇑ a

Department of Biomedical Sciences, University of ‘‘G. D’Annunzio’’ of Chieti-Pescara, Italy Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran c Department of Gynecology & Obstetrics, University of Rome ‘‘Sapienza’’, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 1 June 2012 Accepted 18 October 2012

a b s t r a c t Metastasis is the leading cause of cancer death, yet it is mechanistically considered a very inefficient process suggesting the presence of some sort of (e.g. systemic) routes for fuelling the process. The pre-metastatic niche formation is described as one such metastasis promoting route. Now, the emerging potentials of tumor-derived microvesicles (TDMVs), not only in formulating the pre-metastatic niche, but also conferring neoplastic phenotypes onto normal cells, has integrated new concepts into the field. Here, we note as an ancillary proposition that, exerting functional disturbances in other sites, TDMVs (we have termed them metastasomes) may aid foundation of the secondary lesions via two seemingly interrelated models: (i) tumor-organ-training (TOTr), training a proper niche for the growth of the disseminated tumor cells; (ii) tumor-organ-targeting (TOTa), contribution to the propagation of the transformed phenotype via direct or indirect (TOTr-mediated disturbed stroma) transformation and/or heightened growth/survival states of the normal resident cells in the secondary organs. Respecting the high content of the RNA molecules (particularly microRNAs) identified in the secretory MVs, they may play crucial parts in such ‘‘malignant trait’’ spreading system. That is, the interactions between tumor tissue-specific RNA signatures, being transferred via metastasomes, and the cell-type/tissue-specific RNA stockrooms in other areas may settle a unique outcome in each organ. Thus, serving as tumor-organ matchmakers, the RNA molecules may also play substantial roles in the seeding and tropism of the process. Ó 2012 Elsevier Ltd. All rights reserved.

Introduction The term ‘‘metastasis’’ was initially created in 1829 by Jean Claude Recamier and is defined as ‘‘the transfer of disease from one organ or part to another not directly connected to it’’ [1]. Malignant disease can be dated back to Egyptians (1500 BC) and latter to the Greek physicians in the time of Hippocrates (5th century BC) who detected fatal secondary lesions in breast cancer patients. Although such lesions were primarily considered as ‘‘independent tumors’’ arisen from the spread of ‘‘toxic humors’’, with the discovery of cell as the basic unit of organisms, this theory was abandoned and the secondary lesions were supposed to arise from the migration and seeding of tumor cells from primary sites into the others (reviewed in Ref. [2]). Since then, despite the several (e.g. seed-soil) models [3], the mechanism of tumor metastasis has remained enigmatic. In fact, based on the current models, a proposed metastasis founder cell must go through the sequential ⇑ Corresponding author. Address: Department of Biomedical Sciences, University of ‘‘G. D’Annunzio’’, Via dei Vestini 31, 66100 Chieti, Italy. Tel.: +39 0871 355 6732; fax: +39 0871 355 6707. E-mail address: [email protected] (S. Iacobelli). 0306-9877/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2012.10.011

series of inefficient steps (separation from the primary tumor, intravasation, survival in the circulation, extravasation, and successful colonization in the secondary organs) to found a clinically detectable metastasis [3]. Two questions can be raised here: first, how can metastasis become the main cause of cancer death if it is a very inefficient process? Second, how could the disseminated tumor cells (DTCs) stay dormant even for decades in the eccentric and tumor-suppressive microenvironments of the ectopic regions [4,5] to give then rise to tumor recurrence? Together these conundrums, with the notion that the metastasis founder cell is yet unknown [6], may force a major rethinking of the process. Perhaps, we are missing some parts of the landscape that might be due to our reductionistic views. In this situation, putting all the possibilities together to unravel the matter of metastasis becomes important, as it will provide a base for definition of more effective treatment options and therapeutic strategies. It is increasingly becoming evident that the tumor ‘‘cell’’ migration from primary site into distant organs is a simplistic view or only a part of the whole panorama of the cancer ‘‘disease’’ dissemination process. Recently, the interplay between primary tumor and secondary organs via circulation has emerged as an essential component of tumor metastasis [6,7]. While cancer cells can

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crosstalk with their surroundings by simple interactions using nucleotides, lipids, small peptides, or proteins [8], a primary tumor may employ more sophisticated mechanisms to achieve efficient interplays with distant areas. One such route which has recently been the focus of attentions is provided by tumor-derived microvesicles (TDMVs), serving as to condition the target organs for metastatic growth [7]. Microvesicle (MV) is a collective term for a broad range of membrane particles including exosomes, shedding vesicles, and apoptotic blebs which are released from and taken up (by fusion, endocytosis, or target-receptor interaction, e.g. via tetraspanins) by almost all cell-types, including cancer cells [9–12]. MVs have been identified in many body fluids, including blood, breast milk, cerebrospinal fluid, saliva, and urine [13–15]. They can be isolated or tracked by ultracentrifugation, sucrose-gradient centrifugation floatation, immune-affinity enrichment procedures, and confocal microscopy [16]. MVs carry various cargos from their mother cells, including lipids, specific proteins and nucleic acids (DNA, mRNAs, and miRNAs) which can be translated into functions in the recipient cells [17–19]. Proteins, mRNA, and miRNA may be sorted into some MVs such as exosomes in a selective and energydependent manner [20–22]. Exosomes are a subset of MVs, 40–100 nm in diameter which are formed in specific subcellular compartments called microvesicular bodies (MVBs) [23]. Prostasomes, equivalent to exosomes, improve sperm cell fertilization ability via transferring signaling molecules from prostatic acinar cells to sperms [11]. Remarkably, the tumorigenic factors encased in the circulating TDMVs can be translated into neoplastic phenotypes in the recipient cells [24,25]. These remarks imply that the conditioning of metastatic sites for the growth of cancer (metastatic founder) cells is likely the simple(st) impact that can be attributed to the TDMVs, that is, they may also prompt transformed phenotypes upon the normal recipient cells, hence, foundation of de novo tumors in the secondary organs. So with this perspective, here we note as an ancillary proposition that metastasis likely stems from the transfer of ‘‘malignant traits’’ from primary tumors into other sites via TDMVs. The hypothesis of metastasome We speculate that TDMVs carrying malignant traits, we call them metastasomes, promote a series of cell biological actions in other sites throughout the body of cancer patients that finally lead to the development of secondary lesions in vulnerable areas. Now, describing the concept of metastasome we propose two mechanistic models for tumor metastasis: first, ‘‘tumor-organ-training (TOTr)’’, modulation of the microenvironment of the target sites to serve as hospitable hosts for the alien DTCs, formation of premetastatic niche; second, ‘‘tumor-organ-targeting (TOTa)’’, contribution to the propagation of the transformed phenotypes upon normal resident cells, thereby foundation of de novo and ‘‘second primary’’ tumors in the target organs. The metastasomes A growing body of evidence shows that many cancer-related cell-biological and clinical events are associated with the accelerated rates of MVs secretion from cancer cells [7]. It is noteworthy that the apical-basal polarity and careful arrangement within the underlying basement membrane and neighboring cells are principles of epithelial cells and warrants their normal and stable homeostasis [26] (note that about 80% of the life-threatening cancers – carcinomas – arise from the epithelial tissues [3]). A consequence of such organization is the oriented and controlled trafficking of the transport vesicles by epithelial cells (e.g. exocytosis/secretion of prostasomes/MVs by the prostatic/breast acinar

cells into their lumens) [27,28]. The exocyst complex which is located at the adherens junctions (AJs) in the epithelial cells also plays crucial role in the polarized secretion of the exosomes, so that, mutation of its components results in the cytoplasmic accumulation of the transport vesicles [29]. It can be anticipated that loss of tissue architecture, cell polarity, and AJs in carcinomas may give rise to intrusions in the amounts, contents, and direction of secretion of the MVs by epithelial cells. Accordingly, an active and constitutive shedding of the GFP-labeled MVs from the MDAMB231 and U87 human cancer cells, but not from the normal NIH 3T3 cells, have been explored [25]. The oncogenic mutations in some cancer genes such as EGFR and K-ras may also promote secretion of the exosomes with invasive potentials from cancer cells [30,31]. Besides, hyperactivation of the Rho-dependent signaling pathway promotes tumor development via fuelling of the MV formation from cancer cells [32]. From a clinical standpoint also, in the plasma samples of the patients with colorectal (CRC), lung, melanoma, ovarian, and prostate cancers higher levels of prostasomes/exosomes, compared to those of the normal controls, have been detected [33–37]. The high levels of plasma exosomes have been associated with poorly differentiated tumors and shorter disease overall survival in CRC [34] and the degree of malignancy in ovarian cancer [37]. Even high levels of circulating HER2-bearing exosomes in the HER2-positive breast cancer patients may play some mechanistic roles in the development of resistance to HER2-targeted therapy [38]. Furthermore, as tumor progression proceeds, its microenvironment becomes acidified which, as a hallmark of tumor malignancy, enhances not only the release of MVs, but also their uptake efficiencies by cancer [39] and likely the surrounding stromal cells. This scenario, which is attributable to the higher membrane sphingolipids (sphingomyelin/ganglioside GM3) in TDMVs generated in the acidic environment [39], can establish a self-amplifying positive feedback loop between cancer and the ‘‘surrounding’’ stromal cells. For instance, activated tumor associated macrophages (TAMs) release miRNAs within exosomes that promote the invasive potentials of breast cancer cells [40]. Indeed, TDMVs can facilitate tumor growth and progression by modulating the immune system, i.e. by inhibiting tumor-suppressing T cells and hindering or educating the differentiation of bone marrow derived cells (BMDCs) [7,41–44]. TDMVs (exosomes) can also trigger differentiation of the surrounding fibroblasts to myofibroblasts which support tumor progression and metastasis [45]. It can be anticipated that circulating TDMVs are not the products of carcinoma cells only; indeed, they represent the interplays between a heterogeneous population of cells within the tumor mass, ‘‘tumor organ’’ (Fig. 1). The same tumor yet contains various subclones of neoplastic cells [46], each likely with distinct MV-release capacity or releases MVs with diverse invasive potentials. The major breakthrough in understanding the role of MVs in cancer is provided by the studies uncovering proinvasive and cell cycle-related factors, and oncogenic proteins, DNAs, mRNAs, and miRNAs within TDMVs, enabling them to confer malignant traits onto normal cells or boost the tumorigenic potential of cancer cells [17,22,24,25,30,33,47–49]. Consistent with the potential relevance of this notion, synergic treatment of endothelial cells by human squamous carcinoma cell (A431)-derived MVs along with an inhibitor of MV-uptake (Diannexin) has led to reduction of their tumor promoting effects in the xenograft tumor in mice [50]. Therefore, it can be theorized that, equivalent to the so-called ‘‘Trojan horse mechanism’’ of infection (MV-mediated spread of certain infections, e.g. HIV or prions) [20], TDMVs may act as efficient delivery vehicles that aid tumor spread by paracrine diffusion of ‘‘malignant traits’’ throughout the body of cancer patients, thus, corresponding to oncosomes (carrying oncogenic factors) [30] we coined them ‘‘metastasomes’’.

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Fig. 1. The metastasome model: (1) a symbolic primary tumor (tumor organ) comprised of cancer cells and various types of the stromal cells including fibroblasts, myofibroblasts, endothelial cells, adipocytes, and BMDCs (mesenchymal stem cells, macrophage, and other immune cells). The MV-mediated interplays between cancer and the surrounding ‘‘reactive’’ stromal cells are fueled by several hallmarks, including oncogenic mutations and microenvironmental acidification. (2) Mechanistic models for metastasomes: (A) Tumor-organ-training (TOTr): other sites become conditioned by metastasomes to serve as hospitable hosts for the alien DTCs, (B) Tumor-organ-targeting (TOTa): TDMVs contribute to the propagation of the transformed phenotype upon the normal competent cells in other sites, and the integrative model suggesting that the organ conditioning functions of TDMVs may foster changes in the normal stromal background of other sites in a way that gauge transformed phenotype upon the normal resident cell lineages (C) or derail the carefully controlled architectures surrounded ‘‘occult tumors’’ (D1), e.g. by stimulation of angiogenesis (D2), allowing their further growth into frank lesions. Abbreviations: CTC (circulating tumor cell), DTC (disseminated tumor cell), BMDCs (bone-marrow derived cells), TDMVs (tumor-derived microvesicles).

Two models for tumor spread via metastasomes The metastasomes carrying ‘‘malignant traits’’ may spread through the circulation of cancer patients and functionally affect all over. However, certain organs are intrinsically prone (congenital soils) or preordained for growing of the secondary lesions [2]. Two models can be proposed for such crusade of tumor metastasis: the Tumor-Organ-Training (TOTr) model and the Tumor-Organ-Targeting (TOTa) model. Tumor-organ-training (TOTr) model The stromal backgrounds of other sites may become conditioned by TDMVs for growth of the disseminated tumor cells (DTCs), formation of a ‘‘pre-metastatic niche’’ [7] (Fig. 1A). This scenario is supported by the observation that ‘‘melanoma exosomal messenger system’’ conditions mouse lymph nodes for melanoma metastasis [51]. Melanoma exosomes (collected from the 48 h cultured mouse B16 melanoma cells) induce the expression of a set of metastatic genes (e.g. stabilin-1, integrin aV, and some extracellular matrix and angiogenic factors) in remote (from the injection site) sentinel lymph nodes of C57/BL6 mice to assist melanoma cells recruitment, trapping, and growth [52]. Attending in the training of lymph node and lung, pancreatic cancer cell-derived exosomes can also facilitate metastasis of the even poorly metastatic ASMLkd cells in rat [12]. In addition, subsets of metastasis promoting MVs with stem cell properties have been capable of

inducing the pre-metastatic niche formation (via angiogenesis) and lung metastasis in SCID mice. These MVs contain mRNAs of growth and angiogenic factors (e.g. VEGF, FGF2, angiopoietin1, ephrin-A3, MMP2, and MMP9) and some miRNAs (miR-200c, miR-92, miR-141, miR-29a, miR-650, and miR-151) which had previously been described in tumor progression, metastasis, and poor prognosis in various cancers [53]. Notably, it was earlier documented that factors secreted by metastatic cancer cells induce the expression of fibronectin (by fibroblasts) in the target organs to enhance the recruitment of BMDCs and training a pre-metastatic niche [54]. Also, according to the ‘‘parallel model’’ of metastasis (please see Ref. [6]), dialogues between primary tumor and secondary organs seem credible and it appears as if without such collaboration it would be unlikely for some secondary tumors to grow as detectable ‘‘early metastases’’. Thus, the aforesaid studies [12,52,53] strongly suggest that TDMVs can serve as vehicles to bring tumors into contact with other sites to aid metastasis formation. Tumor-organ-targeting (TOTa) model Given the tumorigenic factors enveloped in the circulating TDMVs and their chance to be theoretically taken up and translated by every cell throughout the body [24,30], it is tempting to speculate that some recipient normal cells acquire malignant traits, e.g. heightened resistance to apoptosis, increased rate of growth and proliferation, and attainment of transformed phenotypes (Fig. 1B). The strongest evidence is provided by a landmark

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study by Antonyak and colleagues [25]. They have demonstrated that the MDAMB231 and U87 cancer cell derived MVs can confer transformed phenotype (anchorage-independent growth and enhanced survival) upon the normal fibroblastic (NIH 3T3) and epithelial (MCF10A) cells [25]. Likewise, some other reports have implicated TDMVs containing oncogenic factors in the promotion of tumorigenic potentials of the recipient cells. For example, TDMVs containing EGFR can be taken up and elicit EGFR-depended angiogenic responses in the endothelial cells [50]; colorectal cancer cell-derived MVs enriched in the cell cycle-related mRNAs boost the endothelial cell proliferation [48]; and application of the malignant ascites-derived exosomes from ovarian cancer patients to the xenografted-carcinoma mice promotes their tumor growth [47]. Besides, hepatocellular carcinoma (HCC) cell-derived exosomes have been able to promote the anchorage independent growth of the transformed cells through miRNA-mediated targeting of the transforming growth factor b activated kinase 1 (TAK1), a gene involved in the development of HCC [22]. MVs collected from cultures of the different human primary tumor cells or established cell lines have also been able to promote the growth and survival of the same cancer cells when added back to them [24,30,49,55]. These observations suggest that circulating TDMVs may aid tumor spread in other ways, i.e. by loaning transformed phenotypes to the normal cell lineages in other sites [25]. As such, the distribution of ‘‘malignant traits’’ via TDMVs would seem to be a major component of tumor metastasis. From anatomical standpoint, the capillaries are too small (typically 3–8 lM in diameter) to allow the passage and circulation of cancer cells which are 20–30 lM in diameter. They are designed to allow the passage of the highly deformable red blood cells (about 7 lM in size) and WBCs which are smaller than many cancer cells [56]. However, the small sizes of MVs [57] may allow their voyage and interaction either with the local stroma or everywhere throughout the body of cancer patients. When portrayed in this way, the metastasomes encompassing ‘‘malignant traits’’ might be considered as ‘‘seeds’’ that migrate and found metastases. Melanoma exosomes have also been resembled to ‘‘seeds’’ and the sentinel lymph nodes to ‘‘soils’’ in the ‘‘melanoma exosome messenger system’’ [52]. This way, the metastases would also seem to be ‘‘true parallel’’ and de novo tumors. Apparently, our TOTa model would basically seem to be similar to the genometastasis model which states that the cell-free DNAs (cf-DNAs) originated from primary tumors and released into the circulation of cancer patients may become taken up by the normal cells in other sites, leading to their transformation and, hence, the development of ‘‘second primary tumors’’ [58]. This theory which had been a resurrection of debate among physicians in the nineteenth century has still kept its space in the field [59,60]. However, it has neither been able to explain the tropism of metastases, nor the morphological similarities of metastases to the primary tumors [59]. Recently, some evidence supports the concept of TDMVsmediated horizontal transfer of (oncogenic) DNAs to normal cells and their potential to induce cell transformation [60–62]. This notion which is corresponding to our TOTa model may not only rationalize the organ tropism of tumor metastasis (potential diverse MV-uptake efficiencies by each organ, please see also the matchmaking model in this article), but it may also provide some descriptions for the morphological similarities observed between metastases and the primary tumors [59] (encompassing a large number of signaling molecules, TDMVs may deliver/dictate tumor-specific ‘‘malignant phenotypes’’ upon the target sites). Towards an integrative model The two models described above may coincide in some ways. It is well-known that alterations in the matricellular composition and structures can switch the stem cell differentiation [63]. Malig-

nancy is also accompanied by substantial alterations in the surrounding stroma [4]. Abnormal stroma can yet induce tumorigenic growth and genomic instability at normal cells and; indeed, reverting the stromal abnormality can reverse the neoplastic phenotype of even the mutation-bearing cancer cells [4,64]. Therefore, in a capable context, TDMVs may interfere with the normal stromal backgrounds of other sites (TOTr) in a way that gauges neoplastic phenotype (TOTa) onto their normal resident (e.g. stem) cells (Fig. 1C). In this situation, the metastasomes may function as double-edged metastasis-driving swords. In addition and/or alternatively, it is relevant to conceive that the organ conditioning function of TDMVs in other sites (TOTr) may derail the carefully controlled architectures surrounded the ‘‘occult tumors’’ (if existed in other sites of the cancer patients), thus allowing their further growths into frank lesions (Fig. 1D). Occult tumors are those at the very earliest stages which have been discovered through microscopic analysis of the autopsies taken from died individuals of unrelated reasons, that is, they have also been considered as ‘‘cancers without disease’’ [64,65]. These malignant, but unnoticed by the hosts, lesions have been common in breast, prostate, and some other studied organs (reviewed in Ref. [64]). The adjacent normal tissues, serving as intrinsic barriers for the recruitment of new blood supplies by these lesions, have been described as the stabilizing effect behind their dormant state [65]. They may thus switch into the lethal expanding mass of tumors if overcome the angiogenic barrier. Interestingly, angiogenic factors have frequently been discovered in TDMVs and it seems that stimulation of angiogenesis is a common ability by which they foster pre-metastatic niche (TOTr) [50,51,53,66]. For example, cancer cell-derived MVs enriched in the angiogenic mRNAs/proteins have been capable of stimulating the angiogenic pathways, formation of the endothelial spheroids and sprouts, and angiogenesis [24,50,51,53,67]. RNA molecules: body language and metastasis matchmakers Among various cargos packaged in the secretory MVs, the RNA molecules sound special in the MVs-mediated remote cell–cell communications. Thousands of RNA transcripts, including functional mRNA, miRNA, and some other types of non-coding RNAs have been identified in the secretory MVs [17,23,24,68]. The RNase treatments of TDMVs have also significantly reduced their biological effects [20,53]. Remarkably, the MVs-mediated mother-infant transfer of mRNA and miRNAs through breast feeding may play roles, respectively, in the compensation of the infant’s genetic defect [69] and maturation of its immune system [15]. Recently, it has even come to light that some plant/food-derived miRNAs, likely transferred via MVs, participate in the regulation of mammalian genes, a cross-kingdom gene regulatory system [70]. Thus, the RNA molecules possess high ranks in the biological functions attributed to the secretory MVs. From a systems biology standpoint, the presence of a common body language functionally bridging different tissue and organs via circulation (like hormones) to establish a systemic homeostasis in multicellular organisms seems credible. Such language may need to have widespread data: (i) accessibility; (ii) portability; and (iii) translatability. The microvesicular RNAs, particularly miRNAs [71,72], may properly warrant these means. As an example, the mRNA molecules can be translated into multiple copies of proteins in the recipient cells; thus, the exchange of mRNA molecules between cells must be more effective than the corresponding proteins [23]. Most interesting is a recently emerged concept, the competitive endogenous RNA (ceRNA) dialect, describing the communication of all types of RNA transcripts through their regulatory elements called ‘‘microRNA response elements’’ (MREs). Thus, owning alphabetical means in this RNA language along with their

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cific ‘‘contents’’ (e.g. RNA profile) with the ability to confer malignant traits onto certain sites. Even expression profiles of different regions of the same renal-cell tumor have been controversial in predicting the disease prognosis [46]. Thus, the pre-existing tissue/cellular conditions (the type and genetic backgrounds of cancer initiating and/or stem cells) may dictate the tropism of metastasomes. Interestingly, exosomes derived from two different ovarian cancer cell lines have been able to activate diverse signaling pathways in the same adipose tissue-derived mesenchymal stem cell [81]. It will be also interesting to examine the tropic effects of MVs derived from same/various cancer-types onto the cell lineages from same/various tissue-types. On the second hand, the ‘‘contexts’’ of the secondary organs may also play substantial roles in the metastatic tropism. For instance, tissue specific subcellular RNA localization and repertoire, which would likely interact with the RNA profile of the primary tumors (via metastasomes), may play complementary roles in the process (note that after export from nucleus, the mRNA molecules undergo precise and active sorting and localization process within cytoplasm [82] and localization of the mRNAs in the right place at the right time is crucial for defining cell shape and polarization as well as their responses to local cues [83]). Furthermore, many miRNAs act in a contextdependent manner. For example, in ovarian, thyroid, and oral squamous cell carcinoma, miR-125b plays as tumor suppressor via inhibition of cell proliferation and cell cycle progression, while in the neuroblastoma cells it suppresses apoptosis and in prostatic cancer cells enhances cell proliferation and invasion (reviewed in Ref. [77]). Such controversial mean has also been noticed for miR-181a, miR-181c, and miR-220 in various cancer types [77].

combinatorial mode of function, miRNAs mediate a broad regulatory network across the transcriptome [73]. It can be conferred that of the cargos being transferred via MVs, at least the MRE containing RNAs (as sponges) and miRNAs (as alphabets; please see Ref. [73]) may affect the free miRNA pool and ceRNA language of the recipient cells, exerting widespread influences onto their physiology. It is noteworthy that some miRNA-mediated silencing machineries are also localized at the endosomal compartments (e.g. RISC complex in MVBs), where are the main site of the traffic routes between cellular exterior environment and the internal organelles [74], and they have close collaborations with the RNA granules [75]. Mother nature might have established such an organization to: (i) make use of the oldest biological (RNA) dialect [76] through this critical cellular gateway for fine-tuning and remote bridging the physiological routes in the multicellular organisms (note that RNA granules are also considered as remnants of the ancient RNA language [75]), (ii) support the responsiveness of post-transcriptional control to the environmental signals, and (iii) promote the specificity and kinetic efficiency of the reactions [74]. Thus, circulating (microvesicular) RNAs may serve as efficient tools, like hormones [23,77], by which a remote organ-organ communication route can be established in multicellular organisms. Putting the metastasome model in this context, microvesicular RNAs may play matchmaking roles between primary tumors and the secondary organs, hence, hold essential parts in metastatic seeding and tropism. On one hand, the little overlaps between lung and bone metastases signatures of breast tumors [78,79], along with the coherence of different tumor types in bone metastasis signature [80], suggest that TDMVs may contain tumor (tissue)-spe-

1. A Primary Tumor

Primary tumor with extensively unbalanced homeostasis (e.g. neoplastic ceRNA language)

2. A Target Organ

3. The Possible Outcomes

Neoplastic

Normal

Normal site with balanced homeostasis (e.g. normal ceRNA language)

Neoplastic

Considerably deviated homeostasis

A

Normal

Slightly deviated homeostasis

Balanced homeostasis

B

C

Fig. 2. Tumor-organ matchmaking model; once a tumor developed in a tissue, its normal homeostasis becomes transformed; hence, the TDMVs being released (from the tumor organ; Fig. 1) into the circulation contain neoplastic signatures likely unique to/representative of that specific tumor with the potentials to affect other organs at various extents. If we consider the homeostasis of every organ as a swing game which is in a balanced state in the normal condition, the interaction between a tumor/tissue-specific (unbalanced) expression signature (e.g. neoplastic ceRNA language; via metastasomes) and those (balanced) of the other areas (their tissue/cell type-specific RNA stockrooms) would result in certain extent of deviation from their balanced state in each site (A, B, or C). For example, a certain primary tumor may prompt extensive deviations onto the homeostasis of only definite organs (A; e.g. prostatic cancer mostly metastasizes to bone), while almost no or ignorable effects onto other organs (C; e.g. prostatic cancer rarely diffuses to lung). On the other hand, a secondary organ would be competent to metastatic growth driven from certain tumors (e.g. liver is mostly colonized by the gastrointestinal tract cancer). A tumor (e.g. breast) may exhibit different signatures in terms of metastases to diverse organs (e.g. bone and lung). On the contrary, various tumors may show similar signatures respecting the metastasis to the same organ (bone). Conceivably, in a tissue/organ-dependent manner, a primary tumor may exert a broad range of functional influences (via TDMVs) onto other sites (through diverse mechanisms; Fig. 1, each yet likely with distinct outcome), ranged from ‘‘early metastasis’’ (A; possibly those aggressive tumors with detectable metastases at or just after the disease diagnosis, e.g. pancreatic cancer), predisposition to metastatic growth over time (B; likely those tumors with late metastases, e.g. breast and prostatic cancers), up to perhaps ignorable or even tumor suppressive effects (C). This tumor-organ interacting model which is corresponding to the seed-soil hypothesis may properly explain the metastasis seeding and tropism.

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Consequently, the metastasomes (containing miRNAs) may not necessarily render same effects onto other sites as those in the original tumor tissues. In some cancers also clinically explosive metastatic growths soon after resection of the primary tumors have been observed [84,85], suggesting the presence of negative impacts from primary tumors onto the previously initiated micrometastases [5]. Altogether, it can be envisaged that the interactions between tumor tissue-specific RNA signature (via metastasomes) and the exclusive RNA stockrooms in other sites may settle a unique outcome in each organ which we have illustrated as swing game in Fig. 2. Accordingly, depending on the type/invasiveness of the primary tumors, the derived metastasomes may drive deviations from the balanced homeostasis of a specific target organ at various extents. This notion can also be considered for different organs being targeted by the same primary tumor (Fig. 2). Comparison of the expression signatures of the paired primary tumor (or TDMVs)/(normal or metastatic) target tissue, using experimental or in silico studies, may give clues about every possible correlation between them and the feasibility of this ‘‘tumor-organ-matchmaking’’ model. Overall, beyond target-ligand interaction (e.g. via tetraspanins [12]) and various MV-uptake efficacies by each organ, metastatic seeding and tropism may be determined by both ‘‘content’’ of the metastasomes and ‘‘context’’ of the target organs, a notion in coherence with the seed-soil hypothesis.

Conclusion and perspectives As outlined in this supplementary model, ‘‘malignancy’’ may be spread throughout the body of cancer patients via circulating TDMVs. It might be more realistic to incorporate a systems biology approach and corresponding to its definition, consider metastasis as the ‘‘disease transfer’’ [1] which might be attained through diverse ways. Thus, ‘‘cell migration’’ as the only/vital component that must be accomplished for fulfillment of the process is likely a stringent outlook. Particularly, the organization and structure of the cells become fundamentally transformed in the neoplastic development and cancer is a disease of tissue, not cell [4]. Moreover, we now know that even in some normal circumstances, cells lose their typical cellularities during the differentiation processes to carry out their specialized functions (e.g. enucleated red blood cells, multiploid hepatocytes and syncytial myocytes). Therefore, the spread of ‘‘malignant traits’’ may appear as a principal, but less addressed, component of tumor metastasis without which (TOTr) even the migration and seeding of tumor cell may not lead to successful metastatic outgrowths. It should be noted that, although we have featured the RNA molecules as matchmakers, this does not mean that we are intended to disapprove the importance of other microvesicular or non-microvesicular components derived from primary tumors. For example, cytokines, proteins, and specially (oncogenic) DNAs identified in the TDMVs [17,86,87] may play their own parts; that is actually, a complex signature (not only the RNA profiles) might be on the playground. Interestingly, an intelligent program (ExoCarta: www.exocarta.org) has recently been developed for construction of a compendium of the proteins and RNA molecules identified in the exosomes [88]. Besides, the transfer of functional miRNAs through gap-junctions has emerged as an effective cell–cell communication route [89]. Overall, TDMVs and their RNA contents hold significant promises for the achievement of effective remote interplays. It is also important to keep in mind that there is currently no consensus on the definition and classification of the circulating microvesicles [90]. For this reason, it is hard to postulate that which types of TDMVs may have the major contribution in the metastasome model (though a combination of diverse types of

them might be on the playground). Technical challenges in dealing with the isolation of MV constructs as well as the presence of various types of MVs with similar sizes, have made it difficult in experimental procedures with their sorting [16]. Even the current sorting methodologies which are primarily based on the immune-affinity enrichment procedures face with some drawbacks. For example, the cleavage of EpCAM (an exosome surface marker) by circulating proteases in breast cancer patients has been troublesome [91]. Confocal microscopy has also the detection limitation (200 nm) which makes the detection and tracking of the smaller vesicles such as exosomes difficult [16]. Hence, future collaborations for defining and classifying MVs and designing methodologies towards their accurate isolation/sorting as well as the model systems for unraveling their tumorigenic potentials may provide fresh insights in the field. From clinical standpoints, it might be interesting to study the potentials of metastasomes in the synchronous vs. metachronous, early vs. late, or local vs. distant metastases. Which model (TOTr or TOTa) might better describe the tumors with late (e.g. breast and prostate) or very early (e.g. lung and pancreatic) metastases? What contribution may the metastasomes have in the growing field of the cancer stem cell? Future studies may give clues about these conundrums. In any case, what attracted our attention with the metastasomes regarding their ostensibly ‘‘toxic effects’’ onto other sites is the possibility of revisiting the oldest metastatic model which had been explained by Hippocrates (500 year BC), the ‘‘humoral theory’’ [2]. However, the intention of this perspective is not to disapprove the prevailing models of metastasis, but to expand the field and pursue strategies for incorporating new ideas into the existing body of knowledge. We hope our models improve scientists’ understanding of tumor metastasis. Conflict of interest statement None declared. Acknowledgements This study was supported by Funds from the University of ‘‘G. D’Annunzio’’ of Chieti-Pescara, Italy and by the Consorzio Interuniversitario Nazionale per la Bio-Oncologia (CINBO), Italy. References [1] Talmadge JE. AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer Res 2010;70:5649–69. [2] Nguyen MJ. Genetic determinants of cancer metastasis. Nat Rev Genet 2007;8:341–52. [3] Valastyan S, Weinberg RA. Tumor metastasis: molecular insights and evolving paradigms. Cell 2011;147:275–92. [4] Bissell MJ. Putting tumours in context. Nat Rev Cancer 2001;1:46–54. [5] Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science 2011;331:1559–64. [6] Klein CA. Parallel progression of primary tumours and metastases. Nature Rev. Cancer 2009;9:302–12. [7] Peinado H, Lavotshkin S, Lyden D. The secreted factors responsible for premetastatic niche formation: old sayings and new thoughts. Semin Cancer Biol 2011;21:139–46. [8] Joyce PJ. Microenvironmental regulation of metastasis. Nat Rev Cancer 2009;9:239–52. [9] Bobrie A, Colombo M, Raposo G, Thery C. Exosome secretion: molecular mechanisms and roles in immune responses. Traffic 2011. [10] Muralidharan-Chari V, Clancy JW, Sedgwick A, D’Souza-Schorey C. Microvesicles: mediators of extracellular communication during cancer progression. J Cell Sci 2010;123:1603–11. [11] Fabiani R, Johansson L, Lundkvist O, Ronquist G. Enhanced recruitment of motile spermatozoa by prostasome inclusion in swim-up medium. Hum Reprod 1994;9:1485–9. [12] Jung T, Castellana D, Klingbeil P, Cuesta Hernandez I, Vitacolonna M, Orlicky DJ. CD44v6 dependence of premetastatic niche preparation by exosomes. Neoplasia 2009;11:1093–105.

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