Effects of exosomes on pre-metastatic niche formation in tumors

Tumor metastasis usually indicates a poor prognosis and is the leading cause of cancer-related death [1]. Thus, meaningful research is being done on how to prevent tumor metastasis. In recent years, the concept of a pre-metastatic niche has provided new ideas to prevent tumor metastasis. The pre-metastatic niche refers to the microenvironment, which is well prepared for tumor cells to colonize in and disseminate to distant organ sites. Lyden firstly proposed the idea of the pre-metastatic niche [2]. The significance of the pre-metastatic niche has received increasing attention in recent years [3, 4]. Cao summarized the characteristics and four stages of the pre-metastatic niche. The key components of the pre-metastatic niche include tumor-derived secreted factors (TDSFs), extracellular vesicles (EVs), bone marrow-derived cells (BMDCs), suppressive immune cells and host stromal cells [5].

Exosomes are extracellular vesicles with a diameter of 30–150 nm [6‐8]. Exosomes were found in blood, urine, saliva and other bodily fluids [9]. As a key player in intercellular communication, exosomes transmit information through their cargo levels, including proteins, lipids, DNA, messenger RNA and microRNAs [10‐15]. Exosome-mediated intercellular communication mainly occurs in the following three ways: First, the exosome membrane protein can bind to the target cell membrane protein, thereby activating the signal pathway in the target cell. Second, in the extracellular matrix, a protease cleaves the exosome membrane protein, which then binds to receptors on the cell membrane to activate the signaling pathway within the cell. Third, the exosome membrane can directly fuse with the target cell membrane, causing nonselective release of the proteins, mRNA and microRNA of the exosomes [16, 17]. Recently, reports have shown that the formation of a pre-metastatic niche depends on tumor-derived exosomes [2, 12, 18‐20]. The function of exosomes depends on the type of cells from which they are derived [21, 22]. Studies have shown that tumor-derived exosomes are involved in the exchange of genetic information between tumor cells and basal cells, resulting in the production of a large number of new blood vessels, which promotes tumor growth and invasion [23, 24].

This article summarizes the role of exosomes in the pre-metastasis niche, to identify new means of cancer immunotherapy. Screening for biomarkers contained within exosomes can provide diagnostic and prognostic value.

Chronic inflammation is a driving force for tumor development and metastasis. Thus, the local inflammatory microenvironment is one of the basic factors for the formation of a pre-metastatic niche.

Hoshino reported the regulatory effect of integrins (ITGs) on the proinflammatory factor S100. They identified differentially expressed genes (DEGs) by RNA sequencing in Kupffer cells (KCs) treated with different cell-derived exosomes. The results showed that S100A8 and S100P were upregulated more than four-fold after treatment with BxPC-3-LiT exosomes compared to exosomes produced by BxPC-3-LiT ITGβ5KD (BxPC-3-LiT cells with knocked down ITGβ5 expression). After treatment of WI-38 fibroblasts with 4175-LuT exosomes, S100A4, S100A6, S100A10, S100A11, S100A13 and S100A16 were upregulated five-fold compared to the 4175-LuT ITGβ4KD (4175-LuT cells with knocked down ITGβ4 expression) exosomes. Thus, exosome integrins could upregulate the expression of proinflammatory S100 molecules in the distant tissue microenvironment. How do tumor-derived exosomes upregulate the expression of proinflammatory S100? The activation of Src, and its subsequent phosphorylation, may be the main pathway [25].

The local inflammatory microenvironment can induce tumor cells to produce tumor-derived secreted factors (TDSFs), such as vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-α), transforming growth factor-β (TGF-β) and interleukin (IL). These TDSFs in turn affect myeloid cells through paracrine means to stimulate their migration to future pre-metastatic niche formation sites [26]. Under the stimulation of TDSFs, host stromal cells in the pre-metastatic niche may upregulate the expression of inflammatory factors. BMDCs or immune cells are recruited to the pre-metastatic niche and accelerate the secretion of inflammatory factors. In addition, tumor-derived exosomes carry inflammatory factors into the bloodstream, which in turn reach the pre-metastatic niche. Through the above possible ways, an inflammatory microenvironment favorable for tumors is finally formed in the pre-metastatic niche.

The microenvironment prior to metastasis can increase angiogenesis and vascular permeability to facilitate metastasis. Studies have reported that exosomes released from hypoxic tumors are more likely to cause angiogenesis and vascular leakage [50, 51].

Mesenchymal-derived microvesicles activate endothelial cells via Akt phosphorylation and promote the formation of a metastatic vascular microenvironment [52]. Human kidney cancer stem cell-derived CD105-positive microvesicles can also stimulate angiogenesis, thereby promoting the formation of the pre-metastatic niche [53]. In addition, the exosome surface expresses soluble E-cadherin, which promotes tumor angiogenesis [54]. Furthermore, exosomes can express carbonic anhydrase 9 to promote angiogenesis [55]. In the pre-metastasis niche, new blood vessels not only transport TDSFs and EVs but also help bring circulating tumor cells to secondary sites. Then, exosomes begin to increase vascular permeability.

Peinado found that exosomes from the highly malignant B16-F10 enhanced lung endothelial permeability in mice compared to exosomes from nonmetastatic cell lines [19]. Melanoma-derived exosomes also induce vascular leakage and receptor tyrosine kinase Met to reprogram bone marrow progenitor cells in the niche [19]. It was observed by exosome tracing that 1833-BoT and 4175-LuT exosomes promoted pulmonary vascular leakage 24 h after injection [25]. MiR-105 secreted by metastatic breast cancer cells may disrupt the vascular endothelial barrier in the early metastatic niche, thereby increasing the vascular permeability of distant organs and promoting metastasis [56]. In addition, breast cancer cells can express matrix metalloproteinase (MMP) and cyclooxygenase. These enzymes promote vascular remodeling and increase vascular permeability to accelerate tumor cell metastasis [57].

A recent study showed that exosome secreted by colorectal cancer (CRC) cells was rich in miR-25-3p, which promoted angiogenesis and disrupted the tight junctions of vascular endothelial cells by targeting KLF2 and KLF4 [58]. In addition, the miR-25-3p of CRC cell-derived exosomes significantly induced vascular leakage and CRC metastasis in the liver and lung of mice [58]. Tumor-derived exosomes are considered to be the major drivers of the pre-metastatic niche. The above studies demonstrated that exosomes were involved in angiogenesis and increased vascular permeability in order to facilitate the formation of the pre-metastatic niche. Before the tumor is transferred, a microenvironment suitable for tumor metastasis has been created for tumor metastasis. This is another evidence that tumor-derived exosomes promote tumor metastasis.

If the tumor cells are regarded as seeds and the pre-metastasis niche is regarded as soil, then the exosomes are similar to fertilizers, which can make the barren land fertile and facilitate the colonization of tumor cells [59‐63]. This hypothesis helps us understand exosome-mediated organ-specific metastasis.

Hoshino found that tumor-derived exosomes were important factors in organ-specific metastasis. Tumor-derived exosomes prepare a favorable microenvironment at future metastatic sites, thereby mediating nonrandom transfer patterns. First, future metastatic sites uptake exosomes. Second, exosomes redirect metastatic distribution. Third, ITGs on the surface of exosomes determine organotropism. Quantitative mass spectrometry and western blot analysis as well as qualitative mass spectrometry showed that ITGα6 was present in lung-directed exosomes, while ITGβ5 was present in liver-directed exosomes. These results indicate that the different expression of exosome ITGs is the basis of organotropism. Fourth, different cells absorb tropic exosomes. Lung-tropic 4175 exosomes mainly colocalized with S100A4-positive fibroblasts in the lung. By contrast, pancreatic cancer exosomes derived from BxPC-3-LiT cells mainly fused with Kupffer cells (Fig. 2). These data demonstrate that specific tissue-resident stromal cells differentially uptake tumor exosomes in metastatic target organs. Finally, exosome tropism requires ITGβ4 and ITGβ5. After knocking down ITGβ4 expression in 4175-LuT cells (4175β4KD), a more than threefold reduction in labeled ITGβ4KD exosomes in the lung was observed. Conversely, ITGβ4 overexpression in 1833-BoT exosomes was sufficient to increase exosome uptake in the lung. Similarly, ITGβ5 knockdown in BxPC-3-LiT exosomes decreased liver uptake by sevenfold compared with uptake by control BxPC-3-LiT exosomes. The above results demonstrate that exosome ITGs are responsible for organ-specific metastasis [25].

The above evidence proves that tumor cells prepare the receiving organs by releasing exosomes. Cancer cells spread from the origin to distant organs through blood circulation. The process of dissemination is not random, and cancer cells will preferentially look for specific organs under the guidance of exosomes and build nests there. This kind of destination-seeking behavior involves the interaction of “seeds” and “soil”. Seeds can fertilize the “soil” through exosomes before they reach the soil, consequently preparing for tumor metastasis.

The matrix environment of the pre-metastatic niche is mainly composed of fibroblasts, endothelial cells and extracellular matrix (ECM). Fibroblasts not only produce inflammatory and growth factors but also express fibronectin (FN) and matrix metalloproteinase (MMP) [64]. Moreover, endothelial cells secrete proinflammatory factors S100A8 and S100A9 [65]. A prominent part of the pre-metastasis matrix of liver cancer is hepatic stellate cells, which can promote ECM deposition and upregulate the expression of VEGF, IL-1a and TGF-β [66, 67] . Inflammatory factors produced by stromal cells may contribute to the formation of local inflammatory microenvironments. Growth factors secreted by stromal cells may promote angiogenesis in the pre-metastatic niche. In addition, hypoxia is also an important factor of ECM. Hypoxia can induce the expression of HIF, VEGF and G-CSF, thereby promoting the formation of a pre-metastatic niche [68, 69].

Melanoma-derived exosomes can reprogram human adult dermal fibroblasts (HADF) to cause extracellular acidification [70]. Multiple types of BMDCs promote matrix remodeling in the pre-metastatic niche, and upregulation of fibronectin (FN) [2, 71]. Costa-Silva showed that exosome macrophage migration inhibitory factor (MIF) induces the release of TGF-β from Kupffer cells, which in turn promotes the production of FN in hepatic stellate cells (HSTCs). FN deposits subsequently promote the stagnation of bone marrow-derived macrophages and neutrophils in the liver, completing the formation of the pre-metastatic niche [72]. Exosomes expressing ITGα6β4 and ITGα6β1 co-localize with laminin, and exosomes expressing ITGαvβ5 colocalize with fibronectin [25]. Therefore, we hypothesize that specific exosome integrins may interact with extracellular matrices, and that deposited laminin and fibronectin may help increase the adhesion of extracellular matrices to facilitate colonization of circulating tumor cells.

Tumor-derived exosomes have been reported to contain DNA fragments that provide exosome origin information. Once released into the peripheral cycle, the information contained in DNA fragments may direct the metastatic behavior of circulating tumor cells [73, 74]. Mutant genes were found in serum exosomes from patients with pancreatic cancer. For example, p53 and KRAS mutations have been verified [75]. In addition, DNA fragments with the ability to amplify the c-Myc gene were found in medulloblastoma cell-derived exosomes [76].

In addition, exosome proteins have been identified to be associated with the cell cycle, cell signaling process, cytoskeleton formation, apoptosis, oxidative stress, focal adhesions formation, and cell mobility [77]. Exosome integrins play an important role in the pre-metastatic niche, which has been described in detail above. Studies have shown that osteopontin derived from tumor microbubbles contributes to the colonization of tumor cells and BMDC mobilization [78].

Furthermore, microRNAs associated with metastasis in exosomes have also been reported [79]. Tumor-secreted microRNAs bind to TLR8 in human immune cells, triggering tumor metastasis [80]. Brain astrocyte-derived exosomes mediate PTEN targeting of miRNA-19a, thereby promoting PTEN silencing in tumor cells. Decreased PTEN upregulates the expression of chemokine CCL2, which recruits bone marrow cells in the brain [81]. Exosomes derived from prostate cancer contain differentially expressed genes (DEGs), such as miR-21-5p and miR-139-5p. These microRNAs derived from exosomes cooperate to adjust the pre-metastatic niche [82]. Studies have reported that lncRNAs have important functions for pre-metastatic niches [83]. Multiple studies have shown that lncRNAs can be used as biomarkers for tumor diagnosis [84‐86] . Circular RNA (circRNA) is a closed loop structure, and its expression is more stable and less prone to degradation. Circular RNA has spatiotemporal specificity and is highly conserved among species. Recently, the role of circRNA in exosomes has been reported. In the future, the function of circRNA in the pre-metastatic niche may be gradually excavated.

From the above studies, we conclude that both coding and non-coding RNA derived from exosomes promote the formation of pre-metastatic niches. At the present, the induction of pre-metastatic niche formation by exosome miRNAs has attracted wide attention. In the future, exosome-derived lncRNA and circRNA will also show their prominence.

In recent years, research on finding biomarkers for detecting diseases has grown exponentially. Some studies have used exosomes containing non-coding RNA as potential biomarkers for detecting different malignancies. MiRNAs isolated from tumor-derived exosomes are more stable and therefore considered to be more reliable biomarkers. In addition, exosome-derived lncRNA and circRNA are also fascinating potential biomarkers.

In summary, exosomes play multiple roles in the development of pre-metastatic niches. Exosomes can not only promote the upregulation of inflammatory molecules, induce angiogenesis and infiltration, and regulate immune function in both directions but also determine organotropism metastasis and cause extracellular matrix remodeling [103] (Fig. 3).

Many studies have confirmed that exosomes can promote the formation of pre-metastatic niches. However, the functions, mechanisms, and contribution rates of exosomes remain to be further explored. The problems that need to be solved urgently include 1. What is the difference in effect between tumor-derived exosomes and immune cell- or stromal cell-derived exosomes on the pre-metastatic niche? 2. The composition of exosomes is complex, so what role do these components play? Is there an interaction between the different components? 3. After the primary tumor is surgically removed, does the effect of the exosomes on the pre-metastatic niche terminate? 4. What is the reliability and validity of using exosomes as biomarkers to judge tumor metastasis? 5. How can the theoretical basis of exosomes and pre-metastatic niches be applied to clinical treatment?

To conclude, exosomes play an essential role in the pre-metastatic niche, and further exploration of the function of exosomes in the pre-metastatic niche will help to determine treatments in the early stage of cancer. In addition, circulating exosomes, as biomarkers, have great potential in liquid diagnostics. As exosomes have the biological role of transporting cargo, exosomes may be used as a carrier for targeted drugs to accurately destroy tumor cells [104]. In the future, tumors are expected to become as chronic as diseases such as diabetes. Just as people with diabetes monitor blood glucose with a blood glucose meter, tumor patients will be able to track tumor progression through exosome biomarkers.