In the process of cancer progression, cancer cells acquire essential biological properties, including angiogenesis, host immune system modulation, and metastasis. Additionally, the development of drug resistance is a serious challenge in cancer treatment. Recent research has revealed that cancer-derived EVs are partially responsible for these biological processes. The following sections will review EV-mediated angiogenesis, immune system modulation, metastasis, and drug resistance in RCC (Figure 1).
Figure 1. The major roles of EVs in RCC progression. EVs secreted from RCC cells contribute to cancer progression through angiogenesis, host immune modulation, metastasis, and drug resistance. MVB, multivesicular body.
2. Angiogenesis in RCC and EVs
ccRCC is the most common type of RCC and typically presents with hypervascular tumors. ccRCC is genetically characterized by the inactivation of the von Hippel–Lindau (VHL) gene. The VHL gene is located on chromosome 3p25 and regulates hypoxia response pathways 
. The VHL protein (pVHL) is a component of the E3 ubiquitin ligase complex that targets hypoxia-inducible factor 1a and 2a (HIF-1a and HIF-2a) for polyubiquitylation and subsequent proteasomal degradation. HIF-1a and HIF-2a are transcription factors that bind to the hypoxia response element (HRE), activating a myriad of genes involved in hypoxia adaptation 
. In most cases of ccRCC, pVHL is inactivated, resulting in the accumulation of HIF-1a and HIF-2a and continual activation of HIF target genes, despite normal oxygen levels 
. One of the most common targets of HIF1 is vascular endothelial growth factor (VEGF), which induces angiogenesis. Therefore, tyrosine kinase inhibitors (TKIs), which inhibit the VEGF/VEGF receptor signal pathway, are frequently used for anti-angiogenic therapy in clinical practice. However, the response rate ranges from 10% to 31%, indicating that a substantial proportion of ccRCC cases show primary resistance to TKIs 
. In addition to VHL loss of function, miRNAs reportedly contribute to VHL inactivation. EVs secreted from RCC contain miR-92a, which targets VHL mRNA. Valera et al. found that miR-92a expression levels are inversely correlated with VHL mRNA levels in ccRCC tissue and concluded that miR-92a regulates the expression level of VHL through mRNA silencing 
Besides the classical VHL/HIF/VEGF pathway, recent studies have revealed that ccRCC secretes EVs that promote angiogenesis. Horie et al. demonstrated that exosomal carbonic anhydrase 9 (CA Ⅸ) released from RCC cell lines promotes angiogenesis in vitro and that the levels of CA Ⅸ in exosomes are elevated by hypoxic treatment. CA Ⅸ expression is regulated by HIF1 in response to hypoxia 
. Grange et al. reported that RCC cells expressing CD105, a popular stemness marker, release microvesicles that induce angiogenesis through the function of miRNA cargo, including miR-92a 
. The miRNA cargo in RCC-derived EVs induces angiogenesis by silencing mRNAs other than of the VHL gene. Hou et al. reported that exosomes secreted from ccRCC cells promote angiogenesis through miR-27a, which targets secreted frizzled-related protein 1 (SFRP1) mRNA 
, and increased expression of SFRP1 induces angiogenesis 
. In addition to these studies, researchers found that EVs secreted from bone metastatic RCC facilitate angiogenesis and endothelial gap formation in bone marrow in a time-dependent manner and that the angiogenesis observed in the study was partially mediated by aminopeptidase N (APN) located in the EV plasma membrane 
RCC-derived EVs partially mediate angiogenesis, which is one of the most distinct characteristics of RCC. Given that inhibiting angiogenesis using TKIs is a mainstay of RCC treatment, EVs involved in angiogenesis could be a promising treatment target.
3. The Role of EVs in Modulation of the Host Immune System
In the 1950s, the concept of cancer immunosurveillance was proposed by M.F. Burnet. He suggested that the human immune system recognizes cancer cells as non-self due to cancer antigens displayed on the surfaces of antigen-presenting cells (APCs), contributing to the suppression of tumor development 
. Classically, APCs include dendritic cells, macrophages, Langerhans cells, and B cells. In 1996, Raposo et al. first described B cells releasing EVs containing MHC class Ⅱ 
. Using immunoelectron microscopy, they observed that intraluminal vesicles containing MHC class Ⅱ were released upon fusion with the plasma membrane. Moreover, they found that EVs released from human and murine B lymphocytes induced an antigen-specific MHC class Ⅱ restricted T cell response 
. More recently, Schreiber RD et al. developed the concept of cancer immunoediting, which refers to the process in which immunity promotes cancer progression as well as eradicates cancer cells 
. Cancer immunoediting consists of three processes: elimination, equilibrium, and escape. In the elimination phase, immunity suppresses nascent tumor growth. In the equilibrium phase, tumor cells start to evade antitumor immunity due to the lower immunogenicity induced by accumulating mutations. However, in this state, tumor outgrowth does not occur because the balance between tumor growth and tumor elimination is maintained by immune cells 
. In the escape phase, tumor cells can evade immune surveillance. In this phase, intercellular communications between tumor and immune cells that are mediated by EVs play critical roles 
. There are some studies describing RCC-derived EVs contributing to immune escape. Grange et al. stated that EVs secreted from RCC cells, especially CD105+
RCC cells, inhibit DC maturation and the T cell immune response through the function of HLA g 
. Moreover, they demonstrated that HLA g blockade leads to the restoration of DC differentiation. Macrophage polarization is one of the critical changes in the tumor microenvironment that favors cancer progression 
. RCC cells release EVs, promoting a shift in the macrophage subpopulation from M1 macrophages, which suppress tumor proliferation, to M2 macrophages, which suppress the antitumor immune response 
. It is widely known that tumor cells express PD-L1, which binds to PD-1 on T cell surfaces, inducing immune suppression and leading to cancer progression 
. Intriguingly, Chen et al. first discovered in 2018 that EVs secreted from malignant melanoma carry PD-L1 on their membrane surfaces 
. Recent studies have demonstrated that various cancer types secrete EVs with PD-L1 on their surfaces 
. To date, there are no studies demonstrating the function of PD-L1 in RCC-derived EVs. However, Qin et al. found that miR-224-5p in EVs contributes to the stability of PD-L1 in RCC cells, suggesting that RCC-derived EVs can induce immune evasion 
. Xu et al. showed that RCC-derived EVs induce immunosuppression through the inhibition of T cell proliferation. They found that Fas ligands on the surfaces of RCC-derived EVs are responsible for activating the apoptotic pathway in T cells 
. Diao et al. described the potential effect of RCC-derived EVs on myeloid-derived suppressor cells (MDSCs), which have immune-suppressive effects on adaptive immune responses in cancer. They demonstrated that heat shock protein 70 (Hsp70) packed in EVs secreted from RenCa cells, a murine renal cancer cell line, mediate immune suppression by triggering Stat3 phosphorylation in MDSC 
. Together, RCC cells secrete EVs that cause immune suppression, suggesting that EVs are possibly involved in the process underlying the acquisition of resistance to immunotherapy. It has been reported that EVs secreted from melanoma cells expressing PD-L1 (PD-L1+
EVs) induce immunosuppression by targeting PD1+
T cells. PD1+
T cells secrete IFN-g, which induces upregulation of PD-L1 levels on EVs 
. This result suggests that PD-L1+
EVs could impact the efficacy of ICI. There are no published studies describing the impact of PD-L1+
EVs secreted from RCC on the efficacy of ICI treatments. However, considering that immunotherapy has been a common treatment option for metastatic RCC, further research on cancer immunoediting is exceptionally significant in RCC. Thus, elucidation of the roles played by RCC-derived EVs in this process will provide new insights into the development of novel therapeutic strategies.
4. Roles of EVs in RCC Metastasis
Given the poor clinical outcomes of mRCC patients, deciphering the mechanism underlying metastasis formation is of great significance 
It is well known that the formation of a premetastatic niche, a microenvironment favoring circulating tumor cell attachment, colonization, and growth, is a crucial step in metastasis formation 
. In this process, EVs secreted from metastatic cancer cells play essential roles in communicating with stromal cells at the metastatic site 
. Mesenchymal stem cells (MSCs) are also a key component of the metastatic niche 
. Lindoso et al. found that cancer stem cells (CSC) of RCC release EVs, inducing phenotypic changes in MSCs and resulting in tumor progression 
The process of tumor cell dissemination from the primary site to distant organs involves five steps: local invasion, intravasation, surviving in circulation, extravasation, and colonization at the metastatic site 
. In the initial step of metastasis, tumor cells detach from the primary site and invade surrounding tissue. This process involves the epithelial–mesenchymal transition (EMT) 
. Jin et al. found that RCC-derived EVs promote cancer cell migration, invasion, and lung metastasis by shuttling metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a lncRNA that is well known to facilitate cancer metastasis 
. Wang et al. demonstrated that CSCs of ccRCC release EVs that induce EMT via the function of miR-19b-3p, with the potential to facilitate lung metastasis 
. Intravasation refers to the step during which tumor cells enter the circulation by crossing the endothelium. Extravasation is another key process in cancer metastasis, where circulating tumor cells break through the barriers of endothelial cells to migrate into stroma at metastatic sites 
. The enhanced vascular permeability caused by a damaged vascular endothelium is involved in these processes 
. Jingushi et al. performed proteomic analysis of EVs released from RCC tissue, identifying azurocidin 1 (AZU1) as a functional protein that is comparatively enriched in EVs from RCC tissue than from neighboring normal tissue. In their study, RCC-derived EVs induced an enhancement in vascular permeability through the function of AZU1 
. Xuan et al. found decreased levels of miR-549a in the EVs secreted from TKI-resistant RCC cells. miR-549a in EVs regulates HIF-1a expression in vascular endothelial cells, contributing to metastasis by promoting angiogenesis and enhancing vascular permeability 
In summary, recent research has revealed that RCC-derived EVs are involved in every step of cancer metastasis.
5. Roles of EVs in RCC Drug Resistance
In the last two decades, newly developed treatment options for RCC, including targeted therapy and immunotherapy, have contributed to improved clinical outcomes 
. Therefore, it is critically important to clarify the molecular basis for resistance to these drugs in RCC to contribute to the development of novel biomarkers of responses and the identification of therapeutic targets.
Some researchers have reported differences in EV cargo between resistant and sensitive cells, suggesting that RCC-derived EVs could play roles in drug resistance 
. Xuan et al. demonstrated that there is decreased miR-549a cargo of the EVs secreted from TKI-resistant RCC cells compared with those from TKI-naïve RCC cells 
. Moreover, EVs secreted from resistant RCC cells carry biomolecules contributing to cell survival and proliferation during anticancer drug treatments. Zhang et al. indicated that EVs isolated from the culture supernatant of drug-resistant RCC cells contain abundant STAT3 compared with EVs secreted from sensitive cells. They concluded that resistant RCC cells generate EVs that promote cell survival and proliferation in the presence of anticancer drugs by activating the mTOR–ERK–STAT–NF-kb signaling pathway 
. Qu et al. demonstrated that EVs secreted from sunitinib-resistant RCC cells disseminated sunitinib resistance to sensitive cells through the function of lncRNA, which activates AXL and c-MET signals via competitive binding to miR-34/miR-449 
. According to a recent study by Pan et al., RCC-derived EVs contribute to sunitinib resistance by transferring insulin growth factor-like family member 2 antisense 1 (IGFL2-AS1), a novel lncRNA inducing enhanced autophagy 
. Together, EVs transfer drug resistance from resistant cells to sensitive cells by carrying biomolecules that allow sensitive cells to survive and proliferate during antitumor treatments. This phenomenon is known as horizontal transfer. While the mechanisms of sunitinib resistance induced by RCC-derived EVs have been extensively investigated, the relationship between RCC-derived EVs and resistance to other types of TKI remains unknown. Ishibashi et al. demonstrated in vitro that RCC cells exposed to sunitinib, sorafenib, or pazopanib increased their secretion of interleukin 6, which is thought to play a key role in the development of TKI resistance 
. Their findings imply that these drugs may develop TKI cross-resistance. Therefore, future research should concentrate on EV-related mechanisms of TKI cross-resistance.
Compared to the classical immunotherapy era, the current standard care for advanced or metastatic RCC, such as TKI, ICI, and their combination, has shown better clinical outcomes, with a response rate of 42–71% 
. However, a substantial proportion of patients show primary or acquired resistance to these treatments. Therefore, deciphering the mechanisms underlying drug resistance to current immunotherapy is paramount for improving clinical outcomes.
6. Potential of EV-Targeting Treatment
EVs are potential cancer therapeutic targets due to their involvement in multiple pathways that lead to cancer progression. Several substances that prevent EV formation and release have been discovered in recent studies 
. Calpeptin, manumycin A, and Y27632 are compounds that reportedly inhibit EV formation. Calpeptin targets calpains, which are calcium-dependent cysteine proteases that are primarily responsible for microvesicle formation. Accordingly, calpeptin reduces the volume of microvesicles that cells secrete 
. Manumycin A inhibits small GTPases from the Ras superfamily that are involved in exosome production and release. Ras regulates multiple cell functions, such as cell differentiation, cell proliferation, adhesion, migration, cytoskeletal integrity, apoptosis, and exosome release. Manumycin A, in combination with GW4869, which is known as an nSMase inhibitor, resulted in a further reduction in exosome release 
. Y27632 decreases microvesicle production and release by inhibiting a Rho-associated protein kinase (ROCK) that mediates signals acting on the cytoskeleton. These agents are proven to reduce EV secretion in prostate cancer, breast cancer, and ovarian cancer cells 
. To date, no published studies are reporting on the inhibition of EV secretion from RCC cells by these agents. In addition, some compounds suppress EV release by affecting lipid metabolism 
. Since these agents affect physiological cellular functions, further investigations to determine potential adverse effects are essential in relation to clinical application.