Cancer is the global leading cause of death. Early diagnosis is critical for its timely treatment and prognosis. Nevertheless, many cancers lack specific and effective diagnostic markers, resulting in missed treatment opportunities. Therefore, there is an urgent need for more effective and less invasive alternative markers for early diagnosis, individualized treatment strategies, and precise prognostic estimation.

Extracellular vesicles are a variety of membranous vesicles released by cells (Xiao et al., 2019). EVs secretion is mediated by hematopoietic and non-hematopoietic cells, including reticulocytes, B lymphocytes, T cells, epithelial cells, astrocytes, etc. (Laulagnier et al., 2004; Fader et al., 2005; Mignot et al., 2006). Besides, EVs have been detected in most body fluids, including urine, amniotic fluid, blood, serum, saliva, ascites, breast milk, cerebrospinal fluid, and nasal secretions (Keller et al., 2007; Li et al., 2008; Taylor and Gercel-Taylor, 2008). They regulate intercellular communication by transporting their contents. The EVs contents depict the phenotypic state of parental cells. EVs-mediated intercellular communication regulates normal physiological and pathological processes of several diseases, including cancer (Barteneva et al., 2013; Kucharzewska and Belting, 2013; Meckes, 2015).

EVs are found in most body fluids, highly stable with their contents similar to parental cells; therefore, they harbor significant potential as liquid biopsy specimens for various diseases (Rak, 2013; Hornick et al., 2015; Li et al., 2017a). Specifically, cancer-derived EVs may serve as biomarkers for early cancer detection since they carry biomolecules that indicate genetic or signaling alterations in the originating cancer cells (Li et al., 2015; Melo et al., 2015; Tang and Wong, 2015). Studies on the mechanisms by which EVs proteins regulate tumor progression and a summary of their feasibility as tumor markers have reached maturity. Nonetheless, studies on how to achieve the clinical use of EVs proteins in tumor diagnosis as well as prognostic assessment remain largely unexplored. Therefore, this review focuses on the use of EVs proteins in the diagnosis of various cancer types.

Extracellular vesicles are a collective term for tiny vesicles with membrane structures that are actively secreted by cells. EVs were classified into exosomes, microvesicles and apoptotic vesicles depending on the formation process and size. Exosomes are formed by the fusion of multivesicular bodies with cell membranes and are 40–200 nm in diameter; microvesicles are formed by the outgrowth of cell membranes and are 200–2000 nm in diameter; apoptotic vesicles are formed by the atrophy and fragmentation of cells and are 500–2000 nm in diameter (Raposo and Stoorvogel, 2013). EVs are produced inside the cell through the endosomal pathway before being released into the extracellular space (Latifkar et al., 2019; Kalluri and LeBleu, 2020) (Figure 1A). First, the plasma membrane of the donor cell invaginates, forming early endonucleosomes. Thereafter, early endosomes mature into late endonucleosomes. During maturation, their membranes invaginate to form intraluminal vesicles (ILVs). Notably, endonucleosomes with ILVs are usually referred to as multivesicular bodies (MVBs). During the formation of MVB, bioactive molecules (e.g. proteins, mRNA, miRNA, lncRNA, and CircRNA) are packaged into the ILV by the endosomal sorting complex necessary for the transport (ESCRT)-dependent and ESCRT non-dependent pathways (Hessvik and Llorente, 2018). Eventually, ILVs are released into the extracellular space (EVs) when MVBs fuse with the plasma membrane. However, the mechanisms that drive EVs formation and secretion remain largely unknown due to different cell types and their states.

EVs carry various molecular contents hinged on their source and state (Duijvesz et al., 2011) (Figure 1B). For instance, EVs contain proteins associated with their biogenesis, including CD9, CD63, CD81, and TSG101, Alix, and Rab family proteins. All these molecules are routinely used as EVs marker proteins. Moreover, EVs harbor numerous nucleic acids, including mRNA, DNA, microRNA (miRNA), long non-coding RNA (LncRNA), etc. (Jeppesen et al., 2019). Also, lipids including cholesterol, phospholipids, glycerophospholipids, and sphingolipids are vital components of EVs. They form a bilayer structure and preserve their steady state (Skotland et al., 2017). At the same time, tumor cells may release more EVs into the microenvironment than normal cells, resulting in high levels of circulating EVs.

In cancer development, EVs-mediated intercellular communication is crucial in remodeling the tumor microenvironment and the formation of pre-metastatic ecological niches. Since tumor cells can establish strong communication with neighboring and distant cells, the tumor microenvironment (TME) modulates their growth and metastasis. The TME contains different components, including extracellular matrix (ECM), endothelial cells, cancer-associated fibroblasts (CAF), immune cells, and mesenchymal stem cells (Luga et al., 2012; Zhao et al., 2016; Mao et al., 2017; Morad and Moses, 2019; Thakur et al., 2020). Primary tumor cell-derived EVs induce the conversion of fibroblasts into metalloproteinase (MMP)-secreting myofibroblasts, in turn degrading ECM((Janowska-Wieczorek et al., 2005)). Additionally, these EVs stimulate neointima formation by activating macrophages in TME, hence generating an ecological niche for inflammation (Sanchez et al., 2016). Also, EVs induce epithelial-mesenchymal transition (EMT), during which epithelial cells lose their intercellular adhesion and separate from the tumor (Welton et al., 2010). This promotes the spread of cancer cells, i.e., one of the hallmarks of metastasis (An et al., 2015; Becker et al., 2016; Kim et al., 2020).

EVs regulate cancer progression, metastasis, and treatment outcomes. Besides, they promote cancer initiation, growth, progression, and resistance. EVs transfer oncogenic proteins and nucleic acids as well as interact with the tumor microenvironment (Zhang et al., 2015; Kosaka, 2016) (Figure 2). First, EVs promote angiogenesis and metastasis (Figure 2A). EVs uptake upregulates angiogenesis-related genes, resulting in enhanced endothelial cell proliferation, migration, and sprouting (Fraser et al., 2016). Cancer EVs are responsible for matrix activation, causing an angiogenic switch and increasing vascular permeability. Also, EVs promote metastasis by targeting epithelial-mesenchymal transition and forming a pre-metastatic ecological niche (Sceneay et al., 2013; Khalyfa et al., 2016). Secondly, EVs promote the formation of cancer-associated fibroblasts (Gu et al., 2012) (Figure 2B). With continuous supply, EVs from breast cancer cells MDA-MB 231 and glioblastoma cells U87 induce the transformation of recipient fibroblasts (Antonyak et al., 2011).

Furthermore, EVs mediate immune escape and production of an immunosuppressive environment, which is crucial in cancer pathogenesis (Figure 2C). EVs have been shown to induce apoptosis in cytotoxic T cells, regulatory T cell expansion, M2 polarization in macrophages, and suppression of cytotoxicity in natural killer cells (Condamine and Gabrilovich, 2011; Yang et al., 2012). Lastly, EVs shield cancer cells from the cytotoxic effects of chemotherapeutic agents and transfer chemoresistant properties to nearby cells (Wang et al., 2014) (Figure 2D). Stromal cell-derived EVs mediate the therapeutic resistance pathway in breast cancer cells by activating the pattern recognition receptor RIG-1 (Boelens et al., 2014).

EVs have attracted research interest due to their role in shuttling specific tumor markers in solid tumors. Unlike tumor-free individuals, cancer patients have higher concentrations of EVs proteins. Moreover, tumor EVs have robust information on cancer biology (Peinado et al., 2012). With the advent of proteomics techniques and means of EVs protein analysis, studies on EVs proteins have rapidly increased. Table 1 summarizes various body fluid-derived EVs proteins diagnosed in cancer.

EVs proteins have been fronted as new diagnostic and prognostic indicators for various cancers. They may serve as biomarkers for breast cancer and melanoma. A significant increase in survivin levels has been reported in serum EVs from 40 breast cancer patients, and survivin-2B potentially acts as a diagnostic or prognostic marker for breast cancer (Khan et al., 2014). Melanoma-derived EVs promote metastasis by stimulating bone marrow-derived progenitor cells to prepare metastatic ecotopes. Wolfers et al. discovered that EVs secreted by melanoma harbor intact cancer antigens that activate CD8⁺ T cells and exhibit anticancer activity when absorbed by dendritic cells (Wolfers et al., 2001). Additionally, Logozzi et al. suggested that CD63 and caveolin-1 in plasma EVs can act as a protein marker for melanoma (Logozzi et al., 2009).

Accumulating studies provide strong evidence for the use of these EVs-based protein markers for early cancer detection and even predict clinical outcomes. EVs proteins are directly derived from their secreting cells. EVs proteins obtained from cancer cells are emerging as novel biomarkers for cancer surveillance and efficacy evaluation according to the following characteristics (Xiao et al., 2019) Cancer-related lipids, proteins, RNA, and DNA in EVs can be used for cancer detection (Penfornis et al., 2016). (Mignot et al., 2006) EVs are small in size, can easily pass through the body tissue barrier, and are widely present in various body fluids, thereby easily detectable in clinical settings (Boukouris and Mathivanan, 2015). (Fader et al., 2005) The lipid bilayer membrane structure of EVs shields their contents from enzymatic degradation in blood circulation (Laulagnier et al., 2004). Blood composition is complex, and specific proteins secreted by cancer cells are diluted in the blood, therefore cancer proteins are not easily detectable at an early stage or low levels (He and Zeng, 2016). Of note, more than 10⁹ EVs are present in each milliliter of human blood. Based on these characteristics, the detection of EVs proteins has significant potential as a biomarker for cancer diagnosis and prognostic evaluation (Kugeratski et al., 2021). Rab GTPases, a large family of small GTPases that control membrane identity and EVs budding, uncoating, motility and fusion through the recruitment of effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases and motors (Stenmark, 2009). In addition, EVs proteins are used in cancer diagnosis as well as in a number of other diseases. Fraser et al. explored leucine-rich repeat kinase 2 (LRRK2) as a biomarker in urinary EVs obtained from patients with Parkinson’s disease and discovered that ser-1292 LRRK2 is closely associated with PD (Fraser et al., 2016). Wang et al. (Wang et al., 2019) conducted a proteomic analysis of urine-derived EVs in PD patients vs. HC. Among all proteins discovered in urine- EVs, only two (SNAP23 and calbindin), were highly expressed in PD patients vs. HC. Therefore, the expression of these two proteins potentially represents a valuable non-invasive biomarker for PD.

Nonetheless, obtaining pure and homogeneous EVs for comprehensive analysis remains a challenge, thereby limiting the clinical use of EVs proteins. The most difficult aspect of EVs research is their isolation and acquisition. At present, EVs are primarily obtained via ultracentrifugation (Coumans et al., 2017), precipitation (Mateescu et al., 2017), and immunocapture methods (Yamamoto et al., 2018); the former is unspecific enough for clinical use, whereas the latter may introduce bias and contamination of serum/plasma proteins. The development of reproducible isolation and extremely sensitive identification technologies effectively integrate data from various laboratories and improve their viability for clinical applications. ISEV recommends that each preparation of EVs be (Xiao et al., 2019) defined by quantitative measures of the source of EVs (e.g., number of secreting cells, volume of biofluid, mass of tissue); (Mignot et al., 2006) characterized to the extent possible to determine abundance of EVs (total particle number and/or protein or lipid content); (Fader et al., 2005) tested for presence of components associated with EV subtypes or EVs generically, depending on the specificity one wishes to achieve; (Laulagnier et al., 2004) tested for the presence of non-vesicular, co-isolated components (Thery et al., 2018). Excitingly, a method has been developed to capture EVs directly from plasma, serum or urine using a variety of EVs proteins. This method requires simple sample preparation without the need to isolate vesicles (Cho et al., 2019). Therefore, the future research strategies of EVs proteins may be divided into two types (Xiao et al., 2019) Isolation and purification of EVs for further study of EVs proteins. This method is limited by the difficulty of obtaining high purity sEVs with current technology (Mignot et al., 2006). Direct capture of EVs in body fluids by immunocapture method and use for protein analysis. However, this method requires antibodies specific for membrane proteins, and the sensitivity of the antibodies used and possible inhibitors of the reaction can affect the accuracy of the results. It is something to look forward to whether EVs in body fluids can be classified according to their proteins like blood cells. As such, the future use of EVs as early cancer detection and prognostic biomarkers of cancer will be a novel intervention to defeat cancer.