In this study, we performed a quantitative comparison of the total particles circulating in the serum derived from HC and patients with CC. We determined the particle concentration and size distribution of the total particles in serum using NTA. The levels of EV particles released in human body fluids, particularly in blood, may themselves represent a diagnostic marker of cancer development and differentiate between patients with and without cancer. Our experiment determined the total number of serum particles in both the HC and CC groups. The results showed that both HC and CC had similar particle sizes in the range of 101–150 nm. However, the mode sizes of the particles in HC were significantly larger than those in CC. In addition to particle size, the particle concentrations of CC were marginally higher than those in the HC group. Consistent with recent clinical evidence, plasma particle levels were significantly higher in patients with cancer than those in healthy controls [18–21]. According to a proteomic study of EVs derived from breast cancer, a single cancer cell can release more than 104 particles per day [22, 23].
Further, we investigated the EV protein profiles derived from the serum of HC and patients with CC. We isolated and purified EV particles using ultracentrifugation combined with SEC. We determined the surface markers of EV and visualized the morphology and size of EV particles using an electron microscope. As expected, the combination of ultracentrifugation and SEC successfully isolated sEVs. From the immunoblotting results, the abundance of EV markers, including CD63, CD9, and CD81, indicates the successful isolation of EVs from serum samples. Moreover, TEM images revealed a typical morphology and size range of sEVs. Interestingly, we successfully purified the EVs from abundant serum proteins, as shown in the TEM image, wherein slightly precipitated abundant proteins were detected.
Here, after isolating and purifying the EVs, we explored the potential of using EVs as circulating biomarkers for the detection and classification of CC by comparing its EV cargo to that from healthy serum. In this study, we focused on identifying protein biomarkers to improve the diagnostic efficiency in patients with CC. We characterized the total EV proteins derived from CC and HC sera using LC-MS/MS. The data obtained were used to create a Venn diagram. The Venn diagram shows an overview of the patterns of both unique and co-expressed proteins. We found that three and 18 genes were uniquely expressed in the HC and CC groups, respectively. Moreover, 3185 proteins were co-expressed in both the CC and HC groups.
Proteins obtained from both the groups were analyzed. The metabolic pathway of the total proteins in each group was similar in many pathways. We further investigated the pathways of 18 unique proteins found in CC. The 18 unique EVs proteins were COX5A, SGSM3, TUFM, ZNF185, CHD2, PHF10, ETS1, CYP51A1, IPO5, USP34, TSHB, VWA3B, ABTB2, L, ERI3, PARP1, ATRX, and ADAM32. COX5A (cytochrome c oxidase), is a complexed enzyme located in the mitochondria which plays a major role in metabolic processes [24], including ATP production [25]. Therefore, COX5A expression is associated with many cancers, including colorectal, breast, prostate, and lung cancer [26–29]. Moreover, several mechanisms including the proliferation of cancer cells and angiogenesis are involved in COX5A expression [30–32]. IPO5 is also expressed in colorectal cancer cells and plays a role in promoting the proliferation and tumorigenesis of cancer cells [34]. Furthermore, the IPO5 test with logistic regression analysis revealed that COX5A is the best individual potential biomarker for differentiating cancer cases from healthy controls [37]. Moreover, our findings revealed CYP51A1 or lanosterol 14α-demethylase expression in EVs derived from CC, which was not present in the HC group. CYP51A1 is a member of the cytochrome P450 family contributes to cholesterol biosynthesis [38]. Consequently, CYP51A1 binding to KRAS leads to the activation of the MAPK cascade. An abnormal MAPK cascade can lead to increased or uncontrolled cell proliferation and apoptosis resistance events [39]. Interestingly, a correlated pathway with unique EVs proteins in CC showed that their association with angiogenesis, FAS signaling, VEGF, and RAS signaling pathways. Pathway analysis inferred that angiogenesis is the creation of new vessels that supply cancer cell growth. VEGF is the most crucial factor in the initial angiogenesis process [40]. Uniquely, the proteins found in CC were also associated with the Ras signaling pathway. Ras proteins are crucial mediators of several malignant characteristics in transformed cancer cells. Ras proteins function as activators in many cancers [41]. Targeting the Ras pathway to inhibit tumor growth is a promising therapeutic development [41]. However, to confirm the clinical applicability of unique EVs proteins in CC, a validation step is required. Thus, this result indicates that the unique expression of CC EV proteins may be considered as a developmental signal in patients with CC.
In addition, 3185 proteins were co-expressed in both CC and HC, with upregulated and downregulated expression in CC. DEPs were analyzed using a multilevel enriched gene ontology (GO) analysis. The top 25 upregulated proteins were mainly involved in cellular and metabolic processes. Cellular processes are required for homeostasis. EVs contribute to many pathways of both physiological and pathological homeostasis through cellular communication, such as the excretion of nucleic acids from cells [43]. Both up- and down-regulated proteins in CC showed components in binding and catalytic activity of cellular processes and were similar in cellular component analysis. Other studies of proteomic profiling in EVs derived from the blood samples of females with breast cancer revealed that 80% of EV proteins were involved in protein binding, consistent with our findings and other cancers, including ovarian and colorectal cancer [44–47]. Additionally, pathway analysis of differential expression of up-and downregulated proteins in CC was different in the CC group compared to that in the HC group. For upregulated proteins, inflammation, mediated by chemokine and cytokine signaling pathways, is a major pathway correlating with these groups of proteins. Chemokines are a group of soluble factors that play crucial roles in immune regulation during inflammatory response processes and in defense against foreign pathogens. Chemokines also regulate many biological processes such as angiogenesis and stem cell migration during embryo development. The chemokine signaling pathway is regulated in diverse cellular processes, such as angiogenesis, epithelial cell proliferation, and survival [42, 43]. Intriguingly, chemokines are critical for cancer development and are highly functional in the tumor microenvironment [44]. Thus, in summary, we assume that chemokine signaling can be utilized to target and deliver chemokines in cancer treatment.
Furthermore, pathway analysis of downregulated proteins in CC showed major components in the endothelin (ET1) and Wnt signaling pathways. ET1 signaling promotes cell proliferation, epithelial mesenchymal transition process, and drug resistance in a context-dependent manner. Hence, the ET1 pathway is critical in cancer therapy. Moreover, ET1 receptors are expressed on tumor-associated cells, such as blood, endothelial, fibroblast, and inflammatory cells, which contribute to cancer progression [45, 46]. However, our findings demonstrated that ET1 protein levels decreased in EVs derived from CC serum, which is inconsistent with other reports. To support the above findings and a previous review, the evidence showed that ET1 proteins are mostly secreted into plasma [47]. Moreover, in hepatocellular and prostate cancers, the plasma levels of ET1 have been determined in patients with several solid tumors [48, 49]. In addition, the Wnt/β-catenin signaling pathway is conserved and plays a role in diverse physiological processes including proliferation, differentiation, apoptosis, invasion, migration, and homeostasis. Aberrant Wnt signaling regulation leads to carcinogenesis [50–52]. The detailed GO analysis revealed that downregulated EV proteins were enriched in the Wnt signaling pathway. Moreover, the current study highlights the crucial role of EVs in the carrier function of most signaling molecules in critical processes, such as cancer development. Moreover, EVs transport and release are correlated with Wnt signaling proteins in both homeostasis and cancer conditions [53]. Moreover, EVs transport activates the Wnt signaling during disease progression. A recent study found that Wnt protein is located on the surface of EVs and activates Wnt signaling on the surface of target cells [54].
EPs analysis was performed to identify the discovered proteins using the UALCAN database. For upregulated proteins, 10 out of 25 EV proteins, including MIPOL1, C1QB, ZNF480, SVIL, MYO3B, JMY, ZNF560, CCDC13, DNAJC25, and NADSYN1, were found in CC tissue, as reported in TCGA data. C1QB, MYO3B, and NADSYN1 proteins showed significantly increased expression levels in CC tissues compared to those with normal tissues. C1Q is part of the subcomponent of the classical complement pathway, which allows the activation of the complement pathway. Corrales et al. provided evidence that C1Q is locally expressed in the stromal and vascular endothelium of many human tumors and acts as a tumor-promoting factor by favoring adhesion, migration, and proliferation of cancer cells, as well as angiogenesis and metastasis [55, 56]. However, the roles of C1Q8, MYO3B, and NADSYN1 in the oncogenesis of CC remains unclear [57].
For downregulated proteins, nine out of 25 EV proteins, including COPB2, MAFK, OR13C9, PIK3C2, OR2T11, PLCB4, RAB12, VIP, and ZNF841 were also found in CC tissue, as reported in the TCGA data. MAFK, PIK3C2, PLCB4, RAB12, and VIP showed a decreasing trend, with no significant difference in CC tissue compared to normal tissue, which is in line with our findings. However, some proteins, such as OR2T11, showed no expression in both CC and normal tissues but showed a decreasing trend in EV-derived CC serum compared to that in EV-derived HC serum.
Our finding of EV proteins derived from serum and reported data in CC tissues showed both consistent and discrepant results. However, it is possible that some proteins from the cells of origin may migrate to other distant cells through EV vehicles; thus, these trafficking proteins can be detected as EV carriers [58, 59].
Thus, our findings provide initial data, which is crucial for further studies to develop combination panel markers instead of using a single biomarker for clinical application.