Epithelial cells are immotile, polarised cells arranged in a single-cell “cobblestone” monolayer or in multi-layered sheets (22). Epithelial cells are connected by intercellular junctions and interact with an intact basement membrane (BM) through integrin receptors, thus movement is quite restricted (23, 24). There are four different types of intercellular junctions; (I) adherens; (II) tight; (III) gap; and (IV) desmosomes (25), and the expression of these junctions is critical in describing the epithelial phenotype (Table 1). Adherens junctions play a role in regulating the cytoskeleton and stabilising adhesive connections for effective intercellular signalling (26). The formation of the adherens junction results in the establishment of the tight junction (26), which forms cell-cell barriers to limit paracellular transport of molecules (27). Gap junctions not only have adhesive qualities, but also allow intercellular passive transport of ions and small molecules (28). Desmosomes attach to the cytoskeleton, thus provide strength to tissues for resistance against mechanical stress (29, 30). The intercellular junctions provide the structure and rigidity required for the primary function of epithelial cells to line the surfaces of body cavities (31). In addition to contributing to structural integrity, intercellular junctions are composed of key protein complexes that control epithelial function (24). The expression of these key proteins maintains epithelial cell integrity and adhesive properties, and prevents differentiation (32, 33). Importantly, these biochemical traits of an epithelial cell restrict cancer cells from entering into the metastatic cascade, and it is the loss and destabilisation of these key proteins that contributes to EMT and thereby cancer metastasis (3-5, 34).

A significant event that occurs during EMT is the downregulation of epithelial cadherin (E-cadherin). E-cadherin is a major transmembrane glycoprotein of the adherens-type junction. E-cadherin is a critical component in intercellular adhesion, inducing the formation of both the adherens junction and desmosomes by pairing cadherins in lateral epithelial cells (24, 26, 35, 36). E-cadherin also binds proteins of the cytoplasm called catenins, including p120 catenin, β-catenin, and α-catenin (26). This allows cadherins to connect to the cytoskeleton and be involved in signalling pathways (24, 26). Cleavage and subsequent degradation of E-cadherin prevents interaction with β-catenin. As a result, β-catenin may translocate to the nucleus with p120 catenin for transcriptional activation of Wnt genes, which drives EMT (25, 37, 38). Desmosomes are very similar to adherens junctions, with desmosomal cadherin proteins connected to intermediate filaments through desmoplaskin, which are disrupted during EMT (25). The main components of tight junctions are the family of transmembrane proteins, occludin and claudins, and the intracellular scaffold protein, zonula occludens 1 (ZO-1) (27). Occludin and claudins regulate ion selection and permeability of the intercellular pathway connecting adjacent cells, whereas ZO-1 binds to the cytoskeleton and proteins of the adherens and tight junctions (26). Downregulation of these proteins in EMT results in the loss of the epithelial cell polarity (39). Furthermore EMT decreases the expression of connexin – a major protein in gap junctions – which causes a loss of junction integrity (25). The inhibition of the expression of these proteins disrupts the epithelial phenotype and leads to the onset of EMT.

EMT is associated with normal, homeostatic events that are spatially and temporally regulated (24, 40). EMT occurs in normal developmental processes such as embryogenesis (22), embryo implantation during pregnancy (41) and organ development (36). EMT also occurs in wound healing, tissue regeneration, and organ fibrosis (36). These processes are initiated to recruit and activate fibroblasts to aid in the healing of tissues that have undergone trauma and inflammation (36). However, in the state of disease, EMT is hijacked and results in disturbing epithelial integrity and producing mesenchymal cells that sustain and exacerbate the disease (24). In the cancer setting, EMT is activated in order to produce cancer cells that exhibit both epithelial and mesenchymal qualities or cells with only a mesenchymal phenotype to propel cell invasion and metastasis (36).

Prior to EMT initiation, cells that will undergo EMT must be primed and conditioned towards a mesenchymal phenotype. For example, cell division may cease so that the cytoskeleton can be used to drive the changes in cell morphology and motility required for EMT (42). This induces major changes in gene expression necessary for EMT initiation (42). Temporal and spatial patterning of the epithelial region encourages morphogenic rearrangement to enable cell transportation to the EMT site (43). It also ensures that the integrity of the remaining epithelium is uncompromised (42). Following this, intercellular junctions and cell-BM connections must be disrupted (24). Dissolution of adherens, tight, gap junctions and desmosomes results in the loss of BM integrity (44). The disruption of these connections allows cells undergoing EMT to detach from the epithelial structure and for the remaining cells to close the gap (24). The detached cells then undergo cytoskeletal changes and differentiate into spindle-shaped mesenchymal cells (42).

Transformation into a mesenchymal phenotype results in the loss of apical and basal polarity, and the acquisition of an elongated morphology allows for fluid cellular movement, thus enables migration (45). A complete transition is characterised by changes in (i) cell morphology; (ii) functionality markers; and (iii) differentiation. Classical mesenchymal markers include N-cadherin, vimentin and fibronectin (Table 1). Downregulation of E-cadherin results in the upregulation of the mesenchymal N-cadherin, facilitating the transition of the cell towards a mesenchymal phenotype, increasing migration and invasion (25). This is commonly referred to as ‘cadherin switching’ and is an important stage in EMT-driven metastasis. Vimentin is a cytoskeletal intermediate filament protein that induces changes in cell morphology, migration and adhesion (46). Fibronectin is an extracellular glycoprotein that acts as a scaffold for the fibrillar ECM of mesenchymal cells (47). EMT results in enhanced cell motility, migratory potential, invasiveness, resistance to apoptosis and alterations of cell-ECM interactions and ECM components (20, 24). The degradation of the BM and formation of a mesenchymal cell marks the completion of an EMT (20).

Depending on the tissue or signalling mechanisms, a ‘partial’ or ‘quasi’ EMT of epithelial cells may take place. This is when epithelial cells lose only some of their traits or exhibit both epithelial and mesenchymal traits. Cancer cells that exhibit this partial EMT phenotype have the ability to move as clusters, which can become more aggressive than the cancer cells that have undergone complete EMT (48). Cells in the clusters exhibit enhanced tumour-promoting properties as they are resistant to apoptotic mechanisms, can exit the bloodstream in a more efficient manner and increase plasticity (49). This poses a problem for detection of partial EMT cancer cells as epithelial and mesenchymal markers may be expressed equally or at varying levels.

There is a significant association between metastatic, mesenchymal cell-derived exosomes and EMT initiation in epithelial cells, which has been demonstrated by numerous in vitro studies (52-57, 93) (Table 2, Figure 2). A study on hepatocellular carcinoma (HCC) demonstrated that exosomes derived from highly metastatic HCC cells were taken up by HCC cells with low metastatic propensity (54). This resulted in the recipient cells undergoing EMT through the activation of the MAPK/ERK pathway, associated with a more malignant phenotype and facilitating HCC progression (54). The ability of the low metastatic cells to migrate, form colonies, follow chemotaxis and invade was significantly increased, co-occurring with high expression of the mesenchymal markers α-SMA and vimentin, and a low expression of the epithelial marker E-cadherin (54). Similarly, another study found that both melanoma and lung cancer-derived exosomes increased the migratory and invasive capacity of primary melanocytes, suggesting that effects of cancer-derived exosomes is not limited to certain cancer types (55). The invasive phenotype of exosome-treated melanocytes is regulated by let-7i, which may induce its effects through LIN28B and HMGA2 (55). These two targets have been suggested to contribute to the EMT process (55, 132). Two EMT-related miRNAs, miR-191 and let-7a, were significantly upregulated in the serum exosomes of stage I melanoma patients, compared to healthy control patients, suggesting that these miRNAs are potential biomarkers for early-stage melanoma (55). This study also showed treatment of primary melanocytes with melanoma-derived exosomes caused an upregulation of SNAI2 and ZEB2, which led to the subsequent downregulation of E-cadherin and upregulation of vimentin (55). Furthermore, in lung cancer, it has been established that treatment of epithelial lung cancer cells with exosomes derived from mesenchymal lung cancer cells resulted in their transition to a metastatic, mesenchymal phenotype (52, 53). The epithelial cells gained an elongated, spindle-like shape, and had exhibited increased migratory and invasive abilities. Also, the downregulation of E-cadherin, and upregulation of N-cadherin and vimentin was observed (52, 53). In addition to these findings, numerous miRNA were differentially expressed in the mesenchymal cell-derived exosomes (52). Interestingly, it was found that the most enriched pathways represented by the miRNA were significantly associated with EMT factors, such as TGF-β and intercellular junctions (52). This study suggests that these differentially expressed miRNAs could serve as EMT biomarkers in lung cancer (52).

Figure 2

Exosomes derived from metastatic, mesenchymal cells carry pro-EMT factors to recipient epithelial cells, consequently inducing EMT. Pro-EMT factors include Vimentin, TGF-β, β-catenin, interleukins-6 and -17, and several miRNA. Uptake of these factors result in the activation of EMT pathways and subsequently EMT transcription factors. Numbers listed refer to citations in the reference list.

The EMT process and the transformation of lung cancer cells into a metastatic phenotype has also been induced in human bronchial epithelial cells (HBECs) by exosomes derived from the serum of late stage lung cancer patients (53). In vitro application of the serum-derived exosomes resulted in increased migration and invasion of the HBECs along with decreased expression of E-cadherin and ZO-1, and increased activity of N-cadherin and vimentin. Knockdown of exosomal vimentin reduced cell migration which suggests that vimentin may behave as an activator of exosome-mediated metastasis in lung cancer (53). Similar findings have been reported in a study that assessed the risk of pregnancy-associated breast cancer (56). Exosomes derived from healthy human milk were found to express significantly increased concentrations of TGF-β2, which, when incubated with benign and malignant epithelial breast cancer cells, led to the initiation of EMT (56). Morphological changes were observed in the benign and malignant cells, with the loss of the cytoskeletal structure and disruption of the intercellular junctions. This was accompanied by the loss of E-cadherin and increase in α-SMA and vimentin (56). These results suggest that women who secrete high amounts of TGF-β2 in their breast milk-derived exosomes may be at an elevated risk of breast cancer (56). Increased concentrations of TGF-β2 have also been described in exosomes derived from hypoxic prostate cancer cells compared to exosomes derived from normoxic prostate cancer cells. In addition to TGF-β2, pro-EMT factors IL-6 and β-catenin were also significantly expressed in the hypoxic exosomes. This resulted in the downregulation of E-cadherin and upregulation of β-catenin in recipient prostate cancer cells along with increased invasiveness, movement and stemness (127).

Currently, there is a paucity of in vivo studies and reports in the literature showcasing the effects of cancer-derived exosomes on EMT and metastasis. It has been shown that exosomes derived from a highly metastatic pancreatic cancer cell line can cause an increase in primary tumour volume in mice (133). Injection of these exosomes also caused a greater metastatic burden and cancer cell metastasis to various organs compared to control mice and mice injected with exosomes derived from poorly metastatic cells (133). In breast cancer, infusion of exosomes derived from the serum of tumour-bearing mice into wildtype mice resulted in tumour formation and increased metastasis of the tumour (60). The tumours themselves and the tumour-derived exosomes displayed an altered cytokine profile compared to exosomes derived from the wildtype mice, with IL-6 and IL-17 significantly upregulated (60). Inhibition of IL-6 and IL-17 resulted in the attenuation of exosome-induced micrometastases in the lung and draining lymph nodes (60). It has been demonstrated in in vitro studies that IL-6 and IL-17 drive EMT in breast, oesophageal, lung and brain cancer (134-140). This growing body of in vitro and in vivo evidence demonstrating exosomes as key mediators of EMT and metastasis suggests a direction for potential translation into the clinical field. However, there is a need for more extensive in vivo investigations, as it is imperative to understand the role of exosomes in physiological settings before clinical application aimed at the improvement of cancer treatments.

Research has shown that exosome uptake can modify recipient cells to adopt a therapy-resistant phenotype (18, 142, 145-150). A study on human breast cancer cells demonstrated that exosomes derived from cells resistant to the drugs tamoxifen (MCF-7/T) and metformin (MCF-7/M), induced resistance to these drugs in the parental MCF-7 cells (145). Exosome-induced resistant MCF-7 cells were characterised by the downregulation of E-cadherin, and activation of NF-κB, SNAI1 and AKT. Interestingly, addition of parental MCF-7-derived exosomes had no effect on the resistant properties of MCF-7/T and MCF-7/M cells (145). Another study on epithelial ovarian cancer (EOC) showed that exosomes derived from hypoxic macrophages increased the resistance of EOC cells to cisplatin, linking the impact of the primary tumour microenvironment on the interaction between infiltrating immune cells and cancer cells (146). These exosomes were highly enriched with miR-223, which increased cisplatin resistance through the PTEN-PI3K/AKT pathways, both in vivo in mice, and in vitro. In EOC patients, those with a high expression of HIF-1α had higher intertumoural levels of miR-223. Furthermore, circulating exosomal miR-223 levels were closely associated with EOC recurrence. Intriguingly, it has been shown that EMT is regulated by the miR-223 pathway (146). In pancreatic cancer cells, downregulation of miR-223 reverses EMT in cells resistant to gemcitabine (147). Overall, these studies suggest that exosomal content can influence chemotherapy responses in cancer by modifying EMT.

Another interesting phenomenon is that radiation triggers an immune response that causes immune-derived exosomes to promote metastasis. Irradiated T cell-derived exosomes caused oesophageal cancer cells to gain a migratory and invasive phenotype (148). The higher the radiation dose, the more invasive the cancer cells were. The metastatic-like phenotype of the cells was associated with an upregulation of NF-κB, SNAIL, and β-catenin (148). Activation of NF-κB is associated with the stabilisation of SNAIL, which is known to suppress E-cadherin expression (151). The onset of EMT is induced by activation of Wnt signaling which prevents GSK-3β from phosphorylating β-catenin and SNAIL. The combined effect of these two factors promotes cancer cell survival during dissemination and invasion (152). The findings of these studies suggest that exosomes induce EMT to facilitate therapeutic resistance to radio- and chemotherapies.

Many studies demonstrating therapy resistance are strongly associated with CSCs (18, 142, 143). A key attribute of CSCs is their ability to enter dormancy, then re-emerge into the circulation, metastasise and form a secondary tumour by undergoing MET (142). Our previous work was first to demonstrate that exosomes derived from oncogenically-transformed, mesenchymal HBECs can transfer chemoresistant traits to, and induce a CSC-like phenotype in recipient untransformed HBECs (18). The mesenchymal HBECs displayed an elongated, spindle-like morphology, along with a decrease in the expression of CDH1, and an increase in the expression of SNAI1, SNAI2, TWIST, ZEB1 and ZEB2. Additionally, the mesenchymal HBECs exhibited significantly elevated resistance to commonly used lung cancer therapies, cisplatin, gemcitabine, and a combination of cisplatin and gemcitabine treatment, compared to the epithelial HBECs. Exosomes derived from the chemoresistant, mesenchymal oncogenic HBECs promoted resistance to gemcitabine and the combination of cisplatin and gemcitabine, in the epithelial, untransformed HBECs. The treatment of exosomes also increased expression of ZEB1 and TWIST1, and promoted “stemness” by shifting the cells towards a CSC-like CD24^low/CD44^high phenotype (18). Another study showed that exosomes derived from breast CSCs and cells resistant to doxorubicin and paclitaxel promoted EMT-mediated chemoresistance of the recipient sensitive breast cancer cells (149). The exosomes derived from both the CSCs and chemoresistant cells were highly enriched with miR-155, which was transferred to the recipient cells. It has been suggested that miR-155 acts as a regulator of EMT and CSCs as it targets FOXO3a and regulates the loss of C/EBP-β, which can result in the loss of TGF-β (149, 153). These exosomes also induced an increase in the mRNA levels of SLUG, SNAIL, SOX9, BMI1 and EZH2, alongside a decrease in E-CAD, TGF-β and FOXO-3a in the recipient sensitive cells. BMI1 and EZH2 are stemness-related transcription factors (149). These results show that the acquisition of a CSC-like phenotype is a major contributing factor of chemoresistance.

Another causative element of chemoresistance is tumour microenvironment pH. An acidic microenvironment has been associated with poor patient prognosis, suppressing the function of cytotoxic lymphocytes and NK cells, and a therapy-resistant phenotype (70, 142). Acidic environments are thought to significantly increase exosome release and facilitate uptake of exosomes by recipient cells in vitro (154-156). Extracellular acidity affects the mechanisms of anticancer therapies that are weak base drugs (157). Cellular uptake of weak base drugs is reduced as a high intracellular pH causes an influx of H⁺ ions from the extracellular space into the cell (157, 158). Weak bases are ionised in acidic environments which reduces its ability to permeate cell membranes (157). The acidic tumour microenvironment also assists exosomes in promoting therapy resistance (142). Some cancer-derived exosomes express ATP-binding cassette (ABC) transporters (159). Exosomal ABC transporters have shown to sequester chemotherapeutic drugs into exosomes (142). The chemotherapeutic drug docetaxel, used for the treatment of breast and prostate cancer, can actually increase the number of exosomal ABC transporters (159). It has also been revealed that cisplatin is sequestered into melanoma exosomes in a pH-dependent manner (160). Chemoresistance is often seen in breast cancer as the multidrug pump ABCG2 is localised in the membrane of breast cancer-derived exosomes (150). Expression of ABCG2 mediates multidrug resistance as it enables sequestration of the drugs mitoxantrone and topotecan into the exosomal lumen. The PI3K-AKT signalling pathway regulates ABCG2, as inhibition of this pathway causes ABCG2 to relocalise to the cytoplasm, thus restores breast cancer cell drug sensitivity (150).

There are not many studies on the relationship between exosomes, EMT and cancer recurrence. One study looked at the effects of highly metastatic exosomes on tumour recurrence in hepatic cellular carcinoma (HCC) (54). Surgical resection is the primary treatment for HCC patients who do not have cirrhosis (54, 161). Despite resection, the five-year risk of recurrence is 70%, which often arises within two years after surgery (162). Injection of highly metastatic HCC cell-derived exosomes into the tail vein of mice resulted in recurrence in the remnant liver in 100% of the mice, compared to the control group in which only 40% experienced recurrence (54). Tumour size and weight was also significantly higher in the group injected with the exosomes compared to the control group. As mentioned earlier these exosomes induced EMT in HCC cells via MAPK/ERK signalling (54). Furthermore, a study on colorectal cancer uncovered that there was a significantly higher count of GPC1⁺ plasma exosomes in CRC patients with relapse, compared to patients without relapse (163). Moreover, patients that died with relapse compared to patients that survived with relapse, and patients that survived with relapse compared to patients that survived without relapse had altered levels of GPC1⁺ plasma exosomes (163). There was also an increasing trend with GPC1⁺ plasma exosomes in patients who relapsed nine months post-surgery. In order to investigate the role of GPC1 in cancer recurrence, GPC1 was overexpressed in CRC cells, which resulted in decreased E-cadherin, increased vimentin and upregulated SNAI1 and SLUG expression, ultimately causing increased migratory and invasion abilities (163). These findings suggest that the upregulation of GPC1 in plasma exosomes may be involved in CRC relapse through induction of EMT (163). A study on urothelial bladder cancer (UBC) found that exosomes derived from the urine of UBC patients had significantly increased expression of the lncRNA HOX transcript antisense RNA (HOTAIR) (164). HOTAIR aids tumour initiation and progression, and is closely linked with poor prognosis in various cancers (165-167). Knockdown of HOTAIR decreased migration and invasion of UBC cell lines (164). It also resulted in the reduction of SNAI1 and ZEB1, TWIST1, MMP1, LAMB3 and LAMC2 and increased expression of ZO1 (164). Previous studies have shown that a high level of expression of HOTAIR is associated with cancer recurrence and even has the potential to act as a biomarker for recurrence in HCC, bladder cancer (165-167). These retrospective studies examining patient-derived exosomal cargo are beneficial for understanding the pathogenesis of cancer recurrence and there is a great urgency for additions to the literature.

Together, these studies have highlighted the involvement of cancer-derived exosomes in the initiation and promotion of EMT, metastasis, therapy-resistance and cancer recurrence (Table 2). Whether exosomes are the driving force behind these factors still requires more research, however, it is evident that exosomes are important contributing factors. In order to determine this, future studies will need to confirm that the effects observed are a true representation of exosomes and not of other elements of the tumour microenvironment, for example by inhibiting exosome release. It is essential that more animal and patient studies are conducted in order to determine the alterations in exosomal cargo induced by cancer. Identifying these changes in exosomal cargo may provide insight into the disease and act as potential biomarkers of cancer to guide prospective patient studies. Early detection of cancer onset, metastatic progression, therapy-resistance and recurrence would allow for early intervention and tailored therapies, thus the requirement for accurate biomarkers is essential. The rapid progression of cancer going undetected can be attributed to the current tissue biopsy and imaging methods used for diagnosis and prognosis. Real-time detection of exosomes using liquid biopsies promises being a suitable, better alternative.

Circulating tumour cells are cancer cells that detach from the primary tumour, invade the BM and surrounding tissues, and disseminate into blood vessels (6, 175). CTCs are considered “predestined sources” of metastasis (175), representing the stage between the acquisition of an invasive phenotype and the formation of metastatic sites (176). EMT transcription factors, such as SNAIL, SLUG, TWIST and SIP1 promote the survival and formation of CTCs by preventing apoptosis, escaping senescence, and enhancing invasiveness and intravasation (175, 176). Circulating tumour DNA (ctDNA) is released by apoptotic and necrotic tumour cells (177). ctDNA therefore contains the entire tumour genome, serving as reservoirs of genetic mutations and alterations (177). It has been proposed that ctDNA may induce oncogenic alterations and promote transformation of normal, non-cancer cells, thus contributing to metastasis (178). Therefore, CTCs and ctDNA have become of interest for their potential to act as diagnostic, prognostic and predictive biomarkers (172). Although CTCs and ctDNA have potential as liquid biopsy biomarkers, there are many limitations.

CTCs and ctDNA are difficult to detect as they are rapidly cleared from bodily fluids due to their short half-lives of 1-2.5 hours and less than 1.5 hours, respectively (179, 180). Up to 99.9% of CTCs go undetected by the current CTC assay methods available, as CTCs are only released into the blood at low concentrations (1-10 CTC/mL), often enter dormancy and are easily clogged in small blood vessels (181, 182). Typically, 7.5 mL of blood is extracted from patients to be used for CTC experimentation. One study analysed the differences in the detection of CTCs derived from 7.5 mL of blood and 30 mL of blood, in 15 patients with colorectal liver metastasis (183). Using the CellSearch^® System, it was revealed that a median of 1 CTC was detected in the 7.5mL samples and a median of only 2 CTCs was detected in the 30mL sample (183). The CellSearch^® System recommends a minimum CTC count for certain types of cancer for assay specificity and prognostic relevance (184). Therefore, patients with a low CTC count are excluded for clinical application, although they may have clinical relevance. During early stage disease, few cancer cells are dying, hence a very low level of ctDNA circulates in the blood (185). This poses a major problem for early cancer detection (186). Similarly to CTC detection methods, ctDNA assays can generate false-positive and false-negative errors (187-189). CTCs and ctDNA abundances are often below detection thresholds after cancer therapies, however, this is not necessarily an indicator of a complete removal of the cancer (190). Although CTCs and ctDNA may act as an alternative to the traditional tissue biopsy, they require validation in large for clinical application of these biomarkers in advanced cancer and numerous obstacles are yet to be overcome.

Exosomes provide significant advantages over CTCs and ctDNA, making them a good candidate for liquid biopsy methods (174). The most important feature of exosomes is that they sensitively reflect the phenotype of the primary tumour in real-time, thus are an accurate representation of tumour heterogeneity (174, 191). Unlike CTCs and ctDNA, exosomes are present in most bodily fluids at high concentrations during all stages of cancer, as exosome release is an active process (174, 182, 192). This allows for disease monitoring over extended time periods (182). Exosomes are stable and can be preserved and maintained in blood ex vivo. Their stability allows for the protection of their complex cargo derived from the tissue of origin (168, 182). This stability, and the presence of EMT associated nucleic acids and proteins within exosomes provides a unique insight into EMT and potential metastasis of the primary tumour, allowing for potentially earlier and more targeted therapy (191). Only a small volume of blood is required for their highly sensitive detection in early-stage disease (168, 182). Studies have identified exosome content derived from cancer patients as potential cancer biomarkers. One study reported elevated levels of GPC1 in exosomes derived from the serum of pancreatic ductal adenocarcinoma patients, compared to healthy donors. These GPC1⁺ exosomes had a sensitivity and specificity of 100% for all stages of pancreatic cancer, demonstrating its potential as a liquid biopsy biomarker for early cancer detection (193). As mentioned earlier, GPC1 plays a role in the progression of EMT, which correlates with the mesenchymal phenotype often found in pancreatic tumours (163, 194). The unique features of exosomes make them a promising source of cancer biomarkers for early diagnosis and prognosis, monitoring metastatic progression and assessing treatment responses (191).