A CTC cluster-based model
Multicellular CTC clusters have been identified (18,20), with up to 100 tumor cells found in one cluster (21). Currently, only a few studies have reported the successful detection of CTC clusters, while single CTCs have been universally found in corresponding studies (20–22). We believe that the amount of CTC clusters is largely underestimated by current detection methods, as the majority of these methods use a strategy of surface protein-targeted antibody-based capture. Since the ratio of the superficial area to the volume decreases as the volume increases, the efficiency of CTC capture by antibody-targeting surface proteins is greatly reduced for CTC clusters compared with a single CTC. Therefore, the actual amount of CTC clusters in a patient is underestimated, and the role of CTC clusters in tumor metastasis is largely unknown. CTC detection methods applying novel principles may improve the efficacy of CTC cluster capture and shed light on an in-depth understanding of CTCs in tumor metastasis.
The origin of CTC clusters remains elusive. At present, there are at least three possible origins (Fig. 1). The first is that CTC clusters directly break off from the primary tumor as a result of the shearing force of the blood. Tumor emboli can often be found in patients with late-stage cancer. Solid tumors tend to invade vessels to obtain abundant oxygen and nutrients for cell metabolism and proliferation-associated biosynthesis. Clinical studies have confirmed that patients with vessel invasion by tumors had an increased risk of recurrence and a poorer prognosis compared with those who had tumor-free vessels in a range of cancers, including breast cancer, hepatocellular carcinoma and papillary thyroid carcinoma (23–25). In addition, in patients without pre-operative metastasis, resection of the tumor-invaded vessels conferred a survival benefit, with a similar long-term prognosis compared with patients without vessel invasion by tumors (26,27). Thus, removal of tumor emboli and tumor-invaded vessels can eliminate a critical origin of CTCs and minimize the possibility and extent of tumor metastasis, as well as tumor-associated mortality.
The second origin of CTC clusters can be the result of single CTC proliferation. Although there is a lack of strong evidence regarding the potential of CTCs to resist immune clearance, CTCs were previously found to be associated with impaired immune function in patients (28,29). In addition, certain single CTCs can survive for a considerable time in the circulatory system due to their probable potential of passing through capillaries and proliferating to form CTC clusters. Thus, for certain single CTCs, this process may continue until a big enough CTC cluster has been established and intercepted by vessels with small diameters.
The third origin of CTC clusters is the aggregation of single CTCs (30). Although it is difficult to monitor the formation of CTC clusters by single CTCs in real time in vivo, existing evidence may indirectly support this idea. For instance, normal cells are programmed to die if they are detached from neighboring cells and the extracellular matrix, this is known as anoikis (31). The proportion of non-apoptotic CTCs is essential to the formation of metastases (13). However, tumor cells resist anoikis partially by forming cell clusters (32,33). Notably, characteristics of EMT, including upregulation of vimentin expression and downregulation of E-cadherin expression, were found to be associated with anoikis (34,35). Meanwhile, EMT was also shown to play an important role in CTC cluster formation (21). These complicated phenomena suggest that CTC cluster formation may be a strategy adopted by tumor cells to resist anoikis. On the other hand, it should also be noted that the existence of CTCs is significantly associated with vessel thrombosis (36), which leads to embolism or thrombosis, particularly in microvessels. The formation and special biological characteristics of CTCs have been proven to be responsible for the hypercoagulability and subsequent thrombosis of patients (36); however, the unique physical trait of being cell cluster is also logically associated with a difficulty in passing through the capillaries. In addition, thrombosis and thrombolysis in microvessels can frequently occur in patients with hypercoagulability. Ongoing thrombosis can induce stenosis of vessel lumina, which tends to intercept CTC clusters. Numerous changes occur under these conditions. Embolism or complete thrombosis in microvessels can cause local hypoxia, which has been proven to induce EMT in various cancers, including hepatocellular carcinoma, gastric cancer, pancreatic cancer, breast cancer and colon cancer (37–41). EMT of CTCs may contribute to tumor cell extravasation from vessels into tissues. This model does not require CTCs to permanently sustain mesenchymal characteristics in the circulatory system, and fits well with the fact that the majority of CTCs detected show epithelial characteristics. In addition, the marked change in the inflammation-immune-cytokine local environment induced by platelet aggregation and subsequent cascades can also upregulate certain EMT-triggering factors, such as TGF-β (21). These changes provide an initial impetus for CTCs to extravasate from vessels into tissues.
CTC clusters may have different characteristics according to their origins. For example, CTC clusters directly from the primary tumor may contain evidently more epithelial traits (if the primary tumor is an epithelial malignancy), while those aggregated by single CTCs may tend to be mesenchymal. In addition, heterogeneity may exist within a CTC cluster if it contains a large number of cells. Little is known about the physical or biochemical details of these CTC clusters, including the oxygen and nutrient supply, and the distribution (42).
Single CTC model
To date, the majority of detected CTCs have been single cells and their existence has been reported to be associated with tumor metastasis, treatment response and the long-term prognosis of patients (43–45). However, only certain patients with malignancies exhibited CTCs in their peripheral blood (46). Even in patients with detectable metastases, CTCs could only be detected in half of them (47). The threshold of positive and negative results of CTC detection by successfully detecting one or two CTCs in a 7.5-ml blood sample is also non-robust. Considering the discrepancy of detection of single CTCs (early stage) and tumor metastasis (late stage) regarding the stage of the disease, certain single CTCs are likely to survive in the circulatory system for a long time or alternatively undergo dormancy in tissues or organs (6). Although few CTCs can stay for a long time in the blood (6), the survival and proliferation of CTCs in ectopic organs demands certain necessary prerequisites. Based on the present point of view, abundant cells are prerequisites of tumor inoculation in ectopic organs. Recently, it was demonstrated that the CTC clusters had 23- to 50-fold increased metastatic potential compared with single CTC cells (48).
Cancer stem cells (CSCs), which have a potent capacity for tumor initiation, require a certain number of cells to form tumors subcutaneously in nude mice (49). Although CTCs have a number of the characteristics of CSCs, a certain number of CTCs is also necessary for the formation of tumor metastases (50). From this point of view, single CTCs can barely effectively generate tumor metastases due to a lack of enough cells. A small number of tumor cells in ectopic organs are easily removed by the local immune system or die due to insufficient self-support in the early stage of metastasis (51). While it is impossible that hundreds or thousands of single CTCs independently extravasate from the same anatomical site of vessels and aggregate together in ectopic organs, the role of CTC clusters, rather than single CTCs, provides a more logical and rational explanation for the initial formation of tumor metastasis.