*2.1.2 Intravasation*

It is a vital and indispensable step for cancer cells to disseminate to distant organs during which cancer cells infiltrate into the vascular or lymphatic wall and then enter circulation, becoming circulating tumor cells (CTCs) and potential metastatic seeds. The formation of new blood vessels around cancer cells has a great influence on cancer cells entering the circulatory system, thus understanding the various mechanisms of neoangiogenesis stimulated by cancer cells in local microenvironment will help us comprehend intravasation. Vascular endothelial growth factor (VEGF), a highly bioactive functional glycoprotein, promotes blood vessel growth and lymphatic vessels, which plays an irreplaceable role in angiogenesis. However, the neo-vasculatures generated by cancer cells increase capillary permeability compared with the blood vessels produced by normal cells and tissues [32]. During the lung metastasis of breast cancer, VEGF/VEGF receptor 2 (VEGFR2) and its target proteins such as ERK1/2, Src, and FAK regulate neo-angiogenesis and blood vessel permeability to enhance metastasis [33].

On the other hand, a bunch of studies reveal intravasation can be improved by boosting the penetrability of cancer cells to pass the barrier of endothelial cells. For example, secretion of epidermal growth factor (EGF) by TAMs enhances the intravasation of breast cancer cells [34]. Additionally, the TGF-β enhances mammary cancer intravasation by increasing carcinoma cell penetration of micro-vessel walls or more generally strengthening invasiveness [35]. What's more, in melanoma, the migration of cancer cells to endothelial cells and intravasation are promoted via endothelialderived SLIT2 protein and its receptor ROBO1 [36]; activated Notch1 receptors (N1ICD) can promote neutrophil infiltration into the tumor, the intravasation of cancer cells and postsurgical metastasis [37]. In the study of Wei et al., increased IL-6 from TAMs is observed and can promote the invasiveness of cancer cells through the STAT3/miR-506-3p/FoxQ1 axis, then increases CCL2 level to boost the recruitment of macrophages. Besides, the authors suggested that there exists a feedback loop between TAMs and cancer cells, which was essential for the EMT and intravasation into the blood vessels [38].

### *2.1.3 Circulation*

Once cancer cells have successfully entered lymph and blood, these malignant cells have the chance to disseminate throughout the body. In blood and lymphatic vessels, these cancer cells must escape the killing of immune cells and physical damage from hemodynamic shear forces to survive. In general, CTCs are in a dormant state that can cause relapse and poor prognosis for patients. This is because conventional surgery, radiotherapy, and chemotherapy are powerless against these CTCs in the blood, lymph, and body fluids, as well as dormant cancer cells, further leading to a decrease in immunity and the rapid growth and metastasis of hidden CTCs.

Many studies have verified the prognostic role and value of CTCs in the early and metastatic stages of cancer by measuring biomarkers [39]. An informative metaanalysis including 1847 patients with colorectal cancer under chemotherapy studied by Huang et al., demonstrated the high expression of CTCs in the bloodstream has a positive correlation with decreased progression-free survival (PFS) (hazard ratios = 2.500, 95% CI [1.746–3.580], P < 0.001) [40]. Moreover, CTCs in blood samples of 100 patients with head and neck squamous cell carcinoma were enriched and isolated and the PFS and overall survival of these patients were observed and recorded. The result showed a worse prognosis like decreased PFS and overall survival in CTCs-high patients [41]. Rink et al. also observed patients with ≥1CTCs C per 7.5 ml of blood in distant metastatic bladder cancer shortened the time of disease recurrence and cancer-specific death, resulting in worse clinical outcomes [42]. With the improvement of technologies and the depth of research, plenty of CTCs-related biomarkers are uncovered. At present, a set of biomarkers has been applied to detect CTCs in various cancers. Lin D et al. summarized the CTC-related biomarkers in different cancers [43]. EpCAM as the most common marker can be found in most cancer (i.e., breast cancer, liver cancer, prostate cancer, kidney cancer, melanoma, bladder cancer), which is because most cancers originate from the epithelium [44]. Just like EpCAM, human epidermal growth factor receptor-2 (HER-2), estrogen receptor (ER), prostate-specific membrane antigen (PSMA), and folate receptor (FR) also have been applied to detect CTCs in some cancers, with outstanding clinical significance [45–49].

In addition to CTCs-related biomarkers, the mechanism by which CTCs escape the detrimental shear stress and anoikis in the circulatory system is becoming clearer. There is evidence that CTCs in the blood can stay away from immune cells' killing to increase survivability by bounding tightly to blood constituents like neutrophils, myeloid-derived suppressor cells (MDSCs), CAFs, or platelet [50, 51]. A few years ago, Szczerba et al. found the concentration of CTCs and neutrophils have a significant correlation in animal models and patients with breast cancer, which displays greater metastatic potential and higher gene expression involving cell proliferation. They thought the binding of CTC and neutrophil is possibly mediated by vascular cell adhesion molecule [52]. Besides, Spicer et al. suggested neutrophils could directly adhere to CTCs by the neutrophil Mac-1/ICAM-1, which becomes a bridge between cancer cells and the liver to accelerate CTCs extravasation and colonization [53]. Neutrophils can also enhance metastasis in an indirect manner by trapping CTCs in the circulation through neutrophil extracellular traps (NETs) [54]. In several in vivo experiments, liver or lung NETs were found to collect cancer cells to promote distant metastases by a transmembrane protein named coiled-coil domain

containing 25 (CCDC25) to activate the ILK-β-parvin pathway, leading to enhance cell motility [55].

TAMs play crucial roles in the mechanical adhesiveness and endurance of CTCs, which contribute to the formation of protective cell clusters and the resistance to shear stress [56]. Liu et al. proposed that CTCs interacting with adhesive immune cells like MDSCs could create a defensive shield to allow evasion of immune surveillance, facilitating distant metastatic lesions [57]. Sprouse et al. found reactive oxygen species (ROS) from MDSCs could activate the Notch pathway in CTCs, promoting CTCs proliferation [58]. In addition, CAFs could protect CTCs from the fluid shear forces in the peripheral blood via intercellular contact and soluble derived factors in prostate cancer [59]. As an important component in blood, platelet also supports the survival and metastasis of CTCs in a CTCs-platelet cluster manner. Platelets have been shown to help CTCs evade attack by NK cells by creating a surface shield and normal MHC-I [60], or by downregulating natural killer group 2 member D (NKG2D) and its ligands, further stimulating glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) to exert functions in NK cells [61–63]. Furthermore, platelets involve the adhesion process of endothelial cells. The attachment between platelets and CTCs is enhanced by integrin αIIbβ3 and P-selectin (platelet adhesion receptors), in which supports the strong adherence of CTCs to the endothelial wall [64–66].
