**2.9. Details of ECM invasion**

It is possible to estimate the site of extravasation of tumor cells and the distribution of the metastases in the organs by looking at the site of the primary tumor and the vascular or lymphatic drainage (**Figure 4**). Most tumors metastasize in the first organ they encounter in the capillary bed upon entering the circulation. However, natural drainage paths may not easily explain the distribution of metastases in many cases. Some tumors such as lung cancers frequently metastasize in the adrenals, while they almost never spread to the skeletal muscle [65]. This organ tropism may be associated with the following described mechanisms:

32 Tumor Metastasis

**Figure 4.** ECM role in tumor angiogenesis, lymphangiogenesis. Angiogenesis and lymphangiogenesis depend on the ECM. Tumor cells produce various components, including VEGF and angiogenic and antiangiogenic ECM fragments, to regulate blood vessel formation (stage 1). During branch initiation, endothelial cells secrete proteases to break down the basement membrane to grow out (stage 2). The outgrowth process of endothelial branching is propelled by at least two groups of cells: tip cells, which lead the migration toward the angiogenic chemoattractant source, and stalk cells, which depend on the ECM and its derivatives to survive and proliferate to provide building blocks for vessel forma‐ tion (stage 3). Additionally, ECM components participate in cell migration and other aspects of tubulogenesis of blood vessels. Although details remain unclear, lymphangiogenesis depends on the ECM and, together with angiogenesis,

1. The expression of the ligands in the tumor cells and preferably of the adhesion molecules present in the endothelium of the target organs. 2. The expression of chemokines and their receptors. Chemokines contribute to the guided movements of leukocytes (chemotaxis), and cancer cells appear as cells which utilize similar tricks in order to settle in special tissues. Chemokine receptors named CXCR4 and CCR7 have a high expression in human breast cancer. The ligands of these receptors (CXCK12 and CCL21) are present in high amounts only in the organs where breast cancer cells have metastasized. Based on this observation, it was claimed

provides routes for cancer cell metastasis and immune cell infiltration (see ref. [5]).

that the blockage of chemokine receptors may limit metastases [44, 66].

Willis et al. drew attention to the astuteness in the invasion of the devilish hidden cancer cells in the review they published [7]. Many groups reached the conclusion that cancer cells acquire an amoeboid phenotype characterized by insensitivity to proteinous inhibitors and surpass type I collagen barriers [12]. Now we know that a wide spectrum of types of cancer cells are definitely dependent on MT1‐MMP when they are faced with cross‐linked Type I collagen barriers [12, 71].

Still, when cancer cells encounter structural barriers, they hold the potential to adapt them‐ selves to a protease‐dependent position. Although there is limited information on the size of ECM pores, it is estimated via confocal reflection microscope that micropores range between of 40–10 μm<sup>2</sup> and macropores of 40–1000 μm<sup>2</sup> inside *in vivo* tissues [72–74]. These results increase the probability indicating that the collagen structure combined in an *in vitro* setting may not be repeated in a complex *in vivo* setting.

However, it should be noted that the defects in the migration of vascular smooth muscle cells, adipocytes differentiation, and stem cell origin displayed an *in vitro* setting duplication with the use of dense acid extracted type I collagen hydrogels in MT1‐MMP‐targeted mice [75–77]. Interestingly, the diameter of collagen fibers at *in vivo* neoplastic fields matched with the self‐ polymerized collagen hydrogels prepared in acid extractor type I collagen under standard conditions [78].

These results led to the thought that cancer cells may rapidly migrate to precleared tunnels via the proteolytic pathway through proteinaceous‐independent processes similar to those in the *in vitro* setting [11, 79–81].

#### **2.10. Role of ECM and ECM‐associated proteins in metastasis**

As cancer cells accumulate mutations or other molecular signals during the metastatic process, they are predisposed to become more easily malignant and lose contact with the surrounding cells and ECM in the primary tumor. These surrounding cells provide the opportunity for invasion. Thus, ECM and ECM‐associated adhesion proteins play a critical role in the meta‐ static process [82]. Therefore, Zacharia et al. [83] published a review describing roles of the new molecules named migfilin, mitogen‐inducible gene‐2 (Mig‐2), and Ras suppressor‐1 (RSU‐ 1) in the cell‐ECM adhesion fields. The authors reached the conclusion that cell‐ECM adhesion proteins are predisposed to function such as adaptor proteins in the form of multiple protein‐ protein interaction in the cell‐ECM adhesion fields.

Even though the different effects in various types of cancer cells were discussed, they added that cell adhesion, which is crucial in terms of cell metastasis in many cases, supported cell invasion and apoptosis.
