ACCESO AL MERCADO DE EXPORTACIÓN

Once suitably mutated single cancer cells and oligoclonal clusters derived from the same primary tumour (Aceto et al., 2014) have invaded the tissue far enough to find themselves adjacent to a lymph or blood vessel (whether in the form of or individually), they can potentially intravasate into the blood system through the basal laminae of blood vessels. The exact mechanism of intravasation into the vasculature is still unclear, mainly because it shows to be difficult to establish tumour models in which intravasation and cancer cell shedding can be observed (Bockhorn et al., 2007). While there is experimental evidence suggesting that a subset of cancer cell lines may only be able to access the blood vessels indirectly via prior intravasation into the lymph vessels, the spread to distant sites in the body ultimately happens by dissemination through the blood vessels (Wong and Hynes, 2006; Lambert et al., 2017). Also, two main intravasation modes —active versus passive intravasation—are proposed in the biological literature. These are likely not mutually exclusive (Cavallaro and Christofori, 2001; Bockhorn et al., 2007; Jie et al., 2017). The active intravasation hypothesis postulates that cancer cells crawl towards and into vessels actively with the help of MDEs while being led by chemokine and nutrient gradients. Passive intravasation, on the other hand, implies a more accidental shedding of cancer cells via newly formed immature vessels, which are fragile and may collapse due to trauma or under the physical pressure caused by rapid tumour expansion.

The following overview of evidence for active and passive mechanisms in cancer cell intravasation was collected by Bockhorn et al. (2007). It highlights that there exists evi- dence for both the active and passive intravasation hypothesis. In Bockhorn et al. (2007), each of the bullet points below is supported by up to five studies.

Evidence for active intravasation:

• cytoskeletal activity associated with metastasis; • integrin upregulation involved in metastasis; • accumulation of mutations needed for metastasis;

• transient, microenvironment-induced changes in gene profile; • MMPs produced by metastatic cells;

• tumour microenvironment can induce migration-related pathways via hypoxia and

Evidence for passive intravasation: • most shed cells are non-viable; • shed cells are not clonogenic; • blood vessels have fragile walls; • solid stress collapses vessels;

• loss of cell-cell and cell-matrix adhesion associated with shedding and metastasis; • tumour microenvironment confers survival advantages to random cells via hypoxia

and other stresses.

A particular focus of this thesis is the role of mesenchymal-like cancer cell phenotypes in contrast to epithelial-like phenotypes—Can cells of one phenotype intravasate without the presence of the other?

A study by Tsuji et al. (2009), the results of which are visually explained in Fig- ure 2.12, sheds some light on this question. Using a mouse model, the study examined cancer cells that had undergone EMT—and were thus of mesenchymal phenotype—and cancer cells of epithelial phenotype with regards to their intravasation success. Suc- cessful intravasation was measured as the cells’ ability to penetrate blood vessels once they had been transplanted into the mice subcutaneously. While tumours consisting of mesenchymal-like cancer cells only were able to intravasate (top row of Figure 2.12), those consisting of epithelial-like cancer cells only were not (third row of Figure 2.12). Simul- taneous subcutaneous injection of the two cell types resulted in successful intravasation of both cell types (fifth row of Figure 2.12).

The difference between mesenchymal-like and epithelial-like cancer cells, as described in Section 2.3.2 together with the above-explained differentiation between active and passive intravasation gives rise to three entry modes of cancer cells into the vasculature. These are further explained in Francart et al. (2018):

• Single MDE-expressing mesenchymal-like cancer cells actively enter the blood ves-

sels and thereafter disseminate as single circulating tumour cells (CTCs).

• Cancer cells of epithelial and of mesenchymal phenotype cooperate in the sense

that mesenchymal-like cancer cells allow epithelial-like cancer cells to enter the vasculature together with or shortly after them. Mesenchymal-like cells express the MDEs required to degrade the vessels’ basal laminae. This allows for co-invasion of the epithelial-like cancer cells in the vicinity. Thus, both mesenchymal-like and epithelial-like cancer cells enter the blood system jointly as a cluster.

• Any single cancer cell or cancer cell clusters near a ruptured blood vessel intravasate

via the passive entry mode.

These entry mechanisms are depicted—left to right—in Figure 2.7 along the upper left blood vessel wall.

Figure 2.12: Visual representation of results from mouse model by Tsuji et al. (2009) suggesting synergetic effects of cells of mesenchymal and ep- ithelial phenotypes in metastatic spreading. (A) Mesenchymal-type cells alone did intravasate into the bloodstream but did not form lung metastases; (B) Epithelial- type cells alone were unable to intravasate but, if injected directly into the bloodstream, could extravasate and metastasise; (C) Jointly subcutaneously injected epithelial-like and mesenchymal-like cells invaded the tissue locally, intravasated, extravasated and success- fully colonised as metastases; (D) Proposed synergetic model of cancer cell metastasis: Mesenchymal-like cells invade into surrounding tissue clearing the path for cells of ep- ithelial phenotype to invade and intravasate. Cell types jointly enter the circulation but only the epithelial-like cancer cells can successfully colonise at distant organs. Reproduced from Tsuji et al. (2009) with permission from the American Association for Cancer Re- search.

Figure 2.13: Cancer cells in the blood system. Once single cancer cells or cancer cell clusters have intravasated, several mechanisms—both to aid the cancer cells (e.g. platelets covering cell surface, neutrophils that enhance extravasation through NET expression, and MMP secretion) and to destroy them (e.g. physical stresses; attacks by NK cells)—come into action. Reproduced from Lambert et al. (2017) with permission from Elsevier Inc.