Tumor pathogenesis is primarily attributed to tumor invasiveness, which is largely determined by the migratory behavior of cancer cells towards and into the surrounding tissue (). Intravital MPM was instrumental in revealing two distinct modes of cancer cell motility, as distinguished by morphological changes during cell movement [39
]. The migration of cells with spindle-like shape, termed mesenchymal or fibroblastic migration, proceeds via pseudo-pod protrusion at the leading edge, followed by focal attachment to the ECM, and detachment of the trailing edge. To pass through the fibrous ECM networks without inflicting excessive distortion to the rigid nucleus, cancer cells created openings between the fibers to then squeeze through the openings. Indeed, the mesenchymal migration was associated with ECM degradation and formation of tube-like tracks lined with ECM degradation products. The dynamics of ECM degradation and cell translation depended on a matrix metalloproteinase (MMP) activity at the focal attachment points as well as integrin-driven adhesion. Interestingly, upon inhibiting extracellular proteases, tumor cells underwent a mesenchymal-amoeboid transition. In particular, they acquired a more spherical morphology, became highly deformable, and instead of digesting their way through the ECM, moved in a propulsive, amoeboid manner by cytoplasmic streaming and squeezing [41
]. The amoeboid movement was associated with short-lived and weak interactions with the substrate, allowing the cells to reach higher velocities. The process of mesenchymal-amoeboid motility transition could be involved in increasing tumor invasiveness and, if it enables cancer cells crossing into the blood circulation, it could increase metastasis.
Collective cell movement refers to coordinated translational motility of cell sheets, aggregates, or clusters migrating as a functional unit [42
]. Multiphoton imaging of a sarcoma tumor margin revealed that cancer cells could proceed into the surrounding tissue in a collective manner by assembling and migrating together in highly asymmetrical multicellular strands [17
]. The cells at the leading edge were highly motile, guiding the cells that followed, whereas the cells in the inner and rear regions of the collective movement unit were pulled passively. The pack’s movement through the collagen network was facilitated by membrane-bound MMPs on the anterior cells. The MMP activity cleaved collagen fibers in such a way that the fibers become aligned parallel to cell body to form tracks of least mechanical resistance, thus facilitating the passage of the cells that followed [45
]. The consecutively passing cells further amplified the process, such that large macrotracks formed that guided the collective invasion. In addition to MMPs, the joint movement also depended on the activity of β1 integrin. Upon blocking both β1 integrins and MMPs, the collective cell motility pattern was abolished, transitioning to more random singular motility; a similar phenomenon was also triggered by TGFβ signaling [43
]. Interestingly, the ECM-derived peptides that were produced by tumor-mediated proteolysis further stimulated cancer cell motility, suggesting a chemokinetic positive feedback loop that could enhance cancer cell migration and tissue invasion [40
The acquisition of migratory behavior by cancer cells is suspected to contribute to metastasis, a poorly understood process of remote tissue colonization requiring, at an initial stage, cancer cell intravasation into the bloodstream or the lymphatics. Interestingly, intravital tracking of quantum dot-labeled tumor cells in mice showed that, in addition to the hematogenous and lymphatic routes, cancer cells can use primo-vessels—semitransparent fluid-conducting multi-lumen channels—to disseminate and form metastases in the peritoneum [48
]. Using MPM, the migratory capacity of non-metastatic and metastatic cells was compared in primary mammary carcinoma tumors [49
]. The cells of the metastatic line MTLn3 interacted more often with collagen fibers than the non-metastatic MTC cells, despite higher collagen content in the non-metastatic tumors. Although their migration rates were similar (3.4 μm/min), the metastasis-prone MTLn3 cells tended to move in a more linear fashion along collagen fibers, stopping whenever fibers were missing. The metastatic capacity also correlated with the ability of tumor cells to reorganize the collagen networks [23
]. In the vicinity of blood vessels, the metastasis-prone MTLn3 cells polarized and migrated toward the vessels, indicating sensitivity to a guiding gradient—a behavior that contrasted with mostly random motility of non-metastatic MTC cells. Epidermal growth factor receptor (EGFR) could be involved in the cell guidance since metastatic tumors had high levels of EGFR and responded to EGF gradients [49
], which originated from tumor stroma and vasculature [47
]. Indeed, over-expression of EGFR in MTC cells increased their chemotactic responses to EGFR ligand in correlation with the metastatic potential [51
]. Only few MTC cells were observed entering the blood vessels, and those that did underwent fragmentation due to the hydrodynamic shearing of pseudo-pods that penetrated into the blood stream, resulting in the cell’s death by apoptosis. The intravasating MTLn3 cells, in contrast, remained intact, their mechanical stability being greater due to increased cytokeratin content. The results of the experiments with the implanted MTLn3 and MTC cell lines, which were further corroborated in spontaneous tumor models [52
], support the notion that the acquisition of metastatic potential by cancerous cells can be in part attributed to a coordinated deregulation of multiple pathways involved in directional cell motility, including chemotaxis, ECM-mediated guidance, and cellular polarization.
Intravital imaging showed a spatial heterogeneity in the ability of tumor cells to migrate within tumors [53
]. Using a chronic cranial window, the motility of red fluorescent protein (RFP)-expressing glioma was recorded in vivo in relation to blood vessels, which were visualized by intravenous injection of high molecular weight fluorescein isothiocyanate-dextran. Tracked over 48 h, the cells migrated predominantly away from the tumor center (84% of cells), stretching out along the abluminal site of the endothelial cells of brain microvessels (91% of cells). The speed of perivascularly moving cells, while relatively slow (0.024 μm/min), was 3.6-fold higher than the cells positioned away from capillaries. The perivascular cancer cell motility coincided with vessel remodeling, capillary dilation, vessel splitting, and glomeruloid bodies (loops) formation.
The mechanisms of tumor invasiveness were further corroborated in a breast cancer window model [54
]. Orthotopically implanted mammary tumor MTLn3 cells expressed the photoswitchable protein Dendra2, whose fluorescence can be changed from green to red by intense UV illumination, thus allowing individual cells to be marked. In the region that lacked discernable blood vessels, UV-photomarked cancer cells stayed close together, whereas in the vascularized regions, photomarked cancer cells spread out over a larger area, demonstrating a higher migratory potential. A faster drop in the number of photo-marked cells in the vascularized region suggested ongoing intravasation. It is thus likely that tumor vascularization can increase its metastatic potential by affecting cancer cell migration.
To study the mechanisms of cancer cell extravasation, leukemic cells were injected intravenously and followed in the bone marrow. Circulating metastatic cancer cells extravasated preferentially in distinct “hot spot” sites that were demarcated by increased concentration of the vascular cell adhesion molecule E-selectin and the SDF-1 chemokine [55
]. Interestingly, not only leukemic cells but also hematopoietic stem/progenitor cells and lymphocytes used the same hot spots to arrive in bone marrow. SDF-1 binding to its receptor CXCR4 was critical, since CXCR4 blockade and/or downregulation inhibited cancer engraftment.