Invasion and metastasis

What is malignancy and metastasis?

Malignant neoplasia is distinguished from benign neoplasia by the acquired ability of malignant cells to spread from their point of origin. This occurs via metastasis - the process by which tumour cells disseminate to distant sites to establish discontinuous secondary tumours. Metastases account for 90% of cancer mortality - compromises organ function, internal bleeding, affects endocrine and nervous systems, cachexia. Tumour types and animal species have very different propensity (tendency) to form metastases - for example, melanoma is quick. Tumours may generate metastases throughout the body or some may have a more defined anatomical preference for metastases. For example, prostate cancer tends to metastasise in bones.

How does metastasis occur?

Metastasis is a cascade of events; detach from original location and breach of basement membrane, migration through the surrounding stroma, enter blood through crossing the endothelial cells via intravasation (some spread through lymphatics, nerves, and outside of blood vessels) and then they travel through the blood/lymph vessel (as clusters). In order to travel through the blood/lymph vessel they have to survive in this foreign environment. As the blood vessels narrow, the tumour cells tend to get stuck in capillary networks, and then they extravasate into the tissue (may require adaptation to survive the new environment). Secondary tumours are then formed via proliferation to form a new colony of cells.

What is the structure of epithelium?

Epithelial cells sit atop a specialised layer of ECM known as the basement membrane. They cover our organs and are specialised to carry out a specific function. They are also highly polarised. The BM comprises multiple proteins such as laminin, type IV collagen, proteoglycans such as perlecan and nidogen. To a cell it is a major physical impediment to the invasion of neoplastic epithelial cells. Epithelial cells are riveted to the basement membrane by specialized adhesion complexes termed hemidesmosomes. These comprise integrin extracellular adhesion molecules (heterodimers) that cross-link components of the basement membrane, notably laminin, to the intermediate filament cytoskeleton comprising cytokeratins. Furthermore, epithelial cells are tightly bonded to each other through multiple junctional complexes to form a barrier that limits diffusion and provides mechanical strength.

What are the obstacles tumour cells must overcome to invade?

There are several obstacles to overcome for a cell to become locally invasive;

• Reduce cell-cell adhesion often involving the downregulation of E-cadherin (main adhesion molecule in cell-cell adhesions).

• Proteolytic degradation of the BM using specialised subcellular structures called invadopodia.

• Acquisition of a mobile phenotype - often involves adhesion to the stromal ECM requiring altered integrin expression, cytoskeletal reorganisation, propulsive force often driven by actomyosin contraction and actin polymerisation, and proteolytic degradation of the stromal ECM done by matrix metalloproteinases.

• This invasive phenotype results from inappropriate expression/activity on oncogenes. It causes an epithelial-to-mesenchymal transition (EMT) of the cells whereby they gain the mobile morphology.

How does down-regulation of E-cadherin occur?

Downregulation of E-cadherin is either by a deletion mutation in some or epigenetic mechanism through methylation in many cancers. E-cadherin is the major adhesion molecule mediating epithelial cell-cell adhesion. Its ectodomain binds homotypically. Its cytoplasmic domain is cross-linked to actin filaments through adaptor molecules called catenins. While normal epithelia express abundant E-cadherin, invasive carcinoma cells express very little.

What are ivadopodia?

Invadopodia are organelles which are out-pockets of the cell membrane driven by actin filaments. At the tips, there is secretion of matrix metalloproteinases which degrade the ECM.

What is mesenchymal migration?

Mesenchymal mechanism of migration is through integrins which attach to the ECM (extracellular) and actin (intracellular). They pull the cell along. They are heterodimers consisting of an alpha and beta subunit - they bind specific ECM components. Experimental evidence for the role of integrins in metastasis is provided by experiments where injection of RGD tripeptides used to interfere with integrin binding to the ECM blocks metastasis of cancer cells inoculated in a mouse host.

Propulsive force is generated by cytoskeletal reorganisation. At the leading edge, actin polymerisation and cross-linking results in a sheet-like protrusion which adheres to the substrate. Contraction of the cell body by myosin sliding actin filaments and detachment of the cell rear result in forward propulsion. Lamellipodia are structures found at the leading edge of the cell formed mainly by actin filaments. These structures help cells to move by extending the cytoplasm forward, creating new “anchor sites” to allow the back of the cell to retract creating net movement. Filopodia are thin protrusions that help the cell to sense the surroundings of the cell. Stress fibres are what allow contraction of the cell body.

What enzymes drive actin reorganisation?

Actin cytoskeletal reorganisation is driven by Rho GTPases. There are 3 main families; Rac, Rho and Cdc42. 

How are Rho molecules and how do they work?

Rho molecules are members of the Ras superfamily of small GTPases. These are ~21 kDa peptides with intrinsic GTP hydrolase activity that cycle between an inactive GDP bound state and active GTP bound state. Thus Rho molecules too are molecular switches that can be deactivated by GAPs which accelerate hydrolysis and activated by GEFs which catalyse GTP loading. They can also be inactivated by sequestration from the plasma membrane by binding to Rho GDI.

• Staining a quiescent cell shows a very stable cytoskeleton. Staining a cell with Rac induction shows a lot more actin polymerisation therefore lamellipodia, and there are more focal contacts via vinculin. With Cdc42, there are more filopodia via actin and vinculin distribution has now changed but still coincides with organised actin at filopodia. When Rho is activated, cells form stress fibres, which act as tensing cables that allow the cell to retract the cell, and, after lamellipodia formation, create net movement.

• In reality, this is not going on all the time all around the cell, but rather there are more stress fibres in the cell body, more actin polymerisation at the leading edge etc.

What are the roles of proteases in invasion?

Proteases play key roles in invasiveness by degrading the ECM. The proteolytic activity occurs at the leading edge of the cell. Proteases are bound either to the membrane as integral membrane proteins, or to the outer leaflet of the membrane via a GPI-anchor, or to other membrane proteins (receptors). Proteases are frequently synthesised as inactive proenzymes which are activated by proteolytic cleavage. During the metastatic process, invading cells encounter very different matrices. Depending on the tools they possess (types of proteases, capacity to contract, form lamellipodia…) they can sort those obstacles more or less efficiently. Protease inhibitors have been used experimentally and show that the nuclei cause cells to become trapped in the BM since it is the largest organelle.

What are the alternatives to mesenchymal migration?

Alternatives to mesenchymal invasion include amoeboid (integrin independent) and collective cell invasion (a few mesenchymal cells lead and create a trail of degraded ECM for other cells to migrate through).

What is intravasation and why is it dangerous?

Intravasation entails degrading the blood vessel basement membrane in the circulation. Tumour cells must be able to resist apoptosis due to loss of ECM attachment (anoikis), as well as survive shear stress and predation by immune cells. The vast majority perish in circulation. Clustering is one way they can survive - cells in the centre are less affected by the environment.

What is the final step of metastasis?

Tumour cells can not deform as readily as blood cells and are many times their dimension. Thus they often get lodged in capillary beds. This is followed within minutes by platelets forming a microembolism. The cancer cells denudes the overlying endothelium and attaches to the basement membrane. The microembolism is dissolved within a couple of days. Cancer cells then begin to proliferate within the lumen of the capillary before erupting into the surrounding tissue (rather than migrate out).

Why is metastasis inefficient?

Metastasis is a highly inefficient process. Only the minority of cells capable of local invasion enter the circulation, the majority of these die. Of those that survive at a distant site, only the minority give rise to clinically significant tumours. The majority become dormant or fail to thrive, that is, they only form micrometastases, due to inadequate blood supply. Dormancy may persist for decades, and residual disease in the form of micrometastases affects prognosis. 

Why do some metastases have secondary site preferences?

Some metastases have preference for secondary sites. The determinant sites are affected by pattern blood flow, tissue ‘tropism’ and homing signals. 

• Pattern blood flow - cancer cells will typically become trapped in the first capillary bed they come across - colorectal cancer tends to form metastases in the liver - the blood from the large intestine goes straight to the liver. 

• Tissue tropism - only certain tissues will have similar tropic signals as present as the primary site to support the growth of escaped tumour cells.

• Homing signals - cancers express receptors for chemoattractants called chemokines therefore they drive a homing response to the source of the chemoattractant.

Why is it difficult to treat metastasis?

The problems with targeting metastasis include occult disease (many undetectable micrometastases) and successful metastasis (body is full of large secondary tumours). 

• Various rationally designed antimetastatic compounds trigger measurable responses in preclinical preventative settings where treatment is initiated prior to the formation of primary tumors or metastases. These include protease inhibitors, inhibitors of cytoskeletal reorganisation etc. Because carcinoma patients frequently already harbor significant numbers of disseminated tumor cells at the time of initial disease presentation, they do not work clinically.