Tumour microenvironment - non-cellular stroma

What are the main components of cancer?

Cancer is dominated by non-neoplastic stromal cells and non-cells (ECM).

What is the ECM and what is its composition and roles?

The ECM is a reinforced composite of structural proteins (collagen [most present], laminin, fibronectin, etc.) that are organised as fibrillar structures embedded within a viscoelastic gel containing proteoglycans, glycoproteins, water, growth factors and other metabolites secreted by cells. It provides the structural support and biochemical signals for multicellular tissues.

ECM constitutes up to 60% of the tumour mass in solid tumours. Collagen deposition results in desmoplasia, which is strongly linked to poor prognosis.

What is the tumour stroma composed of and how does it change?

The tumour stroma mainly consists of the BM, fibroblasts, ECM, immune cells and vasculature. Although most host cells in the stroma possess certain tumour-suppressing abilities, the stroma will change during malignancy and eventually promote growth, invasion and metastasis.

What are glycosaminoglycans?

GAGs are highly negatively charged polysaccharides. The charge is due to the presence of sulfate and carboxyl groups. It attracts cations. The high cation concentration in the ECM creates an osmotic pressure gradient, therefore water moves into the ECM by osmosis. Water imparts gel-like properties which resists compression. This is important in cartilage. In solid tumours, these properties are changed to become more stiff. 

How do integrins regulate cell movement?

Integrins are important in coordinating cell movement. At the front of the cell, there is a protrusion called lamellipodium, driven by cytoskeletal reorganisation. Integrins are important in adhering the front of the cell to the substrate beneath it. At the same time, integrins release at the back of the cells. The same cycle is repeated over and over again, moving the cell forward in a stepwise fashion. In this way, a cell’s external attachments can help regulate its behaviour – and even its survival.

What is the role of fibronectin and integrin?

Fibronectin and integrins help cells attach to the ECM. Integrins function as transmembrane heterodimers (alpha and beta subunit - 18 α and 8 β subunit flavours that combine to make 24 integrin heterodimers in humans). Different combinations of subunits allow integrins to bind to different components of the ECM. The integrin molecule transmits tension across the plasma membrane: it is anchored inside the cell via adaptor proteins to the actin cytoskeleton and externally via fibronectin to other extracellular matrix proteins, such as the collagen fibrils. 

• Fibronectin serves as a molecular bridge between integrins and the ECM. They are parallel homodimers joined by a disulphide bridge. It has 2 key binding sites; integrin site and ECM binding site.

How are integrins dynamically regulated?

Integrins can be dynamically regulated - their activation state is regulated allosterically (the shape of the integrin determines whether it is active or not). Inactive - the extracellular domain is collapsed against the membrane and its intracellular regions are bound together (hard for molecules to bind). Active - the extracellular face becomes extended and accessible, and intracellular domains separate. Both α and β subunits can switch between a folded, inactive form and an extended, active form. An integrin protein switches to an active conformation when it binds to molecules on either side of the plasma membrane such as an ECM molecule or intracellular adaptor. In both cases, the conformational change alters the integrin so that its opposite end rapidly forms a counterbalancing attachment to the appropriate structure. In this way, the integrin establishes a reversible mechanical linkage across the plasma membrane.

• Integrins make and break connections between the cell and the ECM, allowing cells to move through tissues. Some integrins also bind to counter-receptors on other cells (e.g. ICAM, VCAM) to enable cell-cell adhesion.

How are recruitment of stromal cells and generation of ECM rate limiting steps?

Recruitment of stromal cells and generation of ECM are important rate limiting steps in tumour formation. As tumour progression proceeds, the fibroblast-rich stroma is replaced by myofibroblasts which generate desmoplastic stroma. Myofibroblasts predict clinical progression of cancer - inverse relationship between stromal myofibroblast density and patient survival time. 

How is the ECM remodelled?

Cells degrade the ECM as well as make it. Remodelling of the ECM is important - ECM must be degraded to allow cells to pass between endothelial cells such as immune infiltration. There are 2 main groups of ECM degrading enzymes; MMPs which require a bound Ca2+ or Zn2+ for activity, and serine proteases which have conserved serine residues in the active site. Some are high substrate-specific. Localised degradation (not everywhere) of ECM maintains overall ECM structure but creates enough space for migrating cells. Degradation also released a variety of growth factors.

• Activation of fibroblasts by TGF-beta and conversion into myofibroblasts induces release of MMPs. 

How are ECM-degrading proteases regulated?

Activity of ECM-degrading proteases is kept tightly localised. Some proteases are secreted in an inactive form, and a localised activator converts them into active form (only secreted where degradation required). Some proteases are confined by cell surface receptors - for example, urokinase-type plasminogen activator is found at the growing tips of some migrating cells and is elevated in breast tumours. Some proteases are inhibited by the actions of locally secreted inhibitors, such as tissue inhibitors MMPs (TIMPs).

What are invadopodia?

Invadopodia are dynamic, actin-rich membrane protrusions that degrade the ECM through local deposition of proteases that are key to cell invasion.

When does true malignancy begin and what ways does this occur?

True malignancy begins when the tumour cells penetrate or destroy the basal lamina and invade the underlying connective tissue. There are 2 ways in which tumours can initiate breakage of ECM; non-proteolytic but force-mediated (cytoskeletal tension) and proteolytic. Proteases are used to degrade ECM that forms a barrier to cell migration, clearing the path for tumour cells to move away from the relatively contained primary tumour. Invading tumour cells actually migrate along aligned collagen fibres that form tracks to facilitate tumour cell migration. There are clinical trials underway of cancer therapies targeting ECM-degrading enzymes, such as MMPs.

What are the definitions of invasion and metastasis?

• Invasion - penetration of surrounding tissue and migration of cells into neighbouring tissue.

• Metastasis- spread of cancer cells to tissues beyond the primary tumour leading to the formation of secondary tumours.

What is invasion and what follows it in metastasis?

The focus in this unit is the invasion step of the metastatic cascade. Invasion is driven by changes in cell adhesion, enhanced cancer cell migration and ECM degradation. This is followed by intravasation, circulation, extravasation and colonisation.

What is epithelial-to-mesenchymal transition and why is it important in cancer?

Epithelial-to-mesenchymal transition is a key process that allows cancer cells to develop a more migratory phenotype. They are irregular shaped, loss of apico-basal polarity, gain of front-back polarity, more dynamic cell-ECM adhesions, lamellipodia and filopodia, loss of cell-cell junctions, and motility. EMT is found in developing tissues (e.g. neural crest cells in neural tube development). EMT is also aberrantly activated under pathological conditions such as cancer, where it is associated with cancer cell invasion and dissemination.

Lots of common cancers arise in epithelial tissues (~80%) - termed carcinomas. EMT is a crucial cellular programme that enables polarized epithelial cells to transition towards a mesenchymal phenotype with increased cellular motility. A benign epithelial tumour remains bounded by the basal lamina, whereas a malignant epithelial tumour contains migratory cells that invade through the basal lamina, which destroys the integrity of the tissue.

What causes EMT and what happens during it?

EMT is caused by an external event like a change in GFs, TGF-B/SMAD, Wnt/beta-catenin, integrins and inflammation events (NF-KB). This causes intracellular events via heterotypic signalling. It causes the downregulation of E-cadherin and epithelial genes like mucin, cytokeratin, ZO-1, and surfactant proteins. It also causes the upregulation of mesenchymal genes like N-cadherin, smooth muscle actin, collagen I, vimentin and FSP1/S1004A. There are master TFs involved in the mesenchymal phenotype which become activated - include; Snail, Slug, Twist and Zeb1.

There is translocation of beta-catenin in EMT. Cells at the tumour centre express it at the plasma membrane (and diffusely in the cytoplasm). Cells at the invasive front (migrating) express it in the nucleus - nuclear localisation of beta-catenin induces expression of EMT genes. This is because loss of E-cadherin from the membrane liberates beta-catenin molecules.

What are cadherins?

Cadherins mediate cell-cell contacts. 2 cadherins found on neighbouring cells bind together to keep them close. Downregulation is a factor during EMT. Their cytoplasmic tail also has adaptor proteins (alpha and beta catenin). When cadherins are lost, these adaptor proteins are now free inside the cells, so tend to move into the nucleus where they act as TFs and activate mesenchymal genes.

What is the role of calpain?

E-cadherin protein has a half-life of 5-10 hrs and is highly susceptible to proteolytic degradation. Caspases are proteolytic enzymes activated during apoptosis. They degrade numerous essential proteins, including β-catenin and α-catenin This leads to apoptotic cell death. β-catenin and E-cadherin are also substrates for calpain-mediated proteolytic cleavage. Thus, calpains (Ca2+-dependent cysteine proteases) may also modulate cadherin-dependent cell-cell adhesions. Calpain is upregulated in breast tumour, renal cell carcinoma, chronic lymphocytic leukemia B cells. 

• In renal cell carcinoma, elevated levels of calpain-1 correlate with tumour metastases.

How is calpain activated?

Caplain is switched on in response to the activation of the oncogene v-Src. Calpain substrates are involved in the regulation of cell adhesions (calpain inhibitors suppress migration). Caplain substrates are also involved in cell proliferation (p53, p27, cyclin D - inhibitors suppress proliferation). Calpain expression is upregulated in renal cell carcinomas that metastasise to peripheral lymph nodes, and breast cancers. Caplain-2 is proposed as a target for limiting prostate cancer invasion.

What therapies impact tumour invasion?

Current therapies that impact tumour invasion upregulate the function of adherence junctions, promoting cell–cell adhesion and preventing tumour cell invasion. These are mediated by upregulated E-cadherin, α-catenin and β-catenin gene expression, dephosphorylation of β-catenin, increased stabilization of β-catenin at cell–cell adhesions. Currently there are no therapies specifically targeting metastasis. Some examples of therapies targeting invasion include:

• Aspirin can restore E-cadherin dependent cell–cell adhesion (unclear mechanism).

• MMP inhibitors inhibit tumour-induced degradation of ECM proteins. Used in combination therapies (counteract migration mode switch). 

• EGFR inhibitors inhibit tumour growth and metastasis. 

• VEGFR inhibitors inhibit angiogenesis, tumour growth and metastasis. 

• Src inhibitors inhibit tumour growth and metastasis. 

• Cilengitide, a peptide, cyclo(-RGDfV-), is selective for αv integrins.

What are the modes of cancer migration?

There are 3 modes of cancer cell migration. Mesenchymal migration has a front-back polarity, it involves the cytoskeleton to move (actin is tightly regulated) and involves Rho-GTPases (Rac/Rho/CDc42). At the front, all 3 members are involved, Rac is involved in the formation of new adhesions, at the rear end, Rho plays a role in removing adhesions and then contracting the cell body to propel itself forward. MMPs are targeted to the front of the cell to remove the ECM to move. Amoeboid migration is not polarised and pretty rounded. It migrates by protruding blebs (bulges of membrane) to squeeze through gaps. It also involves MMPs and actin remodeling but not to the same extent as mesenchymal. Collective migration is where sets of cells are moving together (retention of cell-cell contacts), with leader cells often at the front (likely mesenchymal - secrete MMPs etc).

Why are laminin and stromal cells important for controlling invasion?

Laminin forms the basement membrane and often needs to be broken down for tumours to start migrating and invading. For example, samples taken from patients with high-grade rectal carcinoma showed that those with intact BMs had zero metastasis, even following up after 5 years. Prognosis for a patient is better prior to the basement membrane breaking down. 

Stromal cells are also key for control of invasion. In a mouse model of intestinal carcinogenesis, colon adenoma cells recruit bone marrow-derived CD43+ myeloid cells to the invasive front. These secrete MMP-2 and -9 to enable collective invasion of the neoplastic cells into the mesenchymal layers of the stroma.

What is Src?

Src is an enzyme that regulates integrin complexes. It is a non-RTK that disrupts normal epithelial structure (cell-cell contacts) and promotes an invasive phenotype in cells (controls the assembly and turnover of focal adhesions between integrin and ECM). Src activity in colon and breast cancer correlates with tumour progression. Inhibition of Src reduces invasion, and delays dissemination and progression of the disease.

What are some examples of signalling pathways that have dual roles in tumour progression?

There are signalling pathways which have dual roles in tumour progression. One example is TGF-beta. Another example is SASP; in precancerous tissues, the effects of SASP are predominantly immunosuppressive (autocrine and paracrine senescence and induction of immunosurveillance). In advanced cancerous tissues, SASP factors from stromal cells such as CAFs can promote tumour growth. The last example is fibroblasts. In early stages, fibroblasts drive proliferation of mutated cancer cells. During development, CAFs can protect against tumour invasiveness. In advanced stages, CAFs can be reprogrammed to allow tumour cell growth. As the tumour evolves, CAFs reorganise the TME to promote angiogenesis and EMT.