Lecture 17: Invasion and Metastasis

Metastasis

• The most deadly aspect of neoplasia is its ability to infiltrate and invade other tissues, spreading around the body, forming 2° tumours → distinguishes malignant neoplasias from benign neoplasias → spread from point of origin

• The process by which process by which tumour cells disseminate to distant sites to establish discontinuous secondary tumours, either throughout the body or with a defined anatomical preference

  • Commonly occurs in melanoma

• Propensity to form differs between species and cancers

Why Do Metastses Account for 90% Cancer Mortality Rate

• This is due to the spread of tumours around the tissue being deleterious

• It compromises organ function and can lead to internal bleeding as it grows through blood vessels;

• It can affect nervous and endocrine systems and cause cachexia (general malaise)

  • muscle and adipose tissue wasting seen in advanced stages of cancer

  • associated with the high metabolic demands of cancer and inflammation associated with cancers and cytokine build-up

Difficulty Targeting Metastatic Cancers

• One of the most important cancer hallmarks, but it is poorly understood due to its complex nature.

• There are no effective therapies that block the metastasis cascade.

• Secondary tumours (2°) can be treated like primary tumours (1°), but they are less responsive to chemotherapy, and treatments are less effective at later stages of cancer.

Invasion Metastasis Cascade

• Multi-step process/ cascade of events → movement through the BM, migration thorugh interstitial matrix and in/out blood vessels and colonising tissues

• 1. Detachment from the original location and breach the basement membrane

• 2. Migrate through the stroma/ interstitial matrix

• 3. Cross the endothelial layer – intravasation – enter the circulation

• 4. Travel through blood/ lymph

• 5. Extravastae into Tissue

• 6. Establish New Clonoy of Cells

Detachment From The Original Location and Breach of BM

• The first step in the metastasis cascade

• This define the cancer as a malignancy rather than a benign neoplasia

Migration Through the Stromal/Interstitial Matrix

• Cancer cells can migrate as single cells in files→ can come together collectively as a broader front of cells

• Cells can have elongated or rounded mophologies

  • 2nd step in metastatic cascade

Intravastation

• The spread of cancer cells to form 2° tumours via the circulation →haematogenous spread

  • Cancer cells can also migrate along nerves or on the outside of blood vessel → neurotropism/ angiotropic

• Tumour cells degrade the blood vessel basement membrane, releasing invadopodia that allow cells to burrow through the BM and enter the blood circulation.

Movement Through Blood/ Lymph

• Most recognised form of dispersion

• Many challenges are present in the vasculature making this process difficult

• Cancer can travel through the vasculature as solitary cells but commonly travel through the blood in small clusters – benefits their survival

• Small clusters of cells can contain heterotypic cells e.g. fibroblasts or can aggregate with cells from the blood e.g. platelets and neutrophils

• As the calibre of the vessels narrows, there is a mismatch between the diameter of the tumour cells and their plasticity – tumour cells get stuck in the capillary network

Extravastae

• Tumours escape from circulation to colonise the tissue and then enter organs where they may resume growth again, forming a colony (tumour)

• Occurs via the remodelling of the vasculature around them → previously believed to crawl out)

  • The critical step is the formation of microemboli

Requirements For Metastasiss

• cell migration – to move from the original site to different depots e.g. blood / out blood to tissue to form a colony

• ability to invade and remodel the ECM

• intravasation – move into the circulation

• dissemination through the circulation

• extravasation – move out vessel

• survival in inappropriate tissue contexts – may not resemble site of origin – tumour may require adaption to survive

Epithelia

• Specialised layers of cells which line organs

  • Located on the surface or sit within the lumen of the organ

• They can have a protective, absorptive or secretory function

  • Highly polarised cells → apical and basal side

  • Sit atop a specialised layer of ECM → BM/ basal lamina

Basement Membrane/Basal Lamina

• A specialised layer of extracellular matrix that epithelia sit atop

• It is a complex extracellular matrix comprised of multiple proteins such as laminin, type IV collagen, and proteoglycans such as perlecan and nidogen.

• It is very dense and resembles an impervious sheet → interstitial matrix sites below with and is an open weave, with gaps present to which cells can bypass

  • Carcinomas metastasis by breaking through this

Hemidesmosomes

• Special junctional complexes that rivet cells to the basement membrane

• It is comprised of integrin a6, B4 receptors for the laminins in the ECM

• On the intracellular side the bind to adaptor proteins and link to intermediate filaments e.g. keratins

• Stable structure that provides adhesive strength for epithelial cells sitting on the basement membrane → not easy for cell to migrate when attached to BM

Intercellular Junctoin

• Cell-cell adhesion complexes that firmly attach one cell to another

Tight Junction

• Cell-cell adhesion complexes located on the apical side

• They act as a barrier to transcellular diffusion

• Occludins are present → membranes held very close together

• help the membrane segregate proteins or direct trafficking → involved in apical basal polarity

Adherens Junctions

• Cell cell adhesion complexes that provide mechanical strength in adhesion

Cell-Cell Adhesion Complexes/ Jucntion

• They all have a similar role in sticking cells together e.g desmosomes and gap junction

Acquisition of Local Invasiveness

• Reduced cell-cell adhesion

  • Down-regulation of E-cadherin – main adhesion protein

• Proteolytic degradation of the basement membrane

  • Invadopodia are specialised subcellular structures which perform this role

• Acquisition of a motile phenotype

  • Adhesion to the stromal ECM/ interstitial matrix → requires altered integrin expression

• Cytoskeletal reorganisation

• Propulsive force → actomyosin contraction and actin polymerisation

• Proteolytic degradation of the stromal ECM – if it appears too thick to move through

  • role for matrix metalloproteinases

Invasive Phenotypes

• They arise from the inappropriate expression or activity of oncogenes, leading to changes in cell morphology, motility, and adhesion.

• Key features

  • Transition from non-motile epithelium to motile, morphologically altered cells.

  • Examples shown using MDCK cells:

Key morphological changes in MDCK cells + V12Ras (Oncogene)

• Changes morphology and adhesion

  • Cells become solitary

  • Elongated shape with pseudopodia at the front and rounded at the back

  • Sparsely distributed

    • invasive phenotype

• Typical appearance: Refractile edges, columnar shape, cobblestone appearance

Epithelial-to-Mesenchymal Transition (EMT)

• A fundamental invasive hallmark and the most well known pheontypic change that occurs in transformed cells

• Occurs during embryogenesis and wound healing, converting epithelial cells to mesenchymal cells → co-opted programme

Mesenchyma Morphology

• Fibroblast-like, fusiform shape.

• Leading edge with pseudopodia/lamellipodia, trailing body round.

MCDK Cells Treated With HGF

• The growth factor acts via an RTK (CMET) to activate Ras

  • Cells begin as cyst (balls of cells), treatment with HGF, causes Ras expression and the formation of the branched structures – invade through the matrix

Reduced Cell-Cell Adhesion

• Associated with the down-regulation of E-cadherin via:

  • Deletion e.g. gastric cancers

  • epigenetic mechanisms through methylation of DNA

• When 1• tumours and metastases are stained for E-cadherin - decrease in brown intensity

Structure of Normal Breast Tissue

• Composed of breast ducts

• Tubues of epithelial cells are highly orgaised

• They are stained for e-cadherin - brown colour

Invadipodia

• Cellular organelles responsible for movement through the basement membrane.

• They are formed by out-pocketing of the plasma membrane, driven by actin filaments.

• At the tips of the actin filaments, matrix metalloproteases (proteases that degrade extracellular matrix proteins) are actively secreted to widen the gaps in the basement membrane (Widen further when invadopodia inflate)

  • These MMPs can have different morphologies e.g. cone shape or ballon structures

MMP Delivery to the Ends of Invadipodia

• A rouge GTPase that stimulates the actin polymerisation of invadopodia

• It triggers certain proteins e.g. WASP and ARP23 → responsible for stimulation actin polymerisation and bundling filaments together

  • Trafficking of secretory vesicles occurs down these filaments, delivering MMPs

Role of Integrins in Mesenchymal Migration

• Migration often involves "grabbing onto something and pulling itself along" (e.g., ECM).

• Multiple migration styles exist, with EMT focusing on mesenchymal migration.

• Integrins (α,β heterodimers) bind to ECM components to pull cells forward.

  • heterodimers each have different specificity for matrix components

• On the intracellular side, integrins bind adaptor proteins (e.g., α-actinin and vinculin) to link with actin filaments, helping organise the cytoskeleton and direct cell movement

aVB6

• a vitronectin fibronectin recepto

• It is upregulated in breast cancer, as cells interact with the EMC

RGD Tripeptide

• A soluble peptide used to block the interactions of integrin with fibronectin (used in melanoma mouse model)

• The soluble peptide competes the binding of integrins to the ECM, by competing with their binding motif – blocking the cells ability to interact with the matrix, blocking metastasis

  • Potential to target integrins as a method for blocking the casacade

     

Actin Cytoskeletal Reorganisation and Propulsive Force Generation

• Propulsive force in mesenchymal migration is generated by myosin motor protein moving actin filaments overreach other shortening the body length of the cell

  • It occurs in a highly orchestrated fashion      

• Mesenchymal cells show a front-rear polarisation

Front End of Mesenchymal Cells

• The leading edge

• In 2D, lamellipodia (fan-shaped) and in 3D, pseudopodia (thin and long).

• It requires adhesion to the matrix, where actin polymerisation extends the membrane and makes new contacts via integrins, which clusters forming focal contacts.

Rear End of Mesenchymal Cells

• Cell body becomes rounded as non-muscle myosin pulls actin filaments over each other, breaking rear attachments and forming new ones at the front.

Laemellipodia at the Leading Edge

• They largely driven by actin polymerisation

• At the leading edge the membrane folds back on itself forming membrane ruffles

• Filopodia present

Actin Polymerisation and Depolymerisation in Lamellipodia

• Lamellipodia are actin-rich structures

• Actin filaments have a growing and shrinking end driven by the treadmilling of actin monomers

• The addition of monomers to the front pushes the membrane forward and can generate some propulsive force

Filopodia

• Actin-rich, sensing structures that follow hepatotactic gradients or gradients of extracellular matrix proteins or chemotaxis agents e.g. chemokines

Stress Fibres

• Actin rich elements and a feature of mesenchymal cells

• Allow contraction of the cellular body

Rho GTPases

• Ras-like molecules that stimulate changes in actin/ myosin and the reorganisation of the cytoskeleton

• Part of the Ras superfamily and are small monomeric GTPases

  • Rac, Rho and CDC42 are the best characterised

Regulation of Rho GTPase Activity

• They are small GTPases with a lipid at the C-terminal that allows them to be embedded in the membrane

• When bound to GTP they are active

  • Stimulated by various GEFs and inhibited by GAPs

• When bound to the GDP they are inactive

Quiescent Cells (NIH3T3 Fibroblasts)

• Cells have a simple stable cytoskeleton

• They are not stimulated by a serum to move around

  • Classical polygonal morphology – not many stress fibres

  • Some cortical actin present providing physical support to the membrane

  • No front rear polarity

• Small focal spots of vinculin seen (integrin adaptor protien)

Injection of Rac

• A GTPase that induces lamellipodia and focal contacts

• When it is injected into cells, it causes a dramatic change in cell morphology and actin organisation

• More actin polymerisation in the cell cortex and the appearance of frills

  • Cell no longer has front-rear polarisation as it has been flooded with the GTPAase → no directional cues, with leading edges forming on all surfaces of the cell

• Leading edges associted with the organisation of integrins into focal contacts

Injection of Cdc42

• Induces the development of filopodia or Invadopodia (thinner membrane protrusion)

• Lots of cortical actin but no real leading edge

• Intense build-up of focal contact (focal adhesion when mature) at the tips of the filopodia

Injection of Rho

• Induces stress fibres and focal contacts - focal contacts anchor the stress fibres

Role of Rho GTPases (Rac, Rho, Cdc42)

• In a migrating cell, not all activities occur uniformly across the cell.

• Stress fibers are more prominent in the body of the cell.

• Actin polymerisation occurs at the front of the cell, with filopodia branching out to help decide the direction of movement.

• The activation of these proteins is highly organised and regulates different aspects of cell movement.

How Do Proteases Contribute To Cell Invasivness

• Cells navigate and must degrade the extracellular matrix (ECM) to move through it. The interstitial matrix has thicker fibres that need to be broken down.

• Cells attach to the matrix to pull themselves forward but also degrade it to remove obstacles.

• Proteolytic activity is focused at the leading edge of the cell, proteases are membrane-anchored rather than secreted.

  • Secreted proteases can bind to receptors on the cell and become activated.

• Proteolytic activity concentrates at the tips of invadopodia or pseudopodia, breaking down the matrix when encountering thick regions.

• MMPs degrade the matrix and are part of a large family related to ADAM proteases → bind to zinc ions for activity.

  • Some MMPS are transmembrane/ GPI anchored or secreted that can bind to receptors

• Secreted MMPs are activated by MT1-MMPs through cleavage.

• Other Proteases e.g. Serine proteases - UPAR and plasminogen-contribute to degradation, binding to receptors to concentrate their activity.

Cell Migration through the Interstitial Matrix:

• Membrane Extension: The cell moves forward via protrusion at the leading edge.

• Cell Body becomes round.

• Detachment at the Back:

• Collagen is cleaved - fibres can be detected using an antibody

  • Cleavage of fibres exposes a new epitope, visualisable with antibodies.

  • Cleavage is localised to the leading edge and cell wake.

• Pinch Point Near the Nucleus:

  • Cell is "caught" in collagen loop.

  • If collagen is not cleaved, the cell can't progress.

• Importance of Membrane Degradation: Focused degradation is critical for cell movement.

Proteinase Inbitiors

• Blockage of MMP5 blocks collagen degradation

• Without collagen, cleavage, migration is hindered

Pinch Point

• Present near the nucleus, where the is cause in a loop of collagen

• If it is not cleaved, the cell is not able to progress

  • Highlights the importance of membrane degradation - it must occur in a focused way to allow migration

Ameboid Invasion

• Proteinase Independent Integrin Independent Invasion - an alternative to mesenchyma invasion

• Driven by membrane blebbing and squeezing the membrane with actin-myosin to form fluid-filled pockets that allow cells to pass through gaps in the matrix

Collective Invasion

• An alternative to mesenchymal invasion

• The presence of mesenchymal leader cells that form a trail through the matrix via proteolysis - the cells behind are epithelial and forming cell-cell junctions

Different Paradigms of Invasion

• It explains why efforts largely focused on mesenchymal migration aren’t effective

• When treating cells undergoing mesenchymal migration with protease inhibitors, they can transform in amoeboid cells

  • Mesenchymal to ameboid transition – an escape mechanism – shows the plasticity of cancer cells

Challenges to Tumour Cells in Circulation

• Shear Stress: Cells face high blood flow and immune cell predation, resulting in many dying.

• Apoptosis: Loss of ECM attachment makes cells more vulnerable to apoptosis (programmed cell death), leading to cell death in most cases.

• Anoikis Resistance: Cells must resist anoikis (detachment-induced cell death) to survive.

• Some cells survive by clustering together, where cells in the centre of the cluster are shielded from shear stress.

  • Clusters can become trapped in capillaries, blocking blood flow.

Importance of Platelets

• Cancer cells associate with platelets

• They act as a source of nourishment allowing tumour cells to proliferative in the blood vessel and secrete proteases to degrade the basement membrane, with the blood vessel remodelling itself around the tumour so that the tumour ends up on the outside of the vessel

Dormancies

• Cells surviving and extravasating the circulation don’t turn into tumours as they are no longer in a permissive environment and are no longer activated by the angiogenic switch → can’t stimulate neovasculature

  • Most of these cells form micrometastases

Implication os Micrometastasis in Organ Transplantation

• The dormancy can be sat in tissues for decades

• Individuals successfully treated for cancer may become organ donors

• When the organ is transplanted, the recipient who is immune suppressed can carry the ‘seeds’ for cancer

Micrometastasis

• Small clusters of cancer cells present at the time of primary tumor removal and are below the resolution of medical imaging.

• They may cause cancer recurrence decades later.

• There are no easy biomarkers for identification, and can only be detected using techniques like bone marrow biopsies and staining.

• Results in later diagnoses as if present they are beneath the resolution of medical imaging, making early detection challenging.

• Chemotherapy is used as adjuvant therapy to destroy as many micrometastases as possible and prevent recurrence.

Outcome For Patients With Micrometastisis

• Patients tend to die earlier compared to those with less or no micrometastasis, who are more likely to undergo successful interventions against the primary cancer.

Tamoxifen

• An oestrogen antagonists that revolutionised the survival of breast cancer in women

  • It blocks oestrogens ability to drive the proliferation od breast cancer

• It is well tolerated and can reduce the reoccurence

Tropism

• The pattern of where metastasis forms - explained through circulatory patterns with tumours transported in the circulation and trapped at the first capillary bed

  • e.g. colorectal cancer metastasises to the liver

  • e.g. prostate cancer metastasises to the bones

  • e.G. melanoma metastasises widely but predominantly to the lungs

Colorectal Cancer Metastasis and Circulatory Patterns

• Cancer cells and tumours will enter the hepatic portal system and will drain into the liver → site where metastasis for in C.C

Breast Cancer Metastasis and Circulatory Patterns

• Cancer cells enter the thoracic lymphatic duct will enter the heart via the venous supply to then be dispersed around the body-wide distribution of metastasis e.g. bone and lungs

Stephen Paget Seed and Soil Hypothesis

• Likend circulating cancer cells to seeds →a plant goes to seed, its seeds are carried in all directions, but they can only live and grow if they land on congenial soil’

• Only certain tissue will have similar trophic signals as present at the primary site to support the growth of escaped tumour cells - > only grow in certain tissue with special features, nutrients, trophic and growth factors that are compatible.

  • Metastatic cells create a niche to establish a tumour

Pre-Metastaic Niche

• Primary tumours secreted extracellular vesicles that condition certain tissues e.g. the lungs to see localised changes in the lung e.g. recruitment of immune cells, remodelling of the matrix to provide a receptive space within the tissue to receive a metastatic cell

  • Creates a niche to receive a cell

• Shows how the distribution of metastasis isn’t random

2-Fold Clinical Problem In Targeting The Invasion Metastasis Cacade

• Occult disease with minimal micrometastasis: need to identify and remove small metastases.

• Body full of metastasis: large tumours present in multiple organs.

Challenges with current therapeutics Targeting the Invasion Metastasis Cascade

• Protease inhibitors, actin/cytoskeleton inhibitors block early invasion steps but don’t effectively remove micrometastasis or macrometastasis.

  • don’t function effectively clinically

Potential Theraputic Sucess in Targeting Invasion Metastasis Cascade

• Treating small lesions of micrometastasis to prevent growth into macrometastasis.

• Tamoxifen and TGFB inhibitors can block this progression.

Difficulties in Targeting Micrometastasis Regression

• Targeted therapies (e.g., MEC, BRAF inhibitors) may work on secondary tumours to an extent but face issues with resistance and tumour adaptation.