3.6. Invasion & metastasis I & II

Question 1

What is invasion?

Answer 1

In carcinomas, spread of the tumour through the basement membrane is the defining feature. In other types of cancer this is more difficult to define.

Question 2

What is the importance of invasion?

Answer 2

• This is the first step towards spread of the cancer
• Metastasis cannot occur until the tumour has invaded through the basement membrane
• Invasion can damage and thus compromise the function of neighbouring structures

Question 3

What is metastasis?

Answer 3

1. The process whereby malignant tumours spread from their site of origin (primary tumour) to form other tumours (secondary tumours / metastases) at distant sites.
   * Any tumour that metastasisesis malignant; however, not all malignant tumours will metastasise

Question 4

what are the routes of metastasis?

Answer 4

1. Lymphatic
2. Haematogenous
3. Transcoelomic
4. Implantation

Question 5

explain lymphatic spread

Answer 5

• via the lymphatics, to regional nodes
• Carcinomas tend to invade lymphatics and spread to lymph nodes at an early stage.
  Once in the lymphatics:
  •form emboli, or
  •form a continuous growth along the lymphatic –“lymphatic permeation”.
  1. Emboli travel in the normal direction of lymph flow via afferent lymphatics to the nodes; lodge in subcapsular sinus at the periphery of the node; extend from there to replace the node.
  2. Tumour cells can then travel via efferent lymphatics to more central nodes; may drain into the blood stream via the thoracic duct.
  3. Retrograde embolism may also occur, and emboli may travel against normal direction of flow in blockage (e.g. Troisier’ssign).

Question 6

what is Virchow’s node?

Answer 6

left supraclavicular node

Question 7

what is Troisier’s sign?

Answer 7

palpable Virchow’s node in abdominal cancer

Question 8

what is haematogenous spread?

Answer 8

• by the blood stream, to form secondary deposits in organs perfused by blood containing malignant cells.
• Malignant tumours may enter the circulation by invading the blood vessels (++thin-walled veins) directly, or draining from the lymphatic system into the blood via the thoracic duct.

Question 9

what is transcoelomic spread?

Answer 9

through body cavities; in pleural, pericardial and peritoneal cavities, where result is often a malignant effusion.

Question 10

what is transcoelomic metastasis?

Answer 10

• Causes an exudative (protein-rich) effusion; may contain fibrin and / or be bloodstained.
• Neoplastic cells are present in the effusion –cytological examination is often helpful in diagnosis.

Question 11

what are the two main phases of the metastatic cascade?

Answer 11

1. Invasionof the extracellular matrix and intravasationinto the vessel
2. Vascular disseminationand homingof tumour cells

Question 12

describe the multiple sequential steps of the metastatic cascade, with reference to the molecular components at play in each step

Answer 12

1. detachment of tumour cells from their neighbours
2. attachment to matrix components –> crawl through matrix to blood vessels
3. invasion of surrounding connective tissue to reach blood vessels and lymphatics by means of degradation of the extracellular matrix –> need to digest basement membrane to get to lumen
4. intravasation into the lumen of vessels
5. evasion of host defense mechanisms (e.g. natural killer cells in the blood) –> only cancer cells that can protect themselves will metastasise
6. adherence to the endothelium at a remote location
7. extravasation of the cells from the vessel lumen into the surrounding tissue
8. colonization can occur where the tumour cells grow into a large tumour deposit/ metastatic tumour known as ‘Macrometastasis’

Question 13

describe the process of invasion of the extracellular matrix

Answer 13

1. Downregulation of E cadherins with subsequent loss of adherens junctions and release of βcatenin which can then act as a transcription factor, is a major event in most carcinomas
2. To get through the ECM, tumour cells must adhere to matrix components. There is altered attachment of tumour cells to laminin and fibronectin (increased density and expression of different integrins in tumour vs normal cells).
3. Active enzymatic degradation of ECM components creates passageways for migration. Tumour cells secrete proteases / induce stromal cells to produce these. Most NB = Matrix Metalloproteinases (MMP9, MMP2).
4. Cleavage products of collagens and proteoglycans also have growth-promoting, angiogenic and chemotactic activities.

Question 14

describe the process of vascular dissemination & evasion of the immune system

Answer 14

Once in the circulation, tumour cells aggregate in clumps -either

(i) homotypic adhesions to one another or
(ii) heterotypic adhesions to blood cells e.g. platelets.

Question 15

what is the homing of tumour cells

Answer 15

•At a distant site there is adhesion to endothelium.
•The site of distant implantation is determined by a number of factors
-from anatomic location to specific ligands on endothelial cells at these sites, and chemokine receptors expressed by cancer cells themselves.

Question 16

what is extravasation

Answer 16

This refers to the process by which cells leave the vessel and enter the new tissue. They may do this individually orlodge as clusters in a small vessel and eventually grow out through the vessel wall. This process requires MMPs to dissolve the basement membrane of the vessel and make space for the cancer cells at the new site.

Question 17

describe the preparation of the metastatic site by the primary tumour

Answer 17

cancer cells shed little vesicles known as exosomes which contain proteins, mRNA, miRNA which get taken up by certain tissues and influence composition of microenvironments in these areas –> make it favourable for metastatic cell growth

Question 18

what is colonisation

Answer 18

• the growth of the new micrometastasis into a large mass of cells.
• It is the most difficult part of the metastatic process –millions of cells enter the bloodstream but very few actually form detectable metastases.
• Processes required include adaptation to a new microenvironment, co-option of new stromal cells (see part 2) and induction of angiogenesis.

Question 19

list the components of the tumour microenvironment

Answer 19

-This is also known collectively as the tumour STROMA
1. Cells–other cancer cells as well as different types of non-cancerous cells
*Non-cancerous cells
•All the cells that make up blood and lymph vessels –endothelial cells, pericytesetc.
•Cancer-associated fibroblasts (fibroblasts and myofibroblasts)
•Inflammatory cells –innate and adaptive -macrophages, neutrophils, different classes of lymphocytes, mast cells
2. Extracellular matrix
3. Soluble factors e.g. Growth factors, cytokines

Question 20

what are Cancer-associated fibroblasts (CAFs)

Answer 20

•“Normal” fibroblasts
AND
•“Myofibroblasts” –contain actin.
Note the association with these cells in wound healing.

Question 21

what is the function of CAFs

Answer 21

1. secrete enzymes which remodel the microenvironment e.g. matrix metalloproteinases, as well as the collagen and other constituents of the ECM.
2. They are also an active part of the cytokine network, just as in wound healing, with crosstalk between them and the cancer cells (keratinocyte equivalents).

Question 22

what is the role of M1 macrophage in cancer

Answer 22

1. acute response to bacterial infection
2. coordinates anti-cancer immune response
3. INHIBITS CANCER GROWTH

Question 23

what is the role of M2 macrophage in cancer

Answer 23

1. supports angiogenesis
2. suppresses immune function
3. PROMOTE CANCER GROWTH

Question 24

discuss some of the major features of wound healing

Answer 24

• dependent on macrophages & cross-talk between epithelial cells, immune cells & fibroblasts as well as endothelial cells
  1. proliferation of endothelial cells (keratinocytes in the case of skin)
  2. migration of epithelial cells (EMT-dependent)
• motility
• changes in adhesion
• alteration (& digestion by MMPs of the ECM)
  3. Fibroplasia
  4. Angiogenesis

Question 25

what triggers the angiogenic switch

Answer 25

occurs when cells (such as the macrophages) start to liberate large amounts of VEGF from the extracellular matrix (where it is being stored)

Question 26

what is Epithelial to Mesenchymal Transition (EMT)

Answer 26

It is a programme which is already encoded within the DNA of every cell –it just needs to be switched on by the production of transcription factors in response to signalling pathways.

Question 27

in which 3 situations does EMT occur?

Answer 27

1. Embryogenesis–the formation of mesoderm and endoderm at the primitive streak, neural crest cells.
2. Wound healing –epithelial cells become motile and highly proliferative
3. Invasion and metastasis by motile cancer cells

Question 28

list the ‘LOSS OF’ features of the EMT

Answer 28

1. Tight and adherensjunctions (E-cadherin)
2. Polarity
3. Epithelial gene expression

Question 29

list the ‘GAIN OF’ features of the EMT

Answer 29

1. Shape change
2. Motility
3. Invasiveness
4. Resistance to apoptosis
5. Mesenchymal gene transcription
6. N-cadherin
   (E cadherin replaced by N-cadherin–> allows for adhesion to completely different cells (e.g. fibroblasts)
7. Protease secretion (breakdown ECM)
8. Stem cell-like traits
9. Altered integrin expression
10. PDGF receptor expression

Question 30

describe EMT in cancer

Answer 30

• Appears to be necessary for invasion and metastasis –currently the leading hypothesis to explain these processes.
• EMT-inducing signals from the stroma e.g. TGF-βand growth factors, lead to the synthesis of transcription factors which then coordinate the process.
• The loss of E cadherin and thus subsequent β-catenin signalling in the nucleus (Wnt pathway) appears to be very important.
• Transition to mesenchyme may not be complete i.e. the cell is not completely epithelial or mesenchymal

Question 31

what if the function of expression of N-cadherin?

Answer 31

allows cancer cells to attach to stromal cells instead of each other

Question 32

Where do the EMT and MET fit into the metastatic cascade?

Answer 32

(refer to pg 37 of notes II)

1. EMT relevant from clonal expansion, growth, diversification & angiogenesis until extravasation
2. MET relevant from metastatic deposit until growth

Question 33

Do all the cells in a cancer behave the same?

Answer 33

no.

Question 34

explain cellular heterogeneity in cancer

Answer 34

1. Genetic instability leads to the formation of sub-clones over time
2. Differences exist in cancer cells’ capacity to initiate metastases –not only because of different mutations. There are behavioural differences even amongst genetically similar cells
3. An important concept –the cancer stem cell

Question 35

describe the cancer stem cell

Answer 35

• Very variable in number, even in the same type of tumour
• Have similar features to other stem cells, including infrequent cell division
• The induction of EMT transcription factor production in carcinoma appears to be closely related to the stem cell state. Wnt-type signalling from the release of βcatenin may contribute.