tumour microenvironment - balancing growth

Question 1

importance of vasculature in growth of all types of tissue

Answer 1

• tissues with high metabolic rates develop very high densities of capillaries, so most cells can directly contact a capillary
• cells need oxygen supply, nutrient supply, metabolic waste removal, carbon dioxide removal
  -so cells generally try to be as close as possible to the vasculature.

Question 2

tumour genesis

Answer 2

one key aspect is that the genetics of the cancer cells are not controlling how this tissue morphology is set up.
- So actually what tumors have to do is to recruit blood vessels to them and to remodel that tumor microenvironment
- and they essentially designing their own vasculature as the tumor grows

Question 3

what makes up the vasculature

Answer 3

• blood vascular endothelial cells
• pericytes
• lymphatic endothelial cells

Question 4

blood vascular endothelial cells

Answer 4

the cell types that form the blood vessels

when endothelial cells are activated they start to deform the morphology and form a curved shape that curves around and joins with other endothelial cells - forms a tube
And what’s really important about this structure is these intercellular cleft - these connections between adjacent endothelial cells
- So these are formed by tight junctions, lots of adhesion proteins in these intercellular connections, and it’s really key for how these endothelial cells could control the passage of various molecules and cells out of the bloodstream. Into the surrounding interstitial tissue

Question 5

how do tumour endothelial cells differ from normal endothelial cells

Answer 5

• In endothelial cells that are associated with the tumor microenvironment There is often a reduced amount of adhesion proteins at the cell surface and that has a direct effect on the ability of these endothelial cells to join together to make these intercellular cleft here.
• And the implication of that is that the precise control of passage of proteins and cells across these barriers is lost.
  ○ There’s a dysregulation of this barrier function
  • The other feature of endothelial cells in the tumor microenvironment is that they often express immune checkpoint molecules.
• this increased level of immune checkpoint molecules suppresses the anti-tumor immune response.
  ○ For example, there are fewer cd8 positive T cells entering the tumor microenvironment, which means that fewer tumor cells are being killed by the T Cell immunity and the tumor can grow more.

Question 6

pericytes

Answer 6

• surround blood vessels
• embedded in the basement membrane of vessels and adjacent to endothelial cells
• support the permeability and maturation of the vasculature
• in tumours, impaired interaction of pericytes and endothelial cells contributes to a leaky and dysfunctional tumour vasculature
• also interact with other stromal cells and cancer cells via paracrine mechanisms: modulation of the tumour microenvironment

Question 7

lymphatic endothelial cells

Answer 7

• form the walls of lymphatic vessels
• lymph ducts drain fluid between cells into venous circulation
• lymph ducts also allow antigen presenting immune cells to access lymph nodes
• a dissemination route for cancer cells in addition to blood vessels
• recently recognised as direct regulators of anti tumour immunity
• can present tumour antigens but also immune checkpoint molecules

Question 8

how close do tumour cells need to be to blood vessels

Answer 8

if tumor cells are within that 200 microns of a capillary or a blood vessel, They appear to grow quite well Whereas Pathologists tended not to see well growing tumors that were further than about 0.2 millimeters from a blood vessel.

Question 9

why do tumours need to be so close to a vessel

Answer 9

in close proximity to either vessel, we’ve got high levels of oxygen.
- So this is because there’s oxygen diffusing through the capillary endothelial cells and
is able to oxygenate that close environment to the blood vessel.

But as you move further away from the vessel in either direction, you get to this point that is quite far away from the vessel and the oxygen pressure has dropped dramatically.
- So oxygen is not able to diffuse through to this distance as well.
- And we get this region of hypoxia at some distance from the blood vessels.
- going to impair cell growth in general and tumor cell growth as well.

Question 10

hypoxic and anoxic environments

Answer 10

is hypoxic and anoxic environments Starts to trigger a process of necrosis and this is where you can see all the cells collapsing in on themselves. The tissue is collapsing in on itself and dying.

Question 11

how can tumour cells try to avoid this hypoxic environment and continue to grow - 1

Answer 11

-early in tumour progression, clusters of tumour cells grow to 0.2 mm
- as the tumor starts to grow and there’s insufficient vasculature in that environment, Then we get we start to get hypoxia - trigger p53 dependent apoptosis.
- to try and counteract this tumour cells will secrete vascular endothelial growth factor vegf.
- when secreted, The ECM that is surrounding all the cell’s is able to tether that growth factor to the ECM.
And when vegf is sequestered into the Matrix like that other cells aren’t able to detect the vegf. It’s unable to stimulate any processes

Question 12

how can tumour cells try to avoid this hypoxic environment and continue to grow - 2

Answer 12

then tumor cells will acquire the ability to recruit other cells and we call this an angiogenic switch + Generally, these are inflammatory cells
- when the tumor cells are able to recruit these into the TME, These inflammatory cells then secrete An enzyme that can degrade The extracellular Matrix.
- a protease called Matrix metalloproteinase 9
- when MMP chops up the extracellular Matrix into smaller parts the vegf that was locked into the Matrix can then become released
- And vegf can then stimulate the recruitment of endothelial cells and activation of endothelial cells to start to change their morphology to curve around to join it with other endothelial cells and form these capillary vessels.
- You’ve then got angiogenesis in the local tumor microenvironment

Question 13

growth factor signalling in tumorigenesis

Answer 13

there are several families of growth factor receptors.
- They have extracellular domains that bind the growth factor.
- They have intracellular tyrosine kinase domain that triggers phosphorylation events and cell signaling events intracellularly.
- many of these are important in tumor Genesis
- platelet-derived growth factor pdgf and Vegf receptor

Question 14

vascular endothelial growth factor

Answer 14

• production governed by the availability of oxygen:
• the VHL protein and its partners sense intracellular oxygen tension
• in hypoxia, this complex allows functional HIF- 1 alpha and HIF-1beta transcription factors to accumulate
• HIF-1 drives expression of angiogenesis related genes
• a major HIF-1 - induced gene product is VEGF - (VEGF-A and VEGF-B proteins can be produced; VEGF-A predominates in angiogenesis
• VEGF an be synthesized by tumor cells, but actually other cell types can also synthesize vegf - macrophages and activated cancer Associated fibroblasts, too.

Question 15

so what happens once the vegf has been secreted and then released from the
Matrix

Answer 15

endothelial cells detect these growth factors such as vegf
- the vegf triggers the endothelial cells to proliferate so there’s more endothelial cells and secondly to deform to form these cylindrical walls to form Intercellular cleft with other endothelial cells to form these cylinders
- capillaries then become addicted to high concentrations of growth factors like vegf. So they are always trying to move up the concentration gradient towards higher concentrations of vegf.
- so that allows the capillaries to develop and grow towards these high vegf concentrations.
- capillaries can penetrate through existing tissues to try and get to these higher concentrations of growth factor.

Question 16

tumour capillaries

Answer 16

often in tumors capillaries are much wider than normal capillaries

The organization early on is quite neat and branched whereas towards the end as the tumor grows larger and larger, There’s much more haphazard arrangement of capillaries

Question 17

leakiness in tumour capillaries

Answer 17

• barriers are much more poorly maintained and There are large rips and tears.
• endothelial cells are not contacting each other perfectly and forming tight seals.
• we think that too much vegf signaling can actually cause this separation of endothelial cell membranes.
  
  • So the tumors are obviously using vegf signaling to try and Trigger more blood vessel formation. But if that goes too far and there’s excess signaling what that is causing is this disruption of the barrier formation and leakiness.

Question 18

growth factor PDGF

Answer 18

the growth factor pdgf is also important for controlled capillary formation.
- can also go wrong in cancer.
- in a normal situation when you’ve got your capillary forming These endothelial cells can secrete pdgf b and the pdgf can fall into the local extracellular Matrix and pericytes in the area can detect this pdgf and that brings them towards the endothelial cells and then they’re able to bind to the endothelial cells and support the capillary structure.
- in cases where there are mutations in pdgf b, certain mutations mean that this pdgf can’t be retained in the local extracellular Matrix and it just diffuses away into the tissue. So there’s no local concentration of pdgf anymore and pericytes are unable to be recruited as efficiently
○ and so what that means is you don’t get the support on the capillary.

Question 19

high intratumoral hydrostatic pressure

Answer 19

• at least 3 mechanisms contribute to relatively high fluid pressure in tumours
  1. tumour associated capillaries leak fluid into the parenchymal space of the tumour
  2. expansion of cancer cell populations causes collapse of lymphatic vessels, disrupting fluid drainage within the cores of solid tumours
  3. PDGF released by many carcinoma cells induces contraction of stromal fibroblasts, squeezing out interstital fluid

Question 20

what does high intratumoral hydrostatic pressure cause

Answer 20

high fluid pressure in the tumour complicates administration of anti-cancer drugs as they are not transferred down the pressure gradient from the circulation to the interstitial spaces of the tumour: drugs stay in the circulation

Question 21

strategies to reduce this hydrostatic pressure

Answer 21

one example leans on the fact that excess vegf is causing this permeability of the blood vessels.
- in a normal tissue, there’s a normal flow of blood through the tissue and as the tumor starts to grow larger and larger this blood supply becomes less regular and less efficient, The capillaries are truncated and dysregulated as the tumor gets bigger and There aren’t enough capillary, So there’s hypoxic regions of the tumor
- in mice with tumours, treatment with anti - VEGF monoclonal antibodyy returns vasculature to a normal configuration and reduces interstitial tumour pressure

Question 22

anti - VEGF monoclonal antibody treatment - importance of balance

Answer 22

this therapeutic approach has to be keenly balanced.
- Because if there is not enough vegf therapy, then there will be no change to that irregular and inefficient blood flow through the tumor.
- But it there is too much suppression of vegf signaling then actually there won’t be enough blood flowing through the tumor so theres more hypoxia
○ And then hypoxia can actually induce invasiveness of cancer cells.
○ So the cancer cells will start to invade through and away from the tumor and start to trigger metastatic Cascade.

to achieve normalisation, the dosing is important, and the timing of the drug treatments are important - must be optimised according to each patient

Question 23

cellular senescence

Answer 23

a typically irreversible form of proliferative arrest
- senescent cells remain metabolically active but lose the ability to re enter the active cell cycle
- induced by nutrient deprivation, DNA damage, organelle damage, oncogene-induced signalling
- characterised by presence of markers of cell cycle arrest (e.g. CDK inhibitors p15, p16, p21)
- characterised by absence of the proliferation marker Ki67

Question 24

replicative senescence

Answer 24

• induced after a finite number of cell divisions of normal cells under normal conditions
• driven by telomere shortening in humans

Question 25

stress-induced premature senescence

Answer 25

• triggered by high levels of oxidative stress or other genotoxic stresses

Question 26

oncogene induced senescence

Answer 26

• triggered by oncogene activation
• a major anti - tumour mechanism

Question 27

SASP

Answer 27

in addition to shutting down cell division, senescence triggers changes in cell morphology and activation of the senescence-associated secretory phenotype (SASP)

SASP involves release of chemokines, cytokines and proteases, which vary dependent on cell type
- SASP factors from senescent fibroblasts have been shown to induce cancer cell proliferation and invasion in culture

Question 28

positive and negative of SASP

Answer 28

positive - long viewed as a protective mechanism against cancer; senescence can limit malignant progression

negatives - in other contexts, senescence can stimulate tumour formation and malignant progression

Question 29

context dependent SASP

Answer 29

• SASP can reinforce senescence in an autocrine manner
• SASP can alter adjacent cell migration or proliferation in a paracrine manner
• can promote angiogenesis
• can enhance the immunosuppressive microenvironment

complexity of SASP highlights importance of identifying SASP- generating cells for therapeutic targeting

Question 30

SASP in cancer therapy

Answer 30

the activation of cyclin D and CDK4/6, which play pivotal roles in the transition from G1 to S phase, is a feature of many cell types, particularly breast cancer cells
- both have been targeted therapeutically
e.g. CDK4/6 inhibitors are used to treat ER+ and HER2- breast cancer
- since CDK4/6 inhibitors mimic the function of p16, induction of cellular senescence is a likely outsome of this treamtnet
- in PDAC, CDK4/6 inhibitors induce SASP
- increased vascularity and immune response