Modelling cancer Flashcards
1
Q
What processes are occur during cancer development?
A
Many. Oncogene/tumour suppressor mutation, angiogenesis, immune system can repress growth, extracellular matrix can promote growth and inhibit movement. Makes cancer very difficult to model.
2
Q
What are the questions we ask about tumour origins?
A
What genetic changes occur and whether the order matters
If one or many cells initiate the tumour
If it involves differentiation or dedifferentiation
How carcinogens can effect things
Passenger mutations/driver mutations
3
Q
How can cancer be investigated in the lab?
A
From tissue biopsy (small, bad quality DNA)
Adding oncogenes to primary cells
Using cell lines (have been grown for decades, questionable as to what they are)
Genetic data from Cancer Genome Project (see what mutations cancers have in common)
4
Q
What in vitro model systems can be used to model cancer?
A
Cells from patients - don’t know if mutations are from patient or from adaptation to growth in culture. Can use to do gene targeting and engineering
Engineered cell lines - express oncogenes. Are they the correct cell type?
Colony forming assays - use blood cells
Primary cells from humans - hard to obtain unless can get from blood or tonsil. High genetic variability
Primary cells from mice
5
Q
What are the problems with modelling cancer in tissue culture?
A
Ignores the contribution from the system such as the immune system, ability to metastasise, extracellular matrix, angiogenesis, hormones, growth factors.
6
Q
How can mice used to model cancer?
A
Can use primary cells in tissue culture
Can do mouse mimics (where you get a tumour but the process of getting to the tumour is not the same as in humans)
Can do mouse models (where process is the same)
7
Q
How can we prove a gene product is an oncogene?
A
Put a transgene into a mouse with a tissue specific promoter and see if get a tumour.
Problems are to do with copy number variations and sites of integration (could be chimeric) - in one study one line of mice had 10 copies of the transgene and died quickly whilst another had only 1 and showed a process more similar to human cancer (however these studies are more difficult to get funding for and are expensive). Also had different tumour phenotypes.
8
Q
How can translocations be modelled?
A
Can make conditional (to ensure expression is in the correct cell type), inducible (to ensure translocation ‘occurs’ at the right time) genetically modified mice. Gene dosage should also be considered e.g. reciprocal translocations - which one is important? May reduce gene dosage of a haploinsufficient tumour suppressor
9
Q
How can Cre-lox be used to model chromosome translocations?
A
Is tissue specific (express Cre from a tissue specific promoter). Use loxP sites to turn off/turn on (if have a stop cassette that is deleted) gene expression. Having loxP sites on different promoters mimics a translocation in different cell types. Used this to find that Mll:Af9 is an oncogene in myeloid cells but not T cells. Mll:Enl is malignant in T cells.
10
Q
How can CRISPR-Cas9 be used to model cancer?
A
Can use to make chromosome translocations by inducing double strand breaks at certain points.
11
Q
How can oncogene addiction be modelled?
A
Make an inducible, transient mouse - link transgene to doxycycline dose in a specific tissue. Give mouse doxycycline then withdraw. Tumours have been shown to be addicted to Ras and Myc (tumours apoptose upon withdrawal). Shows that drugs that target oncogenes could cause tumour regression.
12
Q
How can the necessity of the inactivation of tumour suppressor genes be modelled?
A
Use KO p53 mice - KO by putting a stop codon in the gene, flanked by loxP cassettes. Irradiate to get a tumour. Reactivate p53 by turning on Cre (e.g. with tamoxifen), find that tumour regresses.
13
Q
What are tumour initiating cells?
A
Cell of origin of the tumour, not necessarily responsible for tumour maintenance.
14
Q
What are cancer stem cells?
A
Tumour propagating cells, may be distinct from tumour initiating cell. Have reproductive capacity and can self renew. May produce progeny that can’t reproduce
15
Q
What is the stochastic model of tumour growth?
A
Different populations in the tumour all contribute to growth
16
Q
What is the cancer stem cell model for tumour growth?
A
Only some (one population, the cancer stem cells) of the cells in the tumour contribute to tumour growth, the others don’t.
17
Q
What are the different strains of immunocompromised mice?
A
SCID - deletion in DNA PK gene for TCR and Ig rearrangement. Have NK cells
NOD/SCID - non-obese diabetic severe combined immunodeficiency. Also have defects in NK cells. Short lifespan due to reactivation of a provirus
NOD/SCID/beta2 microglobulin null - nearly no NK function, short life span
NSG - no T, B or NK cell activity. No leakiness or tumour incidence.
18
Q
Describe origination of Acute Myeloid Leukaemia
A
Found a SCID Leukaemia Initiating Cell (by looking at surface markers). Was able to initiate human haematopoiesis in immunocompromised mice. Got differentiation into leukaemia blasts suggesting a hierarchy for this cancer. Cancer stem cell likely to be a transformed tissue-specific stem cell.
19
Q
How has BCR-ABL addiction been studied?
A
In mouse models found that inactivation of BCR-ABL lead to reversion of the leukaemia. Therefore developed drug inhibitors (e.g Imatinib) that were rushed through clinical trials. Found that disease relapsed due to a mutated form of BCR-ABL found in leukaemia stem cells. Now, new inhibitors are being developed and research is being done to find the mechanism of leukaemia stem cell resistance (e.g. expression level, drug efflux, niche environment, not dependent on BCR-ABL)
20
Q
What models can be used to test new drugs?
A
Nude mouse xenografts
Patient derived xenografts in NSG mice
Genetically engineered mouse models (may not be truly representative of human disease and may take time to develop a tumour)
21
Q
Why do people get referred to cancer genetics clinics?
A
People with family histories of breast, ovary and colon cancers
If have a relative with cancer (especially early onset) increases chance of cancer
Certain mutations carry loads of risk e.g. FAP
22
Q
What is FAP?
A
Familial adenomatous polyposis coli. Get polyps throughout the colon and maybe upper GI tract. Average age of cancer development is 15 years of having polyps. Screen for FAP by colonoscopy and do genetic tests to discharge mutation negative individuals. Caused by a mutation in the APC, often truncations in centre of the gene. 2 hit model of tumorigenesis
23
Q
What is MYH?
A
Family with 3 colorectal cancers, but markers didn’t segregate with disease as expected (e.g. like in FAP). Found mutations in the APC different to those in sporadic cancer. Found mutation in the BER DNA repair machinery at MYH. Was a missense mutation with a less severe phenotype than FAP
24
Q
What is Lynch syndrome?
A
Hereditary non-polyposis colorectal cancer. Autosomal dominant with an early onset, tumours on the right hand side of colon (have to do full colon screen). Also get other cancers (endometrial in women, ovarian, stomach). Is due to mutations in MLH1 or MSH2 (and some rarer mutations) which are involved in mismatch repair
25
How is Lynch syndrome diagnosed?
In past used Amsterdam criteria - 3 people with colorectal cancer, one below age of 50. These were then relaxed to include other cancers, but didn't pick up de novo mutations. Now we do micro satellite testing and immunohistochemistry against MSH2 and MLH1. Can have sporadic cancers with these mutations as well esp. MLH1 - test young people with these cancers.
26
How can cancer be prevented in people with Lynch syndrome?
Surveillance to detect tumours at an early age and remove polyp
Found that people with arthritis get less cancer, theorised to be due to aspirin. Found that giving a high dose of aspirin to people with Lynch syndrome significantly reduces cancer - don't know how, looking at different doses.
27
What preventative treatments exist for families with BRCA1 or BRCA2 mutations?
Mastectomy
Oophorectomy (may also reduce the risk of breast cancer)
Birth control pill can reduce risk of ovarian cancer
Lots of screening
28
At what point do we test for BRCA1/2 mutations?
Look at how much disease on one side of the family and how many unaffected relatives there are. Also look at age of diagnosis - older is more likely to be sporadic. Also must check disease actually is cancer as can be inaccurately reported.
Have to be careful, as these cancers only affect women, so can be passed through men and turn up in a woman with no family history
Can do a Manchester score to determine whether to test, but as testing is cheap now criteria have been widened
29
How can BRCA1/2 cancers be treated?
By inhibiting PARP - another pathway of DNA repair. Cells need at least one pathway to survive.
30
How are inherited cancers molecularly classified?
By function - gate keeper genes that regulate cell growth/division (tumour suppressors or photo-oncogenes) OR caretaker genes that help maintain the integrity of the genome (include DNA repair enzymes where mutations lead to genomic instability and increased likelihood of a gatekeeper gene mutation; cause cancer indirectly)
31
How are inherited cancers clinically classified?
Rare familial cancer syndromes - distinctive phenotypes or clustering of rare cancers
Genetic predisposition to common cancers - no phenotypic markers, familial clustering, early age of onset, multiple primary tumours
Sporadic cases - new muttons for high penetrance genes, high risk polygenic cases
32
What is pheochromocytoma?
A tumour arising from the adrenal medulla that secretes catecholamine.
33
Describe the genetics of pheochromocytoma
One third is genetic
25% of sporadic cases may have a gremlin mutation
11 genes are implicated - of which the most frequent are SDHB, SDHD and VHL mutations.
34
What is paraganglioma?
Can be extra-adrenal - catecholamine secreting tumour arising from sympathetic ganglia
Can be head and neck - non functional tumour arising from parasympathetic ganglia. Similar genetics to pheochromocytoma
35
Describe the genetics of renal cell carcinoma
Found higher risk of RCC if sibling or parent has it. 3% of patients have a positive family history. Most inherited cases are dominant.
36
What is multiple endocrine neoplasia 2?
An autosomal dominant cancer. Is a medullary thyroid carcinoma (MTC) and hyperplasia of thyroid C cells. Is a pheomochromocytoma. 30% of sporadic MTC have gremlin RET mutations - incomplete penetrance. Do DNA analysis at 5yrs old and thyroidectomy to prevent thyroid cancer
37
What are the different types of Multiple Endocrine Neoplasia?
MEN 2A: MTC + pheochromocytoma + presence of parathyroid hormone
MEN 2B: MTC + pheochromocytoma + developmental anomalies
Familial MTC - no pheo
38
What is the genetic cause of multiple endocrine neoplasia 2?
Inappropriate activation of RET. Can either be in the extracellular domain to cause inappropriate dimerisation (e.g. each monomer has 2 cysteines in which form a cysteine bond in the molecule. If one of these is mutated e.g. to an arginine, then a cysteine bond can form between monomers instead to make a constituent dimer) or intracellular in the tyrosine kinase domain to cause inappropriate activation. Is a protooncogene.
39
What is Hereditary Papillary RCC type 1?
Renal cell carcinoma. Is very rare dominantly inherited cancer with multiple tumours of incomplete penetrance. Get germ line activation of MET, an RTK (a protooncogene)
40
How can hereditary papillary RCC type 1 be treated?
MET inhibitors e.g. foretinib (also inhibits other receptors)
41
What is neurofibromatosis type 1?
An autosomal dominant cancer which occurs from a germ line mutation in the NF1 tumour suppressor gene. Get cutaneous neurofibromas, Lisch nodules and axillary freckling and a 2% risk of pheochromocytoma. Don't do genetic testing
42
What is von Hippel-Lindau disease?
An autosomal dominantly inherited disease. Get risk of retinal angiomas, cerebellar haemangioblastomas, clear cell renal cell carcinoma,, pheochromocytoma and other cats and tumours.
43
How is von Hippel-Lindau disease managed?
Annual eye exam from age 2
2-3 yearly brain scans from 15
Annual abdominal MRI from 16
24hr urine catecholamines
44
What mutations causes von Hippel-Lindau disease?
Found in VHL, familial pheochromocytoma and recessive polycythaemia. Encodes 2 proteins on 3p25. Found in many sporadic RCC as well.
45
How was the function of the VHL gene identified?
Has little significant homology. VHL tumours are known to be very vascular. Looked for interacting proteins. Found overexpresssion of hypoxic response genes in tumours and early lesions. Found that pVHL is a ubiquitin ligase that regulates HIF-1 and HIF-2 stability
46
How can von Hippel-Lindau disease be treated?
With kinase inhibitors targeting hypoxic response proteins such as VEGF
47
What is Birt-Hogg-Dube syndrome?
Skin fibrofolliculomas on head, neck and upper chest. Lung cysts and renal tumours with variable histopathology. Life time risk of 30% for renal cancer. Have mutations in FLCN in germ line in majority of cases
48
What does FLCN do
Follicullin. Is a tumour suppressor gene, which is poorly characterised. Found that it is implicated in the mTOR signalling pathway in mice (if inactivated, signalling increased). Has also been linked to TGFbeta signalling and enhanced affect of HIF
49
What is Reed syndrome?
Hereditary leiomyomatosis and RCC. Is dominantly inherited with incomplete penetrance. Get multiple uterine fibroids and cutaneous leiomyomatosis. Screen for RCC with MRI (tends to be unilateral, solitary and aggressive). Due to mutations in fumarate hydratase (also associated with pheochromocytoma)
50
How is Reed syndrome monitored?
Predictive testing from 8-10 years
Renal screening by MRI annually
Prompt treatment of tumours with surgical excision
51
How does the Krebs cycle influence hypoxic signalling?
Fumarate inhibits PHDs which oxidate HIFalpha. This prevents the degradation of HIFalpha by VHL and causes pseudohypoxic signalling. Fumarate can accumulate due to loss of fumarate hydratase.
52
How are inherited renal cell carcinomas evaluated?
Detailed family history is taken and cancer is confirmed. Histopathology used to see what kinds of cancer are found (e.g. clear/non clear or papillary type ½)
Look for features of a specific syndrome
If a syndromic cause is suspected then testing of genes.
If there are 2 close relatives with RCC (with bilateral/multicentric disease or early onset as an added risk factor) then may suspect non-syndromic inherited RCC - look at chromosomes and do molecular genetic analysis looking at renal cancer gene panel
53
What is the deterministic/stochastic cancer model?
Each cell in the cancer has a defined chance of replicating the cancer in a different environment. In the right conditions every cell has a chance of replicatingi
54
What is the cancer stem cell model?
A defined population in the cancer (thought to be small and rare) can replicate the cancer in a different model. Can also replicate the variation in the original cancer. Is accepted as true for haematological cancers; is controversial for some solid organ cancers.
55
What is the evidence for the cancer stem cell model?
Dick 1997: transferred human tumours to immunocompromised mice and separated cells based on hierarchy. Found that CD34+/CD38- (closer to stem cells) caused leukaemia but CD34+/CD38+ didn't. Similar experiments have been done for other cancers.
56
What is the evidence against the cancer stem cell model?
2 papers were published using similar assays to papers in which CSC was 'proven'. Showed that cancer stem cells weren't necessarily rare (⅓) and that they would have to be highly frequent
57
How can the evidence for and against the cancer stem cell model be reconciled?
Relate to normal tissue hierarchy - not all tissues have the same hierarchy. Melanoma is derived from neural cells were there is no tissue hierarchy found.
58
What is the significance of the cancer stem cell model for metastasis?
Cells have to leave the primary tumour, go into the blood/lymph and initiate cancer at a new site. Cancers tend to spread to specific areas, is unsure why. To initiate a new cancer, cancer stem cells are required.
59
What is the significance of cancer stem cells for relapse?
Need cancer stem cells for relapse as they generate all the other cell types in the tumours. Can relapse at a primary or distant site and are often accompanied by resistance to therapies.
60
What is the significance of cancer stem cell in the resistance to therapies?
Often find low levels of resistance in the primary tumours (from deep sequencing). Could also generate resistance during chemotherapy as it involves DNA damage.
61
What is the significance of cancer stem cells for therapy?
We are good at treating primary cancer (surgery, radiotherapy, chemotherapy, immunotherapy). Risk of death is from metastasis and relapse, not the primary tumour. They are often more advanced (mutations) than the primary tumour and are treatment resistant. They arise from cancer stem cells.
62
What are the pros for cancer of forming from a stem cell?
Cancer needs endless replication ability (stem cell has unlimited self renewal)
Needs to accumulate mutations (can't do if cell differentiates, apoptoses and dies)
63
What are the cons for cancer of forming from a stem cell?
Well protected as are required to sustain and maintain the organism - have the best DNA repair mechanisms and trigger happy response to DNA damage (are more likely to undergo apoptosis). Often quiescent (not ideal metabolically)
64
What are the pros for cancer forming from a progenitor/other cell?
Genome is less well protected
Metabolically more active
Can make cancers in mice by genetically mutating progenitor cells
65
What are the cons for cancer forming from a progenitor/other cell?
Very limited self renewal - needs to acquire this.
66
What is the evidence for committed progenitors developing cancer?
Can generate acute myeloid leukaemia stem cells form committed progenitor cells - gain mutations that confers self-renewal to become a leukaemia stem cell (get many leukaemic blasts, no further differentiation to terminal myeloid cells)
Looking at VDJ recombination - can see cancer cells are mature (have rearranged). Are transformed fully mature lymphoid cells with some antigenic memory
Target of transformation depends on the resulting cancer
67
How do mutations accumulate in the cancer stem cell hypothesis?
Find premalignant conditions in which normal somatic tissue acquires mutations as you get older that are associated with cancer
Common disease mutations show definitive mutations for myeloid malignancies (DNMT3A, TET2, ASXL1). Also have hazard risk mutations which give risk of cancer and other disease that affects normal white cell function
68
Describe the remission of acute leukaemic patients
Are 'morphologically remission' - no cancer cells under the microscope. However still have some 'normal' cells with cancer mutations which are precursors that don't go away with treatment - some mutations weren't seen in the primary disease but are associated with relapse. These preleukaemic cells are resistant to therapy
69
How can cancer stem cells develop?
Cancer is a stepwise accumulation of mutations (some of which increase the probability of more mutations e.g. decreasing apoptosis). Downstream cells could be a cancer stem cells (transformed later in differentiation)
70
How can cancer stem cells be targeted?
Need treatments that are selective and specific so normal stem cells aren't harmed. Also need to not drive cancer evolution in the wrong way with therapy - need good targeting mechanisms.
Also have to be aware that cancer stem cells are moving targets - may be downstream differentiated cells that are transformed due to accumulation of mutations.
Need to be able to differentiate between cancer stem cell and normal cell - more knowledge!
71
What is Imantinib?
A tyrosine kinase inhibitor of BCR-ABL (in chronic myeloid leukaemia). Doesn't target the cancer stem cell, so need life long expensive drug therapy.
72
What are BET inhibitors?
Target multiple areas of malignant transcription and are selective as cancer cells are dependent on certain transcription pathways. Have short lived efficacy as there is resistance from cancer stem cells - the drug inhibits a protein-protein interaction; mutations up regulate other transcriptional pathways such as beta catenin.
73
How can surface proteins of cancer stem cells be targeted?
Differences between cancer stem cells and normal stem cells can be exploited e.g. acute myeloid leukaemia up regulates CD47 (a don't eat me signal to the immune system), normally only found on stem cells in the bone marrow and those circulating in the blood. Could be a target in AML.
74
What are the components of the tumour microenvironment?
```
Stroma (supporting cells, have many pro tumour roles)
Immune cells
Blood vessels
Lymphatic vessels
Fibroblasts
```
75
What antigen presenting cells are important in the tumour microenvironment?
Macrophages and dendritic cells
76
What do macrophages and dendritic cells do in the early stages of tumour development?
Microenvironment is less complicated
Growth is recognised as abnormal, get a wound healing response
Involves proinflammataory cytokines (IFNgamma, TNFalpha) to recruit APCs which are phagocytotic and cytotoxic
M and DC are recruited by growth factors secreted by the tumour e.g. VEGF
APCs present tumour antigens to T cells which then produce more immunogenic cytokines to activate other things
Also produce superoxide anions and nitrogen free radicals
77
What is the ideal response in early tumour development?
Necrosis/apoptosis in the centre of the tumour results in the pick up of tumour antigens by APCs. These go through lymph to present to T cells which then kill the tumour. Often doesn't happen properly.
78
What is the function of macrophages and dendritic cells in late tumour development?
Can be tumour promoting e.g. producing growth factors
Are poorly phagocytic
Low expression of MHC so can't present tumour antigen
Low levels of co-immunostimulatory molecules e.g. second signal to T cells doesn't occur
Express negative signals (PDL 1 or 2 that tell T cells to die
Don't produce immunogenic factors (do suppressive factors instead)
79
What suppressive factors are expressed by macrophages and dendritic cells late in tumour development?
IDO - removes tryptophan from T cells and induces apoptosis
Arginase - T cells don't respond to antigen and apopsose
IL-10
TGFbeta
80
What are the anti-tumour functions of T cells?
Kill tumour cells through cytotoxic granules and receptor engagement (FasL-Fas; PDL1-PD1)
T regulatory cells inhibit CD8 T cells
High ratio of CD8 T cells is associated with a good prognosis
81
What are the pro-tumour functions of T cells?
T cells express high levels of PD1 and Fas and are killed by cells expressing the ligands
T cells are exhausted and poorly cytotoxic
T cells are blocked from accessing the tumour due to the stroma.
82
What are the functions of blood vessels in tumour development?
Provide nutrients and oxygen to the tumour
Get immune cells to the tumour
Are a route of metastasis
83
What are the characteristics of tumour blood vessels
Abnormal, less functional
Grow very quickly
Leaky - cells surrounding aren't normal
Chaotic - badly organised, not normal branching
Blood doesn't flow properly - poor perfusion, lots of hypoxia leading to more angiogenesis
84
What are some angiogenic factors?
VEGF-A, FGF-2, CXCL12
85
What cells stimulate angiogenesis?
Could be hypoxic tumour cells or immune cells (macrophages, neutrophils) and fibroblasts
86
How do endothelial cells modulate the immune response?
Express FasL on their surface, killing immune cells (only in the tumour tissue so there are fewer T cells in the tumour)
87
What is the function of lymphatic vessels in tumour development?
Collect fluid form leaky blood vessels and take to lymph. Active route of metastasis. Does immune cell trafficking. Are associated with lymph node metastasis and poor patient prognosis
88
How is lymphangigogenesis stimulated?
By tumour cells and immune cells and fibroblasts through VEGF-C, VEGF-D, FGF and VEGF-A
89
What is the importance of drainage through the lymph for tumour development?
If cells experience flow, cell changes its behaviour - fibroblasts secret growth factors and chemokines such as CCL21 (get a gradient as flow is different across the tumour)
90
How is the lymph involved in metastasis?
Has an active role and modulates the metastatic environment. Changes cell surface expression to set up new niches
91
How does the lymph modulate the immune system in cancer?
Expresses PDL-1 and other negative ligands. Can prevent immune cells leaving the tumour to trigger the immune response (control of entry in to lymphatic system, interact with trafficking of APCs).
92
What is the function of fibroblasts in tumour development?
Very abundant stromal cells
Are scaffold cells and produce extra cellular matrix and growth factors. Emerging theories that they may contribute to immune evasion by secreting factors that modulate the immune response and interacting with and killing T cells
93
What is the significance of fibroblasts producing ECM in tumour development?
ECM is protein rich gel which surrounds the cell. Composed of collagen and proteoglycans. As tumour grows, more collagen is included so it stiffens. This promotes tumour cell invasion (use as a scaffold to move along). It is remodelled in response to flow (e.g. from lymphatics) and invasive tumour cells use this to move. As the ECM breaks down, growth factors are released as tumour cells escape
94
What is the source of fibroblasts in the tumour microenvironment?
Tissue resident
Bone marrow
Endothelial cells that have changed to fibroblasts
95
What is an experiment that shows how ECM stiffness can lead to tumour development?
Breast cancer cells in a soft gel form spheroid structures
In a stiffer gel, spheroid structures fill in
In the stiffest gel, changes occur in the basement membrane which then breaks and cells become more mesenchymal and start to invade
96
What is the association between lymphatics and fibroblasts?
Fibres of the gel of the ECM are random in static conditions
If add flow, fibroblasts and fibres align perpendicular to the tumour - this begins cross linking and stiffening of the gel
Can be seen in breast cancer; is associated with poor prognosis
97
How are growth factors released from the ECM?
Growth factors such as VEGF and TGFbeta stick to matrix components which are released when they break down
98
How do tumour cells use ECM remodelling to their advantage?
Pull on matrix environment to aid leaving of tumour
Use matrix as a scaffold to move along
ECM changes the way cells behave
Fibroblasts make holes for tumour cells to follow (break down matrix and remodel to make tunnels for tumour cells)
99
How can the supporting cells of tumours be studied?
Can't do in isolation - need a multidiscipline approach with combinations of modelling techniques (in silico, vitro, vivo - mouse models).
100
How can heterogeneity of fibroblasts be studied?
Makes it very complicated - look on the single cell level. Inject a green mouse with a tumour cell, then remove the tumour and other stromal cell. Use FACS to sort fibroblasts into 96 wells. See a distinct population of the majority of fibroblasts which changes as the tumour develops. Can sequence wells to find out more then look at targeting in vitro and in vivo.
101
How do fibroblasts modulate the immune response?
Scavenge dead tumour cells - chop up debris and instead of clearing it, they express it on the cell surface in the presence of a negative ligand. Get lots of expression of negative immune regulatory ligands. Fibroblasts end up killing the T cells they encounter, allowing tumour cells to grow better.
102
What is the role of fibroblasts in pancreatic cancer?
Controversial - one paper showed they restrained then tumour (upon deletion tumour was more aggressive), other showed they protected the tumour by T cell deletion. Difference could be down to the models being different (e.g. 2 types of fibroblasts) or that our models for the tumour microenvironment aren't stringent enough yet
103
Why is it important to develop models that represent the tumour microenvironment properly?
Often have drugs that work well in vitro and in vivo (immunocompromised mice) but fail in clinical trials. Could be due to the different environment - stiffness of the tissues, cells behaving differently when grown alone
104
How can the tumour microenvironment be represented in vitro?
In a flask, tumours + supporting cells form clusters
| If grow in 3D culture, get more organisation which may be more representative of tumours
105
What is the effect of Gemcitabine on tumours in mouse models?
Depends on the model - if inject a pancreatic tumour into a mouse, find that gemcitabine reduces tumour growth. However, if tumour has the same mutations but is triggered (and developed properly) instead of injected, then there is no response to gemcitabine - could be due to a properly developed tumour microenvironment.
106
How can the tumour microenvironment inhibit the intended effects of drugs?
Could be a physical barrier to drugs (is dense)
Abnormal vasculature may result in poor delivery
Immune cells and fibroblasts may metabolise drugs and release non-toxic break down products (adding fibroblasts reduces effect of chemotherapy)
As tumour evolves and molecular profile changes, targeted therapy fails
Immune cells can be non-responsive to therapies
107
How can T cells in the tumour microenvironment be targeted?
Immune check point inhibitors
Reactivate T cells by switching off negative signals
Engineer T cells to react to tumour antigen
108
How can antigen presenting cells in the tumour microenvironment be targeted?
Inhibit recruitment of cells
Deplete APCs when in tumour e.g. Clodronatae kills cells when it is phagocytksed
Reprogram APCs to be anti-tumour (Imiquimod, Vadimezan)
Engineer dendritic cells
109
How can blood vessels in the tumour microenvironment be targeted?
Anti angiogenesis therapies (anti VEGF agents such as bevacizumab, ramucirumab)
Inhibit angiogenesis and normalise vessels - has a very small window of opportunity, if get wrong can lead to more angiogenesis and metastasis
Nanoparticles to take advantage of leaky properties (improve delivery)
Inhibit lymphatic vessels and growth to prevent metastasis
110
How can fibroblasts and ECM in the tumour microenvironment be targeted?
Stromal targeting agents such as sonic hedgehog inhibitors and matrix destabilisation to prevent cross linking and stiffening
Harness stromal proporties e.g. conjugating drugs to increase size and retention in the tumour (Nab paclitaxel)
111
What recent trials have gone ahead to target the tumour microenvironment?
Targeting checkpoint inhibitors
IL-2 to stimulate immune cell proliferation (have to be careful as can get uncontrolled activation)
Immune inhibitors e.g. kinase inhibitors
Many more
112
What are the implications to treatment of tumour heterogeneity?
Cancer can adapt as it evolves
If know history of the cancer, may be able to predict the future - taxonomy of cancer is first step towards personalised treatment
Patients die as cancer becomes resistant and metastasises.
113
What is inter and intra-tumoural heterogeneity?
Inter - variability among individuals (diff tumours in diff patients)
Intra - variability within a tumour
114
What are the implications to research of tumour heterogeneity?
Need tools that capture the heterogeneous nature - cell lines don't evolve in the same way (or at all) - need better tools
115
What are patient derived xenografts?
Tumour for a patient implanted into a mouse. Can implant lots of tumours and se if they grow - found that this captured all the features of the original tumour and population (looked at molecular diversity)
116
How can xenografts be used for clinical trials?
Very expensive and ethical issues of doing trials in a mouse. Instead, move the tumours into a 96 well plate and screen for many trials - this can be used to guide clinical decisions and personalised medicine.
117
What factors impact cancer evolution?
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Microenvironment
Nutrients
Immune system
Mutational rate
Drift
```
118
What are the mechanisms for diversity within a tumour?
Numerical chromosomal instability
Somatic mutagenesis
Structural chromosomal instability
Epigenetic heterogeneity
119
What is evolution by drift?
Instead of mutations being selected for, they are carried along the way by chance and accumulate. Tumour keeps replicating and accumulating mutations
120
How can drift vs Darwinian selection be studied?
Difficult as have to study the phenotype rather than the genotype
121
What are the different modes of evolution?
Kategis - area ina chromosome with loads of point mutations, isn't gradual
Genome doubling
Chromotripsis - lots of chromosomal alterations in a specific area
122
What is contingent evolution?
The idea that evolution isn't random and that the next step is dependent on the previous step - would mean that all cancers are very very different
123
What is convergent evolution?
The idea that there are laws that govern evolution and not every branch is possible e.g. if p53 is already mutated, can't mutate another DNA damage sensor or there would be too much
124
What kind of evolution do you get in cancer?
Both contingent and convergent.
Example of convergence (or parallel evolution): in renal carcinoma, VHL is mutated first, then get branches. However, each branch has a different de-activvation of SETD2 - law is need VHL off then SETD2 off
125
What is the evo index in cancer evolution?
D#delta#. D is diversity, delta is change over time. Is a way of classifying tumours. A low diversity tumour (D1) may either be quiescent (delta1) or accumulate clones (D2delta2). A high diversity tumour (D2) may undergo a selective sweep (D1delta2) or have slow turnover (D2delta1). Gives an indication of how environment influences evolution
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What is the impact of evolution on therapeutics?
Tumours become resistant to drugs used. Often find that the resistance mutation was present from the start - if we know this then we can predict patient relapse. The more diverse the tumour the more chance of resistance to targeted treatments
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What strategies of therapy can be employed to deal with evolution in cancer?
Don't aim to kill all the cells, keep them constrained for as long as possible - on and off therapy
Need to understand the phenotype, not the genotype - what are mutations converging towards?
Can block proteins that cause many mutations such as the immune system.
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What are clonal and sub clonal mutations?
Clonal mutations are in all the cells in the cancer, sub clonal mutations are only in part of the cancer and aid evolution
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How does the number of sub clones vary over time in a tumour?
Depends on the tumour - can evolve linearly. As tumour is treated, get an increased number of sub clones as there is an environmental bottle neck - more mutations are generated in the hope that one will overcome the treatment
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Why are phylogenetic trees useful in cancer?
Represent the diversity and evolution over time. Are clinically useful as can see the 'trunk' of the tree - if can treat here, can kill the whole tree. However, there are very few common mutations in the trunk.
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What is unusual about lung and melanoma phylogenetic cancer tree?
Have some of the highest mutations as have a clear carcinogen associated - cells have been acquiring mutations prior to transformation. Therefore, begin with many mutations but aren't very diverse.
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What are the future steps for research into evolution for drug resistance?
Can mimic the process in models e.g. xenografts. Issues are that the genomic profile in the original tumour and the resistance mutation are the same - mutation is in epigenetics (histone modifications etc). Can do single cell sorting with different antibodies to study.
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What are the stages of metastasis?
Invasion - tumour spread to blood/lymph
Intravasation - tumour cells in the blood/lymph
Arrest in micro vessels of an organ
Extravasation - tumour cells leave blood/lymph at distant sites and colonise. Survival after this is the limiting step.
Proliferation in new organ
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What static models can be used to study metastasis?
Animal models - spontaneous (induce a tumour, see if metastasis) or experimental (inject cells into tail vein, see where they go)
Cell culture - see if cells invade/form colonies/display metastatic properties through a porous membrane. Can also do motility assays
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What are the assumptions in in vitro models of metastasis?
Metastatic cells have unique properties not in the malignant cell - isn't clear that metastatic cells are invasive; model may not be relevant - malignant breast cells don't invade collagen gels whilst normal ones do. Also, is unclear if malignant cells are abnormally motile.
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What are dynamic experimental models that can be used to study metastasis?
Intravital imaging - detect fluorescence and use it to study location, motility, adhesion and interactions off cells over time.
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What has been observed through intravital imaging?
Macrophages and tumour cells and endothelial cells form a structure that p umps tumour cells into the blood stream
Tumour cells get into structures already present and use as a 'highway' e.g. muscle and nerve fibres
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What is the Fidler classic heterogeneity experiment?
Plated one cell per well and grew up into a colony. Each was injected into a mouse and metastatic potential was observed. The results suggested that only some of the cells in the primary tumour had strong metastatic potential. Fidler suggested that only a few cells in the primary tumour could metastasise but all the cells in the metastatic tumour could
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What is the problem with the Fidler conclusions?
When the Fidler experiment is run using cells from the metastatic tumour, get the same rates of metastasis as with cells from the primary tumour.
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What is the Kerbel clonality experiment?
Create primary tumours with a mass of cells, each market with a distinct retrovirus insert. Found that cells required to create the primary tumour (dominant clone) are generally those required for metastasis (are found in the metastases)
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What is Bernards and Weinberg profiling argument?
That there are no genes and genetic changes that are specific for metastasis - suggested it occurs by 'accident' - chance of a cell escaping into the vasculature is based on the tumour position not the genetics.
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What is known about the sequence heterogeneity of metastasis?
Multi genome sequencing was done of cells in primary and metastatic tumours - found regions of the primary tumour were similar to the metastasis and that diversity in the primary tumour predicts diversity in the metastasis. When looking at driver mutations in pancreatic cancer found that there was little difference between primary and metastasis. When looking in renal carcinoma found that many mutations are sub clonal but all were in the same pathway.
A crispr screen showed that many of the genes lost in tumour progression were the same as those needed to become metastatic.
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What was the experiment done by van der Weyden to find genes involved in metastasis?
Looked in a collection of knockout mice - looked for more or less metastasis into the lungs. Found that genes that affected metastasis were involved in the immune system. Found that genes that when deleted resulted in higher NK and effector T cells in the lungs had less metastasis (more tumour cells were killed)
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How can cancer be monitored with CTC and ctDNA
Circulating tumour cells and circulating tumour DNA. Can monitor fluctuation and predict relapse and look for progression of disease. ctDNA is much more sensitive - but need to know mutational landscape so primers can be designed for deep sequencing of the blood
