L7 - Invasion and Metastasis II Flashcards
What causes most of the cancer related deaths
Metastases
Metastatic dissemination requires
EMT
Describe what happens with EMT
Loss of adhesive protpoerities and acquire fibroblast like morphology and increased motility
What does EMT compass a range of
Hybrid states
What were the research questions put forward by the team who wrote the paper
Are mechanisms regulating pEMT and cEMT the same?
What are the programes underying the differences in epithelial cell plasticity an cell migration
To what extend does P-EMT contribute to each phenotype
What is PDAC
Pancreatic ductal adrenocarconoma
What are the mutations seen in PDAC
Activating mutation of K-Ras - G12D
Loss of allele of p53
Describe the progression of PDAC
Progresses quickly to an invasive and metastatic state
What system was used to make a PDAC mouse mutant
Cre-Lox
Describe the transgenics used to produce a PDAC model
KPCY –>
KRas - GoF G12D
Loss of 1 allele of p53
YFP
ONE MOUSE
Expressing cre under a pdx1 (pancreatic) promoter
ONE MOUSE
Floxed stop codon US of KRasG15D
Floxed P53
Floxed stop codon US of YFP
Describe what happens when the mutant mice are crossed
Cre is active in the pancreas
Removal of US stop condon - K-Ras mutant - is expressed
Removal of US stop condon -
Same for YFP
Cleavage at loxP sites - between p53
What do all transgenic PDAC cells express
YFP
Describe the expression of ECadherin in the PDAC cells
Fail to express ECad
What does M-ECAD mean
Membranous E-Cad
What is the differentiation state of M-CAD+ and M-CAD- cells
+ –> well differeniated
- –> Poorly differentiated
Describe how it was possible to sort cells into those that are M-Cad+/-
FACS to sort YFP+
Stain M-ECAD
FACS to sort M-ECAD
RNA sequencing
Describe what the principle component analysis of the M-Cad+/- cells shows
No huge difference between M-Cad+/- in the same tumour
Two clusters present
Main difference between different tumours
Describe the two clusters seen
Expression of E genes is very low and cells have acquired new M genes
Expressions of E genes is hig and M genes have been acquired
What were the two clusters named
C-EMT
P-EMT
What was seen when looking at mRNA of E genes in C -EMT cells
No mRNA found from epithelial genes
What was seen when looking at mRNA of E genes in P-EMT cells
How does this compare to protein expression
Still mRNA from epithelial genes
Found with protein -
CELLS M-ECAD- STILL EXPRESS E-CADHERIN even though it isn’t present at the membrane
What were the methods used to be able to visualise ECAD at the membrane and IC
FIRST STAIN
Without permeabilising - antibody only stains membrane
SECOND STAIN
Fix and permeabilised, second colour will label the IC ECAD
Desribe what was seen when staining cells that was C-EMT
No signal
NO ECAD EXPRESSION
Describe what was seen when staining cells that were P-EMT
Cells without M-ECAD has high levels of INTERNALISED E-CAD - its just that this wasnt at the membrane
So the P-EMT phenotype comes from _________ and NOT ___________
Protein relocalisation
NOT
Transcriptional repression
What was the second test for internalisation
Immunostaining roud 2
In P-EMT cell see punctate staining - consistent with internalisation
C-EMT used
Transcriptional repression
Describe the normal trafficking of E-Cadherin
ECAD undergoes cycles of endocytosis and recycling
In endosomes marked by Rab GTPase proteins
Rab5
Ealry endosomes
Rab7
Late endosomes
Rab11
Recycling endosomes
How was the identity of the IC compartments discovered
Looking for colocalisation with the Rab proteins - markers of the various types of endosomes
What did ECad colocalise with
What didnt it coloc with
Conclusions
Coloc with Rab 11
No coloc with 5 and 7
Suggests ECad is stuck in a recycling endosome
What is a future direction from the paper
Interogating the moelccular mechanisms that cause ECad to become stuck in the recycling endosomes
How was migration visualised
Tumour spheres generated from P-EMT and C-EMT
Embed these spheres into a 3D matrix (matrigel)
Can then perform live cell imaging to examine this invasive behaviour
Describe how C-EMT cells migrate
Spindle like protrusions
Single cells invade
Describe how P-EMT cells invade
Retention of cell-cell contacts
Invade as a collective group
Multi cellular clusters that delaminate from the primary cell mass
Describe how the morphology of P-EMT cells when migrating is different
No protrusive activity
More like amebooid single cell migration
Describe the efficiency of the migraiton and invasion process
Single cell - looks like every cell in the sphereis making protrusions
Collective cell migration - looks like only a few of the cells are able to make it away from the tumour sphere
Describe how it was seen how c_EMT and P-EMT cells migrate
Orhtoptic implantation of PDAC cells (P/C-EMT) into immunodeficient mice
Look for circulating tumour cells (CTCs) hen look to see if these are single cells or clusters
What is an issue with the orthoptic implantation of C/P-EMT expt
How do we know that the cells entered the blood this way
What do C-EMT cells migrate as
Mainly as single cells
What do P-EMT cells migrate as
Half and half
What does the orthoptic implantation of C/P-EMT expt not look into
Havent said if difference in invasive ability of cell types
If more invasion in one of the subtypes then expect higher number of cells in that subtype
Describe what was seen when staining CTC single cells for ECad
No ECAD
Describe what was seen when staining CTC clusters for ECad
No ECad at outer membrane
Some ECad at the cell junctions between the clusters
Why is CTC clusters having ECAD at junctions an advantage
Fascilitates cluster migratory behaviour
Describe if you could predict C-EMT or P-EMT based on the RNA component - what would you expect
Predict c-EMT - didnt express ECad or express it at low levels
Predict p-EMT - very high expression even in cells without M-ECAD
Double stain to identify M-ECAD/I-ECAD
Predict P-EMT - high proportion of I-ECAD
In humans there are ___ programs of EMT
What are they?
One through loss
One through relocalisation
What was seen when the expts were performed on breast cancer cells
Also in breast cancer - partial EMT where E-CAD is internalised
What was interesting about the EMT programs and the types of breast cancer
Different EMT programs leads to a different type of cancer forming
What type of BC forms from complete EMT
Basal subtype
Worst prognosis
What type of BC forms from the partial EMT
Luminal A/B subtype
If each subtype has a specific EMT what could be done?
Target therapies based on the EMT program
