L7 - Invasion and Metastasis II

Question 1

What causes most of the cancer related deaths

Answer 1

Metastases

Question 2

Metastatic dissemination requires

Answer 2

EMT

Question 3

Describe what happens with EMT

Answer 3

Loss of adhesive protpoerities and acquire fibroblast like morphology and increased motility

Question 4

What does EMT compass a range of

Answer 4

Hybrid states

Question 5

What were the research questions put forward by the team who wrote the paper

Answer 5

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

Question 6

What is PDAC

Answer 6

Pancreatic ductal adrenocarconoma

Question 7

What are the mutations seen in PDAC

Answer 7

Activating mutation of K-Ras - G12D

Loss of allele of p53

Question 8

Describe the progression of PDAC

Answer 8

Progresses quickly to an invasive and metastatic state

Question 9

What system was used to make a PDAC mouse mutant

Answer 9

Cre-Lox

Question 10

Describe the transgenics used to produce a PDAC model

Answer 10

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

Question 11

Describe what happens when the mutant mice are crossed

Answer 11

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

Question 12

What do all transgenic PDAC cells express

Answer 12

YFP

Question 13

Describe the expression of ECadherin in the PDAC cells

Answer 13

Fail to express ECad

Question 14

What does M-ECAD mean

Answer 14

Membranous E-Cad

Question 15

What is the differentiation state of M-CAD+ and M-CAD- cells

Answer 15

+ –> well differeniated

• –> Poorly differentiated

Question 16

Describe how it was possible to sort cells into those that are M-Cad+/-

Answer 16

FACS to sort YFP+
Stain M-ECAD
FACS to sort M-ECAD
RNA sequencing

Question 17

Describe what the principle component analysis of the M-Cad+/- cells shows

Answer 17

No huge difference between M-Cad+/- in the same tumour

Two clusters present
Main difference between different tumours

Question 18

Describe the two clusters seen

Answer 18

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

Question 19

What were the two clusters named

Answer 19

C-EMT

P-EMT

Question 20

What was seen when looking at mRNA of E genes in C -EMT cells

Answer 20

No mRNA found from epithelial genes

Question 21

What was seen when looking at mRNA of E genes in P-EMT cells

How does this compare to protein expression

Answer 21

Still mRNA from epithelial genes

Found with protein -

CELLS M-ECAD- STILL EXPRESS E-CADHERIN even though it isn’t present at the membrane

Question 22

What were the methods used to be able to visualise ECAD at the membrane and IC

Answer 22

FIRST STAIN
Without permeabilising - antibody only stains membrane

SECOND STAIN
Fix and permeabilised, second colour will label the IC ECAD

Question 23

Desribe what was seen when staining cells that was C-EMT

Answer 23

No signal

NO ECAD EXPRESSION

Question 24

Describe what was seen when staining cells that were P-EMT

Answer 24

Cells without M-ECAD has high levels of INTERNALISED E-CAD - its just that this wasnt at the membrane

Question 25

So the P-EMT phenotype comes from _________ and NOT ___________

Answer 25

Protein relocalisation NOT Transcriptional repression

Question 26

What was the second test for internalisation

Answer 26

Immunostaining roud 2 In P-EMT cell see punctate staining - consistent with internalisation

Question 27

C-EMT used

Answer 27

Transcriptional repression

Question 28

Describe the normal trafficking of E-Cadherin

Answer 28

ECAD undergoes cycles of endocytosis and recycling | In endosomes marked by Rab GTPase proteins

Question 29

Rab5

Answer 29

Ealry endosomes

Question 30

Rab7

Answer 30

Late endosomes

Question 31

Rab11

Answer 31

Recycling endosomes

Question 32

How was the identity of the IC compartments discovered

Answer 32

Looking for colocalisation with the Rab proteins - markers of the various types of endosomes

Question 33

What did ECad colocalise with What didnt it coloc with Conclusions

Answer 33

Coloc with Rab 11 No coloc with 5 and 7 Suggests ECad is stuck in a recycling endosome

Question 34

What is a future direction from the paper

Answer 34

Interogating the moelccular mechanisms that cause ECad to become stuck in the recycling endosomes

Question 35

How was migration visualised

Answer 35

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

Question 36

Describe how C-EMT cells migrate

Answer 36

Spindle like protrusions | Single cells invade

Question 37

Describe how P-EMT cells invade

Answer 37

Retention of cell-cell contacts Invade as a collective group Multi cellular clusters that delaminate from the primary cell mass

Question 38

Describe how the morphology of P-EMT cells when migrating is different

Answer 38

No protrusive activity | More like amebooid single cell migration

Question 39

Describe the efficiency of the migraiton and invasion process

Answer 39

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

Question 40

Describe how it was seen how c_EMT and P-EMT cells migrate

Answer 40

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

Question 41

What is an issue with the orthoptic implantation of C/P-EMT expt

Answer 41

How do we know that the cells entered the blood this way

Question 42

What do C-EMT cells migrate as

Answer 42

Mainly as single cells

Question 43

What do P-EMT cells migrate as

Answer 43

Half and half

Question 44

What does the orthoptic implantation of C/P-EMT expt not look into

Answer 44

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

Question 45

Describe what was seen when staining CTC single cells for ECad

Answer 45

No ECAD

Question 46

Describe what was seen when staining CTC clusters for ECad

Answer 46

No ECad at outer membrane | Some ECad at the cell junctions between the clusters

Question 47

Why is CTC clusters having ECAD at junctions an advantage

Answer 47

Fascilitates cluster migratory behaviour

Question 48

Describe if you could predict C-EMT or P-EMT based on the RNA component - what would you expect

Answer 48

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

Question 49

In humans there are ___ programs of EMT What are they?

Answer 49

One through loss One through relocalisation

Question 50

What was seen when the expts were performed on breast cancer cells

Answer 50

Also in breast cancer - partial EMT where E-CAD is internalised

Question 51

What was interesting about the EMT programs and the types of breast cancer

Answer 51

Different EMT programs leads to a different type of cancer forming

Question 52

What type of BC forms from complete EMT

Answer 52

Basal subtype | Worst prognosis

Question 53

What type of BC forms from the partial EMT

Answer 53

Luminal A/B subtype

Question 54

If each subtype has a specific EMT what could be done?

Answer 54

Target therapies based on the EMT program