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BMS379 - Cancer Biology > L7 - Invasion and Metastasis II > Flashcards

L7 - Invasion and Metastasis II Flashcards

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1
Q

What causes most of the cancer related deaths

A

Metastases

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2
Q

Metastatic dissemination requires

A

EMT

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3
Q

Describe what happens with EMT

A

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

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4
Q

What does EMT compass a range of

A

Hybrid states

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5
Q

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

A

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

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6
Q

What is PDAC

A

Pancreatic ductal adrenocarconoma

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7
Q

What are the mutations seen in PDAC

A

Activating mutation of K-Ras - G12D

Loss of allele of p53

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8
Q

Describe the progression of PDAC

A

Progresses quickly to an invasive and metastatic state

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9
Q

What system was used to make a PDAC mouse mutant

A

Cre-Lox

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10
Q

Describe the transgenics used to produce a PDAC model

A

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

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11
Q

Describe what happens when the mutant mice are crossed

A

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

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12
Q

What do all transgenic PDAC cells express

A

YFP

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13
Q

Describe the expression of ECadherin in the PDAC cells

A

Fail to express ECad

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14
Q

What does M-ECAD mean

A

Membranous E-Cad

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15
Q

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

A

+ –> well differeniated

  • –> Poorly differentiated
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16
Q

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

A

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

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17
Q

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

A

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

Two clusters present
Main difference between different tumours

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18
Q

Describe the two clusters seen

A

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

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19
Q

What were the two clusters named

A

C-EMT

P-EMT

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20
Q

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

A

No mRNA found from epithelial genes

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21
Q

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

How does this compare to protein expression

A

Still mRNA from epithelial genes

Found with protein -

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

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22
Q

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

A

FIRST STAIN
Without permeabilising - antibody only stains membrane

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

23
Q

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

A

No signal

NO ECAD EXPRESSION

24
Q

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

A

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

25
Q

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

A

Protein relocalisation
NOT
Transcriptional repression

26
Q

What was the second test for internalisation

A

Immunostaining roud 2

In P-EMT cell see punctate staining - consistent with internalisation

27
Q

C-EMT used

A

Transcriptional repression

28
Q

Describe the normal trafficking of E-Cadherin

A

ECAD undergoes cycles of endocytosis and recycling

In endosomes marked by Rab GTPase proteins

29
Q

Rab5

A

Ealry endosomes

30
Q

Rab7

A

Late endosomes

31
Q

Rab11

A

Recycling endosomes

32
Q

How was the identity of the IC compartments discovered

A

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

33
Q

What did ECad colocalise with

What didnt it coloc with

Conclusions

A

Coloc with Rab 11

No coloc with 5 and 7

Suggests ECad is stuck in a recycling endosome

34
Q

What is a future direction from the paper

A

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

35
Q

How was migration visualised

A

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

36
Q

Describe how C-EMT cells migrate

A

Spindle like protrusions

Single cells invade

37
Q

Describe how P-EMT cells invade

A

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

38
Q

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

A

No protrusive activity

More like amebooid single cell migration

39
Q

Describe the efficiency of the migraiton and invasion process

A

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

40
Q

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

A

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

41
Q

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

A

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

42
Q

What do C-EMT cells migrate as

A

Mainly as single cells

43
Q

What do P-EMT cells migrate as

A

Half and half

44
Q

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

A

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

45
Q

Describe what was seen when staining CTC single cells for ECad

A

No ECAD

46
Q

Describe what was seen when staining CTC clusters for ECad

A

No ECad at outer membrane

Some ECad at the cell junctions between the clusters

47
Q

Why is CTC clusters having ECAD at junctions an advantage

A

Fascilitates cluster migratory behaviour

48
Q

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

A

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

49
Q

In humans there are ___ programs of EMT

What are they?

A

One through loss

One through relocalisation

50
Q

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

A

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

51
Q

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

A

Different EMT programs leads to a different type of cancer forming

52
Q

What type of BC forms from complete EMT

A

Basal subtype

Worst prognosis

53
Q

What type of BC forms from the partial EMT

A

Luminal A/B subtype

54
Q

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

A

Target therapies based on the EMT program

BMS379 - Cancer Biology (11 decks)

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