Download Immune response to multiple stimuli

Immune response to multiple stimuli
Ann Babtie
17th December 2014
17th December 2014
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Zebrafish larvae model for innate immune response
Translucent, genetically tractable, only innate immunity
Juliane — immune cell migration to wounds
(Immunology and Cell Biology, 2013):
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Innate immune response to cancer
Pre-neoplastic cells trigger inflammatory response in zebrafish
Collaboration with Paul Martin’s group, Bristol University
(Feng et al, PLoS Biology 2010 & Current Biology 2012)
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Immune response to wound and cancer in vivo
Data from Laura Ward, Bristol lab
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Feng et al, PLoS Biology 2010
Neutrophils migrate to both wounds and cancer cells
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Feng et al, PLoS Biology 2010
Cancer triggers chronic (non-resolving) inflammation
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Feng et al, PLoS Biology 2010
Neutrophils attracted by (stochastic) H2 O2 signal
Leukocytes physically interact with cancer cells
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Feng et al, Current Biology 2012
Leukocyte-derived prostaglandin E2 promotes tumor growth
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ph
ils
+ neu
PG tro
E2 ph
ils
tro
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-n
co
nt
ro
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Cancer cell count reduced by Cox-2 or mPGES inhibitors,
neutrophil knockdown, EP1 receptor inhibition ....
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Project aim
What are spatio-temporal dynamics of chemoattractants?
What is spatial range of signal from cancer cells?
Threshold for immune cell to move from wound to cancer?
Does wound visit impact leukocyte response to cancer cell?
How do immune cells behave around different cancer types?
Do neutrophils & macrophages influence each other?
What are molecular mediators of cell behaviour?
....
Tools
Tracking software, cell migration statistics, models
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Cell tracking software
1) Original image
2) Thresholding
6) Reduce each cell to point 0
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3) Binarised image
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4) Labelling 5) Combine z-­‐stacks 0
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z
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z
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y
y
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(pymol example)
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Cell tracking software — problems
Over/under-segmentation of cells
Short cell tracks
Trade-off between noise and time-resolution
A) macrophages
C) neutrophils (good time-resolution)
B) cancer cells
D) cancer cells (good time-resolution)
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Cell tracking software — planned changes
Record cell areas (and “volumes”) to help segmentation
Use cell volumes to help tracking
Calculate full cell-cell distance matrix for adjacent times
t3
t3
t2
t1
t2
t1
Alternative tracking algorithms (e.g. keyhole, multiple paths)
Correct for fish movement over time
How to validate?
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Cell tracking software — adapt alternative?
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Cell migration statistics
Are there spatial/temporal patterns for cell migration features?
Distance from wound/cancer
Step size
Direction of movement
Sliding window averages
Level of bias?
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300
500
0 100
Distance / um
0 100
0 1
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120
Min dis
50
100
length (no. timepoints)
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0 100 500 300
Distance / um Distance / um
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5
10
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length (no. timepoints)
0 500
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300
Distance / um
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0
0 500
100
0
0
0
40
Frequency
Frequency
100
50
150
Frequency
50
80
40
Frequency
150
100
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Distance / um
Distance / um
120
Frequency
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30
50
Frequency
80 120
20
0 100
500
Step (um)
0 050
5 10
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40
Frequency
0
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max. radius (um)
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800
Frequency
1200
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Short trajectories
150
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0 500
100
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Distance / um
Trajectory lengths
Distance to wound, t Min
41−80
Min distance to cancer, t 41−80
distance to cancer,
t 1−40
Distance
to wound, t 81−120
Trajectory Step sizes Cell size Distance to wound, t 1−40
lengths 0
Frequency
300
Distance / um
0
Fre
0
0 100
Step sizes
Neutrophils (3174 cells)
0 50
40
Fre
40
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0
Cell migration statistics — problems & plans
500
20
0 1
!me 0 100
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Distance / um
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Distance / um
Distance to wound, t 41−80
0 500
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300
0 100 500 300
Distance / umDistance / um
300
0 100 500 300
Distance / um Distance / um
Min distance to cancer,
Distancet 41−80
to wound, t Min
121−167
Distance to wound, t 81−120
distance to cancer, t 81−120
100
0
50
Frequency
80
40
Frequency
0
0
0 500
100
Min dis
120
100 150
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Frequency
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Frequency
0
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Frequency
0
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0 50
Frequency
80 120
0
40
Frequency
80
40
Frequency
0
120
Min distance to cancer,
Distancet 1−40
to wound, t Min
81−120
Min distance to cancer, t 81−120
Distance to wound, t 41−80
distance to cancer,
t 41−80
Distance
to wound, t 121−167
120
Distance to wound, t 1−40
0 500
100
300
Distance / um
500
Min distance to cancer, t 121−167
50
Frequency
80
40
Frequency
50
Frequency
Frequency
80
40
Frequency
40
Frequency
Broken trajectories — improve tracking software
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100 150
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Developmental guidelines dominate — image different section
0 100
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Distance / um
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Distance to wound, t 81−120
0 100
300
Distance / um
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0
0 500
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How to compare videos — normalise by cancer cell density?
pre/post-wound video?
300
0 100 500 300
Distance / umDistance / um
0 500
100
300
500
Distance / um
distance to cancer, t 121−167
Immune response to multipleMinstimuli
Min distance to cancer, t 81−120
Distance to wound, t 121−167
0 100
300
500
Distance / um
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0 1
Models — stochastic population simulations
Can we use stochastic models to analyse cell population dynamics?
Define species and reactions
Simulate using Gillespie’s algorithm
from à to 1.  Ø N 2.  N Ø 3.  C C* 4.  C* C 5.  N+C NC 6.  N+C* NC* 7.  NC NC* 8.  NC* NC 9.  NC N + C 10. NC* N + C* 11. N NW 12. NW N 17th December 2014
N NW C NC Immune response to multiple stimuli
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Models — stochastic population simulations
Define species (populations) by spatial groups — comparison
between videos?
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Biological questions?
What are spatio-temporal dynamics of chemoattractants?
What is spatial range of signal from cancer cells?
Threshold for immune cell to move from wound to cancer?
Does wound visit impact leukocyte response to cancer cell?
How do immune cells behave around different cancer types?
Do neutrophils & macrophages influence each other?
What are molecular mediators of cell behaviour?
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