The final presentation of my PhD defense!

1. CC BY-SA 4.0
TOWARDS AN OPEN DATA EXCHANGE ECOSYSTEM:
FORGING A NEW PATH FOR CELL MIGRATION DATA
ANALYSIS AND MINING
18 October 2016 public PhD defense - paola masuzzo

3. OpenCV; http://www.xinkaiwu.com/index.html

4. CC BY-SA 4.0
Introduction to cell migration
Research problems
Results
CellMissy: an automated tool for cell migration
A new CellMissy module for single-cell analysis
Engineering features to describe stochasticity
Towards an open data exchange ecosystem
Conclusions and future perspectives

5. CC BY-SA 4.0
Introduction to cell migration
Research problems
Results
CellMissy: an automated tool for cell migration
A new CellMissy module for single-cell analysis
Engineering features to describe stochasticity
Towards an open data exchange ecosystem
Conclusions and future perspectives

6. CC BY-SA 4.0
Cell migration is necessary for many
physiological functions
Basement
membrane
Wound
Migrating
epithelial cells

7. CC BY-SA 4.0
Cell migration is necessary for many
physiological functions
Blood
vessel
Site of tissue injury
Migrating
neutrophil
Basement
membrane
Wound
Migrating
epithelial cells

8. CC BY-SA 4.0
Unfortunately, it is also implicated in many
diseases, such as metastatic cancer

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The cytoskeleton is the key structural
framework responsible for cell migration
Adapted from Herzog et al., Cell Bio Lab Handbook, 1994

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The cytoskeleton is the key structural
framework responsible for cell migration
Adapted from Herzog et al., Cell Bio Lab Handbook, 1994

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Different actin filament structures are
essential for cell migration
Actin network
Filopodia
Lamellipodium
movement
Leading edge
2D migration

12. CC BY-SA 4.0
Different actin filament structures are
essential for cell migration
Actin network
Filopodia
Lamellipodium
movement
Leading edge
2D migration 3D invasion
Basement
membrane
Epithelial cell
Tumor cell
Extracellular matrix
Invadopodia
Protruding bleb
Lamellipodia or
pseudopodia

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Cell translocation depends on a cyclic interplay
between cell adhesion and de-adhesion

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A typical experimental workflow for a cell
migration study is composed of diverse steps
Servier Medical Art, CC-BY 3.0; Cell Image Library, CC-BY 3.0
sample
preparation
image
acquisition
image
processing
data
analysis

15. CC BY-SA 4.0
A typical experimental workflow for a cell
migration study is composed of diverse steps
Servier Medical Art, CC-BY 3.0; Cell Image Library, CC-BY 3.0
sample
preparation
image
acquisition
image
processing
data
analysis

16. CC BY-SA 4.0
Several assays are available for in vitro
assessment of cell migration
Servier Medical Art, CC-BY 3.0; Cell Image Library, CC-BY 3.0
sample
preparation
image
acquisition
image
processing
data
analysis
Wound-healing assay
pipette tip
scratch
Cell-exclusion zone assay
cell-free
zone
silicone
stopper
Spheroid assay
multicellular
spheroid

17. CC BY-SA 4.0
Live-cell phase-contrast and fluorescence
microscopy generates quantifiable data
Servier Medical Art, CC-BY 3.0; Cell Image Library, Public Domain; Mierke et al., 2011
sample
preparation
image
acquisition
image
processing
data
analysis

18. CC BY-SA 4.0
Time-lapse video microscopy captures
cell migration dynamics
time

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Image processing is a multi-step operation
comprising segmentation and tracking
Servier Medical Art, CC-BY 3.0; Cell Image Library, CC-BY 3.0, Harder et al., 2015
image
pre-processing
cell
tracking
cell
segmentation
sample
preparation
image
acquisition
image
processing
data
analysis

20. CC BY-SA 4.0
Quantitative parameters are then extracted
for cell sheet and single-cell trajectories
Image processed with CELLMIA, UGent (Van Troys M, Ampe C) and DciLabs
area in time
µm²/min
coordinates (x, y, t)
µm/min

21. CC BY-SA 4.0
Ultimately, data analysis enables
interpretation of the experiment
Servier Medical Art, CC-BY 3.0; Cell Image Library, CC-BY 3.0; O’ Brien et al., 2014
sample
preparation
image
acquisition
image
processing
data
analysis

22. CC BY-SA 4.0
The Ghent platform enables automation of
high-throughput cell migration experiments
Phase-contrast
live-cell imaging
time-lapse: 16-48 h
interval: 15-20 min
Adapted from Lynn Huyck, PhD thesis, 2012 (promoter Van Troys M)

23. CC BY-SA 4.0
Images are automatically processed
Adapted from Lynn Huyck, PhD thesis, 2012 (promoter Van Troys M); images processed with CELLMIA, UGent and DciLabs
t = 0h t = 24h
t = 0h t = 36h

24. CC BY-SA 4.0
Such high-throughput experiments produce
complex and rich data sets
Servier Medical Art, CC-BY 3.0; Cell Image Library, CC-BY 3.0
sample
preparation
image
acquisition
image
processing
data
analysis
• paper laboratory
notebooks
• electronic
laboratory
notebooks
• spreadsheets
• text files
• protocols
• papers...
• raw files
• XML files
• proprietary
microscope or
acquisition
software files 
ND2 for Nikon, LIF
for Leica, OIB or
OIF for Olympus,
LSM or ZVI for
Zeiss
• image files with
pixel values and
metadata
• png, jpeg, tiff, avi
• text files
describing
processing
algorithms
• text files
describing
extracted features
• graphs, plots
• analysis pipelines
• text files
describing
computational
algorithms...

25. CC BY-SA 4.0
Introduction to cell migration
Research problems
Results
CellMissy: an automated tool for cell migration
A new CellMissy module for single-cell analysis
Engineering features to describe stochasticity
Towards an open data exchange ecosystem
Conclusions and future perspectives

26. CC BY-SA 4.0
The overall objective of this PhD is to
advance bioinformatics for cell migration
Cell migration experiments have become de facto
high-throughput, but bioinformatics has lagged behind
Due to lack of automated systems and appropriate
algorithms, a big proportion of cell migration data is
still not exploited
The heterogeneity of the field hampers open data
exchange, impeding advanced data analysis and mining

27. CC BY-SA 4.0
Introduction to cell migration
Research problems
Results
CellMissy: an automated tool for cell migration
A new CellMissy module for single-cell analysis
Engineering features to describe stochasticity
Towards an open data exchange ecosystem
Conclusions and future perspectives

28. CC BY-SA 4.0
CellMissy is our open-source tool for cell
migration data management and analysis
0 3h 6h
wound
cells
Experiment
Data Analyzer
Data Loader
Collective cell migration Single-cell migration
Experiment Manager
Masuzzo et al., Bioinformatics, 2013; https://github.com/compomics/cellmissy

29. CC BY-SA 4.0
CellMissy guides and captures the
experimental setup

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This experimental setup encloses detailed
metadata annotation

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CellMissy can automatically import all the
data and metadata

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All the data are then stored in a structured
way in a relational database

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CellMissy enables efficient data exploration
and analysis
time (min)
Area(μm2
)
wound area
cell-covered area

34. CC BY-SA 4.0
A primary focus of data analysis is statistical
comparison of samples
analysis report with graphs,
tables and results
cell sheet velocity (µm²/min)

35. CC BY-SA 4.0
Introduction to cell migration
Research problems
Results
CellMissy: an automated tool for cell migration
A new CellMissy module for single-cell analysis
Engineering features to describe stochasticity
Towards an open data exchange ecosystem
Conclusions and future perspectives

36. CC BY-SA 4.0
Cell migration can occur in both collective
and individual fashion
Collective
migration
Multicellular
streaming
Mesenchymal
Amoeboid
(blebs)
Amoeboid
(pseudopodia,
filopodia)
INDIVIDUAL
MIGRATION
COLLECTIVE
MIGRATION
Adapted from Friedl et al., J. Exp. Med., 2010

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Many informative parameters can be derived
from single-cell trajectories
Masuzzo et al., under review, 2016
x
y
single cell

38. CC BY-SA 4.0
Many informative parameters can be derived
from single-cell trajectories
Masuzzo et al., under review, 2016
Euclidean
distance
Cumulative
distance
x
y
single cell

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Many informative parameters can be derived
from single-cell trajectories
Masuzzo et al., under review, 2016
Euclidean
distance
Cumulative
distance
x
y
single cell parameter mathematical description
di: instantaneous
displacement of the
cell centroid between
adjacent time points
𝑑𝑖 = 𝑥𝑖+1 − 𝑥𝑖
2 + 𝑦𝑖+1 − 𝑦𝑖
2
si: instantaneous speed
between adjacent time
points
𝑠𝑖 = 𝑑(𝑝𝑖, 𝑝𝑖+1) ∆𝑡
αi: turning angle
between consecutive
steps
𝛼𝑖 = 𝑡𝑎𝑛−1 𝑦𝑖+1 − 𝑦𝑖 𝑥𝑖+1 − 𝑥𝑖
dtot: cumulative
distance, total distance
travelled
𝑑 𝑡𝑜𝑡 =
𝑖=1
𝑁−1
𝑑 𝑝𝑖, 𝑝𝑖+1
dnet: Euclidean distance,
net distance travelled
𝑑 𝑛𝑒𝑡 = 𝑑 𝑝1, 𝑝 𝑁
ep_dr: end-point
directionality ratio
(confinement ratio,
meandering index)
𝑒𝑝_𝑑𝑟 = 𝑑 𝑛𝑒𝑡 𝑑 𝑡𝑜𝑡
MD: median
displacement
𝑀𝐷 = 𝑚𝑒𝑑𝑖𝑎𝑛 𝑑𝑖
MS: median speed 𝑀𝑆 = 𝑚𝑒𝑑𝑖𝑎𝑛 𝑠𝑖
MTA: median turning
angle
𝑀𝑇𝐴 = 𝑚𝑒𝑑𝑖𝑎𝑛 𝛼𝑖

40. CC BY-SA 4.0
Step-centric parameters are aggregated
values of all migration steps

41. CC BY-SA 4.0
Trajectory-centric parameters are instead
computed on each cell and then averaged for the
cell population
...
trajectory 1
trajectory 2
trajectory 3
trajectory 4
trajectory 5

42. CC BY-SA 4.0
The new single-cell module allows for both
these computations to take place
Masuzzo et al., under review, 2016
...
trajectory 1
trajectory 2
trajectory 3
trajectory 4
trajectory 5
trajectory-centric parameters
trajectory displacement (µm)
density
step displacement (µm)
density
pool of migration steps
step-centric parameters

43. CC BY-SA 4.0
A flexible two-step filtering criterion is
implemented for data quality control
Masuzzo et al., under review, 2016

44. CC BY-SA 4.0
Introduction to cell migration
Research problems
Results
CellMissy: an automated tool for cell migration
A new CellMissy module for single-cell analysis
Engineering features to describe stochasticity
Towards an open data exchange ecosystem
Conclusions and future perspectives

45. CC BY-SA 4.0
More advanced features are needed to
describe the complexity of the phenomenon
Masuzzo et al., in preparation, 2016

46. CC BY-SA 4.0
The enclosing circle set is a new way to
describe local structure of trajectories
Masuzzo et al., in preparation, 2016
radius: 6 µm
nr_circles: 14
direction
of motion

47. CC BY-SA 4.0
The enclosing circle set is a new way to
describe local structure of trajectories
Masuzzo et al., in preparation, 2016
radius: 6 µm
nr_circles: 14
direction
of motion
radius: 3 µm
nr_circles: 22
direction
of motion

48. CC BY-SA 4.0
The fractal dimension is derived from the
enclosing circle set
𝐹𝐷 𝑆 = lim
𝑟→0
log )𝑁(𝑟 log 1
𝑟
Masuzzo et al., in preparation, 2016
FD=0.35 FD=0.83

49. CC BY-SA 4.0
Introduction to cell migration
Research problems
Results
CellMissy: an automated tool for cell migration
A new CellMissy module for single-cell analysis
Engineering features to describe stochasticity
Towards an open data exchange ecosystem
Conclusions and future perspectives

50. CC BY-SA 4.0
Data and metadata exchange options are
already available in CellMissy
lab A

51. CC BY-SA 4.0
Data and metadata exchange options are
already available in CellMissy
lab A lab B

52. CC BY-SA 4.0
Data and metadata exchange options are
already available in CellMissy
lab B
This is one file in CellMissy! (≈10 MB)
lab A

53. CC BY-SA 4.0
But we can easily extend this concept
to a bigger scale
Data
Repository
Local Software

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The seed of this idea was planted in the field
Friedl et al., Nature Reviews, 2012

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It only needed some water to grow
Cell migration workshop, Ghent, March 2014; Masuzzo et al., Trends in Cell Biology, 2015

56. CC BY-SA 4.0
An open data exchange ecosystem for cell
migration research is now on its way
Masuzzo et al., Trends in Cell Biology, 2015

57. CC BY-SA 4.0
An open data exchange ecosystem for cell
migration research is now on its way
Masuzzo et al., Trends in Cell Biology, 2015

58. CC BY-SA 4.0
An open data exchange ecosystem for cell
migration research is now on its way
Masuzzo et al., Trends in Cell Biology, 2015

59. CC BY-SA 4.0
This open data ecosystem falls into the
broader context of open science
Knoth and Pontika, Open Science Taxonomy, figshare, 2015

60. CC BY-SA 4.0
Open access is another key factor in the
open science equation
Knoth and Pontika, Open Science Taxonomy, figshare, 2015

61. CC BY-SA 4.0
The impacts of open access are very broad
and affect many areas
Tennant, Masuzzo et al., F1000Research, 2016; Wikimedia Commons, Public Domain

62. CC BY-SA 4.0
Publish your work open access can bring you
enormous benefits
CC-BY Danny Kingsley & Sarah Brown

63. CC BY-SA 4.0
Publish your work open access can bring you
enormous benefits
CC-BY Danny Kingsley & Sarah Brown

64. CC BY-SA 4.0
Publish your work open access can bring you
enormous benefits
CC-BY Danny Kingsley & Sarah Brown

65. CC BY-SA 4.0
Open access also allows automatic
knowledge extraction through text mining
automatically detect a set of core information reported when
describing cell migration experiments
check for nomenclature consistency, the use of common terms or
ontologies to describe the same concept
construct a knowledge map to describe the state-of-the-art, especially
in terms of cell motility-related compounds and cancer cell lines

66. CC BY-SA 4.0
Introduction to cell migration
Research problems
Results
CellMissy: an automated tool for cell migration
A new CellMissy module for single-cell analysis
Engineering features to describe stochasticity
Towards an open data exchange ecosystem
Conclusions and future perspectives

67. CC BY-SA 4.0
This PhD has tackled key bioinformatics
challenges in cell migration research
CellMissy is the first free and open-source tool for the
management, annotation and storage of cell migration
experiments
A new dedicated module, together with novel features,
enable detailed and more complex quantification of
single-cell migration experiments
International research efforts are currently spent towards
the establishment of an open data exchange ecosystem,
opening the way to more advanced data analysis and
mining strategies

68. CC BY-SA 4.0
These results have paved the way to even
more exciting opportunities
CellMissy has already been extended with dose-response
analysis capabilities, and more development is planned to
allow meta-analyses to take place
Further engineering, validation, and selection of single-cell
migration features is planned; these features will then be
used to automatically detect and classify migratory
phenotypes
Joined efforts of MULTIMOT and the CMSO will ultimately
enable global data dissemination in the field, allowing data
re-use, re-discovery and re-purpose

69. CC BY-SA 4.0