intravital microscopy of cancer cell invasion and therapy ... · efficient excitation of...
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Microscopical Imaging of the Cell, Dept. Cell Biology
Radboud University Nijmegen, The Netherlands
David H. Koch Center
The University of Texas MD Anderson Cancer Center
Intravital microscopy of cancer cell invasion
and therapy response
Peter Friedl
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Cancer progression and metastasis: niche business
Hanahan & Weinberg, Cell, 2011
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Near-infrared and infrared multiphoton microscopy
3 Titanium:Sapphire lasers (700 – 1060 nm)
2 Optical Parametric Oscillators (1060 – 1400 nm)
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Nearinfrared / infrared-excited
multiphoton microscopy
Efficient excitation of red-shifted fluorophores
(RFP, mCherry, Neptun, E2-Crimson, AlexaFluor 660...)
80 - 100% deeper tissue penetration
Low phototoxicity (<100 mW)
Third harmonic generation to visualize interfaces
Combined fluorescence intensity with lifetime detection
In development: Multiphoton endomicroscopy of deep
tissue regions
Andresen et al., Curr. Opin. Biotechnol. (2009); Weigelin et al., IntraVital (2012)
Bakker et al., Meth. Enzymol. (2012); Alexander et al., Curr. Opin. Cell Biol. (2013)
Third harmonic generation
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S0
S1
3
(ex) = 1200 nm
OPO
(THG) = 400 nm
forward
detector
backward
detector
Fat cells
Third harmonic generation (THG)
Scatter process:
unification of 3
photons
triple frequency
scattered by tissue
Generated by:
Interfaces
Inhomogeneities
Dependent on size and orientation of heterogeneities relative to light path
*
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Combined THG / SHG / fluorescence intensity imaging
H2B-GFP
(tumor
cells)
THG
(nerve
bundles)
THG
(fat
cells)
SHG
(fibrillar
collagen)
1180 n
m
393 n
m
Ex. Em.
1180 n
m
590 n
m
Ex. Em.
Third harmonic
generation
Second harmonic
generation
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Nonlinear
microscopy of
biomaterials
in vivo
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Higher harmonic microscopy of medical implant materials
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http://explantinfo.com
Foreign body response: fibrotic encapsulation of implants
Fibrotic
silicone
breast
implant
(after
surgical
removal)
Silicone implant (ruptured) Fibrotic capsule
http://pathologyoutlines.com
Collagen bundles
Silicone
Polyurethane
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Higher harmonic microscopy of the FBR response
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Interstitial cells (monocytes/macrophages)
Scaffold-associated multinucleated giant cells
PCL fiber
Neovessel
Giantcell
Fibrotic capsule
Monocyte/macrophage
Macrophages and giant cells:
friends or foes?
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VEGF producing M1 macrophages and giant cells
IRF5 (M1) CD163 (M2)
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UntreatedClodronate
VEGF TrapClodr.+VT
Interfering with the foreign body response
Fibroticcapsule
Regular dermalcollagen
GF
P+
ce
llsD
extr
an
SH
G T
HG
SH
G, d
28
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Foreign body response: desmoplasia-like progression
Dondossola et al., Nat. Biomed. Engineering (2017)
2
1
Fibrosis
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Foreign body response: desmoplasia-like progression
Dondossola et al., Nat. Biomed. Engineering (2017)
22 11
2
1
Fibrosis
Intervention options
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Monitoring
tumor invasion
and metastasis
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Needle
Tumor cells
Connective
tissue
Border
of metal chamber
Histon 2B-EGFP
Red cytoplasm (DsRed2, E2-Crimson)
Alexander et al., Histochem. Cell Biol. (2008)
Epidermis
Blood vessels
Muscle strand
Cover slip
Interface
Tumor cells
Modified dorsal skin-fold chamber model
Dermis
Xenograft tumors: HT-1080, MV3
Syngenic tumors: B16/F10, MFS
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d1
Dynamics of tumor neoangiogenesis and invasion
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d3 Onset of invasion
Dynamics of tumor neoangiogenesis and invasion
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d5 Angiogenic switch
Dynamics of tumor neoangiogenesis and invasion
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d7 Exponential growth
Dynamics of tumor neoangiogenesis and invasion
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Friedl & Alexander
Cell (2011)
Diversity of
invasion
programs
in vivo
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Wolf et al., Nat. Cell Biol. (2007)
Friedl et al., Nat. Cell Biol. (2012)
Aceto et al., Cell, 2014
Cheung et al., PNAS, 2016
Cheung & Ewald, Science, 2016
Collective cancer invasion and metastasis
K14
Plakoglobin
EPCAM
seeding
50x
CTC cluster from peripheral bloodDuctal breast carcinoma (clinical sample)
E-cadherin Vimentin DAPI
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Microenvironmental
context:
principles of
guidance by tissue
structures
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Liotta, LA, Trygvason K, et al., Nature (1980)
Liotta, LA Rhodes Memorial Award Lecture, Cancer Res. (1986)
First three-step model of invasive cell migration
Step 1: Attachment Step 2: Dissolution Step 3 Locomotion
Type IV collagenase (MMP2, MMP9)
Laminin
Laminin receptor
Type IV collagen
Deduced paradigm:
Proteolytic degradation of extracellular matrix is
required for cell migration into tissue
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Linear / confined Broad / diffuse
Two types of collective invasion patterns in response
to connective tissue organization
50 µm
E2-Crimson SHG THG
AlexaFluor750-dextran H2B/eGFPWeigelin et al., IntraVital (2012)
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SHGE2-Crimson
MergeTHG
Multicellular streaming along aligned fibrillar collagen
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Tissue geometry dictates invasion pattern
Weigelin et al., Intravital (2012)
Te Boekhorst et al., Annu. Rev. Cell Dev. Biol. (2016)
Non-destructive migration along preexisting interfaces
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Collective invasion
protects moving cells
from mechanical
assault
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Identification of mechanical challenge in vivo
NLS
NLS NLS
Transient rupture ofthe nuclear envelope
Collaboration with Jan Lammerding, Cornell Univ.
Denais et al., Science (2016)
Mechanical stress sensing: translocation
of NLS-GFP
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Identification of mechanical challenge in vivo
NLS
NLS NLS
Transient rupture ofthe nuclear envelope
Collaboration with Jan Lammerding, Cornell Univ.
Denais et al., Science (2016)
Mechanical stress sensing: translocation
of NLS-GFP
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NLS-GFP H2B-mCherry NLS-GFP
SHG 70kDa-dextran
Mitosis
Sin
gle
cell
Thin
str
and
Thic
k s
trand
Number of cells observed:
Single cells: 22
Thick strands: 417
Thin strands: 82
4 independent mice
0
10
20
30
Cells
with
nucle
ar
ruptu
re(%
)
Tissue regions imposing mechanical challenge in vivo
**
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Adaptation of migration mode and tissue patterns
Weigelin et al., Intravital (2012)
Denais et al. Science (2016)
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Track widening or not widening
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Infrastructure enabling mobility: tubes and tracks
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Vessel
Muscle strand
EC
Collagen I
Pericyte
Multicellular
tumor
strand
Macrophage
Basemant
membrane
Fibroblast
Integrins MMPCadherins Chemokine/Growth factor & receptorEphrin/Eph
The invasion niche
- Aligned tracks of least resistance (microchannels)
- Multiple interfaces: collagen fibers, BM, cell surfaces, GAGs
- Nearby nutrient / oxygen supply
- Constitutive trafficking routes for passenger cells
Guided migration along "tissue highways"
GAGs
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Conclusions
• Tissue building blocks and interfaces enabling
nondestructive guidance for cancer cell invasion
• Invasion is coupled to accelerated DNA damage response
and enhances cancer cell survival during radiation therapy
• Tumor cell elimination by cytotoxic T lymphocytes is greatly
enhanced by CTL cooperation and additive cytotoxicity along
migration-supporting tissue tracks
Intravital microscopy has enabled understanding of
key questions:
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Cell Dynamics Laboratory / MIC
Stephanie
Alexander
Bettina
Weigelin
Volker Andresen
Gert-Jan Bakker
Anna
Häger
Esther
Wagena
Steve Alexander
Eleonora
Dondossola
Imaging Section
Michael Weiger WeigerJack Fransen
Manon
Vullings
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Michael Flentje, Stephan Kissler
U Würzburg
Collaborators
Uta Flucke, Willeke Bloxxk,
Jan Bussink
U Nijmegen
Simon Goodman
Merck Darmstadt
Funding
Erik Bushong, Mark Ellisman
UC San Diego
Jan Lammerding
Cornell Univ.
Grudrun Köhl, Ed Geissler
U Regensburg
Robert M. Hoffman
UC San Diego
Dietmar Hutmacher, Boris Holzapfel
Brisbane