High content screening for 3D cell migration and invasion technologies and applications report

IntroThe current technology in this field can be categorized into three categories: monolayer (co)
culture in/on gel, 3D spheroids (co) culture in/on gel, and microfluidic devices. Monolayer culture
requires the least skill/time/hardware/money. 3D spheroid culture requires automated confocal
microscopy with advanced imaging software. Microfluidic devices are not an option to most ordinary
biomedical research labs.
1. Monolayer of cells in/on gel
This consists of seeding a monolayer of cells in or on gels. A filter (Boyden Chamber) can
optionally be added. The most common implementation is monolayer on gel, with filter. Then,
the invasiveness of the cancer cells can be easily quantified by the number of cells that passed
through the filter at the bottom of the gel. The contents and the thickness of the gel can be
easily varied. Phenotypic screening is easy. Cells can be fluorescently labelled and with
Fluoroblok technology (opaque filter), the number of cells that passed through can be read with
a bottom-reading plate fluorescence reader. Alternatively, a normal filter can be used and the
gel removed. The plate is then visualized under a microscope. The benefits include short time
requirement, easy quantification and variation of parameters, and well-defined cell migration
starting point. Drawbacks include only endpoint monitoring, which might lead to misleading
results. However, the cross sections of the gels can be subsequently analyzed, yielding better
results.

Monolayer invasion assay on gel can be also done without filter. The gel cross section could be
examined for the migration of cells. This could be done through confocal microscopy or
sectioning of the gel. Current high-throughput applications of this is quite limited. Alternatively,
the gel could be fluorescently labelled (gelatin degradation assay), and the areas that lacked
fluorescence is where the cells ate through. Cells can also be tracked by other methods,
including automated confocal microscopy with advanced imaging analysis. ImarisTrack is a
commercially available software for 3D cell tracking.

Monolayer invasion assay in gel can be used for asymmetric measurement of different gel
environment factors on tumor invasiveness. The monolayer is seeded in between two gels, and
the upper and bottom gel environment may be varied. The cross section can be taken, or
confocal microscopy can be done. The high-throughput potential of this technique and the one
mentioned above will depend largely upon automated 3D imaging technology and processing
software.
2
A recent protocol for 3D imaging using GFP reporter is promising.
3
Also, the Platypus
Pro assay can be used. Its basically an exclusion zone assay stuck in between two layers of BME
gel. Kinetic and live-imaging and high-throughput is possible.

2. Spheroids in/on gel
Monolayer 3D models do not allow extensive cell-cell interactions and simulation of other
factors like hypoxia and chemical gradients. The mechanisms of metastasis, EMT, and
angiogenesis can be modeled in 3D and are different from 2D, closer to in vivo, and has been
extensively reviewed.
13
Tumor cells can be grown to form spheroids. Spheroids of cancer cells
can be grown in or on gel, which might or might not contain suspension of spheroid cultures of
other cells (fibroblasts, etc.). 3D spheroids can also be formed from hanging-drop methods or
micro-carriers under the force of gravity. The table above gives a list of all 3D culture methods
with pros and cons.
20
Alternatively, spheroids of normal cells can also be grown in a cancer cell
culture. Recently, magnetic 3D spheroids have been developed.
7
Cell viability assays and
protein/RNA/gene expression assays can be done pretty similarly to 2D cultures with
fluorescence/luminescence plate reader/rt PCR, microarrays respectively.
6, 16
3D HCS results for
all sorts of cancers using these easier assays have been reviewed extensively.
17
Invasion and
migration tendencies under various environmental parameters can be measured better using
more sophisticated HCS/microscopy approaches, if one does not want to resort to cutting cross
sections in cryostat or paraffin. Microscopy of 3D spheroids samples in gel are extremely hard
because of high light scattering. A more detailed description of all microscopy options and a
promising option called light sheet fluorescence microscopy has been reviewed.

The only current decent approach in this field, I guess, is automated confocal microscopes or
HCS with confocal (INCell, Operetta), with software for large Z-stack data sets.
5
(Imaris, Volocity,
Fiji) For example, in a recent study to establish comprehensive 3D prostate cancer invasion
models, they used a Zeiss Axiovert 200 M confocal microscope with spinning disc confocal unit
Yokogawa CSU22 and a Zeiss Plan-Neofluar 5* objective, with SlideBook 4.2.0.7, NIH ImageJ, and
VTT Acca. Time lapse images were created using Incucyte, ImageJ and VTT Acca.
8
(This was not a
HCS study) Here is another example of Confocal microscopy/ software combo (Imaris) that
studied the effects of EGF on metastasis speed
12
. High content screening technological advances
in this area is very promising. (Not mentioned in the table copied above)

One recent study laid out the groundwork for automated and high-throughput generation of
uniform-sized tumor spheroids and efficient liquid handling. The protocol is demonstrated to be
compatible with migration, invasion, and co-culture assays. The efficiency of various different
cancer cell lines in forming spheroids are also examined. Imaging methods using a Celigo
Imaging Cytometer are also explored.
23
Another recent study detailed an imaging process using
a GFP expression reporter that can be used to quantify the cross-sectional area of GFP positive
cells in real time, effectively eliminating the need to take cross sections.
3, 15
An article that talks
about one such developed analyzer (I dont think its commercial) and the algorithms used:
PCaAnalyser: A 2D-Image Analysis Based Module for Effective Determination of Prostate Cancer
Progression in 3D Culture.

An alternative to all this, of course, is on-gel spheroids instead of in-gel. All you have to do is to
grow the spheroids via hang-drop, agar-coat, low-attachment, microcarrier, or whatever
method, and then seed onto ECM, then fill well with culture. Conventional brightfield or
fluorescence microscopy is sufficient.
22
A pretty good review of all the 2D, 3D assay methods
with good tables can be found here:
25


3. Microfluidics

Recent microfluidics approaches have facilitated the precise control of the tumor micro-
environment, with potential applications to HCS. 3D models with microfluidics is a booming field
and holds a lot of promises. 3D models with microfluidics can lower reagents costs, facilitate
HTS, and more closely mimic the in vivo environment. 3D cell culture can be grown and
maneuvered in gel, and immobilized in the bioreactor using temperature variation. The PDMS
can be functionalized with different stuff to generate different patterns of cells and change
adhesion properties. The wiring can mimic the circulation in vivo. Microfluidics systems for HTP
drug screening has been demonstrated to be feasible. Various pumping schemes and flow
division schemes have to be considered. Because of the laminar flow and diffusion-dependent
mass transfer, chemo-gradients can easily be generated in microfluidics, this allows the easy
administering of different drug concentrations to the same batch of cells. (A continuous flow is
required which might ramp up reagent costs) On-chip HTS drug screening, and anti-cancer drug
screening have been designed and done. A HTS strategy creates tiny drug spots on a
membrane beneath the cells that can act on the gel/cell surface layer above. On-chip anti-
cancer drug HTS was done using the cytotoxicity assay and hanging-drop spheroids.
11, 21

Because chemo-gradients can easily be generated in microfluidics, chemotaxis, and therefore
cancer metastasis can be studied.
24


Microfluidics technologies have also
been used to generate 3D tumor
spheroids. (left)
10
Another interesting
application is the development of a
microfluidic transwell insert that can
be used to generate a chemical
gradient across a well.
19


Different microfluidic models for cancer cell extravasation, intravasation, and ECM invasion have
been developed. A recent study at MIT reconstructed the vascular endothelial lining/ ECM /
tumor microenvironment, and provided a crude, but working model. (endothelial cells and
cancer cells separated by thin ECM lining) Chemical gradients of EGF or other signals
were integrated. The model was visualized using confocal microscopy. It was shown that
macrophages or TNF-alpha stimulation was helpful to cancer intravasation. Many techniques
from 3D invasion were used, like cell tracking (Imaris).
9
Another recently developed model of
the local tumor spheroid microenvironment is slightly more sophisticated, and comprehensive
migration assays and drug treatment studies were done using simple inverted microscopy. Two
upper channels representing cancer and blood vessel are connected by a lower channel
representing the stroma. The upper and lower channels are separated by a membrane. A
chemical gradient was maintained across the lower channels.
14



Similarly, a model for extravasation was also recent developed. Basically, cells were allowed to
migrate through an endothelial lining from a continuous flow pipe (vessel) to gel filled
compartments filled with chemokines (organ). Again, very crude, but works. Fluorescent
microscopy was subsequently done.
18



Another model for cancer extravasation made use of narrow gaps that colonizing cells have to
squeeze through. The micro-gaps were coated with Matrigel, and all the channels are lined
with endothelial cells. The migratory capability and viability of control cancer cells and cells that
squeezed through were compared, and it was shown that deformation has a strong effect on
cancer cells biological capability. Not surprisingly, it was shown that both the endothelial lining
and Matrigel inhibited extravasation. Just an optical microscope was used.
4



A recent model used a microfluidics device to study EMT-blocking compounds. This relatively
simple microfluidic device was just employed to maintain a chemical gradient. 3D and 2D
migratory assays were used with confocal microscopy and subsequent analysis. 12 drugs were
also tested for their effects on cell migration.
1

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