Intravital imaging of
through a mammary
Dmitriy Kedrin1,4, Bojana Gligorijevic1,2,4,
Jeffrey Wyckoff1,2, Vladislav V Verkhusha1,2,
John Condeelis1,2, Jeffrey E Segall1&
Jacco van Rheenen1–3
We report a technique to evaluate the same tumor
microenvironment over multiple intravital imaging sessions
in living mice. We optically marked individual tumor cells
expressing photoswitchable proteins in an orthotopic mammary
carcinoma and followed them for extended periods through
a mammary imaging window. We found that two distinct
microenvironments in the same orthotopic mammary
tumor affected differently the invasion and intravasation
of tumor cells.
The early steps of metastasis are characterized by tumor cells
invading the stroma (invasion) and entering the blood (intravasa-
tion)1,2. Short-term tracking of individual cells inside fluorescent
tumors by intravital imaging has revealed dramatic heterogeneity
in tumor cell invasion and intravasation3. However, long-term
tracking of individual cells is required to quantify these behaviors
and to determine the fates of cells in specific tumor microenviron-
ments. Imaging techniques that rely on surgical dissection to
expose the imaging site have limitations for long-term experiments
such as (i) tissue dehydration and impaired thermoregulatory
(ii) possible effects of prolonged anesthesia exposure, and (iii) a
tumors through a dorsal skinfold chamber4. The use of dorsal
skinfold chambers, however, limits the experiments to tumor
models based on cell lines and, for many tumors, a non-orthotopic
environment. For example, invasion and intravasation of breast
tumor cells is highly dependent on the specific local microenviron-
ment5, which may not exist in the nonmammary environments
such as the dorsal skinfold chamber site4. Here we describe the
integration of a mammary imaging window (MIW) with photo-
switchable fluorescent protein labeling. This technique enabled
tracking of selected tumor cell subpopulations in different breast
tumor microenvironments and monitoring of cell migration for
more than 24 h.
To image orthotopic (in the natural, mammary gland environ-
ment) breast tumors intravitally at high resolution for prolonged
times, we developed a MIW that can be placed on top of the
mammary gland of a mouse (Fig. 1a). The protocol for these
animal studies was approved by the Institutional Animal Care and
Use Committee for the Albert Einstein College of Medicine. The
MIW contains two plastic rings that form a mount for a glass
the skin, and the glass coverslip assures the optimal working
distance and refraction index for high-resolution imaging (for
details on equipment used for imaging, see Supplementary
Fig. 1 online). Whereas surgical dissection of the skin overlaying
the imaging site allows for several hours of data collection and is
usually a terminal procedure, imaging through the MIWextended
the imaging time to multiple days (up to 21 d). Implantation of
the MIW did not alter inflammation or the microenvironment
(measured 24 h after implantation), or tumor growth over 9 d
(Supplementary Fig. 2 online). However, expression of the photo-
switchable protein we used, Dendra2, may alter blood-vessel
density (Supplementary Fig. 2c).
To locate thesamesubpopulationofcellsforcell tracking ineach
ofthe imaging sessions,reference pointswere required6.Inthe fast-
changing tissue topology of the tumor, the use of fixed reference
points is limited, and therefore we used photoswitchable fluores-
cent proteins7,8as photomarkers of the cells of interest. These
proteins are a new group of GFP-like fluorophores that allow
labeling and tracking of a single cell or a group of cells9–12. We
stably expressed the photoswitchable protein Dendra2 in the
metastatic breast cancer line MTLn3. Dendra2 resembles GFP in
its spectrum before photoswitching, but exposure to blue light (for
example, 405 nm) can induce an irreversible red shift 4150 nm in
photoswitch, the red fluorescence stably increases up to 250-fold
both in vitro (Fig. 1b) and in vivo (Fig. 1c), resulting in red/green
contrast ofup to850and allowing ustotrackcellsmarkedthis way.
Five days after photoswitching, the red fluorescence of the photo-
switched cells was still 31-fold higher than the red fluorescence of
nonswitched cells, which enabled us to recognize the highlighted
cells in vivo for extended amounts of time after the photoswitch
We photoswitched regions of the Dendra2-MTLn3 tumor
containing one to hundreds of cells and imaged them through the
RECEIVED 25 AUGUST; ACCEPTED 10 OCTOBER; PUBLISHED ONLINE 9 NOVEMBER 2008; DOI:10.1038/NMETH.1269
1DepartmentofAnatomyandStructural Biology,2Gruss LipperBiophotonicsCenter,AlbertEinstein CollegeofMedicineofYeshiva University, 1300MorrisParkAvenue,
Bronx, New York 10461, USA.3Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Uppsalalaan 8, 3584CT
Utrecht, The Netherlands.4These authors contributed equally to this work. Correspondence should be addressed to J.v.R. (firstname.lastname@example.org), J.C.
(email@example.com) or J.E.S. (firstname.lastname@example.org).
NATURE METHODS | VOL.5 NO.12 | DECEMBER 2008 | 1019
© 2008 Nature Publishing Group http://www.nature.com/naturemethods
Fig. 3 online). As cells in the tumor migrated and invaded, the
distribution of these cells relative to blood vessels and other tumor
cells changed over time. By selectively photoswitching the fluoro-
phore in a group of cells (Fig. 1e), we visualized changes in
distribution of cells in the tumor microenvironment. Twenty four
hours after photo-switching, we recorded images of the non-
photoswitched tumor cells (Dendra2 green fluorescence), photo-
switched cells (Dendra2 red fluorescence), extracellular matrix
(collagen was visualized by reflectance in Fig. 2a or second-harmo-
nic-generation imaging in Supplementary Movie 3 online) and
blood vessels (fluorescent dextrans were used for vessel labeling;
Supplementary Movie 4 online). Notably, some photoswitched
regions showed dramatic migration and invasion of the surround-
ing microenvironment (Fig. 2a).
The tumor perivascular microenvironment (tumor tissue
surrounding blood vessels) was enriched in tumor-associated
macrophages and extracellular matrix. This microenvironment
supports metastatic behavior, including inhibition of proliferation
and stimulation of migration, invasion and intravasation5,14.
This suggests the existence of distinct mammary tumor
0 50 100
0 20 406080100
406080 100Before 01
Figure 1 | The MIW allows for long-term, high-resolution imaging of the
orthotopic tumors. (a) To assemble the MIW, a coverslip was mounted on
a plastic frame consisting of two plastic rings (top) and was surgically
implanted on top of the mammary gland or mammary tumor of a mouse
(bottom). (b,c) Average changes in fluorescence for Dendra2, as measured
in a region of interest, in cells in vitro (b) or in vivo after injection of the
Dendra2-expressing MTLn3 tumor cells into the abdominal mammary fat
pad (c) upon photoswitching. The values were normalized to the highest
fluorescence level in the red fluorescence channel and the initial fluorescence
level in the green fluorescence channel. Insets, ratio of the non-normalized
red/green fluorescence. (d) Cells in Dendra2-tumors were photoswitched
through the MIW and the red fluorescence was quantified before
photoswitching (before), immediately after photoswitching (0 d) and at
indicated times thereafter. The values were normalized to the red fluorescence
level before photoswitching. (e) Photoswitching of Dendra2-expressing MTLn3
tumor cells in vivo in regions of interest ranging from one cell (left; scale bar,
10 mm) to hundreds of cells (right; scale bar, 75 mm) through the MIW. Shown
are combined images from the green and red fluorescence channels using an
’or’ function. Only the pixels in the red channel that are above background are
shown, and for all other pixels, the green channel is shown.
0 h 6 h
6 h24 h
Number of cells
Figure 2 | Photoswitching through the MIW is a tool for studying orthotopic tumor
microenvironments. (a) Images of tumor cells labeled with Dendra2 (red and green),
Texas Red–dextran for blood vessels (blue) and reflectance (purple) for extracellular
matrix (ECM) (left). The merge of all channels is shown on the right. (b,c) Fluorescence
images of nonphotoswitched cells (green) and photoswitched cells (red) 0, 6 and 24 h
after the photoswitch in avascular (b) and vascular (c) microenvironments (visible vessel
indicated by white dotted lines). Shown are combined images from the green and red
fluorescence channels as in Figure 1e. (d,e) The relative infiltration areas (areas where
cells were found; d) and numbers (e) of photoswitched cells over time in vascular and
avascular microenvironments. Error bars, s.e.m.; n 4 20; *P o 0.05. (f) Detection of
photoswitched cells in the lung. Lungs of a mouse, in which a large vascular area
(20–40 mm2) of the primary tumor was photoswitched, were examined ex vivo 24 h
after photoswitching in green and red fluorescence channels by epifluorescence
microscopy. Arrow, red tumor cell photoswitched in and disseminated from the primary tumor. We determined 0.009 ± 0.007 (s.e.m.) red cells and 1.4 ± 0.33
green-fluorescent cells per mm2lung, resulting in a green/red ratio of 152 ± 0.81. Scale bars, 30 mm (a–c) and 20 mm (f).
1020 | VOL.5 NO.12 | DECEMBER 2008 | NATURE METHODS
© 2008 Nature Publishing Group http://www.nature.com/naturemethods
microenvironments within the same tumor with different rates of Download full-text
invasion andintravasation. However,the long-term implicationsof
these observations required the ability to image distinct micro-
environments inside the same mammary gland over a 24-h period.
To quantify invasion and intravasation within distinct mammary
gland microenvironments, we photoswitched square regions
(B300 cells) in different tumor microenvironments of the same
orthotopically grown tumor (Supplementary Fig. 3), focusing on
regions lacking and containing detectable blood vessels (Fig. 2b,c).
We located the photoswitched red cells by acquiring z-dimension
6 and 24 h after photoswitching. In regions not containing
detectable vessels there was limited migration, and the number of
photoswitched cells increased (Fig. 2b,d,e), suggesting that this
microenvironment does not support metastatic behavior. In con-
trast, photoswitched cells in the vascular microenvironment infil-
trated larger areas (Fig. 2c,d) and even migrated to sites outside of
the field of view. Moreover, photoswitched cells in this vascular
microenvironment lined up along the blood vessel (Fig. 2c), with a
concomitant decrease in the number of red-fluorescent tumor
ells and the appearance of red-fluorescent tumor cells in the lung
(Fig. 2c,e,f and Supplementary Methods online). From these
experiments we concluded that cell behavior is determined by the
surrounding microenvironment, and that the vascular microenvir-
onment promotes invasion and intravasation of tumor cells.
Although the existence of different tumor microenvironments
has been reported previously5, the quantitative analysis of such
microenvironments in the same tumor with spatial and temporal
resolution is not possible with previous techniques. The combina-
tion of photoswitchable proteins with the MIW allowed for such
analysis, as a distinct group of cells can be photomarked in any
location in the primary tumor and trackedover time without long-
term anesthesia. Furthermore, the high stability of Dendra2
allowed us to freeze-fix the tissues and analyze them by microscopy
without additional labeling. A limitation of Dendra2 is that a
as violet light causes a switch, 4,6-diamidino-2-phenylindole
(DAPI) stains should only be used after imaging other wavelengths
as DAPI imaging would cause all green-fluorescent cells to then
become red-fluorescent. Nevertheless, through the MIW the for-
be followed for days, which is not possible in surgically dissected
areas. This also opens the possibility to study fluorescently tagged
proteins that have lethal effects if stably expressed but can be
studied in transiently transfected cells. For example, transiently
transfected cells expressing membrane-targeted GFP and injected
into the mammary gland can be imaged with high resolution
through the MIW (Supplementary Fig. 4a online). Although this
would also be possible with the dorsal skinfold chamber4, imaging
through the MIW allows studies of cell behavior in their physio-
logical breast microenvironments and moreover, the MIW tech-
nology can be extended to tumors of transgenic origin, such as the
MMTV-PyMT tumor model (Supplementary Fig. 4b). Such
tumor models allow the investigation of different stages of tumor
progression15in contrast to cell line–derived xenografts. The
combined use of MIW and photomarking cells to revisit chosen
subpopulations of cells is an important capability not only in
tumor studies but any studies related to cell motility and morpho-
genesis. Visualization of infectious agents and immune responses,
or the progression of chronic inflammation, are other examples of
the potential applications of the technique described here.
Note: Supplementary information is available on the Nature Methods website.
This work was supported by US Department of Defense (BC061403 to D.K.), US
National Institutes of Health (U54GM064346 to J.v.R.; CA100324 to J.C., J.E.S.
and J.W.; U54CA126511 to J.C. and B.G.; and GM070358 and GM073913 to V.V.V.).
We thank the staff of the Analytical Imaging Facility and D. Entenberg for help
with microscopy, the immunohistochemistry facility for help with histology,
M. Rottenkolber for help in fabrication of the imaging box, J. Pollard
(Albert Einstein College of Medicine) for providing the F4/80 antibody,
S. Garofalo for technical assistance, and members of the Condeelis,
Segall, Cox and Verkhusha laboratories for discussions.
Published online at http://www.nature.com/naturemethods/
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