ARTICLE SERIES: Imaging Commentary 299

IntroductionComplete surgical resection of the primary tumor is still one of themost efficient therapies for cancer. Unfortunately, cancer canprogress to a stage at which tumor cells leave the primary tumorand spread to a distant organ to form a secondary tumor, a processreferred to as metastasis. Complications caused by metastases arethe major cause of cancer-related death, but this process is not fullyunderstood.

Initially, it was thought that spread of tumor cells and subsequentmetastasis formation are late events in tumor progression; however,more recently it was realized that it might instead be an early event(Hüsemann et al., 2008; Klein, 2009). In either case, tumor cellshave to acquire certain traits that allow them to escape from theprimary tumor site and home in on and colonize a secondary site(Fig. 1). These gained properties, such as survival, invasivenessand motility, allow tumor cells to move into the surrounding tissue,where they enter blood or lymph vessels (Talmadge and Fidler,2010; Wyckoff et al., 2000; Wyckoff et al., 2007). Once incirculation, tumor cells are transported to a secondary site, wherethey can grow out to form metastatic foci or become dormant(Chambers et al., 2002; Chambers et al., 1995; Nguyen et al.,2009; Talmadge and Fidler, 2010). For these colonization events totake place, tumor cells need to be able to respond tochemoattractants and growth factors, and survive in the newenvironment. The requirement for these traits might vary duringtumor progression or among different tumor types, and theacquisition of these traits does not follow a particular order (Chiangand Massague, 2008; Nguyen et al., 2009). It is worth noting thatonly a small fraction of the cells present in the primary tumor havethe necessary characteristics to escape from the primary site and

colonize a secondary site, which renders metastasis an inefficientprocess (Chambers et al., 2002). To investigate these dynamicprocesses underlying metastasis, most studies rely on techniquesthat are only able to provide a static view, such as histochemistry,visual inspection of tumor size and end-stage measurements (e.g.the number of metastatic foci). In addition, these techniques analyzelarge numbers of cells, which obscures the signaling properties andactivities of individual cells. This results in loss of crucialinformation concerning the adaptive properties of the few tumorcells that spread and form metastases.

Recent advances in optical methods have made it possible tovisualize the metastatic process at a subcellular resolution in realtime in vivo. By the 19th century, microscopes were being used toimage tissue in living animals (e.g. Wagner, 1839), a techniquereferred to as intravital microscopy (IVM). In these early days,most IVM studies could only examine the vasculature and themicrocirculation, because the optics available at that time and lackof contrast limited the visualization of other tissues. In the 1950s,the visualization of metastasis was pioneered in a rabbit ear chamber(Wood, 1958). Major breakthroughs in this field occurred in the1990s, when intravital imaging techniques improved considerablyand genetic tumor models of rodents that expressed fluorescentproteins (FPs) became available. Since then, IVM has evolved intoan important tool for investigating the processes underlying cancerand metastasis (see IVM images in Fig. 1) (Chishima et al., 1997;Farina et al., 1998; MacDonald et al., 1992; Naumov et al., 1999).

Recently, a number of new IVM techniques have becomeavailable with different properties in relation to imaging depth,resolution, timescale and applications (Table 1) [for an extensivereview on in vivo optical microscopy methods, see Ntziachristos

SummaryMetastasis, the process by which cells spread from the primary tumor to a distant site to form secondary tumors, is still not fullyunderstood. Although histological techniques have provided important information, they give only a static image and thus compromiseinterpretation of this dynamic process. New advances in intravital microscopy (IVM), such as two-photon microscopy, imaging chambers,and multicolor and fluorescent resonance energy transfer imaging, have recently been used to visualize the behavior of single metastasizingcells at subcellular resolution over several days, yielding new and unexpected insights into this process. For example, IVM studies showedthat tumor cells can switch between multiple invasion strategies in response to various densities of extracellular matrix. Moreover, otherIVM studies showed that tumor cell migration and blood entry take place not only at the invasive front, but also within the tumor massat tumor-associated vessels that lack an intact basement membrane. In this Commentary, we will give an overview of the recent advancesin high-resolution IVM techniques and discuss some of the latest insights in the metastasis field obtained with IVM.

Key words: Intravital imaging, Intravital microscopy, Cancer, Invasion, Metastasis

Journal of Cell Science 124, 299-310 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.072728

Intravital microscopy: new insights into metastasis oftumorsEvelyne Beerling1,*, Laila Ritsma1,*, Nienke Vrisekoop2, Patrick W. B. Derksen3–5 and Jacco van Rheenen1,‡

1Hubrecht Institute-KNAW & University Medical Center Utrecht, Uppsalalaan 8, Utrecht 3584CT, The Netherlands2Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health,Bethesda, MD 20892, USA3Department of Medical Oncology, University Medical Center Utrecht, Utrecht 3584CX, The Netherlands4Cancer Center, University Medical Center Utrecht, Utrecht 3584CX, The Netherlands5Department of Pathology, University Medical Center Utrecht, Utrecht 3584CX, The Netherlands*These authors contributed equally to this work‡Author for correspondence (j.vanrheenen@hubrecht.eu)

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(Ntziachristos, 2010)]. Techniques that are based on fluorescencemicroscopy provide (sub)cellular resolution and have successfullybeen used to image single cells in living organisms in theirnatural environment, such as in fruit flies, zebrafish, nematodesand small rodents (Fouquet et al., 2009; Ghosh and Hope, 2010;Le Dévédec et al., 2010; Stoletov et al., 2010). In thisCommentary, we will focus on high-resolution IVM techniquesused in small rodents (rats and mice), as they are the most widelyused model organisms to study cancer. We will describe theadvances in high-resolution IVM techniques that have made itpossible to directly visualize the various steps of metastasis atcellular resolution (see Fig. 1). We will also discuss the newinsights into the process of tumor cell invasion that have beenobtained with such IVM studies.

Advances in IVMHigh-resolution IVM techniquesThere are several high-resolution IVM techniques, including opticalfrequency domain imaging (OFDI), spinning disk confocalmicroscopy and multiphoton microscopy (Table 1). In OFDI, anoptical beam is focused into the tissue and reflected light is detectedafter interference with a reference beam. The amplitude and phaseof the reflected signal as a function of time are then used to derivethe optical scattering properties and thereby the tissue structure atdifferent depths. OFDI has been used to study angiogenesis,lymphangiogenesis and tissue viability (Vakoc et al., 2009). Theadvantage of this technique is that it enables the imaging ofsubstantial volumes of tissue over prolonged periods without theneed for contrast agents.

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Table 1. Overview of different types of intravital imagingTechnique Imaging depth Resolution Costa Contrast reagents Comments

Ultrasound >5 cm 50 µm ++ Microbubbles Used clinically for vascular andinterventional imaging.

Magnetic resonance imaging Unlimited 10 100 µm +++ Magnetic particles Used clinically. High contrast in soft tissue.CT Unlimited 50 µm ++ Iodinated molecules Used clinically. High contrast in lungs and

bones.PET and SPECT Unlimited 0.3 mm +++ Radioactive

compoundsImaging of labeled antibodies, proteins and

peptides.Bioluminescence >2 cm >1 mm ++ Luciferins Imaging of tumor growth and gene

expression.Whole-body imaging >2 mm Cellular (10 30 µm) + Fluorophores User-friendly equipment.OFDI >1 mm Subcellular (<15 µm) +++ – No exogenous contrast reagents. No

photobleaching and no phototoxicity.Spinning disk confocal imaging <100 µm Subcellular (<0.5 µm) ++ Fluorophores Rapid imaging. Low photobleaching and

toxicity. User-friendly equipment.Multiphoton (MP) imaging <1000 mm Subcellular (<0.5 mm) +++ Fluorophores SHG. Low photobleaching and toxicity (for

details, see Box 1). The commerciallyavailable microscopes are user friendly.

aCosts range from low (+) to high (+++). CT, X-ray computed tomography; PET, positron emission tomography; OFDI, optical frequency domain imaging; SPECT, single-photon emission computed tomography.

Intravasation

Extravasation

Outgrowth

Cancer cell Basement membraneEndothelial cell Type I collagenRed blood cell Interstitial matrix

Invasion

TraitsEscapeinvasivenessmotilitysurvival

TraitsColonizationattachmentchemosensingsurvival

C

Epithelial cell

sationsa

Extravasation

IntravasIntravas

CCCC

TraitsEscape:invasivenessmotilitysurvival

TraitsColonization:attachmentchemosensingsurvival

t=0 h t=3 h

C

Key

Transport byblood

Fig. 1. IVM of individual steps of metastasis. The schematicillustrations aim to provide a simplified overview of the metastaticprocess. To metastasize, tumor cells (green) have to escape from theprimary tumor and colonize a distant site. During this process, cellsemploy traits, such as invasiveness, motility, attachment,chemosensing and survival, that allow them to detach from theprimary tumor, invade the interstitial matrix (purple), overcome thebarrier of the BM (blue), enter the blood (red), be transported to adistant site, exit the blood and finally grow out to form metastaticfoci. IVM can be used to image metastatic processes, as illustratedby the IVM images of tumor cells (green), type I collagen (purple)and blood (red). IVM images A and B represent different time pointsof invasion of a polyoma middle T (PyMT) mammary tumor. IVMimage C shows the tumor cells present in a vessel that collects bloodfrom a C26 colorectal tumor and is one time point of supplementarymaterial Movie 1. Scale bar: 10m.

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At present, a number of mouse tumor models are available inwhich various FPs are expressed. For the imaging of tumors thatexpress FPs, spinning disk confocal microscopy and multiphotonmicroscopy have been the IVM techniques of choice. In spinningdisk confocal microscopy, visible light is used to excite FPs. Here,emitted out-of-focus light (outside the optical section) is eliminatedby multiple pinholes, resulting in increased Z-resolution and contrastenhancement, which makes subcellular structures more easily visible.A spinning disk confocal microscope can acquire high-resolutionimages at high speed and low phototoxicity, therefore allowing thelong-term intravital imaging of tumors (up to 30 hours) (Egeblad etal., 2008; Lohela and Werb, 2010). The downside of spinning diskmicroscopy is that the visible light used for excitation is scattered inthe tissue, which limits its imaging depth to 100 m. By contrast,the short pulses of infrared (IR) light used in multiphoton microscopy,with a typical wavelength range of 800–1000 nm, penetrate thetissue more than tenfold deeper compared with spinning diskmicroscopy (see Box 1) (Theer et al., 2003). An additional advantageof IR excitation is its ability to create a second harmonic generation(SHG) signal from non-centrosymmetric structures with highlyordered repeats, such as collagen type I fibers, an extracellularmatrix component (Campagnola et al., 2002). In contrast tofluorescence microscopy, SHG does not involve the excitation offluorophores and hence the signal does not suffer fromphotobleaching (for reviews, see Helmchen and Denk, 2005; Mohleret al., 2003; Zipfel et al., 2003b). SHG microscopy has been used tostudy the role of fibrillar collagen in tissues and organs, includingskin, gut and breast (e.g. Wolf et al., 2009).

The combination of high-resolution acquisition of fluorescenttissues and deep tissue penetration has made multiphoton IVM thetechnique of choice for most research groups that exploit intravitalimaging for tumor cell biology (for more details, see Box 1).

Advanced detection methods for simultaneous imaging ofmultiple cell types and moleculesUsing multicolor IVM, different cell types and molecules can bevisualized simultaneously, which has made it possible to study theinteraction between different cell types and molecules in real time(Fig. 2A) (Bajénoff et al., 2006; Boissonnas et al., 2007; Castellinoet al., 2006; Egeblad et al., 2008; Egen et al., 2008; Farina et al.,1998; Goswami et al., 2005; Lohela and Werb, 2010; Massberg etal., 2007; Wyckoff et al., 2007). Since the introduction of the greenfluorescent protein (GFP) (Chalfie et al., 1994; Heim et al., 2005;Shimomura et al., 1962) and its color variants, cyan fluorescentprotein (CFP), yellow fluorescent protein (YFP) and red fluorescentprotein (RFP) (Giepmans et al., 2006), together with the morerecently discovered FPs [e.g. photoswitchable proteins (Piatkevichand Verkhusha, 2009)], several cell types can now be geneticallylabeled and visualized in tumors using, for example, CFP and YFP(e.g. Sahai et al., 2005). In addition, tumor cells labeled with an FPcan also be imaged at the same time as cells that are labeled withfluorescent dyes [e.g. dextran-labeled macrophages (Fig. 2)]. Aspecific way to label immune cells (often lymphocytes) is to isolatethem and load them with fluorescent tracker dyes before transferringthem back into the recipient animal. The disadvantage to thisapproach is, however, that the fluorescently labeled cells willconstitute only a small fraction of the population compared withtheir endogenous, non-labeled counterparts. A simple but functionalapproach to accomplish contrast between different cell types andtissues is intravenous or subcutaneous injection of cell-type-specificfluorescent antibodies or other targeted tracers [e.g. for lymphatics,

see Alexander et al. (Alexander et al., 2008) and McElroy et al.(McElroy et al., 2008); for Gr1+ myeloid cells, see Egeblad et al.(Egeblad et al., 2008)]. Nevertheless, it should be noted that thesesignals are short lived, whereas FP signals persist. However, amajor disadvantage of FPs is the difficulty in translating their useinto patients. A general problem with multicolor IVM is that theexcitation (particularly in multiphoton excitation) and emissionspectra of most FPs and dyes overlap, and some FPs and dyes canonly be successfully distinguished when complicated algorithmsare used (Jalink and van Rheenen, 2009). Alternatively, FPs anddyes can be discriminated based on their unique fluorescencelifetime (the average time an excited fluorophore is in its excitedstate), which can be measured with fluorescence lifetime imagingmicroscopy (FLIM) (see Box 2) (Bastiaens and Squire, 1999).Using FLIM increases the number of different fluorophores andthus cells that can be imaged simultaneously.

In addition to discriminating spectral overlap betweenfluorophores, FLIM is also used to distinguish between theautofluorescence of various tissue types (see Box 2).Autofluorescence is the intrinsic property of cells to emitfluorescence upon excitation and is caused by its endogenousconstituents, such as tryptophan, pyridinic coenzymes (i.e.NADPH), flavin coenzymes, melanin and elastin, which all havefluorescent properties [e.g. Monici and El-Gewely, 2005; Zipfelet al., 2003a]. Because most of the autofluorescent tissue

301Advances in intravital microscopy

Box 1. Multiphoton microscopy.Multiphoton excitation is based on the (almost) simultaneousabsorption of two low-energy IR photons, whose combinedenergies induce fluorescence in the visible range (see Figure,panel A). Simultaneous absorption of two photons by afluorophore is an extremely rare event and multiphoton excitationtherefore requires high excitation powers (i.e. a dense photonflux). IR lasers achieve this by condensing all photons over a timespan of 12.5 ns into a 100 fs pulse (thus concentrating the photondensity 1.25 � 105 times). Although the overall power is low (i.e.low number of photons per second), the photon density per pulseis sufficiently high to cause efficient fluorophore excitation in thefocal plane (see Figure, panel B). Beyond the focal plane,however, the photon density is too low for simultaneousabsorption by a fluorophore to occur (see Figure, panel B) (Dunnand Young, 2006; Helmchen and Denk, 2005; Zipfel et al.,2003b). Therefore, in contrast to confocal microscopy, excitationis strictly confined to the focal plane, thus avoiding the need for apinhole to delete out-of-focus light (see Figure, panel B). Thisgreatly enhances the detection efficiency, as the in-focus emittedlight can be detected close to the objective, without the need totravel back through the scanner optical path (with loss of tissue-scattered emission at the pinhole and further losses at eachfilter) (Centonze and White, 1998). Furthermore, deep tissuepenetration, reduced excitation of surrounding tissue, increaseddetection and low-energy IR light give the additional advantage oflow phototoxicity and low photobleaching (Eggeling et al., 2005).

Box 1 Fig. Two-photon versus single-photon excitation.

Fluorescence

ZS0

S1

Single photon

Multiphoton

Absorption Emission

Single photon beam

Multiphoton beam

ExcitationA B

Objective

Objective

X

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components exhibit comparable excitation and emission spectra,the resulting fluorescence levels are similar and cannot be usedto distinguish tissues. However, as the lifetime of autofluorescencediffers between different cell types, autofluorescence lifetimemeasurements have successfully been used to distinguish healthytissues from tumor tissues (Galletly et al., 2008; Provenzano etal., 2009).

Intravital imaging of proteins, proteases, signalingpathways and cellular processes in vivoImaging of molecular processes in living mice has been achievedby multiphoton IVM of FP fusion proteins and injectable dyes.High-resolution IVM of cells that express a fusion of FP with areceptor [e.g. ErbB2 (Dennis et al., 2007)], an adhesion molecule[e.g. E-cadherin (Serrel et al., 2009)], or an actin- or myosin-binding protein (Philippar et al., 2008; Wyckoff et al., 2006) hasbeen used to study intracellular signaling in single tumor cells. Inaddition to FP fusion proteins, many biosensors that are based onmeasuring a change in fluorescent resonance energy transfer(FRET) (see Box 2) can be used to study intracellular signaling invivo. The number of available biosensors is rapidly increasing, andspecific FRET biosensors are already available to measure thelevels of second messengers, protein–protein interactions or theactivation status of proteins (e.g. Rho) [for a review on biosensors,see Morris (Morris, 2010) and VanEngelenburg and Palmer(VanEngelenburg and Palmer, 2008)]. However, as the field ofFRET biosensors is still an emerging area, only a few researchgroups have thus far used this powerful technique in vivo. Forexample, FRET biosensors have been used to measure theproteolytic activity of calpain in muscles (Stockholm et al., 2005),changes in calcium concentration and myosin light-chain kinaseactivity in arteries (Zhang et al., 2010), and the induction of

apoptosis in the skin (Bins et al., 2007), colorectal carcinomas andlymphomas (Breart et al., 2008; Keese et al., 2010).

In particular, the apoptosis sensor has been of interest for tumorbiology, because tumor cells have to overcome many apoptoticsignals during several steps of metastasis. The FRET apoptosissensor consists of a CFP that is separated from a YFP by a caspase-3 recognition sequence (Fig. 2B). Caspase-3 activity cleaves CFPfrom YFP, leading to a loss of FRET efficiency. Indeed, when weinduced apoptosis in single keratinocytes in the skin of livingmice, a caspase-3-mediated loss of FRET efficiency is observed,which shows that caspase-3 activity is a good gauge of apoptosisinduction in vivo (Fig. 2C,D) (Bins et al., 2007). Keese andcolleagues used FRET IVM of this biosensor to study the inductionof apoptosis resistance upon chemotherapy (Keese et al., 2007;Keese et al., 2010). Moreover, Breart and colleagues used thisbiosensor to study apoptosis of tumor cells by cytotoxic Tlymphocytes (CTLs) and showed that a CTL kills, on average, onetumor cell every 6 hours (Breart et al., 2008).

Many synthetic fluorescent probes have been developed to studythe intracellular and extracellular of activity of proteases such asmatrix metalloproteinases (MMPs) and cathepsins in tumors. Theinjectable probes that have been used to measure protease activityin tumors include autoquenched protease substrates that fluoresceupon cleavage (Weissleder et al., 1999), proteolytic beacons thatlose FRET upon cleavage (e.g. McIntyre et al., 2010; Scherer etal., 2008) and fluorescently labeled activatable cell-penetratingpeptides (ACCPs) that translocate into cells upon cleavage (Olsonet al., 2009; Olson et al., 2010). The disadvantage of these probesis that chemical knowledge is required to synthesize them, althoughrecent commercialization of some of these probes has overcomethis problem. It is also important to note that phagocytic cells, suchas macrophages, might take up some of the probes despite not

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C Y C Y

FRET

No FRET

B

C

Caspase-3

A Macrophage Tumor cell Collagen I (SHG)

D CFP YFP YFP:CFP

0

2

Apo

ptos

is

Caspase-3 biosensor + apoptosis inducer

Caspase-3 biosensor

No

apop

tosi

s

Fig. 2. Multicolor and FRET IVM. Caspase-3 activity and cell–cell interactions can be imaged using multicolor and FRET IVM.(A)Schematic representation (left) and corresponding multicolorIVM image (zoom and overview on the right) of a colorectal C26tumor. Merged images of Dendra2-expressing tumor cells (green),70 kDa Texas-Red-labeled macrophages (red) and type I collagen(purple; SHG) illustrate the interaction between macrophages andtumor cells. (B)Cartoon of a caspase-3 FRET biosensor tomeasure caspase-3 activity during apoptosis induction. Here, CFP(C) and YFP (Y) are in close proximity, resulting in efficientFRET. When caspase-3 is activated in the cell by induction ofapoptosis, the sensor is cleaved, leading to increased distancebetween CFP and YFP with a subsequent loss of FRET. This lossis observed as increased CFP fluorescence and decreased YFPfluorescence. (C)The IVM image shows a large overview of theskin of a mouse in which single keratinocytes are transfected byDNA tattooing with the caspase-3 FRET biosensor and achemical-sensitive inducer of apoptosis. The arrows point toexamples of cells that are transfected with the caspase-3 sensor, inwhich the apoptosis status can be imaged using FRET. (D)Usingthe system shown in B, IVM images of CFP and YFP are acquiredupon CFP excitation and FRET is expressed as a ratio of YFPover CFP (see color bar on the right). A striking drop in FRET isobserved upon chemical induction of apoptosis (lower row ofimages), illustrating the measurement of signaling events in vivo.All scale bars: 10m.

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having any proteolytic activity, which complicates the interpretationof high-resolution IMV images. Nevertheless, IVM of fluorescentlytagged proteins, and of genetic and synthetic fluorescent probeshas become a great tool to visualize protein activity, signalingpathways and cellular processes during several steps of metastasis.

Long-term intravital imaging over several daysIVM has contributed to the visualization of the behavior of tumorcells in living mice. However, most IVM studies only monitor short-term movement of cancer cells because they use tumor dissection,which effectively limits experimental observations to a few hoursafter surgery [typically around 4 hours and after optimization up to24 hours (Egeblad et al., 2008)]. Given the small number of tumorcells that display high motility and subsequently migrate and invadeover periods that last up to days, long-term IVM techniques areneeded to provide valuable information regarding these processes.By the 1990s, Lehr and co-workers had developed the dorsal skinfoldchamber, which is surgically implanted on the back of a mouse,allowing multiple tumor-imaging sessions over several days(Alexander et al., 2008; Brown et al., 2001; Lehr et al., 1993). Sincethen, orthotopic chambers (i.e. in their natural environment) havebeen developed, including the so-called ‘mammary imaging window’(MIW) for mammary tumors that we developed (Fig. 3A,B)(Gligorijevic et al., 2009; Kedrin et al., 2008; Shan et al., 2003) andthe ‘cranial imaging window’ for brain tumors (Yuan et al., 1994).These imaging windows have made it possible to visualize a numberof metastatic processes over several days, including the formation ofnew blood vessels (Abdul-Karim et al., 2003; Farhadi et al., 2005;Fukumura et al., 2010), tumor cell invasion and intravasation (Fig.3C) (Kedrin et al., 2008), and tumor cell colonization at a distant site(Kienast et al., 2010).

Tracking of individual tumor cells over several imagingsessionsTo enable long-term tracking of individual cells, reference pointssuch as vascular and extracellular matrix (ECM) structures in healthy

tissues have been used to enable retracing of the previously imagedarea over several imaging sessions (Bins et al., 2007; Hayashi et al.,2007; Kimura et al., 2010; Perentes et al., 2009; Yamauchi et al.,2006; Yang et al., 2002). However, when tumorigenic tissues areobserved in which the topology changes dynamically, these initialreference points might change and cannot be used for retracing. Tobe able to track the dynamics of areas of interest, tumor cells havebeen photomarked using photoconvertable fluorophores, such asKaede (Ando et al., 2002) and Dendra2 (Gurskaya et al., 2006).These fluorophores have absorption and emission spectra that aresignificantly red shifted by violet light. For example, we have usedthis property of Dendra2 to mark regions of interest in a tumor overseveral days with single-cell precision (Fig. 3C) (Gligorijevic et al.,2009; Kedrin et al., 2008) and were thus able to track a largepopulation of cells over multiple imaging sessions.

Taken together, we are now able to detect fluorescence at highresolution, deep inside living animals. By combining imagingchambers and photomarking of regions of interest in the tumor,single tumor cells can be tracked and visualized over several days.By using FRET IVM, dynamic changes in subcellular signalingevents that are required for metastasis can be imaged over time,whereas multicolor IVM allows the visualization of cell–cell andcell–molecule interactions. These recent advances in intravitalimaging open the field to new and exciting discoveries.

Important new insights into tumor cell biologyRecent IVM studies using the above-described techniques visualizedhow tumor cells exit the tumor and spread to a secondary site,generating new important insights into this process. For example, itis generally believed that cells at the tumor border have to invadethe surrounding tissue, where they traverse physical barriers of theECM and endothelial cells before they enter the circulation to betransported to a secondary site. Interestingly, IVM studies show amuch more dynamic picture, in which cells switch between differentinvasion strategies to exit the primary tumor as a reaction to physicalECM barriers or intracellular signaling, as discussed below.

303Advances in intravital microscopy

Box 2. Fluorescence lifetime imaging microscopy to distinguish spectrally overlapping fluorophores.FLIM produces images that are based on the fluorescence lifetime of a fluorophore, rather than on its intensity and color (Gerritsen et al.,2009; Wouters et al., 2001). A major advantage of the FLIM technology is that fluorophores that have overlapping emission spectra (seeFigure, panel A) can be distinguished, a feature that is not possible with an intensity image. When fluorophores are excited with afemtosecond IR pulse, the fluorescence willdecay exponentially with a rate that isdetermined by the fluorescence lifetime, whichis close to zero for SHG (see Figure, panel B)and around 2 ns for Dendra2 (see Figure, panelC). In every pixel of a FLIM image, thefluorescence decay curve is measured andused to calculate the fluorescence lifetime.Many pixels of the image contain bothfluorophores, and therefore will have an averagelifetime that depends on the fraction of eachfluorophore that is present in that pixel (seeFigure, panel D). A FLIM image can bevisualized by color coding the fluorescencelifetime (see color bar next to the FLIM image).In the FLIM image, fluorophores with distinctfluorescence lifetimes, such as SHG andDendra2, will be color coded differently and cantherefore be distinguished.

Box 2 Fig. Fluorescence lifetime imaging.

Wavelength

Flu

ores

cenc

e

Flu

ores

cenc

e

Time

Excitation pulse

Intensity image FLIM image

2 ns

Overlappingemissionspectra

SHG

Dendra2

Fast decay(short lifetime)of SHG

Col

or c

ode

of li

fetim

e

Flu

ores

cenc

e

Time

Slow decay(long lifetime)of Dendra2

Excitation pulse

Time

Flu

ores

cenc

e Average of afast and slowdecay

Excitation pulse

0.2 ns

A B C

D

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Hematogenous versus lymphatic tumor cell spreadTumor cells can exit the primary site by entering either lymphaticvessels or blood vessels. Interestingly, IVM of lymphatic vessels,which drain labeled macromolecules, shows that lymphatic vesselsinside hyperplastic lesions and the tumor mass are non-functionalor even absent (Hagendoorn et al., 2006; Padera et al., 2002). Bycontrast, regions at the tumor margin contain functional lymphaticvessels and thus might be the starting point for lymphatic metastasis(Padera et al., 2002). Cells that metastasize through the lymphaticsystem will first be transported to the draining lymph node (Fig.4A). From there, the tumor cells flow through efferent lymphaticvessels to the heart and into the blood system and, consequently,are transported to distant sites where they can form metastatic foci(Fig. 4A). Although the presence of tumor cells in regional lymphnodes is recognized as a prognostic factor for breast cancer andother cancers (Hartveit, 1979; Hartveit, 1984; Sleeman and Thiele,2009; Tanis et al., 2001), it remains elusive whether tumor cellsmetastasize from the lymph nodes or whether their presence inlymph nodes merely reflects their intrinsic invasiveness (Das andSkobe, 2008). Therefore, it is unknown whether tumor cells usethe lymphatic route, the hematogenous (blood) route, or indeedboth, to metastasize to other organs.

To address this question, IVM has been used to visualize thesignaling pathways that are required for the entry of tumor cellsinto lymphatic or blood vessels (Farina et al., 1998; Giampieri etal., 2009; Hagendoorn et al., 2006; Hoffman, 2009; Kedrin et al.,2008; Wyckoff et al., 2007). For this purpose, Gaimpieri andcolleagues engineered MTLn3 mammary tumor cells to express

Smad2 fused to GFP, which translocates to the nucleus in responseto transforming growth factor (TGF) (Giampieri et al., 2009),a well-studied inducer of tumor cell motility (Leivonen and Kähäri,2007). Using high-resolution IVM of MTLn3 mammary tumors,they showed that TGF signaling is active in cells that move assingle units, but not in those that move cohesively as cell chains.(Giampieri et al., 2009; Giampieri et al., 2010) (Fig. 4A). Inaddition, they showed that single moving cells enter the bloodvessels, whereas collectively moving cells enter lymphatic vessels.As the motility of single cells entering the blood vessels is TGFdependent, blood entry, but not lymphatic entry, is blocked uponTGF inhibition (Giampieri et al., 2009). Interestingly, the numberof tumor cells drained from the heart, which corresponds to theadded effects of hematogenous and lymphatic spread (see mergingpoint in Fig. 4A), also decreases upon inhibition of TGF signaling(Giampieri et al., 2009). This suggests that tumor cells in thesetumor models use the hematogenous route to metastasize to otherorgans, as TGF signaling is only required for the entry of tumorcells into blood vessels.

These studies clearly illustrate that IVM of signaling pathwayscan provide crucial information regarding the spread of tumorcells, but future research is necessary to determine whether theseobservations are also true for other tumor models.

The role of proteolysis in invasionThe proteolytic activity of peptidases is important for severalaspects of metastasis, including tumor growth, apoptosis,angiogenesis and invasion (Kessenbrock et al., 2010). The roles of

304 Journal of Cell Science 124 (3)

Day 3 Day 4 Days 5–21

MIW surgery

Recoveryin a cage

Recoveryin a cage

Recoveryin a cage

IVM IVM IVM

3 4 5

Dorsal view Lateral view

MIW

Intr

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oral

inva

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A B

C

Plastic frame

Cover glass

Suture

Day 0

Days after surgery

Intr

atum

oral

intr

avas

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Fig. 3. IVM over several days using anMIW. Experimental setup for repeatedIVM imaging of live mice over severaldays using an MIW. (A)Schematicillustration of an MIW consisting of aplastic frame and a cover glass (topimage), which is surgically placed on amammary tumor (lower image).(B)After implantation of the MIW at day0, the mouse recovers for two days. Thisis followed by repeated IVM sessions inthe subsequent days. (C)IVM images ofa Dendra2-expressing colorectal C26tumor, in which green represents thenon-switched Dendra2 and red theswitched Dendra2. At day 3, tumor cellsthat are present either close to (upperimages) or surrounding (lower images) ablood vessel are photolabeled in a squareregion by photoswitching Dendra2 fromgreen to red using violet illumination.Note that the combination of using aMIW and photomarking of tumor cellsallows imaging regions to be retraced insubsequent imaging sessions, and thusthe motility and intravasation to bevisualized. Also note the highintratumoral motility (top images) andthe loss of red-shifted tumor cells byintravasation (lower images). Scale bars:10m.

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peptidases, such as MMPs, include remodeling of the ECM andcleavage of numerous other substrates, such as growth-factor-binding proteins, cell adhesion molecules, receptor tyrosine kinasesand other proteinases (Egeblad and Werb, 2002; Kessenbrock etal., 2010). Many of these roles are thought to be crucial for tumorcells to be able to overcome the physical barriers of dense ECMlayers when they invade surrounding tissues and enter the blood(Christofori, 2006; Rowe and Weiss, 2008; Tanjore and Kalluri,2006). These ECM layers include a three-dimensional fibrillarmeshwork of interstitial tissues and the two-dimensional sheet ofthe basement membrane (BM), which underlies epithelial andendothelial tissues (Fig. 1) (Kalluri, 2003; LeBleu et al., 2007;Rowe and Weiss, 2009). The BM is thought to act as a particularlyeffective physical barrier owing to its small pore size (~50 nm).However, it is not clear whether the breakdown of these ECMlayers is always required for tumor cell invasion and intravasation.

IVM experiments show that proteolytic activity supports thecollective invasion of tumor cells (e.g. Friedl and Gilmour, 2009;Giampieri et al., 2009). For example, cancer-associated fibroblastshave been found to remodel the interstitial ECM meshwork, throughboth peptidase- and force-mediated matrix remodeling. This formstracks through the matrix, which enable tumor cells to collectivelymove into the surrounding tissue (Gaggioli et al., 2007; Giampieriet al., 2009). Macrophages have been shown to assist in the entryof tumor cells into the circulation by producing chemokines andpeptidases, leading to BM breakdown and tumor cell motility(Goswami et al., 2005; Wyckoff et al., 2007). Alternatively, tumorcells might degrade the endothelial BM themselves. In vitro, manytumor cells can form actin-rich structures, the so-calledinvadopodia, which locally degrade the ECM (Poincloux et al.,2009; Stylli et al., 2008). However, a corresponding function oreven the very existence of such invadopodia in vivo has not yetbeen established. With the recent advances in high-resolution IVMtechniques and protease probes, these questions might be answeredsoon.

Interestingly, using IVM, several laboratories have nowestablished that ECM degradation is not always required fortumor cell invasion through dense ECM layers. For instance,Wolf et al. showed that, upon inhibition of MMPs, tumor cellscan still invade connective collagen matrices by switching froma mysenchymal mode of migration to an amoeboid mode ofmigration (Wolf et al., 2003). Both in vitro and in vivoexperiments have shown that mysenchymal migration ischaracterized by secretion of proteases and an elongated polarizedcell morphology, whereas amoeboid movement is characterizedby a non-polarized flexible round morphology of the cell anddoes not require any proteolytic activity (Wolf et al., 2003; Wolfet al., 2007; Wyckoff et al., 2007; Sahai and Marshall, 2003). Theamoeboid mode of migration enables cells to squeeze throughsmall gaps by forming bleb-like structures, thereby pushing thesefibers out of the way without the requirement for proteolyticactivity (Pinner and Sahai, 2008), allowing them to migrate withinmammary tumors with high velocity (Wyckoff et al., 2006;Wyckoff et al., 2007). Although amoeboid movement does notdepend on ECM layer breakdown, it might be facilitated by otherpeptidase-dependent processes, such as the activation ofchemokines. Nevertheless, these data illustrate that a tumor cellcan adapt its invasion strategy, that is, its mode of migration, tothe surrounding microenvironment.

Invasion and intravasation within the tumor massIn static histological samples, the tumor cells that are located in thesurrounding tissue close to the invasive front of the tumor areidentified as motile tumor cells. As the presence of these cells inthe surrounding tissue correlates with poor prognosis, it is generallythought that tumor cells escape the tumor by invading the tumorstroma (Christofori, 2006; Rowe and Weiss, 2008; Tanjore andKalluri, 2006). However, the contribution of motile cells within thetumor mass, which also enter the vessels, has either not beenstudied or simply been overlooked, as the motility and intravasation

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Fig. 4. Single moving tumor cells of MTLn3 tumors use the hematogenous route to metastasize to other organs. (A)Simplified schematic overview ofspreading routes from a breast tumor to the lung (L). At the primary tumor site, tumor cells can enter the blood either directly (blue route) or through lymphaticvessels (purple route), which drain into the blood at a merging point located just before the heart (H), from where the blood is then transported into the lung. UsingIVM of the invasive MTLn3 tumor model, it has been shown that cells move either as single cells, which leave the primary tumor using the hematogenous route, oras collective chains using the lymphatic route. (B)Inhibition of TFGsignaling leads to inhibition of invasion by single moving cells and a subsequent drop in thenumber of tumor cells in the heart, whereas the collective movement to lymphatic vessels remains unaltered. These experiments show that, in this invasivemammary carcinoma model, tumor cells use the hematogenous (blood) route to metastasize to other organs.

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of this population of cells cannot be visualized in static histologicalimages.

With the recent high-resolution IVM studies in mice, intratumoralmotility and intravasation can now be directly visualized in realtime (Fig. 5) (Farina et al., 1998; Giampieri et al., 2009; Kedrin etal., 2008; Wyckoff et al., 2000). In an elegant study using multicolorspinning disk microscopy, Egeblad and co-workers showed thatstromal cells and leukocytes (including T cells, dendritic cells,myeloid cells and fibroblasts) exhibit higher motility at the tumorstroma of mammary tumors than within the tumor mass (Egebladet al., 2008; Lohela and Werb, 2010). However, the motility oftumor cells has not been visualized in this study. The extent ofintratumoral motility became clear when we tracked tumor cellsthrough MIWs over long periods of time. By photomarking asubset of tumor cells, we observed intratumoral cell movement andentry into the vessels within the tumor mass (Fig. 3C) (Gligorijevicet al., 2009; Kedrin et al., 2008). Importantly, in contrast to tumorcells present in the tumor stroma, invading and intravasating tumorcells within the tumor mass no longer encounter physical barriers,such as the dense epithelial and endothelial BMs. Vessels that areembedded within the tumor mass, referred to as tumor-associatedvessels, are less organized and less solid compared with normalvasculature (Fukumura et al., 2010; Jain, 1988). In fact, owing tothe lack of an intact BM and irregularly organized endothelial cellsand pericytes, these vessels have been shown to be leakier thanvessels of healthy tissues (Baluk et al., 2003; Chang et al., 2000;Fukumura et al., 2010; Jain, 2003; Kessenbrock et al., 2010).

Therefore, tumor-associated vessels within the tumor mass aremore easily accessible to tumor cells, thereby alleviating theintravasation process (see cartoon in Fig. 5).

IVM also shows that, in addition to active entry of cells intotumor-associated blood vessels, tumor cells can also accidentallyenter the blood stream when they are positioned close to a poorlystructured tumor-associated vessel that collapses upon elevatedinterstitial fluid pressure caused by proliferating cancer cells. As aresult of the collapse, big clumps of cells from the surroundingtissue shed into the lumen of the collapsed vessel and aretransported through the vasculature (Baluk et al., 2003; Chang etal., 2000; Fukumura et al., 2010; Jain, 2003; Kessenbrock et al.,2010).

Taken together, IVM has shown that both active and inactiveentry of tumor cells into the vasculature occurs within the tumormass, implying that tumor cell migration and intravasation do notonly take place at the invasive front. Therefore, intratumoralmotility and intravasation should be considered an important escaperoute for tumor cells. This idea also explains the observation thatmany patients who have metastatic outgrowth show no BMbreakdown and therefore no invasion at the invasive front at theprimary tumor site; this was initially explained by a resynthesis ofremoved BM (Dingemans, 1988).

Conclusions and future perspectivesIn this Commentary, we have focused on the advances in IVMand have given examples of its usefulness in studying the

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Fig. 5. Direct entry of tumor cells intotumor-associated vessels as shown by IVM.Tumor cell migration and intravasation takeplace not only at the invasive front, but alsodeep within the tumor mass. The upper rowof IVM images, in which the mouse invasivelobular carcinoma tumor cells are in greenand the SHG signal is in purple, shows highmotility within the tumor mass. The arrowspoint to the position of motile cells at time (t)zero (see also supplementary materialMovie 3). The row of images below showsmaximum projections of Z-stacks with a totaldepth of 50m. In the IVM images, greenrepresents C26 colorectal tumor cells andpurple represents SHG. This time series ofthe maximum projections shows thedisappearance of a tumor cell, which is nolonger present in the Z-stack because it entersa tumor-associated vessel and is transportedout of sight by blood (see circle;supplementary material Movie 2). Thecartoon shows a simplified model of leakytumor-associated vessels within tumors,which lack an intact BM and through whichtumor cells can enter the blood. Scale bars:10m.

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metastatic process. So far, most of the advances have resulted inthe ability to image the earlier steps of metastasis, includingmigration, invasion and intravasation. Only a small number ofstudies have attempted to image cells in organs that are prone tometastasis, such as the lungs (Kimura et al., 2010), bone marrow(Sipkins et al., 2005), lymph nodes (Mempel et al., 2004) andspleen (Grayson et al., 2001). Unfortunately, these experimentsrely on surgical dissection to expose the imaging site, whichhampers long-term imaging and therefore the visualization ofcolonization and dormancy. The use of the cranial imagingwindow has overcome these problems for the brain (Kienast etal., 2010) and, to expand this advance to other organs, the nextgeneration of imaging chambers should aim to visualize thespleen, liver and lymph nodes. IVM of colonization will help toanswer many unresolved questions, such as why tumor cellscolonize a specific organ without the formation of coloniesin other organs, how long tumor cells stay dormant andwhat factors can reactivate them (e.g. angiogenesis, growthfactors).

Although IVM has been successful in providing new insightsinto the early steps of metastasis, most studies are onlyobservational. In the next few years, it will be important to movethe field from observational IVM to experiments that can alsocharacterize the underlying molecular processes. For example, newcancer models should be imaged that have been engineered tomanipulate the behavior of cancer cells by inducible expression ofoncogenes or signaling proteins (Le Dévédec et al., 2010).Developments in photo-manipulation will further help the transitionto experimentally driven studies, such as laser-induced release andactivation of caged compounds (NPE-caged cAMP) or oftranscription. Moreover, high-resolution and FRET IVM ofbiosensors are important tools to study molecular processes, suchas cell–cell communication, intracellular signaling events and thegain of traits that are required for cells to metastasize. For example,a FRET probe for the proto-oncogenic tyrosine kinase Src (Ting etal., 2001) can be used to study the regulation of integrin–cytoskeleton interactions during cell motility, and a reactive oxygenspecies (ROS)-sensitive FRET probe that responds to hypoxia(Guzy et al., 2005) might be important for studying the influenceof the tumor microenvironment during escape and colonization.Unfortunately, most FRET biosensors have a low signal-to-noiseratio and are not sensitive enough to be used in IVM experiments.Biosensor development is an expanding field, and the next-generation probes should aim to increase the signal-to-noise ratioand to probe for proteolytic activity, proliferation, adhesion andsenescence.

Given the increasing number of genetically engineered tumorcell lines and fluorescent reporter mice, the transcriptional anddifferentiation state of cells that metastasize can also be studied byIVM. For example, Pinner and colleagues engineered melanomatumor cells to express GFP driven by the Brn-2 promoter, andshowed that melanoma cells can switch from a less differentiatedstate to a differentiated state when these cells exit the primarytumor and enter the secondary site (Pinner and Sahai, 2008). Thesetypes of IVM experiments will be able to answer long-standingquestions in the field, such as whether cells can transiently switchfrom an epithelial to a mesenchymal phenotype (EMT) duringmetastasis, or whether the metastasizing tumor cells have a stemcell phenotype.

As discussed in this Commentary, IVM is an important tool togain knowledge of tumor cell dynamics during metastasis, which

will enable the development of more powerful strategies fortreatment of human cancer. Although xenograft cancer models inimmune-deficient mice have been used extensively for IVM, theseare end-stage tumors that do not recapitulate the naturaldevelopment and morphology of common human cancers (e.g.Cardiff et al., 2000). More importantly, they lack particularinteractions with the microenvironment, which contains key players,such as T cells, that influence tumor development and metastasis(Condeelis and Pollard, 2006; de Visser and Coussens, 2006;DeNardo et al., 2009; Orimo and Weinberg, 2006). Novel cancermodels have recently been developed and result in mice that havea spectrum of tumors, which strongly resembles human tumordevelopment and progression in the breast (Derksen et al., 2006;Jonkers et al., 2001; Olive et al., 2004; Xu et al., 2001), pancreas(Hingorani et al., 2003), lung (Jongsma et al., 2008; Meuwissen etal., 2003) and brain (Llaguno et al., 2008; Xiao et al., 2002). Eventhough the incorporation of fluorophores will be technicallychallenging, these models will undoubtedly advance thetranslational aspects of future intravital imaging experiments.

We thank Johan de Rooij, Onno Kranenburg and Stephan Huveneersfor critically reading this manuscript, Tom Schonewille for technicalassistance, and Anko de Graaff and the Hubrecht Imaging Center forimaging support. This work was supported by VIDI fellowships91710330 (J.v.R. and E.B.) and 91796318 (P.W.B.D.) from the DutchOrganization of Scientific Research (NWO), a grant from the DutchCancer Society (KWF: HUBR 2009-4621) (L.R.) and an equipmentgrant (175.010.2007.00) from the Dutch Organization of ScientificResearch (NWO). N.V. was supported by the intramural researchprogram of NIAID, NIH. Deposited in PMC for release after 12months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/3/299/DC1

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310 Journal of Cell Science 124 (3)

Commentaries and Cell Science at a GlanceJCS Commentaries highlight and critically discuss recent and exciting findings that will interest those who workin cell biology, molecular biology, genetics and related disciplines, whereas Cell Science at a Glance posterarticles are short primers that act as an introduction to an area of cell biology, and include a large poster andaccompanying text.

Both of these article types, designed to appeal to specialists and nonspecialists alike, are commissioned fromleading figures in the field and are subject to rigorous peer-review and in-house editorial appraisal. Each issueof the journal usually contains at least one of each article type. JCS thus provides readers with more than 50topical pieces each year, which cover the complete spectrum of cell science. The following are just some of theareas that will be covered in JCS over the coming months:

Cell Science at a GlanceChaperone-mediated autophagy at a glance Ana Maria CuervoThe role of Rho GTPases in organising the actin cytoskeleton Ed ManserNucleoskeleton mechanics at a glance Kris DahlSirtuins at a glance Leonard Guarente

CommentariesIsoaspartate-dependent molecular switches for integrin–ligand recognition Angelo Corti defining thecrossover/non-crossover decision in meiosis Simon J. BoultonCohesin loading and sliding Frank UhlmannChromatin folding: from biology to polymer models and back Dieter W. HeermannER glutathione- and non-glutathione-based oxidant control Christian Appenzeller-Herzog

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