A P P L I C AT I O N N O T E

Pre-clinical Imaging
Authors
Jeffrey D. Peterson, Ph.D.
Kristine O. Vasquez
PerkinElmer, Inc.
Waltham, MA USA

Quantitative Pre-clinicalFluorescence Imagingof Cancer Metastasis
to the Lung andResponse to Therapy

Abstract

Quantitative pre-clinical fluorescenceimaging transcends the boundaries oftraditional optical imaging of biologicalstructures and physiology by providing
information at the molecular level about
disease states and therapeutic response.
PerkinElmer’s proprietary FluorescentPre-clinical Imaging Agents and FMT®
(Fluorescence Molecular Tomography)
Quantitative Pre-clinical Imaging Systemrepresent powerful tools for research anddrug development in the imaging of biological processes and pharmaceuticalactivity in living animals. Traditional mouse models of cancer rely primarily on
ex vivo measurements of disease morphology and histologic analysis for theassessment of tumor burden. These measurements of disease may be distantfrom the actual biological targets of interest and can be time consuming, expensive,
and impractical for measuring local disease biology in context. Further, thesemetrics do not enable measurements in living animals and merely focus onchanges in shape or size within the affected tissue. In contrast, by using
PerkinElmer’s near infrared (NIR) imaging agents in combination with the FMTsystem, the biological processes that change with disease progression andtherapeutic response, rather than alterations in morphology, can be visualized
non-invasively over time. In this set of studies, ProSense® 750 FluorescentPre-clinical Imaging Agent, a protease-activatable NIR probe, effectively imaged

and quantitated the abnormal increase in protease activity
associated with tumor aggression and growth within thelungs of BALB/c mice receiving an intravenous injection of
4T1 mouse breast adenocarcinoma cells. The responses todifferent chemotherapy treatments were quantitatively
measured as a reduction in protease activity that correlatedwith a decreased tumor burden as assessed by histology.
These results indicate that tomographic fluorescence imaging
datasets can be established in pre-clinical drug development
programs as standards for measuring disease progression
and therapeutic response.

Materials and Methods

On the day of imaging, mice were anesthetized, depilatedto minimize interference with the fluorescent signal, andpositioned in the FMT system imaging chamber. The FMT
system’s low-power laser diode light source trans-illuminatedthe tomographic scan region of interest with fluorescentsignal captured via a cooled CCD camera fitted with theappropriate optical filters. The collected fluorescence data
was reconstructed by TrueQuant™ software for the quantitationof 3-dimensional fluorescence signal within the lung region.
The mean fluorescence (in nM) and total amount of fluorescence (in pmoles) were generated for all studies.

4T1 Cancer Metastasis Model

Ex vivo imaging

Four to five week-old female BALB/c mice were purchasedfrom Charles River Laboratories® (Wilmington, MA) andmaintained in a pathogen-free animal facility with water andlow-fluorescence mouse chow (Harlan Tekland®, Madison,WI). Handling of mice and experimental procedures werein accordance with PerkinElmer IACUC guidelines and veterinarian requirements for animal care and use. To induce
cancer metastasis, BALB/c mice were IV injected with 5x105
4T1 mouse breast adenocarcinoma cells/100 µL (ATCC®,Manassas, VA). These conditions were optimized to generatemeasurable lung metastases in the majority of mice within
2 weeks as assessed by tumor burden (determined by lungweight) and fluorescent imaging (described below). The
presence of metastatic nodules within the lung parenchyma
was confirmed by histology after the lungs were fixed inbuffered formalin, sectioned and stained with Hematoxylin
and Eosin.

At the termination of each study (day 14) mice weresacrificed and lungs collected, weighed, and assessed
macroscopically. Lungs were also imaged on a fluorescenceplanar imaging system to corroborate the non-invasive invivo finding. Lungs were subsequently embedded in OCT,frozen, and cut into 7 mm sections which were immediately
mounted in DAPI- containing medium for assessment oftissue fluorescence localization.

Chemotherapeutic regimen

Introduction

5-Fluorouracil (5-FU Sigma-Aldrich , St Louis, MO) is a
fluorinated pyrimidine that is metabolized, in vivo, to yield
5-fluoro-deoxyuridine monophosphate and other metabolites that block DNA synthesis and cause the production offaulty RNA. 5-FU was resuspended in deionized sterile waterand injected intraperitoneally (i.p.) 1x/day starting on theday of tumor cell injection. Mice received 5-FU in combination
with 2’-deoxyinosine (dIno, Sigma-Aldrich®), a deoxyribose
1-phosphate precursor with the ability to potentiate 5-FU
cytotoxicity and enhance apoptosis. Mice were injected
2x/day i.p. with 1.6 g/kg dIno and 4.4 – 17.5 mg/kg/day
5-FU, for 5 consecutive days. Mice were injected with
ProSense 750 agent on day 11 and imaged 24 hrs lateron the FMT system as described above.
®

Imaging agents
At different timepoints after implantation of mice with 4T1
cells, mice were injected with ProSense 750 agent (to detecttumor-related cathepsin activity), IntegriSense™ 680 agent(to detect avb3 integrin expression), or AngioSense® 680
agent (to detect vascular leak) and imaged by the FMT 2500 LX
system 24 hours later.
2Lung Imaging Protocol

Statistical analysis
Data are presented as the means ± the standard error of themean (SEM). Significance analysis of in vivo lung fluorescence
was conducted using a two-tailed unpaired Student t testwhen 2 groups were analyzed or nonparametric ANOVA
when 3 or more groups were compared (StatView®, SASInstitute, Cary, NC). Probability values of < 0.05 wereconsidered significant.

Metastatic breast cancer remains a clinical challenge todaywith 210,000 new breast cancer cases per year reported in2010 in the United States alone (American Cancer Society®).
In 10% of breast cancer diagnoses, the cancer has alreadymetastasized to distant organs in the body, such as bone,liver, and lung, decreasing the 5-year relative survival rate to20%. Thus, it is essential to develop robust in vivo models
that can help dissect the metastatic process and assist in theeffective development of targeted therapeutic agents.
Over the last 100 years there have been numerous efforts
to develop in vivo breast cancer models. These efforts havefocused on the establishment of solid tumor models as wellas metastatic models that closely mimic the clinical presentationof breast cancer. The establishment of these models, in addition
to the development of accurate imaging technologies, havehelped in expanding our understanding of tumorigenesis,
tumor-host interactions, tumor progression and metastasis.
New technologies are also now routinely applied to theelucidation of pharmacologic mechanism of action of new
drug entities.

In the present study, we used a well established 4T1 mouse
mammary carcinoma model that yields highly invasive metastases to the lungs of normal BALB/c mice upon IV injection.
Using the FMT 2500LX system, in conjunction with threedifferent NIR fluorescent probes; we longitudinally tracked
and characterized the pulmonary metastatic spread of breast
cancer to the lungs as well as quantitated the anti-tumor
effect of the chemotherapeutic agent 5-fluorouracil (5-FU).
Our non-invasive imaging results were robust and correlated
well with terminal assessments such as changes in gross lungweight, ex vivo tissue imaging, and histologic assessment.
Such an imaging approach should help to visualize andquantitate steps in cancer progression or metastatic cascadein vivo, and will be beneficial in developing novel therapeutictreatments.

metastases were also confirmed by histology after fixation informalin, sectioning and Hematoxylin and Eosin staining. In
situ localization of ProSense agent fluorescence in tissue wasperformed on frozen sections.
Imaging results show that disease is readily detected in micereceiving injections of 4T1 cells, but only when using theFMT 2500 LX system’s tomographic capability and not bywhole body 2D imaging. Control mice showed little or no
signal other background fluorescence associated with agent
metabolism (liver, kidney – not shown). Ex vivo assessmentof tissues agreed well with in vivo imaging and quantitation.
Monitoring kinetics of lung metastasis by FMT
Mice were injected/imaged with ProSense agent at four
different times following 4T1 cell injection to monitor diseaseprogression. Un-injected mice served as controls (time 0).
All imaging was performed 24 hours following intravenous
agent injection on the FMT system. No signal could be
detected in the lung region of non tumor-injected mice.
However, as soon as 1 week following tumor cell injection,
discrete areas of near infrared fluorescent ProSense agent
signal could be identified in the lung, increasing over time.
Quantitation of ProSense agent signal showed statisticallyhigher mean pmols and fluorescent volume (mm3) in 4T1bearing mice as compared to un-injected mice (*p < 0.01;**p < 0.005), with earliest detection of differences seen at
7 days. Total fluorescent signal increased over the next week,reaching peak values on day 14, but the volume offluorescence per lung did not change much. Results generally
agreed with lung weight assessment (which assesses acomposite of tissue weight, pulmonary edema, and tumorweight), however, as expected, imaging better reflected thekinetics of tumor growth in vivo.

in vivo
FRI

Tomography

ex vivo
TissueHistology

4T1 metastasis model
Figure 1 depicts the schematic of the model used in thisstudy, a protocol optimized to generate high incidence oflung metastasis by 14 days. Figure 2 represents the resultsof a study in which BALB/c mice were injected intravenouslywith 5x105 mouse mammary 4T1 cells. Two weeks later,mice were injected intravenously with the ProSense 750
agent and imaged 24 hours later with the FMT system togenerate non-invasive, quantitative imaging. At the endof the imaging session, mice were sacrificed and their lungs
observed for macroscopic evidence of abnormalities. Inaddition, lung tissues were imaged in 2D to validate thelevels of ProSense agent fluorescent signal in tissue. LungControlResults

4T1-injected

Figure 1. 4T1 lung metastasis model.

Two weeks following injection of 5x105 4T1 cells, mice were injectedwith ProSense 750 agent and imaged by the FMT system 24 hours later.
Liver, GI tract, and bladder regions were not imaged.
Figure 2. FMT 2500 LX system imaging of tumor cathepsin activity.
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Assessing the 5-FU dose response using the FMT system

At different times after inoculation with 4T1 cell, mice were injectedwith ProSense 750 agent and imaged by the FMT system 24 hours later.
Analysis of tomographic lung datasets was performed using thresholds
based on negative control animals. ProSense agent fluorescence volumeand total picomoles within the lungs of 4T1 recipients were quantified
by FMT system software.
Figure 3. Quantitation of tumor cathepsin activity.

To monitor the effect of 5-FU on tumor burden by in vivo
fluorescence imaging, mice were IV injected with 5x105 4T1
cells and dosed with increasing concentrations of 5-FU (4.375,8.75, and 17.5 mg/kg/day, n = 5 mice per group) on the sameday. Mice received 5-FU for an additional 4 days. On day 10,
mice were IV injected with ProSense 750 agent and imaged
24 hours later on the FMT system. As shown in Figure 4,
tomographic fluorescent signal in the lung region decreased
with increasing doses of 5-FU. Figure 5 (left panel) shows asignificant decrease of 46% and 63% with doses of 8.75 and17.5 mg 5-FU/kg/day (p = 0.02 and p = 0.003, respectively
as compared to no treatment; p = 0.04 between 4.375 and8.75 mg/kg/day and p = 0.006 between 8.75 and 17.5 mg/kg/day). Fluorescence volume (center panel) provides quantitative
data regarding the volume of lung affected by disease, withhigh dose treatment decreasing this by as approximately 50%.
Immediately following the imaging session, mice weresacrificed and their lungs dissected and weighed. With
progressively increasing tumor burden, as assessed by lungweight (right panel), a fair correlation between in vivo
fluorescence and lung weight was observed (r2=0.71),
however total fluorescence showed much greater sensitivity
as a readout of drug efficacy.

Whole animal FMT and tissue FRI Imaging

FMT Quantitation

Mice received 5-Fluorouracil (5-FU) + Deoxyinosine (dIno), at doses and dosing regimens indicated, beginning one day following 4T1 cell injection.
On day 13, mice received a dose of ProSense 750 agent and were imaged by the FMT system 24 hours later.
Figure 4. Quantitation of 5-FU efficacy with ProSense 750 agent.
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At different timepoints after inoculation with either 0.5x105 or 5x105
4T1 cells, mice were injected with ProSense 750 agent (cathepsin activity),IntegriSense 680 agent (avb3 integrin expression), or AngioSense 680
agent (vascular leak) and imaged by FMT 2500 LX system 24 hours later.
Two-color representation of FMT 2500 LX system tomography and FMT2500 LX system FRI imaging shows regions of biological co-localization.

Mice received an i.p injection of a small molecule integrin antagonist(25 mpk/mouse/b.i.d.) starting at day 0, starting 1 hour prior to 4T1
cell injection. On days 9 and 13, mice received a combined dose ofAngioSense 680 agent and ProSense 750 agent and were imaged by theFMT system 24 hours later.

Figure 5. Multiplex FMT 2500 LX system quantitation of vascular leak,cathepsin activity, and integrin expression.

Figure 6. Multiplex FMT 2500 LX system quantitation of integrin agonist
expression.

Multiplex Fluorescence Imaging

Quantitation of tumor fluorescence in the lung for these
two studies (Figure 6) also incorporated earlier imaging times,
showing a clear progression in signal for both high dose andlow dose studies. Imaging results for ProSense agent andIntegriSense agent correlated well with lung weights, but
the AngioSense agent showed a high background in normal
tissue and delayed detection of tumor-induced changes.

To assess the ability of the FMT 2500 LX system to provide
dual imaging with simultaneous injection of two imaging
agents, both in vivo and in post-mortem tissues (2D), mice
were IV injected with 4T1 cells [either 0.5x105 (“low 4T1 celltransfer”) or 5x105 (“high 4T1 cell transfer”)] and imaged
with combinations of ProSense agent/AngioSense agent orProSense agent/IntegriSense agent, respectively (Figure 5).
The high cell transfer study (upper panels) recapitulated
results from Figures 2 and 4, with robust ProSense agent
imaging results achieved 14 days after cell transfer. Dual color
representation of ProSense agent (tumor and inflammatorycells) and AngioSense agent (vascular leak) revealed widespread distribution of both agents throughout the lungs
with considerable overlap in the localization of these agentsas expected. Using a low 4T1 cell dose (lower panels) anda combination of ProSense agent and IntegriSense agent(tumor, vascular, and inflammatory cells), optimal imaging
signal was delayed to day 18 and there were more regionsof distinct localization of the two agents. Ex vivo 2D imaging
further showed a more nodular distribution of signal, albeit
still well distributed widely throughout the lungs.

Conclusions
Mouse models of cancer metastasis rely predominantly on exvivo tissue weight, nodule counts, and/or histologic analysis
for the assessment of tumor burden. To assess the feasibilityof non-invasive, quantitative imaging of deep tissue metastases,
we used different NIR imaging agents to image vascular
leak, upregulation of cathepsins and increases in integrin
expression, known to occur in cancer progression andmetastasis. We found that 4T1 cell injection led to diffuse,wide-spread metastases within the lungs of recipient mice,
rather than distinct small metastases. Severity of the diseaseprogression was modified using different 4T1 cell doses.
Tomographic imaging allowed the quantitation of fluorescentagents accumulating in the tumors, with this quantitation

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yielding better results than typical lung weight assessment.
Treatment efficacy with either 5-FU or a small molecule
integrin antagonist led to robust and quantitative changes,
with the additional capacity of measuring multiple biologicalchanges simultaneously. These findings were confirmed by 2D
imaging of ex vivo tissue and by histology. With this model wehave established the potential utility of quantitative fluorescence
tomography in preclinical drug discovery in the challenging areaof deep tissue metastasis.

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July 2012

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