IVISense™

FLUORESCENT PROBE

SELECTOR GUIDEENTER .

Oncology Series

TUMOR BURDEN

FolateMMPMETABOLIC ACTIVITYREAGENTS DESIGNED FOR YOUR IN VIVOIMAGING RESEARCH IS HOW PRECLINICALADVANCEMENTS ARE MADE.

Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase

ABOUT THIS GUIDEThe goal of in vivo fluorescence molecular imaging is to enable non-invasive visualization and quantificationof cellular and biological functioning to better understand and characterize disease processes and treatment
effects earlier within the context of a biological system.
This can be achieved using biologically specific, validated fluorescent probes. In cancer research, welldefined fluorescent probes can be used to detect specific oncological biomarkers and protease activity, as
well as drive cancer therapies.
It’s essential to understand how a probe’s structure, size, specificity, and physiochemical properties cansignificantly impact its pharmacokinetics, biodistribution, and metabolic clearance characteristics. These
properties, designed and fine-tuned to best suit a certain biology and optimal imaging time point, must be
carefully considered in relation to the biological and temporal characteristics of one’s model.

INFLAMMATION
Hydroxyapatite

CELL DEATH

With an emphasis on proper probe selection and careful study design, non-invasive assessment of in vivo
biological readouts can be maximized to detect early indicators of standard endpoints and realize moremeaningful research results.

TransferrinThis selector guide for IVISense™ fluorescent probes is a powerful tool to help advance your oncology
research. By matching probe properties to specific biology and biomarker characteristics, you canbetter understand how imaging and quantification of early biological changes associated with diseasedevelopment, therapeutic efficacy, and drug-induced tissue changes can be realized.
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TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US.

Also called tumor load, tumor burden is a measurement of the amount of cancer present. It can be an
important factor for determining the efficacy of a treatment program and provides a basis for which tocompare different treatments.
Often assessed using bioluminescence, tumor burden is essentially a non-biological readout relying on
gross overt changes in tumor size in order to assess tumor progression or response to therapy and is a lateindicator of treatment efficacy. Fluorescence molecular imaging methods can be used to provide sensitive
detection of treatment efficacy sooner. Depending on your experimental design, it may be of value toconsider other application modules in this Probe Selector Tool (such as Metabolic Activity), as they may
provide useful ways to detect changes in tumor biology that occur prior to overt changes in tumor volume.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Also called tumor load, tumor burden is a measurement of the amount of cancer present. It can be an
important factor for determining the efficacy of a treatment program and provides a basis for which to
MEASUREMENT
OF THE AMOUNT OF CANCER PRESENT
comparedifferent treatments.
Tumorburdenusing
is a calculated
volume thatcanbe expressedin mma3.non-biological
Total tumor burden
is thesumonof allOften
assessedbioluminescence,tumor
burdenis essentiallyreadout
relyingmeasurements
to obtain
tumor andvolume,or onmeasuredtumor volumes.
modelsrely tumor
on ex vivo
grossovert changes
in tumorTraditional
size in order
to assess
progression
or responseto therapy
is a latevivo longitudinaltumor
focusingsensitiveon changes incaliper measurementsof widthFluorescenceand length toassess inimaging
indicatorof treatment efficacy.
molecular
methods canbe burden,
used to provide
shape orof
size
of subcutaneoustumors.Depending
However, relying
onexperimental
gross measurement
tumor
to assessdetection
treatment
efficacy sooner.
on yourdesign, of
it maybesizeof value
to treatment
efficacyothercan bemisleading,asProbeinfiltrating
inflammatory
cells,
calcifiedparts, andnecrotic
may
obscureconsidermodulesin thisSelectorTool (such as
Metabolic
Activity),as they
maytissue
provideusefultreatment
effects
on viabletumor
tissue.that
Imagingsuchchanges
as optical,
microCTor ultrasound are moreways
to detectchangesin tumor
biologyoccur modalities
prior to overt
in tumor
volume.
reliable options for evaluating tumor burden, especially for deep tumor measurement where in vivo caliper
measurements are not possible.
MORE INFO .

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

In the case of optical imaging, bioluminescent imaging (BLI) results generally corroborate conventional
caliper measurements quite well, but importantly BLI also takes into account areas of the tumor that may befibrotic or necrotic. This makes BLI more accurate in measuring viable tumor mass. However, 2D BLI can bequantitatively variable for deep tumors due to attenuation of signal when depth of the signal varies between
subjects. Optical tomographic (3D imaging) methods, including both bioluminescence and fluorescence, not only
enable an adjusted view of intensity according to its depth and provides better anatomical context for scientificinterpretation, but can also be used to achieve more accurate quantification for calculating tumor volume.
Fluorescent probes such as those described in the recommended probes section, or luciferin substrate, can beused effectively with PerkinElmer’s IVIS® tomographic (3D) optical imaging systems for accurate longitudinaltumor burden assessment.

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TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium

TUMOR SIZE AND LOCATION
Large Tumors: Palpable, well-established tumors can be imaged using the fluorescent agentsdescribed in the recommended probes section.
Small Tumors: Detection of tumors smaller than 100 mm3 is possible but is dependent on tumor location.
Tumors located distal from the route of metabolism (the region with the greatest background signal) will
be more clearly detected due to the reduced background signal.

αvβ3 Integrin
Integrin

PDX TUMORS AND VASCULARITY

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

When planning a tumor imaging strategy, consider the vascularization status of the tumor. Vascularizationis required for the survival of implanted tumors, but this may take some time to develop, and fluorescentprobes may not be as effective immediately following implantation. One approach to consider for earlymonitoring of tumor implantation is to label with IVISense 680 Cell Labeling Dye. This will allow the tissue
surface to be labeled and may provide useful information early on regarding localization and efficiency of
implantation (though it will not assess the viability of the tissue).
Once tumor growth is established, fluorescent probes are very effective in assessing growth, metabolism,protease activity, inflammation, cell death, hypoxia, and other important aspects of tumor biology.

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As transfection of patient derived xenograft (PDX) tumors with a luc construct is difficult and generally
considered undesirable, fluorescence is an appropriate imaging method, and a variety of imaging probes,
specific for different aspects of tumor biology, are available to assess growth and response to treatment.

.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

TUMOR SIZE AND LOCATION
Large Tumors: Palpable, well-established tumors can be imaged using the fluorescent agentsdescribed in the recommended probes section.

ROUTE OF METABOLISM /BACKGROUNDTISSUESon tumor
location.
Small Tumors: Detection of tumors smaller than 100 mm is possible but is dependentPROBE

METASTASISEndothelium
αvβ3 Integrin
Integrin

Tumors located distal from the route of metabolism (the region with the greatest background signal) will
IVISense Cat B 680 FAST
Cathepsin B
Salivary Glands > Liver, Kidneys
be more clearly detected due to the reduced background signal.
IVISense Cat B 750 FAST

Cathepsin B

MMP 645 FAST
Matrix Metalloproteinase family
PDXIVISense
TUMORSAND VASCULARITY

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

IVISense MMP 680

Matrix Metalloproteinase family

Salivary Glands > Liver, Kidneys
Liver > Kidneys
Liver

As transfection of patient derived xenograft (PDX) tumors with a luc construct is difficult and generally
IVISenseundesirable,
MMP 750 FAST
Metalloproteinase> Kidneys
considered
fluorescence is anMatrix
appropriate
imagingfamily
method, and Livera varietyof imaging probes,
specific
for different
aspectsare proteaseavailable
to assess growth Liverand response to treatment.
IVISense
Pan Cathepsin
680 of tumor biology,
CathepsinfamilyPan aCathepsin
750
familyliver,tumor.
intestine
WhenIVISense
planning
tumor imaging
strategy,Cathepsin
considerprotease
the vascularization
status Lowof theVascularizationis required
survival750
of FAST
implanted tumors,butthis maytake some timeLow
to develop,
and fluorescent
IVISensefor
PantheCathepsin
Cathepsin
proteasefamilyliver, bladderprobes may not be as effective immediately following implantation. One approach to consider for early
IVISenseof
Integrin
Receptor645
αvβ3with
Integrin
Bladder, Kidneys
monitoring
tumor
implantation
is to label
the cell tracking agent VivoTrack™
680. This will allow
the tissue
surfacetoReceptor
be labeledprovideuseful information early onKidneys,
regarding
localization and
IVISense
Integrin
680 and may αvβ3
Integrin
Intestines
efficiency of implantation (though it will not assess the viability of the tissue).
IVISense Integrin Receptor 750

αvβ3 Integrin

KidneysOnce tumor growth is established, fluorescent probes are very effective in assessing growth, metabolism,protease activity, inflammation, cell death, hypoxia, and other important aspects of tumor biology.

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BIOMARKER SPECIFICITY
3.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium

TUMOR SIZE AND LOCATION
Large
Tumors:well-establishedtumorsbesoluble
imageddyeusing
thefluorescentagents
IVISense
680Palpable,
fluorescentcell labelling dye
is a canwater
thatenablesa simpleand easy method
described
intherecommended
probessection.
for labelling cell membranes. IVISsense 680 cell labeling dye is specially formulated for excellent solubility
in the absence of organic solvents such as ethanol or DMSO. The agent intercalates into cell membranes,
Small
Tumors:a Detectionof tumorssmallerthan
mm3 isinpossible
vivo. but is dependent on tumor location.
providingbright signal
by whichcells canbe100
trackedTumors located distal from the route of metabolism (the region with the greatest background signal) will
be more clearly detected due to the reduced background signal.

αvβ3 Integrin
Integrin

REQUIREMENTS
EFFECTIVE
CELL LABELINGPDX
TUMORSFORAND
VASCULARITY

DRUG RESISTANCEIVISENSE 680 CELL LABELING DYE CHARACTERISTICS

Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrinsolublein aqueousissolution- doesneed to transition
As transfection of patient derived xenograft (PDX) tumorsHighlywith aluc constructdifficultand not
generally
from organic
to aqueous
solution of
upon
additionprobes,
to the cellconsidered
undesirable,
fluorescence is an appropriate imaging
method,and a variety
imaging
Uniform celllabeling
suspension, allowing quick dispersion into cell medium andspecific for different aspects of tumor biology, are available
to assess growth and response to treatment.
uniform cell labelingLong planningcellular residencetime
Signal is retainedin dividing
cells
in vitroVascularization
for more than 7 days
When
a tumorimaging strategy, consider the vascularization
statusof thetumor.
is required for the survival of implanted tumors, but this may take some time to develop, and fluorescent
Does not use DMSO or ethanol, known to adversely affect cellular
Minimalimpactviability andimmediately
functionprobesmay
not onbecellas effective
following implantation.
One approach to consider for earlyfunctionmonitoring of tumor implantation is to label with the cell tracking agent VivoTrack™ 680. This will allow
Near infrared wavelength for optimal light penetration of
the tissue surface to be labeled and may provide useful informationon 696
regarding
localization andFluorescence early
emission:nm
tissueefficiency of implantation (though it will not assess the viability of the tissue).
Effective longitudinal in vivo labeling of:Capableof labelingrange of cells
T cells,inmacrophages,
and many
other
Once
tumor
growthbroad
is established,fluorescent probes are stem
very cells,
effectiveassessing growth,
metabolism,
cell types

protease activity, inflammation, cell death, hypoxia, and other important aspects of tumor biology.

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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase

MMP

CELL DEATH

Transferrin

RESOURCESCONTACT US.

αvβ3 INTEGRIN

Matrix metalloproteases (MMPs) are calcium-dependent, zinc-containing endopeptidases that play a variety ofroles in tissue remodeling by degrading the extracellular matrix. Present in healthy biological tissues, they are
activated extracellularly by multiple levels of control that strictly regulate their activation. In models of disease,
MMP activation can go unchecked, potentially wreaking havoc on essential structural proteins. In cancer
models, MMPs tend to localize in tumor margins, playing an important role in tumor metastasis.
The ability to differentiate between active and inactive MMPs at the site of disease, and not just detect thepresence of MMPs in general, can reveal distinctive local biology as well as assess therapeutic efficacy ofspecific drugs prior to overt biological changes. IVISense MMP fluorescent probes are able to detect a broad
range of active MMPs including MMP 2, 3, 7, 9, 12, and 13, to track tumor progression, or evaluate thepotential therapeutic efficacy of drugs targeting the underlying mechanisms involved.

INFLAMMATION
Hydroxyapatite

CATHEPSIN

VIEW IMAGE .

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

αvβ3 INTEGRIN

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Matrix metalloproteases are active in regions of tissue remodeling, in this case highly expressed andlocalized at the invasive tumor margins. IVISense MMP fluorescent activation is represented in the figureon the right.
©2019, PerkinElmer, Inc. All rights reserved

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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE

MMP

CATHEPSIN

αvβ3 INTEGRIN

Associated with tumor aggression, cathepsins are a family of proteases that are pivotal components ofsignaling pathways which can enhance cancer cell growth and inflammation. Within the human lysosomal
cysteine cathepsin family, there are 11 members, including cathepsin B, L, S, and Plasmin. As a family, cysteine
cathepsins are involved in the response to anticancer therapy within the tumor microenvironment and play
crucial roles in the development of resistance to therapeutics. In particular, cathepsin B expression correlateswith the invasive and metastatic capabilities of many tumors, such as in breast cancer where high expression
levels of cathepsin B have been linked to highly aggressive tumors. Cathepsin B is also upregulated in a variety
of inflammatory cells (including eosinophils, neutrophils, and macrophages).

Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

For fast, non-invasive in vivo imaging of tumor status and progression via protease expression, NIR fluorescentimaging probes such as IVISense Pan Cathepsin and IVISense Cat B are available for evaluating potential
anticancer therapies. IVISense Pan Cathepsin imaging probes are versatile activated agents, able to detectand measure the expression and activity of key cancer-associated proteases and can be used to monitor theactivity of these proteases in real time. IVISense Cat B fluorescent probes are selectively cleaved by cathepsin B
proteinases for specific and rapid detection of tumors associated with cathepsin B activity.

Transferrin
VIEW IMAGE .

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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

MMP

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Inflammation in response to tumor
growth involves proteases andcysteine cathepsins expressed byinflammatory cells. High levels ofcathepsin expression in cancer cells
also correlate to cancer metastatic
potential. IVISense Pan Cathepsin
fluorescent activation is represented
in the figure on the right.

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CATHEPSIN

αvβ3 INTEGRINTUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase

MMP

In particular, αvβ3 integrin is significantly upregulated in tumor cells and activated endothelial cells during
neoangiogenesis. As a distinct biomarker of tumor burden and progression, αvβ3 integrin can be imaged using
IVISense Integrin Receptor NIR fluorescent imaging probe. The IVISense Integrin Receptor probe provides
excellent tumor definition with minimal off-target distribution due to its high affinity (Kd = 4.2 nM) andselectivity 5-20x over RDG peptide-based agents. With improved circulation half-life and specificity, IVISense
Integrin Receptor probe enables detection and measurement of αvβ3 integrins in vivo.

Hydroxyapatite
VIEW IMAGE .
Transferrin

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αvβ3 INTEGRIN

Integrins are cell surface signaling molecules that are essential for regulating communication between cells
and their microenvironment. Critically involved in the cells’ ability to adhere to the extracellular matrix as wellas in cellular migration, they are important to maintaining the integrity of healthy tissue and are abnormally
expressed in diseases such as cancer where integrin expression correlates with tumor aggressiveness..

INFLAMMATIONCELL DEATH

CATHEPSINTUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

αvβ3 INTEGRIN

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

αvβ3 integrin is significantly upregulated in tumor cells and correlates with tumor aggressiveness.
IVISense Integrin Receptor fluorescent imaging of fine tumor definition is represented in the figureon the right.

©2019, PerkinElmer, Inc. All rights reserved

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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

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BIOPROFILING TUMOR BURDEN AND PROTEASE ACTIVITY:
IDENTIFYING CHARACTERISTIC DIFFERENCES BETWEEN TUMOR TYPES
Critical to understanding the biology of a model is to know when a particular biomarker is active. Cancer
biomarkers may be up- or down-regulated at different times, as different factors may influence theirexpression during the growth of a tumor. To illustrate the potential of broad tumor profiling, the followingcase study uses two different tumors imaged with different fluorescent agents. These agents are capableof detecting protease activity or surface receptor expression of markers for assessing tumor cell burden. As
shown in the case study, fluorescence quantification with multiple probes can illustrate the importance ofgiving consideration to characteristic differences in rates of protease expression in tumors for appropriate
probe selection, as well as likely prove valuable for the full biological characterization of tumors.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

CASE STUDY: EFFECTS OF PROTEASES ON TUMOR GROWTH
AND IMPLANTATION EFFICIENCIES
PerkinElmer’s IVISense NIR fluorescent in vivo imaging probes are important tools to non-invasively detectand quantify biomarkers such as protease activity (MMPs and cathepsins) and indicators of tumor burden(integrin). Groups of HeLa and 4T1 tumor-bearing mice were injected with two pairs of imaging agents;
IVISense MMP 680, IVISense Pan Cathepsin 750, and IVISense Osteo 680 & IVISense Integrin Receptor 750(IVISense Osteo was used as a control agent not expected to localize appreciably to tumor tissue) to detect
relative differences in protease activity (associated with tumor progression and aggression) and integrin
expression (general tumor burden marker) in HeLa and 4T1 tumors.
Fluorescence quantification of in vivo imaging probes shows that the two different tumor types were quite
similar in IVISense Integrin Receptor, indicating similar levels of integrin receptor expression. In contrast,
HeLa cells were 50-70% lower in protease activity as assessed by IVISense MMP and IVISense Pan Cathepsin
imaging. This lower protease activity in HeLa cells may account for the slower initial growth rate of HeLa
tumors (see graph on the right), suggesting the importance of proteases for growth early on, following theimplantation of cells. At later time points, the HeLa tumors rapidly achieve the size of the 4T1 tumors.

Transferrin
VIEW GRAPHSRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

CASE STUDY: EFFECTS OF PROTEASES ON TUMOR GROWTH
AND IMPLANTATION EFFICIENCIES
PerkinElmer’s NIR in vivo imaging agents are important tools to non-invasively detect and quantify
biomarkers such as protease activity (MMPs and cathepsins) and indicators of tumor burden (integrin).
Groups of HeLa and 4T1 tumor-bearing mice were injected with two pairs of imaging agents; MMPSense®
680 & ProSense® 750EX, and OsteoSense® 680EX & IntegriSense™ 750 (OsteoSense® was used as a control
agent not expected to localize appreciably to tumor tissue) to detect relative differences in protease activity(associated with tumor progression and aggression) and integrin expression (general tumor burden marker)
in HeLa and 4T1 tumors.
Fluorescence quantification of in vivo imaging probes shows that the two different tumor types were quite
similar in IntegriSense™, indicating similar levels of integrin receptor expression. In contrast, HeLa cells were
50-70% lower in protease activity as assessed by MMPSense® and ProSense® imaging. This lower proteaseactivity in HeLa cells may account for the slower initial growth rate of HeLa tumors (see right-hand chart),
suggesting the importance of proteases for growth early on, following the implantation of cells. At later
Growth Rates. Caliper measurements oftime points, the HeLa tumors rapidly achieve the size of the 4T1Tumor
tumors.
4T1-luc2 and HeLa-luc tumors were measured
Fluorescence Quantification of in vivo imaging
over time to assess growth kinetics. Length andprobes in HeLa and 4T1 cell lines Imaged on
width measurements were used to calculate
the IVIS® Spectrum optical imaging system.
VIEW CHARTS .
tumor volumes in mm3.

Application Note: Peterson et al., Multiplex 2D Imaging of NIR
Molecular Imaging Agents on the IVIS® SpectrumCT and FMT® 4000.

RESOURCES© 2015-2020, PerkinElmer, Inc. All rights reserved.

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BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISBY CHARACTERIZATIONIVISense Probe

Phosphatidylserine

METASTASISEndothelium" Cat B 680 FAST

33,000" Cat B 750 FAST

23,000" MMP 645 FAST

43,000" MMP 680

αvβ3 Integrin
Integrin

DRUG RESISTANCE
BombesinMW(g/mol)" MMP 750 FAST

~450,000
43,000" Pan Cathepsin 680~400,000" Pan Cathepsin 750~450,000" Pan Cathepsin 750 FAST

22,500± Integrin Receptor 645

1,250± Integrin Receptor 680

1,432± Integrin Receptor 750

1,278

BY APPLICATION
Specificity

In Vivo Biology Use

Cathepsin B

Selectively cleaved by cathepsin
B proteinases upregulated intumors and inflammatory cells.

Matrix
Metalloprotease

Cleaved by multiple
metalloproteinases associatedwith many disease areas including
cancer, inflammation, andcardiovascular disease.

Cathepsin
proteases

Pan-cathepsin activatable agent
that detects abnormal
upregulation of cathepsin
secretion associated with cancer
and inflammation.

αVβ3 Integrin

Detection of integrin αvβ3
expression in tumors andneovasculature.

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Mouse Dose(25 g)

Sold As(Unit Size)

Optimal Imaging
Time (h)2 nmol24 nmol

6-24 h

4 nmol

48 nmol

6-24 h

4 nmol

48 nmol

24 h (6-24)

2 nmol20 nmol

24 h (24-36)

2 nmol24 nmol

24 h (12-24)

2 nmol20 nmol

24 h (24-48)

2 nmol24 nmol

24 h

4 nmol

48 nmol

6-24 h2 nmol24 nmol

6-24 h2 nmol24 nmol

24 h

2 nmol24 nmol

24 h" Activatable probes are optically silent upon injection and become highly fluorescent following protease-mediated activation. Activatable FAST

agents are designed with a novel small molecule architecture that confers an accelerated pharmacokinetic profile with earlier imaging time points.

± Targeted probes actively target and bind to distinct biomarkers with highly specific targeting to key biological mechanisms.

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BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISBY CHARACTERIZATIONBY CHARACTERIZATIONIVISense

Cat B 680 FAST
Cat B 750 FAST

MMP 680

MMP 645 FAST
MMP 750 FAST

Pan Cathepsin 680
Pan Cathepsin 750

Pan Cathepsin
750 FAST

Integrin Receptor 645
Integrin Receptor 680
Integrin Receptor 750

Biomarker specificity

Cathepsin B

Matrix Metalloproteasefamily proteases

Matrix
Metalloproteasefamily proteases

Cathepsin family
proteases

Cathepsin family
proteases

αvβ3 Integrin

Phosphatidylserine

METASTASISEndothelium

BY APPLICATION

Applications

αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION

Early Tumor
Assessment

+++

+++

+++

+++

+++

++++

Metastasis Imaging+++/- due to liverinterference+++/- PC680 (liver)++ PC750++++

Image Tumor
Vascularity

-

-

-

-

-

+++

Assessing
Treatment Efficacy

+++

+++

+++

+++

+++

+++

Probe Characteristics

CELL DEATH

Hydroxyapatite

Imaging Time Post
Probe Injection

6-24 hr

24 hr (24-36)

24 hr (6-24)
24 hr (12-24)

24 hr (24-48)

6-24 hr

6-24 hr
24 hr
24 hr

Transferrin

Tumor Washout

3 days

6-7 days

6-7 days

6-7 days

3 days

6-7 days
14 days
4-6 days

Summary of probes and applications. An overview of results, including practical information regarding optimal imaging/washout time and response totreatments (from internal and external, published and unpublished, research). Performance is based on a subjective scale (+, ++, +++, ++++) that takes into
account the signal intensity, kinetics, background, washout, and general utility.

RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATHRESOURCESCONTACT US.

Cancer is highly adept at hijacking cellular pathways to gain a biological advantage. Metabolic
reprogramming exploits the system of biochemical processes within cells, altering pathways to meetthe increased demand for energy, synthesis, and proliferation that is characteristic of tumor growth.
Biomarkers of these altered pathways offer opportunities to study not only tumor progression, but also
to specifically assess therapies that target critical pathways in tumor metabolism. Detecting biomarker
fluctuations by in vivo fluorescence imaging can provide an early indication of changes in tumor
metabolism and growth, prior to overt changes in tumor burden as detected by traditional caliper
assessment or standard bioluminescence.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATHRESOURCESCONTACT US.

IMAGING HEPATIC TUMORS
Iron is necessary for cellular metabolism in cells throughout the body. In healthy tissue, cellular ironhomeostasis is maintained in part by transferrin receptors. In tumor cells, increased metabolic demandfor iron requires the number of transferrin receptors on the cell surface to increase. Therefore, increased
expression of transferrin receptors can be exploited as a biomarker of tumor metabolism and effectively
targeted for fluorescent in vivo imaging.
When developing an imaging strategy for a tumor in the liver, the main site of iron storage in the body, it
is important to choose a probe with a non-interfering route of metabolism (the region with the greatest
background signal). As some probes are metabolized through the liver, choosing a probe that metabolizes
away from the liver will allow for better detection due to reduced background signal.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium

MEASUREMENTOFTUMORS
THE AMOUNT OF CANCER PRESENTIMAGINGHEPATIC
Iron is necessary for cellular metabolism in cells throughout the body. In healthy tissue, cellular ironhomeostasis
transferrin receptors.
In tumorcells,OF
increased
metabolicdemandPROBE* is maintained in part byBIOMARKER
SPECIFICITYROUTE
METABOLISM/ BACKGROUNDTISSUESfor iron requires the number of transferrin receptors on the cell surface to increase. Therefore, increased
expressionof transferrin
receptors canbereceptor
exploited as a biomarker
of tumor metabolism and effectively
IVISense Folate
Receptor 680
FolateKidneytargeted for fluorescent in vivo imaging.
IVISense Bombesin Receptor 680

αvβ3 Integrin
DRUG RESISTANCE
Bombesin

Bombesin receptor

Pancreas, kidney

Whendeveloping
imaging
for a tumor
in the liver, themainsite of iron storage in the body, it
IVISense
Transferrinan
Receptor
750strategy
Transferrin
receptor
Liver,
kidneyis important to choose a probe with a non-interfering route of metabolism (the region with the greatest
background signal). As some probes are metabolized through the liver, choosing a probe that metabolizes*Other probes , such as IVISense Pan Cathepsin 750 FAST & 750, IVISense MMP 750 FAST, and IVISense Integrin Receptor, also
away from the liver will allow for better detection due to reduced background signal.

metabolize away from the liver, and are capable of imaging hepatic tumors for biological applications other than detecting biomarkers

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATHRESOURCESCONTACT US.

of metabolic activity (for example, detecting biomarkers of inflammation or tumor burden and progression). See the Fluorescent
Agents Reference Table in the Resources section for a full list of probes and their specificity, routes of metabolism, and more.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATHRESOURCESCONTACT US.

FOLATEBOMBESIN

TRANSFERRIN

Folate is essential for cellular metabolism and DNA synthesis, methylation, and repair. Rapidly proliferating
cancer cells have increased demand for folate to maintain their accelerated DNA synthesis; thus overexpressionof folate receptors (FRs) is a recognized biomarker of many tumors, though tumors can differ in theirdependence on folate metabolism.
The ability to quantify FR expression to distinguish between high and low FR expressing tumors is important
for the efficient delivery of chemotherapeutic agents, drug carriers, photosensitizers, and diagnostic reporters.
IVISense Folate Receptor NIR fluorescent probe enables the non-invasive visualization and quantification of FR
expression on highly metabolic cancer cells.

VIEW IMAGE .

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATIONFOLATEBOMBESIN

FOLATE RECEPTOR (FR) EXPRESSION IN CANCER

TRANSFERRIN

Folate is essential for cellular metabolism and DNA synthesis,
methylation,and repair.onRapidly
Determining
tumor dependence
folate proliferating
metabolism cancancer cells have increased demand for folate to maintaintheir acceleratedDNA
synthesis;
thus overexpression
be importantbecause folic
acid(the synthetic
form of folate)of folate receptors (FRs) is a recognized biomarker of
manythoughtumors ascandiffer intheir targetinghas
beentumors,successfully
exploiteda cancer
specific
dependence on folate metabolism.
moiety for the efficient delivery of various treatments. Thus
the level of FR expression for a given tumor is critical to
The ability to quantify FR expression to distinguish between
highlow
FR expressing
is importantthe successof andthesetreatments,
sincetumors
weak FR-expressing
for the efficient delivery of chemotherapeutic agents,
drug will
carriers,
photosensitizers,
and diagnostictherapies.
cancers
not respondwell to folate-targetedreporters. FolateRSense™, a folate receptor targeted NIR fluorescent imaging agent, enables the non-invasive
visualization and quantification of FR expression onBelow
highlydemonstrates
metabolic cancer
howcells.
IVISense Folate Receptor 680(FR680) probe was used to determine high, medium, andlow FR expression levels in three different cancer cell lines.
VIEW IMAGE .

Hydroxyapatite
KB, HeLa or HT-29 tumor bearing mice were injected withTransferrin
Transferrin

CELL DEATH

FR680 in the absence or presence of 100x excess folic acid andimaged tomographically 4h later.
A. Shown are 4 representative mice per group.
B. Quantification of FR680 signal in tumors. Results show
folate-specific blockade of FR680 accumulation in thetumors, even in the case of low-expressing tumor lines. This is

RESOURCES

attributed to the presence of FR-expressing inflammatory cells.

CONTACT US.

©2019, PerkinElmer, Inc. All rights reserved

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISFOLATEBOMBESIN

TRANSFERRIN

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATH

Depending on the tissue of origin, tumors overexpress metabolic receptors that can be used as
biomarkers of cancer progression. The image above illustrates surface membrane receptors for folate.
The panel on the right illustrates fluorescence for the folate receptor, showing less activity in dead,hypoxic, or poorly vascularized regions, where metabolic activity is reduced.
©2019, PerkinElmer, Inc. All rights reserved

GO BACKRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATIONFOLATEBOMBESIN

TRANSFERRIN

Bombesin is a 14-amino acid peptide originally isolated from the European fire-belly toad (Bombina
bombina). Bombesin-like peptides and bombesin receptors (BBRs) have been the subject of investigation
for the past two decades because of their involvement in cancer cell energy metabolism and proliferation.
Mammalian bombesin analogs, such as gastrin-releasing peptide (GRP), can promote cell growth, malignant
transformation, and tumor differentiation, such that many types of human cancer, including prostate, breast,
lung, CNS, gastric, colon, and renal, show upregulated expression of BBRs on the surface of tumor cells.*
As potential targets for drug delivery, further exploration of these receptors is warranted. IVISense Bombesin
Receptor NIR fluorescent imaging probe, a novel NIR fluorescent imaging agent, is comprised of a 7-aminoacid bombesin peptide analog, an NIR fluorophore, and a pharmacokinetic modifier to improve its plasma
availability, and is an ideal probe to target and quantify upregulation of bombesin receptors in vivo
associated with tumor proliferation.

Hydroxyapatite
VIEW IMAGE .
Transferrin
Transferrin

CELL DEATH*Tseng et al. Fluorescence imaging of bombesin and transferrin receptor expression is comparable to 18F-FDG PET inearly detection of sorafenib-induced changes in tumor metabolism. PLoS ONE 12(8): 2017. e0182689.

RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISFOLATEBOMBESIN

TRANSFERRIN

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATH

Depending on the tissue of origin, tumors overexpress metabolic receptors that can be used as
biomarkers of cancer progression. The image above illustrates surface membrane receptors forbombesin. The panel on the right illustrates fluorescence for the bombesin receptor, showing less activity
in dead, hypoxic, or poorly vascularized regions, where metabolic activity is reduced.
©2019, PerkinElmer, Inc. All rights reserved

GO BACKRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

FOLATE

Transferrin receptors can serve as a useful biomarker for imaging changes in tumor metabolism and biology
prior to overt physical changes, thus making it possible to evaluate potential cancer drugs earlier. IVISense
Transferrin Receptor fluorescent probe is compromised of PEGylate recombinant transferrin and a NIR
fluorescent reporter, is specific for cell surface transferrin receptors and can be used to detect early effects ofdrugs designed to inhibit tumor metabolism.

VIEW IMAGE .
Hydroxyapatite

Transferrin
Transferrin

CELL DEATHRESOURCESCONTACT US.

TRANSFERRINTransferrin is an important serum protein that mediates iron transport into cells via the transferrin receptor
in the cell membrane. This function is essential for cell growth and metabolism. Increased transferrin receptor
levels are associated with an altered metabolic need for iron, such as in many cancers where increased ironlevels are required to support the demands of tumor growth.

Elastase
INFLAMMATION

BOMBESIN

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATIONFOLATERECEPTOR EXPRESSION
BOMBESIN
TRANSFERRININ CANCER

Increased iron metabolism is linked to tumor progression as well as drug resistance and immune evasion.
Treatment
methods
may involve
decreasingiron
stores,a methodcurrently
being
studied,orTransferrin
is an important
serum
protein intracellularthat mediates
irontransport
into cells
via thetransferrin
receptoralternatively,
iron overload,
is toxic
to cells forandcellcangrowthcause aandformof regulated
cell deathin cancerreceptor
in the cell membrane.
Thiswhichfunction
is essential
metabolism.
Increasedtransferrin
growths
called‘ferroptosis’*.
levels areassociatedwith an altered metabolic need for iron, such as in many cancers where increased ironlevels are required to support the demands of tumor growth.
The table below from Brown et al, shows two types of transferrin receptors identified and their relevance incancer.
Iron transport
proteins
as wellas transferrin-based
like
IVISense
ReceptorbindtoTransferrin
receptors
can serve
as a usefulbiomarker for probes
imagingchangesin Transferrin
tumor metabolism
andbiologybothtransferrin
receptorschanges,
TfR1 and
TfR2.
prior
to overt physicalthusmaking it possible to evaluate potential cancer drugs earlier. TransferrinVivo™, a NIR fluorescent imaging agent compromised of PEGylate recombinant transferrin and a NIR
fluorescent reporter,is specificsurface transferrin
be used toearly effects ofExpressionof forIroncellMetabolism
Relatedreceptors
Proteins andandcanRelevance
to detect
Cancer*drugs designed to inhibit tumor metabolism.

Protein

VIEW IMAGE .
Hydroxyapatite

Transferrin receptor 1(TfR1)

Transferrin
Transferrin

CELL DEATH

Transferrin receptor 2(TfR2)

Sample typeRelevance in cancer

Cell lines

Overexpressed in breast, colon, prostate, leukemia, andesophageal cancer cells.

Tissue samples

Elevated in esophageal, colon, and lung tumors vs. normal tissues.
Expression was elevated with increasing stage of liver cancer andcorrelated with poor prognosis of glioma and breast cancers.

Serum

Higher in prostate cancer patients than healthy controls.

Cell lines

Upregulated in ovarian, colon, and glioblastoma cancer cell lines.

Tissue samples

Expression correlated with high tumor grade, but inversely
correlated with prognosis of glioblastoma and leukemia. Expressedin a proportion (~26%) of colon cancers.

RESOURCES*Brown et al. Altered Iron Metabolism and Impact in Cancer Biology, Metastasis, and Immunology.
Front Oncol. 2020;10:476.

CONTACT US.

TRANSFERRINTUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISFOLATEBOMBESIN

TRANSFERRIN

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATH

Depending on the tissue of origin, tumors overexpress metabolic receptors that can be used as biomarkers ofcancer progression. The image above illustrates surface membrane receptors for Transferrin. The panel on
the right illustrates fluorescence for the transferrin receptor, showing less activity in dead, hypoxic, or poorly
vascularized regions, where metabolic activity is reduced.
©2019, PerkinElmer, Inc. All rights reserved

GO BACKRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE

FOLATE RECEPTOR PROBE UPTAKEAND THE EFFECTS OF INFLAMMATORY CELLS
Folate metabolism occurs in inflammatory cells as well, and folate receptors (FRs) are overexpressed on
activated (but not resting or quiescent) macrophages.1 In drug efficacy studies, where tumor cells may bedamaged by the effects of a drug, inflammation will increase in the affected area as a phagocytic response
to dead and dying cells. As tumor folate uptake decreases, folate uptake by inflammatory cells in the area
may increase, leading to either no change in total FR probe uptake or an increase overall upon treatment.
As such, it is important to have a full understanding of the biology of a tumor, as a very inflamed tumor
may exhibit paradoxical FR imaging results.

Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATHRESOURCESCONTACT US

1. Low PS, Henne WA, Doorneweerd DD. Discovery and Development of Folic-Acid-Based Receptor Targetingfor Imaging and Therapy of Cancer and Inflammatory Diseases. Acc Chem Res. 2008; 41:120-129.

.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATIONBEYOND GLUCOSE: OTHER INDICATORS OF ALTERED TUMOR METABOLISM
Called the “Warburg Effect”, glycolysis under aerobic conditions is an important method by which tumors
alter their metabolic pathways to support their growth and proliferation.
Although glucose uptake is often the focus when interrogating tumor metabolism, other biomarkers areaffected by changes in tumor metabolism and can be exploited as indicators of altered cellular metabolism.
Many are fairly ubiquitous biomarkers that are relevant in most if not all cancers. Upregulated to glean
nutrients from their environment and provide for their increased metabolic needs, these surface receptors
can serve as highly useful biomarkers with remarkable capability as early indicators of tumor
treatment efficacy.

FLUORESCENCE IMAGING IS COMPARABLE TO 18F-FDG PET IN EARLY
DETECTION OF CHANGES IN TUMOR METABOLISM

Hydroxyapatite

Transferrin
Transferrin

CELL DEATH

In this case study, researchers tested a multiplex optical imaging strategy in a tumor xenograft model todemonstrate how metabolic fluorescence imaging can have remarkable agreement with PET imaging,showing it’s potential in preclinical application as an additional method for detecting drug-induced
metabolic changes in tumors.

READ MORE .
RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase

Sorafenib treatment of HCT116-luc2 tumors was imaged with bioluminescence, fluorescence, and
18
F-FDG PET. Fluorescence imaging used two probes: IVISense Bombesin Receptor 680 (BombesinRSense
680) and IVISense Transferrin Receptor 750 (Transferrin-Vivo 750). Results show the capability of the two
fluorescent probes to detect metabolic changes in tumors in the first 3 days after treatment, prior to
overt changes in tumor volume, while no overt changes were detected in bioluminescence in the same
period. Fluorescent probe detection of early tumor metabolic changes was corroborated by 18F-FDG PET,
showing that biomarkers can be used as predictive efficacy markers in fluorescence imaging to indicate
pharmacologic response shortly after treatment, and prior to surrogate or clinical endpoints.
This study demonstrated the synergistic use of multimodal imaging to depict a comprehensive pictureof the tumor response to drug treatment. More importantly, results indicate the ability of fluorescentimaging probes to detect pharmacologic response prior to surrogate or clinical endpoints. Fluorescentprobe detection of metabolic disturbances that occurred within 2-3 days was corroborated by 18F-FDG
PET, the gold standard in metabolic imaging.

INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATHRESOURCESCONTACT US.

GO BACKTUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATH

Sorafenib
treatment SUBTLE
of HCT116-luc2
tumors was imaged with bioluminescence, fluorescence, andMONITORING
TUMOR
18F-FDG
PET. Fluorescenceimaging
two probes: BombesinRSense™ 680 and Transferrin-Vivo™ 750.
METABOLICCHANGES
INusedRESPONSE
Results show the capability of the two fluorescent probes to detect metabolic changes in tumors in
TO LOW DOSE SORAFENIB
the first 3 days after treatment, prior to overt changes in tumor volume, while no overt changes weredetectedbioluminescencein the same period. Fluorescent probe detection of early tumor metabolic
Metabolicinchanges
in HCT116-luc2changes
was corroboratedby IVISense
18F-FDG PET, showing that biomarkers can be used as predictive efficacy
tumors werevisualized using
markers
inReceptor
fluorescenceimaging
to indicate pharmacologic response shortly after treatment, and prior to
Bombesin
680 and
IVISense
surrogate
clinical endpoints.
TransferrinorReceptor
750.
This
study demonstrated
thearesynergistic
A: Representative
mouse images
shown use of multimodal imaging to depict a comprehensive picture
for the2D fluorescenceimaging
IVISenseoftumor response
toofdrug
treatment. More importantly, results indicate the ability of fluorescent
Bombesin probes
Receptorto
680(BombesinRSense
680) response prior to surrogate or clinical endpoints. Fluorescentimaging
detectpharmacologic
tumor uptake, with quantitation of tumor
probe
detection of metabolic disturbances that occurred within 2-3 days was corroborated by 18F-FDG
fluorescent signal graphed below. Tumor
PET, the gold standard in metabolic imaging.
regions of interest (ROI) are represented bysolid line circles, and background control ROI
are represented by dotted line circles.

GO BACK

B: Representative mice are shown for IVISense
Transferrin Receptor 750 (Transferrin-Vivo 750)
uptake in tumors, with quantitation of tumor
fluorescent signal graphed below.

DIAGRAM 1

RESOURCESCONTACT US

DIAGRAM 2©2019, PerkinElmer, Inc. All rights reserved.

Peterson, J. et al. Fluorescence imaging of bombesin and transferrin receptor expression is comparable to 18F-FDG PET in early
detection of sorafenib-induced changes in tumor metabolism. PLoS One. 2017; 12(8): e0182689.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATH

Sorafenib
treatmentof HCT116-luc2 tumors was imaged with bioluminescence, fluorescence, and
SUMMARYANALYSIS
18F-FDG
PET. Fluorescenceimaging used two probes: BombesinRSense™ 680 and Transferrin-Vivo™ 750.
OF TUMOR
VOLUME:
Results show the capability of the two fluorescent probes to detect metabolic changes in tumors in
BIOLUMINESCENCE,
the first 3 days after treatment, prior to overt changes in tumor volume, while no overt changes were
FLUORESCENCE, AND
detected in bioluminescence in the same period. Fluorescent probe detection of early tumor metabolic
PET IMAGINGRESULTSchanges
was corroborated
by 18F-FDG PET, showing that biomarkers can be used as predictive efficacy
markers
in fluorescenceimaging to indicate pharmacologic response shortly after treatment, and prior to
NORMALIZEDFOR
surrogate
or clinical endpoints.
COMPARISON
This
the synergistic use of multimodal imaging to depict a comprehensive picture
A: study
Tumordemonstrated
volumeof the tumor response to drug treatment. More importantly, results indicate the ability of fluorescentB:
BLI signal
imagingprobes to detect pharmacologic response prior to surrogate or clinical endpoints. Fluorescentprobe detection of metabolic disturbances that occurred within 2-3 days was corroborated by 18F-FDGC:
PET signal
PET, the gold standard in metabolic imaging.
D, E: IVISense Bombesin Receptor 680 and
IVISense Transferrin Receptor 750
fluorescenceimaging signals respectively.
GO
BACK
Tumor metabolic changes due topharmacologic response weredetected by fluorescent probe by
day 3, corroborated by 18F-FDG PETDIAGRAM 1

RESOURCESCONTACT US

DIAGRAM 2©2019, PerkinElmer, Inc. All rights reserved.

Peterson, J. et al. Fluorescence imaging of bombesin and transferrin receptor expression is comparable to 18F-FDG PET in early
detection of sorafenib-induced changes in tumor metabolism. PLoS One. 2017; 12(8): e0182689.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISBY CHARACTERIZATIONIVISense Probe

BIOPROFILING
Mouse Dose(25 g)

Sold As(Unit Size)

Optimal Imaging
Time (h)Detection of folate receptor
protein upregulation formonitoring tumor growth andmetabolism.

2 nmol24 nmol

6 h (6-24)

Bombesinreceptor

Detects upregulation ofbombesin receptors on
tumor cells associated withincreased tumor
proliferation.

2 nmol24 nmol

24 hTransferrin
receptor

Detects upregulation oftransferrin receptors on
tumor cells associated withincreased tumor
metabolism.

2 nmol24 nmol

24 h (6-24)

MW (g/mol)

Specificity

In Vivo Biology Use± Folate Receptor 680

1,606

Folate receptor± Bombesin Receptor 680

24,000± Transferrin Receptor 750

106,000

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

Transferrin
Transferrin

CELL DEATHRESOURCESCONTACT US.

± Targeted Probes actively target and bind to distinct biomarkers with highly specific targeting to key biological mechanisms.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateFolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

BY CHARACTERIZATIONBY CHARACTERIZATIONMETABOLISM AND THE TUMOR MICROENVIRONMENT
Tumors exist within the context of their microenvironment. In addition to tracking biomarkers of metabolism, the ability
to non-invasively measure tumor microenvironment biology can offer a more complete picture of treatment-induced
biological changes, very early on prior to over tumor regression.
PerkinElmer offers a fluorescent Tumor Metabolism Panel consisting of a carefully curated collection of probes to detect
multiple aspects of tumor biology. Including probes at various wavelengths (680 nm and 750 nm), the panel offers
the opportunity for multiplex imaging of appropriate probe combinations to maximize information gained from yourresearch studies.

Elastase

Tumor Metabolism Panel

INFLAMMATION

Set of Fluorescent Probes Bundled by Application

Hydroxyapatite

IVISense Probe
Vascular 680

Transferrin
Transferrin

CELL DEATH.

In Vivo Biology Use
Leaks into sites of edema

Bombesin Receptor 680
Transferrin Receptor 750

106,000 Transferrin receptor

Annexin-V 750

35,000

Folate Receptor 680

RESOURCES

MW
Specificity(g/mol)~70,000 Vascularity

Cleaved by multiple metalloproteinases Matrix Metalloproteasesecreted markers of neutrophils,family proteases
macrophages, and mast cells
Cathepsin family
Images lysosomal marker in a variety of~400,000
proteases
inflammatory cells
Targets
marker on inflammatory cells;
1,606 Folate receptor
metabolic marker on tumor cells
24,000 Bombesin receptor
Targets metabolic marker on tumor cells

MMP 750 FAST
Pan Cathepsin 680

CONTACT US

BIOPROFILING

43,000Targets iron metabolism marker on tumor cells

Phosphatidyl exposure Detects surface phosphatidylserine increaseon cells
on dying cells, Death/apoptosis

Mouse Dose(25g)
2 nmolAmount(Unit Size)
24 nmol

Optimal Imaging
Time (h)
24 h

Probe
Clearance
6-7 d

2 nmol24 nmol

24 h (12-24)

6-7 d

2 nmol20 nmol

24 h (24-48)

6-7 d

2 nmol24 nmol

6 h (6-24)

6-7 d

2 nmol24 nmol

24 h

6-7 d

2 nmol24 nmol

24 (6-24) h

4d

100 uL

1 mL

2h

3d

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
Endothelium

αvβ3 Integrin
DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US.

Angiogenesis describes the formation of new blood vessels from pre-existing vessels to meet the demand for
nutrients in metabolically active tissue. Tumor angiogenesis refers to the process for establishing vasculature
specifically to meet the increased nutrient demands of a growing tumor. Unlike healthy angiogenesis, tumor
vasculature can be impaired due to oncogenic mutations in proteins that normally join the endothelial cells
together to form the lining of blood vessel walls, resulting in increased vascular permeability. Fluids are able
to leak out and pool in surrounding tissues, contributing to tumor metastasis, as well as providing an early
indication of tumor progression.
Distinct vascular changes can also characterize inflammation. In acute inflammation, functional changes suchas vasodilation and increased vascular permeability are prevalent. These short-term changes are induced by theactions of inflammatory mediators, allowing the appropriate cells, cytokines, and other factors to have accessto tissue sites in need of wound healing or protection from infection or other injurious agents. Prolonged
immune response to the continued presence of such agents, or likewise the initiation of tumor development,
which is known to resemble the process of wound healing, can result in chronic inflammation. The “woundlike” aspects of early tumor growth, where proteinases degrade the basal membrane surrounding tumor cells,
allow cancerous cells to come in contact with stroma. Normal wound healing involves such stromal–epithelialinteractions. However, multi-factor, inflammatory-mediated interaction between cancer cells and theirmicroenvironment abnormally alters the composition of the stroma as the tumor develops, causing structuralchanges to the vasculature. This has been recognized as an opportunity for therapeutic intervention bytargeting these effects on the tumor stroma, such as with angiogenesis inhibition therapies.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
Endothelium

αvβ3 Integrin

THE KINETICS OF INFLAMMATIONThe biology of acute edema, with peak neutrophilia and vascular leak occurring very early on, canbest be imaged by a probe that clears quickly from the tissue, with a short tissue half-life and apeak signal at 1-3 hours after injection.
Once these peaks subside, neutrophils are replaced by a macrophage influx as vascular leak begins
to resolve. This phase of inflammation, driven less by edema and more by cellular activity, can best
be imaged using a probe with a long tissue half-life that will accumulate in the leaky areas andhave a peak signal at 24 hours after injection.

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US.

Understanding the kinetics of inflammation and aligning with the characteristic blood and tissue
half-life of the probe, as well as the timing of the injection, are important considerations forproper imaging of inflammation.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESREVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONS

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
Endothelium

αvβ3 Integrin

PROBE
HALF-LIFE& INJECTION TIMINGTHE
KINETICSOF INFLAMMATIONThe biology of acute edema, with peak neutrophilia and vascular leak occurring very early on, can
BLOOD
TISSUE
OPTIMALIMAGINGbest
be imaged by a probe thatclearsquickly fromthe tissue,
with a shorttissuehalf-life and aROUTE OF METABOLISM/
PROBE*AGENT
TYPE
HALF-LIFE HALF-LIFE
TIME AFTER INJECTIONBACKGROUND TISSUE(S)peak signal at 1-3 hours after injection.
24 h(ImagingofOnce these peaks subside, neutrophils are replaced by a macrophage influx
as vascularleak begins
PEGylated large
7h
72 h
vasculature for up to 4
Low liver lungto resolve. This phase of inflammation,
scaffold driven less by edema and more by cellular activity, can best
IVISense Vascular 750
h; tumor accumulation
be imaged using a probe with a long tissue half-life that will accumulate
in theat 24h) leaky areas and
IVISense Vascular 680

have a peak signal at 24 hours after injection.
IVISense Vascular NP 680

DRUG RESISTANCE
Bombesin

ElastaseLong term tissue
accumulation
Understanding
the kinetics of inflammation and aligning with the characteristic blood and tissue
IVISense Vascular NP 750
Nanoparticle

CELL DEATH

Transferrin

RESOURCESCONTACT US.

>100 h24 hhalf-life of the probe, as well as the timing of the injection, are important considerations forAlbumin-binding
properimaging
IVISense
Edema of
680inflammation.
1.5 h5h
3 h (1-3)
Bladdersmall molecule

INFLAMMATION
Hydroxyapatite

20 h

IVISense Tomato Lectin 680

Fluorescently
labeled tomato
lectin

2h

>48 h

6h

Overall vascular
background*Many other probes, such as IVISense Pan Cathepsin, IVISense MMP, IVISense Neutrophil Elastase, or IVISense Integrin Receptor,
are capable of imaging aspects of inflammation by detecting biomarkers other than vascular leak. See the Fluorescent Agents
Reference Table in the References section for a full list of probes and their specificity, routes of metabolism, and more.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
Endothelium

αvβ3 Integrin
DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

ENDOTHELIUM

Endothelium is comprised of a single layer of endothelial cells and forms the lining of blood vessels
throughout the body. Angiogenic vessels that sprout to support tumor growth will often develop
abnormally, with leaks in the endothelial layer due to oncogenic mutations which impair the proteins thatjoin the endothelial cells together. This increased vascular permeability allows fluid to leak out and pool insurrounding tissues.
Many fluorescent vascular imaging agents have no specificity and work by passively circulating in the blood,
such as IVISense Vascular NIR fluorescent probe, which can image vascularity and vascular leak. However,agents can also be designed to image vasculature by targeting the endothelium, binding the glycoproteins
expressed on the luminal surfaces.
Tomato (Lycopersicon esculentum) lectin, a single polypeptide glycoprotein, can be conjugated to a fluor andvalidated to bind the sugar-containing proteins present on the surface of endothelial cells. IVISense Tomato
Lectin 680 NIR fluorescent probe preferentially labels endothelial cells and can be used to non-invasively
assess tumor vasculature in response to an anti-angiogenic drug, or to assess vascular burden as anotherbiological consideration.

VIEW IMAGE .

RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISENDOTHELIUM

Phosphatidylserine

METASTASISEndothelium
Endothelium

αvβ3 Integrin
DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Endothelial cells line the inside of blood vessels and can be targeted using fluorescent probes to image
vasculature and tumor-related angiogenesis. IVISense Tomato Lectin fluorescent activation is represented
in the figure on the right.
©2019, PerkinElmer, Inc. All rights reserved

GO BACKRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
Endothelium

αvβ3 Integrin
DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Antiangiogenic agents have become a promising therapeutic pursuit in cancer therapy. Early in thedevelopment of these agents, it was thought that anti-angiogenesis strategies worked by blocking
tumor angiogenesis, and that this effect could then limit the effectiveness of the agent itself, as wellas other adjunct therapies - a paradoxical remodeling of tumor vasculature.1,2 However, data has sincedemonstrated that anti-angiogenic drug treatments normalize vasculature, allowing for improveddelivery of drugs to the tumor.2
This selective transient pruning of poorly formed vessels leads to a temporary improvement of bloodflow and oxygen delivery1, with vessel structure that yields less vascular leak around the tumor over thecourse of treatment. Additionally, vascular changes can result in improved delivery of chemotherapy
and a corresponding increase in efficacy. These phenomena may be functionally demonstrated using
fluorescent vascular imaging agents, providing powerful biological characterization of tumor biology
and the effects of treatment, including a normalized vascular bed and tumor regression.

Transferrin

1. Rajabi M, Mousa SA. The Role of Angiogenesis in Cancer Treatment. Biomedicines. 2017 Jun 21;5(2):34.
2. Ackermann et al. Adnectin CT-322 inhibits tumor growth and affects microvascular architecture and functionin Colo205 tumor xenografts. Int J Oncol. 2011 Jan;38(1):71-80.

RESOURCESCONTACT USLONGITUDINAL IMAGING OF FUNCTIONAL CHANGES IN TUMORMICROVASCULATURE.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
Endothelium

αvβ3 Integrin
DRUG RESISTANCEIMAGED USING FLUORESCENCE
MOLECULAR TOMOGRAPHYElastase

A: Growth curves of Colo205 tumors on nude micetreated with vehicle (n=12) or CT-322 (n=12).

Hydroxyapatite

Transferrin

B: Tumor vascularity, AngioSense fluorescencequantitation (pmol) on days 1, 7, 14 and 21. Notethe increase in fluorescence on days 7-21 in thevehicles that is paralleled by increases in tumor size.
Non-tumor associated fluorescence was assessed inadjacent background flank areas within each mousescan (contralateral).
C: Tumor Vascularity, IVISense Vascular (AngioSense)
fluorescence images acquired 24 h post injection.
Representative controls vs. CT-322 treated mice.

RESOURCESCONTACT US

In this study by Ackerman, et al., the researchers used IVISense Vascular 680 (AngioSense 680) fluorescent probe
and fluorescence tomography, to establish that the maximal anti-vascular effect of a potent antiangiogenic
drug, CT-322, occurred within 24 h after the first drug administration, and this response mechanistically
preceded the inhibition of tumor progression. The measurement of functional vascular leakiness with avascular probe provided data that could not be easily achieved with histology or vascular corrosion casting.

Bombesin

INFLAMMATIONCELL DEATH

CASE STUDY: REDUCED ACCUMULATION OF VASCULAR IMAGING PROBE
AFTER A SINGLE DOSE OF ANTIANGIOGENIC DRUG SUGGESTS REDUCEDTUMOR VASCULAR PERMEABILITY.

Ackermann et al. Adnectin CT-322 inhibits tumor growth and affects microvascular architectureand function in Colo205 tumor xenografts. Int J Oncol. 2011 Jan;38(1):71-80.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISBY CHARACTERIZATIONBY CHARACTERIZATIONMW

IVISense Probe

Phosphatidylserine Vascular 680
 Vascular 750

METASTASISEndothelium
Endotheliumë Vascular NP 680
ë Vascular NP 750

αvβ3 Integrin
DRUG RESISTANCE
Bombesin Edema 680±Tomato Lectin 680

BY APPLICATION

Sold As(Unit Size)

Optimal Imaging Time (h)2 nmol24 nmol

24 h(Imaging of vasculature for up
to 4 h; tumor accumulation at
24 h)

Imaging of vascularity, perfusion, and vascularpermeability. Long pharmacokinetic profile.

100 uL

100 nmol(0.5 mL)

24 hPassive; non-specificsystemic circulation

Imaging of vascularity, perfusion, and vascularpermeability. Short pharmacokinetic profile.

4 nmol40 nmol

3 h (1-3)

Endothelial
glycoproteins

Vascular burden assessment through binding ofglycoprotein N-actylglucosamines on thesurface of vascular endothelial cells.

2 nmol24 nmol

6hSpecificity

In Vivo Biology Use

Passive; non-specificsystemic circulation

Imaging of vascularity, perfusion, and vascularpermeability.

~4,000,000
Passive; non-specific(20-50 nm
systemic circulation
diameter)~1,540~72,000(g/mol)~70,000

BIOPROFILING
Mouse Dose(25 g)

 Vascular and Physiological Probes are a range of highly fluorescent in vivo imaging molecules that remain highly stable and localized in the anatomy for various periods of time to enable imaging

Elastase
INFLAMMATION

ë Highly fluorescent near infrared nanoparticles specifically designed for in vivo imaging. They remain localized in the vasculature for extended periods and enable imaging of blood vessels andHydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US

of disease physiology, vasculature, vascular permeability and angiogenesis.

.

angiogenesis.

± Targeted Probes actively target and bind to distinct biomarkers, with highly specific targeting to key biological mechanisms.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISBY CHARACTERIZATIONBY APPLICATIONBIOPROFILINGBY CHARACTERIZATION

Each vascular probe can detect vascularity in cancer and inflammation, and may be especially suitable for:
Phosphatidylserine

METASTASISEndothelium
Endothelium

Tumor Imaging - Orthotopic mouse breast cancer
IVISense Vascular probes provide superior signal to background.

Acute Edema Imaging - Mouse model of acute, inflammatory paw edema
αvβ3 Integrin

IVISense Edema 680 provides superior imaging capability.

Intravital Microscopy - Assessment of vessel morphometry

DRUG RESISTANCE
Bombesin

IVISense Vascular NP fluorescent nanoparticles are designed as highly robust agents for this application; Vascular NP shows the ability
to image both tumors and inflammation with long term accumulation at the imaging site (can be a benefit for time course studies).

Elastase

IVISense Probe

Hydroxyapatite

Biomarker specificity

Edema 680

Vascular 680
Vascular 750

Vascular NP 680
Vascular NP 750

Passive; non-specificsystemic circulation

Passive; non-specificsystemic circulation

Passive; non-specificsystemic circulation

Endothelial glycoproteins

+

+++

++

++

Tumor Imaging Time3h

24 - 48 h

24 - 48 h

6-24 h (stable label ofvasculature)

Tumor Washout

48 h

144 h

>192 h>192 hResponse To Anti-Angiogenic Agents

nd

+++

nd

nd

Acute Edema Imaging

+++

+

+

-

Tissue Imaging Time

3h

24 h

24 h

NA

Tissue Washout

48 h

144 h

>192 hNA

Intravital Microscopy

++

+++

+++

+++

5 – 15 min

5 – 60 min

5 min – 3 h

6-24 hINFLAMMATIONCELL DEATH

Transferrin

RESOURCESCONTACT US.

Tumor Imaging

Optimal Imaging TimeTomato Lectin 680

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISBY CHARACTERIZATIONBY APPLICATIONBY CHARACTERIZATION

Phosphatidylserine

METASTASISEndothelium
Endothelium

PerkinElmer offers a fluorescent Vascular Panel consisting of a carefully curated collection of probes to detect multiple
aspects of vascular disease. Including probes at various wavelengths (680 nm and 750 nm), the panel offers theopportunity for multiplex imaging of appropriate probe combinations to maximize information gained from yourresearch studies.

Vascular Panel

αvβ3 Integrin
DRUG RESISTANCE
Bombesin

Set of Fluorescent Probes Bundled by Application
IVISense ProbeMW(g/mol)Specificity

In Vivo Biology Use

Mouse Dose
(25g)

Amount(Unit Size)

Optimal Imaging
Time (h)Probe
Clearance

Vascular 680~70,000

Vascularity

Leaks into sites of edema

2 nmol24 nmol

24 h

6-7 d

43,000Matrix
Metalloproteasefamily proteases

Cleaved by multiple
metalloproteinases - secreted
2 nmolmarkers of neutrophils,
macrophages, and mast cells

24 nmol

24 h (12-24)6-7 dαVβ3 integrin

Detection of integrin αvβ3
expression in tumors andneovasculature.

2 nmol24 nmol

24 h

14 d

Cathepsin B

Selectively cleaved bycathepsin B proteinases
upregulated in tumors andinflammatory cells.

2 nmol24 nmol

6-24 h3dCathepsin family
proteases

Versatile pan-cathepsinactivatable agent

4 nmol

48 nmol

6-24 h6-7 d

Elastase
INFLAMMATION

MMP 750 FAST

Hydroxyapatite

CELL DEATH

Transferrin

Integrin Receptor 680

Cat B 680 FAST

1,432

33,000Pan Cathepsin 750 FAST 22,500

RESOURCESCONTACT US

BIOPROFILING.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

THE HALLMARKS OF METASTASIS1
The leading cause of morbidity and mortality in cancer patients is metastasis; when tumor cells spread from a
primary tumor and establish secondary tumors at distant sites in the body. Tumors form when subpopulationsof cells with genomic instability undergo morphological and biochemical changes, becoming neoplastic. The
cells may maintain their neoplastic state, proliferating abnormally while never progressing to malignancy. The
complex process leading to malignancy and metastasis begins long before a primary tumor is detectable. Someof the neoplastic cells acquire additional traits, including aberrant active cytoskeletal rearrangement, that givethem the ability to move around neighboring cells. Upregulation of a variety of proteases can allow tumor cells
to degrade extracellular matrix, disrupt the basement membrane, and penetrate underlying stroma. This sets
the stage for possible dissemination and growth into other tissues.
Although invasion is intrinsic to metastasis, the ability to penetrate the stroma by itself is not nearly sufficient
to ensure metastasis will occur. Some tumors remain only locally invasive, never spreading to a secondary site.
In other instances, as many as 4 x 106 cells per gram of tumor can disseminate per day, yet the overwhelming
majority of these cells fail to colonize. Though metastatic potential exists on a spectrum and is not binary, none
the less if an invasive cell cannot complete any of the subsequent steps in the metastatic cascade, it will notform a metastasis. To successfully metastasize, in addition to motility and invasion, a cell must further acquire
the abilities to modulate the secondary site or local microenvironments, maintain plasticity as needed, andcolonize secondary tissues, proposed by Welch and Hurst as the four hallmarks of metastasis.

RESOURCESCONTACT US.

1. Welch , Danny R. and Hurst, Douglas R. Defining the Hallmarks of Metastasis. Cancer Res
June 15, 2019 (79) (12) 3011-3027.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US.

DETECTING METASTASES
Tumors must generally reach a certain size (~100-1000 mm3) before becoming biologically and physiologically
active at a detectable level. Though it presents an imaging challenge, detection of small metastatic
tumors can be done and is best accomplished by choosing an imaging probe that aligns with the operant
biology within the tumor. For example, the biological complexity of large tumors/metastases will include
angiogenesis, inflammation, hypoxia, inflammation, and necrosis as well as upregulation of a numberof additional biomarkers. In contrast, very small metastases may show very few of these biologicalabnormalities, so probes for specific tumor markers or early biological abnormalities may be most effective.
Imaging probes should be designed and validated to possess characteristics (clearance rate and route of
metabolism) biologically appropriate for tumor/metastasis imaging and specific for relevant biomarkers toyield the best results. As the tumor grows, it becomes easier to detect, due to size and biological complexity,
increasing the range of possible imaging probes to quantify such biological changes in real time.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESREVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONS

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

CLEARANCEMETASTASESRATES AND ROUTES OF METABOLISM
DETECTING
Tumors must generally reach a certain size (~100-1000 mm3) before becoming biologically andphysiologically active at a detectablelevel. Though BLOOD
it presents an imaging
detectionof small
BIOMARKER
TISSUEchallenge,ROUTE
OF METABOLISM/
IVISENSE PROBE*
HALF-LIFE
HALF-LIFEBACKGROUNDTISSUESmetastatic tumors can be doneSPECIFICITY
and is best accomplishedby choosing
an imaging probethat aligns withthe operant biology within the tumor. For example, the biological complexity of large tumors/metastases
B 680 FAST
willCatincludeangiogenesis, inflammation,andas wellSalivary
as upregulationof KidneysaCathepsin B hypoxia, inflammation,
1 hr
36necrosis
hrGlands > Liver,
Cat B 750 FAST
number of additional biomarkers. In contrast, very small metastases may show very few of these biological
Cat K 680 FAST
Cathepsin
K markers30
36 hr
Liver effective.
abnormalities,
so probes for specific
tumor
ormin
early biologicalabnormalitiesKidneys
may be> most
Pan Cathepsin 750

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

Cathepsin

5 hr

72 hr

Low liver, intestine

Integrin Receptor 645
Integrin Receptor 680
Integrin Receptor 750

αVβ3 Integrin

10 min10 min30 min

48 hr
24 hr
24 hr

Bladder, Kidneys
Kidneys, Intestines
KidneysBombesin Receptor 680

Bombesin receptor

1.5 hr

96 hr

Pancreas, Kidney

Hydroxyapatite

5-10 min5-10 min5-10 min~30 days
7-10 days
7-10 days

Bladder

Imagingprobes750
shouldand validated topossess characteristics(clearance
route of
Pan Cathepsin
FAST be designed
proteases
1 hr
36 hr
Lowrateliver,and
bladder
metabolism) biologically appropriate for tumor/metastasis imaging and specific for relevant biomarkers to
MMP
FAST
Liver > Kidneys
yield
the645best
results. As the tumor
Matrixgrows, it becomes easier to detect, due to size and biological complexity,
MMP 680
5 hr
72 hr
Liver
Metalloproteaseincreasing
the
range
of
possible
imagingprobestoquantify
suchbiologicalchangesin realtime.
Liver> Kidneys
MMP 750 FAST

Osteo 680
Osteo 750
Osteo 800

RESOURCESCONTACT US*IVISense Pan Cathepsin 680 is not included in this table because it is not recommended for imaging small
tumors due to higher background signal in the liver.

.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATIONMMPRESOURCESCONTACT US.

BOMBESIN

HYDROXYAPATITE

The ability to detect active MMPs upregulated and secreted at the site of metastasis can reveal distinctive local
biology as well as assess therapeutic efficacy of specific drugs, sometimes prior to overt biological changes.
MMPSense® NIR fluorescent imaging agents are able to detect a broad range of active MMPs, including
MMP 2, 3, 7, 9, 12, and 13, to track tumor progression or evaluate the potential therapeutic efficacy of drugs
targeting the underlying mechanisms involved.

VIEW IMAGE .
Transferrin

αvβ3 INTEGRIN

Matrix metalloproteases (MMPs) are calcium-dependent, zinc-containing endopeptidases that play a variety ofroles in tissue remodeling by degrading the extracellular matrix. Normally under strict regulation, controlledlevels of matrix degradation are necessary to maintain healthy biological tissues. However, when cell signaling
pathways are co-opted by tumor cells, abnormal activation of MMPs can restructure tissues in ways thatfacilitate cellular dispersal and tumor cell invasion. Tending to localize in tumor margins, MMPs play animportant role in tumor metastasis and correlate with advanced cancer.

Hydroxyapatite
Hydroxyapatite

CELL DEATH

CATHEPSINTUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITE

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

Matrix metalloproteases are active in regions of tissue remodeling, in this case highly expressed andlocalized at the invasive tumor margins. IVISSense MMP fluorescent activation is represented in the
figure on the right.
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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISMMPCATHEPSIN

αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITE

Associated with tumor aggression, cathepsins are a family of mostly lysosomal proteases that are pivotal
components of signaling pathways which can enhance cancer cell growth and inflammation and contribute to
matrix degradation, thereby facilitating tumor cell invasion.

Endothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE

Cathepsin B: Correlates with invasive and metastatic capabilities of many tumors, such as breast cancer
where high expression levels have been linked to highly aggressive tumors; also upregulated in a variety of
inflammatory cells (including eosinophils, neutrophils, and macrophages) capable of exerting either anti- or
pro-cancer effects and contributing to motility and cellular invasion.

Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

Cathepsin K: A lysosomal cysteine protease expressed in osteoclasts, chondrocytes, and synovial fibroblasts;
involved in bone resorption and collagen degradation; high levels of expression are auseful marker for breast cancer and bone metastases.
IVISense Pan Cathepsin fluorescent imaging probes: versatile pan-cathepsin-activated agents.
IVISense Cat B fluorescent imaging probes: selectively cleaved by cathepsin B proteinases.
IVISense Cat K fluorescent imaging probes: selectively cleaved by cathepsin K proteinases.
For specific detection of tumors with associated cathepsin activity, NIR fluorescent imaging probes IVISense
Pan Cathepsin, Cat B, and Cat K enable non-invasive longitudinal imaging of tumor progression via
protease expression.

VIEW IMAGE .

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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

Above: Osteolytic lesions form when tumor cells inbone help drive osteoblast precursors to increasedifferentiation into osteoclasts, causing osteoclastic
bone resorption to exceed osteoblastic
bone formation.
A: Representation of cancer cells and inflammatory
cells as imaged using IVISense Cat B.
B: IVISense Cat K detects cathepsin K as expressedin osteoclasts.
C: IVISense Pan Cathepsin is a versatile imaging
probe that detects cathepsin proteases as expressedby tumor cells, inflammatory cells, and osteoclasts.

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αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITETUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

CATHEPSIN

αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITE

Integrins are cell surface signaling molecules that are essential for regulating communication between
cells and their microenvironment. Key to maintaining the integrity of healthy tissue, integrins are criticallyinvolved in the cells’ ability to adhere to the extracellular matrix. Likewise, they are key components in cellularmigration, and thus are implicated in nearly every step of cancer progression including metastasis. Alteredintegrin expression can support cancer cell migration and invasion, playing an important role in metastaticcolonization and facilitating survival of anchorage-independent circulating tumor cells.1
In particular, αvβ3 integrin is significantly upregulated in activated endothelial cells during neoangiogenesis,
required early on for colonized cells to establish a secondary location. As a distinct biomarker of tumor
progression, αvβ3 integrin expression can be imaged using IVISense Integrin Receptor, an NIR fluorescentimaging probe. IVISense Integrin Receptor probe provides excellent tumor definition with minimal off-target
distribution due to its high affinity (Kd = 4.2 nM) and selectivity 5-20x over RDG peptide-based agents. With
improved circulation half-life and specificity, it enables detection and measurement of αvβ3 integrins in vivo.

VIEW IMAGE .

1. Hamidi, H., Ivaska, J. Every step of the way: integrins in cancer progression and metastasis. Nat Rev Cancer 18, 533–548 (2018).

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MMP.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITE

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

αvβ3 integrin is significantly upregulated in tumor cells and correlates with tumor aggressiveness.
IVISense Integrin Receptor fluorescent imaging of fine tumor definition is represented in the figureon the right.

©2019, PerkinElmer, Inc. All rights reserved

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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

CATHEPSIN

αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITE

Bombesin is a 14-amino acid peptide originally isolated from the European fire-belly toad (Bombina
bombina). Bombesin-like peptides and bombesin receptors (BBRs) have been the subject of investigation
for the past two decades because of their involvement in cancer cell energy metabolism and proliferation.
Mammalian bombesin analogs, such as gastrin-releasing peptide (GRP), can promote cell growth, malignant
transformation, and tumor differentiation, such that many types of human cancer, including prostate, breast,
lung, CNS, gastric, colon, and renal, show upregulated expression of BBRs on the surface of tumor cells.1
As potential targets for drug delivery, further exploration of these receptors is warranted. IVISense Bombesin
Receptor, a novel NIR fluorescent imaging probe, is comprised of a 7-amino acid bombesin peptide analog,
an NIR fluorophore, and a pharmacokinetic modifier to improve its plasma availability. This is an ideal probe
to target and quantify upregulation of bombesin receptors in vivo, associated with tumor proliferation andmetabolic activity, and this marker downregulates rapidly under anti-metabolic treatment regimen, like is seen
in 18F-FDG PET imaging.1

VIEW IMAGE .

1. Tseng et al. Fluorescence imaging of bombesin and transferrin receptor expression is comparable to 18F-FDG PET in early
detection of sorafenib-induced changes in tumor metabolism. PLoS ONE 12(8): 2017. e0182689.

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MMP.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITE

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

Depending on the tissue of origin, tumors overexpress metabolic receptors that can be used as
biomarkers of cancer progression. The image above illustrates surface membrane receptors forbombesin. The panel on the right illustrates fluorescence for the bombesin receptor, showing less activity
in dead, hypoxic, or poorly vascularized regions, where metabolic activity is reduced.
©2019, PerkinElmer, Inc. All rights reserved

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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISCATHEPSIN

αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITE

The inorganic component of bone is mainly comprised of hydroxyapatite (HA), the major mineral product ofosteoblasts for new bone formation. HA levels can serve as a useful biomarker for osteoblast activity, whereabnormal exposure of HA can indicate a disturbance to bone homeostasis, such as in bone metastasis.

Endothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

Bone is one of the most frequent sites for tumor metastasis and is the prevailing site for breast and prostatecancer metastasis. The metastatic site is not random; dispersed cells can only grow in environments that are
favorable for colonization. As such, the bone microenvironment appears to provide an especially conducive
niche that enables the somewhat distinct process of metastasis as it occurs in bone, from what can be a very
long dormancy to reactivation to cellular proliferation.
IVISense Osteo NIR fluorescent bisphosphonate imaging probes bind with high affinity to HA both in vitro andin vivo, using subtherapeutic doses for imaging. IVISense Osteo fluorescent probes have been shown to bind toactive osteoblastic and osteoclastic surfaces of bone1, enabling in vivo detection, measurement, and monitoringof skeletal changes and progression of bone metastasis.

VIEW IMAGE .

1. Kozloff, K.M., Weissleder, R. and Mahmood, U. (2007), Noninvasive Optical Detection of Bone Mineral. J Bone Miner Res,
22: 1208-1216.

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MMP.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

αvβ3 INTEGRINBOMBESIN

HYDROXYAPATITE

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

Osteolytic lesions form when tumor cells in bone help drive osteoblast precursors to increase differentiation into
multinucleated osteoclasts, causing osteoclastic bone resorption to exceed osteoblastic bone formation.
Hydroxyapatite (HA) is exposed during bone resorption and can be a biomarker of osteoclast activity. IVISense
Osteo binds to HA, enabling in vivo detection of skeletal remodeling as shown in the image on the right.
©2019, PerkinElmer, Inc. All rights reserved

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TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Healthy bone is maintained by a strict balance of continuous bone formation and resorption. When this dynamic
equilibrium is upset, an increase or decrease in activity of one cellular player may be better understood by alsocharacterizing changes in its functional counterpart.
Bone cells (i.e. osteoblasts, producing hydroxyapatite for new bone formation, and osteoclasts, secreting cysteine
proteases, including cathepsin K, for bone resorption) are influenced by cancer cells in mainly two ways1: mostcommonly, tumor cells in bone will drive osteoblast precursors to increase differentiation into multinucleatedosteoclasts while also inhibiting osteoblasts. The resulting prevalence of osteoclastic bone resorption over
osteoblastic bone formation leads to osteolytic lesions.
A less common occurrence is when cancer cells stimulate osteoblast precursors to increase differentiation into
osteoblasts, tipping the balance toward more new bone deposition, or osteoblastic lesions. These bulging growths
have abnormal architecture which weakens the structural integrity of the bone despite its increased mass. Althoughosteolytic lesions are more commonly seen than osteoblastic lesions, neither is exclusive and often will occur
together in the same individual with bone metastasis.1

Transferrin

Fluorescence molecular imaging of bone cancer can detect changes in the rates of bone resorption and formation invivo for a more complete characterization of disease progression or therapeutic effectiveness. The ability of a drug
to block the influence of cancer cells by either increasing bone formation or slowing bone resorption (in the caseof osteolytic lesions) can be tracked by quantifying and comparing changes in the levels of exposed hydroxyapatite(HA) to changes in cathepsin K (cat K) expression. When tracked together in the same animal, the effects of a drug
can be more clearly defined with a deeper understanding of the mechanisms at play.

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QUANTIFYING AND COMPARING COMPLIMENTARY BIOMARKERS AS
INDICATORS OF TREATMENT RESPONSE.

1. Ottewell PD. The role of osteoblasts in bone metastasis. J Bone Oncol. 2016;5(3):124-127.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE

CASE STUDY: MULTIMODALITY IMAGING OF TUMOR AND BONE RESPONSE IN A
MOUSE MODEL OF BONY METASTASIS1
Recent research focusing on targeted agents that disrupt specific intricately-involved signaling pathways in cancer
presents both possibilities and challenges. The highly complex effects of these treatments involve numerous
mechanisms, making them difficult to characterize and sometimes eliciting a treatment response with no drastic
morphologic changes. In this case study, the authors explored imaging methods that possess the sensitivity andspecificity needed to measure the affects of these targeted agents. By using fluorescence molecular imaging in
conjunction with other imaging methods, the authors were able to quantify the response to two drugs in a mousemodel of osteolytic bony metastasis; docetaxel, a chemotherapy drug used to kill cancer cells by halting cell division,and zoledronic acid (ZA), a bisphosphonate used to slow the effects of osteoclasts on bone.

Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

The results showed significant bone remodeling changes at weeks 2 and 4 in the docetaxel group, and no
significant change detected in the ZA or control group. For the IVISense Cat K signal, positive change indicatedbone loss, while negative change indicated bone growth. The IVISense Osteo signal was interpreted to mean the
inverse. The comparison of the IVISense Osteo signal to the Cat K signal provided the context for a very elegant
approach to delineating drug effects.
Using multiple imaging modalities and cleverly pairing IVISense Cat K and IVISense Osteo probes to understand thecomplex interplay of osteolytic and osteoblastic activity, the authors were able to characterize changes occurring
under different treatment regimens.

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Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored using a multimodal approach that
included functional assessment by fluorescence imaging. After treatment with either docetaxel and ZA, each animal
was imaged with two probes. IVISense Osteo 800 fluorescent probe was used to image the amount of exposedhydroxyapatite (HA) resulting from both osteoblastic and osteoclastic activity, and IVISense Cat K 680 FAST was used
to image cathepsin K expressed by osteoclasts.

.

1. Hoff BA, Chughtai K, Jeon YH, et al. Multimodality imaging of tumor and bone response in a mouse modelof bony metastasis. Transl Oncol. 2012;5(6):415-421.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCES

Figure 1: Bar plots of fluorescent signals in thetumor-bearing leg from (A) IVISense Osteo 800(OsteoSense 800) and (B) IVISense Cat K 680
FAST are presented as values normalized by the
non-tumor bearing leg. The ZA group showedno significant difference from controls witheither fluorescent probe, but the docetaxelgroup showed significant increases in IVISense
Osteo 800 uptake (A, black bar) on weeks 2 and
4 and a significant drop in IVISense Cat K 680
FAST signal (B, black bar) on week 4. Asterisk
indicates a significant difference from thecontrol group (P < .05).

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Figure 2: Assessment of osteoblastic activity, bone integrity, and tumor burden
by multi-modality imaging. (A) Fluorescence image using OsteoSense 800 clearly
identifies the right leg (magenta circle) as having increased osteoblastic activity
relative to the left (control) leg. (B) The extend of bone damage as a result of thetumor is clearly observed in the microCT surface rendering of the right tibia (Right)
with metastatic cancer. The healthy tibia (Left) shows no appreciable loss in bonedensity. (C) Finally, the location (magenta contour) and extent of tumor burden
can be assessed by magnetic resonance imaging (MRI) of a tumor (~10 μL) in theright tibia. The MRI data is presented as individual slices going from the foot (topleft) to the mid-region of the femur (bottom-right). The location of the knee isindicated by the yellow asterisk. All of these images were acquired in the same
animals approximately 25 days post-implantation. Image courtesy of Craig Galban,
PhD, University of Michigan.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

BY CHARACTERIZATIONIVISense
IVISense
Probe
Probe

INFLAMMATION
Hydroxyapatite
Hydroxyapatite

CELL DEATH

Transferrin

8,500
8,500 Cathepsin
Cathepsin
K K

Selectively
cleavedby cathepsin
Selectively
cleavedby cathepsin
K K
proteinases
proteinases
upregulated
upregulated
in osteoclasts
in osteoclasts
tumor-associatedmacrophages.
macrophages.
andandtumor-associated

23,00023,000

24 24
nmol
nmol

6-24
6-24
h h

4 nmol
4 nmol

48 48
nmol
nmol

6-24
6-24
h h

2 nmol2 nmol

24 24
nmol
nmol

6-24
6-24
h h

2 nmol2 nmol

20 20
nmol
nmol

24 24
h (24-48)
h (24-48)

2 nmol2 nmol

24 24
nmol
nmol

24 24
h h

4 nmol
4 nmol

48 48
nmol
nmol

6-24
6-24
h h

Cleavedby multiple
metalloproteinases
Cleavedby multiple
metalloproteinases
MatrixMatrix~450,000~450,000
associatedmanydiseaseareas
associatedwithwithmanydiseaseareas
Metalloprotease
Metalloproteaseincludingcancerandinflammation.
includingcancerandinflammation.
43,00043,0004 nmol
4 nmol

48 48
nmol
nmol

24 24
h (6-24)h (6-24)2 nmol2 nmol

20 20
nmol
nmol

24 24
h (24-36)
h (24-36)

2 nmol2 nmol

24 24
nmol
nmol

24 24
h (12-24)
h (12-24)

1,250
1,250

2 nmol2 nmol

24 24
nmol
nmol

6-24
6-24
h h

2 nmol2 nmol

24 24
nmol
nmol

24 24
h h

2 nmol2 nmol

24 24
nmol
nmol

24 24
h h

2 nmol2 nmol

24 24
nmol
nmol

24 24
h h

2 nmol2 nmol

24 24
nmol
nmol

3-24
3-24
h h

4 nmol
4 nmol

48 48
nmol
nmol

3-24
3-24
h h

2 nmol2 nmol

24 24
nmol
nmol

3-24
3-24
h h

~400,000~400,000
Cathepsin##
PanPanCathepsin
Cathepsin
750750~450,000~450,000Cathepsin
proteases
proteases
22,500##PanPanCathepsin
Cathepsin
750750FAST
FAST 22,500

##
MMP
MMP
750750
FAST
FAST

±±
Integrin
Integrin
Receptor
Receptor
645645±±Integrin
Integrin
Receptor
Receptor
680680±±Integrin
Integrin
Receptor
Receptor
750750±±Bombesin
Bombesin
Receptor
Receptor
680680

Mouse
Mouse
Dose
Dose Sold
Sold
AsAs Optimal
OptimalImaging
Imaging(25(25
g) g)
(Unit
(Unit
Size)
Size)
Time
Time
(h)(h)
2 nmol2 nmol##PanPanCathepsin
Cathepsin
680680

±±
Osteo
Osteo
680680
±±
Osteo
Osteo
750750
±±
Osteo
Osteo
800800Pan-cathepsin
Pan-cathepsin
activatable
activatable
agent
agent
thatthatdetects
detects
abnormalabnormalupregulation
upregulation
of ofcathepsin
secretion
associatedwithwithcathepsin
secretion
associatedcancerinflammation.
cancerandandinflammation.

43,00043,000Detection
Detectionof integrinof integrin
αVβ3
αVβ3
expression
expression
in in
1,432
1,432 αVβ3
αVβ3
Integrin
Integrintumorsneovasculature.
neovasculature.
tumorsandand1,278
1,278
Bombesin
Bombesin
24,00024,000receptor
receptor
1,471
1,471

Detects
upregulationof bombesin
Detects
upregulationof bombesin
receptors
tumor
cells
associatedreceptors
on on
tumor
cells
associatedwithwith
increased
tumor
proliferation.
increased
tumor
proliferation.

Binds
exposedhydroxyapatite
(HA),
Binds
exposedhydroxyapatite
(HA),
a a
biomarker
biomarker
for forosteoblast
osteoblast
activity;
activity;
abnormalabnormal
1,101
1,101 Hydroxyapatite
Hydroxyapatite
HAHA
levelscancanindicatebonelevelsindicatebone
damage/disease,suchasmetastasis.
in metastasis.
damage/disease,
such
as in
1,281
1,281

##
Activatable
Activatableprobesprobesare areoptically
optically
silentsilentupon
upon
injection
injection
andandbecomebecomehighlyhighlyfluorescentfluorescent
following
followingprotease-mediatedprotease-mediatedactivation.
activation.
Activatable
Activatable
FAST
FAST
agentsagentsare aredesigneddesigned
withwith
a novela novelsmall
small
molecule
moleculearchitecturearchitecture
thatthatconfers
confers
an accelerated
an accelerated
pharmacokinetic
pharmacokinetic
profileprofilewithwithearlierearlier
imaging
imaging
timetimepoints.
points.

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Cathepsin
Cathepsin
B B##
CatCat
B 750
B 750
FAST
FAST

##
MMP
MMP
645645FAST
FAST

In Vivo
Biology
Use
In Vivo
Biology
Use
Selectively
cleavedby cathepsin
B B
Selectively
cleavedby cathepsin
proteinases
proteinases
upregulated
upregulated
in tumorsin tumorsandandinflammatory
cells
inflammatory
cells

33,00033,000##MMP
MMP
680680

Elastase

MW
MW
Specificity
Specificity(g/mol)(g/mol)##
CatCat
B 680
B 680
FAST
FAST

##
CatCat
K 680
K 680
FAST
FAST

BY APPLICATION±Targeted±Targetedprobesprobesactively
activelytargettargetandandbindbind
to distinct
to distinct
biomarkers
biomarkers
withwithhighlyhighlyspecific
specific
targetingtargetingto keyto keybiologicalbiologicalmechanisms.
mechanisms.
.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin
Integrin

DRUG RESISTANCE
Bombesin
Bombesin

Elastase
INFLAMMATIONBY CHARACTERIZATIONBY APPLICATIONIVISense
Probe
Cat B
Cat K
BY CHARACTERIZATION
680 FAST
750 FAST

Available Versions

Pan Cathepsin

680

MMP

750 FAST
750

680

Integrin Receptor(αVβ3)

Bombesin
Receptor

Osteo(Hydroxyapatite)

645
680
750

680

680
750
800

645 FAST
750 FAST

680

Applications
Early Tumor
Assessment

+++

+

+++

+++

+++

+++

++++

+++

+

Metastasis
Imaging++Boneresorption:+++++/- due to liverinterference+++/- due to liverinterference

++

++

+++

Bone growth orresorption:
++++

Tumor
Microenvironment

-

+++

-

-

-

-

-

-

+++

Image Tumor
Vascularity

-

-

-

-

-

-

+++

-

-

Assessing
Treatment Efficacy

+++

+++

+++

+++

+++

+++

+++

++++

+++

Probe Characteristics

CELL DEATH

Hydroxyapatite
Hydroxyapatite

Imaging Time Post
Probe Injection

6-24 hr

24 hr (24-36)

24 hr (24-48)

6-24 hr
24 hr (24-48)

24 hr (24-36)

24 hr (6-24)
24 hr (12-24)

6-24 hr
24 hr
24 hr

24 hr

3-24 hrTransferrin

Tumor Washout

3 days

6-7 days

6-7 days

3 days
6-7 days

6-7 days

6-7 days

6-7 days
14 days
4-6 days

6-7 days

Preimage subtraction

Summary of probes and applications. An overview of results, including practical information regarding optimal imaging/washout time and response to treatments (from internal and external,
published and unpublished, research). Performance is based on a subjective scale (+, ++, +++, ++++) that takes into account the signal intensity, kinetics, background, washout, and generalutility.

RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Therapeutic resistance is a complex process that can arise from multiple origins. It occurs in all cancer types and in all
therapy methods, including chemotherapy, immuno-therapy, and novel targeted drug therapy.1 Broadly, tumor cells canacquire drug resistance as an adaptive response during treatment or it can be intrinsic to the cells, existing prior to treatment and manifesting in ways that allow some cells to survive and proliferate while others die.
Tumor microenvironment plays a critical role in the development of therapeutic resistance and in malignant progression.
Most notably, tumor hypoxia and acidosis have been identified as conditions that profoundly influence cancer cellbiology and lead to inhibition of the therapeutic response. Hypoxia is a pathological condition where tissue is deprivedof adequate oxygen. In normal cells, hypoxia typically leads to cell death. However, in cancer cells, hypoxic conditions
can directly or indirectly enable some cells to survive and proliferate. Hypoxia can induce epigenomic and genomicchanges which, among other adaptations, can enable cells to survive the poor nutrient supply of the harsh tumor
microenvironment by altering the tumor metabolism. This can function as a mechanism of drug resistance, allowing cells
to evade drugs that preferentially target rapidly dividing cells. Under selective pressure from therapy, surviving cells can
go on to form subpopulations of resistant cells that can lead to clinical relapse.
Acidosis of the tumor microenvironment is an important epigenomic change that occurs in response to hypoxia, and thisis regulated by the expression of carbonic anhydrase IX (CA IX). Rarely expressed in normal human tissue, CA IX is highly
over-expressed in many cancer types in response to low oxygen, which is driven by HIF-1α, one of a family of transcription
factors called hypoxia-inducible factors (HIFs).2 Although this makes CA IX an excellent biomarker for hypoxia, the mainrole of CA IX is in promoting an acidic extracellular environment, and acidosis of the microenvironment that facilitatestumor cell growth, invasion, survival, and mechanisms of drug resistance. Research using CA IX inhibitors is helping
to better define and establish the importance of CA IX in metastasis, poor prognosis, and resistance to therapeuticinterventions, making it a highly relevant biomarker in the study of hypoxia and acidosis in cancer therapy.3
1. Wang X, Zhang H, Chen X. Drug resistance and combating drug resistance in cancer. Cancer Drug
Resist 2019;2:141-160.

RESOURCESCONTACT US

2. Al Tameemi Wafaa, Dale Tina P., Al-Jumaily Rakad M. Kh, Forsyth Nicholas R. Hypoxia-modified cancer
cell metabolism. Frontiers in Cell and Developmental Biology 2019; 7:4.

.

3. Andreucci E, et al. The carbonic anhydrase inhibitor SLC-0111 sensitises cancer cells to conventional
chemotherapy. J Enzyme Inhib Med Chem 2019; 34(1):117-123.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Therapeutic resistance is a complex process that can arise from multiple origins. It occurs in all cancer types and in all
therapymethods,
includingchemotherapy,
immuno-therapy, and novel targeted drug therapy1. Broadly, tumor cells can
ORIGINSOF
THERAPEUTICRESISTANCE
acquire drug resistance as an adaptive response during treatment or it can be intrinsic to the cells, existing prior to treatment and manifesting in ways that allow some cells to survive and proliferate while others die.
Unravelingthe mechanisms
Tumor
microenvironment
plays thata critical role in the development of therapeutic resistance and in malignant progression.
underliedrug
resistance
cancercouldhave been identified as conditions that profoundly influence cancer cell
Most
notably,tumor
hypoxiainand
acidosis
potentiallydisableone of cancer’s
most
biologyand leadto inhibitionof the therapeutic
response. Hypoxia is a pathological condition where tissue is deprived
significantoxygen.
biologicaldefenses.
of adequate
In normal
cells, hypoxia typically leads to cell death. However, in cancer cells, hypoxic conditions
can directly or indirectly enable some cells to survive and proliferate. Hypoxia can induce epigenomic and genomic
This graphic
demonstrates
the many can enable cells to survive the poor nutrient supply of the harsh tumor
changeswhich, among
other adaptations,routes that can byleadtomicroenvironment
altering
the tumor metabolism. This can function as a mechanism of drug resistance, allowing cells
drug resistance,theto evadedrugs that andpreferentiallytarget rapidly dividing cells. Under selective pressure from therapy, surviving cells can
feedbackthat can of resistant cells that can lead to clinical relapse.
go on
to formloops
subpopulations
support it.
Acidosis of the tumor microenvironment is an important epigenomic change that occurs in response to hypoxia, and thisis regulated by the expression of carbonic anhydrase IX (CA IX). Rarely expressed in normal human tissue, CA IX is highly
over-expressed in many cancer types in response to low oxygen, which is driven by HIF-1α, one of a family of transcription
factors called hypoxia-inducible factors (HIFs)2. Although this makes CA IX an excellent biomarker for hypoxia, the mainrole of CA IX is in promoting an acidic extracellular environment, and acidosis of the microenvironment that facilitatestumor cell growth, invasion, survival, and mechanisms of drug resistance. Research using CA IX inhibitors is helping
to better define and establish the importance of CA IX in metastasis, poor prognosis, and resistance to therapeuticinterventions, making it a highly relevant biomarker in the study of hypoxia and acidosis in cancer therapy.3

1. Wang X, Zhang H, Chen X. Drug resistance and combating drug resistance in cancer. Cancer Drug Resist 2019;2:141-160.

RESOURCES

2. Al Tameemi Wafaa, Dale Tina P., Al-Jumaily Rakad M. Kh, Forsyth Nicholas R. Hypoxia-modified cancer cell metabolism.
Frontiers in Cell and Developmental Biology 2019; 7:4.

©2021, PerkinElmer, Inc. All rights reserved

CONTACT US.

3. Andreucci E, et al. The carbonic anhydrase inhibitor SLC-0111 sensitises cancer cells to conventional chemotherapy. J Enzyme
Inhib Med Chem 2019; 34(1):117-123

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESIMAGING CA IX AS A MARKER FOR HYPOXIA IN SMALL TUMORS1While most tumors are expected to have some regions of hypoxia, quantifying increased expression of carbonic
anhydrase IX (CA IX) in relatively small tumors, which are reported to be less hypoxic, may present a challenge.
However, CA IX expression can be measured
in mice in response to forced hypoxia by
maintaining the mice in a low oxygen (8%O2) environment. When subsequently imaged
with the CA IX-specific probe IVISense
Hypoxia CA IX 680 (HypoxiSense 680), thismethod was shown to yield readouts that
correlated with ex vivo images of excised
tumor, confirming that changes in CA IX
expression levels as a result of lower oxygen
levels, even in small tumors, can be quantified
non-invasively by fluorescence imaging.
By imaging ex vivo tissue sections, IVISense
Hypoxia CA IX 680 was shown to be
specifically localized in hypoxic regions of thetumors. These results also indicate the efficacy
of Hypoxia CA IX 680 to pene-trate poorly
vascularized tumor tissue, a critical property
for a non-invasive hypoxia agent.

The effect of breathing 8% O2 on IVISense Hypoxia CA IX 680 (HypoxiSense 680) tumor signal in
HeLa xenografts (Bao, et al).
Left: Two groups of mice (n = 4 per group) were used to assess the effect of oxygen breathinglevels on Hypoxia CA IX 680 labeling of small tumors. Top Left shows representative fluorescence
molecular tomography 3D images of mice breathing normal air (left) and breathing low oxygen(center) showing the intensity differences of Hypoxia CA IX 680 signals within the tumors.
Right: Quantitative analysis of Hypoxia CA IX 680 tumor signals in mice breathing normal or low
oxygen, showing greater tumor Hypoxia CA IX 680 signals in mice breathing low oxygen thanthat of mice breathing normal air.

CONTACT US.

1. Bao B, Groves K, Zhang J, Handy E, Kennedy P, Cuneo G, et al. (2012) In Vivo Imaging and Quantification of
Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia. PLoS ONE 7(11): e50860.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase

CARBONIC ANHYDRASE IX

Carbonic anhydrase IX (CA IX) is a transmembrane cell surface enzyme that catalyzes the reversible
conversion of CO2 into bicarbonate and a proton. Normally expressed in only a few human tissues, CA IX is atumor-associated protein that serves as an important biomarker in the study of both hypoxia and acidosis intumor progression and response to drug therapy.
Expression of CA IX is primarily regulated at the transcriptional level by hypoxia-inducible factor-1α (HIF-1α).1
When expressed, CA IX helps produce and maintain an intracellular pH favorable for cell growth and survival,
while at the same time contributing to an increasingly acidic extracellular space (acidosis). This not only
facilitates tumor progression and invasiveness, but also promotes conditions for cells to develop mechanismsof drug resistance such as epigenomic and genomic changes, metabolic changes, drug inactivation and drug
efflux capabilities, quiescence, and cell death inhibition.

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

VIEW IMAGE .

1. Tafreshi NK, Lloyd MC, Bui MM, Gillies RJ, Morse DL. Carbonic anhydrase IX as an imaging and therapeutic target for tumors
and metastases. Subcell Biochem. 2014;75:221-254.

RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESISCARBONIC ANHYDRASE IX

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Carbonic Anhydrase IX (CA IX) is highly expressed in tumor cells in response to a low oxygen environment, yielding
significant intracellular pH changes and extracellular acidosis to favor cancer survival. IVISense Hypoxia CA IX fluorescentimaging of hypoxic regions is represented on the right.

©2021, PerkinElmer, Inc. All rights reserved

GO BACKRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

CA IX EXPRESSION1
Present in few normal tissues, CA IX expression is mainly limited to the stomach and gallbladder epithelia. It is
upregulated in some but not all cancer cell lines, while very often being highly expressed in spontaneously arising,
generally more aggressive tumors.
Among those known to upregulate CA IX expression are CNS, head & neck, lung, breast, colon, cervical, ovarian,
prostate, renal cancers, leukemia, and melanoma. In addition, a variety of other tumors show increased focal
expression of CA IX, occurring in 90% of cervical, glioblastoma, and basal cell carcinomas, as well as in 25– 30% of
non-invasive and invasive ductal breast cancers.

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US.

1. Bao B, Groves K, Zhang J, Handy E, Kennedy P, Cuneo G, et al. (2012) In Vivo Imaging and Quantification of
Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia. PLoS ONE 7(11): e50860.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

In glioma, hypoxia is considered the major driving force for tumor angiogenesis. Since inhibiting angiogenesis impairs
tumor progression, understanding the molecular players involved in hypoxia-mediated angiogenesis could yieldnovel therapies.
The cellular response to low oxygen concentrations is regulated by hypoxia-inducible factors (HIFs). In hypoxic cancer
cells, the HIF pathway is up-regulated, including HIF-1α, the master gene for cellular response to hypoxia. Under normoxic
conditions, HIF-1α is rapidly degraded. However, under hypoxic conditions, it can escape proteolysis, altering a multitudeof downstream genes to promote cellular survival and proliferation. Among those genes is the long non-coding RNA(lncRNA) H19, in which the microRNA, miR675-5p, is embedded. Another gene expressed downstream from HIF-1α iscarbonic anhydrase IX (CA IX) which has been shown to be a biomarker of HIF-1α activity.
The authors here demonstrate that miR675-5p is a hypoxia-regulated microRNA able, by itself, to mimic a low oxygen
condition in normoxia, and was found to be: i) essential for hypoxia establishment, ii) a stimulator of hypoxia-mediatedangiogenesis and iii) a promoter of glioma progression.
As part of this demonstration, the authors generated a glioma orthotropic murine model using U251-HRE-mCherry cells.
The mice were divided into two groups treated with either an miRNA675-5p mimic or a scramble. The mice were imaged
to detect mCherry fluorescence to indicate hypoxia, and fluorescent probes to quantify two biomarkers; CA IX whichindicates HIF-1α expression, and the neo-angiogenesis biomarker αvβ3 integrin which indicates neoangiogenesis. Using
IVISense Hypoxia CA IX (HypoxiSense) and IVISense Integrin Receptor (IntegriSense) probes respectively, image acquisitions
showed the induction of CA IX expression in mimic mice as well as increased αvβ3 integrin expression. These findings werein agreement with increased hypoxia as indicated by mCherry fluorescence, and an increase in HIF-1 activity and neoangiogenesis stimulation, as shown by immunohistochemistry and CT scan.
Mice were subsequently treated with an inhibitor of miRNA675-5p and imaged in the same way. The data indicate thatthe inhibition of miRNA675-5p in vivo can i) turn off tumor molecular response to hypoxia ii) constrain angiogenesis andiii) block hypoxia-mediated tumor growth, thus suggesting a therapeutic role for miRNA675-5p inhibitor in glioblastoma.
See figures

RESOURCESCONTACT US

CASE STUDY: IMAGING DOWNSTREAM BIOLOGY TO INTERROGATE A NEW
THERAPEUTIC TARGET FOR TREATING GLIOMA1.

1. Lo Dico A, Costa V, Martelli C, Diceglie C, Rajata F, Rizzo A, Mancone C, Tripodi M, Ottobrini L, Alessandro R,
Conigliaro A. MiR675-5p Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma.
Theranostics. 2016 May 8;6(8):1105-18.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESIS

Phosphatidylserine

CASE STUDY: IMAGING DOWNSTREAM BIOLOGY TO INTERROGATE A NEW
THERAPEUTIC TARGET FOR TREATING GLIOMA1
FIGURE 1

FIGURE 2

In glioma, hypoxia is considered the major driving force for tumor angiogenesis. Since inhibiting angiogenesis impairs
tumor progression, understanding the molecular players involved in hypoxia-mediated angiogenesis could yieldnovel therapies.

miR675-5p in vivo is able to enhance

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

The cellular response to low oxygen concentrations is regulated by hypoxia-inducible factors (HIFs). In hypoxic cancer
hypoxia establishment and neocells, the HIF pathway is up-regulated, including HIF-1α, the master gene for cellular response to hypoxia. Under normoxic
angiogenesis.
conditions,
HIF-1α is rapidly degraded. However, under hypoxic conditions, it can escape proteolysis, altering a multitudeof downstream genes to promote cellular survival and proliferation. Among those genes is the long non-coding RNA(C) Left panel: Representative 2D images of(lncRNA)
H19, in which the microRNA, miR675-5p, is embedded. Another gene expressed downstream from HIF-1α ismice co-injected with IVISense Hypoxia CA IX
carbonic anhydrase IX (CA IX) which has been shown to be a biomarker of HIF-1α activity.
680 (HypoxiSense, BlueHot scale) and IVISense
Integrin Receptor 750 (IntegriSense, YellowHot
The authors
here demonstrate that miR675-5p is a hypoxia-regulated microRNA able, by itself, to mimic a low oxygen
scale) and axial images of IVISense Integrin
condition in normoxia, and was found to be: i) essential for hypoxia establishment, ii) a stimulator of hypoxia-mediatedReceptor 750 co-registered with CT scan. Allangiogenesis
and iii) a promoter of glioma progression.
scans were performed at the final time point(day 19).

As part of this demonstration, the authors generated a glioma orthotropic murine model using U251-HRE-mCherrycells. The mice were divided into two groups treated with either an miRNA675-5p mimic or a scramble. The mice were(C) Right panel: Quantification of IVISense
imagedto detect mCherry fluorescence to indicate hypoxia, and fluorescent probes to quantify two biomarkers; CA IX
Hypoxia CA IX 680 and IVISense Integrin
which indicates HIF-1α expression, and the neo-angiogenesis biomarker αvβ3 integrin which indicates neoangiogenesis.
750 fluorescence by ROI analysis for
UsingReceptor
IVISense
Hypoxia CA IX (HypoxiSense) and Integrin Receptor (IntegriSense) probes respectively, image acquisitions
scramble and mimic-treated mice. Data are
showed the induction of CA IX expression in mimic mice as well as increased αvβ3 integrin expression. These findings wereexpressed as average radiance efficiency [(photons/s/cm^2/steradian)/(μW/cm^2)].
in agreementwith increased hypoxia as indicated by mCherry fluorescence, and an increase in HIF-1α activity and neoangiogenesis stimulation, as shown by immunohistochemistry and CT scan.
(D) Left panel: Representative (n=5) 2D mCherry fluorescent signal (rainbow scale) of U251-HRE-mCherry tumors in scramble
and miR675-5p mimic-treated mice at day 19.

Mice were subsequently treated with an inhibitor of miRNA675-5p and imaged in the same way. The data indicate thatthe inhibition of miRNA675-5p in vivo can i) turn off tumor molecular response to hypoxia ii) constrain angiogenesis and(D) Right panel: Graphical representation of mCherry activity at the end of treatment. Data are presented as averageiii) block
hypoxia-mediated tumor growth, thus suggesting a therapeutic role for miRNA675-5p inhibitor in glioblastoma.
radiance efficiency [(photons/s/cm^2/steradian)/(μW/cm^2). Images are presented with the same scale bar. Mimic-treated mice
See figures

RESOURCESCONTACT US

vs scramble- treated mice * p<0.05; **p<0.01; ***p<0.001.

.

1. Lo Dico A, Costa V, Martelli C, Diceglie C, Rajata F, Rizzo A, Mancone C, Tripodi M, Ottobrini L, Alessandro R, Conigliaro A. MiR675-5p
Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma. Theranostics. 2016 May 8;6(8):1105-18.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESIS

Phosphatidylserine

CASE STUDY: IMAGING DOWNSTREAM BIOLOGY TO INTERROGATE A NEW
THERAPEUTIC TARGET FOR TREATING GLIOMA1
FIGURE 1

FIGURE 2

In glioma, hypoxia is considered the major driving force for tumor angiogenesis. Since inhibiting angiogenesis impairs
tumor progression, understanding the molecular players involved in hypoxia-mediated angiogenesis could yieldnovel therapies.

miR675-5p inhibitor counteracts

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

The cellular response to low oxygen concentrations is regulated by hypoxia-inducible factors (HIFs). In hypoxic cancer
hypoxia-mediated angiogenesis in vivo.
cells, the HIF pathway is up-regulated, including HIF-1α, the master gene for cellular response to hypoxia. Under normoxic
conditions, HIF-1α is rapidly degraded. However, under hypoxic conditions, it can escape proteolysis, altering a multitude(A) Left Panel: Representative (n=5) 2D imagesof downstream genes to promote cellular survival and proliferation. Among those genes is the long non-coding RNAof scramble or miR675-5p inhibitor-treated mice(lncRNA) H19, in which the microRNA, miR675-5p, is embedded. Another gene expressed downstream from HIF-1α is
co-injected with IVISense Hypoxia CA IX 680
carbonic anhydrase IX (CA IX) which has been shown to be a biomarker of HIF-1α activity.
(HypoxiSense, BlueHot scale) and IVISense Integrin
Receptor 750 (IntegriSense, YellowHot scale) and
The authors here demonstrate that miR675-5p is a hypoxia-regulated microRNA able, by itself, to mimic a low oxygen
axial images of IVISense Integrin Receptor 750 cocondition in normoxia, and was found to be: i) essential for hypoxia establishment, ii) a stimulator of hypoxia-mediatedregistered with CT scan. All scans were performed
angiogenesis and iii) a promoter of glioma progression.
at the final time point (day 28).

As part of this demonstration, the authors generated a glioma orthotropic murine model using U251-HRE-mCherry(A) Right panel: Quantification of IVISense Hypoxia
cells. The mice were divided into two groups treated with either an miRNA675-5p mimic or a scramble. The mice were
CA IX 680 and IVISense Integrin Receptor 750
imaged to detect mCherry fluorescence to indicate hypoxia, and fluorescent probes to quantify two biomarkers; CA IX
fluorescence by ROI analysis for scramble andwhich indicates HIF-1α expression, and the neo-angiogenesis biomarker αvβ3 integrin which indicates neoangiogenesis.
miR675-5p inhibitor-treated mice (considering
Using IVISense Hypoxia CA IX (HypoxiSense) and Integrin Receptor (IntegriSense) probes respectively, image acquisitions
five mice for each treatment). Data are expressed
showed the induction of CA IX expression in mimic mice as well as increased αvβ3 integrin expression. These findings wereas average radiance efficiency [(photons/s/cm^2/in agreement with increased hypoxia as indicated by mCherry fluorescence, and an increase in HIF-1α activity and neosteradian)/(μW/cm^2)].
angiogenesis stimulation, as shown by immunohistochemistry and CT scan.
(B) Left Panel: Representative (n=5) 2D mCherry fluorescent signal (rainbow scale) of U251-HRE-mCherry tumors in scramble

Mice were subsequently treated with an inhibitor of miRNA675-5p and imaged in the same way. The data indicate thatand inhibitor-treated mice (one of five mouse for each treatment) at day 28. Images are presented with the same scale bar.
the inhibition of miRNA675-5p in vivo can i) turn off tumor molecular response to hypoxia ii) constrain angiogenesis andiii) block hypoxia-mediated tumor growth, thus suggesting a therapeutic role for miRNA675-5p inhibitor in glioblastoma.
(B) Right panel: Graphical representation of mCherry activity at the end of treatment considering five mice for each
See figures

RESOURCESCONTACT US

treatment. Data are presented as the average radiance efficiency [(photons/s/cm^2/steradian)/(μW/cm^2). Images arepresented with the same scale bar. Inhibitor-treated mice vs scramble-treated mice * p<0.05; **p<0.01; ***p<0.001.

.

1. Lo Dico A, Costa V, Martelli C, Diceglie C, Rajata F, Rizzo A, Mancone C, Tripodi M, Ottobrini L, Alessandro R, Conigliaro A. MiR675-5p
Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma. Theranostics. 2016 May 8;6(8):1105-18.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Carbonic
AnhydraseIX
IX
Anhydrase

ANGIOGENESISBY CHARACTERIZATIONIVISense ProbeMW(g/mol)Specificity

In Vivo Biology Use

1,500

Carbonic
Anhydrase IX

Detection of carbonic
anhydrase 9 (CA IX) protein
expressed in many tumor
cell lines undergoing hypoxia
and acidosis.

Phosphatidylserine± Hypoxia CA IX 680
METASTASISEndothelium

Mouse Dose(25 g)

Sold As(Unit Size)

Optimal Imaging Time (h)2 nmol24 nmol

24±Targeted probes actively target and bind to distinct biomarkers with highly specific targeting to key biological mechanisms.
αvβ3 Integrin
DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATIONThe current view of inflammation during carcinogenesis is a complex dichotomy of opposing processes:
immunosurveillance and anti-tumorigenic immune functioning are countered by pro-tumorigenic inflammation,
which promotes cancer by blocking anti-tumor immunity and shaping the tumor microenvironment toward amore tumor-permissive state.1 Additionally, inflammation can both contribute to and be driven by tumor growth.
It is well known that long-standing inflammation that is secondary to chronic infection or repeated tissue
insult can predispose to cancer.2 The reverse is also true, where “wound-like” pre-malignant tumors trigger atissue repair response, eliciting pro-inflammatory factors which in later stages of tumor growth can function topromote cancer under direct control of the tumor itself.2
Regardless of its initiation, long-term ‘smoldering’ inflammation in the tumor microenvironment has manytumor-promoting effects, such as aiding in the proliferation and survival of malignant cells, promoting
angiogenesis and metastasis, subverting adaptive immune responses, and altering responses to hormones andchemotherapeutic agents.3
A deeper understanding of the role of cancer-related inflammation, including the identification of new targets
and more effective approaches to mediating the inflammatory response, is central to the development of
improved diagnosis and novel treatments.

Hydroxyapatite

CELL DEATH

Transferrin

1. Greten FR, Grivennikov SI. Inflammation and Cancer: Triggers, Mechanisms, and Consequences.
Immunity. 2019; 51(1):27-41.

RESOURCES

2. Rakoff-Nahoum S. Why cancer and inflammation? Yale J Biol Med. 2006; Dec;79(3-4):123-30.

CONTACT US.

3. Mantovani, A., Allavena, P., Sica, A. et al. Cancer-related inflammation. Nature. 2008; 454: 436–444.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US

A critical biological consideration with in vivo imaging is the timing of a particular biomarker’s activity. Diseasebiomarkers may be up- or down-regulated at different times, as different factors may influence their expression
during disease progression. Thus, understanding when the biomarker is operant so that the corresponding
imaging probe can be optimized is paramount. A useful example of probes scheduled to longitudinally detectmutable inflammatory markers was devised based on each probes’ potential use in imaging atherosclerosis in
ApoE-deficient mice. It is worth noting that atherosclerotic plaques start in much the same way as many cancers,
and the link in patterns of progressive inflammation is currently being investigated.
Atherosclerosis and cancer are both chronic, multifactorial diseases, sharing not only several important
molecular pathways but also many etiological and mechanistical processes from the very early stages ofdevelopment up to the advanced forms in both pathologies.1 In atherogenesis, small sub-endothelial deposits
of monocyte-derived macrophages cause tissue insult which then drives an inflammatory cascade, recruiting
additional macrophages and further propagating the inflammatory process. This initiating tissue insult, alongwith other factors such as uncontrolled cell proliferation and oxidative stress, bares considerable similarities tothe neoplastic process of cancer.1
Research with the aim to identify molecular ties between the phenomenon that closely correlates
atherosclerosis and cancer is ongoing, with the hope that this new understanding could lead to novel
treatments, such as a cancer treatment currently in clinical trials which may also reduce the inflammation
responsible for atherosclerosis.2

1. Tapia-Vieyra JV, Delgado-Coello B, Mas-Oliva J. Atherosclerosis and Cancer; A Resemblance with Far-reaching
Implications. Archives of Medical Research, 2017; 48(1): 12-26.

.

2. Jarr K, et al. Effect of CD47 Blockade on Vascular Inflammation. N Engl J Med, 2021; 384:382-383.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

PROBE
USE INconsideration
A LONGITUDINALIMAGINGSTUDY:
A
critical biologicalin in vivo imaging
is the timingof a particular biomarker’s activity. Diseasebiomarkers
may beup- or down-regulated
at different
times, as different factors may influence their expression
POTENTIALIMAGINGOF DISEASEPROGRESSION
duringdisease progression. Thus,
understanding
the biomarker is operantso that the
1 corresponding
IN APOE-DEFICIENTMICE
FED HIGHwhen
CHOLESTEROLDIET (HCD)
imaging probe can be optimized is paramount. A useful example of probes scheduled to longitudinally detectmutable inflammatory markers was devised based on each probes’ potential use in imaging atherosclerosis in
IVISense
ApoE-deficientmice. It is worth noting that atherosclerotic plaques start inmuch Probe
the same way as many cancers,
Time on
MMP FAST
IntegrinPan
Annexin-V
HCD the link in patterns
Concurrent
Biology2
andof progressive
inflammation
is currently
being investigated.
MMP
Cathepsin
Receptor
Cat B FAST (MMPSense(MMPSense)(ProSense)
FAST)(IntegriSense)(weeks)(AnnexinVivo)

Cat K FAST

Atherosclerosis
and cancer
are both chronic, multifactorial diseases, sharing not only several important• Moncyte
adhesion
4-6• Sporadic
foam
cells
molecular pathways
but
also
many etiological and mechanistical processes from the very early stages ofdevelopment
to foamthe advanced
• up
Early
cell lesions forms in both pathologies.1 In atherogenesis, small sub-endothelial deposits
8-10• Subendothelial
progressionof monocyte-derived
macrophagescause tissue insult which then drives an inflammatory cascade, recruiting
additional
macrophages
further propagating the inflammatory process. This initiating tissue insult, along• Intermediateandlesions
10-15• Smoothmusclecell proliferation
with other factors
such
as uncontrolledcell proliferation and oxidative stress, bares considerable similarities to1• process
Larger fibrousplaquesthe 15
neoplastic
of cancer.
•

Small necrotic cores•

Advanced plaques•Partial medial destruction

20-30
Researchwithaim toidentify molecular ties between the phenomenon that closely correlates• theIncreased
necrosis
atherosclerosis
and cancer
is ongoing,• Larger,advancedplaques with the hope that this new understanding could lead to novel• Progression
to coronary
arteries
treatments,such
as a cancer
treatment
currently in clinical trials which may also reduce the inflammation
30-40• Larger
necrotic cores2
responsible foratherosclerosis.

Relevant Targets:
Broad or selective (Cat B, Cat K) cathepsin activity
Matrix metalloprotease (MMP) activity

αVβ3 integrin expression
Apoptosis (phosphatidyl-serine)RESOURCES
1. 1.
Tapia-Vieyra
Peterson, JV,
JD. Delgado-CoelloNon-Invasive Quantitative
B, Mas-Oliva
In J.Vivo
Atherosclerosis
Imaging of Atherosclerosis
and Cancer; A Resemblance
Disease Progression
with Far-reaching
and Treatment
Implications.
Response in ApoE Deficient Mice using FluorescentArchives
Molecularof Medical
Tomography
Research,and2017,NIR Fluorescent
Volume 48, Pre-clinicalIssue 1, Pages
Imaging
12-26.Agents. Application Note. PerkinElmer, Inc. Waltham, MA.

CONTACT US

.

2. 2.
JarrNakashima
K, et al. Effectet al.,
ofApoE-deficient
CD47 BlockadeMice
on Vascular
DevelopInflammation.
Lesions of All N
phasesEngl Jof
Med,
Atherosclerosis
Jan 2021, 384:382-383.
Throughout the Arterial Tree, Arterioscler Thromb 1994, 14(1):133-40.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

MMP

CATHEPSIN

ELASTASE

Matrix metalloproteases (MMPs) are known to cleave numerous cell surface proteins in remodeling theextracellular matrix but have been shown to also have a role in modulating the immune response via theirproteolytic regulatory functions. Expressed at low levels under homeostasis, MMP expression is markedly
increased in inflammatory and cancer cells during inflammatory processes including infection, wound healing,
and cancer. Increasing research is showing that MMPs are not just a downstream consequence of a generalized
inflammatory process, but rather are critical factors in the overall regulation of the pattern, type, and durationof immune responses.1

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION

The ability to detect MMPs can reveal distinctive local biology as well as assess therapeutic efficacy of specificdrugs, sometimes prior to overt biological changes. IVISense MMP NIR fluorescent imaging probes are able
to detect a broad range of active MMPs, including MMP 2, 3, 7, 9, 12, and 13, to evaluate the potential
therapeutic efficacy of drugs targeting the underlying mechanisms involved.

Hydroxyapatite
VIEW IMAGE .
CELL DEATH

Transferrin

RESOURCES
1. Smigiel KS, Parks WC. Matrix Metalloproteinases and Leukocyte Activation. Prog Mol Biol Transl Sci. 2017;147:167-195.

CONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

ELASTASE

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Matrix metalloproteases are active in regions of tissue remodeling, in this case highly expressed andsecreted at the invasive tumor margins as well as by inflammatory cells.

©2019, PerkinElmer, Inc. All rights reserved

GO BACKRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISMMPCATHEPSIN

ELASTASE

Cathepsins are a family of proteases that are pivotal components of signaling pathways which can enhancecancer cell growth and inflammation, and contribute to matrix degradation, thereby facilitating tumor cell
invasion.

Endothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

Cathepsin B: Upregulated in a variety of cancers as well as infiltrating inflammatory cells (including
eosinophils, neutrophils, and macrophages), cathepsins are capable of exerting either anti- or pro-cancer
effects. They may initiate proteolytic pathways crucial for invasion by inflammatory breast cancer, the mostlethal form of primary breast cancer.1
Cathepsin K: A lysosomal cysteine protease involved in bone resorption and collagen degradation, andexpressed in osteoclasts, certain tumor-associated macrophages (TAMs), and other cells, such as bone marrow
macrophages which are known to mediate the inflammatory process in the bone microenvironment.

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

IVISense Pan Cathepsin fluorescent imaging probes: versatile pan-cathepsin-activated agents.
IVISense Cat B fluorescent imaging probes: selectively cleaved by cathepsin B proteinases.
IVISense Cat K fluorescent imaging probes: selectively cleaved by cathepsin K proteinases.
For specific detection of tumors with associated cathepsin activity, NIR fluorescent imaging agents ProSense,
Cat B, and Cat K enable non-invasive longitudinal imaging of tumor progression via protease expression.
VIEW IMAGE .

RESOURCESCONTACT US

1. Nouh, M.A., Mohamed, M.M., El-Shinawi, M. et al. Cathepsin B: a potential prognostic marker for inflammatory breast
cancer. J Transl Med. 2011; 9(1).

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Above: Osteolytic lesions form when tumor cells inbone help drive osteoblast precursors to increasedifferentiation into osteoclasts, causing osteoclastic
bone resorption to exceed osteoblastic
bone formation.
A: Representation of cancer cells and inflammatory
cells as imaged using IVISense Cat B.
B: IVISense Cat K detects cathepsin K as expressedin osteoclasts.
C: IVISense Pan Cathepsin is a versatile imaging
probe that detects cathepsin proteases as expressedby tumor cells, inflammatory cells, and osteoclasts.

GO BACKRESOURCESCONTACT US.

ELASTASE

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

MMP

CATHEPSIN

ELASTASE

Neutrophil elastase (NE), a major product of neutrophils, is an especially potent proteolytic enzyme, normallystored in vesicles in an inactive form prior to degranulation or to the release of neutrophil extracellular
traps (NETs), a process of cell death called NETosis whereby a network of extracellular fibers dotted with NE
as arsenal against an injurious agent is released from the cell. Extensively studied as bacterial-responsive,
expendable innate immune cells, neutrophils have recently been found to be more heterogeneous than
was traditionally thought and to possess a longer survival time1, sparking renewed interest in the study ofneutrophil biology.
Neutrophils are frequently found within tumors, prompting adoption of the term ‘tumor-associated neutrophil’(TAN). TANs are less well characterized than their counterpart, ‘tumor-associated macrophages’ (TAMs), buthave been shown to have both pro- and anti-tumor effects.2 Found to perform previously unsuspected tasks,
neutrophil functionality may include modulating the activity of other immune cells, such as either boosting or
suppressing T-cell activation. Multiple other tumor-promoting functions of TANs have been described, such asencouraging cellular proliferation, tumor angiogenesis, metastasis, and immunosuppression.2
IVISense Neutrophil Elastase 680 FAST is a near infrared fluorescent in vivo imaging probe that can detect NE
post-degranulation or NETosis. (See Biomarker Considerations for further discussion of the in vivo study oftumor-associated neutrophil activity.)
VIEW IMAGE .
1. Carlos R. Neutrophil: A Cell with Many Roles in Inflammation or Several Cell Types? Frontiers in Physiology. 2018; Vol. 9
2. Elliot A, Myllymaki H, Feng Y. Inflammatory Responses during Tumor Initiation: From Zebrafish Transgenic Models ofCancer to Evidence from Mouse and Man. Cells. 2020;9(4) 1018

RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISMMPCATHEPSIN

ELASTASE

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Tissue damage from inflammatory bowel disease can lead to abnormal cell growth with the potential to
become invasive cancer. Neutrophil Elastase activation by extracellular NE and NETs is represented in the
figure on the right.

Transferrin©2019, PerkinElmer, Inc. All rights reserved

GO BACKRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Neutrophils play an important role in shaping the tumor microenvironment. Upon degranulation, they release
enzymes including neutrophil elastase (NE) which significantly promotes cancer cell proliferation, metastasis, andtherapy resistance.1
A well-designed in vivo study of neutrophils requires a full understanding of the animal model and the kinetics ofdegranulation and NETosis as biomarkers of neutrophil activity. It is important to note that there are situations inwhich neutrophils are present, but degranulation has not yet occurred. In some models, neutrophil degranulation
is a coordinated occurrence, while in others the degranulation is sporadic. This means one should plan their study
design carefully, as it can be easy to miss the optimal imaging time for NE activity.
Apart from neutrophils, cells such as tumor cells, B cells, lymphocytes, and macrophages present in the tumor
microenvironment also secrete regulatory factors to facilitate cancer progression. Monitoring neutrophils as part ofan imaging study to monitor the overall innate immune response may provide a true sense of the ratio of activatedneutrophils to other immune cells involved. This could help researchers gain a deeper understanding of thepathological process of the inflammatory response, with the ultimate goal of regulating neutrophil recruitment for
better clinical outcomes.
IVISense Neutrophil Elastase 680 FAST is a near infrared fluorescent in vivo imaging probe that can detect NE postdegranulation or NETosis. A highly specific probe, it can detect active NE in the extra cellular matrix.

RESOURCESCONTACT US.

1. Wu L, Saxena S, Awaji M, Singh RK. Tumor-Associated Neutrophils in Cancer: Going Pro. Cancers (Basel).
2019;11(4):564.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

CASE STUDY: IMAGING THE EFFECTS OF N-ACETYLCYSTEINE (NAC) ANTIOXIDANT ON
CHRONIC INFLAMMATORY PATHOLOGY RESULTING FROM EPSTEIN-BARR VIRUS1
Numerous studies have established a link between chronic inflammation and increased cancer risk. When the
inflammatory response is persistently activated or fails to resolve, chronic inflammation can result, leading to tissue
damage and promoting conditions favorable for cancer development. Certain viral infections can induce persistent
inflammation, generating high levels of reactive oxygen and nitrogen species which then perpetuate the inflammatory
response and cause damage to a wide range of biomolecules. One such virus is the Epstein-Barr virus (EBV) which
commonly results in chronic inflammatory post-viral disease.
In this study, the effects of long-term administration of NAC, an antioxidant involved in redox regulation, on chronic
inflammation was investigated. Specifically, researchers used a transgenic mouse model of chronic inflammationassociated carcinogenesis induced by the viral-encoded latent membrane protein 1 (LMP1) of EBV. LMP1 acts as a proinflammatory factor with multiple oncogenic properties, including the ability to activate proliferative, cell survival, andinflammatory pathways. It was found that treatment with NAC resulted in a significant reduction of leukocyte infiltrate
in inflamed tissue, an amelioration of disease effects, and a delay in the chronic progressive inflammatory phenotype as
quantified by in vivo imaging using IVISense Pan Cathepsin 680 (ProSense 680).
Given that targeting any one chemokine or cytokine in the inflammatory process may have limited effect and/or
unwanted side effects, the researchers instead sought to assess the ability of a readily available, non-toxic antioxidant toinhibit the pernicious cycle of inflammation, oxidative stress, and tissue damage, showing that NAC could be considered
as a treatment to alleviate chronic inflammatory pathologies, including post-viral disease. While the use of antioxidants
in cancer treatment and prevention is much debated and the effects are likely to be cancer-type dependent, this study
supports the idea that NAC in the treatment of inflammation-associated and specifically EBV/LMP1-associated cancers
could be a valid area for further exploration.
See figures

RESOURCESCONTACT US.

1. Gao X, Lampraki EM, Al-Khalidi S, Qureshi MA, Desai R, et al. N-acetylcysteine (NAC) ameliorates Epstein-Barrvirus latent membrane protein 1 induced chronic inflammation. 2017; PLoS ONE 12(12).

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

CASE STUDY: IMAGING THE EFFECTS OF N-ACETYLCYSTEINE (NAC) ANTIOXIDANT ON
CHRONIC INFLAMMATORY PATHOLOGY RESULTING FROM EPSTEIN-BARR VIRUS1
Numerous studies have established a link between chronic inflammation and increased cancer risk. When the
inflammatory response is persistently activated or fails to resolve, chronic inflammation can result, leading to tissue
damage and promoting conditions favorable for cancer development. Certain viral infections can induce persistent
inflammation, generating high levels of reactive oxygen and nitrogen species which then perpetuate the inflammatory
response and cause damage to a wide range of biomolecules. One such virus is the Epstein-Barr virus (EBV) which
commonly results in chronic inflammatory post-viral disease.
In this study, the effects of long-term administration of NAC, an antioxidant involved in redox regulation, on chronic
inflammation was investigated. Specifically, researchers used a transgenic mouse model of chronic inflammationassociated carcinogenesis induced by the viral-encoded latent membrane protein 1 (LMP1) of EBV. LMP1 acts as a proinflammatory factor with multiple oncogenic properties, including the ability to activate proliferative, cell survival, andinflammatory pathways. It was found that treatment with NAC resulted in a significant reduction of leukocyte infiltrate
in inflamed tissue, an amelioration of disease effects, and a delay in the chronic progressive inflammatory phenotype as
quantified by in vivo imaging using IVISense Pan Cathepsin 680 (ProSense 680).

IVISense Pan Cathepsin 680 was used to show
N-acetylcysteine(NAC)
the in the inflammatory process may have limited effect and/or
Given
that targeting any
onetreatment
chemokinereduces
or cytokine
quantifiablelevels theof chronic
inflammation
in to assess the ability of a readily available, non-toxic antioxidant tounwantedside effects,
researchers
instead sought
the the
L2LMP1transgenic
mice.
inhibitpernicious
cycle ofinflammation, oxidative stress, and tissue damage, showing that NAC could be considered
as a treatment to alleviate chronic inflammatory pathologies, including post-viral disease. While the use of antioxidants
Left: Typicalepifluorescentimage taken
hoursdebatedafter IV and the effects are likely to be cancer-type dependent, this study
in cancer
treatment
and prevention
is 24
much
injectionof idea
IVISense
Pan
Cathepsin
into a transgenic
supports
the
that
NAC
in the680
treatmentof inflammation-associated and specifically EBV/LMP1-associated cancers
L2LMP1at phenotypicstageexploration.
5 (St5) and negative
could
be amousevalid areafor further
sibling control (NSC); color scale shows radiant efficiency
displayed ((photons/sec/cm2/sr)/(μW/cm2)).
Seerange
figures
Right: Graphs depicting the mean (with SD error bars) radiant efficiency (Y axis) observed in mice in increasing age groups (X axis)
imaged at 24 and 30 hours (as indicated) post injection of 1nmol/mouse IVISense Pan Cathepsin 680. Four groups were examined,
transgenic (Tg) and NSC mice, either untreated or treated with NAC. N = 2 to N = 8 for each age and group.

RESOURCESCONTACT US.

1. Gao X, Lampraki EM, Al-Khalidi S, Qureshi MA, Desai R, et al. N-acetylcysteine (NAC) ameliorates Epstein-Barr virus latent membraneprotein 1 induced chronic inflammation. 2017. PLOS ONE 12(12).

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESISBY CHARACTERIZATIONBY CHARACTERIZATION
MW
MW

IVISense
IVISense
Probe
Probe
Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
Elastase

INFLAMMATION
Hydroxyapatite

Transferrin

In Vivo
Biology
Use
In Vivo
Biology
Use

Cathepsin
Cathepsin
B B

Selectively
cleavedby cathepsin
Selectively
cleavedby cathepsin
B B
proteinases
proteinases
upregulated
upregulated
in tumorsin tumorsandandinflammatory
inflammatory
cells
cells

8,500
8,500 Cathepsin
Cathepsin
K K

Selectively
cleavedby bycathepsin
K K
Selectively
cleavedcathepsin
proteinases
upregulated
in osteoclasts
proteinases
upregulated
in osteoclasts
tumor
associatedmacrophages.
andandtumor
associatedmacrophages.

(g/mol)(g/mol)33,00033,000##
CatCat
B 750
B 750
FAST
FAST

23,00023,000##PanPanCathepsin
Cathepsin
680680~400,000~400,000

Cathepsin~450,000~450,000Cathepsin
proteases
proteases
22,50022,500##PanPanCathepsin
Cathepsin
750750FAST
FAST##
PanPanCathepsin
Cathepsin
750750##MMP
MMP645645
FAST
FAST
##
MMP
MMP
680680##
MMP
MMP
750750FAST
FAST##
Neutrophil
Neutrophil
Elastase
Elastase
680680FAST
FAST

CELL DEATH

Specificity
Specificity##
CatCat
B 680
B 680
FAST
FAST

##
CatCat
K 680
K 680
FAST
FAST

BIOPROFILING

Pan-cathepsin
Pan-cathepsin
activatable
activatable
agent
agent
thatthatdetects
detects
abnormalabnormalupregulation
upregulation
of ofcathepsincathepsin
secretion
secretion
associatedassociatedwithwithcancerinflammation.
cancerandandinflammation.

43,00043,000Cleavedby multiple
metalloproteinases
Cleavedby multiple
metalloproteinases
MatrixMatrix~450,000~450,000
associatedmanydiseaseareas
associatedwithwithmanydiseaseareas
Metalloprotease
Metalloproteaseincludingcancerinflammation.
includingcancerandandinflammation.
43,00043,00043,00043,000

Neutrophil
Neutrophil
Elastase
Elastase

Selectively
cleavedby elastaseproduced
Selectively
cleavedby elastaseproduced
by byactivated
neutrophils
duringacute
activated
neutrophils
duringacute
inflammation.
inflammation.

Mouse
Mouse
Dose
Dose Sold
OptimalImaging
Imaging
Sold
AsAs Optimal(25(25
g) g)
(Unit
(Unit
Size)
Size)
Time
Time
(h)(h)
2 nmol2 nmol

24 24
nmol
nmol

6-24
6-24
h h

4 nmol
4 nmol

48 48
nmol
nmol

6-24
6-24
h h

2 nmol2 nmol

24 24
nmol
nmol

6-24
6-24
h h

2 nmol2 nmol

20 20
nmol
nmol

24 24
h (24-48)
h (24-48)

2 nmol2 nmol

24 24
nmol
nmol

24 24
h h

4 nmol
4 nmol

48 48
nmol
nmol

6-24
6-24
h h

4 nmol
4 nmol

48 48
nmol
nmol

24 24
h (6-24)h (6-24)2 nmol2 nmol

20 20
nmol
nmol

24 24
h (24-36)
h (24-36)

2 nmol2 nmol

24 24
nmol
nmol

24 24
h (12-24)
h (12-24)

4 nmol
4 nmol

48 nmol
48 nmol

3-63-6h h##Activatable
Activatableprobesprobesare areoptically
optically
silentsilentupon
upon
injection
injection
andandbecomebecomehighlyhighlyfluorescentfluorescent
following
followingprotease-mediatedprotease-mediatedactivation.
activation.
Activatable
Activatable
FAST
FAST
agentsagentsare aredesigneddesigned
withwith
a novela novelsmall
small
molecule
moleculearchitecturearchitecture
thatthatconfers
confers
an accelerated
an accelerated
pharmacokinetic
pharmacokinetic
profileprofilewithwithearlierearlier
imaging
imaging
timetimepoints.
points.

RESOURCESCONTACT US.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin

Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium

BY CHARACTERIZATIONBIOPROFILINGBY CHARACTERIZATION
PerkinElmer offers a fluorescent Inflammation Panel consisting of a carefully curated collection of probes to detect multiple
aspects of inflammation. Including probes at various wavelengths (680nm and 750nm), the panel offers the opportunity for
multiplex imaging of appropriate probe combinations to maximize information gained from your research results.

Inflammation Panel

αvβ3 Integrin

Set of Fluorescent Probes Bundled by Application

IVISense Probe

DRUG RESISTANCE
BombesinMW(g/mol)Specificity

CONTACT US

Probe
Clearance

2 nmol24 nmol

24 h

6-7 d

NA

Folate Receptor 680

1,606

Folate Receptor

Detection of Folate Receptor protein
upregulation for monitoring tumor
growth and metabolism.

2 nmol24 nmol

6 h (6-24)

6-7 d

22,500Cathepsin proteases

Pan-cathepsin activatable agent that
detects abnormal upregulation ofcathepsin secretion associated withcancer and inflammation.

4 nmol

48 nmol

6-24 h3d

MMP 750 FAST

43,000Cleaved by multiple
metalloproteinases associated with
Matrix Metalloprotease
many disease areas including
cancer and inflammation.

4 nmol

48 nmol

24 h (12-24)

6-7 dIntegrin Receptor 750

1,278

αvβ3 integrin

Detection of integrin αvβ3
expression in tumors andneovasculature.

2 nmol24 nmol

24 h

4-6 days

Neutrophil Elastase
680 FAST

43,000

Neutrophil Elastase

Selectively cleaved by elastaseproduced by activated neutrophils
during acute inflammation.

4 nmol

48 nmol

3-6 h2dTransferrin.

Optimal Imaging
Time (h)~70,000

Pan Cathepsin 750
FAST

RESOURCES

Amount (Unit
Size)

Vascular 680

Hydroxyapatite

CELL DEATH

Mouse Dose(25 g)

Imaging of vascularity, perfusion,and vascular permeability. Imaging
of vasculature for up to 4 h; tumor
accumulation at 24 h.

Elastase
Elastase

INFLAMMATIONIn Vivo Biology Use

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase

Cell death is a fundamental process in all multicellular organisms. For many years it was considered to occur in
one of two basic forms – apoptosis, defined as programmed cell death, or necrosis, defined as unregulated cell
death. Research has since shown that the dichotomy is far less distinct than once thought, and there is still debate
regarding the actual number of death modes and their categorization.1 Study of modalities of cell death has
progressed to the point where it is now understood that necrosis is subject to genetic and chemical manipulation
as a regulated process under certain circumstances.2,3 As new forms of regulated cell death are characterized,
it is clear that some forms have characteristics that challenge the long-held dogma regarding the distinguishingfeatures once thought unique to apoptosis.
For cells undergoing apoptosis, exposure of phosphatidyl serine (PS) on the outer plasma membrane serves asa signal to phagocytes, marking the cell for efferocytosis. Long considered a feature only of apoptotic cells, PS
exposure has recently been reported in non-apoptotic forms of regulated cell death as well, such as necroptosis3,
a programmed form of necrosis. Indeed, much remains to be understood regarding different modes of cell death,and some notions which have been firmly engrained in cell death research may still be found to have morenuance than was once thought and may uncover new possibilities for cancer treatment.

INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

1. Liu, X., Yang, W., Guan, Z. et al. There are only four basic modes of cell death, although there are many
ad-hoc variants adapted to different situations. Cell Biosci. 2018; 8, 6

RESOURCESCONTACT US

2. Shlomovitz, I., Speir, M. & Gerlic, M. Flipping the dogma – phosphatidylserine in non-apoptotic cell
death. Cell Commun Signal. 2019; 17:139.

3. Linkermann, A., & Green, D. R. Necroptosis. New Eng Jrnl Med. 2014; 370(5): 455-465.

TUMOR BURDEN

BIOLOGY

BIOMARKERS

REVIEW & SIGNIFICANCEBIOLOGY CONSIDERATIONSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

IMAGING CELL DEATH TO EVALUATE DRUG EFFICACY IN CANCER
As we deepen our understanding of the underlying mechanisms of different modes of cell death, finding ways
to exploit these pathways with novel cancer therapies is a valid pursuit.
In vivo studies using IVISense Annexin-V 750 (AV750), an NIR fluorescent probe that detects and quantifies
externalized phosphatidyl serine (PS) as a marker of cell death, to evaluate cancer drug treatment results may
employ different imaging approaches. With some tumor models, it may be desirable to assess AV750 results
in several different ways and at different time points to gain a more thorough understanding that may not be
fully conveyed by absolute signal alone or may be confounded by imaging too early or too late.

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Depending on the imaging time point post treatment and on the drug, treatment effects could present as avery dynamic process with potentially 25% of animals exhibiting increasing tumor AV750 signal after a single
cytotoxic drug treatment, which may increase to ~75-100% of animals after a second or third dose. Means ofassessment may include:• Imaging general diffuse signal across the tumor• Imaging tumor collapse with bright, focal apoptotic/necrotic regions• Imaging high signal across the tumor
Though all of these cell death readouts are efficacious, they may quantitatively differ, therefore it is essential tounderstand the operant biology at the time of imaging and to maximize time-points accordingly.

RESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

PHOSPHATIDYL SERINE
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Phosphatidyl serine (PS) is an anionic phospholipid that, under homeostatic conditions, is sequestered by cells
to the inner leaflet of their plasma membrane. During the induction of some forms of cell death, PS becomes
externalized on the outer leaflet of the cell lipid bilayer where it functions as a signal for engulfment byphagocytic cells.
Although cell surface exposure of PS remains to be determined in all known forms of cell death (of which someestimate there are upwards of 34 proposed cell death modes1), it has been documented in several programmed
forms, specifically in apoptosis, ferroptosis, and necroptosis.2 When occurring in apoptosis, the immunologicalresult of PS exposure is the release of immune dampening signals that avert an auto-immune response in normal
tissues. This immune dampening ability can be exploited by cancer cells, where PS signaling may be highly
dysregulated in the tumor microenvironment.3

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

In other forms of cell death, such as necrosis, pro‐inflammatory proteins and compounds are often involved,
resulting in a cascade of inflammation and tissue damage. Currently, our knowledge regarding the clearance ofcells undergoing different types of cell death as well as how these cell deaths are decoded by the immune systemremains limited.2 However, it is reasonable to suggest that targeting PS activity could augment anti-cancer immune
therapy, and in fact, multiple strategies have been and are being developed.3 IVISense Annexin-V fluorescentprobe can be used for non-invasive PS exposure research and in vivo evaluation of potential anticancer therapies.

VIEW IMAGE .
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1. Liu, X., Yang, W., Guan, Z. et al. There are only four basic modes of cell death, although
there are many ad-hoc variants adapted to different situations. Cell Biosci. 2018; 8, 6
2. Klöditz, K., Fadeel, B. Three cell deaths and a funeral: macrophage clearance of cells
undergoing distinct modes of cell death. Cell Death Discovery. 2019; 65.
3. Dayoub, A.S., Brekken, R.A. TIMs, TAMs, and PS- antibody targeting: implications for cancer
immunotherapy. Cell Commun Signal. 2020; 18, 29.

.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin

PHOSPHATIDYL SERINE
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

Normally confined to the inner leaflet of the cellular membrane, phosphatidyl serine (PS) distribution
is disrupted and becomes exposed on the surface of cells when programmed cell death causes themembrane to become highly disordered. IVISense Annexin-V 750 fluorescent imaging of exposed PS isrepresented on the right.
©2019, PerkinElmer, Inc. All rights reserved

GO BACKRESOURCESCONTACT US.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Though effective in treating many types of cancer, chemotherapy can produce severe side effects such as
suppression of immune function and damage to heart muscle, gastrointestinal tract, and liver. If extensive enough,tissue injury can be a major reason for late-stage termination of drug discovery research projects. Therefore, it is
becoming more important to integrate safety/toxicology assessments earlier in the drug development process.
There are a variety of traditional serum markers tailored mechanistically to specific tissues, however there are no
current noninvasive assessment tools capable of looking broadly at in situ biological changes, such as cell death,inflammation, and vascular and metabolic changes, in target and non-target tissues as indicators of chemical insult. To address this need, one study found that a cocktail of in vivo imaging probes was a powerful tool for cancer
drug efficacy/tox screening. In this study, HT-29-bearing nude mice were given a single dose of 5-Fluorouracil (5FU) at low and high doses. Mice were tomographically imaged using a cocktail of IVISense probes: Annexin-V 750/ MMP 750 / Transferrin Receptor 750 (AMT 750), combined with IVISense Vascular 680. The very short treatment
regimen showed no apparent effects on tumor mass but increasing signal from both Vascular 680 and AMT 750
was detected in heart, liver, and lungs, with the heart showing the most dramatic increase (>20-fold). The acutenature of the response, and the absence of histologic inflammation, suggested that AV 750 was detecting tissue
apoptotic changes. The reversible nature of the biological changes at 24h suggested that this multi-probe approach could be a sensitive imaging strategy for detecting early tissue toxicity.

Transferrin

For a more complete characterization of drug efficacy/toxicity, multiple biomarkers can be tracked concurrently.
By quantifying and comparing changes in the levels of exposed phosphatidyl serine, matrix metalloproteinase(MMP), transferrin receptor expression, and changes in vascularity, researchers were able to detect early tissue
biological changes resulting from injury. This may provide a robust approach to the simultaneous assessment ofdrug-induced efficacy and collective tissue injury in mice, allowing earlier and more efficient therapeutic screening
in drug discovery.

RESOURCESCONTACT US

CONCURRENTLY TRACKED BIOMARKERS AS EARLY INDICATORS OF
DRUG-INDUCED TISSUE INJURY.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

CONCURRENTLY TRACKED BIOMARKERS AS EARLY INDICATORS OF
DRUG-INDUCED TISSUE INJURY
1COMPLIMENTARY BIOMARKERS AS INDICATORS OF DRUG-INDUCED TISSUE INJURY

Though effective in treating many types of cancer, chemotherapy can produce severe side effects such as
suppression of immune function and damage to heart muscle, gastrointestinal tract, and liver. If extensive enough,tissue injury can be a major reason for late-stage termination of drug discovery research projects. Therefore, it is
becoming more important to integrate safety/toxicology assessments earlier in the drug development process.
There are a variety of traditional serum markers tailored mechanistically to specific tissues, however there are no
current noninvasive assessment tools capable of looking broadly at in situ biological changes, such as cell death,inflammation, and vascular and metabolic changes, in target and non-target tissues as indicators of chemical insult. To address this need, one study found that a cocktail of in vivo imaging probes was a powerful tool for cancer
drug efficacy/tox screening.1 In this study, HT-29-bearing nude mice were given a single dose of 5-Fluorouracil (5FU) at low and high doses. Mice were tomographically imaged using a cocktail of IVISense probes: Annexin-V 750/ MMP 750 / Transferrin Receptor 750 (AMT 750), combined with IVISense Vascular 680. The very short treatment
regimen showed no apparent effects on tumor mass but increasing signal from both Vascular 680 and AMT 750
was detected in heart, liver, and lungs, with the heart showing the most dramatic increase (>20-fold). The acutenature of the response, and the absence of histologic inflammation, suggested that AV 750 was detecting tissue
apoptotic changes. The reversible nature of the biological changes at 24h suggested that this multi-probe approach could be a sensitive imaging strategy for detecting early tissue toxicity.
HT-29 tumor bearing mice were treated with PBS, 50 mg/kg, or 100 mg/kd of 5-fluorouracil, and 2h later injectedwithimaging
agents. (A)of
Mice
were
injected withmultiple
luciferinbiomarkers
12-15 min prior
to tracked
imagingconcurrently.
(at 24h) for
For a fluorescence
more completecharacterization
drug
efficacy/toxicity,
can be(B)levels
AMT of
750/ IVISense
Vascular 680(AngioSense
680) fluorescent signal
tumor
bioluminescence(IVIS® SpectrumCT).
By
quantifying
and comparing
changes in the
exposedphosphatidylserine,matrix metalloproteinase(MMP),
transferrin
receptor
expression,and changes
in vascularity,
researchers
wereable to detectof
earlytissue(C) 2-color
representations
imaging
distribution
was assessedusing
fluorescence
tomography(FMT® 4000).
biologicalchangesresultingfrominjury.
This
may provide
a robust approachto thedatasets
simultaneousassessment
datasets wererenderedusing
1 voxelslicesthrough
the fluorescence
tomographicof indicatedtissueofdrug-induced
efficacy images
and collective
tissueinjuryin mice,allowing
earliermore
efficienttherapeutic
screeningregions. Red outlined
indicate
tissues
showing
obviouschangesin andsignalintensity
relativeto controls.
in drug discovery.

RESOURCESCONTACT US.

1. Vasquez, Kristine O., Peterson, Jeffrey D. Combined efficacy and toxicity imaging following acute 5-FU
1. Vasquez, Kristine O., Peterson, Jeffrey D. Combined efficacy and toxicity imaging following acute 5-FU
treatment of HT-29 tumor xenografts. Poster (WMIC)
treatment of HT-29 tumor xenografts. Poster; PerkinElmer website, Resources.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

CASE STUDY: VALIDATING A FOCUSED ULTRASOUND ANTI-CANCER TREATMENTBY IMAGING CELL DEATH1
Triggerable drug delivery systems allow for precisely placed and highly localized anti-cancer treatment withthe potential advantage of lowering the systemically administered chemotherapeutic dose. In this study,
researchers prepared novel MR-imageable thermosensitive liposomes (iTSL) with triggerable release properties
that encapsulated doxorubicin (DOX). The resulting iTSL-DOX allowed for focused ultrasound (FUS)-mediatedspatiotemporal tumor drug delivery in tumor-bearing mice with direct clinical relevance. The affects of thetreatment on cancer cell viability were verified by monitoring the tumors in vivo with IVISense Annexin-V 750(Annexin Vivo 750).
Using an MDA-MB-231 murine model, researchers designed a study that assessed iTSL-DOX + FUS treatment in two
groups of animals, each bearing two tumors (one on each side). In one group of double-tumor mice, no iTSL-DOX
was administered. The left side tumor received no FUS while the right side was treated with FUS. In the second
group, mice recieved iTSL-DOX, while the left and right tumors were respectively not treated or treated with FUS
in the same way as the first group.
48 hours after the first group recieved treatment with iTSL-DOX + FUS, the mice were injected with IVISense
Annexin-V 750. The second group were sacrificed at the same time point with tumors excised and stained.
IVISense Annexin-V 750 signal was stronger in the FUS-treated tumor to a large effect compared to the FUSuntreated tumor, indicating that the combination of iTSL-DOX and FUS enhanced tumor cell apoptosis, andhistology confirmed these results, showing necrotic areas induced by the treatment. Noninvasive in vivo imaging
with IVISense Annexin-V was able to detect iTSL-DOX + FUS cell death localized in the tumor, suggesting that the
drug was released only after FUS, and that the high local dose was efficient in killing tumor cells without systemic
toxicity, thereby increasing chemotherapies’ therapeutic index.
See figures

RESOURCESCONTACT US.

1. Amrahli M, Centelles M, Cressey P, Prusevicius M, Gedroyc W, Xu XY, So PW, Wright M, Thanou M.
MR-labelled liposomes and focused ultrasound for spatiotemporally controlled drug release in triple
negative breast cancers in mice. Nanotheranostics. 2021; 5(2):125-142.

TUMOR BURDEN

BIOLOGY

BIOMARKERSRECOMMENDED PROBESRELEVANCEBIOMARKER CONSIDERATIONSCASE STUDY

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US.

CASE STUDY: IMAGING THE EFFECTS OF N-ACETYLCYSTEINE (NAC) ANTIOXIDANT ON
CHRONIC INFLAMMATORY PATHOLOGY RESULTING FROM EPSTEIN-BARR VIRUS1
Numerous studies have established a link between chronic inflammation and increased cancer risk. When the
inflammatory response is persistently activated or fails to resolve, chronic inflammation can result, leading to tissue
damage and promoting conditions favorable for cancer development. Certain viral infections can induce persistent
inflammation, generating high levels of reactive oxygen and nitrogen species which then perpetuate the inflammatory
response and cause damage to a wide range of biomolecules. One such virus is the Epstein-Barr virus (EBV) which
commonly results in chronic inflammatory post-viral disease.
In this study, the effects of long-term administration of NAC, an antioxidant involved in redox regulation, on chronic
inflammation was investigated. Specifically, researchers used a transgenic mouse model of chronic inflammationassociated carcinogenesis induced by the viral-encoded latent membrane protein 1 (LMP1) of EBV. LMP1 acts as a proinflammatory factor with multiple oncogenic properties, including the ability to activate proliferative, cell survival, andinflammatory pathways. It was found that treatment with NAC resulted in a significant reduction of leukocyte infiltrate
in inflamed tissue, an amelioration of disease effects, and a delay in the chronic progressive inflammatory phenotype as
quantified by in vivo imaging using IVISense Pan Cathepsin 680 (ProSense 680).
Given that targeting any one chemokine or cytokine in the inflammatory process may have limited effect and/or
unwanted side effects, the researchers instead sought to assess the ability of a readily available, non-toxic antioxidant toinhibit the pernicious cycle of inflammation, oxidative stress, and tissue damage, showing that NAC could be considered
as a treatment to alleviate chronic inflammatory pathologies, including post-viral disease. While the use of antioxidants
in cancer treatment and prevention is much debated and the effects are likely to be cancer-type dependent, this study
supports the idea that NAC in the treatment of inflammation-associated and specifically EBV/LMP1-associated cancers
could be a valid area for further exploration.
Assessing necrosis and apoptosis 48 h after treatment. (A) Histology (H & E sections), tumors were excised 48 h
Seeafter
figures
the treatment. Slices (4 μm) of MDA-MB-231 tumors untreated and (B) treated with iTSL-DOX and focusedultrasound (FUS) were stained with haematoxylin and eosin. The necrotic areas induced by the treatment are
indicated by the arrows. Large necrotic areas could only be found when the combination of iTSL and hyperthermia
was applied; (C) Apoptosis monitoring in living mice two days post treatment using IVISense Annexin-V 750, withimages recorded 3-4 h after the administration. Significant increases in apoptosis were apparent on the right side
where FUS had been applied.

1. Gao X, Lampraki EM, Al-Khalidi S, Qureshi MA, Desai R, et al. N-acetylcysteine (NAC) ameliorates Epstein-Barr virus latent membraneprotein 1 induced chronic inflammation. 2017. PLOS ONE 12(12).

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESISBY CHARACTERIZATIONBY CHARACTERIZATION
MW
Probe

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US.

±IVISense Annexin-V 750(g/mol)35,000BIOPROFILING
Specificity

In Vivo Biology Use

Targeting of membrane
Phosphatidyl
phosphatidyl serine exposedserine exposure on cells undergoingon cells
apoptosis/necrosis/otherforms of cell death.

Mouse Dose(25 g)

Sold As(Unit Size)

Optimal Imaging
Time (h)100 μL

1 mL

2±Targeted probes actively target and bind to distinct biomarkers with highly specific targeting to key biological mechanisms.

BIOLOGYTUMOR BURDEN

BIOMARKERSRECOMMENDED PROBES

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine
Phosphatidylserine
METASTASISEndothelium

BY CHARACTERIZATIONBY CHARACTERIZATION

PerkinElmer offers a fluorescent Toxicology Panel consisting of a carefully curated collection of probes to detect
multiple aspects of toxicity, including cell death. With probes at various wavelengths (680nm and 750nm),
the panel offers the opportunity for multiplex imaging of appropriate probe combinations to maximize
information gained from your research results.

αvβ3 Integrin

Toxicology PanelSet of Fluorescent Probes Bundled by Application

DRUG RESISTANCE
Bombesin

Elastase

Hydroxyapatite

CELL DEATH

Transferrin

RESOURCES.

Mouse Dose(25 g)

Amount(Unit Size)

Optimal Imaging
Time (h)Probe
Clearance

Vascular probe. Leaks into sites ofvascular damage or inflammation.

2 nmol24 nmol

24

6-7 d

Detects upregulation of transferrin
106,000 Transferrin receptor receptors on tumor cells. Marker foriron metabolism in liver.

2 nmol24 nmol

24 (6-24)

96 h

4 nmol

48 nmol

24 (12-24)

6-7 d

IVISense Probe

MW
Specificity(g/mol)

In Vivo Biology Use

Vascular 680~70,000 NA

Transferrin Receptor 750

INFLAMMATIONCONTACT US

BIOPROFILING

Cleaved by multiple metalloproteinases. Secreted markers ofstellate cells, Kupffer cells,
macrophages, and neutrophils.

MMP 750 FAST

43,000Matrix
Metalloprotease

Annexin-V 750

35,000Cell death/apoptosis probe. Marker on
Phosphatidyl serine
inflammatory cells; metabolic marker
exposure on cells
on tumor cells.

100 μL

1 mL

2

3d

Renin 680 FAST

43,000Renin proteaseactivity

2 nmol24 nmol

24 (12-24)

4dRenin activatable, marker of tissue
renin-angiotensin system activity.

RESOURCES
TUMOR BURDEN

FolateMMPMETABOLIC ACTIVITYANGIOGENESISCathepsinBEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING

VIEW

Carbonic
Anhydrase IXAPPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)

VIEW

Phosphatidylserine

PROBE REFERENCE TABLESMETASTASISEndothelium

Characterization of the full portfolio of IVISense probes:
Activatable, Targeted, Vascular

VIEW

αvβ3 Integrin

REFERENCES

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US

.

VIEW

RESOURCES
TUMOR
FolateBESTBURDEN
PRACTICES IN FLUORESCENCE
MOLECULAR IMAGINGPREVIOUSNEXT

MMP
METABOLIC ACTIVITY
Cathepsin

VIEW
BEST PRACTICES IN FLUORESCENCEIMAGING
PerkinElmerMOLECULAR
has a variety of NIR
fluorescent imaging probes that can beused to:Carbonic
Anhydrase IXANGIOGENESISAPPROACHES TO FLUORESCENCE
MOLECULAR IMAGING (ILLUSTRATION)• Study complex biology

Phosphatidylserine•

Multiplex up to 4 imaging probes•

Perform deep-tissue imaging

PROBE REFERENCE TABLESMETASTASISEndothelium

Combine with bioluminescent microCT, and PET imaging for
Characterization of the full portfolioof IVISense
multimodal
studies probes:
•

VIEW

VIEW

Activatable, Targeted, Vascular
αvβ3 Integrin

Fluorescence molecular imaging is the visualization of cellular and biological
DRUG
RESISTANCEREFERENCES
functionin vivo to gain deeper insights into disease processes
andBombesintreatment effects.
Elastase

One key aspect of successful in vivo imaging is using carefully selected, validated
INFLAMMATION
probes. Importantly, probes with high specificity and biologically appropriate
characteristics, such as clearance
rate and route of metabolism, will enable you
Hydroxyapatite
to gain unique and meaningful biological information from your fluorescentimaging studies.
CELL DEATH

Transferrin

PerkinElmer’s fluorescent imaging probes are developed through an extensive
R&D process, designed to incorporate drug-like biodistribution properties, andvalidated for optimal target delivery and performance. By considering both thebiology of interest and the probe characteristics, you can design imaging studies
that yield better quantification of early biological changes, and allow you to
RESOURCES“see” biological activity in a living animal.
CONTACT US.

DESIGNING AN EFFECTIVE STUDY
VIEW

Fluorescence molecular imaging approaches can differ depending on
your ultimate goal. The following are key steps to designing a fluorescentimaging study:
1. Set Clear Goals for Your Study, and Identify Which Approach Will
Maximize Your Research•

Give careful consideration to which imaging approach will provide themost appropriate benefits (see Approaches to Fluorescence Molecular
Imaging – Illustration).

For example, is your primary goal to explore early disease, or latedisease? Waiting until the end of the study to understand the limited
biology available at that time can miss a lot of biology that has alreadyhappened. With the proper imaging approach, it’s possible to draw usefulRESOURCES
TUMOR
FolateBESTBURDEN
PRACTICES IN FLUORESCENCE
MOLECULAR IMAGINGPREVIOUSNEXT

MMP
METABOLIC
ACTIVITY
distinctions
between tissues when looking at critical early
timepoints,
where IN FLUORESCENCE
disease markers
may yield IMAGING
early biological information to characterize
VIEW
BEST
PRACTICES
MOLECULAR
Cathepsintissues have similar status and only subtle but critical biological differences.
disease onset and progression, whereas probes for detecting treatment
Later imaging sees only gross overt physiological changes from disease, withmarkers may provide data predictive of non-imaging terminal
Carbonic
gross overt biological differences. Thus, at later points, it becomes harder toreadouts.
Anhydrase IX
VIEW
APPROACHESTOFLUORESCENCE
MOLECULAR IMAGING (ILLUSTRATION)ANGIOGENESIS
make meaningful comparisons, given the very different biological landscapes• Many of our probes are designed to be universally applicable for mostbeing comparing.
diseases, which usually manifest some component of inflammation,
Phosphatidylserine

vascular damage, metabolic changes, or cell death – aspects of disease
that our probes are acutely capable of detecting. There may not
2. Know Your Animal Model’s Biology Up Front (Cellular Players, Kinetics)
VIEW
METASTASISEndothelium
be a “tailored”
probe probes:
to your target biology, however other moreof IVISense• The timing and/or kinetics of biology is important. If youCharacterization
are imaging cells of the full portfolio
downstream probes may be effective.
Activatable,
Vascularthat don’t arrive on the scene until a certain time, then it
is important Targeted,to plan

PROBE REFERENCE TABLES

your probe injection andtheimaging window to coincide with when the
αvβ3Integrin
biology is operant.

REFERENCES

DRUG RESISTANCE
Bombesin

5. Choose Proper Imaging Controls•

3. Identify Optimal Probe Objectives
What do you need your probe to be able to do? Examples of probe objectives
Elastase
could be to bind a specific cell surface receptor, or fluoresce only in the
INFLAMMATION
presence of a specific protease or biomarker.

Appropriate study design should include both positive control andnegative control (i.e. un-diseased) mice injected with probe(s).

VIEW

•

•

Hydroxyapatite
If antibodies are how youplan to perform your imaging, be cautious about
using intact antibodies as probes.

CELL DEATH•

Consider probe characteristics such as route of metabolism, time frame frominjection to imaging, or tissue washout time (Refer to Technical Datasheets on
website).
Test probes to identify best imaging strategies, timepoints, and analysis
RESOURCES Depending on your imaging strategy, choosing probes to detectapproaches.
CONTACT US•

Use fluorescent probes suitable for ex vivo imaging and even frozen
sections for microscopy with appropriate excitation/emission spectra.

Transferrin

4. Understand Probe Performance for Alignment with Your Model•

6. Confirm Results In Vivo with Careful In Situ And/Or Ex Vivo Tissue
Corroboration.

7. Validate Imaging Results Against “Gold Standard” Non-Imaging
Metrics•

Proper imaging results will correlate with or sometimes surpass current“gold standard” readouts, such as histology.

RESOURCES
TUMOR
FolateBESTBURDEN
PRACTICES IN FLUORESCENCE
MOLECULAR IMAGINGPREVIOUSNEXT

MMP
METABOLIC ACTIVITY
CathepsinBEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING

GENERATING A PRECLINICAL IMAGING MODEL

Carbonic
Anhydrase
When adaptingMOLECULAR
an animal modelIMAGING
for in vivo fluorescentimaging, it is
APPROACHESTO FLUORESCENCE(ILLUSTRATION)
In new model development,
it is IX
essential to validate yourimaging
ANGIOGENESIS

results with non-imaging ex vivo observations/quantifications aswell as with establishedPhosphatidylserine“gold standard” readouts.

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VIEW

important to know as much as possible about the biology and its kinetics inyour model. Important factors include:

PROBE REFERENCE TABLESMETASTASISEndothelium

IMAGING WITH INTACT ANTIBODIES• Time to first biological changes; time to overt clinical disease
Characterization of thefull portfolio of IVISense probes:
If using a therapeutic treatment, do you understand the biological
Activatable, Targeted,•Vascularchanges that will be induced?

αvβ3 Integrin

Though easy to label and highly target specific, intact
antibodies have several disadvantages, including: REFERENCES
DRUG RESISTANCE•

Bombesin•

A very long circulating half-life•

Elastase
Poor tissue penetration

INFLAMMATION•

A tendency for the Fc receptor to bind phagocytic cells
Hydroxyapatite

For these reasons, accumulation of intact antibodies in offtarget biology is possible, which raises concerns regarding
CELL DEATH
Transferrin
misinterpretation of experimental results. Ab fragments
are a better choice, and can be labeled with PerkinElmer’s
IVISense® Fluorescent Dyes, designed to have minimal
interaction with biological tissue.

RESOURCESCONTACT US

.

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For example, in cancer, cytotoxic drug activity may be detected byimaging cell death such as IVISense™ Annexin-V, whereas an antiVIEW
angiogenic efficacy may be best detected with a vascular probe such as
IVISense Vascular.

•

Optimal probe(s) injection time•

Deep tissue or superficial tissue imaging; 2D vs 3D (or ex vivo) imaging•

The anatomical region to be imaged and its proximity to difficult areas,such as liver, kidney, or bladder, where some probes will metabolize.
This can cause high background signal, and should be a consideration inprobe selection.

•

The cellular players in the model (i.e. neutrophils, macrophages,
lymphocytes, tumor cells, bacteria, hepatocytes, etc.)•

Some probes will be obvious choices based on the cells involved (e.g.
IVISense Pan Cathepsin or IVISense MMP for inflammatory cells)

RESOURCES
TUMOR
FolateBESTBURDEN
PRACTICES IN FLUORESCENCE
MOLECULAR IMAGINGPREVIOUSNEXT

MMP
METABOLIC ACTIVITY
CathepsinBEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING

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Hair removal can be performed under
PROPER IMAGING TECHNIQUE AND
injectable or inhaled anesthesia. Care should
ANALYSIS CONSIDERATIONS
Carbonic
be taken to remove hair from an area larger
Anhydrase IX
VIEW
APPROACHES TO FLUORESCENCE MOLECULAR IMAGING(ILLUSTRATION)
than just theregion of focus to assure
ANGIOGENESIS
In conjunction with properly selected, validated
that target and surrounding backgroundprobes, an emphasis onPhosphatidylserine(i) effective study design, (ii)fluorescence can be captured. For tomounderstanding your imaging model, and, (iii) careful PROBE REFERENCE TABLES
graphic imaging, hair must be removed fromimaging technique as detailed
in the next section, you
front, sides, and back for the region of focus.
VIEW
METASTASISEndothelium
Characterization of the full portfolio of IVISense probes:
can maximize your in vivo biological readouts to detect
Activatable, Targeted, Vascular
early indicators of standard endpoints, and realize
Once animals are properly anesthetized,
αvβ3results.
Integrinmore meaningful research
apply depilatory cream (NairTM lotion, Churchand Dwight Co., Inc., Princeton, NJ) thicklyVIEWon
DRUG RESISTANCEREFERENCES
Best Practice Techniques
for In Vivo Imaging:
Bombesinhair over the imaging region of each mouse.
Rinse off thoroughly with warm water and• Depilation
reapply until all hair has been removed.
• Consistent Animal Positioning
Elastase

INFLAMMATION• Injection Method• Proper Analysis

Hydroxyapatite

CELLDEPILATION
DEATH

Transferrin

To minimize light scattering and absorption influorescence imaging, hair should be removed fromthe appropriate body region of all mice. Figure 1
demonstrates the significance of proper depilation. HairRESOURCES
removalcan be performed under proper depilation.
CONTACT US.

Figure 1. The image above demonstrates
the impact of no depilation vs. depilation
on epi-fluorescence signal.

IMPORTANT: Rinsing must be done carefully
and thoroughly to minimize any introductionof skin lesions that can cause imaging
artifacts. Take care to ensure that themouse does not swallow or breathe in any
water during rinsing. Once mouse has been
depilated fully, move to a heating pad or toits original cage.

RESOURCES
TUMOR
FolateBESTBURDEN
PRACTICES IN FLUORESCENCE
MOLECULAR IMAGINGPREVIOUSNEXT

MMP
METABOLIC ACTIVITY
Cathepsin

CONSISTENT ANIMALPOSITIONING
Carbonic
Anhydrase IXANGIOGENESISBEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING

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INJECTION METHODAPPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)

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Animal positioning can influence the epi-fluorescence signal, as• For most of the probes, intraperitoneal (IP) or subcutaneous (SC) injection will either
demonstrated in FigurePhosphatidylserine
2. It is important to ensure that proper
not work at all or will be extremely variable with high injection site signal. All probes
positioning based on tumor location is consistently used.
be injected systemically either through the retro-orbital plexus or the tail vein.
PROBE REFERENCE must
TABLESMETASTASISEndothelium

Characterization of the full portfolio of IVISense probes:
Activatable, Targeted, Vascular

αvβ3 Integrin

REFERENCES

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US

.

VIEW

Figure 2. The images tothe right demonstrate theimpact of animal positioning
VIEW
on epi-fluorescence signal.

RESOURCES
TUMOR
FolateBESTBURDEN
PRACTICES IN FLUORESCENCE
MOLECULAR IMAGINGPREVIOUSNEXT

MMP
METABOLIC ACTIVITY
Cathepsin

PROPER ANALYSISBEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING

Carbonic
Anhydrase
IX
Consider Figure 3 below,
showing hypotheticalin which there
APPROACHES TO FLUORESCENCE
MOLECULARIMAGINGdatasets(ILLUSTRATION)
When analyzing fluorescenceimagingdata (even without
ANGIOGENESIS

VIEW

VIEW

are untreated animals and treated animals but the examples differ only intaking fluorescent background into account) the results arebackground levels. Assuming there is no background yields a calculation ofvalid and will provide robust
and statistically significant data.
Phosphatidylserine
44% inhibition (Example 1), however Examples 2 and 3 show the impact of a
However, although fluorescent imaging probes can provide
PROBE REFERENCE TABLES
modest background and a high background, with percent inhibition raising to
excellent targeting of disease biology, they also distribute widely
VIEW
METASTASISEndothelium
Characterization
of
the
full
portfolio
of
IVISense
probes:57%
and80%
respectively.
Takingbackgroundinto
account
in
youranalysisthroughout the body and can vary in routes of clearance andActivatable, Targeted,
will Vascularprovide you with higher precision and improved data interpretation.
kinetics of background Interference. In some cases (depending
αvβ3 Integrin
on the intensity of the signal
atthe target) more that 50% of the
VIEW
DRUG RESISTANCEREFERENCES
signal in the target region could
Bombesin
be attributed to backgroundcontribution. This means that some
sort of objective and consistent
Elastase
INFLAMMATION
approach for correcting data forbackground contribution is needed
Hydroxyapatite
for proper interpretation.
CELLThis
DEATH
Transferrin
is particularly importantwhen

making other calculations, such as %inhibition, background, or ratios of
two different imaging probes.
Figure 3. Proper image analysis can greatly impact interpretation of study results.
RESOURCESCONTACT US.

RESOURCES
TUMOR
FolateBESTBURDEN
PRACTICES IN FLUORESCENCE
MOLECULAR IMAGINGPREVIOUSNEXT

MMP
METABOLIC ACTIVITY
CathepsinBEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING

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PROTOCOL FOR MONOPLEX, MULTIPLEX,
Single Probe Imaging
AND LONGITUDINALIMAGING WITH
Carbonic
Anhydrase IXPROBES
VIEW
APPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)
IVISENSE FLUORESCENTANGIOGENESIS
1. Prepare the imaging probe according to included instructions. Note
Phosphatidylserine

Study Preparation

PROBE REFERENCE TABLES

that systemic injection is required, either through the retro-orbital
plexus or the tail vein.

2. Place a heating pad beneath the anesthesia induction chamber to keep

VIEW
METASTASIS
the body temperature
the mice constant. Be careful not to overheat.
Characterizationof the full portfolioof IVISense ofprobes:
1. Two weeks before theEndothelium
imaging study, switch mice to low
fluorescence

Anesthetize the first mouse by placing it in a gas anesthesia induction
Activatable,chow. Regular mouse chow contains chlorophyll that auto
fluoresces Targeted, Vascularchamber.
around 700 nm whichαvβ3
can Integrininterfere with imaging.
3. Remove the mouse from the induction chamber when it appears
2. On the study day, it is essential to prepare ahead of time for optimal
VIEW
DRUG RESISTANCEREFERENCES
completely anesthetized, and confirm the depth of anesthesia throughresults. Group and number the mice to be injected andimaged. Remember
Bombesinunresponsiveness to toe pinch.
that appropriate study design should include both positive control andnegative control (i.e. un-diseased) mice injected with probe(s).
3. Mouse hair removal isElastaseessential for sensitive, high quality fluorescence

INFLAMMATION
imaging. Either genetically hairless mice (SKH-1E) or normal, haired

mice (BALB/c, C57BL/6,Hydroxyapatiteetc.) with depilation must be used for optimal
fluorescence tomographic imaging. Follow the depilation procedure above
for haired mice.
CELL
Transferrin
4. DEATH
Establish the readinessof the imaging system by checking the anesthesia

chamber and connections to the system. Activate the anesthesia, setting
evaporator to the appropriate settings for your particular set-up.
5. Make sure you know ahead of time the proper positioning of the mouse

you will use to facilitate acquisition of the best quality data for yourparticular animal model.

RESOURCESCONTACT US.

4. Inject the appropriate volume of probe (100 - 150 μL, as per eachprobe’s specific instructions) via the retro-orbital plexus (or tail vein) of
the anesthetized mouse. Record the injection time.
5. Return the mouse to the cage for recovery and go to the next mousefor injection.
6. Repeat steps 1 - 6 until all mice are injected.
7. Imaging is performed at the suggested time(s) using a single

excitation/emission filter pair optimal for the wavelength of the probe
wavelength of the probe to be imaged (see table). Anesthetize miceusing inhaled anesthesia and place them carefully in the appropriateorientation in the imaging system. Multiple images can be acquiredwith little or no concern for photobleaching of the probes.

RESOURCES
TUMOR
FolateBESTBURDEN
PRACTICES IN FLUORESCENCE
MOLECULAR IMAGINGPREVIOUSNEXT

MMP
METABOLIC ACTIVITY
Cathepsin

Two Probe Imaging

BEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING

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Longitudinal Imaging

Carbonic
1. PerkinElmerprobes are wellcharacterizedwith respect to tissue
1. Prepare the first imaging
probeIXas described in thatAPPROACHES
probe’s instructions.
Anhydrase
TO FLUORESCENCE
MOLECULARIMAGING(ILLUSTRATION)ANGIOGENESIS

Note that systemic injection is required, either through the retro-orbital
plexus or the tail vein.Phosphatidylserine

PROBE REFERENCE TABLES

VIEW

clearance kinetics, providing guidance for longitudinal imaging
strategies. Depending on the probe, reinjection generally can be
performed three to seven days following the first image acquisition.

To minimize injection volume, either use the Probe 1 solution to solubilize
2. Imaging bone turnover with IVISense Osteo 680 requires a different
VIEW
METASTASISEndothelium
Characterization
of
the
full
portfolio
of
IVISense
probes:
Probe 2 or make each probe at half-volumes for mixing. (Note: Bearstrategy due to the very long tissue clearance kinetics. Secondary imaging
Activatable,
Targeted,
Vascularin mind that three of the probes come in 10X liquid form: IVISense
time points must be performed using a pre-imaging strategy; briefly,
Annexin-V 750, MMP αvβ3680, Integrin
and Pan Cathpsin 680). Multiple specificmice should be imaged immediately prior to each additional probe
strategies for preparation are possible, but it is ideal to keep mouse
injection to allow subtraction correction of additional imaging datasets.
VIEW
DRUG RESISTANCE
injection volumes under 250 μL.
REFERENCES
Bombesin

2. Prepare and inject mice as described above in Single Probe Imaging.
3. Imaging should be performed as described in step 7 above, but for both

Elastase
680 nm and 750 nm using
the appropriate excitation and emission filter
INFLAMMATION
pairs. Correct times for acquisition should be noted; although most of theprobes are optimal for
24 h imaging, some have flexibility to be imaged
Hydroxyapatite
earlier, while others should only be imaged earlier. For example, IVISense
Annexin-V 750 imaging is optimal for most applications at 2 hours,
CELL DEATH
whereas Pan Cathepsin
680 is optimal at 24 hours. You can either image
Transferrin
both wavelengths at both 2 hours and 24 hours, or you can image 750 nm
at 2 hours and 680 nm at 24 hours.
4. Multiple repeat acquisitions can be performed with little or no concern forphotobleaching of the probes.
RESOURCESCONTACT US.

RESOURCES
TUMOR
BURDEN
FolateAPPROACHESTO FLUORESCENCE
MOLECULAR IMAGINGMMPMETABOLIC ACTIVITYANGIOGENESISCathepsinBEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING

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Carbonic
Anhydrase IXAPPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)

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Phosphatidylserine

PROBE REFERENCE TABLESMETASTASISEndothelium

Characterization of the full portfolio of IVISense probes:
Activatable, Targeted, Vascular

VIEW

αvβ3 Integrin

REFERENCES

DRUG RESISTANCE
Bombesin

Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US*Multiplex Imaging (See “Two Probe Imaging” in Best Practices in Fluorescence Molecular Imaging for a detailed protocol.).

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PROBE REFERENCE TABLE— ACTIVATABLE PROBESTUMOR BURDENPREVIOUS

NEXT

FolateNOTE: Blood t1/2 in hours or fractions of hours

Cat B 680 FAST
Cat B 750 FAST
Cat K 680 FAST

NEV11100

MMP 645 FASTDRUG RESISTANCE

8,500

MMP 680

NEV10168

MMP 750 FASTElastase

Cathepsin K

Bombesin~450,000

Cleaved by multiple metalloproteinases
REFERENCES

associated with many disease areas including
cancer and inflammation.

43,000

Neutrophil Elastase
43,000680 FAST
Hydroxyapatite

NEV10003

Pan Cathepsin 680

Neutrophil
Elastase

Selectively cleaved by elastase produced by
activated neutrophils during acute inflammation.

~400,000

Transferrin

NEV10001EX

Pan Cathepsin 750~450,000

NEV11171

Pan Cathepsin 750
FAST

22,500

NEV11079

Renin 680 FAST

43,000RESOURCES

Matrix
Metalloprotease

Cathepsin
proteases

Renin
Protease

atherosclerosis)

nmol

4 nmol; (8 nmol
atherosclerosis)

12
nmol

Pan-cathepsin activatable agent that detects
abnormal upregulation of cathepsin secretion
associated with cancer and inflammation.

Activated by normal kidney renin and upregulated
renin-angiotensin system (RAS) activity.

CONTACT US

.

IV

1

36

6-24

3d

IV

1

36

6-24

3d

Salivary glands >liver, kidneys* Animal injection times should be staggered to meet imaging timepoint(s) +/- 0.5 h.

1/2

36

5

72

4 nmol12-40nmol

2 nmol

6-20
nmol

IV

5

72

2 nmol

6-20
nmol

IV

5

72

4 nmol

12-40
nmol

IV

3

12

2 nmol

6-20
nmol

IV

12

72

2 nmol

6-20
nmol

IV

5

4 nmol

12-40
nmol

IV

2 nmol

6-20
nmol

IV

Activatable probes are optically silent upon injection and become highly fluorescent following protease-mediated activation.

VIEW

SalivaryVIEW
glands >liver, kidneys

Selectively cleaved by cathepsin K proteinases
Characterization of the full portfolio of IVISense probes:
6-20
upregulated in osteoclasts and tumor-associated
2 nmolIV
nmol
Activatable,
Targeted,
Vascularmacrophages.

43,000

NEV11169

CELL DEATH

Selectively cleaved by cathepsin B proteinases
upregulated in tumors and inflammatory cells.

PROBE REFERENCE TABLES

αvβ3 Integrin

NEV10126

INFLAMMATION

Cathepsin B

23,000Endothelium

NEV11000APPROACHES TO FLUORESCENCE MOLECULARIMAGING(ILLUSTRATION)
2 nmol; (4 nmol
6-20

33,000

Phosphatidylserine

BEST
IN FLUORESCENCE MOLECULAR IMAGINGIn VivoPRACTICES
Biology Usee ofRout bolism/
Meta ground
Backue(s)Tiss

Carbonic
Anhydrase IX

e of
Rout

METASTASISSpecificity

50g)

NEV11098

MW(g/mol)5g)

ANGIOGENESIS
NEV11112

IVISense Probe
Cathepsin

(2
ose
Rat D

Cat #

(2
ose
se D
Mou

METABOLIC ACTIVITY

on
jecti
Rein letemalOpti – CompTime rance
Clea
ing
Imag
mal
Opti (h)*
Time
)
/2 (h
ue t1
Tiss
2 (h)
d t1/
Bloo
tion
Injec

MMP

IV

VIEW

6-24

3d

Kidney > liver

24

6-7 d

Liver > kidneys(6-24)

VIEW

24

6-7 d

Liver

24

6-7 d

Liver > kidneys

3-6

2d

Bladder > liver,intestines

24

6-7 d

Liver

72

24

6-7 d

Low liver,intestine

1

36

6-24

3d

Low liver, bladder

7

48

24

4d

Kidney, lung, liver(12-24)(24-36)(24-48)(12-24)

PROBE REFERENCE TABLE— TARGETED PROBESTUMOR BURDENRESOURCESPREVIOUS

NEXT

FolateNOTE: Blood t1/2 in minutes

NEV10090

Bombesin Receptor
680

35,000

Phosphatidylserine

METASTASIS

24,000Endothelium

NEV10040

Folate Receptor 680

NEV11070

Hypoxia CA IXαvβ3
680 Integrin
1,500

DRUG RESISTANCE

1,606

NEV10640

Integrin Receptor
645
1,250
Bombesin

NEV10645

Integrin Receptor 680

1,432

Elastase

NEV10873
Integrin Receptor 750
INFLAMMATION

1,278

NEV10020EX

Osteo 680

Hydroxyapatite
1,471

NEV10053EX

Osteo 750

1,101

NEV11105

Osteo 800

1,281

NEV10060

Tomato Lectin 680~72,000

CELL DEATH

Transferrin
NEV10091
RESOURCES
Receptor 750

Transferrin

106,000

Phosphatidylserine exposure on cells
Bombesinreceptor
Folate receptor

Targeting of membraneTO
phosphatidylserineAPPROACHESFLUORESCENCE
MOLECULAR IMAGING (ILLUSTRATION)
300

exposed on cells undergoing apoptosis/necrosis/
100 μLIV
μL
other forms of cell death.
Detects upregulation of bombesin receptors on tumor
6-20
2 nmolIVPROBE
REFERENCE
TABLES
cells associated with increased tumor proliferation.
nmol
Detectionof folate receptor ofproteinfor
6-20
Characterization
theupregulation
full portfolio
2 of
nmolIVISense probes: IVmonitoringtumor growthand metabolism.
nmol
Activatable,
Targeted,
VascularDetection of carbonic anhydrase 9 (CA IX) protein
Carbonic
6-20
expressed in many tumor cell lines undergoing
2 nmolIV
Anhydrase IX
nmol
hypoxia and acidosis.
REFERENCES
6-20
IV
2 nmolnmol (IP)αVβ3
Detection of integrin αVβ3 expression in tumors
6-20
IV
2 nmolIntegrin
and neovasculature.
nmol (IP)12-40
IV
2 nmolnmol (IP)6IV
2 nmolnmol (IP)Binds exposed hydroxyapatite (HA), a biomarker
Hydroxyfor osteoblast activity; abnormal HA levels can
12IV
4 nmolapatiteindicate bone damage/disease, such as in
nmol (IP)metastasis.
6IV
2 nmolnmol (IP)
Vascular burden assessment through binding ofEndothelial
6glycoprotein N-actylglucosamines on the surface of
2 nmolIV
glycoproteinsnmolvascular endothelial cells.
Transferrin
Detects upregulation of transferrin receptors on
6-20
2 nmolIV
receptor
tumor cells associated with tumor metabolism.
nmol

e of
Rout bolism/
Meta ground
Backue(s)TissAnnexin-V 750

e of
Rout

NEV11053

BEST
IN FLUORESCENCE MOLECULAR IMAGINGIn VivoPRACTICES
Biology Use
50g)

Carbonic
Anhydrase IX

Specificity

5g)

ANGIOGENESIS

MW(g/mol)IVISense Probe
Cathepsin

(2
ose
Rat D

Cat #

(2
ose
se D
Mou

METABOLIC ACTIVITY

on
jecti
Rein letemalOpti – CompTime rance
Clea
ing
Imag
mal
Opti (h)*
Time
)
/2 (h
ue t1
Tiss
2 (h)
d t1/
Bloo
tion
Injec

MMP

VIEW

VIEW

15

14

2

3d

Kidneys (high),
liver

90

96

24

6-7 d

Pancreas,kidneys

5

>168

6

6-7 d

Kidneys

2

12

24

6-7 d

Kidneys

10

48

6-24

6-7 d

Bladder, kidneys

10

24

24

14 d

Kidneys,intestines

30

24

24

4-6 d

Kidneys

5- ~30
10 days3-24

Preimage
subtraction

Bladder

5- 7-1010 days3-24

Preimage
subtraction

Bladder

5- 7-1010 days3-24

Preimage
subtraction

Bladder

2

>48

6

4-6 d

Overall vascular
background

10

24

24

96 h

Liver, kidney

Targeted probes actively target and bind to distinct biomarkers with highly specific targeting to key biological mechanisms.
CONTACT
US injection. times should be staggered to meet imaging timepoint(s) +/- 0.5 h.
* Animal** Some probes can be administered IP, but they may leave a residual peritoneal signal that could interfere with imaging.

(6-24)(6-24)VIEW

VIEW

RESOURCES

PROBE REFERENCE TABLE— VASCULAR & PHYSIOLOGICAL PROBESTUMOR BURDENPREVIOUS

NEXT

FolateNOTE: Blood t1/2 in hours or fractions of hours

Edema 680

Endothelium~1,540αvβ3 Integrin

NEV11121
Gastrointestinal 750
DRUG RESISTANCE~40,000

NA
Passive;
non-specificsystemic
circulationNA

GFR 680~6,000NA

Elastase
INFLAMMATION

NEV10054EX

Vascular 680
Hydroxyapatite~70,000

NEV10011EX
CELL DEATH

NEV10149

NEV10150

Vascular 750
Transferrin

Vascular NP 680
Vascular NP 750~4,000,000(20-50 nm
diameter)

Small molecule fluorescent agent that can be used
as a control for vascular permeability imaging.

50 μg

150500 μg

IV

1.5 5-103

96 hBladder

Kinetic
5 min3h

3-5 h

Stomach,
VIEW
intestines

PROBE REFERENCE TABLES
Characterizationof theportfolio of IVISense probes:
Imagingof vascularity, perfusion,
andfullvascular
12-40
4 nmolIVpermeability.
Short pharmacokinetic
profile.
nmol
Activatable,
Targeted, Vascular
Monitoring of gastric emptying/motility.

0.25 nmol

0.75OralNA
2.5
gavagenmol

Quantitative assessment of renal Glomerular
Filtration Rate (GFR), as an indicator of renal
toxicity or injury.

2 nmol6-20
nmol

REFERENCES

Passive;
non-specificsystemic
circulation

Imaging of vascularity, perfusion, and vascularpermeability.

2 nmolPassive;
non-specificsystemic
circulation

Imaging of vascularity, perfusion, and vascularpermeability. Long pharmacokinetic profile.

200 μL6-20
nmol3001000
μL

IV

NA

(1-3)

Bladder

VIEW

See protocol on website to inKidneys
clude Optimal Reinjection Time.
24

7
IV

72
16

IV

VIEW

0.5

0.2

Not determined

APPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)

Bombesin

NEV30000

VIEW

Not determined

NEV10116

Acute VascularPhosphatidylserine
750
1,086
(1mg, 5mg)e ofRout bolism/
Meta ground
Backue(s)Tiss

METASTASIS(1mg, 5mg)

e of
Rout

NEV10118,
NEV10177

Carbonic
IX
Acute VascularAnhydrase
680

1,646

BEST
In VivoPRACTICES
Biology Use IN FLUORESCENCE MOLECULAR IMAGING
50g)

NEV10130

Specificity

5g)

NEV10117,ANGIOGENESIS

MW(g/mol)IVISense Probe
Cathepsin

(2
ose
Rat D

Cat #

(2
ose
se D
Mou

METABOLIC ACTIVITY

on
jecti
Rein letemalOpti – CompTime rance
Clea
ing
Imag
mal
Opti (h)*
Time
)
/2 (h
ue t1
Tiss
2 (h)
d t1/
Bloo
tion
Injec

MMP

20 >100(imagevasculaturefor up to
4h; Tumor
accumulation
at 24h)24

6-7 d

Preimage
subtraction

Low liver ling

Long term tissue
accumulation

Vascular & Physiological Probes are a range of highly fluorescent in vivo imaging molecules that remain highly stable and localized in the anatomy for various
RESOURCES
periods
of time to enable imaging of disease physiology, vasculature, vascular permeability, and angiogenesis.
IVISense Vascular NP 680 & 750 probes are highly fluorescent near infrared nanoparticles specifically designed for in vivo imaging. They remain localized in thevasculature
forUS
extended
CONTACT. periods and enable imaging of blood vessels and angiogenesis.
* Animal injection times should be staggered to meet imaging timepoint(s) +/- 0.5 h.

RESOURCES
TUMOR
BURDEN
FolateLITERATURE
REFERENCESPREVIOUSNEXT

MMP
METABOLIC
ACTIVITY
Ackermann
et al. Adnectin CT-322 inhibits tumor growth and affects microvascular architecture and function in Colo205 tumor xenografts. Int J Oncol. 2011;BEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING
Cathepsin
Jan;38(1):71-80.

VIEW

CarbonicAl Tameemi Wafaa, Dale Tina
P., Al-Jumaily
Rakad M. Kh, Forsyth Nicholas R. Hypoxia-modified cancer cell metabolism. Frontiers in Cell and Developmental Biology
Anhydrase
IX
VIEW
APPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)ANGIOGENESIS
2019; 7:4.
Phosphatidylserine

Amrahli M, Centelles M, Cressey P, Prusevicius M, Gedroyc W,
Xu XY,REFERENCE
So PW, Wright TABLES
M, Thanou M. MR-labelled liposomes and focused ultrasound for spatiotemporally
PROBE
controlled drug release in triple negative breast cancers in mice. Nanotheranostics. 2021; 5(2):125-142.
METASTASISEndothelium

Characterization of the full portfolio of IVISense probes:
Activatable, Targeted, Vascular

VIEW

Andreucci E, et al. The carbonic anhydrase inhibitor SLC-0111 sensitises cancer cells to conventional chemotherapy. J Enzyme Inhib Med Chem 2019; 34(1):117-123.
αvβ3 Integrin

Bao RESISTANCE
B, Groves K, Zhang J, Handy E, Kennedy P, Cuneo G, etREFERENCES
al. In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker
of
VIEW
DRUG
Bombesin
Tumor Hypoxia. PLoS ONE. 2012;
7(11): e50860.
Elastase
Brown et al. Altered Iron Metabolism
and Impact in Cancer Biology, Metastasis, and Immunology. Front Oncol. 2020;10:476.
INFLAMMATION
Hydroxyapatite
Carlos R. Neutrophil: A Cell with
Many Roles in Inflammation or Several Cell Types? Frontiers in Physiology, 2018; Vol. 9.

Dayoub,A.S., Brekken, R.A. Transferrin
TIMs, TAMs, and PS- antibody targeting: implications for cancer immunotherapy. Cell Commun Signal. 2020; 18, 29.
CELL DEATH
Elliot A, Myllymaki H, Feng Y. Inflammatory Responses during Tumor Initiation: From Zebrafish Transgenic Models of Cancer to Evidence from Mouse and Man. Cells.
2020;9(4):1018.
Gao X, Lampraki EM, Al-Khalidi S, Qureshi MA, Desai R, et al. N-acetylcysteine (NAC) ameliorates Epstein-Barr virus latent membrane protein 1 induced chronic
RESOURCES
inflammation. 2017; PLoS ONE 12(12).
CONTACT US.

RESOURCES
TUMOR
BURDEN
FolateLITERATURE
REFERENCESPREVIOUSNEXT

MMP
METABOLIC
ACTIVITY
Greten FR,
Grivennikov SI. Inflammation and Cancer: Triggers, Mechanisms, and Consequences. Immunity. 2019;51(1):27-41.
BEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING
Cathepsin

VIEW

Hamidi, H., Ivaska, J. Every step of the way: integrins in cancer progression and metastasis. Nat Rev Cancer. 2018; 18:533-548.
Carbonic
Anhydrase IXANGIOGENESISAPPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)

VIEW

Hoff BA, Chughtai K, Jeon YH, et al. Multimodality imaging of tumor and bone response in a mouse model of bony metastasis. Transl Oncol. 2012;5(6):415-421.
Phosphatidylserine

PROBE
REFERENCE
Jarr K, et al. Effect of CD47 Blockade on Vascular Inflammation.
N EnglJ Med, 2021;TABLES
384:382-383.
Characterization of the full portfolio of IVISense probes:
Activatable,
Vascular distinct modes of cell death. Cell Death Discovery. 2019; 5: 65.
Klöditz, K., Fadeel, B. Three cell deaths and a funeral: macrophage
clearanceTargeted,of cells undergoing
METASTASISEndothelium

VIEW

αvβ3 Integrin

Kozloff, K.M., Weissleder, R. and Mahmood, U., Noninvasive Optical Detection of Bone Mineral. J Bone Miner Res. 2007; 22:1208-1216.

REFERENCES

DRUG RESISTANCE

VIEW

Bombesin

Linkermann, A., & Green, D. R. Necroptosis. The New England Jrnl of Med. 2014; 370(5): 455-465.
ElastaseLiu, X., Yang, W., Guan, Z. et al. There are only four basic modes of cell death, although there are many ad-hoc variants adapted to different situations. Cell Biosci. 2018;
INFLAMMATION8, 6.

Hydroxyapatite

Lo Dico A, Costa V, Martelli C, Diceglie C, Rajata F, Rizzo A, Mancone C, Tripodi M, Ottobrini L, Alessandro R, Conigliaro A. MiR675-5p Acts on HIF-1α to Sustain Hypoxic
CELL DEATHA New Therapeutic
Transferrin
Responses:
Strategy for Glioma. Theranostics. 2016; 8;6(8):1105-18.
Low PS, Henne WA, Doorneweerd DD. Discovery and Development of Folic-Acid-Based Receptor Targeting for Imaging and Therapy of Cancer and Inflammatory
Diseases. Acc Chem Res. 2008; 41:120-129.
Mantovani,A., Allavena, P., Sica, A. et al. Cancer-related inflammation. Nature. 2008; 454:436-444.
RESOURCESCONTACT US.

RESOURCES
TUMOR
BURDEN
FolateLITERATURE
REFERENCESPREVIOUSNEXT

MMP
METABOLIC
ACTIVITY
Nakashima
et al., ApoE-deficient Mice Develop Lesions of All phases of Atherosclerosis Throughout the Arterial Tree, Arterioscler Thromb. 1994; 14(1):133-140.
BEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING
Cathepsin

VIEW

Nouh, M.A., Mohamed, M.M., El-Shinawi, M. et al. Cathepsin B: a potential prognostic marker for inflammatory breast cancer. J Transl Med. 2011; 9, 1.
Carbonic
Anhydrase IXANGIOGENESISAPPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)

VIEW

Ottewell PD. The role of osteoblasts in bone metastasis. J Bone Oncol. 2016;5(3):124-127.
Phosphatidylserine

PROBE
REFERENCE
Peterson et al., Multiplex 2D Imaging of NIR Molecular Imaging
Agentson the IVISRTABLES
SpectrumCT and FMT 4000. Application Note, PerkinElmer Inc.
VIEW
Characterization of the full portfolio of IVISense probes:
Activatable,
Targeted,Peterson, J. et al. Fluorescence imaging of bombesin and transferrin
receptor
expressionVascular
is comparable to 18F-FDG PET in early detection of sorafenib-induced changes in
METASTASISEndothelium

tumor metabolism. PLoS One.
2017;
12(8): e0182689.
αvβ3IntegrinPeterson, JD. Non-Invasive Quantitative In Vivo Imaging ofREFERENCES
Atherosclerosis Disease Progression and Treatment Response in ApoE Deficient Mice using Fluorescent
Bombesin
Molecular Tomography and NIR Fluorescent Pre-clinical Imaging Agents. Application Note. PerkinElmer, Inc.

DRUG RESISTANCE

VIEW

Elastase

Rajabi M, Mousa SA. The Role of Angiogenesis in Cancer Treatment. Biomedicines. 2017; Jun 21;5(2):34

INFLAMMATION

Hydroxyapatite

Rakoff-Nahoum S. Why cancer and inflammation? Yale J Biol Med. 2006; 79(3-4):123-30.
CELL DEATHI., Speir, M. & Gerlic,Transferrin
Shlomovitz,
M. Flipping the dogma – phosphatidylserine in non-apoptotic cell death. Cell Commun Signal. 2019; 17:139.

Smigiel KS, Parks WC. Matrix Metalloproteinases and Leukocyte Activation. Prog Mol Biol Transl Sci. 2017;147:167-195.
Tapia-Vieyra JV, Delgado-Coello B, Mas-Oliva J. Atherosclerosis and Cancer; A Resemblance with Far-reaching Implications. Archives of Medical Research, 2017; 48(1): 1226
RESOURCESCONTACT US.

RESOURCES
TUMOR
BURDEN
FolateLITERATURE
REFERENCESPREVIOUSNEXT

MMP
METABOLIC
Tafreshi ACTIVITY
NK, Lloyd MC, Bui MM, Gillies RJ, Morse DL. Carbonic anhydrase IX as an imaging and therapeutic target for tumors and metastases. Subcell Biochem.
BEST PRACTICES IN FLUORESCENCE MOLECULAR IMAGING
Cathepsin
2014;75:221-254.

VIEW

Carbonic

Tseng et al. Fluorescence imaging
of bombesin
and transferrin receptor expression is comparable to 18F-FDG PET in early detection of sorafenib-induced changes in
Anhydrase
IX
VIEW
APPROACHES TO FLUORESCENCE MOLECULAR IMAGING (ILLUSTRATION)ANGIOGENESIS
tumor metabolism. PLoS ONE. 2017; 12(8): e0182689.
Phosphatidylserine

Vasquez, Kristine O., Peterson, Jeffrey D. Combined efficacy
and toxicity
imaging following
PROBE
REFERENCE
TABLESacute 5-FU treatment of HT-29 tumor xenografts. Poster; PerkinElmer Inc.

Characterization of the full portfolio of IVISense probes:
Wang X, Zhang H, Chen X. Drug resistance and combating drug
resistance inTargeted,cancer. Cancer
Drug Resist. 2019;2:141-160.
Activatable,
Vascular
METASTASISEndothelium

VIEW

αvβ3 Integrin

Welch , Danny R. and Hurst, Douglas R. Defining the Hallmarks of Metastasis Cancer Res. 2019;79(12):3011-3027.

REFERENCES

DRUG RESISTANCE
Bombesin

Wu L, Saxena S, Awaji M, Singh RK. Tumor-Associated Neutrophils in Cancer: Going Pro. Cancers (Basel). 2019;11(4):564.
Elastase
INFLAMMATION
Hydroxyapatite

CELL DEATH

Transferrin

RESOURCESCONTACT US

.

VIEW

CONTACT US
TUMOR BURDEN

FolateMMPMETABOLIC ACTIVITY
Cathepsin
Carbonic
Anhydrase IXANGIOGENESIS

Phosphatidylserine

METASTASISEndothelium
αvβ3 Integrin

DRUG RESISTANCE
Bombesin

We can help you design your fluorescence experiment, choose the rightprobe, or interpret your data. Talk to a scientist before starting your next
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Elastase
INFLAMMATION

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Hydroxyapatite

CELL DEATH

Transferrin

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