Wavelength-dependent backscattering measurements for quantitative monitoring of apoptosis, part 1: early and late spectral changes are indicative of the presence of apoptosis in cell cultures.

Abstract

Apoptosis, a form of programmed cell death with unique morphological and biochemical features, is dysregulated in cancer and is activated by many cancer chemotherapeutic drugs. Noninvasive assays for apoptosis in cell cultures can aid in screening of new anticancer agents. We have previously demonstrated that elastic scattering spectroscopy can monitor apoptosis in cell cultures. In this report we present data on monitoring the detailed time-course of scattering changes in a Chinese hamster ovary (CHO) and PC-3 prostate cancer cells treated with staurosporine to induce apoptosis. Changes in the backscattering spectrum are detectable within 10 min, and continue to progress up to 48 h after staurosporine treatment, with the magnitude and kinetics of scattering changes dependent on inducer concentration. Similar responses were observed in CHO cells treated with several other apoptosis-inducing protocols. Early and late scattering changes were observed under conditions shown to induce apoptosis via caspase activity assay and were absent under conditions where apoptosis was not induced. Finally, blocking caspase activity and downstream apoptotic morphology changes prevented late scattering changes. These observations demonstrate that early and late changes in wavelength-dependent backscattering correlate with the presence of apoptosis in cell cultures and that the late changes are specific to apoptosis.

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Wavelength-dependent backscattering
measurements for quantitative
monitoring of apoptosis, Part 1: early and
late spectral changes are indicative of the
presence of apoptosis in cell cultures
Christine S. Mulvey
Kexiong Zhang
Wei-Han Bobby Liu
David J. Waxman
Irving J. Bigio
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Journal of Biomedical Optics 16(11), 117001 (November 2011)
Wavelength-dependent backscattering measurements
for quantitative monitoring of apoptosis, Part 1: early
and late spectral changes are indicative of the presence
of apoptosis in cell cultures
Christine S. Mulvey,aKexiong Zhang,bWei-Han Bobby Liu,aDavid J. Waxman,band Irving J. Bigioa,c
aBoston University, Department of Biomedical Engineering, 44 Cummington Street, Boston, Massachusetts 02215
bBoston University, Department of Biology, 5 Cummington Street, Boston, Massachusetts 02215
cBoston University, Department of Electrical and Computer Engineering, 8 St. Mary’s Street, Boston,
Massachusetts 02215
Abstract. Apoptosis, a form of programmed cell death with unique morphological and biochemical features, is
dysregulated in cancer and is activated by many cancer chemotherapeutic drugs. Noninvasive assays for apoptosis
in cell cultures can aid in screening of new anticancer agents. We have previously demonstrated that elastic
scattering spectroscopy can monitor apoptosis in cell cultures. In this report we present data on monitoring the
detailedtime-courseofscatteringchangesinaChinesehamsterovary(CHO)andPC-3prostatecancercellstreated
with staurosporine to induce apoptosis. Changes in the backscattering spectrum are detectable within 10 min, and
continue to progress up to 48 h after staurosporine treatment, with the magnitude and kinetics of scattering changes
dependent on inducer concentration. Similar responses were observed in CHO cells treated with several other
apoptosis-inducing protocols. Early and late scattering changes were observed under conditions shown to induce
apoptosis via caspase activity assay and were absent under conditions where apoptosis was not induced. Finally,
blockingcaspaseactivityanddownstreamapoptoticmorphologychangespreventedlatescatteringchanges.These
observations demonstrate that early and late changes in wavelength-dependent backscattering correlate with the
presence of apoptosis in cell cultures and that the late changes are specific to apoptosis.C ?2011 Society of Photo-Optical
Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3644389]
Keywords: backscattering; spectroscopy; fiber optic sensors.
Paper 11400R received Jul. 25, 2011; revised manuscript received Sep. 5, 2011; accepted for publication Sep. 8, 2011; published
online Oct. 28, 2011.
1
Apoptosis, a regulated form of cell death, is implicated in the
selective deletion of cells in certain disease states, where a bio-
logical role for deletion is evident.1,2A wide variety of stimuli
and conditions, both physiological and pathological, can trigger
apoptosis. These stimuli include: DNA damage, intracellular
damage, toxins, extracellular signals, heat, free radicals, growth
factor withdrawal, cytokines, loss of matrix attachment, gluco-
corticoids, nitric oxide, oxidative stress, and radiation.3
The role of apoptosis in cancer is so prominent that its eva-
sion is considered to be one of the fundamental hallmarks of
cancer.4Further, it is now well-documented that many cyto-
toxic anticancer agents induce apoptosis, raising the possibility
that defects in the apoptotic programs contribute to treatment
failure.5–7Reed may have put it best: “when it comes to the
successful eradication of cancer cells by nonsurgical means, ul-
timately, all roads lead to apoptosis. Essentially all cytotoxic
anticancer drugs [and other cancer therapies] currently in clini-
cal use, when they work, induce apoptosis of malignant cells”.8
Enhanced apoptosis is also responsible for many of the adverse
effects of chemotherapy in addition to tumor regression.9Due
to the central role of apoptosis in disease management, it would
Introduction
Address all correspondence to: Christine Mulvey, Boston University, Department
ofBiomedicalEngineering,44CummingtonStreet,Boston,Massachusetts02215;
Tel: 617-358-1519; E-mail: cmulvey@bu.edu.
be desirable to have a noninvasive method to serially detect and
monitor apoptosis in patients undergoing conventional cancer
treatments as well as for the development and testing of new
drugs.10
Thedevelopmentofanoninvasivedetectionmethodforapop-
tosis requires knowledge of the pathways and mechanisms that
allow for the biochemical and morphological changes of apop-
tosis. Many of these biochemical and morphological changes
resultfromtheactivationofaseriesofcysteineproteases,called
caspases, which cleave target molecules after certain aspar-
tate residues11and produce the morphological and biochemical
changes characteristic of apoptosis.12–14
Apoptosis is distinguished from other forms of cell death by
its unique micromorphology changes. Perhaps the most widely
recognized apoptotic morphology changes are those that occur
in the cell nucleus. During apoptosis, nuclear material (DNA
and other proteins) collects into condensed, coarsely granular
aggregates, which can be confluent over the entire nucleus, or
localized to crescent-shaped caps.15In most, but not all cases,
the nucleus then breaks up into discrete fragments, in which the
characteristic segmentation of chromatin is maintained.1,16
Dramatic changes in mitochondrial morphology, including
swelling17and shrinkage, can also occur during the early stages
of apoptotic cell death.18After treatment with apoptotic stimuli,
1083-3668/2011/16(11)/117001/10/$25.00 C ?2011 SPIE
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Mulvey et al.: Wavelength-dependent backscattering measurements...
mitochondrial christae remodel from a normal configuration to
a vesicular structure to release cytochrome c into the cytosol.
During this progression, the distribution of mitochondria is also
affected. Fragmentation of the mitochondrial network has been
described in connection with many modes of apoptosis induc-
tion. The mitochondrial network transitions from an intercon-
nected tubular structure to a punctiform, fragmented structure at
early stages of the apoptotic cascade.19
Apoptosis also inducesa processknownas apoptotic volume
decrease (AVD), which occurs before or coincidentally with
nuclear changes, and during which the cell cytoplasm progres-
sively condenses, presumably a consequence of an expulsion of
water.16AVD is accompanied by an increase in cellular density
and is often the earliest visible manifestation of apoptosis.
In vitro apoptosis measurements in cell culture are com-
monly employed in early stage research for screening potential
anticancer agents, using assay methods that exploit some of
the biochemical and morphological features of apoptosis de-
scribed above. These methods include: various types of optical
microscopy, gel electrophoresis, DNA nick-end labeling, mi-
tochondrial assays, and detection of apoptotic proteins. Some
assays utilize flow cytometry, which can quantify the apoptotic
population. All current methods, however, require either the ad-
dition of an exogenous stain or labeling agent, or the physical
disruption of the culture itself.20,21
An ideal assay for apoptosis would allow for assessment of
the apoptotic state of a population of cells without altering the
intracellular environment. Such a method would enable mon-
itoring of a single culture sample (or even a single cell) over
time, rather than requiring a new culture for each time point.
This would allow for a more accurate assessment of cellular
responses and minimize the effects of culture-to-culture biolog-
ical variability. In addition, it would be ideal for this detection
method to be sensitive to very early apoptotic changes, which
could help elucidate the specific apoptotic pathways activated
by the agents under investigation.
Recently, there has been interest in developing optical meth-
ods of detecting apoptosis in cell cultures due to their in-
herent noninvasiveness, with a longer-term goal of in vivo
measurements.22–27Specifically, our group has been develop-
ing elastic scattering spectroscopy (ESS) as a diagnostic tool
for apoptosis. ESS is sensitive to underlying cellular micromor-
phology at the organelle level,28–30so the dramatic morphology
changes of apoptotic cells make apoptosis an attractive target
for monitoring by this method. In recent publications,25,26we
have demonstrated that both angle- and wavelength-dependent
scattering can discriminate between apoptotic and normally
propagating cells. Wavelength-dependent backscattering mea-
surements can detect changes in scattering from Chinese
hamster ovary (CHO) cells 10 to 15 min after treatment with
the apoptosis-inducing agent staurosporine.26Though we are
interested in sensing changes in scattering due to apoptosis as
early in the process as possible, we also wished to investigate
the possibility of later scattering changes, particularly since the
process can progress in culture for 24 h or more. Furthermore,
when developing a new detection method for apoptosis, it is
important to determine whether the new technique is specific to
the apoptotic process.
Having identified a reliable optical signal, we conducted a
seriesofexperimentstodeterminewhethertheobservedchanges
SpectrometerLaptop
Xenon Arc
Lamp
Illuminating Fiber
100 µm
Collecting Fiber
100 µm
sca=179° ±0.6°
Water Bath
37° C
12 Well Plate
Fig. 1 Schematic of instrumentation to measure the wavelength de-
pendence of backscattered light from cell cultures. Broadband light
was delivered to an adherent cell culture by a fiber optic probe. Light
scattered at approximately 179 deg was collected and analyzed.
in optical signature were, in fact, specifically due to apoptosis.
This paper reports on that series of experiments, which verify
the specific correlation of the optical signal with the apoptotic
process. A discussion of the possible biological sources of the
earlyscatteringchangesispresentedinacompanionpublication.
2
2.1
Wavelength-dependent measurements in the near-backward di-
rection were made with instrumentation previously described
and validated.26A schematic is presented in Fig. 1. Briefly,
broadband light from a xenon arc lamp (Hamamatsu, LC5,
L8253) was delivered to cell cultures plated in glass-bottomed
12-well plates (Mat-Tek Corp., P12G-1.5-14-F) using a fiber
probe. The probe consisted of six 100-μm illumination fibers
arranged in a circularly-symmetric pattern around a single
100-μm collection fiber. The probe was positioned above the
sampleatadistancesuchthatthescatteredlightwascollectedat
an angle of approximately 179 deg. A kinematic mount allowed
access to each of the plate’s 12 wells with precise repositioning.
The unpolarized scattered light was transmitted to a spectrom-
eter (Ocean Optics, USB4000) and analyzed. The sample plate
was positioned in a temperature-controlled water bath (37◦C)
with a black surface, both to help maintain culture conditions
and to minimize collected background light.
Two separate measurements were required for each
sample: 1. Isample, the raw spectrum collected from a cell culture
and2.Ibackground,the spectrumcollectedusingall componentsof
thesystemexcludingthecells.Additionally,aspectralresponse,
Ispec.resp., was acquired using a spectrally-flat diffuse reflector
(Spectralon, Labsphere, Inc.), to account for the spectral char-
acteristics of the system. Pixelation noise was reduced using
boxcar smoothing with a width of eight pixels corresponding to
the resolution of the spectrometer (∼1.5 nm). The spectrum of
each cell sample was calculated using the following formula:
Ispectrum= (Isample− Ibackground)/Ispec.resp.. To eliminate depen-
dence on the intensity of incident light or the number of cells
interrogated, the spectra were normalized to the area under the
curve.
Experiment Design
Instrument Design and Measurement Protocol
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Mulvey et al.: Wavelength-dependent backscattering measurements...
2.2
Correlation of ESS Changes to Drug
Concentration
A first measure of specificity for apoptosis was performed to
determine if the observed optical signal is caused by the addi-
tionofstaurosporine.Varyingtheconcentrationofstaurosporine
should alter the extent of apoptosis in a culture. The optical re-
sponse is expected to correlate with the percentage of apoptotic
cells, and if the optical signal is correlated with apoptosis, the
kinetics of optical changes should also increase with increas-
ing dose. To assess this, we performed scattering measurements
in the near-backward direction on CHO cells and confirmed
the occurrence of apoptosis with a standard assay of caspase
activity.
CHO cells were plated in polystyrene culture dishes and
grown in Dulbecco’s modification of eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum and 1% penicillin
and streptomycin. Atmosphere was held at 37◦C and 5% CO2.
Cellswereplatedinglass-bottomed12-wellplatesandgrown
to confluence. At the beginning of the experiment, the growth
medium was aspirated and replaced with clear DMEM supple-
mented with one of the five different concentrations of stau-
rosporine (Sigma-Aldrich, S6942). Each treated sample was
paired with a control sample containing an equivalent volume
of dimethyl sulfoxide (DMSO). Thus, each plate held the five
different concentrations and their corresponding controls, such
that the environmental conditions were the same for all samples
in each set. The scattering measurements described in Sec. 2.1
were made at various times up to 48 h, to capture the onset and
progression of changes in the optical signal.
Apoptotic activity in samples treated with each concentra-
tion of staurosporine was quantified 4 h and 24 h after stau-
rosporine treatment using a luminescent assay for caspase ac-
tivity (Caspase-Glo 3/7 Assay, Promega). Cells were grown
in white-walled 96-well plates (Cliniplate 96-well microplate,
Thermo Fisher Scientific) under the same conditions as those
used in the optical experiments.
2.3
Anewmethodfordetectingapoptosisincellculturesshouldnot
be limited to a specific response in a single cell type. Thus, the
protocol from Sec. 2.2 was performed on PC-3 cells, a human
prostatecancerline,todetermineifthescatteringcharacteristics
of staurosporine-induced apoptosis were similar to that of CHO
cells.31
ESS Measurements in Other Cell Types
2.4
ESS Measurements With Other Apoptosis
Inducers
If the observed optical signal is due to the apoptotic process,
rather than some other feature of staurosporine treatment, dif-
ferentinductionprotocolsshouldproducesimilarapoptoticmor-
phology changes to staurosporine treatment and, therefore, sim-
ilar optical signals. To confirm this, scattering measurements
were performed on multiple sets of CHO cells, each treated
with a different apoptosis inducer. (See list of concentrations in
Sec. 3.3.) Control cultures were also considered, one without
a vehicle for the compounds dissolved in water, and one with
DMSO only. The concentrations used for each inducer were
chosen based on the manufacturer’s data sheets or a literature
search.32Scattering measurements were taken for each culture
at six time points, chosen based on the features of the optical
signal observed for cultures treated with 2 μm staurosporine, to
allow for comparison of magnitude and timescale. The presence
or absence of apoptosis in cultures treated with each inducer
after 4 or 24 h was verified using the caspase activity assay
described in Sec. 2.2.
2.5
Determining Specificity: Blocking the Caspase
Cascade
Many of the classic apoptotic morphology changes, including
chromatin condensation and nuclear fragmentation, are down-
stream of the apoptosis-specific caspase cascade. These down-
stream morphology changes should be prevented if the caspase
cascade is blocked. Inhibition of caspase activity by the use of
broad-spectrum caspase inhibitors results in the inhibition of
apoptosis as assessed by the lack of appearance of apoptotic nu-
clear morphology and oligonucleosomal DNA fragmentation.33
The commonly used caspase inhibitor carbobenzoxy-valyl-
alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD-fmk),
which binds to the catalytic site of the caspase proteases
and can thereby block apoptosis induction, was used in this
study.
We expect that if the features of the optical signal ob-
served in CHO cells following treatment with staurosporine
result from apoptotic morphology changes that are downstream
of the caspase cascade, blocking caspase acitivty, and there-
fore, the downstream morphological changes, should attenuate
the optical response. Conversely, if the optical signal results
from morphological changes upstream of the caspase cascade,
blocking that pathway should have little effect on the optical
response. These measurements should allow identification of
optical features that are necessarily specific to the apoptotic
process. We note that while features due to upstream morphol-
ogy changes may not necessarily be specific to apoptosis, they
may nevertheless be characteristic and yield useful diagnostic
information.
Confluent cultures of CHO cells were treated with one of
the following: a control volume of DMSO; 2 μm staurosporine
in DMSO; 2 μm staurosporine in DMSO plus 50 μm z-VAD-
fmk (Enzo Life Sciences, BML-P416-0001); or 50 μm z-VAD-
fmk alone. Scattering measurements were made at times up to
48 h following treatment. The presence or absence of apoptosis
in cultures treated with staurosporine or staurosporine plus z-
VAD-fmk was determined by the caspase assay described in
Sec. 2.2.
3
3.1
Results
Dose Response: CHO Cells Treated
with Staurosporine
Scattering measurements in the near-backward direction were
performed on six sets of samples. Cultures treated with stau-
rosporine showed both an early optical signal, characterized
(simplistically) by an overall decrease in the (negative) spectral
slope,aswellasalateopticalsignalcharacterizedbyanincrease
in spectral slope. Representative spectra in Fig. 2, taken from
culturestreatedwith2μmstaurosporine(dottedred)andthecor-
respondingcontrol(solidblack)at30mand24hpost-treatment,
respectively, illustrate the early (top) and late optical signals
(bottom). To determine the statistical significance of these scat-
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Mulvey et al.: Wavelength-dependent backscattering measurements...
450500550600 650700
0
0.5
1
1.5x 10
-3
Wavelength (nm)
Normalized Scattered Intensity (a.u.)
Late Optical Signal, 24 h


DMSO only
2 µM Staurosporine
450500550600650 700
0
0.5
1
1.5x 10
-3
Wavelength (nm)
Normalized Scattered Intensity (a.u.)
Early Optical Signal, 30 m


DMSO only
2 µM Staurosporine
Fig. 2 Representative spectra showing early and late optical signals.
Spectra were taken from control cultures containing 0.2% DMSO only
(solid black) and cultures treated with 2 μm staurosporine carried in
DMSO for 30 m and 24 h.
teringchanges,thesloperatiosofESSspectraat450and700nm
were calculated for all cultures of CHO cells treated with either
2 μm staurosporine or 0.2% DMSO (combining results from
Secs. 3.1, 3.3, and 3.4; n = 17). A paired student’s t-test com-
paring the ratio from the DMSO and staurosporine-treated cells
revealed that the spectral slope changes were significant both at
30 m (p < 10−5) and at 24 h (p < 10−8).
Boththekineticsandthemagnitudeofthesesignalswerede-
pendent on staurosporine concentration. These qualitative dose-
response results are summarized in Table 1, where the onset
times of the early and late optical signals, as well as their rel-
ative magnitudes, are indicated. The magnitude of the optical
signal is described qualitatively in terms of the slope differ-
ence of the ESS signal between CHO cells treated with 0.5 μm
staurosporine and the control population.
The cultures treated with 0.5 μm staurosporine showed only
slight changes in optical scattering. The magnitude of both the
early and late optical signals was greater for the cultures treated
with 1 μm staurosporine than for the cultures treated with 0.5
μm staurosporine. The three highest concentrations of stau-
rosporine produced an optical signal with still greater magni-
tude. For concentrations that produced detectable changes in
Table 1 A summary of scattering changes observed in cell cultures (n
= 6) treated with five different concentrations of the apoptosis inducer,
staurosporine. The onset times for early and late scattering changes as
well as relative magnitudes of the changes are indicated for each con-
centration. The relative magnitudes of the effect are expressed in terms
of the arbitrary unit, +, relating to the degree of the slope differences
in ESS signal between CHO cells treated with 0.5 μm staurosporine
and the corresponding control cells.
Staurosporine
concentration
(μm)
Onset early
signal
(m)
Onset late
signal
(h)
Magnitude of
optical
response
0.53012
+
115 10 to 12
++
2
<156 to 8
+++
5
<154 to 6
+++
10
<154 to 6
+++
scattering, faster kinetics was apparent with increasing concen-
tration. The early optical signal occurred after 30 m of treatment
with 0.5 μm staurosporine and after 15 m of treatment with
1 μm staurosporine. The cultures treated with the three higher
concentrations produced early optical signals beginning earlier
than 15 m. The strength of the optical signal at 15 m was greater
for higher concentrations, suggesting progressively early onset
times.
Theonsetofthelateopticalsignalwasalsoacceleratedincul-
tures treated with higher concentrations of staurosporine. The
incubation time required for late signal onset decreased from
12 h in cultures treated with the lowest concentration of stau-
rosporine to 4 to 6 h in cultures treated with 5 and 10 μm
staurosporine. This indicates that the time required for onset of
apoptosis in a number of cells is sufficient to produce detectable
differences in scattering decreases with increasing inducer con-
centration.
The average and range (n = 3) of caspase activity produced
by each concentration of staurosporine or DMSO are shown in
Fig. 3. In each of the control samples, a low level of caspase ac-
tivity was observed, corresponding to a low level of apoptosis.
At 4 h post-treatment, the treated samples show a monotonic
increase of caspase activity with increasing inducer concen-
tration. By 24 h post-treatment, each treated sample displays
similar high levels of caspase activation.
From the correlation between the scattering and caspase ac-
tivity data, it is evident that the magnitude and timing of the
features of the optical signal are correlated with treatment dose
of staurosporine. Furthermore, the caspase activity assay data
also suggests that the optical signal is related to staurosporine-
induced apoptotic activity present in the cultures.
3.2
Dose Response: PC-3 Cells Treated
with Staurosporine
Following the same protocol as for CHO cells, scattering mea-
surements were performed on two sets of PC-3 cultures. The
representativespectrainFig.4,takenfromPC-3culturestreated
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