Architectural heterogeneity in tumors caused by differentiation alters intratumoral drug distribution and affects therapeutic synergy of antiangiogenic organoselenium compound.

Abstract

Tumor differentiation enhances morphologic and microvascular heterogeneity fostering hypoxia that retards intratumoral drug delivery, distribution, and compromise therapeutic efficacy. In this study, the influence of tumor biologic heterogeneity on the interaction between cytotoxic chemotherapy and selenium was examined using a panel of human tumor xenografts representing cancers of the head and neck and lung along with tissue microarray analysis of human surgical samples. Tumor differentiation status, microvessel density, interstitial fluid pressure, vascular phenotype, and drug delivery were correlated with the degree of enhancement of chemotherapeutic efficacy by selenium. Marked potentiation of antitumor activity was observed in H69 tumors that exhibited a well-vascularized, poorly differentiated phenotype. In comparison, modulation of chemotherapeutic efficacy by antiangiogenic selenium was generally lower or absent in well-differentiated tumors with multiple avascular hypoxic, differentiated regions. Tumor histomorphologic heterogeneity was found prevalent in the clinical samples studied and represents a primary and critical physiological barrier to chemotherapy.

Full text

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Hindawi Publishing Corporation
Journal of Oncology
Volume 2010, Article ID 396286, 13 pages
doi:10.1155/2010/396286
Research Article
ArchitecturalHeterogeneity inTumorsCausedby Differentiation
AltersIntratumoral Drug Distribution andAffectsTherapeutic
Synergyof AntiangiogenicOrganoseleniumCompound
Youcef M.Rustum,1K´ arolyT´ oth,1MukundSeshadri,1ArindamSen,2FarukhA.Durrani,1
EmilyStott,1CarlD.Morrison,3Shousong Cao,1and ArupBhattacharya4
1Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
2Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
3Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
4Department of Cancer Prevention & Control, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
Correspondence should be addressed to Arup Bhattacharya, arup.bhattacharya@roswellpark.org
Received 25 December 2009; Accepted 22 January 2010
Academic Editor: Arkadiusz Dudek
Copyright © 2010 Youcef M. Rustum et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Tumor differentiation enhances morphologic and microvascular heterogeneity fostering hypoxia that retards intratumoral drug
delivery, distribution, and compromise therapeutic efficacy. In this study, the influence of tumor biologic heterogeneity on the
interaction between cytotoxic chemotherapy and selenium was examined using a panel of human tumor xenografts representing
cancers of the head and neck and lung along with tissue microarray analysis of human surgical samples. Tumor differentiation
status, microvessel density, interstitial fluid pressure, vascular phenotype, and drug delivery were correlated with the degree of
enhancement of chemotherapeutic efficacy by selenium. Marked potentiation of antitumor activity was observed in H69 tumors
that exhibited a well-vascularized, poorly differentiated phenotype. In comparison, modulation of chemotherapeutic efficacy by
antiangiogenicseleniumwasgenerallylowerorabsentinwell-differentiatedtumorswithmultipleavascularhypoxic,differentiated
regions. Tumor histomorphologic heterogeneity was found prevalent in the clinical samples studied and represents a primary and
critical physiological barrier to chemotherapy.
1.Introduction
Despiteconcertedeffortsformorethanfivedecades,curative
response to chemotherapy remains elusive in majority of
solid malignancies [1]. Efforts in discovering novel anti-
cancer agents without similar efforts in understanding and
overcoming physical barriers within tumors are unlikely
to radically change chemotherapeutic efficacy in the clinic.
Tumor being a heterogeneous three-dimensional composite
ofvariouscomponents,factorsatcoarserphysiologicalscales
can and does influence tumor response [2]. For an optimum
chemotherapeutic efficacy, the drug has to extravasate,
diffuse to distant tumor cells, be transported inside the cells,
bind to the specific target, and induce cell death. Only when
an anticancer agent is able to affect each and every individual
proliferating cancer cells in effectively inducing tumor cell
death, can it result in a complete remission (CR) or a cure.
Factors within the tumor microenvironment that impede
drug delivery and distribution are likely to result in tumor
regrowth and resistance irrespective of the drug’s efficacy in
vitro.
Tumor drug delivery system constituted predominantly
of abnormal vasculature, lack pericyte coverage, have abnor-
mal branching patterns and shunt perfusion, prestasis, stasis
and reversal of flow [3]. This dilated, chaotic and leaky
vasculature with an intermittent or unstable blood flow
contributes to an adverse and high intratumoral interstitial
fluid pressure (IFP) that retards delivery and distribution of
drugsfromthevesselsintothetumor[4].Wehavepreviously
demonstrated that differentiated regions in squamous cell

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2Journal of Oncology
carcinoma and adenocarcinoma do not contain blood vessel
and contribute to heterogeneity in microvessel distribution
that physiologically further retards an optimal intratumoral
drug delivery and distribution [5–7]. Various strategies
for improving drug penetration include improving tumor
blood flow, enhancing vascular permeability, reducing IFP,
and modifying the extracellular matrix [2]. For example,
use of antiangiogenic agents has been shown to result in
a favorable improvement in extent and quality of tumor
perfusion while reducing vascular permeability and tumor
IFP as a result of tumor vascular normalization [5, 7].
While there has been an ongoing effort in improving
tumor drug penetration in order to improve therapeu-
tic efficacy, newer approaches are required to overcome
tumor architectural and morphologic barriers that foster
therapeutic resistance by adversely affecting tumor drug
delivery.
In our earlier studies, we have observed that high
but nontoxic doses of antiangiogenic organoselenium com-
pounds such as 5-methylselenocysteine (MSC) and seleno-
methionine (SLM) act as selective modulators of chemother-
apeutic efficacy of a broad range of anticancer drugs
(irinotecan, taxanes, platinum complexes, doxorubicin and
cyclophosphamide) that are currently used in the clinic [8].
Administration of MSC (0.2mg/mice/day per oral starting
7 days before irinotecan) in combination with irinotecan
(100mg/kgi.v.weekly×4)wasfoundtoenhancetherapeutic
responsefrom20%and30%CRwiththedrugaloneto100%
CR in uniformly well-vascularized poorly differentiated col-
orectal carcinoma HCT-8 and head and neck squamous cell
carcinoma (HNSCC) FaDu, respectively [8]. In contrast, this
therapeutic synergy was less dramatic in well differentiated
xenografts such as the colorectal adenocarcinoma HT-29
and HNSCC A253 (0% and 10% CR with the drug alone
to 20% and 60% CR with the combination, resp.). In
subsequent studies, we demonstrated that this therapeutic
synergy was the result of enhanced tumor drug delivery and
distributionasaconsequenceofanimprovedtumorvascular
normalization and tumor IFP [5, 7].
In this study, we examined the influence of tumor
histologicheterogeneityontheinteractionbetweencytotoxic
chemotherapy and selenium (Se) using a panel of surgical
human tumor xenografts representing cancers of the head
and neck (A253, well differentiated and poorly differentiated
patient tumor derived HNSCC xenografts) and lung (H69,
A549). Tumor differentiation status, microvessel density,
interstitial fluid pressure, tumor blood, volume and perme-
ability were correlated with the degree of enhancement of
chemotherapeutic efficacy by selenium. Additionally, tissue
microarray (TMA) analysis of human surgical samples was
also performed to examine the occurrence and relevance of
the observed biologic heterogeneity.
2.MaterialsandMethods
2.1. Tumor Model. The human cancer cell lines H69 (small
cell lung cancer, SCLC), A549 (nonsmall cell epithelial lung
carcinoma, NSCLC), and A253 (well differentiated head and
neck squamous cell carcinoma) were originally obtained
from American Type Culture Collection (Manassas, VA)
and xenografts were established in ∼8 week old female
athymic nude mice (Foxn1nu, Harlan Sprague Dawley, Inc.
Indianapolis, IN) as previously described [6]. Human poorly
differentiated squamous cell carcinoma (SCC) surgical sam-
ple #17073 (maxillary sinus, PDSCC) and well differentiated
SCC #16653 (larynx, WDSCC) were obtained in house
at Roswell Park Cancer Institute and were maintained
in SCID (C.B-Igh-1bIcrTac-Prkdcscid/Ros) mice. Mice were
assessed for tumor growth using digital vernier calipers for
measuring tumor burden (mm3) using the formulae: 1/2
(L × W2), where L and W are the longest and shortest
axis in mm as per established method [8]. All studies were
performedinaccordancewithInstituteAnimal CareandUse
Committee-approved protocols and each treatment group
had a minimum of 4 animals per group.
2.2. Patient Samples of Cancer. Formalin/paraffin sections
of human cancer surgical TMA containing 0.6mm cores
from head and neck, colorectal, and lung cancer were
studied for presence or absence of differentiated structure,
microvessel distribution (MVD),and tumor hypoxia. Tumor
vesselsweredetectedusingCD34markerforendothelialcells
and hypoxia was determined using carbonic anhydrase IX
(CAIX) staining as per methods described earlier [9].
2.3. Drugs and Chemicals. MSC or SLM (1mg/mL, Sigma,
St. Louis, MO) was administered orally as a sterile
saline solution at the maximum tolerated dose of 0.2mg/
mouse/day [8] or 8mg/kg/day for 14 days, starting at least
3 days after tumor implantation. Taxotere (Sanofi-Aventis,
Bridgewater, NJ) was administered as a single intravenous
dose of 60mg/kg while irinotecan (Pharmacia & Upjohn,
New York, NY) was administered at a weekly schedule of
100 or 200mg/kg ×4. Doxorubicin (Bedford Laboratories,
Bedford, OH) was administered as a single intravenous
dose of 30mg/kg, 24 hours after the MSC dose on day
14. Albumin-GdDTPA was obtained from the Contrast
Media Laboratory, Department of Radiology (Dr. Robert
C. Brasch), University of California at San Francisco (San
Francisco, CA). For the combination chemotherapy with
MSC/SLM, MSC or SLM was given 7 days before therapy
and continued daily for 7 more days after the last dose of the
chemotherapeutic drug.
2.4. Immunohistochemistry. MVD determination was done
using CD31 in mouse and CD34 in human tumor TMAs.
CD31 and alpha-smooth muscle actin (α-SMA) double
staining was used to detect endothelial cells and pericytes,
respectively, as described previously [5]. Briefly, 5–8μm
cryosections were fixed in cold acetone (−20◦C) for 15
minutes and following quenching with endogenous perox-
idase were incubated with rabbit polyclonal SMA antibody
(1μg/mL or 1/500) (Abcam, Cambridge, MA), biotinylated
goat antirabbit secondary antibody (1/250) (Vector Labs)
for 30 minutes, and CD31 antibody (B.D. Biosciences
Pharmingen, Franklin Lakes, NJ) at a concentration of 10μg/
mL for 60 minutes. An isotype-matched negative control

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Journal of Oncology3
was used in all cases. Immunostaining of tumor vasculature
in the TMAs was done using antihuman CD34 (DAKO,
Carpinteria, CA) used at 1/50 dilution for 90 minutes at
room temperature followed by 30 minutes incubation with
Streptavadin complex (Zymed Lab, Inc., San Francisco, CA)
as per methods described earlier [5–7]; CAIX immunos-
taining, the primary antibody M75 (gift from Dr Pastorek,
Institute of Virology, Slovak Republic) was used at 20μg/mL
for 90 minutes followed by 30-minutes incubation with
Streptavadin complex (Zymed Lab, Inc., San Francisco, CA)
as described earlier [5–7]. Hypoxia inducible factor 1-α
(HIF-1α) was detected with a multilayer, amplified method
after antigen retrieval with Target Retrieval Solution (TRS,
Dako Carpenteria, CA) in a pressure cooker. The method
was developed and optimized at our Core Facility [10].
All immunohistochemical interpretation and analysis was
carried out under the supervision of a board-certified and
experienced pathologist (KT).
2.5. Magnetic Resonance Imaging. Tumor-bearing mice were
imaged in a 4.7T horizontal bore MR scanner (GE NMR
Instruments, Fremont, CA). The imaging protocol and data
analysis methods for estimating the vascular volume and
permeability of tumors using the intravascular contrast
agent, albumin-GdDTPA, have been previously described
[11, 12]. The change in tumor T1-relaxation rate of tumors
(ΔR1)wascalculatedoverapproximately30–50minutespost
contrast. ΔR1 values were measured in the kidneys as a
measure of vascular relaxation enhancement and used to
compute vascular volume (y-intercept of the linear regres-
sion line) and permeability (slope of the linear regression
line) tumor/blood fit. Image processing and analysis were
carried out using commercially available medical imaging
software Analyze PC, Version 7.0 (AnalyzeDirect, Lenexa,
KS).
2.6. In Vivo Tumor IFP Measurements. MSC-induced chan-
ges in tumor IFP were monitored in A253, H69, and A549
xenografts in vivo real-time using microcatheter pressure
transducers in externally accessible tumors and in normal
tissue as per method described earlier [5, 7]. The inter-
stitial fluid pressure within the tumor microenvironment
was measured-using a modified “wick-in-needle” technique
using custom-designed instrumentation broadly based upon
an earlier published design [13]. Briefly, measurements were
madewithaMikroTipCatheterTransducer(ModelSPR-524,
Millar Instruments) via a 23.5 gauge wing-tipped infusion
needlecatheter.ThetransducerwasinterfacedtoaPCusinga
pressure control unit (PCU-2000, Millar Instruments) via an
USBanalog-to-digitalconverter(ModelDT9816DataTrans-
lation, Marlboro MA). The software used to acquire the data
was developed in the laboratory using DT Measure Foundry
Ver. 4.0.7 (Data Translation, Marlboro, MA). The needle
was inserted into the tumor, with measurements made every
few millimeters; thus three to six measurements were made
within each tumor, and the average value was used. Prior
to each measurement, the apparatus was aspirated to ensure
that no tissue was clogging the needle, and the instrument
was zeroed. The instrument was calibrated before and after
each experiment to ensure proper function, using a custom
built water-column manometer. Mice with subcutaneous
tumors (N = 4–8 per group) were used to assess the IFP
measurements under anesthesia at time points equivalent to
24 hours post 14 days of MSC.
2.7. Determination of Intratumoral Doxorubicin Distribu-
tion Gradient. The influence of MSC on drug distribution
gradient (N
= 47 or more linear paths 90 pixels long
using multiple sections from tumors) was assessed using
fluorescence microscopy as described earlier [5]. Two hours
post doxorubicin administration, animals were euthanized
and the harvested tumor frozen and ∼5–10μm thick frozen
sections were used. An average of 4 maximum intensity
projection images with a resolution of 0.23μm was acquired
using identical acquisition parameters under 63× objective
of Leica confocal microscope [5] and the digitized images
were used further for image analysis.
2.8. Image Analysis. CD31 positive endothelial cell clusters
in multiple high-power fields (400×) covering non-necrotic
areas of the whole tumor sections were used to determine
MVD. Tumor vascular maturation index (VMI) was derived
bycalculatingthetotalnumberofCD31+α-SMA+areasand
areas positive for CD31 alone in double-stained (CD31/α-
SMA) tissue sections using Analyze (AnalyzeDirect, Over-
landPark, KS) [5]. The mean intensity of doxorubicin
autofluorescence at various distances away from the blood
vessels was calculated using Analyze.
2.9. Statistical Analysis. Results are expressed as mean ±
standard error of the mean and the differences between the
mean of the groups were analyzed using unpaired two-tailed
Student t-test (GraphPad Version 5.00, GraphPad Software,
San Diego, CA). Linear regression analysis was carried out
to determine statistical significance of DCE-MRI-measured
parameters and intratumoral drug distribution gradient. A
P value <.05 considered statistically significant.
3.Results
3.1. Histological Characteristics of Tumor Xenografts. While
H69 (Figure 1(d)) is a poorly differentiated small cell
lung carcinoma, HNSCC A253 (Figure 1(a)) and the non-
small cell lung adenocarcinoma A549 (Figure 1(g)) are
well and moderately differentiated tumors, respectively.
CD31 immunostaining shows H69 (Figure 1(e)) to be uni-
formly well-vascularized while A253 (Figure 1(b)) and A549
(Figure 1(h)) xenografts have differentiated regions that are
devoid of blood vessels and are thus consequently hypoxic
as seen by presence of CAIX immunostaining (arrows,
Figures 1(c) and 1(i), resp.). While very few cells were
found to be CAIX positive in smaller H69 tumors (<1000
mg, Figure 1(f(a))), the larger H69 tumors (>2000 mg,
Figure 1(f(b))) had the characteristic perinecrotic hypoxia.
As shown in Figure 1(j), #17073 is a PDSCC while #16653 is
a WDSCC (Figure 1(m)). The hallmark of surgical PDSCC

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4Journal of Oncology
(a)
(b)(c)
(d)
(e) f(a)f(b)
(g)
(h) (i)
(j)(k) (l)
(m)(n) (o)
H and ECD31 CAIX
A253
H69
A549
#17073
#16653
Figure 1: Photomicrographs of human cancer cell line xenografts A253, H69, A549 along with human HNSCC surgical samples-derived
xenografts #17073 and #16653. (Left panels, H&E; middle panels, CD31 immunostaining to visualize microvessels; right panels, CAIX
immunostaining to visualize tumor hypoxic regions; original magnifications, ×100). Poorly differentiated H69 (d) is uniformly well
vascularised (e) and has no regions of hypoxia (f(a)) in the ∼250 mg tumor but has perinecrotic hypoxic regions in larger (>2000mg)
tumor (f(b), arrow). Similar vascular arrangement (k) is seen in viable regions of the poorly differentiated HNSCC #17073 (PDSCC) which
though has hypoxic regions ((l), arrow) around the many necrotic regions seen in the growing tumor. In contrast, A253 and A549 have
differentiated regions ((a), arrow & (g)) that being avascular ((b), (h), arrows) are hypoxic ((c), (i), arrows). Surgical sample derived well
differentiated HNSCC #16653(WDSCC) has a highly differentiated morphology due to presence of many well differentiated ((m), arrow)
regions that are avascular (n) and hypoxic ((o), arrow).
xenografts was the presence of many necrotic regions
throughout the tumor. These necrotic regions did not
contain viable tumor cells or any vasculature and were sur-
rounded by CAIX positive hypoxic cells (arrow, Figure 1(l)).
Though the other regions in the PDSCC exhibited a uniform
microvessel distribution (Figure 1(k)), these necrotic regions
contribute to a histomorphological heterogeneity that is
not conducive to an optimal intratumoral drug delivery
and distribution. The surgical WDSCC xenograft contained
several well differentiated regions (arrow, Figure 1(m)) that
are without microvessels (Figure 1(n)) and are surrounded
by rims of CAIX positive hypoxic proliferating cells (arrow,
Figure 1(o)).
3.2. Effect of MSC on Tumor Growth. As shown in
Figure 2(a), the HNSCC A253 grew at a faster pace than
the lung xenografts H69 and A549. Treatment with MSC
resulted in a reduction of tumor burden when compared
to the untreated controls (Figure 2(b)) by 30%, 62%, and
36% in A253 (1952 ± 32.07 versus 1360 ± 59.75, N = 6),
H69 (279.5 ± 106.60 versus 105 ± 31.84, N = 4) and
A549 (552.50 ± 60.62 versus 353.40 ± 68.16, N = 4),
respectively, albeit significantly only in A253 (P < .0001).
Untreated PDSCC and WDSCC xenografts grew slower than
control A253 tumors (Figure 2(c)). Treatment with MSC
for 2 weeks did not lead to a significant reduction in
tumor burden when compared to untreated controls in both

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Journal of Oncology5
0
500
1000
1500
2000
Tumor volume (mm3)
5 1015
Days of MSC Rx
A253 control
A253 MSC
H69 control
H69 MSC
A549 control
A549 MSC
(a)
0
500
1000
1500
2000
Tumor volume (mm3)
A253 H69A549
Control
MSC
P = .22
P < .0001
P = .08
(b)
0
500
1000
1500
2000
Tumor volume (mm3)
05 10 15
Days of MSC Rx
17073 control
17073 MSC
16653 control
16653 MSC
(c)
0
500
1000
1500
2000
Tumor volume (mm3)
1707316653
Control
MSC
P = .92
P = .79
(d)
Figure 2: Effect of MSC on tumor growth ((a), (c)) and tumor volume ((b), (d)) at the end of 14 days treatment.
PDSCC (541.9 ± 167.2 versus 561.80 ± 107.30, N = 6) and
WDSCC (363.80 ± 68.53 versus 338.50 ± 65.63, N = 6–8)
xenografts (Figure 2(d)).
3.3. Effect of MSC on MVD, IFP, and Drug Delivery.
Poorly differentiated untreated H69 xenografts were highly
vascularized compared to the untreated well differentiated
HNSCC A253 (P = .002) and NSCLC A549 (P = .008)
tumors. As shown in Figure 3(a), the antiangiogenic effect of
MSC (0.2mg/mouse/day × 14) led to a 59%, 62%, and 6%
reduction in MVD compared to untreated control tumors in
A253 (4.63 ± 0.87 versus 11.23 ± 1.87, P = .03), H69 (9.28
± 0.82 versus 24.29 ± 1.45, P = .008) and A549 (6.60 ± 1.50
versus 7.05 ± 2.23, P = .88) respectively. No differences in
MVD of normal mouse liver tissue were observed (data not
shown). Tumor vascular maturation index (VMI), that is,

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6Journal of Oncology
0
10
20
30
Average microvessel density/HPF 400X
A253H69 A549PDSCCWDSCC
P = .88
P = .03
P = .0008
P = .58
P = .79
Control MSC
(a)
0
20
40
60
80
100
Vascular maturation index (%)
A253 H69 A549 PDSCC WDSCC Liver
P = .04
P = .001
P = .0005
P = .21P = .98
P < .001
ControlMSC
(b)
0
5
10
15
20
Tumor interstitial fluid pressure (mm of Hg)
A253H69A549
P = .18
P = .002
P = .79
Control MSC
(c)
0
25
50
75
100
125
150
175
200
225
250
Doxorubicin fluorescence
signal intensity (a.u.)
0 20
Distance from blood vessels (μm)
4060 80100
H69 control, P = .1
H69 MSC, P = .1
A549 control, P = .14
A549 MSC, P = .14
A253 control, P = .0001
A253 MSC, P = .0001
(d)
0
25
50
75
100
125
150
175
200
225
250
Doxorubicin fluorescence
signal intensity (a.u.)
0 20
Distance from blood vessels (μm)
40 60 80100
PDSCC control
PDSCC MSC
WDSCC control
WDSCC MSC
(e)
Figure 3: (a) Bar graphs show MVD counts per high-power field (HPF) (original magnification, 400×) in the untreated versus MSC-treated
xenografts and surgical samples following 14 days of MSC treatment (0.2mg/mouse/day). Significant reduction in MVD was seen in A253
and H69 xenografts after 14 days of MSC treatment with no changes seen in A549 xenografts or the surgical samples. (b) MSC led to an
improved vascular maturation in A253, H69, A549 xenografts and PDSCC but did not induce any change in WDSCC and normal liver. (c)
Treatment with MSC (0.2mg/mouse/day × 14) led to a lowering of interstitial fluid pressure in A253 but not in H69 and A549 xenografts.
(d) As a consequence, there was a significant improvement in tumor doxorubicin fluorescence intensity gradient in A253 xenografts but not
in H69 or A549 xenografts. (e) MSC did not result in an improvement in tumor doxorubicin fluorescence intensity gradient in the surgical
samples PDSCC and WDSCC.

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Journal of Oncology7
the percentage of endothelial cells associated with pericytes,
showed an increase of 31.38%, 49.93% and 6.38% in A253,
H69 and A549 xenografts, respectively (Figure 3(b)). Tumor
IFP was higher in the untreated HNSCC xenograft A253
compared to the untreated lung tumor xenografts that were
lower by 31% and 71% in H69 and A549, respectively. As
showninFigure 3(c),treatmentwithMSCledtoasignificant
reduction in tumor IFP in A253 compared to the untreated
controls (6.78 ± 0.45 versus 13.26 ± 1.15, P = .002) but
this reduction was less dramatic in the lung xenografts—
H69 (7.38 ± 0.75 versus 9.13 ± 0.84, P
A549 (3.58 ± 0.91 versus 3.89 ± 0.66, P = .79). The
net effect of MSC on tumor vasculature and IFP was a
significantly(P = .0001)increasedintratumoraldoxorubicin
gradient in A253 xenografts while there was no significant
increase in the overall drug gradient in the lung xenografts
(Figure 3(d)). Interestingly, at a distance of ∼90μm from
tumor blood vessels, all the MSC-treated tumors showed a
significantly higher doxorubicin concentration as measured
by the fluorescence intensity compared to the non-MSC-
treated A253 (91.38 ± 4.81 versus 66.90 ± 3.21, P = .001),
H69 (68.06 ± 2.60 versus 59.17 ± 1.41, P = .003), and A549
(62.21 ± 1.84 versus 53.94 ± 1.58, P = .0008) xenografts.
In contrast, the untreated human surgical samples of
HNSCC had a higher MVD (17 ± 0.3 in PDSCC, and 19
± 0.41 in WDSCC) compared to HNSCC A253 xenograft
(11.23 ± 1.87) (Figure 3(a)). Though treatment with MSC
did not lead to a significant reduction in MVD in PDSCC
compared to untreated controls (16 ± 0.13 versus 17 ± 0.3),
there was a significant increase in tumor vascular maturation
(84.39 ± 1.06 versus 76.57 ± 1.48, P < .001) (Figure 3(b)).
In WDSCC, MSC did not lead to any reduction in MVD
(20.90 ± 0.35 versus 19.36 ± 0.4) or improvement in
VMI (86.82 ± 1.45 versus 89.10 ± 1.03). The intratumoral
doxorubicin gradient was not significantly different in both
the surgical samples (Figure 3(e)). At a distance of ∼90μm
from tumor blood vessels, MSC-treated tumors showed a
significantly higher doxorubicin concentration as measured
by the fluorescence intensity compared to the non-MSC
treated PDSCC (56.13 ± 0.94 versus 52.96 ± 0.94, P = .047).
A similar increase was not seen in the well differentiated
WDSCC at ∼90μm from tumor blood vessels. Instead, a
significant increase in doxorubicin fluorescence intensity
was seen at a distance of ∼50μm within the avascular
well differentiated hypoxic foci in the MSC-treated tumors
compared to the non-MSC-treated tumors (63.62 ± 2.11
versus 56.32 ± 1.96, P = .02).
= .18) and
3.4. Influence of Histologic and Vascular Heterogeneity on
Tumor Vascular Response to Selenium. In order to examine
changes in vascular function following selenium treatment,
noninvasive MRI was utilized. Changes in vascular volume
and permeability were estimated in tumor-bearing mice
following treatment with MSC (0.2mg/day×14) and com-
pared to untreated controls. As shown in Figure 4, only
A549 tumors showed a significant reduction (P < .001) in
vascular volume compared to untreated control tumors (n =
4 per group). No significant differences in tumor vascular
volumeandpermeabilitywereobservedbetweencontroland
selenium-treated mice for H69 (P > .05; N = 3 controls,
N = 4 MSC), A253 (P > .05; N = 9 controls, N = 7
MSC), poorly differentiated (P > .05; N = 8 controls,
N = 12 MSC), and the well differentiated patient tumor-
derived HNSCC xenografts (P > .05; N = 6 per group).
3.5. Modulation of Antitumor Activity. We had earlier
reported that MSC (0.2mg/mouse/day p.o. starting 7 days
prior to and continuing daily till end of chemotherapy) in
combination with the maximum tolerated dose of irinotecan
(100mg/kg i.v. weekly × 4) resulted in an increase in cure
rates (CR) in A253 xenografts from 10% with irinotecan
(100mg/kg weekly × 4 i.v.) alone to 60% with the combi-
nation [8]. As can be seen in Figure 5(a), the small cell lung
cancer xenograft H69 gives a 100% CR either with irinotecan
(100 mg/kg weekly × 4 i.v.) alone or in combination with
SLM. The CR with taxotere (60mg/kg × 1 i.v) alone was
80% that increased to 100% when used in combination with
SLM. These high cure rates were obtained when treatments
were initiated at ∼100mm3size of tumor in H69 tumor
xenografts. H69, a uniformly well vascularised tumor with
relatively small pockets of necrosis, is an attractive preclinical
model for determining the influence of tumor morpholog-
ical heterogeneity on synergistic chemotherapeutic efficacy
in combination with antiangiogenic agents. To determine
if this activity of combination treatment was persistent
against larger tumors, studies were carried out using larger
H69 tumors (∼1000–1500 mm3). These large H69 tumors
contain multiple necrotic regions surrounded by hypoxic
CAIX positive tumor cells (Figure 1(f(b)) and, thus, are less
homogeneous compared to the smaller (<1000mg) H69
tumors. As seen in Figure 5(b), a CR of 0% and 20% with
taxotere alone and in combination with SLM and a CR of
20%and60%withirinotecanaloneandincombinationwith
SLM, respectively were achieved. In contrast, the moderately
differentiated non-small cell lung carcinoma A549 had 0%
cures either with the drug alone or in combination with
MSC.
In order to further investigate this therapeutic efficacy of
Se in combination chemotherapy, 2-patient tumor-derived
HNSCC xenografts with varying differentiation status were
used. Both xenografts were strongly positive for hypoxia
as determined by CAIX immunostaining. As can be seen
in Figure 5(c) and Figure 5(d), treatment with irinotecan
(100mg/kg weekly × 4 i.v.) alone gave a cure rate of 0% and
37.5% in WDSCC and PDSCC, respectively. In combination
with MSC there was no significant improvement in CR
except in WDSCC where the CR increased to 14%. Since
our earlier studies [8] have indicated the possibility of dose
escalation to double the normal MTD of irinotecan, due to
the additional chemoprotective properties of MSC, we used
this higher dose of irinotecan (200mg/kg weekly × 4, i.v.) in
another group of mice bearing these human cancer surgical
samples. As can be seen in Figure 5(d), MSC in combination
with this higher irinotecan dose resulted in a higher response
of 75% CR and 25% partial response in PDSCC. No such
improvement in response was seen in the surgical WDSCC.
Thus, enhancement in therapeutic efficacy by antiangiogenic
MSC in combination of chemotherapy is higher in tumors

Page 8

8 Journal of Oncology
0
0.05
0.1
0.15
0.2
0.25
ΔR1tumor/blood
0 10203040 50
Time (min)
(a) H69
0
0.05
0.1
0.15
0.2
0.25
ΔR1tumor/blood
0 1020 3040 50
Time (min)
(b) A549
0
0.05
0.1
0.15
0.2
0.25
ΔR1tumor/blood
010203040 50
Time (min)
(c) A253
0
0.05
0.1
0.15
0.2
0.25
ΔR1tumor/blood
0 10 20
Time (min)
30 40 50
Control
MSC
(d) Patient SCC (Poorly-diff)
0
0.05
0.1
0.15
0.2
0.25
ΔR1tumor/blood
0 1020 3040
Time (min)
Control
MSC
(e) Patient SCC (Well-diff)
Figure 4: Change in relative vascular permeability and vascular volume as a result of treatment with MSC in H69 (a), A549 (b), A253 (c),
PDSCC (d), and WDSCC (e) tumors as assessed by DCE-MRI.
that are relatively more homogeneous versus in tumors that
are highly heterogeneous such as in the WDSCC.
3.6. Patient Samples of HNSCC, Colorectal, and Lung Cancer.
In order to determine the relevance of tumor morphologic
heterogeneity in influencing efficacy of antiangiogenic ther-
apy such as MSC/SLM, we studied the prevalence of archi-
tectural heterogeneity in solid tumor malignancies in terms
of tumor cell differentiation, vascularization, and hypoxia
using TMAs derived from patient surgical cancer specimens
from three different disease sites—head and neck, colorectal,
and lung. As summarized in Table 1, 51% of HNSCCs
are poorly differentiated while 41% of HNSCCs are well
differentiated (41%). Most (47%) HNSCC show presence
of hypoxia marker CAIX. Figures 6(a) and 6(d) shows the
representative tumor vascular distribution (arrow, brown)
in poorly and well differentiated HNSCC with regions of
hypoxia stained for CAIX immunostaining (Figures 6(b)
and 6(e)) and HIF-1α (Figures 6(c) and 6(f)). In colorectal
cancers, the majority were found to be moderately differen-
tiated cancers (79%) and hypoxic (CAIX, 63%) (Table 1).
Figures 6(g) and 6(j) show typical distribution of tumor
MVD (arrow, brown) with regions of hypoxia stained for
CAIX (Figure 6(k), arrow, brown) and HIF-1α (Figure 6(l),
brown) immunostaining in the well differentiated colorectal
cancers.NSCLCalsopresenteditselfpredominantly(86%)as
a moderately differentiated cancer with 41% of tumors being
hypoxic (CAIX positive). Figure 6(m) shows the distribution
of vessels (brown) in lung cancer (arrow, brown) while
Figure 6(n) and 6(o) depict the distribution of hypoxia as
assessed using CAIX (brown, arrow) and HIF-1α, respec-
tively in lung cancers. The majority of solid malignancies
studied was found to have the hallmark of a heterogeneous
tumor architecture that is not conducive for an optimal

Page 9

Journal of Oncology9
0
20
40
60
80
100
Cures (total (%))
Taxotere Irinotecan
Drug alone
Drug + SLM
(a) H69
0
20
40
60
80
100
Cures (total (%))
Taxotere Irinotecan
Drug alone
Drug + SLM
(b) H69
0
10
20
30
40
50
60
70
80
90
100
Response (%)
Complete responsePartial response Non-responders
Irinotecan (100 mg/kg)
MSC + irinotecan (100 mg/kg)
MSC + irinotecan (200 mg/kg)
(c) WDSCC
0
10
20
30
40
50
60
70
80
90
100
Response (%)
Complete response Partial response Non-responders
Irinotecan (100 mg/kg)
MSC + irinotecan (100 mg/kg)
MSC + irinotecan (200 mg/kg)
(d) PDSCC
Figure5:ResponsetocytotoxicdrugsaloneandincombinationwithMSCinH69lungxenograftswhentreatmentwasstartedat ∼100mm3
tumor volume (a), at ∼1000mm3tumor volume (b), and in human surgical samples of HNSCC WDSCC and PDSCC.
intratumoral drug delivery and distribution and, thus, will
not respond optimally to monotherapy or to the synergistic
modulation by an antiangiogenic agents such as MSC/SLM
used in combination setting.
4.Discussion
The effectiveness of traditional and novel anticancer agents
is limited by physical barriers that compromise tumor drug
delivery at therapeutically meaningful concentrations. Our
knowledge and understanding of tumor vasculature and
hypoxia has increased over the past few decades, but if this
understanding is not tied to the morphologic or cellular
heterogeneity arising out of tumor tissue differentiation,
progress in treating solid malignancies will remain inade-
quate.Drugpenetrationinthetumortissuesisbyconvection
and/or diffusion. Convection depends on both hydrostatic
and osmotic pressure gradients between vascular space and
interstitial space, vascular permeability, surface area; and the
volume and structure of extracellular matrix [2]. Diffusion
is determined by concentration gradients. Since tumor IFP
is often elevated to the level of microvascular pressure, it
retards extravasation of macromolecules that must penetrate
distances of up to 200μm within the tumors in order to
reach all viable cells [2]. While many studies have reported
an association between low IFP and therapeutic response
[14,15],someothershavenotfoundsuchassociation[2,16].
Obviously, a lowered IFP by itself may not be the panacea for
improving drug delivery and therapeutic response.

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10Journal of Oncology
(a)(b) (c)
(d)(e)(f)
(g)(h) (i)
(j)(k)(l)
(m)(n)(o)
CD34CAIX HIF-1α
PD HNSCC
WD HNSCC
PD colorectal
WD colorectal
Lung adeno-
carcinoma
Figure 6: Photomicrographs of tumor tissue microarrays (TMAs) from surgical samples of head and neck squamous cell, colorectal, and
lung carcinoma. (Left panels, CD34 immunostaining to visualize microvessels; middle panels, CAIX immunostaining and right panels, HIF-
1α immunostaining (brown, nuclear) to visualize tumor hypoxic regions; original magnifications, ×200). Poorly differentiated TMAs are
relatively uniformly well vascularised ((a), (g), arrows) with some regions of hypoxia as measured by CAIX ((b), arrow, (h)) and HIF-1α
((c), (i)). In contrast, the well differentiated tumors have large differentiated regions that are avascular ((d), (j), (m), arrows) and more
strongly hypoxic as seen with CAIX ((e), (k), (n), arrows) and HIF-1α ((f), (l)) immunostaining. Most lung adenocarcinomas were negative
for HIF-1α staining (o).
Differentiated cells do not lead to tumor propagation
and metastasis but can result in a morphologic heterogeneity
that hinders optimal intratumoral drug delivery [6]. While
many studies have focused on the individual cancer cells,
stromal cells and the vascular network, there are few studies
on the effect of tumor histomorphologic heterogeneity in
drug resistance. Tumor tissue architecture is an important
factor that influences drug delivery. For instance, the higher
cell density in differentiated regions of tumors prevents the
sprouting and growth of blood vessels [6]. This combined
with a reduced interstitial space and volume of extracellu-
lar matrix results in lower intratumoral drug penetration
[2].
SCC and adenocarcinoma are the most common his-
tological types of solid malignancies. In well differenti-
ated forms of SCC, tumor cells nests are often observed
with a closely packed arrangement of individual tumor
cells without stroma or microvessels. Tumor cells in these
differentiated regions (Figure 1(a), arrow) or the lumen
structure (Figure 1(h), arrows) seen in adenocarcinoma do
not receive adequate blood supply causing hypoxia, limited
drug delivery and resulting in sanctuaries for proliferating
cellsthatescapetherapy.Incontrast,thepoorlydifferentiated
partsofthesetumorsorintumorsthatarewhollypoorlydif-
ferentiated, microvessels are uniformly distributed providing
blood supply to most such parts of the tumors. In our study,

Page 11

Journal of Oncology 11
Table 1: Prevalence of histomorphologic heterogeneity in human cancers.
Cancer
disease site
Total
evaluable
tumors
Tumor differentiation status
(% of Total)
Mod. Diff
CAlX immunostaining
(% of Total)
<33% 33–67%
25%8%
Total—47% of all tumors Total—21% of all tumors
28%5% 30%
Total—63% of all tumors Total—21% of all tumors
23%10% 8%
Total—41% of all tumors Total—4% of all tumors
HIF-1α immunostaining
(% of Total)
<33% 33–67%
9% 4%
Well Diff
Poorly Diff
>67%
14%
>67%
8%
Head and
neck
198
41% (72% CAIX+) 8% (50% CAIX+) 51% (38% CAIX+)
Colorectal57
12% (57% CAIX+) 79% (64% CAIX+) 9% (60% CAIX+)
7%2%12%
Lung102
3% (0% CAIX+) 86% (42% CAIX+) 11% (36% CAIX+)
3%1% 0%
MSC treatment led to a lower tumor burden in all three
human tumor xenografts albeit only significantly in A253
xenografts. This effect was not seen in the human surgical
samples of head and neck cancers-PDSCC and WDSCC.
Greater optimal therapeutic synergy of antiangiogenic Se
compounds in combination to anticancer drug was observed
in the morphologically less heterogeneous large human
SCLC cell line xenograft H69 compared to more heteroge-
neous NSLC A549 that had a lower, relatively normalized
MVD, IFP, or an improved drug delivery. In contrast, A253
despite being well differentiated shows a better response
to the combination therapy as reported earlier [8]. This
is as a consequence of MSC-induced vascular modulation
that led to a significant lowering of MVD, an improved
VMI and IFP, thereby resulting in an increased intratumoral
drug gradient. The heterogeneous differentiated human
NSCLC A549 with a lower but relatively matured MVD
did not show a marked modulation to antiangiogenic Se
in terms of reduction in MVD, IFP or an improved VMI.
Hence, it did not respond with a higher efficacy to the
combination chemotherapy with Se. Interestingly, in SCLC
H69, the synergy persisted even when the tumors were
allowed to grow to ∼1–1.5g before the start of therapy. H69,
a representative SCLC with uniformly poorly differentiated,
well-vascularised tumor, contains few small necrotic foci
and had the least morphologic heterogeneity in terms of
its cellular architecture and vascular distribution amongst
the xenografts studied. As shown in Figure 1(d), the more
homogeneous morphologic feature of this tumor facilitates a
better drug penetration and distribution within the tumor.
This translates into a better therapeutic response. We had
earlier reported similar results in the poorly differentiated
humantumorxenograftsHNSCCFaDuandcolorectalHCT-
8 that has a similar uniform vasculalarized homogeneous
morphology and in which treatment with MSC resulted
in a significant modulation of the vascular parameters and
IFP [5, 7]. The antiangiogenic effect in terms of reduction
in MVD was the highest in H69 (62%) and A253 (59%)
and was minimal in A549. Despite a significant reduction
in MVD and a dramatic improvement in VMI (49.93%),
there was no significant difference in IFP or in the vascular
parameters assessed with DCE-MRI in H69 xenografts. In all
threetumorxenografts,treatmentwithMSCledtoincreased
doxorubicin fluorescent intensity at a distance of ∼90μm
from vessel wall. These MSC-induced changes in vascular
parameters and IFP resulted in a significant increase in
response rates in H69 and A253 xenografts. Despite the
minimal reduction in MVD, MRI revealed a statistically
significant reduction in vascular volume of A459 tumors
following selenium treatment (Figure 4). It is plausible
that this dramatic reduction in perfusion contributes to
decreased chemotherapeutic delivery leading to a minimal
therapeuticresponsewithcombinationtherapyinthistumor
model.
SCLC, being poorly differentiated, homogeneous, and
well vascularized, is sensitive to chemotherapy. Regrowth
and resistance in the clinic has been associated with cellular
differentiation in these cancers induced in part by the agents
themselves [17]. Thus, an effective first line chemother-
apeutic regime that can kill most of the cancer cells is
likely to be more efficacious in such tumors and the use of
antiangiogenic agents such as Se in the combinatorial setting
is likely to be the most promising especially in chemo-naive
patients. In contrast, the heterogeneous surgical samples of
HNSCC did not show any significant MVD reduction by Se.
The improvement in VMI was significant but modest (∼8%)
in the poorly differentiated surgical sample #17073 but was
not significant in well differentiated #16653. Both these
surgical samples did not show any MSC-induced increase in
doxorubicin concentration gradient immediately adjacent to
tumor vasculature and MSC did not result in an enhanced
synergistic therapeutic response of irinotecan at its MTD
in both these tumors. Unlike the poorly differentiated and
uniformly vascularized FaDu, HCT8, and H69 xenografts,
PDSCC is relatively less homogeneous due to the presence
of irregular and large regions of hypoxia and necrosis
that does not facilitate therapeutic exposure of drugs in
meaningful concentration to all proliferating cancer cells
(Figure 1(l)). Since Se has chemoprotective effect [8] and
allows for dose escalation to higher than MTD, we treated
a subset of the mice bearing these surgical specimens with
MSC in combination with double the MTD of irinotecan.
At this dose, MSC did result in a significant increase in
CR (75%) in the surgical sample of PDSCC but showed
no change in the surgical sample WDSCC. The histological
structure of WDSCC is extremely heterogeneous due to the
presence of many keratinized well differentiated avascular
(Figure 1(m), arrow), hypoxic regions (Figure 1(o), arrow).

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12Journal of Oncology
Such regions do not allow penetration of an effective drug
in a therapeutically meaningful concentration in the rim
region surrounding these differentiated regions. This causes
proliferating cells within these rims to escape therapy and
allow tumor repopulation and regrowth [6]. Further, the
remnant tumor after therapy generally contained mainly
differentiated regions remaining as survivor—a pointer to
their contribution to drug resistance [6].
As previously reported [18], poorly differentiated tumor
xenografts such as FaDu and HCT-8 have a slightly
faster doubling time (∼2.5 to 3 days) compared to their
well differentiated counterparts such as A253 and HT-29
(∼3.5 days), respectively. In our studies, the PDSCC had
a similar doubling time of 3.5–4 days while the WDSCC
had a doubling time of more than 5 days (Figure 2(c)).
The lung cancer xenografts had a slower proliferation rate
with the poorly differentiated SCLC xenografts H69 showing
a doubling time of 6.1 days [19] while the differentiated
NSCLC was found to have a doubling time of ∼8.3 days
in our studies (Figure 2(a)). It is likely that due to the
presence of more uniformly distributed and often higher
number of blood vessel, the poorly differentiated tumors
tend to proliferate and grow faster. Proliferating cells are
more sensitive to chemotherapy while the uniform dis-
tribution of vessels in poorly differentiated tumors makes
the tumor easily accessible to anticancer drugs especially
after tumor vascular normalization by antiangiogenic agents
including MSC/SLM. From the results, one can surmise that
chemomodulation of an antiangiogenic agent such as MSC
will be most optimal in tumors where it can significantly
reduce MVD, increase VMI, and as a consequence lead to
a reduced IFP. In tumors such as the A549 and WDSCC
where the untreated controls showed a presence of either
high (WDSCC) or a low (A549) MVD, and the addition
of the antiangiogenic agent did not change the VMI, the
chemomodulation effect of antiangiogenic agents in com-
bination chemotherapy is unlikely to lead to a significantly
high response. The higher the degree of tumor morphologic
heterogeneity, the less likely is the synergistic effect of
antiangiogenic agents in combination chemotherapy. There
remains an urgent need in developing novel agents that
can abrogate tumor morphological heterogeneity in order to
increase chemotherapeutic response in the clinic.
Similar to observations by others, our results indicate
that CAIX and HIF-1α do not always co-localize in the same
tumor regions [20, 21]. CAIX is a more robust indicator of
physiologic hypoxia while HIF-1α can often be transiently
upregulated by acute hypoxia or by other non-hypoxia
related factors [22].
TheresultsofourTMAanalysis,evenwithlimited tissue,
indicate presence of histological architectural heterogeneity
in majority of HNSCC, colorectal and lung cancers. This
heterogeneity is not conducive for optimal intratumoral
drug delivery and distribution leading to the suboptimal
chemotherapeutic response seen in the clinic. It is also likely
to compromise synergistic efficacy of antiangiogenic agents
in combination with anticancer agents. Morphologically
homogeneous tumors such as SCLC, FaDu, and HCT-
8 are more likely to respond to antiangiogenic agents in
combinatorial chemotherapy setting especially in chemo-
naive cases.
Currently available antiangiogenic agents are limited by
host tissue toxicity, are cost prohibitive [23], and suffer
from the lacunae that most tumors can easily overcome the
blockade ofa single specificproangiogenic moleculethrough
bypassing onto other proangiogenic markers/pathways. In
fact, recent reports suggest an alarming trend towards
increased metastatic and aggressive disease in the tumors
surviving antiangiogenic therapy in both the clinical and
preclinical model [24, 25]. Since Se is part of the mam-
malian physiology, it is relatively well tolerated and affects
multiple upstream targets such as HIF-1α, Cox-2, and iNos
[26] important for cancer survival and progression and is
likely to have a better success as an antiangiogenic agent
in combination chemotherapy [27]. Inhibition of HIF-1α,
a critical master gene that regulates tumor angiogenesis,
growth, survival, and resistance, has been shown recently
to be through downregulation of reactive oxygen species
and stabilization of prolylhydoxylase 2 and 3 by MSC [28].
This inhibition results in the observed antiangiogenic effects
of MSC besides sensitizing the small but therapy resistant
population of HIF-1α positive, hypoxic cell population to
the cytotoxic effects of the chemotherapeutic agents used
in the combination therapy with MSC [28]. Unlike other
antiangiogenic agents, Se also asserts its anticancer efficacy
independentofantiangiogeniceffectsthroughvariousmech-
anisms such as redox cycling, altering protein-thiol redox
status and methionine mimicry [29]. Since the dose levels of
Se used in the preclinical studies have been attained in the
clinic [30], it is a clinically viable antiangiogenic agent that
can enhance therapeutic efficacy of chemotherapy in various
solid malignancies especially those with a relatively well-
vascularised and homogenous tumor architecture. Further,
the protective effect of Se on healthy tissues from the adverse
effects of cytotoxic drugs [8] allows for drug dose escalation
to higher than their MTDs. Our current data and our earlier
published data [5, 7] demonstrate that a nontoxic dose of Se
is a promising antiangiogenic agent for use in combination
chemotherapy especially against solid tumors with little or
nomorphologicalheterogeneitysuchaswhatisseeninSCLC
H69, HNSCC FaDu and the colorectal cancer HCT-8.
Acknowledgments
Drs. Haikuo Tang and Elizabeth Repasky, Department of
Immunology, Roswell Park Cancer Institute, are acknowl-
edged for generously making the human surgical xenografts
of SCC available. This research is Supported by the
National Cancer Institute Grant 1R21 CA133682-01A2 (A.
Bhattacharya) and a Comprehensive Cancer Center Sup-
port Grant CA016056 from the National Cancer Institute,
Bethesda, Md, USA.
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