TUMOR PHYSIOLOGIC RESPONSE TO COMBRETASTATIN A4 PHOSPHATE
ASSESSED BY MRI
DAWEN ZHAO, M.D., PH.D., LAN JIANG, M.S., ERIC W. HAHN, PH.D., AND RALPH P. MASON, PH.D.
Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX
Purpose: To evaluate the effect of the vascular targeting agent, combretastatin A4 phosphate, on tumor
oxygenation compared with vascular perfusion/permeability.
Methods and Materials:19F MRI oximetry and dynamic contrast-enhanced (DCE)-MRI were used to monitor
tumor oxygenation and perfusion/permeability in syngeneic 13762NF rat breast carcinoma.
Results: A significant drop was found in the mean tumor pO2(23 to 9 mm Hg, p <0.05) within 90 min after
treatment (30 mg/kg of combretastatin A4 phosphate) and a further decrease was observed at 2 h (mean 2 mm
Hg; p <0.01). The initial changes in pO2in the central and peripheral regions were parallel, but by 24 h after
treatment, a significant difference was apparent: the pO2in the periphery had improved significantly, and the
center remained hypoxic. These data are consistent with DCE-MRI, which revealed an ?70% decrease in
perfusion/permeability (initial area under signal-intensity curve) at 2 h (p <0.001). The initial area under
signal-intensity curve recovered fully after 24 h in a thin peripheral region, but not in the tumor center.
Conclusion: The response observed by DCE-MRI, indicating vascular shutdown, paralleled the pO2measure-
ments as expected, but quantitative pO2measurements are potentially important for optimizing the therapeutic
combination of vascular targeting agents with radiotherapy.© 2005 Elsevier Inc.
MRI, Vascular targeting agent, Vasculature, Oxygenation, Breast tumor.
Tumor growth, survival, and metastasis depend critically on
the development of new blood vessels (1). Therefore, ex-
tensive research has focused on developing strategies to
attack the tumor vasculature (1, 2). Tubulin-binding agents
(e.g., combretastatin A4 phosphate [CA4P] and ZD6126)
represent one kind of vascular targeting agent (VTA) (3, 4).
Promising preclinical studies have shown that such agents
selectively cause tumor vascular shutdown and subse-
quently trigger a cascade of tumor cell death in experimen-
tal tumors (4, 5). Although massive necrosis can be induced,
tumors usually regrow from a thin viable rim. Thus, a
combination of VTAs with additional conventional thera-
peutic approaches will be required (6, 7). Several studies
involving the combination of VTAs with radiotherapy (8–
11) or chemotherapeutic agents (12) have shown enhanced
To better understand the mode of action, and hence,
optimize such combinations, in vivo imaging approaches
have been initiated to monitor the physiologic changes
resulting from VTA administration (13–15). Dynamic con-
trast-enhanced (DCE)-MRI based on the transport proper-
ties of gadolinium-diethylenetriamine pentaacetic acid (Gd-
DTPA) is the most commonly used imaging approach to
study tumor vascular perfusion and permeability. DCE-MRI
was included as part of the Phase I clinical trials of CA4P
(16, 17). The results of preclinical and clinical DCE-MRI
studies have shown a reversible change in vascular perfu-
sion in the tumor periphery after a single dose of VTA
(18–21). For combination with radiotherapy, measurement
of tumor oxygen dynamics will be especially important,
because reduced perfusion can induce hypoxia, potentially
modulating the radiation response. A number of studies
have reported an improved response when administering
VTAs after radiotherapy, with the enhancement reduced or
lost, if VTAs were administered before radiotherapy, im-
plying increased hypoxia induced by VTAs (8, 10). Direct
measurements of tissue pO2using the Eppendorf electrode,
conducted by Horsman et al. (22, 23), found increased
hypoxia 3 h after CA4P or ZD6126 administration. We have
Reprint requests to: Ralph P. Mason, Ph.D., C.Sci., C.Chem.,
Department of Radiology, University of Texas Southwestern Med-
ical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9058. Tel:
(214) 648-8926; Fax: (214) 648-2991; E-mail: Ralph.Mason@
Supported by DOD Breast Cancer IDEA Award (DAMD
170310363) (D.Z.), in conjunction with NCI RO1 CA79515/
EB002762 and the Cancer Imaging Program, a P20 Pre-ICMIC
CA86354; MRI experiments were performed at the Mary Nell &
Ralph B. Rogers MR Center—an NIH BTRP No. P41-RR02584.
Acknowledgments—We are grateful to Ammar Adam, and Drs.
Philip Thorpe and Matthew Merritt for technical and collegial
Received Oct 14, 2004, and in revised form Feb 28, 2005.
Accepted for publication Mar 7, 2005.
Int. J. Radiation Oncology Biol. Phys., Vol. 62, No. 3, pp. 872–880, 2005
Copyright © 2005 Elsevier Inc.
Printed in the USA. All rights reserved
0360-3016/05/$–see front matter
developed a method for measuring tumor oxygenation and
dynamics based on19F nuclear magnetic resonance echo
planar imaging after direct intratumoral injection of the
reporter molecule hexafluorobenzene (HFB) called “fluoro-
carbon relaxometry using echo planar imaging for dynamic
oxygen mapping” (FREDOM) (24, 25). This technique pro-
vides pO2measurements at multiple specific locations si-
multaneously within a tumor and reveals the dynamic
changes at individual locations with respect to interven-
tions. We have previously evaluated the tumor oxygen
response to varying interventions such as hyperoxic gas
breathing (26, 27). We also had an anecdotal example of
pO2response to a tumor-selective infarcting agent (28). We
have now applied both DCE-MRI and FREDOM to evalu-
ate tumor perfusion/permeability and oxygen dynamics in
response to CA4P in conjunction with confirmatory histo-
METHODS AND MATERIALS
Rat mammary carcinoma 13762NF was implanted syngenei-
cally in a skin pedicle surgically created on the fore back of Fisher
344 adult female rats (n ? 25, ?150 g, Harlan), as previously
described in detail (29). Of the 25 rats, 9 were used for DCE-MRI,
10 for FREDOM, and 6 for histologic study. The Institutional
Animal Care and Use Committee approved the investigations.
Drug preparation and dosing
CA4P was provided by OXiGENE (Waltham, MA). CA4P was
dissolved in 0.9% saline at a concentration of 30 mg/mL before
each experiment. A single dose of 30 mg/kg CA4P was chosen for
this study, because it is considered a clinically relevant dose (18).
When tumors reached ?1 cm diameter (?0.6 cm3), MRI was
performed using a 4.7 T horizontal bore magnet with a Varian
Unity Inova system. Each rat was given intraperitoneal ketamine
hydrochloride (120 ?L; 100 mg/mL, Aveco, Fort Dodge, IA) as a
relaxant and maintained under general anesthesia (air and 1%
isoflurane, Baxter International, Deerfield, IL). A 27-gauge butter-
fly catheter (Abbott Laboratories, Abbott Park, IL) was placed
intraperitoneally for infusion of CA4P or saline alone. For DCE-
MRI, a tail vein was catheterized using a second 27-gauge butterfly
catheter for contrast agent administration. For oximetry, hexaflu-
orobenzene (50 ?L, Lancaster, Gainesville, FL) was injected di-
rectly into the tumor along two or three tracks in a single central
plane of the tumor, coronal to the rat’s body using a Hamilton
syringe (Reno, NV) with a custom-made, fine, sharp needle (32-
gauge), as previously described in detail (25). A tunable (1H/19F)
volume radiofrequency (RF) coil was placed around the tumor-
bearing pedicle. Each animal was placed on its side in the magnet
with no change in position during the whole study, so that indi-
vidual regions could be tracked. A thermal blanket was used to
maintain body temperature.
Nine tumor-bearing rats were studied before CA4P injection (n
? 6) or saline alone (n ? 3) and 2 and 24 h after treatment. On
each occasion, a series of T1-weighted spin echo images (TR 160
ms, TE 16 ms, field of view 40 ? 40 mm, matrix 128 ? 128, voxel
size 2.0 ? 0.3 ? 0.3 mm, total time for 3 slices 23 s) was acquired
before and after a bolus injection of Gd-DTPA-BMA (injection
within 1 s; 0.1 mmol/kg, Omniscan, Amersham Health, Princeton,
NJ) on three 2-mm-thick cross-sections parallel to the animal. Data
were processed on a voxel-by-voxel basis using software written
by us using Interactive Data Language (IDL), version 5.3/5.4
(Research Systems, Boulder, CO). For each slice, the tumor was
separated into central and peripheral regions. The tumor periphery
was taken to be a 1–2-mm-thick rim aligned around the whole
tumor. Signal intensity vs. time curves were plotted and relative
signal intensity changes (?SI) of each tumor voxel were analyzed
using the equation: (?SI) ? (SIE? SIb)/SIb, where SIErefers to the
enhanced signal intensity in the voxel and SIbis defined as the
average of the baseline images. The area under the normalized
signal intensity-time curve (IAUC) for the first 1.5 min after
Gd-DTPA-BMA injection was integrated.
19F tumor oximetry—FREDOM
A separate cohort of 10 tumors (7 treated and 3 controls) was
used for pO2measurement. A single 4-mm slice parallel to the rat
body containing the strongest fluorine signal was chosen for the
19F MRI pO2studies.1H and19F MR images were acquired using
a spin-echo sequence. Overlaying the19F MR image on the cor-
conventional MRI, tumor oxygenation was estimated on the basis
of19F pulse burst saturation recovery echo planar imaging relax-
ometry of the HFB, as previously described (24). This approach
provided pO2maps with 1.25 mm in plane resolution and 6-?L
voxel size in 6.5 min. The spin-lattice relaxation rate [R1 (s?1) ?
1/T1] was estimated on a voxel-by-voxel basis using a three-
parameter monoexponential function. pO2was estimated using the
relationship pO2mm Hg ? (R1 ? 0.0835)/0.001876 (24). The
data are presented in bins of 5 mm Hg, except for the highest and
lowest bins, which were open ended. Before CA4P or saline
injection, a series of pO2maps was acquired with respect to
respiratory challenge with oxygen: typically, two baseline mea-
surements, three with oxygen and four on return to air. Immedi-
ately after the last (fourth) air measurement, CA4P (30 mg/kg) or
saline (0.15 mL) was injected intraperitoneally. An additional
series of pO2maps was acquired after 10, 30, 60, 90, and 120 min,
and finally another three maps while breathing oxygen. The 24 h
follow-up study comprised two measurements with air and four
with oxygen. The oxygen challenge was included to evaluate
1H image revealed the distribution of HFB. After
Markers of vascular perfusion and endothelium
Six animals were used to study the total blood vessels and
perfused vessels before (n ? 2), 2 h (n ? 2), and 24 h (n ? 2) after
CA4P. The blue fluorescent dye Hoechst 33342 (Molecular
Probes, Eugene, OR) was injected into the tail vein of the anes-
thetized rats at a concentration of 10 mg/kg in 0.9% saline (0.1
mL), and the tumors were excised 1 min later. Tumor specimens
were immediately immersed in liquid nitrogen and then stored at
?80°C. Immediately after cryostat sectioning (6 ?m thick), the
slices were imaged for Hoechst 33342 under ultraviolet wave-
length (330–380 nm). Perfused vessels were determined by count-
ing the total number of structures stained by Hoechst 33342 in four
fields per section selected to show high perfusion and calculating
873Tumor oxygen dynamics after combretastatin A4 phosphate ● D. ZHAO et al.
13. Tozer GM, Prise VE, Wilson J, et al. Combretastatin A-4
phosphate as a tumor vascular-targeting agent: Early effects in
tumors and normal tissues. Cancer Res 1999;59:1626–1634.
14. Kragh M, Quistorff B, Horsman MR, et al. Acute effects of
vascular modifying agents in solid tumors assessed by nonin-
vasive laser Doppler flowmetry and near infrared spectros-
copy. Neoplasia 2002;4:263–267.
15. Goertz DE, Yu JL, Kerbel RS, et al. High-frequency Doppler
ultrasound monitors the effects of antivascular therapy on
tumor blood flow. Cancer Res 2002;62:6371–6375.
16. Rustin GJ, Galbraith SM, Anderson H, et al. Phase I clinical
trial of weekly combretastatin A4 phosphate: Clinical and
pharmacokinetic results. J Clin Oncol 2003;21:2815–2822.
17. Galbraith SM, Maxwell RJ, Lodge MA, et al. Combretastatin
A4 phosphate has tumor antivascular activity in rat and man as
demonstrated by dynamic magnetic resonance imaging. J Clin
18. Prise VE, Honess DJ, Stratford MR, et al. The vascular
response of tumor and normal tissues in the rat to the vascular
targeting agent, combretastatin A-4-phosphate, at clinically
relevant doses. Int J Oncol 2002;21:717–726.
19. Robinson SP, McIntyre DJ, Checkley D, et al. Tumour dose
response to the antivascular agent ZD6126 assessed by mag-
netic resonance imaging. Br J Cancer 2003;88:1592–1597.
20. McIntyre DJ, Robinson SP, Howe FA, et al. Single dose of the
antivascular agent, ZD6126 (N-acetylcolchinol-O-phosphate),
reduces perfusion for at least 96 hours in the GH3 prolacti-
noma rat tumor model. Neoplasia 2004;6:150–157.
21. Padhani AR. MRI for assessing antivascular cancer treat-
ments. Br J Radiol 2003;76(Spec No 1):S60–S80.
22. Horsman MR, Ehrnrooth E, Ladekarl M, et al. The effect of
combretastatin A-4 disodium phosphate in a C3H mouse
mammary carcinoma and a variety of murine spontaneous
tumors. Int J Radiat Oncol Biol Phys 1998;42:895–898.
23. Horsman MR, Murata R. Vascular targeting effects of
ZD6126 in a C3H mouse mammary carcinoma and the en-
hancement of radiation response. Int J Radiat Oncol Biol Phys
24. Hunjan S, Zhao D, Constantinescu A, et al. Tumor oximetry:
Demonstration of an enhanced dynamic mapping procedure
using fluorine-19 echo planar magnetic resonance imaging in
the Dunning prostate R3327-AT1 rat tumor. Int J Radiat
Oncol Biol Phys 2001;49:1097–1108.
25. Zhao D, Jiang L, Mason RP. Measuring changes in tumor
oxygenation. Methods Enzymol 2004;386:378–418.
26. Zhao D, Constantinescu A, Hahn EW, et al. Differential
oxygen dynamics in two diverse Dunning prostate R3327 rat
tumor sublines (MAT-Lu and HI) with respect to growth and
respiratory challenge. Int J Radiat Oncol Biol Phys 2002;
27. Zhao D, Constantinescu A, Hahn EW, et al. Tumor oxygen
dynamics with respect to growth and respiratory challenge:
Investigation of the Dunning prostate R3327-HI tumor. Radiat
28. Mason RP, Ran S, Thorpe PE. Quantitative assessment of
tumor oxygen dynamics: Molecular imaging for prognostic
radiology. J Cell Biochem 2002;87:45–53.
29. Hahn EW, Peschke P, Mason RP, et al. Isolated tumor growth
in a surgically formed skin pedicle in the rat: A new tumor
model for NMR studies. Magn Reson Imaging 1993;11:1007–
30. Pedley RB, Hill SA, Boxer GM, et al. Eradication of colorec-
tal xenografts by combined radioimmunotherapy and combret-
astatin a-4 3-O-phosphate. Cancer Res 2001;61:4716–4722.
31. Song Y, Worden KL, Jiang X, et al. Tumor oxygen dynamics:
Comparison of19F MR EPI and frequency domain NIR spec-
troscopy. Adv Exp Med Biol 2003;530:225–236.
32. Song Y, Constantinescu A, Mason RP. Dynamic breast tumor
oximetry: The development of prognostic radiology. Technol
Cancer Res Treat 2002;1:1–8.
33. Brown JM. The hypoxic cell: A target for selective cancer
therapy—Eighteenth Bruce F. Cain memorial award lecture.
Cancer Res 1999;59:5863–5870.
34. Höckel M, Schlenger K, Aral B, et al. Association between
tumor hypoxia and malignant progression in advanced cancer
of the uterine cervix. Cancer Res 1996;56:4509–4515.
35. Brizel DM, Scully SP, Harrelson JM, et al. Tumor oxygen-
ation predicts for the likelihood of distant metastases in human
soft tissue sarcoma. Cancer Res 1996;56:941–943.
36. Höckel M, Vaupel P. Tumor hypoxia: Definitions and current
clinical, biologic, and molecular aspects. J Natl Cancer Inst
37. Baudelet C, Gallez B. How does blood oxygen level-depen-
dent (BOLD) contrast correlate with oxygen partial pressure
(pO2) inside tumors? Magn Reson Med 2002;48:980–986.
38. Zhao D, Jiang L, Constantinescu A, et al. Evaluation of breast
tumor microcirculation and oxygenation using a combination
of BOLD, DCE and19F MRI [Abstract]. Proc Int Soc Magn
Reson Med 2004;1:222.
39. Beauregard DA, Pedley RB, Hill SA, et al. Differential sen-
sitivity of two adenocarcinoma xenografts to the anti-vascular
drugs combretastatin A4 phosphate and 5,6-dimethylxanthe-
none-4-acetic acid, assessed using MRI and MRS. NMR
40. Le D, Mason RP, Hunjan S, et al. Regional tumor oxygen
dynamics:19F PBSR EPI of hexafluorobenzene. Magn Reson
41. Mason RP, Rodbumrung W, Antich PP. Hexafluorobenzene:
A sensitive19F NMR indicator of tumor oxygenation. NMR
880 I. J. Radiation Oncology ● Biology ● PhysicsVolume 62, Number 3, 2005