2315 Tumor Chemoradiotherapy and Normal Tissue Toxicity Assessment.

Rao V. L. Papineni**, Amit Deorukhkar*, Parmeswaran Diagaradjane*, Sean Orton,

Douglas Vizard, Doulas Wood, William McLaughlin, and Sunil Krishnan*.
Carestream Health, Inc., New Haven, CT and *MD. Anderson Cancer Center, Houston
**E.Mail: rao.papineni1@carestreamhealth.com

Chemoradiotherapy is a standard treatment for locally advanced pancreatic cancer. Available data
support the concept that current chemotherapeutic regimens do not substantially sensitize tumors to radiation 120
100
therapy. Clinical data have shown that newer agents are superior to older agents for advanced pancreatic Control 0 M
cancer. Therefore, we investigated the effects of the combination of these agents with radiation on a Radiation Schema Radiosensitizer (10M) 10 M
pancreatic cancer model, with special emphasis on the likelihood of sensitization of normal tissues Group
Randomize Euthanasia
100 25 M

Relative Absorbance
contributing to increased toxicity. I Vehicle 50 M
We established MiaPaCa pancreatic cancer xenograft tumors on the right thighs of nude mice and II Drug

Survival (%)
III Radiation
randomized them into four groups treated with vehicle, radiosensitizer, gamma-radiation (2Gy x 5 to the Drug + Rad
10 80
IV
tumor alone), and gamma-radiation in combination with radiosensitizer. Bidimensional tumor measurements
Tumor
were made periodically after treatment and the time to doubling of tumor volume was estimated in each inoculation Radiation

cohort. Tumors, both femurs and both thigh musculatures were collected from mice in each cohort and 60
subject to X-ray densitometric analysis and immunohistochemical analysis. Changes in the bone density
resulting from radiation are routinely determined from 2D radiographic images obtained using a 1
commercially available imaging system, KODAK in-Vivo Multispectral FX system and the bone density 40
analysis software.
An ideal radiosensitizer widens the therapeutic window for treatment of locally advanced pancreatic cancer
by improving the radioresponse of tumors without increasing normal tissue toxicity. Such agents need to be 20
tested in animal models where simultaneous assessment of tumor and normal tissue effects are feasible. We
0.1
present early evidence of such a radiosensitizer.
0 2 4 6
0
Pancreatic cancer cell lines MiaPaCa-1, AsPC-1 and Colo-357 were kindly provided by Dr. Ray Radiation Dose (Gy) MiaPaCa-2 Colo-357 AsPC-1
Meyn (MD Anderson Cancer Center). They were cultured in DMEM supplemented with 10% FBS
Pretreatment of the drug displays profound intrinsic radiosensitization Cell Viability assay shows the drug concentrations required for the
and 1% Penicillin-streptomycin (Invitrogen, Grand Island, NY). The radiosensitizer was procured
effects of the cells as determined by Clonogenic Survival Assay. Radiosensitization is far below the levels that result in cell toxicity.
from Sigma, and stock solutions in dimethyl sulfoxide (DMSO) were stored at -20 C, and then
diluted in cell culture medium immediately prior to use.
In vitro cytotoxicity
The effect of radiosensitizer on tumor cells was assessed using the XTT cell proliferation kit (Roche
Applied Science, Indianapolis, IN). Briefly, cells were seeded in 96-well plates (3,000 cells per well) 2.5
and grown overnight. Next day, the medium was aspirated and cells were exposed to different
Left Femur
concentrations of drug. Following 4 h, drug was aspirated; the wells were rinsed and replenished Femur-Tumor Side
with fresh medium. After further 48 h of culture, cell viability was determined by XTT assay. Results
2.0
are expressed as percent cell viability for each concentration of drug with respect to controls (0.1%
DMSO in medium).

Density (g/cm3)
Clonogenic Survival
Cells were treated with vehicle control (DMSO) or 10 M drug for 4 h and then irradiated with a 1.5
137Cs unit at room temperature. Following 3 h, drug was washed, cells were trypsinized and specific
Increased Femur bone density – tumor bearing
cell densities were re-plated in 60-mm petridishes and were incubated for colony formation for 10-14 limb. The bone densities (Left Plot) were
days. Colonies were counted after staining with 0.5% alcoholic crystal violet. The fraction surviving 1.0 determined using the Long-bone Density Software,
a given treatment was calculated with respect to the survival of unirradiated controls (cells treated corrected for both the tissue depth and the effect of
with DMSO or drug alone). that depth upon CaPO4 column density. This is
Animals enabled by modeling a cylindrical symmetry
0.5
Athymic nu/nu mice (4 weeks old) were obtained from the breeding colony of the Department of (Cartoon above) to the analytical X-ray image (Far
Experimental Radiation Oncology at The University of Texas M. D. Anderson Cancer Center. The Left Panel). Estimates provided as CaPO4 mass per
animals were housed five/cage in standard mouse plexiglass cages in a room maintained at constant volume (g/cm3).
temperature and humidity under 12-h light and dark cycles and fed with regular autoclaved chow diet 0.0 X-ray Image ( Far Left Panel):
Zones away from
with water ad libitum. None of the mice exhibited any lesions, and all were tested pathogen free. the Vertebrae:
Zone 1 Zone 2 Zone 3 Zone 4 Zone 5 Zone 6 60 FOV; 5- min exp, 0.8 mm X-ray filter, F-stop 4.0,
Before initiating the experiment, we acclimatized all mice to a pulverized diet for 3 days. Our no binning.
experimental protocol was reviewed and approved by the Institutional Animal Care and Use
Committee.
Xenograft implantation of MiaPaCa-2 cells A representative X-ray image Left Tibia
Tibia-Tumor Side
Conclusions
MiaPaCa-2 cells were harvested from sub-confluent cultures after a brief exposure to 0.25% trypsin of thigh region from Left Tibia (Radius) •Early evidence display the effective
and 0.2% EDTA. Trypsinization was stopped with medium containing 10% fetal bovine serum. The Irradiated mice - imaged Tibia-Tumor Side (Radius) concentrations of the radiosensitizer-the cohort
using the Magnification stage 140
cells were washed once in serum-free medium and resuspended in phosphate-buffered saline (PBS). study is ongoing.
accessory for IS4000 3.0
Only suspensions consisting of single cells with >90% viability were used for the injections. Mice •Tumorigenesis causes profound bone changes in
were anesthetized with ketamine-xylazine solution, and 2 x 106 cells were injected subcutaneously FX/DXS X-ray imaging

Outer Radius Size (%)

(Right Panel). The results mice. Bone density measurements using bench-top
into the right leg of each mouse using a 27-gauge needle and a calibrated push button-controlled 2.5 120 X-ray imaging and non-invasive approaches is
dispensing device (Hamilton Syringe Company, Reno, NV). from the cylindrical fitting
routine using the Lon-Bone ongoing to determine the cause and significance of
Density (g/cm3)

Experimental Protocol these changes.

density software is shown 2.0
One week after implantation, mice were randomized into the following treatment groups (n = 9-10)
beneath the X-ray Image.
based on the tumor volume measured by Vernier calipers: (A) untreated control (corn oil, 100 l 100 •Bone density and morphometric measurements
60 FOV; 5- min exp, 0.8 mm provide a valuable information to assess the tissue
daily); (B) radiation (2 Gy, five times a week; two weeks). For irradiation, animals were anesthetized 1.5
X-ray filter, F-stop 4.0; no toxicity effects during chemoradiotherapy. Future
and immobilized in the treatment position with their right legs extended. Radiation was delivered at
binning, converted to density, studies are necessary to determine if use of drugs
a dose rate of 1.25 Gy/min through a single posterior to anterior collimated 3-cm cobalt beam with a 80
Grid ROI applied to profile 1.0 to counter these changes are essential during the
5-mm bolus placed over the tumor. After the last dose of radiation (see schema), mice were
density. chemoradiotherapy.
sacrificed and half of the tumor tissue was formalin-fixed and paraffin-embedded for IHC and
routine hematoxylin and eosin (H&E) staining. The other half was snap-frozen in liquid nitrogen and 0.5
stored at –80°C. H&E staining confirmed the presence of tumor(s) in each specimen. 60
Bone Density Irradiation results in decreased bone density and bone
The animal were subjected to bone density measurements using a commercially available imaging morphological changes at the tumor bearing half. The 0.0
A Method and Apparatus for Measuring Long Bone Density
system, bench-top multimodal (X-ray and optical) imaging apparatus- KODAK in-Vivo bone density and the outer radius measurements of tibia Controls Irradiation of Small-Animal. Douglas L. Vizard, Douglas Wood, William E. Mclaughlin
Multispectral FX system and the Long-bone density analysis software. (Right Plot). and Rao V. L. Papineni. Carestream Molecular Imaging, New Haven.
Docket 94280 U.S. Patent application (in process).

"Molecular Imaging - Wisdom To See For Maladies To Flee"

Dr. Rao V. L. Papineni