Early therapeutic efficacy of anti–death receptor 5 antibody (TRA-8) combined with gemcitabine was measured using diffusion-weighted magnetic resonance imaging (DWI) in an orthotopic pancreatic tumor model. Groups 1 to 4 of severe combined immunodeficient mice (n = 5–7 per group) bearing orthotopically implanted, luciferase-positive human pancreatic tumors (MIA PaCa-2) were subsequently (4–5 weeks thereafter) injected with saline (control), gemcitabine (120 mg/kg), TRA-8 (200 μg), or TRA-8 combined with gemcitabine, respectively, on day 0. DWI, anatomic magnetic resonance imaging, and bioluminescence imaging were done on days 0, 1, 2, and 3 after treatment. Three tumors from each group were collected randomly on day 3 after imaging, and terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining was done to quantify apoptotic cellularity. At just 1 day after starting therapy, the changes of apparent diffusion coefficient (ADC) in tumor regions for group 3 (TRA-8) and group 4 (TRA-8/Gem) were 21 ± 9% (mean ± SE) and 27 ± 3%, respectively, significantly higher (P < 0.05) than those of group 1 (−1 ± 5%) and group 2 (−2 ± 4%). There was no statistical difference in tumor volumes for the groups at this time. The mean ADC values of groups 2 to 4 gradually increased over 3 days, which were concurrent with tumor volume regressions and bioluminescence signal decreases. Apoptotic cell densities of tumors in groups 1 to 4 were 0.7 ± 0.4%, 0.6 ± 0.2%, 3.1 ± 0.9%, and 4.7 ± 1.0%, respectively, linearly proportional to the ADC changes on day 1. Further, the ADC changes were highly correlated with the previously reported mean survival times of animals treated with the same agents and doses. This study supports the clinical use of DWI for pancreatic tumor patients for early assessment of drug efficacy. [Cancer Res 2008;68(20):8369–76]

Pancreatic cancer has the highest fatality rate of all cancers and is the fourth leading cause of cancer death in the United States in 2007.8

8

Presentation from the American Cancer Society (Cancer Statistics, 2007).

The efficacies of current drugs such as 5-fluorouracil, irinotecan, oxaliplatin, and gemcitabine are modest in most pancreatic cancer patients (15). A therapeutic approach that selectively kills pancreatic cancer would be highly advantageous and may be possible by targeting death receptors expressed on pancreatic tumor cells (6, 7). Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in most tumor cell lines (811) via death receptor 4 and death receptor 5 (1215). Of concern, the significant cytotoxicity of TRAIL in human hepatocytes may limit its clinical application (16). The hepatoxicity of TRAIL may be related to binding to multiple receptors (1720); the limitation could be overcome by using an agonistic antibody targeting only one death receptor.

A novel murine, monomeric monoclonal antibody (TRA-8) was developed specifically to target death receptor 5, predominantly expressed in most cancer cells but not in normal cells (21). The antitumor efficacy of TRA-8 in five pancreatic cancer cell lines has been measured by in vitro cytotoxicity assay; each cell line had a unique sensitivity for TRA-8 (22). Pancreatic tumor cell resistance might be reduced by exposure to additional drugs and/or radiation, which destabilizes the mitochondrial membrane and subsequently releases cytochrome c, leading to the activation of caspase-3 (23, 24). Although combination therapy might be superior to monotherapy, a certain range of therapeutic efficacy is predicted in patients with genetically heterogeneous tumors. Therefore, it would be ideal to determine the degree of tumor response in each individual patient following treatment, and then to adjust therapeutic strategy at the earliest possible time in efforts to improve survival.

Diffusion-weighted magnetic resonance imaging (DWI) has successfully been applied in various cancers to evaluate early response against effective therapy (2527) and has been positively correlated with eventual clinical outcome (28). In the early stage of apoptosis, water in the extracellular space is increased due to apoptotic volume decrease. This quantitative change in water can be measured as the apparent diffusion coefficient (ADC), depicted on DWI with high sensitivity, before visible change of tumor morphology and size. Early assessment of response should enable application of appropriate agents during neoadjuvant chemotherapy. Effective neoadjuvant chemotherapy will result in a decrease of primary tumor size to facilitate surgical tumor removal as well as to prevent potential metastasis.

The aim of this study was to develop a DWI protocol to detect early therapeutic response following treatment with TRA-8 combined with gemcitabine in a mouse model of orthotopic pancreatic tumor, and to correlate the early ADC change with animal survival time. In addition, living tumor mass was monitored by bioluminescence imaging to confirm the killing efficacy by the combined therapy while, simultaneously, the tumor volumes were measured using standard anatomic magnetic resonance imaging; both parameters were compared with the ADC values from repeated DWI. The results show that noninvasive imaging parameters developed in this study accurately reflected the efficacy of the novel combined therapy in pancreatic cancer, and thus may be readily translated to a clinical trial.

Reagents and cell lines. All reagents were from Fisher unless otherwise specified. The human pancreatic cell line MIA PaCa-2 was a gift from Dr. M. Hollingsworth (University of Nebraska, Omaha, NE). MIA PaCa-2 cells were cultured in DMEM (Mediatech, Inc.) with 10% fetal bovine serum (Hyclone). Luciferase-positive MiaPaCa-2 cells were created using the ViraPort retroviral vector, which does not require antibiotics for selection (Stratagene). After viral infection, MiaPaCa-2 cells were diluted to single cells to produce a stable luciferase-positive clone. Single colonies were screened based on luminescence signal obtained with the IVIS-100 system. The luciferase-positive Mia PaCa-2 clone was allowed to proliferate, resulting in the cells used for this study. All MIA PaCa-2 cells reported in this publication were luciferase positive but denoted as only MIA PaCa-2. Luciferin was purchased from Xenogen, Inc. Purified TRA-8 (mouse origin) was provided by Daiichi Sankyo (Tokyo, Japan). Gemcitabine (Eli Lilly and Company) was purchased from the University of Alabama at Birmingham Hospital Pharmacy. Purified mouse IgG1 K isotype control antibody was purchased from SouthernBiotech. Fresh Tc-99m pertechnetate was purchased from Birmingham Nuclear Pharmacy.

HYNIC conjugation and radiolabeling. A fresh 1.8 mmol/L solution of succinimidyl 6-hydrazinonicotinate (HYNIC; courtesy of Dr. Gary Bridger, AnorMED, Inc., Langley, British Columbia, Canada) in dimethylformamide was prepared. Forty picomoles were transferred to glass vials, followed by freezing at −90°C, and then the solutions were vacuum dried using Advantage Benchtop Freeze Dryer (Virtis Co., Inc.) with the shelf temperature at −75°C and trap at −90°C. The vials were sealed under vacuum and kept frozen at −80°C until use. Each vial was reconstituted with 1.0 mL of sodium phosphate buffer (0.15 mol/L, pH 7.8) containing 1 mg of TRA-8 (HYNIC/TRA-8 molar ratio of 6; ref. 29). After a 3-h incubation at room temperature, the mixture was transferred to Slide-A-Lyzer dialysis cassette having 10,000 molecular weight cutoff (Pierce) and then immersed in 1,000 mL PBS (pH 7.4) overnight at 4°C. The HYNIC-modified TRA-8 was labeled with Tc-99m using SnCl2/tricine as the transfer ligand (30), and unbound Tc-99m was removed by G-25 Sephadex size-exclusion chromatography. The radiolabeling yield was ∼60%. Protein concentrations of the collected fractions were measured by Lowry assay (31). The level of Tc-99m binding to TRA-8 was always >96%, as measured by TLC using separate strips eluted with saturated saline and methyl ethyl ketone.

Animal preparation. Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee. Seven groups of female severe combined immunodeficient mice (National Cancer Institute-Frederick Animal Production Program; 4–5 wk old; n = 5 for groups 1 to 3, 6, and 7; n = 7 for group 4; n = 3 for group 5) were used. The procedure for intrapancreatic tumor implantation was as follows: a 1-cm incision was made in the left upper quadrant of the abdomen, and a solution of 2.5 × 106 MIA PaCa-2 cells in 40 μL of DMEM was injected into the tail of the pancreas, whereas the mice were anesthetized using i.p. injection of xylazine and ketamine. The skin and peritoneum was closed in one layer with three interrupted 5-0 Prolene sutures. Four weeks after tumor implantation, abdominal ultrasound imaging was done as described below to select mice with matched tumor size for groups 1 to 5. DWI, anatomic magnetic resonance imaging, and bioluminescence imaging were done for groups 1 to 4 on days 0, 1, 2, and 3, at 4 to 5 wk after tumor cell implantation; groups 1 to 4 were injected with i.v. saline (200 μL), i.p. gemcitabine (120 mg/kg), i.v. TRA-8 (200 μg), and i.v. TRA-8 (200 μg) combined with i.p. gemcitabine (120 mg/kg), respectively, on day 0 after imaging. TRA-8 and gemcitabine were injected mixed with 200 μL of saline. DWI and anatomic magnetic resonance imaging were also done on group 5, a second control group injected with i.v. saline (200 μL), on days 0, 3, and 6. The mean tumor sizes of the groups 1 to 5 were not statistically different at the beginning of therapy. All mice in groups 1 to 4 were sacrificed after imaging on day 3, and histologic analysis [terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining] for tumors was followed. To assess distribution of TRA-8, animals in groups 6 and 7 were i.v. injected with Tc-99m–labeled TRA-8 (4.4 MBq, 9 μg) and Tc-99m–labeled isotype control antibody (3.2 MBq, 10 μg), respectively; single photon emission computed tomography (SPECT) and X-ray computed tomography (CT) were applied at 6 h after injection, followed by biodistribution study at 24 h after injection. The mean tumor weights of the groups 6 and 7 were not statistically different. All animals were anesthetized using isoflurane gas (1–2%) during imaging.

Magnetic resonance imaging. Small animal DWI was done on a Bruker BioSpec 9.4T system (Bruker BioSpin Corp.). The animal was placed on an animal bed equipped with circulating warm water to regulate body temperature during magnetic resonance imaging scans. An orthogonally bent plastic board was used to prevent the transfer of the respiratory motion in chest to abdominal area, as shown in Fig. 1. DWI data were collected using a standard spin echo sequence with two b-factors (5 and 1,000 s/mm2) at three orthogonal gradient directions (x, y, z) because no significant difference between ADC values obtained with two b factors and those with six b factors was measured in previously reported DWI study on glioma (28). The acquisition parameters are as follows: repetition time (TR)/echo time (TE), 2,500/32 ms; diffusion separation time, 16 ms; diffusion gradient duration, 6 ms; 128 × 128 matrix; and a 30 × 30-mm field of view. A total of three to five 1-mm-thick slices (0.2-mm gap) were used to cover tumor regions of interest. Tumor was imaged using a combination of a 1H volume resonator/transmitter and a surface coil receiver (Bruker BioSpin). A magnetic resonance–compatible small animal respiratory gating device (SA Instrument, Inc.) was used during the scans. A test tube filled with 70% ethanol was placed on the animal's skin adjacent to the tumor and imaged concurrently, so that ADC values in the tumor regions were adjusted using the mean ADC value of the standard in efforts to correct ADC quantification error due to body temperature fluctuation (32) or systemic error from the imager.

Figure 1.

Illustration for applying an orthogonally bent plastic board to minimize motion transfer from chest to abdominal area, where tumor is located.

Figure 1.

Illustration for applying an orthogonally bent plastic board to minimize motion transfer from chest to abdominal area, where tumor is located.

Close modal

While the animal was under anesthesia, anatomic magnetic resonance imaging to measure tumor volume was done using a T2-weighted spin echo sequence (RARE) with the following acquisition parameters: repetition time (TR)/echo time (TE), 2,000/48.8 ms; 128 × 128 matrix; and a 30 × 30-mm field of view. Continuous 1-mm thick slices were used to cover the entire tumor region. Total acquisition time for DWI and anatomic magnetic resonance imaging was ∼35 min.

The tumor area was segmented from images obtained with the smaller b factor, based on the signal intensity between the region of interest and background. The ADC value of each pixel of the segmented tumor region was calculated as follows: ADC = ln(I1/I2)/(b2/b1), where I1 was the intensity on the pixel with b1, and I2 was that with b2. The final ADC map was obtained by averaging three ADC maps in three orthogonal directions to acquire orientation-independent ADC values. The segmentation and quantification of 70% ethanol used as the standard were implemented with the same procedure. The final ADC value of a tumor was obtained by averaging all ADC values in the whole tumor region. Tumor volume was measured by summing all voxels inside of the tumor boundary of the anatomic magnetic resonance images. Segmentation of the tumor volume was done using ImageJ version 1.37v (NIH). The ADC quantification and finding best-fitting curves for ADC changes (described in result session) were implemented using computer software developed with Labview version 8.5 (National Instruments Co.).

Ultrasound imaging. Ultrasound imaging was done using a VisualSonics VEVO 660 high-frequency, high-resolution ultrasound instrument with a 40-MHz probe. Animals were placed in the supine position for examination with B-Mode imaging (33). The largest diameter was found in the anterior-posterior plane, and this diameter and a transverse diameter were measured to quantify tumor area (length × width).

SPECT/CT imaging. SPECT/CT imaging was done using a SPECT/CT dual-modality imager (X-SPECT, Gamma Medica-Ideas). In SPECT imaging, a total of 64 projections (data matrix size, 56×56 per projection) were acquired with a 50-s acquisition time per projection using a pinhole collimator with a 1-mm tungsten pinhole insert. The field of view was 43.8 mm, whereas the radius of rotation was 32 mm. Images were reconstructed using an ordered subsets expectation maximization algorithm (8 subsets and 20 iterations). The fourth-order Butterworth digital filtrations (fc, 0.25; fm, 0.15) provided by the vendor software were applied for all SPECT images to enhance the image quality. For the CT system, the X-ray tube was operated at a voltage of 50 kVp and an anode current of 0.6 mA. Two hundred fifty-six projections were acquired to obtain the CT images, and acquisition time per projection was 0.5 s. The coregistration of SPECT and CT images was done using computer software, IDL Virtual Machine (Research System, Inc.). A 60-W heat lamp warmed the bodies of the animals while they were under anesthesia. A consistent color scale was applied to all SPECT images after correction for radioactive decay and dose.

Biodistribution study. Tumor, liver, and blood were collected for each animal; those samples were weighed and the Tc-99m activity was measured using a calibrated gamma-ray counter (MINAXIγ Auto-gamma 5000 Series Gamma Counter, Packard Instrument Company), decay corrected to dosing time, and converted to absolute radioactivity; then the percent of injected dose per gram of each tissue (%ID/g) was determined.

Bioluminescence imaging. Bioluminescence imaging was applied to all mice after magnetic resonance imaging on each day using the IVIS-100 imaging system (Xenogen). Each mouse was i.p. injected with luciferin at a dose of 2.5 mg (0.1 mL) and imaged after 15 min on a temperature-controlled warm bed (37°C) with the following imaging parameters: 5- to 10-s luminescent exposure time, 8 photographic binning, 25-cm axial field of view, and 1 f-stop. The region of interest was drawn manually around the tumor area, and the light emitted from the region of interest was measured using the vendor software. Mice were imaged weekly before selection by bioluminescence and ultrasonography for inclusion in the therapy study. Mice with diffuse bioluminescence signal in the peritoneal cavity (leakage from implantation site, ∼15% of implantations) were excluded from further study. Mice with nonspherical tumors as determined by ultrasonography were also excluded from further study.

Histologic analysis. Three tumors from each group were selected randomly after sacrificing the animals, and TUNEL staining of each tumor tissue was done. Detailed tumor tissue staining procedure is presented in Appendix 1. Two digital pictures (×250) were taken away from areas of necrosis but otherwise randomly for each tumor slice by one investigator blinded to the treatments that each animal had received, using SPOT camera on a Nikon Optiphot-2 microscope (Nikon, Inc.), interfaced with a personal computer and SPOT software. The image analysis software was ImageJ version 1.37v (NIH). The apoptotic (TUNEL) cells were segmented by the signal intensity difference between the target cells and background in each picture, whereas the intensity and minimum particle size thresholds were determined manually and then counted in all the two pictures per tumor. The number of total tumor cells was also counted with the same procedure, and the cell density (apoptotic cell number / total tumor cell number) was calculated. Uneven background intensity was corrected using the “rolling ball” algorithm (34), whereas the radius was manually determined.

Statistical analysis. One-way ANOVA (35) was carried out using SAS version 8.2 (SAS Institute, Inc.) to compare the averaged ADC, bioluminescence, and tumor volume changes in mice without treatment to those treated with TRA-8, gemcitabine, and TRA-8 combined with gemcitabine. ANOVA was also applied to compare the results of TUNEL staining in tumors of mice in the four different groups.

SPECT imaging and biodistribution analysis confirmed the specific uptake of Tc-99m–labeled TRA-8 into orthotopic pancreatic tumor xenografts.Figure 2 shows a photograph of a representative mouse bearing an orthotopic pancreatic tumor (Fig. 2A) and SPECT/CT fused images (coronal view) at 6 hours after injection of Tc-99m-TRA-8 (Fig. 2B) or Tc-99m-isotype control antibody (Fig. 2C) in another mouse with the same color scaling. The dotted circle in each subfigure indicates the tumor region. There was higher tumor uptake of Tc-99m-TRA-8 as compared with the Tc-99m-isotype control antibody. From the biodistribution study, the %ID/g in tumor, liver, and blood at 24 hours after Tc-99m-TRA-8 injection was 20.0 ± 2.2, 11.0 ± 0.6, and 12.9 ± 1.7, respectively, whereas those tissues for mice injected with Tc-99m-isotype control antibody were 9.4 ± 1.8, 12.5 ± 0.7, and 14.7 ± 0.8, respectively. The tumor uptake of Tc-99m-TRA-8 was significantly (P < 0.05) higher than that of the Tc-99m–labeled isotype control antibody.

Figure 2.

Photograph of a representative orthotopic pancreatic tumor (A,) and in vivo SPECT/CT images (coronal view) at 6 h after Tc-99m-TRA-8 (B) or Tc-99m-isotype control antibody (C) injection i.v. The same color scale was applied for both the SPECT images.

Figure 2.

Photograph of a representative orthotopic pancreatic tumor (A,) and in vivo SPECT/CT images (coronal view) at 6 h after Tc-99m-TRA-8 (B) or Tc-99m-isotype control antibody (C) injection i.v. The same color scale was applied for both the SPECT images.

Close modal

A significant ADC increase was detected by DWI at 1 day after combined therapy or TRA-8 monotherapy.Figure 3 shows a representative set of diffusion-weighted magnetic resonance images at b = 5 s/mm2 (Fig. 3A) and b = 1,000 s/mm2 (Fig. 3B) with the same gray scaling and the ADC map calculated from both the images (Fig. 3C). The tube filled with 70% ethanol (standard for ADC quantification) and the tumor regions are indicated with dotted rectangle and circle, respectively, in Fig. 3A. Figure 4A presents the ADC changes (mean ± SE) in tumors (all voxels) for groups 1 to 4 at 1, 2, and 3 days after treatment. The ADC value for the four groups on day 0 averaged 0.00118 ± 0.00006 mm2/s (n = 22). On day 1, the mean ADC changes of groups 3 and 4 were 21 ± 9% and 27 ± 3%, respectively, significantly (P < 0.05) higher than that of group 1 (−1 ± 5%) or group 2 (−2 ± 4%). The mean ADC increases for group 4 on all the 3 days of measurement were higher than those of group 3, but the differences were not significant statistically (P > 0.05). The mean ADC change of group 2 gradually increased and reached 12 ± 9% on day 3, which was statistically different (P < 0.05) than that of group 1. Of interest, the ADC values of group 1 (control) gradually decreased over time, which was confirmed in a repeat experiment (group 5); the mean ADC changes of group 5 (control) were −12 ± 4% and −26 ± 6% at 3 and 6 days after treatment, respectively.

Figure 3.

Representative diffusion-weighted images of a mouse bearing a MIA PaCa-2 tumor orthotopically at two different diffusion-weighting factors, b = 5 s/mm2 (A) and b = 1,000 s/mm2 (B), with constant gray scale. C, ADC map calculated from images A and B. The orthotopic pancreatic tumor and a plastic tube filled with 70% ethanol (a standard for ADC quantification) are indicated with dotted circle and rectangle, respectively (A).

Figure 3.

Representative diffusion-weighted images of a mouse bearing a MIA PaCa-2 tumor orthotopically at two different diffusion-weighting factors, b = 5 s/mm2 (A) and b = 1,000 s/mm2 (B), with constant gray scale. C, ADC map calculated from images A and B. The orthotopic pancreatic tumor and a plastic tube filled with 70% ethanol (a standard for ADC quantification) are indicated with dotted circle and rectangle, respectively (A).

Close modal
Figure 4.

Analyses of tumor response to treatment. Intratumoral ADC change (A), tumor volume change (B), and bioluminescence signal change (C), measured at 1, 2, and 3 d after initiation of therapy, when groups 1 to 4 were untreated and treated with gemcitabine (Gem; 120 mg/kg), TRA-8 (200 μg), and combined therapy, respectively, on day 0. Statistical differences among groups are indicated by different letters on each day.

Figure 4.

Analyses of tumor response to treatment. Intratumoral ADC change (A), tumor volume change (B), and bioluminescence signal change (C), measured at 1, 2, and 3 d after initiation of therapy, when groups 1 to 4 were untreated and treated with gemcitabine (Gem; 120 mg/kg), TRA-8 (200 μg), and combined therapy, respectively, on day 0. Statistical differences among groups are indicated by different letters on each day.

Close modal

Tumor volume and bioluminescence signal changes for groups 1 to 4 are presented in Fig. 4B and C, respectively. Tumor volume changes were not statistically different among any of the groups on day 1, but the mean values for groups 3 and 4 were significantly lower (P < 0.05) than those of groups 1 and 2 on days 2 and 3. Whereas the mean tumor volume of group 2 remained constant, that of group 1 increased 18 ± 14% during the 3-day monitoring period. The tumor volume changes of group 5 (control) were 10 ± 11% and 6 ± 11% on days 3 and 6, respectively. Mean bioluminescence signal changes of both groups 1 and 2 increased ∼250% during the 3 days, whereas those of groups 3 and 4 decreased 89 ± 2% and 88 ± 2%, respectively, during the same time period, comparable with tumor volume decreases of group 3 (69 ± 5%) and group 4 (74 ± 6%).

Early ADC increase was highly correlated with apoptotic cellularity.Figure 5A shows representative photomicrographs of tumor slices (5-μm thickness) following TUNEL staining. Quantifications of apoptotic cell density of groups 1 to 4 are represented in histograms (Fig. 5B). The apoptotic cell densities of groups 3 and 4 were significantly higher than those of the other groups, indicating the superior therapeutic efficacy of TRA-8. Figure 5C shows that the mean apoptotic cell density was linearly proportional to the mean ADC increase measured at 1 day after therapy initiation (shown in Fig. 4A).

Figure 5.

Histologic analyses of tumor response. A, representative TUNEL (×250) staining of MIA PaCa-2 tumors collected at day 3, when saline (untreated), gemcitabine, TRA-8, and TRA-8 combined with gemcitabine were administrated on day 0. An apoptotic cell is indicated with a black arrow in each sub-figure. B, apoptotic cell densities are presented at the four different treatments, whereas different letters above the bars indicate statistical differences among groups. C, apoptotic cell density (shown in B) versus ADC increase over 1 d (shown in Fig. 4A).

Figure 5.

Histologic analyses of tumor response. A, representative TUNEL (×250) staining of MIA PaCa-2 tumors collected at day 3, when saline (untreated), gemcitabine, TRA-8, and TRA-8 combined with gemcitabine were administrated on day 0. An apoptotic cell is indicated with a black arrow in each sub-figure. B, apoptotic cell densities are presented at the four different treatments, whereas different letters above the bars indicate statistical differences among groups. C, apoptotic cell density (shown in B) versus ADC increase over 1 d (shown in Fig. 4A).

Close modal

Early ADC increase was highly correlated with survival time.Figure 6A shows the ADC changes at 1 day after treatment were linearly proportional to the mean survival time, measured in our previous study (22); the mean survival times were 76 ± 3 (mean ± SE), 79 ± 5, 121 ± 4, and 142 ± 7 days, when mice were untreated (control) or treated with two cycles of gemcitabine, TRA-8, and TRA-8 plus gemcitabine, respectively, with the same doses used for the current study. From Fig. 6A, an equation to correlate the early ADC changes and survival times was derived as follows,

Figure 6.

Correlation between early ADC change and survival time. A, mean survival times [from Derosier et al. (22)] versus ADC increases at 1 d after therapy (shown in Fig. 4A). The mean survival times were measured in previous experiment when four groups (n = 10 per group) of severe combined immunodeficient mice bearing MIA PaCa-2 tumors orthotopically were untreated (control) or treated with two cycles consisting of multiple injections of gemcitabine, TRA-8, and TRA-8 combined with gemcitabine. The dose fraction for each group was the same with that in the current study. B, mean ADC changes of four groups (shown in Fig. 4A) for 3 d after therapy initiation and best-fitting nonlinear curves (Eq. B) with four different β values. C, mean survival times versus the curve constants (β).

Figure 6.

Correlation between early ADC change and survival time. A, mean survival times [from Derosier et al. (22)] versus ADC increases at 1 d after therapy (shown in Fig. 4A). The mean survival times were measured in previous experiment when four groups (n = 10 per group) of severe combined immunodeficient mice bearing MIA PaCa-2 tumors orthotopically were untreated (control) or treated with two cycles consisting of multiple injections of gemcitabine, TRA-8, and TRA-8 combined with gemcitabine. The dose fraction for each group was the same with that in the current study. B, mean ADC changes of four groups (shown in Fig. 4A) for 3 d after therapy initiation and best-fitting nonlinear curves (Eq. B) with four different β values. C, mean survival times versus the curve constants (β).

Close modal
\[\mathrm{EST}=2.89\mathrm{ADC}_{1\mathrm{D}}+5.26{\approx}3\mathrm{ADC}_{1\mathrm{D}},\]

where EST is extended survival time (%) by treatment and ADC1D is ADC change (%) at 1 day after therapy initiation. However, one major concern in the application of ADC measurement for early therapy evaluation would be that the water induced by tumor cell killing may not be confined within the tumor boundary but may diffuse away. As more water is generated, a higher oncotic pressure may result, causing an even faster diffusion. Therefore, the prognosis based on Eq. A might be varied according to the imaging time point after starting therapy.

A new, DWI-based imaging biomarker to measure therapeutic efficacy independently from imaging time point was proposed.Figure 4A shows that the amount of ADC increase for group 4 was decreased on each subsequent day after therapy was initiated. If the amount of extracellular water induced by apoptosis were in equilibrium with the water that was diffusing out from the tumor, then the magnitude of ADC increase relative to the baseline value would be constant. Therefore, the ADC increase over time could be modeled by

\[\mathrm{ADC}_{n}={\alpha}(1{-}e^{{\beta}n}),\]

where n is time (day); ADCn is ADC change (%) relative to day 0 at n day after therapy initiation; α is the maximum ADC increase (%); and β is a constant to determine the change rate of ADC increase over time. From the best-fitting curve to the ADC changes of group 4, α was estimated to be 37%. Figure 6B presents four best-fit curves for the average ADC changes in groups 1 to 4, when α was fixed to 37%. The β values were −0.113, 0.096, 0.634, and 1.224 for groups 1 to 4, respectively. Figure 6C shows that the β values were linearly proportional to the mean survival time, and therefore Eq. A can be rewritten to correlate the β values with survival times as follows:

\[\mathrm{EST}=69.7{\beta}+5.26{\approx}70{\beta}\]

.

In summary, the β value obtained from multiple DWI studies could be considered as a new imaging biomarker.

To our knowledge, this is the first report of the application of DWI in an orthotopic pancreatic tumor model. Further, this study shows the feasibility of DWI for early detection of effective therapy of an apoptosis-inducing drug for pancreatic cancer treatment. To date, DWI of orthotopic pancreatic tumor has had limited success due to severe respiratory motion artifact in the abdominal area. To prevent motion transfer from the chest region, each tumor was secured using an orthogonally bent plastic board (Fig. 1). The pressure applied by this board was localized on the abdominal area and did not result in respiratory difficulty or mortality of the animals. No injury or death occurred in the 98 imaging sessions, although each imaging session required ∼1 hour including preparation time. Importantly, most diffusion-weighted magnetic resonance images at even high b values had minor motion artifact, facilitating reproducible ADC quantification. Furthermore, high-frequency ultrasound imaging was used to select mice bearing identical tumor sizes throughout the groups, increasing the accuracy of ADC quantification. In a previous experiment, variable tumor growth was observed in the same orthotopic pancreatic tumor model (22, 36). Larger tumors may contain central necrosis (37, 38), which could increase ADC values for the tumor and be unrelated to drug-induced apoptosis.

In contrast to the significant increases of ADC values at 1 day after therapy in groups 3 and 4, tumor volumes were not changed at this same time. This confirms that extracellular water increase due to apoptosis preceded change in tumor size, and thereby DWI may be more sensitive than the current method (Response Evaluation Criteria in Solid Tumors) to measure early anticancer efficacy following treatment. The decrease of ADC values in the control groups (two experiments) was presumably caused by increased tumor cell density (or fibrous tissue) over time, leading to a decrease of extracellular water.

Although bioluminescence imaging detected significant decrease of emitted light from tumors in groups 3 and 4 at 1 day after initiating therapy, quantification error induced by attenuation of optical light should be considered in days 2 to 3 (i.e., when tumor volume shrank, the distance from the tumor to abdominal surface became correspondingly greater, in general). Bioluminescence signal could be reduced due to contribution of signal attenuation as well as an actual decrease of viable tumor mass.

Early assessment of therapeutic efficacy is essential to prevent unnecessary treatment and optimize therapeutic strategies to extend patients' lives. ADC quantification is a safe and logical approach to establish personalized medicine and can be applied to measure effective neoadjuvant therapy. Eqs. A and C show how to predict the survival of animals that harbor pancreatic cancer following treatment, based on monitoring early ADC change. These equations will need adjustments following more extensive preclinical and clinical studies, to be used for human pancreatic cancer prognosis, but they may serve as prototypes for future versions.

Each tumor was sliced into two pieces, which were then immersed in 10% neutral buffered formalin overnight at room temperature. Tissue sections of 5-μm thickness were cut on an Accu-Cut SRM microtome (Sakura Finetek USA, Inc.). Sections of paraffin-embedded tissue were mounted on Bond-Rite slides from Richard-Allan Scientific and heated at 60° for 2 h. Paraffin was removed from the sections by three changes of xylene and rehydrated through graded alcohols from absolute to 70% for 5 min each.

The TUNEL assay was done with a Chemicon International ApopTag Peroxidase In Situ Detection Kit. The slides were rehydrated as above and pretreated for 1 min in 10 mmol/L glycine (pH 3) with fast cooling after the pressure cooker. The slides were rinsed for a minimum of 2 h with deionized water after the H2O2 quench. The procedure after the quenching was previously described. The chromogen used was 3-3′diaminobenzidine (BioGenex Laboratories, Inc.). After 7 min, the slides were rinsed with water and lightly counterstained with Mayer's hematoxylin. The sections were dehydrated through graded alcohols from 70% to absolute and three xylene baths for 5 min each. The coverslips were mounted with Permount.

H. Kim: Commercial research grant, Daiichi Sankyo. D.E. Morgan: Commercial research grant, Daiichi Sankyo. D.J. Buchsbaum: Commercial research grant and ownership interest in TRA-8, Daiichi Sankyo. J.M. Warram: Commercial research grant, Daiichi Sankyo. J.C. Sellers: Commercial research grant, Daiichi Sankyo. K.R. Zinn: Commercial research grant, Daiichi Sankyo. The other authors disclosed no potential conflicts of interest.

Grant support: Health Services Foundation General Endowment Fund Scholar Award; Research Initiative Pilot Award; Translational Research Pilot Project Program; AACR-Pancreatic Cancer Action Network Career Development Award; Daiichi Sankyo; and NIH grants 5P50CA89019, P20CA10195, and 5P30CA013148.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Karri Folks for assistance with bioluminescence imaging, tumor dissection, and histology, and Dr. Thian Ng for valuable consultation. All experiments complied with current regulatory requirements (including ethics requirements) and laws of the United States of America.

1
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Supplementary data