DCE-MRI Detects Early Vascular Response in Breast Tumor Xenografts Following Anti-DR5 Therapy

Abstract

Purpose

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) measured the early vascular changes after administration of TRA-8, bevacizumab, or TRA-8 combined with bevacizumab in breast tumor xenografts.

Procedures

Groups 1–4 of nude mice bearing human breast carcinoma were injected with phosphate-buffered saline, TRA-8, bevacizumab, and TRA-8 + bevacizumab on day0, respectively. DCE-MRI was performed on days0, 1, 2, and 3, and thereafter tumors were collected for terminal deoxynucleotidyl transferase-mediated dUT nick end labeling and CD31 staining.

Results

DCE-MRI measured a significant K^trans change within 3 days after TRA-8 therapy that correlated with tumor growth arrest, whichwas not shown with statistical significance by histopathology at these early time points posttreatment. The K^trans changes followed quadratic polynomial curves.

Conclusion

DCE-MRI detected significantly lower K^trans levels in breast tumor xenografts following TRA-8 monotherapy or combined therapy with bevacizumab.

Introduction

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via death receptors expressed on cancer cells [1], and its therapeutic application has demonstrated a high efficacy in various animal cancer models [2]. However, TRAIL caused substantial toxicity in human hepatocytes in an ex vivo experiment [3], which raised concerns regarding future clinical applications. The hepatotoxicity may be related to the binding of TRAIL to several receptors including death receptor 4, death receptor 5 (DR5), decoy receptor 1, decoy receptor 2, and osteoprotegerin [4]. Therefore, TRA-8, a monoclonal antibody targeting only DR5, was developed [5]. Recently, the phase I study of the humanized TRA-8 (tigatuzumab) showed that it was well tolerated in 17 cancer patients without dose-limiting toxicities [6]. However, a differential cytotoxic efficacy of TRA-8 was observed for human breast tumor cell lines [7]. While investigating the mechanism of natural and/or acquired resistance for TRA-8, it would be advantageous to determine the TRA-8 sensitivity of a tumor in each individual patient as early as possible during treatment, in order to tailor the therapeutic strategy.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been utilized for early assessment of therapeutic efficacy of cancer drugs by noninvasively measuring pharmacokinetic parameters in tumor microvasculature by quantifying the transfer of a contrast agent from the vascular space to the extravascular–extracellular space over time [8]. DCE-MRI has been well established to assess breast cancer response to various chemotherapies [9, 10] as well as to antiangiogenic drugs [11, 12]. Effective therapies disrupt tumor vascular angiogenesis, leading to a decrease of microvessel density, perfusion, and permeability prior to quantifiable tumor volume decrease or morphology change. Therefore, anti-DR5 therapy is expected to reduce parameters in tumor vasculature, which might be detectable via DCE-MRI at early stages of therapy. Early detection of TRA-8 sensitivity for each breast cancer patient during neoadjuvant therapy should enable prompt adjustment of TRA-8 usage and increase the probability for a favorable outcome by preventing continuance of a failing treatment. TRA-8 can be administrated readily in combination with other chemotherapeutic and/or antiangiogenic agents, since no negative side effects of TRA-8 have been reported to date.

Small molecular weight contrast agents such as gadopentetate dimeglumine (Gd-DTPA), gadodiamide (Gd-DTPA-BMA), and gadoteridol (Gd-HP-DO3A) are extensively used clinically for cancer diagnosis and prognosis purposes [13], and therefore preclinical magnetic resonance (MR) studies using Gd chelate contrast agents may be readily translated to human studies. In order to quantify the transfer of a contrast agent in a tissue microvasculature, the contrast agent concentration in blood plasma, so-called arterial input function (AIF), should be obtained [14]. AIF can be measured by analyzing the signal intensity within a large vessel included in the imaging field of view [15]. However, high temporal resolution is required to accurately measure the AIF, which is also compromised by limited spatial resolution and lower signal-to-noise ratios (SNRs). Spatial resolution should be sufficient to minimize partial volume effect in an AIF measurement and to better assess heterogeneity within neoplastic tissue. Monitoring the signal intensity in a reference region (usually muscle tissue) can obviate the need to measure AIF; this quantification method is called a reference region model [16]. A major weakness of this approach is that the fractional extravascular–extracellular volume in the reference region (v_e,RR)must be assumed. In one study, when the perivertebral muscle tissue was designated as the reference region, the measured v_e,RR was 0.0797±0.0276 (mean ± SD) in nine rats [17].

The purpose of this study was to measure the early changes of volume transfer constant (K^trans) and fractional extravascular–extracellular volume (v_e) in breast carcinoma xenografts following TRA-8, bevacizumab (anti-vascular endothelial growth factor (VEGF) antibody), or combination therapy using DCE-MRI based on a reference region model.

Materials and Methods

Reagents and Cell Lines

Purified TRA-8 (mouse origin) was provided by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Bevacizumab (Avastin, Genentech Inc., South San Francisco, CA, USA) was purchased from the University of Alabama at Birmingham Hospital Pharmacy. All reagents were from Fisher Scientific (Pittsburgh, PA, USA) unless otherwise specified. The luciferase-positive 2LMP cell line (a subclone of human breast cancer cell line MDA-MB-231) was used to enable comparison with earlier diffusion-weighted MR imaging studies that tested the same cell line [18]. This clone was determined to have identical sensitivity to TRA-8 killing, as compared to the parent MDA-MB-231 cell line (data not shown). All 2LMP cells reported in this article were luciferase positive but denoted as only 2LMP. Prohance® (gadoteridol; 5 ml, 0.5 mmol/ml) was purchased from Bracco Diagnostics Inc. (Princeton, NJ, USA).

Animal Preparation

Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee. Four groups of female athymic nude mice (4~5 weeks old, n=6 per group) were implanted with two million cells in culture medium (DMEM, with 2 mM lglutamine, 1 mM Na pyruvate, 10% fetal bovine serum; pH 7.4; 0.2 mL/well) in the right flank subcutaneously. Three weeks after implantation, DCE-MRI and anatomical MRI were performed at days0, 1, 2, and 3 in all mice. Four days prior to imaging, a vascular access port (PennyPort, Access Technologies, Skokie, IL, USA) was subcutaneously implanted on the back of each mouse, and the catheter connected to the port was inserted into a jugular vein, to facilitate repeated intravenous gadoteridol injections. The detailed procedure of port implantation and maintenance is provided in “Appendix A.” The groups of mice were injected intraperitoneally as follows: group 1 with phosphate-buffered saline (PBS), serving as control, group 2 with TRA-8 (0.2 mg), group 3 with bevacizumab (0.1 mg), and group 4 with TRA-8 (0.2 mg) combined with bevacizumab (0.1 mg), on day0, postimaging. All therapeutic doses were administrated in 0.2 ml PBS. All mice (n=24; n=6 per group, four groups) were sacrificed after imaging on day3, but only four tumor tissues of group 1 or 4 (n=4 per group) were selected for further histologic analyses.

MRI

Small-animal MRI was performed on a Bruker BioSpec 9.4 T system (Bruker BioSpin Corp., Billerica, MA, USA). To increase throughput, two animals bearing tumors were imaged simultaneously by positioning them on an animal bed, with the two tumors under a surface coil (Bruker BioSpin). The animal bed was equipped with circulating warm water to regulate body temperature, and anesthesia was maintained using isoflurane (1–2%) during the MRI scans. A 27-gauge needle connected to a sterilized polyurethane tube (outer diameter × inner diameter 0.84 mm×0.34 mm, Strategic Applications Inc., Libertyville, IL, USA) was inserted perpendicularly into the lumen of each port, to deliver gadoteridol. Anatomical MRI to measure tumor volume was performed using a T2-weighted spin echo sequence (RARE) with the following acquisition parameters: repetition time (TR)/echo time (TE)=2,000/34 ms, 128×128 matrix, and a 30×30-mm field of view. One-millimeter-thick slices with a 0.2-mm gap were used to cover the entire tumor region. Then, a T₁ map was acquired with a gradient-echo multiflip-angle approach with the following parameters: TR/TE=115/3 ms, 128×128 matrix, a 30×30-mm field of view, NEX=4, and seven flip angles of 10°, 20°, 30°, 40°, 50°, 60°, and 70°. A maximum of five 1-mm-thick slices (0.2-mm gap) were used to cover the tumor region of interest (ROI). DCE-MRI employed the same acquisition parameters as those above but with the fixed flip angle of 30°. Five baseline images were acquired before gadoteridol injection, and then 20 images were acquired after gadoteridol injection (0.0267 mmol/ml: 0.2 mmol/kg per body weight (BW) for a 20-g mouse, over a period of 15 s with a total injection volume of 0.15 ml). A syringe pump (NE-1600, New Era Pump Systems, Inc., Wantagh, NY, USA) was used to inject gadoteridol at a constant rate (0.01 ml/s).

Image Analysis

The reference region model was employed to calculate volume transfer constant (K^trans) and fractional extravascular–extracellular volume (v_e) [16]. Reference region (RR) model is based on the flow-limited Kety model [14] and uses the signal enhancement in a reference region to remove the need for the AIF as follows,

$$C_{\mathrm{t},\mathrm{R}\mathrm{O}\mathrm{I}}(t)=(K^{\text{trans},\mathrm{R}\mathrm{O}\mathrm{I}}/K^{\text{trans},\mathrm{R}\mathrm{R}})C_{\mathrm{t},\mathrm{R}\mathrm{R}}(t)+(K^{\text{trans},\mathrm{R}\mathrm{O}\mathrm{I}}/v_{\mathrm{e},\mathrm{R}\mathrm{R}})\underset{0}{\overset{t}{\int }}{C}_{\mathrm{t},\mathrm{R}\mathrm{R}}(t\prime )\mathrm{d}t\prime -(K^{\text{trans},\mathrm{R}\mathrm{O}\mathrm{I}}/v_{\mathrm{e},\mathrm{R}\mathrm{O}\mathrm{I}})\underset{0}{\overset{t}{\int }}{C}_{\mathrm{t},\mathrm{R}\mathrm{O}\mathrm{I}}(t\prime )\mathrm{d}t\prime$$ (1)

where C_t,ROI(t), K^trans,ROI, and v_e,ROI are the contrast agent concentration, volume transfer constant, and fractional extravascular–extracellular volume, respectively, in the tumor, while C_t,RR(t), K^trans,RR, and v_e,RR are those in the RR. Thirty-two voxels (two 4×4 voxel windows) located in the perivertebral muscle were selected as the RR, and the v_e,RR was assumed to be constant at 0.08 over the region [17]. The fifth-order polynomial curve fitting into each contrast agent concentration curve was used to suppress a sudden signal variation due to noise or animal motion. The tumor area was segmented from the anatomical MR images using the signal intensity difference between the ROI and background, while the intensity thresholds were determined manually. In addition, the isodistance peripheral region with 0.5-mm thickness beginning from the tumor surface was segmented for each slice, while the random topological structure of the tumor was maintained. After the tumor boundary was detected, the distances from each voxel inside the boundary to all voxels located on the boundary were calculated. The voxels whose minimum distances were less than 0.5 mm were segmented. The K^trans and v_e values averaged in either the whole tumor region or the peripheral tumor region were obtained using Eq. 1. The negative K^trans and v_e values were replaced with 0, and the v_e values larger than 1 were replaced with 1. Segmentation of the whole tumor area was performed using ImageJ, version 1.40 (National Institutes of Health, Bethesda, MD, USA). The K^trans and v_e quantification, peripheral tumor region segmentation, and tumor volume calculation were implemented using computer software developed using Labview, version 8.5 (National Instruments Co., Austin, TX, USA). The best-fitting second-order polynomial curves for K^trans change in the peripheral tumor regions over 3 days following therapy were obtained for groups 2–4, and the “maximum K^trans increase (MaxK)” and “day of maximum K^trans increase (DMaxK)” were retrieved from each curve (n=6 for group 2 or 4; n=4 for group 3, because two curves were diverging). Linear and second-order polynomial regressions were performed using Excel, version 11.3.6 (Microsoft Corporation, Seattle, WA, USA).

Histological Analysis

Terminal deoxynucleotidyl transferase-mediated dUT nick end labeling (TUNEL) and CD31 staining were performed to measure the apoptotic cell and microvessel densities for the tumor tissues of groups 1 and 4 (n=4 per group). The detailed procedure for staining the tumor tissue is presented in “Appendix B.” Digital pictures were taken, specifically avoiding areas of necrosis, but otherwise randomly for each TUNEL-stained slice (n=2 per slice, ×400 magnification) or CD31-stained slice (n=1 per slice, ×250 magnification), using a SPOT camera on a Nikon Optiphot-2 microscope (Nikon inc., Melville, NY, USA), interfaced with a personal computer and SPOT software. The image analysis software was ImageJ, version 1.40 (National Institutes of Health, Bethesda, MD, USA). The apoptotic (TUNEL) cells were segmented by the signal intensity difference between the target cells and background in each picture, while the intensity and minimum particle-size thresholds were determined manually and then counted in 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 “Rolling Ball” algorithm [19], while the radius was manually determined. The CD31-stained area was segmented in the same way, and the area fraction (CD31-stained area/total area), considered as microvessel density, was calculated.

Statistical Analysis

SPSS version 16.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data, and p values less than 0.05 were considered significant. Measurements made among the four groups over 3 days were analyzed using repeated-measures analysis of variance (RM ANOVA) [20]. v_e values in the peripheral region were log-transformed prior to analysis because of nonnormality in the data. When the RM ANOVA computed with four groups and three time periods was significant, multiple pairwise RM ANOVAs [21] were done to determine where the differences occurred. Comparisons for a single measurement were done using one-way ANOVA [22] followed by Tukey’s honestly significant differences test [23]. The Pearson correlation coefficient [24] was used to look at the relationships between tumor volume and K^trans. Data are presented as a mean ± SE (standard error).

Results

Figure 1a, b shows representative DCE MR images at (a) 1 min before and (b) 5 min after gadoteridol injection with the same intensity scale (the maximum value was normalized to 100), while the boundary of the two tumor regions are indicated with the white dotted circles in the subfigure (a). Figure 1c, d presents the (c) K^trans and (d) v_e maps of the left tumor shown in Fig. 1a. Figure 1e shows the contrast-enhancement curves averaged in the RR and the ROI indicated with the white square (4×4 window, 16 pixels) and the black rectangle (2×1 window, 2 pixels), respectively, in the left animal shown in Fig. 1a, together with the best-fitting fifth-order polynomial curves.

Fig. 1.

Representative dynamic contrast-enhanced MR images of two animals at a 1 min before and b 5 min after gadoteridol injection using the same intensity scale (from 0 to 100) when maximum value is normalized to 100, with c K^trans and d v_e maps of the left tumor and e contrast-enhancement curves averaged in the reference region (RR) and region of interest (ROI) indicated with the white square (4×4 window: 16 pixels) and the black rectangle (2×1 window: 2 pixels), respectively, in the left animal shown in a, together with the best-fitting fifth-order polynomial curves. The boundaries of the two tumor regions are indicated with dotted circles in a. The dark central area is necrosis caused by vascular insufficiency.

Figure 2 shows (a, b) the K^trans maps of representative tumors in groups 1–4 at 0 and 3 days posttreatment and (c, d) the K^trans changes of all four groups during 3 days after therapy in (a, c) the whole tumor region and (b, d) the peripheral tumor region. The initial K^trans values at day0 were 0.016±0.001 and 0.027±0.002 min⁻¹ in the whole and peripheral regions, respectively, without statistical difference among groups in either region (p>0.05). After initiation of therapy, the increase of K^trans values was significantly suppressed by TRA-8 monotherapy (group 2), compared with that of control (group 1), in both the whole tumor region (p=0.039) and the peripheral tumor region (p=0.010). The K^trans changes of groups 3 and 4 were significantly lower than those of groups 1 and 2 (p<0.05), whereas no significant difference between groups 3 and 4 was detected (p>0.05), in either region. Of note, the standard errors of K^trans changes on each time point relative to day0 were 32±6% lower for the peripheral tumor region as compared with those in the whole tumor region (n=12; n=3 per group).

Fig. 2.

Representative K^trans maps of groups 1–4 before dosing (day 0) or 3 days posttherapy (day3) in the a whole tumor region or b peripheral tumor region with a constant scale (from 0 to 0.07 min⁻¹). c, d K^trans changes of the four groups during 3 days posttherapy in the c whole tumor region or d peripheral tumor region. Different Greek letters present statistical differences among the groups during 3 days.

Figure 3 shows (a, b) the v_e maps of the same tumors shown in Fig. 2a and (c, d) the v_e changes in (a, c) the whole tumor region or (b, d) the peripheral tumor region. The initial v_e values at day0 were 0.15±0.01 and 0.22±0.01 in whole and peripheral tumor regions, respectively, without statistical difference among groups (p>0.05). The v_e changes during 3 days in whole tumor region were not statistically different among the groups (p=0.267), whereas for the peripheral region, that of group 4 was significantly lower than those of groups 1 (p=0.012) and 2 (p=0.014). The standard errors of v_e changes on each time point relative to day0 were 18±5% lower in the peripheral tumor region than those in the whole tumor region.

Fig. 3.

Representative v_e maps of groups 1–4 before dosing (day 0) or 3 days posttherapy (day 3) in a a whole tumor region or b peripheral tumor region with a constant scale (from 0 to 0.7). c, d v_e changes of the four groups during 3 days posttherapy in c whole tumor region or d peripheral tumor region. Different Greek letters present statistical differences among groups during 3 days.

Figure 4a shows the mean K^trans changes of the four groups during 3 days after therapy in the peripheral region (Fig. 2d), together with the best-fitting second-order poly-nomial curves. The mean K^trans change of the control group increased linearly over time, while those of groups 2–4 followed the second-order polynomial curves with high R² values. Because of the nonlinear characteristic of K^trans change, we calculated two parameters to evaluate vascular regression efficacy of drugs independent of a particular monitoring time point. We refer to these new parameters as MaxK and DMaxK. Figure 4b shows MaxK and DMaxK values retrieved from the second-order polynomial curves fitting to K^trans changes of groups 2–4 in the peripheral tumor region; the lower MaxK or DMaxK value represents the higher efficacy of vascular regression. The MaxK and DMaxK of group 2 were significantly higher than those of groups 3 and 4, but MaxK showed higher significance (p<0.001 for both groups 3 and 4) than DMaxK did (p=0.026 and 0.003 for groups 3 and 4, respectively).

Fig. 4.

a Mean K^trans changes of groups 1–4 during 3 days posttherapy in peripheral tumor region with the best-fitting second-order polynomial curves for the four groups. b Maximum K^trans increases (MaxK) and days of maximum K^trans increases (DMaxK) retrieved from the second-order polynomial curves fitting to the K^trans changes in the peripheral tumor region during 3 days posttherapy of groups 2–4; different Greek letters present statistical differences among the groups (lowercase letters for MaxK, uppercase letters for DMaxK).

Figure 5a shows tumor volume changes of groups 1–4 during 3 days after therapy. The mean tumor volume of the four groups at day0 was 164±20 mm³ without a statistical difference among groups (p=0.361). Of interest, the tumor volume change of group 3 was gradually increased over time similar to that of control and significantly different from that of group 4 (p=0.025), clearly inconsistent with the K^trans and v_e changes of group 3 (Figs. 2 and 3). Figure 5b shows tumor volume changes versus K^trans changes in the peripheral region during 3 days after therapy. When considering only groups 1, 2, and 4, the correlation between them was significant (p=0.007), but the significance disappeared when group 3 was included (p=0.299).

Fig. 5.

a Tumor volume changes of groups 1–4 during 3 days posttherapy, while different Greek letters present statistical differences among the groups. b Tumor volume changes versus K^trans changes in peripheral tumor region of groups 1–4 for 3 days after therapy; data samples of groups 1, 2, and 4 are indicated with black dots, whereas those of group 3 are indicated with gray rectangles (linear regression was performed only for groups 1, 2, and 4).

Figure 6a shows the representative photomicrographs of tumor slices (5-µm thickness) of groups 1 and 4 following CD31 and TUNEL staining, while the microvessels and apoptotic cells are indicated with black arrows. Figure 6b represents quantifications of microvessel and apoptotic cell densities in histograms (n=4 per group). The averaged microvessel density of group 4 was 24% lower than that of group 1 without statistical significance (p=0.508), whereas the K^trans values of the four tumors (peripheral region) of group 4 on day3 were 0.021±0.006 min⁻¹, significantly lower than those of group 1 (0.042±0.004 min⁻¹; p=0.028). The mean apoptotic cell density of group 4 was about fivefold higher than that of group 1, but the difference was not statistically significant either (p=0.221).

Fig. 6.

a Representative CD31 (×250 magnification) and TUNEL (×400 magnification) staining for tumors of groups 1 and 4 collected at day3 after therapy with b histograms of microvessel and apoptotic cell densities (n=4 per group). Representative microvessels and apoptotic cells are indicated with black arrows in each subfigure of a.

Discussion

TRA-8 significantly suppressed K^trans increase within 3 days postadministration in the breast tumor xenografts, while tumor volume did not change. Since the significant therapeutic efficacy of TRA-8 was confirmed for the same tumor type at 6 days postadministration in our previous study [18], DCE-MRI might be considered a better approach to assess early breast tumor response following anti-DR5 therapy than Response Evaluation Criteria in Solid Tumors, a standard method to measure therapeutic response by monitoring tumor dimensions [25], but the effect of K^trans suppression by TRA-8 therapy could be weakened in human breast tumors because DR5 receptors are expressed on human endothelial cells [26]. Since K^trans reflects vascular perfusion and/or permeability, DCE-MRI has been traditionally considered an ideal modality to evaluate antiangiogenic agents, which explains the greater suppression of K^trans increase when treated with bevacizumab. Instead, diffusion-weighted imaging (DWI) has been usually employed for therapy assessment of apoptosis-inducing drugs. In fact, DWI detected significant increase of ADC values at 3 days after TRA-8 injection in our previous study with the same animal model and dosing [18]; the p value of the difference between the treated and control groups was less than 0.001, which is much smaller than that of the K^trans changes in this study (p=0.038). However, it is difficult to assert that DWI is better for evaluating TRA-8 than DCE-MRI because the ADC values change nonlinearly over time as well; the ADC values of the treated group decreased on day6, relative to those on day3, although an additional dose was administrated on day3 [18]. Therefore, it would be better to incorporate both modalities to improve the measurement accuracy. MR spectroscopy (MRS) can also measure breast cancer response following successful treatment within 2–4 days after therapy initiation by quantifying the substantial decrease of choline or phosphomonoester [27] and has been utilized for early response assessment of breast cancer therapy in a neoadjuvant setting [28]. Thus, MRS may enhance the accuracy of therapy evaluation even further when used together with DCE-MRI and DWI. Early detection of a tumor response will enable optimization of the therapeutic strategy for each patient. Breast DCE-MRI, DWI, and MRS can be completed within an hour in a standard clinical imaging protocol. However, a single-voxel MRS cannot analyze heterogeneity of tumor tissues. MR spectroscopic imaging can quantify the amount of metabolites in a two-dimensional matrix [29], but long acquisition time could be a concern. The glucose analog 2-[¹⁸F] fluoro-2-deoxy-d-glucose (FDG) positron emission tomography (PET) may be utilized for early therapy assessment of TRA-8, together with MR modalities [30]; FDG is the most commonly used PET imaging agent, which accumulates inside cells in proportion to the glucose metabolism. Because tumor cells require glycolysis more than normal cells do, FDG accumulates in tumor cells more intensively, and, thereby, FDG PET is extensively used for cancer staging and early detection of therapeutic response. However, due to the limited spatial resolution, this modality suffers from quantification error caused by partial volume effect, especially in small tumors.

Since gadoteridol is a small molecular weight contrast agent (0.56 kDa), the intratumoral K^trans values measured with gadoteridol are mainly affected by vascular perfusion, proportional to the microvessel density. However, the averaged K^trans value at the peripheral tumor region decreased 50% compared with that of control at 3 days after the administration of TRA-8 and bevacizumab with statistical significance, whereas the averaged microvessel density assessed by CD31 staining decreased only 24% during the same time without statistical significance; this difference might be explained by the fact that high-field DCE-MRI assessing vascular response in the entire peripheral tumor region was more sensitive than the histological analysis of a limited tissue area.

Most researchers have delivered a contrast agent to mice through a catheter inserted into a tail vein in DCE-MRI [31, 32]. However, we inserted a port into each mouse surgically to facilitate the transfer of gadoteridol and to save time prior to each imaging session. Three animals bearing the ports were monitored for 3 weeks, with injection of a catheter-rinsing solution (heparin mixed with gadoteridol and saline) every 24 h, and no adverse effects were detected in either the ports (smooth bolus injection through catheter) or animals (normal grooming and body movement). One concern of this approach could be that the weight-based dosing of a contrast agent was difficult due to the dilution in the dead volume (0.05 ml) of the port, and therefore a flat dose of gadoteridol (0.004 mmol in 0.15 ml PBS) was used. The mean animal body weight was 18.3±0.4 g (n=24) when excluding port weight (1.6 g) on day0, so the mean weight-based dose was 0.219±0.004 mmol/kg on that day.

The sequential imaging provided sufficient data to characterize a nonlinear response of tumor K^trans values at an early stage of therapy, which may allow evaluation of vascular regression efficacy of drugs independently of a monitoring time point. The K^trans values of 2LMP tumors were linearly increased when no treatment was applied, and most curves reflecting the change of K^trans values after therapy were converging. However, the fitting model was not based on tumor physiology but on visual assessment. Therefore, additional studies with more breast cancer models including spontaneous and tumorgraft types will need to be performed, to address the physiologic rationale and thereafter to adjust the imaging biomarkers [33, 34]. The clinical translation of this strategy may be achieved without major concerns, considering that gadoteridol is known as a safe agent at the recommended dosage (0.1 mmol/kg) for patients without chronic renal failure [35] and has a short half-life (about 94 min) in the healthy adult [36]. Since repeated clinical MRI can be very expensive and somewhat inconvenient for patients undergoing cancer therapy, an alternative approach could be to determine the optimal posttherapy initiation imaging time point in order to maximize the accuracy of measuring therapeutic response. Determination of this optimal imaging time point will likely require preclinical and clinical studies that may characterize the nonlinear change of the traditional DCE-MRI biomarker (K^trans) via a series of MRI.

Relatively lower K^trans and v_e values in the central tumor region were observed mainly due to central necrosis, as presented in our previous study [37], and thereby the SNR of those values in that region should be lower as well. Therefore, confining the ROI to only the peripheral tumor region is a feasible approach to increase the measurement accuracy, which may explain the lower variability (standard error) of the normalized K^trans and v_e values after analysis of the peripheral region. Several investigators have used the peripheral tumor region analysis, but both the inner and outer boundaries of the peripheral region were determined manually in most studies [31, 38]. However, in our approach, the inner boundary was determined by computer software, maintaining the random topology of the outer boundary and constant thickness around the rim, which increased the segmentation accuracy. Further, the thickness evaluated can be easily adjusted for each tumor type. The viable tumor tissue area can be identified by multispectral analysis, but additional diffusion and transverse relaxation rate (R²) maps need to be acquired [39].

Conclusions

Intratumoral K^trans values were significantly suppressed within 3 days after each therapy, while the variability of K^trans measurements was markedly reduced by peripheral region analysis. The early change in K^trans values followed second-order polynomial curves; based on these nonlinear characteristics, two novel imaging biomarkers were proposed, which may estimate the therapeutic efficacy of an anticancer drug independently of a particular monitoring time point. Reduction in K^trans was accompanied by tumor volume change over the 3-day interval when anti-DR5 therapy was administered either alone or in combination with bevacizumab. When given as monotherapy, bevacizumab suppressed tumor K^trans significantly during 3 days postdosing compared to controls but did not inhibit tumor growth during the same time interval. Bevacizumab targets VEGF, so the antitumor effect by anti-VEGF monotherapy may be slower than those by chemotherapies or anti-DR5 therapy. We stress, therefore, that the therapeutic efficacies of various cancer drugs should not be directly compared with when monitoring K^trans as an imaging biomarker but should be interpreted in a mechanism-dependent manner.

Appendix A

Mouse Port Implantation and Maintenance

Each mouse was anesthetized with an intraperitoneal injection of sodium pentobarbital (60–65 mg/kg BW, Nembutal sodium solution, Abbott Laboratories, North Chicago, IL, USA) in 0.2 ml of saline and placed in supine position on the operative field. A 0.7~1.0-cm incision in an area of the mouse back was made for implanting the mouse port. A subcutaneous pocket was made by insertion of hemostats (micro-mosquito hemostat, Fine Science Tools Inc., Foster City, CA, USA), and then the port was implanted into the pocket. Another skin incision (0.5 cm) was made in the neck area to expose a jugular vein, and a canal was created under the skin between two incisions using a straight forceps. The catheter connected to the port was held and dragged through the canal and was introduced into the jugular vein. The vein was isolated and secured with two 7-0 sutures. After surgery, each mouse was injected intramuscularly with 2 mg/kg BW of buprenorphine hydrochloride (Buprenex, Hospira Inc., Lake Forest, IL, USA) in 0.2 ml of saline for analgesia. The cage containing the mice was then placed on a SoftHeat Heating Pad (Kaz Inc., Southborough, MA, USA) for about 1 h during recovery from surgery. The port was rinsed with heparin (8.6 U/ml) in 0.1 ml of PBS (pH7.4) every 24 h to prevent blood coagulation inside of the catheter. The lumen of the port had 0.05-ml dead volume, so the heparin solution was mixed with gadoteridol (0.0267 mmol/ml) to fill the dead volume with gadoteridol and avoid dilution.

Appendix B

Tumor Tissue Staining

Each tumor was sliced into two pieces and then immersed into 10% neutral-buffered formalin overnight at room temperature. Tissue sections of 5-µm thickness were cut on an Accu-Cut SRM microtome (Sakura, Tokyo, Japan). Sections of paraffin-embedded tissue were mounted on Bond-Rite slides from Richard-Allan Scientific (Kalamazoo, MI, USA) and heated at 60°C 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.

Antigen retrieval was performed with high-temperature treatment with 0.5 M Tris buffer at pH10. H₂O₂ avidin and biotin solutions and 3% goat serum were used to quench peroxidases, block endogenous biotin, and block nonspecific binding. Rabbit polyclonal antibody to CD31 (Abcam Inc., Cambridge, MA, USA) was diluted 1:200 and applied to the tissue at room temperature for 1 h. The secondary antibody was goat antirabbit (Jackson Immuno Research, West Grove, PA, USA) and the label was avidin–HRP (Signet Pathology Systems, Dedham, MA, USA). After the DAB chromagen (BioGenex, San Ramon, CA, USA) was applied, the tissues were counterstained with hematoxylin and the coverslips mounted with Permount.

The TUNEL assay was performed with a Chemicon International ApopTag Peroxidase In Situ Detection kit (Temecula, CA, USA). The slides were rehydrated as above and pretreated for 1 min in 10 mM glycine at pH3 with fast cooling after heating in a pressure cooker. The slides were rinsed for a minimum of 2 h with deionized water after quenching with H₂O₂. The chromagen used was 3-3′diaminobenzidine according to themanufacturer’s protocol (BioGenex, San Ramon, CA, USA). After 7 min, the slides were rinsed with water and lightly counterstained with Mayer’s hematoxylin. The sections were dehydrated through graded alcohols, 70% to absolute ethanol, followed by three xylene rinses for 5 min each. The coverslips were mounted with Permount.