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To investigate the efficacy of polydisulfide-based biodegradable macromolecular contrast agents of different degradability and molecular weight for tumor characterization based on angiogenesis using dynamic contrast enhanced MRI (DCE-MRI).
Biodegradable macromolecular MRI contrast agents, GDCC and GDCP, with molecular weight of 20 and 70 KDa were evaluated for tumor characterization. The DCE-MRI studies were performed in nude mice bearing MDA PCa 2b and PC-3 human prostate tumor xenografts. Tumor angiogenic kinetic parameters, endothelium transfer coefficient (Ktrans) and fractional tumor plasma volume (fPV), were calculated from the DCE-MRI data using a two-compartment model.
There was no significant difference in the fPV values between two tumor models estimated with the same agent except for GDCC-70. The Ktrans values in both tumor models decreased with increasing molecular weight of the agents. GDCC-70 showed a higher Ktrans values than GDCP-70 due to high degradability of the former in both tumor models (p < 0.05). The Ktrans values of MDA PCa 2b tumors were significantly higher than those of PC-3 tumors estimated by Gd(DTPA-BMA), GDCC-20, GDCC-70, GDCP-70, and albumin-(Gd-DTPA) (p < 0.05).
The polydisulfide based biodegradable macromolecular MRI contrast agents are promising in tumor characterization with dynamic contrast enhanced MRI.
Accurate tumor characterization is critical for cancer patient care management. For example, more than 30% of men older than 50 years have microscopic prostatic carcinoma at autopsy, fewer than 10% of them finally develop malignant prostate cancer (1,2) and therefore need aggressive treatment (3). Thus, accurate and reliable non-invasive methods are needed to characterize different tumors and define the malignancy of every individual carcinoma. Tumor angiogenesis has been considered as a valuable parameter for tumor characterization, and it is directly associated with tumor malignancy and metastasis. Tumor angiogenesis can be correlated to tumor vascularity, which is determined by intratumoral vascular permeability and microvessel density (MVD) (4–7). High vessel densities, proliferation of endothelial cells and increased vascular permeability in tumors often result in tumor aggression and poor prognosis (8,9). Dynamic contrast enhanced MRI (DCE-MRI) is a non-invasive imaging modality, which has become an important tool in cancer diagnosis, differentiating benign from malignant lesions and monitoring antiangiogenic therapy (8,10). DCE-MRI acquires MR images repetitively can provide quantitative measurement of tumor vascular parameters such as endothelial transfer coefficient (Ktrans) and fractional tumor plasma volume (fPV) (11,12). These quantitative parameters can be correlated to tumor vascularity and angiogenesis, and can be used for assessing the efficacy of anticancer therapies, including anti-angiogenic therapy (2,13).
It has been reported that the molecular weight or particle size of MRI contrast agents has a significant impact on tumor vascular parameters determined by DCE MRI (11,14,15). MRI contrast agents with different sizes have been investigated for tumor differentiation based on tumor angiogenesis and vascular permeability. Low molecular weight Gd(III)-based contrast agents often overestimated tumor blood volume and vascular permeability because these agents rapidly extravasate from the blood to the extracellular space in tumor tissue (16,17,18). Macromolecular contrast agents have shown better assessment in tumor angiogenesis than low molecular weight contrast agents (19,20). For example, albumin-(Gd-DTPA), a prototype macromolecular MRI contrast agent, was able to differentiate tumor vascular permeability and provided more accurate tumor characterization than the low molecular weight contrast agents (21,22). Good correlation has also been reported between the histological analysis and tumor vascular permeability estimated by DCE-MRI and macromolecular contrast agent, but not for low molecular weight agents (11). However, macromolecular MRI contrast agents are not available for clinical applications because of the safety concerns related to their slow excretion and high tissue accumulation of toxic Gd(III) ions (20,23).
We have recently developed polydisulfide Gd(III) chelates as biodegradable macromolecular MRI contrast agents to facilitate the excretion of Gd(III) chelates after the MRI examinations. These agents initially behave as macromolecular agents and result in superior contrast enhancement in the vasculature and tumor tissues. They can then be degraded in vivo into oligmeric and low molecular weight Gd(III) chelates, which rapidly excrete from the body through renal filtration, resulting in minimal tissue accumulation (24,25), similar to that of low molecular weight contrast agents (23,26,27). The biodegradable macromolecular MRI contrast agents are also effective for evaluation of tumor angiogenesis and vascular permeability with DCE-MRI (28). The purpose of this study was to evaluate the efficacy of the polydisulfide Gd(III) complexes in tumor differentiation with DCE-MRI. Tumor vascular parameters derived from the DCE-MRI data were compared in mice bearing PC-3 and MDA PCa 2b human prostate cancer xenografts.
Gd(DTPA-BMA) (574 Da) was obtained from Nycomed Inc., Princeton, NJ. Gd-DTPA cystamine copolymers (GDCC) and Gd-DTPA cystine copolymers (GDCP) were prepared as previously described (25,26). GDCP is a modified polydisulfide Gd(III) complexes with slower degradation rates than GDCC. They were further fractionated using a Sephacryl S-300 column on a Pharmacia FPLC system (Gaithersburg, MD) to prepare the agents with narrow molecular weight distributions. The apparent molecular weights of the fractions were determined by size exclusion chromatography using poly[N-(2-hydroxypropyl)methacrylamide] as a standard on an AKTA FPLC system (GE Biosciences, Piscataway, NJ). Albumin-(Gd-DTPA) (92 KDa) was prepared as previously described (29). The Gd(III) content in the agents was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES, Perkin Elmer Optima 3100XL).
Human prostate cancer MDA PCa 2b and PC-3 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA) with ATCC number CRL-2422 and CRL-1435, respectively. PC-3 cell line was cultured using ATCC complete growth medium (F-12K medium with 10% fetal bovine serum). MDA PCa 2b cell line was cultured with Kaighns modification of Hams F12 medium (F12K) with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate, supplemented with 25 ng/ml cholera toxin, 10 ng/ml epidermal growth factor, 0.005 mM phosphoethanolamine, 100 pg/ml hydrocortisone, 45 nM selenious acid, 0.005 mg/ml insulin, and 20% fetal bovine serum according to ATCC’s instruction. The MDA PCa 2b cells grew in clumps, formed layers, and had a doubling time of 14 days with great tumorigenicity (30). PC-3 cells had a tumor doubling time of 8.5 days.
Athymic male NCr-nu/nu nude mice at 5 weeks old were purchased from National Cancer Institute at Frederick, MD. Cell suspension of MDA PCa 2b or PC-3 in their preferred medium was mixed with Matrigel matrix (BD Biosciences, San Jose, CA) at a 1:1 ratio. 5×106 cells in 120 μL mixture were inoculated subcutaneously in both right and left sides of the mouse’s hip. DCE-MRI study was performed when the tumors reached about 1 cm in diameter (14 weeks after MDA PCa 2b cell inoculation, 4 weeks after PC-3 inoculation) (31–34).
All images were acquired on a Siemens Trio 3T scanner using the system body coil for RF excitation and a human wrist coil for RF reception. A group of three mice weighing 28 g were used for each agent. Mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (Bedford, OH, 90 mg/kg) and xylazine (St. Joseph, MO, 10 mg/kg). They were then placed prone with the tumors located at the center of a human wrist coil. A tail vein was catheterized using a 30 gauge needle connected to a 2-m long thin tubing filled with heparinized saline. 120 μL of contrast agent was injected via the tubing and 200 μL saline was used to flush the tubing after the injection of contrast agent. The dose for all contrast agents was 0.1 mmol-Gd/kg except that the dose for albumin-Gd-DTPA was 0.03 mmol-Gd/kg as reported in literatures.
Before the injection, 3D fast low angle shot (FLASH) images and 2D axial spin echo (SE) images were acquired. The 3D FLASH image was used to define the regions of interest (ROIs) for 2D SE image. The axial slices in 2D SE images were selected for the acquisition of DCE-MRI data. Dynamic MRI scan was performed using 2D FLASH for a period of 15 min. After a 45 second delay, the contrast agent was administered via the tubing. 2D axial SE scans was acquired at 15 min after the injection. Parameters of the 3D FLASH pulse sequence are: TR/TE = 7.75/2.56 ms, α = 25°, 0.5 mm coronal slice thickness, averages of 4, fat saturation, and 1:21 min scan time. Parameters of the 2D SE pulse sequence are: TR/TE = 400/10 ms, α = 90°, 2 mm axial slice thickness, averages of 2, fat saturation, 8 slices and 1:01 min scan time. Parameters of the 2D FLASH pulse sequence (for dynamic scans) are: TR/TE= 104/4.46 ms, 0.5×0.5×1.5 mm, α= 30°, 1.5 mm axial slice thickness, single acquisition, a total of 10 axial slices (covering majority of the tumor with the last two slices cover the heart), and 11 seconds scan time for a single acquisition.
The 3D FLASH and 2D SE images were reconstructed and analyzed using Osirix (http://homepage.mac.com/rossetantoine/osirix/). A package of programs based on MATLAB (The MathWorks, Inc., Natick, MA) was developed to process dynamic 2D FLASH data in DICOM format. Regions of interest (ROIs) were placed in the whole tumor and in the right ventricle of the heart to obtain signal intensity (SI) in the blood. The average MR signal intensity (SIpre) of the ROIs before the contrast agent injection was used as the baseline and was subtracted from the SI after contrast agent injection (SIpost) to calculate the increase in SI (ΔSI). It is assumed that ΔSI is proportional to the change of the contrast agent concentration, which is a reasonable approximation at low contrast agent concentration (35). The signal-time kinetic data were analyzed using a two-compartment bidirectional exchange kinetic model as shown in the equation
where Cp and CT are the contrast agent concentrations in blood and in the tumor, respectively; Ktrans is the endothelial transfer coefficient; kep is the rate constant of reflux from the extravascular and extracellular space (EES) back to blood; θ is a Laplace operator and fPV is the fractional vascular volume (11). The tumor endothelial transfer coefficient (Ktrans) and fractional tumor plasma volume (fPV) was similarly calculated by the methods as described in the literature (11).
The mice were sacrificed at the end of the experiments. Tumors were collected and fixed in 10% buffered formalin and embedded in paraffin. Tissue sections were cut at 4 μm and prepared on uncharged slides. Some of the tissue sections were stained with hematoxylin and eosin and analyzed by microscopy. The rest of tissue section was immunostained with rabbit polyclonal antibodies to factor VIII antigen. Histological analysis of microvessel density count was performed as described in the literature (36).
Statistical analysis was performed using a student t-test (GraphPad Prism; GraphPad Software, San Diego, CA). P values were two-tailed with a confidence interval of 95%.
The polydisulfide Gd(III) complexes, GDCC and GDCP, with narrow molecular weight distributions and different molecular weights were prepared by fractionation of the polydisulfides with size exclusion chromatography. Table 1 lists the number averaged molecular weight (Mn), weight averaged molecular weight (Mw), polydispersion index (PDI), and T1 relaxivity per complexed Gd(III) ion at 3T of the polymeric contrast agents. The agents with molecular weights of 20 and 70 KDa, GDCC-20, GDCC-70, GDCP-20 and GDCP-70, were chosen to represent low and high molecular weight contrast agents. The apparent weight and number averaged molecular weights of a control macromolecular contrast agent, albumin-(Gd-DTPA), were 44 and 45 KDa, respectively, relative to the linear poly[N-(2-hydroxypropyl)methacrylamide] standards.
Figure 1 shows the representative MR signal intensity-time course in both PC-3 and MDA PCa 2b tumor tissues for each contrast agent. Since the endothelial transfer coefficient (Ktrans) measures the perfusion of the contrast agents from the blood circulation into tumor extracellular space and the fractional tumor plasma volume (fPV) is associated to the initial SI rise in tumor tissue, the DCE-MRI study was focused on the contrast uptake kinetics in the first 15 minutes after the injection. The tumor uptake kinetics varied with the tumor tissues and properties of the contrast agents. After the initial SI rise, the diffusion of contrast agents into the extracellular space was controlled by the contrast agent concentration in the blood and the permeability of the tumor microvessels. The MDA-PCa-2b tumor exhibited more rapid uptake kinetics than the PC-3 tumor for all tested contrast agents. For the same tumor, contrast uptake kinetics slowed down with the increasing molecular weight and decreasing degradability of the agents. GDCP was less degradable than GDCC and showed slower tumor uptake than GDCC with the same molecular weights. Albumin-(Gd-DTPA) was non-degradable and had much slower tumor uptake kinetics than GDCC and GDCP of both molecular weights in both tumors.
Table 2 lists the tumor endothelial transfer coefficient (Ktrans) and fractional tumor plasma volume (fPV) calculated with the two-compartment model from the DCE-MRI data obtained with the contrast agents in both PC-3 and MDA PCa 2b tumors. The comparison of the vascular parameters estimated for PC-3 and MDA PCa 2b tumors by different contrast agents was shown in Fig. 2. Although MDA PCa 2b tumor has a slower growth rate than PC-3 tumor, the Ktrans values of MDA-PCa-2b tumor were significantly higher than those of PC-3 tumor with similar tumor size estimated by all agents except GDCP-20 (p < 0.05). For the same tumor model, the Ktrans value decreased with the increase of molecular weight and decrease of degradability of the contrast agents. The low molecular weight agent Gd(DTPA-BMA) resulted in the highest Ktrans values while the albumin-(Gd-DTPA) gave the lowest Ktrans in both tumor models. The Ktrans values of GDCC-20, GDCP-20, GDCC-70 and GDCP-70 were between those of Gd(DTPA-BMA) and albumin-(Gd-DTPA). GDCP with a slower degradability resulted in smaller Ktrans values than GDCC with similar molecular weight, except GDCP-20 for PC-3 tumor. There was no significant difference in the values of fPV estimated by most of the contrast agents between two tumors. Histological analysis showed that there was no significant difference in the microvessel density between two tumors based on immunostain of factor VIII antigen.
Dynamic contrast enhanced (DCE) MRI non-invasively measures the uptake kinetics of a contrast agent in tumor tissues. The vascular parameters calculated from the uptake kinetics can be used as biomarkers to characterize individual tumors. These biomarkers can be used for non-invasive tumor grade and evaluation of tumor response to anticancer therapies. Low molecular weight contrast agents, which are exclusively used in clinical studies, rapidly extravasate through both tumor blood vessels and normal blood vessels and are not ideal for tumor characterization. Macromolecular contrast agents with molecular weights larger than 20 KDa have limited extravasation through normal blood vessels and can selectively pass through porous tumor blood vessels. They are considered to be effective for more accurate tumor characterization with DCE-MRI. Unfortunately, macromolecular contrast agents are not available for clinical application because of safety concerns. Polydisulfide based biodegradable macromolecular MRI contrast agents have been developed to alleviate the safety concerns (25–27). The biodegradable macromolecular contrast agents can be rapidly excreted from the body after the MRI studies and have showed minimal long-term tissue accumulation comparable that of low molecular weight clinical contrast agents. These agents have shown promises to be further developed for clinical applications, including tumor characterization with DCE-MRI.
In this study, the effectiveness of different biodegradable macromolecular MRI contrast agents in tumor characterization with DCE-MRI were preliminarily investigated in two different mouse tumor models. The results obtained with the biodegradable macromolecular contrast agents were compared to those obtained with a clinical contrast agent, Gd(DTPA-BMA), and a prototype macromolecular contrast agent, albumin-(Gd-DTPA). In both tumor models, the values of the calculated vascular parameters, particularly microvascular permeability Ktrans, showed a dependence on size and degradability of the contrast agents (Fig. 2A). The values estimated by biodegradable macromolecular contrast agents were between those by the small molecular weight contrast agent and those by the non-degradable macromolecular contrast agent. With the same high molecular weight, GDCC-70 resulted in a higher Ktrans values than GDCP-70 in both tumors due to high degradability of the former. However, the difference of biodegradability between GDCC-20 and GDCP-20, which had similar low molecular weight, had not significant effect on the Ktrans values possibly due to their relatively small size and high diffusion rate from plasma to tumor extracellular space.
It appears that Ktrans was a more sensitive parameter than fPV in tumor characterization as shown in Figure 2. There was a significant difference in Ktrans between PC-3 and MDA PCa 2b prostate tumor xenografts estimated by most of the tested contrast agents, including Gd(DTPA-MBA), GDCC-20, GDCC-70, GDCP-70, and albumin-(Gd-DTPA). No significant difference was observed in Ktrans between the two tumor models when assessed by GDCP-20, probably due to a relatively small sample size and large experimental errors among the samples (Fig. 1C). In contrast, the difference of fPV between two tumor models was not significant for most agents except for GDCC-70. The result was consistent to the histological observation that the microvessel density was not significantly different between two tumor models. No clear trend was observed on the influence of the size and degradability of the contrast agents on the fPV values.
The correlation of the Ktrans values to physiology and histology of the tumor models is not clear. Although the MDA PCa 2b tumor xenografts grew slower than the PC-3 tumor xenografts, the Ktrans values of the MDA PCa 2b tumor xenografts were significantly larger than those of the PC-3 tumor xenografts estimated with most of the agents. Similar uptake kinetics was reported previously studies by Kim et al. for two tumor with different agents (31,32). PC-3 cells are androgen-independent and poorly differentiated cells, while MDA PCa 2b cells are androgen-sensitive and relatively well-differentiated cells (37). There might be an association of tumor vascular permeability with the androgen-sensitivity of the cancer. We also observed the MDA PCa 2b tumor xenografts were softer than the PC-3 tumor xenografts when palpated. Further studies are needed to understand the correlation of the correlation of the Ktrans values of the tumor models to their physiology and histology.
The degradation of the biodegradable macromolecular contrast agents might affect accurate data analysis and calculation of vascular parameters due to the change of the relaxivities of the degradation products since the contrast agents degraded into smaller chelates during the process of the DCE-MRI data acquisition. As shown in Table 1, the difference in the T1 relaxivities of biodegradable macromolecular contrast agents of two different molecular weights (20 and 70 KDa) was relatively small and may not be significant in DCE-MRI data analysis because DCE-MRI is not a perfectly quantitative method. The vascular parameters calculated from the DCE-MRI data with the biodegradable macromolecular contrast agents, particularly those with a relatively large size and slow degradation rate, were comparable to those estimated by albumin-(Gd-DTPA) in both tumors without consideration of the change of relaxivities. The biodegradable macromolecular MRI contrast agents, particularly those with high molecular weights, were effective to characterize individual tumors with DCE-MRI.
The results in this study showed that the vascular parameters, particularly Ktrans, measured by DCE-MRI varied with the properties of contrast agents. In general, the Ktrans values of both tumors increased with decreasing molecular weight of the agents and increasing of degradability of biodegradable macromolecular contrast agents. The fPV values showed little dependence on the properties of the agents and tumors. The Ktrans values of MDA PCa 2b tumors measured by most of the contrast agents were higher than those of PC-3 tumors. The biodegradable macromolecular contrast agents with high molecular weights were effective for characterization of individual tumors with DCE-MRI based on tumor vascular permeability Ktrans. The polydisulfide based biodegradable macromolecular MRI contrast agents are promising in tumor characterization with dynamic contrast enhanced MRI..
The authors thank Dr. Yong-En Sun for his technique in the tail vein catheterization, Melody Johnson for the operation of MRI scanner. This work is supported in part by the NIH grant R01 EB00489.