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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Res. Author manuscript; available in PMC 2010 February 1.
Published in final edited form as:
PMCID: PMC2633414
NIHMSID: NIHMS80178

Multi-Parametric Monitoring of Tumor Response to Chemotherapy by Non-Invasive Imaging

Abstract

With the emerging concept of individualized cancer therapy, it becomes crucial to develop methods for the noninvasive assessment of treatment outcome. With this in mind, we designed a novel approach for the comprehensive evaluation of response to chemotherapy with the established agent doxorubicin in a pre-clinical breast cancer model. This approach delivers information not only about change in tumor size but also about target antigen expression. Our strategy relies on a tumor-specific contrast agent (MN-EPPT) targeting the underglycosylated MUC-1 (uMUC-1) tumor antigen, found on over 90% of breast cancers and predictive of chemotherapeutic response. MN-EPPT consists of superparamagnetic iron oxide nanoparticles (MN) for magnetic resonance imaging, modified with Cy5.5 dye (for near-infrared fluorescence optical imaging, NIRF), and conjugated to peptides (EPPT), specifically recognizing uMUC-1. In vivo, treatment of mice bearing orthotopic human breast carcinomas with doxorubicin led to a reduction in tumor mass and resulted in downregulation of uMUC-1. The tumor-specific accumulation of MN-EPPT allowed the assessment of change in tumor volume by noninvasive imaging. Furthermore, in mice injected with MN-EPPT, tumor delta-T2 was significantly reduced after treatment with doxorubicin, indicating a lower accumulation of MN-EPPT and reflecting the reduced expression of uMUC-1. With these studies, we have demonstrated the utility of magnetic resonance imaging for the multiparametric characterization of breast tumor response to chemotherapy. This approach has the potential of significantly advancing our ability to better direct the development of molecularly-targeted individualized therapy protocols, since it permits the monitoring of therapy on a molecular scale.

Keywords: underglycosylated mucin-1, chemotherapy, magnetic resonance imaging, optical imaging, targeted probe

INTRODUCTION

The emergence of molecularly targeted cancer therapies mandates the development of methods to directly determine their efficacy. Non-invasive imaging techniques are currently available for visualizing different pathological conditions. However, their use for cancer monitoring is limited due to the lack of tumor-specific imaging probes. We have previously developed a multi-modal imaging probe (MN-EPPT) targeting the underglycosylated mucin-1 tumor antigen (uMUC-1), which is overexpressed and underglycosylated on over 90% of breast tumors and whose abundance appears to be linked to tumor progression and response to chemotherapy (1). Our tumor-specific multi-modal imaging probe consists of superparamagnetic iron oxide nanoparticles (MN) for magnetic resonance imaging (MRI) modified with Cy5.5 dye (for optical near-infrared imaging, NIRF) and has peptides (EPPT) specifically recognizing uMUC-1 attached to its dextran coat (1). The dual modality approach combines the high-spatial resolution, tomographic capability, and unlimited tissue penetration of MRI with the high sensitivity and low cost of in vivo optical imaging as a validation tool (2).

In the past, we have performed extensive studies validating the utility of MUC-1 as a candidate antigen and specificity of MN-EPPT as a multi-modal targeted probe in various cancer models (1, 3). In our new studies described here, we used this imaging probe for the simultaneous quantitative assessment of tumor volume and target antigen availability in breast tumors before and after chemotherapy. The importance of these studies is underscored by the fact that breast cancer is the second leading cause of cancer-related deaths in women of the western world. In the USA alone over 180 000 new cases are diagnosed each year. Of these patients, approximately 25% will succumb to their disease despite early diagnosis and aggressive therapeutic intervention, possibly due to the inability to optimally tailor-fit treatments on an individualized basis.

As of today, the most reliable predictor of overall breast cancer survival and therapeutic outcome in breast cancer patients is the stage at which the tumor is diagnosed. At the same time, early therapeutic intervention holds promise because of its life-saving potential. Therefore, it is critical to devise ways in which to explore the molecular complexity of breast cancer for early diagnosis and monitoring of treatment progress. There is a wealth of imaging modalities capable of evaluating the tumor and the tumoral response to therapy, including mammography and sonography (47), magnetic resonance imaging (MRI) (811), and scintigraphy or positron emission tomography (PET) (1214). These modalities, however, primarily evaluate tumor volume and metabolism but do not provide dynamic information about the molecular profile of the lesion, which would be a much earlier and more reliable predictor of tumor outcome (4).

Here we have addressed the need to develop new tumor-antigen specific contrast agents in combination with high resolution in vivo imaging methods for the monitoring of response to therapy. We believe that this study is significant because it explores a noninvasive imaging approach to monitor change in tumor size and the relative availability of a tumor antigen (uMUC-1). uMUC-1 provides a unique advantage in this case because it demonstrates predictable changes in phenotype directly linked to breast cancer progression, as well as to therapeutic outcome (15). Considering that related iron oxides are already in clinical use (16), if successful, our studies would establish the feasibility of a multi-parameter method for monitoring breast cancer response to chemotherapy, based on the expression of molecular markers implicated in disease progression.

MATERIALS AND METHODS

Probe Synthesis and Characterization

The uMUC-1-targeted MN-EPPT probe and the scrambled control probe, MN-SCR, were synthesized and characterized as described (1, 3). Peptide sequences were as follows: EPPT= C-AHA-A-R-E-P-P-T-R-T-F-A-Y-W-G-K(FITC); SCR= C-AHA-A-E-G-R-P-TF-P-T-R-A-Y-W-K(FITC). Synthesis resulted in a triple labeled nanoparticle, consisting of FITC on EPPT peptide (for fluorescence microscopy), superparamagnetic iron oxide (MN, a magnetic label for MRI), and Cy5.5 dye (Amersham Biosciences, Piscataway, NJ) attached to the MN (for NIRF optical imaging). Iron concentration and peptide/FITC, Cy5.5 payloads were determined as described in (1). The resultant probe had an average iron concentration of 7.3±0.16 mg/ml, 2.1 Cy5.5 molecules per nanoparticle, and 4.43 peptides per nanoparticle.

In vitro Cell Treatment

To validate the observation that treatment with doxorubicin downregulates uMUC-1 in BT-20 breast adenocarcinoma cells, cells were incubated with a 0.4 µM of doxorubicin-HCl (Sigma, St. Louis, MO) for 48 hrs, as previously described (17). PBS-treated cells served as controls. Following incubation, the cells were analyzed for uMUC-1 expression by quantitative RT-PCR analysis and Western blot, as described below. Alternatively, to assess the relative accumulation of MN-EPPT in these cells, following treatment with doxorubicin, the cells were incubated at 37°C overnight with MN-EPPT or the scrambled control probe, MN-SCR, washed, fixed in 2% paraformaldehyde, suspended in 0.5 ml PCR tubes, and imaged by MRI, as described below. Following the MR imaging session, the cells were transferred to FACS tubes and analyzed by flow cytometry.

Real-Time Quantitative RT-PCR

Total RNA was extracted from breast adenocarcinoma cells and tumor tissues, using the Rneasy Mini kit, according to the manufacturer’s protocol (Qiagen Inc., Valencia, CA). Relative levels of uMUC-1 mRNA were determined by real-time quantitative RT-PCR (TaqMan protocol). TaqMan analysis was performed using an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). The PCR primers and TaqMan probe specific for MUC-1 mRNA were designed using Primer express software 1.5. Primer and Probe sequences were as follows:

Forward primer, 5’-ACAGGTTCTGGTCATGCAAGC-3’ (nucleotides 64–84 in the 5’ non-repetitive region);

Reverse primer, 5’-CTCACAGCATTCTTCTCAGTAGAGCT-3’ (nucleotides 139–164 in the 5’ non-repetitive region);

TaqMan Probe, 5’-FAM-TGGAGAAAAGGAGACTTCGGCTACCCAGA-TAMRA-3’ (nucleotides 96–124 in the 5’ non-repetitive region).

Eukaryotic 18S rRNA TaqMan PDAR Endogenous Control reagent mix (PE Applied Biosystems, Foster City, CA) was used to amplify 18S rRNA as an internal control, according to the manufacturer’s protocol.

Western Blot

For Western blot, total cell extracts were prepared by solubilization of BT-20 breast adenocarcinoma cells with RIPA lysis and extraction buffer (Pierce, Rockford, IL) with added Halt™ Protease Inhibitor Cocktail (Pierce, Rockford, IL).

Protein concentration in the extracts was determined using BCA Protein Assay kit (Pierce, Rockford, IL). Samples were incubated with 1% SDS and 3% 2-mercaptoethanol at 100°C for 5 min. and centrifuged. Approximately 100 µg of total protein was separated on a 12% polyacrylamide gel (SDS-PAGE). Precision Plus Protein Standards/Kaleidoscope (Bio-Rad, Hercules, CA) were used as molecular weight standards. Separated proteins were transferred to PVDF membrane and developed with One-Step TM Advanced Western Kit for mouse primary antibody (Genscript Corp., Piscataway, NJ). Primary anti-Muc-1 mouse monoclonal antibodies specific for the backbone peptide APDTRPAP (VU4H5 clone, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Tubulin mouse monoclonal antibodies as an internal standard (V10178, Biomeda, Foster City, CA) were used at a 200- and a 10,000-fold dilution, respectively.

MR Imaging of Cell Phantoms

In order to show that the specificity of MN-EPPT for uMUC-1 can be used to probe for the downregulation of uMUC-1 by doxorubicin, we performed MR imaging of cell phantoms prepared as described in the previous section. Imaging was performed using a 9.4T Bruker horizontal bore scanner (Billerica, MA) equipped with ParaVision 3.0 software. The imaging protocol consisted of coronal T2 weighted spin echo (SE) pulse sequences with the following parameters: SE TR/TE = 3000/ [8, 16, 24, 32, 40, 48, 56, 64]; FOV = 40 ×40 mm; matrix size = 128 × 128 pixels; slice thickness = 0.5 mm; in-plane resolution = 312×312 µm. Image reconstruction and analysis were performed using Marevisi 3.5 software (Institute for Biodiagnostics, National Research Council, Canada). T2 maps were constructed according to established protocol by fitting the T2 values for each of the eight echo times (TE) to a standard exponential decay curve.

T2 relaxation times were calculated by manually segmenting out the cell pellet on MR images.

Flow Cytometry

Flow cytometry was performed in order to confirm if the MN-EPPT specificity for uMUC-1 translates in reduced probe accumulation in breast adenocarcinoma cells, following uMUC-1 downregulation by doxorubicin. Flow cytometry was performed on cells treated as described in the “In vitro Cell Treatment” section, using a FACSCalibur (Becton Dickinson, San Diego, CA, USA) equipped with the Cell Quest software package (Becton Dickinson, San Diego, CA).

Tumor Model

In order to establish a pre-clinical orthotopic tumor model of human breast cancer, 5–6 wk old female nu/nu mice (n = 10; Massachusetts General Hospital Radiation Oncology breeding facilities) were inoculated in the right mammary fat pad with the uMUC-1-positive human breast adenocarcinoma cell line, BT-20 (American Type Culture Collection, Manassas, VA), as previously described (18).

All animal experiments were performed in compliance with institutional guidelines and according to the animal protocol approved by the Subcommittee on Research Animals Care (SRAC) at Massachusetts General Hospital.

Doxorubicin Treatment

In order to establish a clinically relevant treatment model, we utilized the standard chemotherapeutic agent, doxorubicin (DOX). The treatment protocol involved intravenous injections of 7 mg/kg of DOX in saline solution once weekly for two consecutive weeks beginning ~8 d after tumor implantation once tumors had reached a diameter of 0.5 cm as previously suggested (17). Saline solution-injected animals served as non-treated controls. As previously described, in order to evaluate tumor response to chemotherapy, in vivo imaging was performed one day before the beginning of treatment and one day following the completion of treatment (3).

In vivo MR Imaging

MRI was performed before and 24-hrs after intravenous injection of MN-EPPT or the scrambled control probe, MN-SCR (10 mg Fe/kg), using a 9.4T Bruker horizontal bore scanner (Billerica, MA) equipped with ParaVision 3.0 software using sequences as for in vitro MRI. Image reconstruction and analysis were performed as described for in vitro MRI.

Tumor volumes and tumor T2 relaxation times were calculated by manually segmenting out the tumor on MR images. Quantitative evaluation of differential tumor growth by MRI was based on multislice T2-weighted images. The volume was estimated according to the formula for the volume of an ellipsoid: V = 4/3 π(abc) where a and b are the equatorial radii (along the x and y axes) and c is the polar radius (along the z-axis). For T2 map analysis of relaxation times, the terminal slices were not included in the analysis in order to avoid interference from partial volume effects. Relative MN-EPPT accumulation in the tumors was estimated based on the formula: T2 before injection minus T2 after injection (delta-T2, ms).

In vivo and ex vivo Optical Imaging

In vivo near-infrared fluorescence (NIRF) optical imaging was performed immediately after each MR imaging session. Animals were placed into a whole-mouse imaging system (Imaging Station IS2000MM, Eastman Kodak Company, New Haven, CT) and imaged in the Cy5.5 channel. At the end-point of each experiment following the last imaging session, mice were sacrificed, tumors excised, placed in the optical imaging system and imaged ex vivo. Image analysis was performed using the Kodak 1D™ 3.6.3 Network software. The actual volumes of excised tumors were determined by measuring tumor dimensions ex vivo using calipers.

Immunohistochemistry and in situ apoptosis detection

To detect the accumulation of MN-EPPT in tumors at the microscopic level, we performed correlative immunohistochemistry. Tumors were embedded in Tissue-Tek O.C.T. Compound (Sakura Fineteck, Japan) and snap-frozen in liquid nitrogen. Tumors were then cut into 7-µm frozen sections, fixed in 2% paraformaldehyde, washed, counterstained with VECTASHIELD Mounting Medium with DAPI (Vector) and analyzed by fluorescence microscopy. Microscopy was performed using a Nikon Eclipse 50i fluorescence microscope equipped with an appropriate filter set (Chroma Technology Corporation, Rockingham, VT). Images were acquired using a CCD camera with near infrared sensitivity (SPOT 7.4 Slider RTKE; Diagnostic Instruments, Sterling Heights, MI) and analyzed using SPOT 4.0 advanced version software (Diagnostic Instruments, Sterling Heights, MI). Fluorescence was collected in the green channel for detection of the FITC label on EPPT peptides, blue channel for DAPI and in the NIR channel for detection of the Cy5.5 label on MN nanoparticles.

To evaluate levels of apoptosis in tumor cells, we performed a terminal deoxynucleotidyl transferase –mediated dNTP nick end-labeling (TUNEL) assay (Apoptag Fluorescein In Situ Apoptosis Detection kit, Chemicon International, Temecula, CA) according to the manufacturer's protocol. The nuclei were counterstained with DAPI and examined under the fluorescence microscope.

Statistical Analysis

All data were represented as means ±SEM. Statistical analysis was performed using two-tailed Student’s t test and linear regression where indicated. A p-value ≤0.05 was considered statistically significant.

RESULTS

MN-EPPT Accumulation in an Orthotopic Model of Human Breast Adenocarcinoma Can Be Detected by MRI and NIRF Imaging

In this study, we utilized a pre-clinical orthotopic model of human breast cancer to obtain information about tumor response to the standard chemotherapeutic agent, doxorubicin. As a first step towards this goal, we evaluated whether MN-EPPT accumulates in the orthotopic breast tumor model and whether this accumulation is sufficient to generate detectable contrast on T2-weighted MR and optical images. Eight days after tumor implantation once tumors had reached a diameter of 0.5 cm, female nu/nu mice were injected with the uMUC-1-targeted MN-EPPT probe. Twenty-four hours after probe injection mice were subjected to MR and fluorescence optical imaging. Representative T2-weighted images and their corresponding T2 maps before and 24 h following MN-EPPT injection are shown in Fig. 1. Before injection of the contrast agents, breast tumors appeared with characteristically long T2s (T2 = 46.8±2.0 ms). After administration of MN-EPPT, there was a significant decrease in tumor T2 relaxation time (T2 = 30.9±1.2 ms), compared to the pre-contrast value (p < 0.0011, n = 9; Fig. 1). In vivo NIRF optical imaging was performed immediately after the post-contrast MR session. In mice injected with MN-EPPT, there was a high-intensity fluorescence signal coming from the implanted tumor (4388.8±420.4 RFU; Fig. 2A). Ex vivo imaging of excised tumors (Fig. 2B) revealed bright fluorescence associated with the tumor compared to muscle tissue which was used to define background fluorescence. Furthermore, ex vivo fluorescence microscopy on frozen tumor sections revealed very extensive accumulation of the probe in tumor cells (Fig. 2C). The good colocalization between fluorescence in the NIR channel (Cy5.5, MN) and the green channel (FITC, EPPT peptide) indicated integrity of the probe after persistence of the circulation (Fig. 2C).

FIGURE 1
MR imaging of MN-EPPT accumulation in orthotopic breast adenocarcinomas
FIGURE 2
Optical imaging of MN-EPPT accumulation in orthotopic breast adenocarcinomas

With these experiments, we established that MN-EPPT accumulated in orthotopic breast tumor models, and that this accumulation can be detected by non-invasive MRI and NIRF optical imaging. These findings set the stage for our next studies in which we assessed the utility of the described imaging approach for the monitoring of tumor response to chemotherapy. Since the affinity of MN-EPPT for tumors is a function of its uMUC-1 targeted nature (1, 3), we speculated that the uptake of this probe by tumors would permit not only tumor delineation for the calculation of tumor volume but also measurement of target antigen expression as reflected by tumor T2 relaxation times.

Tracking Change in Tumor Size Following Chemotherapy in a Pre-clinical Orthotopic Model of Breast Cancer

In order to determine if MN-EPPT can serve as a tumor-targeted agent for the monitoring of tumor response to chemotherapy, we treated tumor bearing animals with doxorubicin and compared changes in tumor size as measured by MRI and NIRF optical imaging between experimental mice and saline-treated controls. As seen in Fig. 3, whereas in animals treated with DOX there was an actual reduction in tumor volume following the course of therapy, in saline-treated controls tumor volume increased dramatically. These changes were clearly detectable by MRI (Fig. 3A). Quantitative region-of-interest analysis of relative tumor volume revealed an average decrease of 53% in DOX-treated mice. By contrast, in saline-treated controls, tumor volume increased 9-fold by the end of the experiment (p = 0.0011, n = 6; Fig. 3A).

FIGURE 3
Tracking changes in tumor size following chemotherapy

These findings were independently confirmed by NIRF optical imaging. In this case, the area characterized by near-infrared fluorescence on the NIRF images served as a surrogate for the definition of tumor margins. Visual inspection of NIRF images clearly suggested a differential in tumor growth between experimental and control animals (Fig. 3B). Semi-quantitative analysis of relative tumor size was performed by selecting a region of interest (ROI) based on the section of NIRF fluorescence and measuring the area of the ROI. While in animals treated with DOX, there was a decrease in relative tumor size of 51% in saline-treated controls tumor size increased dramatically (p < 0.001, n = 6; Fig. 3B). In terms of relative measurements of change in tumor size the two modalities were highly correlated (R2 = 0.89; Fig. 3C) and accurately defined true differences between experimental and control tumors, as seen ex vivo (Fig. 3D).

Tracking Change in Tumor Antigen Expression Following Chemotherapy in a Pre-clinical Orthotopic Model of Breast Cancer

There is evidence in the literature that treatment of breast tumor cells with doxorubicin leads to down-regulation of the uMUC-1 antigen (19). With this knowledge, we speculated that, since the accumulation of MN-EPPT in tumors is predicated on its specificity for the uMUC-1 tumor-specific antigen (1, 3) and since the change in T2 relaxation times post-contrast vs. pre-contrast (delta-T2) is a function of local contrast agent distribution and abundance (20), by measuring delta T2 relaxation times we would be able to indirectly extract information about uMUC-1 relative availability as a result of chemotherapeutic treatment. Obtaining such information would be very valuable from a clinical point of view since changes in uMUC-1 expression directly relate to tumor progression, response to therapy, metastatic potential, and survival (2124).

We first validated the observation that treatment with doxorubicin down-regulated uMUC-1 in BT-20 breast adenocarcinoma cells. Cells were incubated with doxorubicin for 48 hrs as previously described (17). Following incubation, the cells were analyzed for uMUC-1 expression by quantitative RT-PCR analysis and Western blot. Both methods confirmed substantial down-regulation of MUC-1 in BT-20 cells following treatment with doxorubicin (Fig. 4A and B). qRT-PCR revealed a significant 60±0.7% down-regulation of the gene (p = 0.028; Fig. 4A). This down-regulation effect was accompanied by the induction of considerable levels of apoptosis in the doxorubicin-treated cells as assessed using TUNEL assay (Fig. 4C). Consistent with the known mechanisms of cytotoxicity of doxorubicin, there was abundant nuclear as well as extranuclear TUNEL staining indicating the formation of multiple micronuclei and loss of nuclear membrane integrity. These events accompany mitotic catastrophe and reflect advanced stages of doxorubicin-induced apoptosis (25) and successful mediation of cytotoxicity by the drug (Fig. 4C). The down-regulation effect was uMUC-1 specific, since house-keeping genes (18s, qRT-PCR and tubulin, Western blot) were not down-regulated (Fig. 4A and B).

FIGURE 4
Effects of doxorubicin on uMUC-1 availability in BT-20 cells - in vitro tests

Next we wanted to see if we could use the uMUC-1-selectivity of MN-EPPT in order to probe for these changes. Populations of cells treated with doxorubicin or PBS as described above were incubated with either PBS, MN-EPPT or a scrambled control probe, MN-SCR (3) that is identical to MN-EPPT except that the targeting EPPT peptide is replaced with a scrambled peptide. Following incubation, cell phantoms were imaged by MRI. As seen in Fig. 5A and B, cells incubated with MN-EPPT displayed significantly lower T2 relaxation times than cells incubated with MN-SCR, further confirming the specificity of MN-EPPT for uMUC-1 (p < 0.0001, n = 6; Fig. 5A and B). Treatment with doxorubicin resulted in significantly higher T2 relaxation times of MN-EPPT incubated cells (p = 0.0003, n = 6; Fig. 5A and B) indicating a lower accumulation of the probe and closely mirroring the reduced expression of the antigen in these cells as demonstrated by qRT-PCR and Western blot. Unlike cells incubated with MN-EPPT in cells incubated with the MN-SCR probe, treatment with doxorubicin did not change the T2 relaxation times of the cells (Fig. 5A and B).

FIGURE 5
MN-EPPT uptake by BT-20 cells - in vitro studies

The findings by MRI were also confirmed using flow cytometry in the FL4 channel (Cy5.5), performed immediately after the MR imaging session (Fig. 5C and D). The relative fluorescence of cells incubated with MN-EPPT was significantly higher than in cells incubated with MN-SCR (p = 0.007; Fig. 5C and D) reflective of the specificity of the probe for uMUC-1. In addition, in cells treated with doxorubicin and further incubated with MN-EPPT, relative fluorescence was lower than in control-treated cells (p = 0.007; Fig. 5C and D) reflecting the reduced availability of the antigen in the presence of doxorubicin. By contrast, when MN-SCR was used as a contrast agent relative fluorescence levels in DOX-treated cells were not different than in controls (Fig. 5C and D). These studies confirmed that MN-EPPT specific accumulation in breast adenocarcinoma cells is a function of relative levels of availability of the target antigen.

These experiments established an in vitro framework for our subsequent in vivo studies aiming to quantitatively assess relative uMUC-1 availability in pre-clinical breast cancer tumors. To accomplish this final goal, we analyzed tumor T2 relaxation times of animals treated with doxorubicin or saline as described in the previous section and calculated tumor delta-T2 values before the beginning and after the completion of treatment. In animals treated with doxorubicin, there was a 25% decrease in delta-T2 post-treatment with doxorubicin (p = 0.04, n = 5; Fig 6A). In control mice treated with saline, there was no significant difference between pre- and post-treatment delta-T2 (Fig. 6A). The observed drop in delta-T2 post-treatment with doxorubicin was indicative of reduced relative MN-EPPT accumulation in the tumors and was accompanied by a corresponding down-regulation of uMUC-1 as seen by qRT-PCR (p = 0.03; Fig. 6B). Unlike mice injected with the targeted, MN-EPPT probe, in control mice injected with MN-SCR as a contrast agent there were no significant differences in delta-T2 between mice treated with doxorubicin and saline (results not shown). These studies corroborated our in vitro findings and further confirmed that MN-EPPT can be used to non-invasively assess relative uMUC-1 availability in breast tumors.

FIGURE 6
MN-EPPT uptake by BT-20 tumors as a function of uMUC-1 expression - in vivo studies

DISCUSSION

The recent past has witnessed considerable progress in the way human tumors are characterized with a particular gain in knowledge of cancer at the molecular level. This has resulted in a shift towards using molecularly targeted therapies for cancer necessitating the development of corresponding molecular tools to determine which patients are most likely to benefit from particular therapies (26). Consequently, the capacity to non-invasively assess tumor anatomy and physiology and at the same time register aberrations in the molecular phenotype of cells is essential for early diagnosis and effective treatment. With these studies, we have demonstrated the utility of magnetic resonance imaging for multi-parametric characterization of tumor response to chemotherapy in a pre-clinical breast cancer model. We have confirmed that MRI and NIRF can be combined to obtain complementary information about anatomic changes in tumor size following treatment using the breast cancer model. In addition, we have established that our contrast agent by virtue of being target-specific for a tumor antigen can provide molecular information about target antigen expression when combined with magnetic resonance imaging as a modality with a high quantitative sensitivity to local contrast agent abundance. This approach represents an example of non-invasive semi-quantitative assessment of therapy-induced changes in tumor molecular expression levels by MRI.

An important point relevant to our studies is the potential for effects other than probe uptake by the tumor such as vascularity and the presence of necrosis to affect the measurements by MRI. Areas of active angiogenesis as well as edema can result in elevated T2 relaxation times, whereas areas of local hemorrhage or necrosis can appear T2 short. We have attempted to eliminate or minimize the influence of these factors by focusing on the delta-T2 parameter, which represents an estimate of the change in T2 after probe injection relative to pre-injection. Assuming a minimal change in anatomical/physiologic tumor characteristics during the time between the pre-injection and post-injection MR measurements, the delta-T2 value reflects relative probe accumulation. Consequently, the development of similar targeted probes with a faster clearance rate (e.g. Gd chelates) and optimal target binding kinetics will permit a further shortening of the time-span between pre- and post-contrast imaging and will even more accurately reflect probe uptake by the tumor.

In summary, this study describes a method for antigen-specific noninvasive breast tumor imaging by MRI. Our strategy can be used in vivo to monitor tumor response to therapy based not only on changes in tumor size, which is a late and often unreliable marker of response to therapy, but also on the expression of a molecular biomarkers. Considering that many newly established and experimental breast cancer therapies are molecularly targeted, similar probes can be developed for the monitoring of the availability of antigens such as her2/neu, the epidermal growth factor receptor (EGFR), the estrogen receptor (ER), and the somatostatin receptor, to name a few. Progress in that direction has already been made (2731). The ultimate implications of the approach include optimization of existing cancer treatment regimens, testing of novel therapeutic paradigms, and development of personalized medicine protocols.

Acknowledgements

The authors would like to thank John Moore for excellent technical support in animal surgery.

Footnotes

1This work was supported in part by NIH grant EB001727 to A.M

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