MRI Detection of Breast Cancer Blood Vessels

Institution: University of California, Irvine
Investigator(s): Orhan Nalcioglu, Ph.D. -
Award Cycle: 1997 (Cycle III) Grant #: 3IB-0028 Award: $149,935
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Imaging, Biomarkers, and Molecular Pathology: improving detection and diagnosis

Initial Award Abstract (1997)
Angiogenesis (blood vessel growth) plays a critical role in breast cancer progression and metastasis (spread in the body); and could be an important target for early detection and therapeutic intervention. Numerous angiogenesis-associated proteins (vascular endothelial growth factor, basic fibroblast growth factor, pleiotrophin, heregulin, and eosinophil cationic proteins) are detectable in human breast cancers. All of these proteins have extensive basic, positively-charged regions having a high affinity for heparin, a polyanionic glycosaminoglycan that is routinely used to block blood clotting. Therefore, we propose to synthesize, purify, characterize, and test a novel compound, heparin-albumin, that will deliver a contrast agent (GdDTPA) suitable for magnetic resonance imaging (MRI) of the new blood vessels that form within breast cancers. MRI is potentially more sensitive than mammography and can be utilized to detect specific breast cancer components. The proposed studies will involve a multidisciplinary collaboration between a university-based physicist and a pathologist.

In the first phase of this project, we will synthesize the new compound (heparin-albumin GdDTPA) using a well-established chemical procedure. After purification of the compound by sequential chromatography, we will then perform detailed characterizations in the laboratory, including measurement of its heparin content and analysis of its binding to a strong anion resin. In the second phase, we will conduct studies initially in mice bearing a human mammary cancer cell line (MCF-7), and subsequently in rats bearing subcutaneous implants of an adenocarcinoma cell line. We will use immunohistochemistry to study the biodistribution of a derivative of the compound (heparin-albumin GdDTPA) at various times after intravenous injection (12 minutes to 3 hours). The third phase of the project will consist of sequential MRI studies in tumor-bearing mice and rats that will receive intravenous injections of the compound. This mimics the conditions that would be used in women either diagnosed with or suspected of having breast tumors or suspect lesions, such as ductal carcinoma in situ (DCIS).

Our aim is to extend these experiments into a multidisciplinary drug-discovery effort to develop a novel strategy for early detection of breast cancer. The overall advantage of this approach is that it avoids the use of radiation and can detect potentially invasive tumors at an early stage. This approach also has the potential of targeting therapeutic compounds to the site of tumor angiogenesis to block the progression of breast cancer.

Final Report (1999)
Our project produced some unexpected results. During the first year of this project, we synthesized and purified a novel contrast agent that was intended to bind to blood vessels in breast cancer in rats and mice. Although the contrast agent met our original quality control criteria for composition, purity, binding activity, and stability in the test tube, the contrast agent failed to produce a significant signal enhancement when injected into rats with breast cancer implants. We now attribute this failure to two separate problems: (1) the contrast agent bound to itself in solution, producing a milky suspension called a colloid; and (2) consequently the amount of signal produced by the contrast agent was insufficient for detection by magnetic resonance imaging.

These negative findings led us to explore another compound during the second year of support, namely protamine-Gd-DTPA. Adenocarcinomas in rats and humans frequently contain perivascular, degranulating mast cells which release heparin. Protamine is a low-molecular weight, cationic polypeptide that binds avidly to heparin. Therefore, we hypothesized that systemically administered protamine may serve as a vehicle to deliver contrast agents to heparin within the tumor. We were able to label protamine with gadolinium (Gd), and this approach yielded a stable compound with a molar relaxivity (12.5 s-1/mM Gd) that is three times higher than the molar relaxivity of a contrast agent that is clinically used for magnetic resonance imaging (Gd-DTPA). Moreover, we also showed that the protamine-Gd compound served as a successful contrast agent for dynamic contrast-enhanced magnetic resonance imaging (MRI) of a mammary adenocarcinoma (R3230AC) implanted into Fischer 344 rats. Sequential cytochemical studies of tissues from additional, tumor-bearing rats that were injected with biotinylated protamine demonstrated that the biotinylated protamine was localized almost exclusively in extracellular, perivascular locations at the periphery of the tumors in a distribution identical to that of mast cells. On the basis of these results, we conclude that protamine was retained in R3230 adenocarcinoma implants in rats and that MRI can be used to monitor the uptake of protamine by the tumor.

These results suggest that protamine should be considered as a possible candidate for experimental modulation of the effects of heparin and mast cells within mammary adenocarcinomas. In addition, another form of protamine, namely protamine sulfate, could serve as a pro-thrombotic drug for its anti-angiogenic effects on breast and ovarian cancer models in rats. This compound neutralizes the heparin released by the degranulating mast cells that are found in the connective tissue regions of most tumors. Our pilot studies indicate that protamine sulfate causes selective thrombosis of blood vessels within tumors. Our finding suggests that protamine and related compounds should be further investigated as possible anti-neoplastic drugs.

Because the protamine-based drugs investigated here are already approved for human use and have well-defined toxicity profiles, success of the tests may readily translate into human studies in the near future.

Protamine an an anti-angiogenesis agent for cancer therapy
Periodical:International Society of Magnetic Resonance in Medicine
Index Medicus:
Authors: Su M-Y, Samoszuk MK, Song Y, and Nalcioglu O
Yr: 1999 Vol: Nbr: Abs: Pg:1326 (abstract)

Characterization and biodistribution of a peptide based MR contrast agent: protamine-Gd-DTPA
Periodical:International Society of Magnetic Resonance in Medicine
Index Medicus:
Authors: Su M-Y, Samoszuk MK, Song A, Najafi A, and Nalcioglu O
Yr: 1999 Vol: Nbr: Abs: Pg:1186 (abstract)