Lymphatic Drainage Imaging of Breast Cancer in Mice by Micro-Magnetic Resonance Lymphangiography, by Using a Nano-Sized Paramagnetic Contrast Agent

Kobayashi H, Kawamoto S, Sakai Y, Choyke PL, Star RA, Brechbiel MW, Sato N, Tagaya Y, Morris JC, and Waldmann TA. Lymphatic drainage imaging of breast cancer in mice by micro-magnetic resonance lymphangiography using a nano-size paramagnetic contrast agent. J Natl Cancer Inst 96: 703–8, 2004.

reast cancer remains a common malignancy, resulting in approximately 45,000 deaths annually in the United States. Lymph node metastasis has major prognostic implications and is a major criterion used for determining adjuvant therapy requirements. Sentinel lymph node (SLN) biopsy has become an increasingly popular surgical procedure to assess SLN disease status. Patients with negative biopsy results can be spared the more extensive and traumatic lymph node dissection, which is associated with substantial short- and long-term sequelae. The two most common methods of assessing the SLN are the use of a blue dye or radioscintigraphy. The former is limited because there is no external means of detecting the node; the latter is limited because of ionizing radiation to the patient, surgeon, and pathologist. Moreover, both methods suffer from poor spatial resolution. Thus, in this study, we focused on the development of an accurate, reliable, and non-invasive magnetic resonance imaging (MRI) method of lymph node assessment.

The use of a macromolecular MRI contrast agent for lymph node assessment is based on a combination of the agent’s physical and pharmacokinetic properties including molecular size and shape, lipo/hydrophilicity, charge, a large molar concentration density of Gd(III), and enhanced molar relaxivity conferred by correlation of rotation with a magnetic field. Investigation of these variables has been facilitated by the creation of a library of nano-sized (generations 2–9 [G2–G9]; PAMAM [polyamidoamine] and DAB [polypropyleneimine] dendrimers) MRI contrast agents.

We developed a dynamic micro-MR mammo-lymphangiography method to visualize lymphatic flow from breast tumor tissue to draining lymph nodes to identify an SLN and to determine the presence/absence of nodal involvement. For this study, we chose a G6 PAMAM dendrimer MRI contrast agent (240 kDa) from our library of dendrimer-based, nano-sized contrast agents. Smaller diameter agents (< 4 nm) penetrate lymphatic capillary membranes and distribute into the interstitial tissue resulting in poor signal-to-background ratios for lymphatic vessels and lymph nodes. Larger molecules (> 13 nm) diffuse slowly and therefore accumulate more slowly in the sentinel nodes necessitating a greater imaging window. We chose the G6 contrast agent (9 nm) to balance these two parameters. That is, it was large enough to be retained, but not too large to inhibit efficient uptake into the lymphatics.

Figure 1. Lymphatic drainage and detected sentinel lymph nodes from the left breast in mice are shown. Detection rates originating from 10 mice for the 3 nodes are indicated.

We imaged lymphatic flow from the mammary gland to draining lymph nodes in mice with implanted or spontaneous (Balb/HER2 transgenic) mammary tumors after peritumoral injection of the dendrimer. The method allowed visualization of lymphatic flow from the mammary gland to both metastatic and non-metastatic draining lymph nodes (Figure 1). The U.S. Food and Drug Administration-approved MRI low molecular weight contrast agent, Gd-[DTPA]-dimeglumine (Magnevist), failed to provide an image of the three nodes that were imaged by using the macromolecular agent. This was confirmed by injection of the image-enhancing agent, Magnevist, first, which only vaguely demonstrated the SLN, followed by injection of the G6 in the same mouse, which demonstrated not only the node but also the lymphatic vessel. Additionally, metastatic tumor growth in lymph nodes was detected as “a lack of filling” in the MRI image.

This micro-MRI methodology has advantages over current practice: 1) high spatial resolution (0.1 mm); 2) high time resolution (1 min/frame), enabling differentiation of a sentinel and second lymph node; 3) three-dimensional cine-display from any direction that potentially provides a precise map of the lymph nodes to surgeons; and 4) no adverse effects from either external or internal radiation. Adverse events from MRI contrast agents are related to dose. In this study, G6 was used at 1/2500 of the clinical Magnevist dose (molar concentration) to obtain images, thereby minimizing potential toxicity. Thus, this dynamic micro-MR mammo-lymphangiography method, using a nano-sized MRI contrast agent, may be translatable to clinical practice and should contribute to improved prognosis and quality of life. Current efforts to refine this agent include dual labeling with Gd(III) and an optical imaging element to optimize preoperative and intra-operative visualization of the sentinel node.

Peter L. Choyke, MD

Hisataka Kobayashi, MD, PhD

Martin W. Brechbiel, PhD
Radiation Oncology Branch
NCI-Bethesda, Bldg. 10/Rm. B3B69
Tel: 301-496-0591
Fax: 301-402-1923
martinwb@mail.nih.gov

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