Death from cancer is usually the result of dissemination of cancer cells from a primary tumor to secondary vital organs, and the formation of metastases. This process involves a series of steps, each of which have become targets of anticancer therapies such as intravasation of cancer cells into the bloodstream or lymphatics, delivery to organs (e.g., liver, lung, bone, brain, and lymph nodes), extravasation of cells into the organ parenchyma, cell proliferation to form secondary tumors, and development of new blood vessels to sustain continued growth (1). Importantly, single metastatic cells (2,3) or prevascular micrometastases (4) may also remain dormant within an organ, persisting until conditions are suitable for proliferation. Therefore, while surgical treatment of the primary tumor may be successful, undetectable dormant single metastatic cells or prevascular micrometastases can remain clinically silent for long periods and may eventually result in tumor formation and patient relapse (3,4).Metastasis to the brain can occur with many tumor types, including breast cancer, lung cancer, and melanoma. For breast cancer patients, the prevalence of brain metastases was historically estimated at 10 -16% with a 1-year survival rate of 20% (5). More recent studies, however, have demonstrated the prevalence of brain metastases in breast cancer patients to be closer to 22-30% (6), suggesting that its incidence may be increasing as a sanctuary site as systemic control improves. Brain metastases are typically treated with stereotactic radiosurgery or surgery with whole-brain radiation, supplemented with corticosteroid therapy for symptomatic relief. Patchell et al. (7) reported that surgery and whole-brain radiation can cure up to 90% of solitary brain metastases, which suggests that undiagnosed micrometastases or dormant cells are responsible for treatment failure. Thus, identification of micrometastatic and dormant brain metastatic tumor cells may facilitate an understanding of their biology and development of therapeutic interventions.For brain metastases of breast cancer, only a handful of experimental model systems have been reported. Yoneda et al. (8) performed six rounds of selection of human MDA-MB-231 breast carcinoma cells for brain metastasis in mice, followed by excision of the lesion and establishment of a cell culture. The resulting MDA-MB-231BR "brain-seeking" clone metastasized to the brain following intracardiac injection in 100% of the mice. Metastasis was identified histologically, which provided only one time point per animal. Clearly, studies of the metastatic process would greatly benefit from techniques that could dynamically monitor metastases from their earliest stage to endstage growth throughout entire organs or animals. This

We have developed a magnetic resonance imaging (MRI) technique for imaging Feridex (superparamagnetic iron oxide [SPIO])-labeled islets of Langerhans using a standard clinical 1.5-Tesla (T) scanner and employing steadystate acquisition imaging sequence (3DFIESTA). Both porcine and rat islets were labeled with SPIO by a transfection technique using a combination of poly-L-lysine and electroporation. Electron microscopy demonstrated presence of SPIO particles within the individual islet cells, including ␤-cells and particles trapped between cell membranes. Our labeling method produced a transfection rate of 860 pg to 3.4 ng iron per islet, dependent on the size of the islet. The labeling procedure did not disrupt either the function or viability of the islets. In vitro 3DFIESTA magnetic resonance images of single-labeled islets corresponded with their optical images. In vivo T2*-weighted scan using 1.5 T detected as few as 200 SPIO-labeled islets transplanted under rat kidney capsule, which correlated with immunohistochemistry of the transplant for insulin and iron. Ex vivo 3DFIESTA images of kidneys containing 200, 800 or 2,000 SPIO-labeled islet isografts showed good correlation between signal loss and increasing numbers of islets. These data provide evidence that islets can be labeled with SPIO and imaged using clinically available 1.5-T MRI. Diabetes

The metastatic cell population, ranging from solitary cells to actively growing metastases, is heterogeneous and unlikely to respond uniformly to treatment. However, quantification of the entire experimental metastatic cell population in whole organs is complicated by requirements of an imaging modality with the large field of view and high spatial resolution necessary to detect both single cells and metastases in the same organ. Thus, it is difficult to assess differential responses of these distinct metastatic populations to therapy. Here, we develop a magnetic resonance imaging (MRI) technique capable of quantifying the full population of metastatic cells in a secondary organ. B16F1 mouse melanoma cells were labeled with micron-sized iron oxide particles (MPIO) and injected into mouse liver via the mesenteric vein. Livers were removed immediately or at day 9 or 11, following doxorubicin or vehicle control treatment, and imaged using a 3T clinical magnetic resonance scanner and custom-built gradient coil. Both metastases (>200 μm) and MPIO-labeled single cells were detected and quantified from MR images as areas of hyperintensity or hypointensity (signal voids), respectively. We found that 1 mg/kg doxorubicin treatment inhibited metastasis growth (n = 11 per group; P = 0.02, t test) but did not decrease the solitary metastatic cell population in the same livers (P > 0.05). Thus, the technique presented here is capable of quickly quantifying the majority of the metastatic cell population, including both growing metastases and solitary cells, in whole liver by MRI and can identify differential responses of growing metastases and solitary cells to therapy. [Cancer Res 2009;69(21):8326-31]

There are several factors that contributed to this important result. First, a greater-than-linear SNR gain was achieved in mouse brain images at 3 T. Second, a reduction in the bandwidth, and the associated increase in repetition time and SNR, produced a dramatic increase in the contrast generated by iron-labeled cells.