It is remarkable that the first four clinical studies of ferumoxides were performed outside the United States (two in Europe, one in Asia, and one in South America) [25
]. In the first study [25
], performed in The Netherlands, investigators examined the use of ferumoxides-labeled dendritic cells. The first injection into a patient was performed on April 26, 2004. The following specific aspects of the study facilitated fast approval by the local institutional review board to initiate the first clinical studies. First, a clinically approved contrast agent (ferumoxides) was used, albeit off label. Second, no secondary (transfection) agent was needed because immature dendritic cells are phagocytic by nature. Third, immature dendritic cells had always been isolated from peripheral blood mononuclear cells with Miltenyi superparamagnetic beads (Miltenyi Biotec) conjugated to specific antibodies for immunomagnetoisolation. Unlike in the United States, removal of these beads from the surface of the cell was not required. The dextran-coated beads are an excellent SPIO MRI contrast agent [29
] and resemble ferumoxides in many ways. Because clinical studies with these beads had already been performed, the argument was made that rather than SPIO beads on the outside of the cell, SPIO inside these cells would be studied. Fourth, studies of 111
In-oxine radionuclide cell tracking [31
] had been performed with the same dendritic cells and melanoma patient population. Fifth, the participants in the study were patients with advanced stage III melanoma, which has a poor prognosis and no other effective therapy. Sixth, the entire draining lymph node bed into which the ferumoxides-labeled cells were injected was resected after 2 weeks.
Two key findings were reported in that first study [25
]. The first was that it is feasible with a routine clinical setup to detect ferumoxides-labeled cells not only in the injected lymph node but also in nearby lymph nodes to which they migrate (). This phenomenon occurred when cells containing approximately 30 pg Fe/cell [32
] were used in 3-T MRI performed with conventional pulse sequences. With labeling with 111
In-oxine in parallel, it was estimated that the sensitivity with the coil setup used at a resolution of 0.5 × 0.5 × 3.5 mm was approximately 15,000 cells [25
]. It also became evident that because of its flexible 3D multiplanar nature, MRI was superior to radionuclide imaging with regard to accurate detection of the number of nodes that contained injected dendritic cells.
Fig. 2 74-year-old man with stage III melanoma who had undergone intranodal injection of autologous dendritic cells primed with synthetic melanoma antigens and labeled with ferumoxides and 111In-oxine. In vivo migration of superparamagnetic iron oxide and 111 (more ...)
The second finding was surprising. Cells had been accidentally misinjected in four of the eight patients who satisfied the end point criteria among the 10 patients enrolled in the study [25
]. This poor injection rate for procedures performed by experienced radiologists was not known until the results of MRI cell tracking became available. On the radionuclide scans, only a cloud of radioactivity was visible in the area of the draining lymph node bed. When the radionuclide scans were cross-referenced with the MR images containing anatomic information, it became clear that the cells had been injected into either surrounding muscle or subcutaneous fat (). An important factor is that the cells were injected under ultrasound guidance. Compared with MRI, ultrasound imaging has poor resolution, and anatomic features sometimes are difficult to interpret. In the case of a particular lymph node, it is common that the tip of the injection catheter pushes the node into the fatty bed without puncture and that once the needle is in the node, it is relatively easy to puncture all the way through.
Fig. 3 35-year-old woman with stage III melanoma. Accuracy of delivery of ferumoxides and 111In-oxine-labeled dendritic cells. (Adapted with permission from )
In general, the clinical benefit of cancer vaccine therapy varies widely. Some patients respond well and some not at all, according to immunostimulatory outcome measures [33
]. When cells are misinjected, there is no response because intrafollicular T cells must form rosettes with dendritic cells to become activated. The results of this first clinical MRI cell-tracking study are testimony to the absolute need for a noninvasive technique that can be used to assess the accuracy of cell injections and preferably to guide the injection itself in real time. MRI-guided cell injections conform to this requirement.
A different approach to monitoring the efficacy of cancer vaccine therapy is to label the tumor vaccine itself with ferumoxides rather than labeling the dendritic cells of interest. This approach, called magnetovaccination [34
], can be used to obtain serial images of sentinel dendritic cells that have homed to draining lymph nodes and have activated T cells. The difference is that the MR images depict dendritic cells that have captured antigen, and are thus the specific immunostimulatory cells of interest, in the course of engulfing both the tumor antigens and the ferumoxides inside the irradiated dying tumor cells used as vaccine [34
]. This process is illustrated in . A gene-transduced autologous tumor vaccine (GVAX, Cell-Genesys) is a clinical investigational cancer vaccine developed at my institution [35
]. In the spring of 2009, discussions began about initiating clinical trials of the vaccine with magnetovaccination.
Fig. 4 Experimental mouse model. Preclinical example of magnetovaccination for MRI tracking of injected dendritic cells. Dendritic cells were labeled in vivo (in situ) after phagocytosis of granulocyte–macrophage colony-stimulating factor (GM-CSF) tumor (more ...)
The second clinical MRI study, performed in Shanghai, China, is a report of two patients with traumatic brain injury [26
]. Autologous neural stem cells, isolated after removal of brain tissue during emergency surgery, were labeled with ferumoxides and a nonclinical-grade unapproved lipofection transfection agent (Effectene, Qiagen). Cells were stereotactically injected near the area of brain injury in one patient (), and gradient-echo MR images were obtained at 3 T. Over time, dynamic changes in the hypointensity were encountered that were attributed to movement of neural stem cells from the injection site to the border zone of the lesion. The signal intensity had disappeared completely 7 weeks after injection, possibly as a result of cell proliferation and dilution of ferumoxides toward undetectable levels. A control case of a patient with brain trauma who received unlabeled cells () showed an absence of hypointense voids. This study clearly showed that it is feasible, at higher field strength, to detect magnetically labeled stem cells in the human brain.
Fig. 5 34-year-old man with traumatic brain injury (A–F) who received stereotactic injection of ferumoxides-labeled neural stem cells and 42-year-old man with traumatic brain injury (G–L) who received unlabeled autologous neural stem cells. MRI (more ...)
The third clinical study [27
] was performed in São Paulo, Brazil, and stands out from the others in terms of the substance used as the MRI contrast agent: it was not designed as a contrast agent. The magnetic particles used to label CD34+
bone marrow stem cells were nonclinical, nonbiodegradable larger microspheres (Dynal Magnetic Beads, Invitrogen) developed solely for magnetic cell separation and bone marrow stem cell purging of tumor cells. Ten patients with chronic spinal cord injury received magnetic bead–labeled bone marrow stem cells, and six patients acting as controls received beads without cells. All injections were into the spinal cord by lumbar puncture.
Serial MR images were obtained at 1 T before and 20 and 35 days after injection (). As in the patient with traumatic brain injury in the study performed in China, migration of labeled cells toward the site of injury was observed over time, and the pattern was absent in the images of the patients who received injections of unlabeled beads. Although these results are significant in terms of ability to visualize cell migration noninvasively over time, there is considerable concern about patient safety with use of unapproved non-clinical-grade, nonbiodegradable contrast agents.
Fig. 6 21-year-old man with chronic spinal cord injury. (Reprinted with permission from )
The fourth and last MRI clinical study [28
] of cell tracking as of this writing was performed in Geneva, Switzerland. In that study, human cadaveric islet cells were labeled with ferucarbotran and transplanted intraportally according to the Edmonton protocol [36
]. T2*-weighted MRI was performed before and at various times after transplantation (). The viability and in vitro and in vivo functions of labeled islet cells were similar to those of control islet cells. All patients became insulin-independent after transplantation. The liver exhibited normal signal intensity on pre-transplantation images, and iron-loaded islet cells were identified after transplantation as hypointense spots within the liver. Many diabetic patients, however, experience spontaneous iron overload that interferes with detection of labeled islets ().
Fig. 7 Findings before and after intraportal transplantation of ferucarbotran-labeled human cadaveric islet cells. (Reprinted with permission from )
In the study performed in Geneva [28
], all patients who received ferucarbotran-labeled islet cells achieved insulin independence, confirming that SPIO labeling of islet cells appears to have no harmful effects on islet cell function. In that study, islet cell–induced spots continued to be identified 6 months after transplantation, but no correlation was found between the number of transplanted islets and the number of spots within the liver. The total number of hypointense spots was low in relation to the number of transplanted islet cells, even though 300,000–600,000 islet cells were transplanted. It was postulated that the sensitivity of detection may be too low for visualization of individual islet cells and that actual visible spots represented multiple islets grouped together. One way to increase sensitivity is to immunoprotect islet cells in semipermeable alginate capsules labeled with ferumoxides. These magnetocapsules, each containing a single islet cell and approximately 80 ng of iron, can be detected at the level of a single capsule [38