Tumour-associated macrophages (TAMs) are associated with tumour growth and metastatic spread.¹ Breast tumours can be comprised of up to 50% TAMs² and their presence is correlated with a poor outcome.³ Cell tracking with MRI can be used to image TAMs. Previous studies have shown that iron oxide particles (USPIO) administered intravenously (IV) are preferentially taken up by TAMs and that signal loss on MR images corresponds to TAMs identified by histopathology.⁴ Quantification of TAMs with iron-based cell tracking is challenging; the degree of signal loss produced by iron labeled cells is only linear at low iron concentrations.⁵ Studies that use iron-based cell tracking usually measure the volume of signal loss or percentage of black pixels, which is influenced by the blooming artifact induced by iron. Fluorine-19 (¹⁹F) MRI is another method being developed to track and quantify cells in vivo. ¹⁹F has some major advantages, with the ability to image perfluorocarbon (PFC)-labeled cells with high specificity due to the lack of endogenous fluorine in biological tissues. Most importantly, ¹⁹F MRI is quantitative, since the signal intensity is linearly related to the number of ¹⁹F-labeled cells. Therefore, it may be used to quantify the number of macrophages in vivo. This study is the first time iron-based and ¹⁹F-based cell tracking methods have been compared for TAM detection and quantification. 4T1 cells were implanted orthotopically into the inguinal mammary fat pad in female BALB/c mice. Four groups of mice were studied. For Groups 1&2, MRI was performed as soon as tumours were palpable (3 days post implantation (p.i.)). For Groups 3&4, MRI was performed 3 weeks p.i. All images were acquired using a balanced steady state free precession (bSSFP) pulse sequence. Iron-Based Cell Tracking: Mice in Groups 1&3 were imaged at 3T pre and 24 hours post IV injection of a USPIO. Spatial resolution was 200x200x200 μm³. ¹⁹F-Based Cell Tracking: Mice in Groups 2&4 were imaged at 9.4T using a dual-tuned ¹H/¹⁹F birdcage coil, at 48 hours post IV injection of a red fluorescent PFC. Spatial resolution was 0.5x0.5x1.0 mm³. A ¹H/¹⁹F overlay was composed for anatomical reference. For Groups 1&3, pre and post iron images were compared to visualize the distribution of signal loss. The percentage of black pixels within the total tumour volume was determined from post iron images. For Groups 3&4, the number of ¹⁹F atoms per mm³ was quantified by relating the detected ¹⁹F signal within the tumour to the signal generated by a reference tube containing a known amount of ¹⁹F atoms and dividing by tumour volume. Tumours were examined using fluorescence microscopy to detect the red fluorescence of the PFC. Perls Prussian Blue (PPB) was used to stain for iron and anti-lectin antibody was used (DAB and green fluorescence) to identify macrophages. Figure 1 shows results for tumours detected at 3 days p.i. (1A) is a post-iron MRI in which the signal loss encompasses nearly the entire tumor volume. Histology (1B&C) shows iron-positive cells and macrophages distributed throughout the tumour. ¹⁹F signal distribution is similar (1D); since ¹⁹F signal is proportional to the number of ¹⁹F atoms, these images provide more information than the post-iron images, revealing that the signal is higher in the central region (white) compared to the periphery (red) at this early time point. The presence of the PFC in the tumour is confirmed with fluorescence microscopy. Figure 2 shows images obtained at 3 weeks p.i. At this time point the signal loss observed post-iron (2B) appears around the periphery of the tumour and corresponds very well with the iron staining (2C). ¹⁹F MRI (2D) locates the ¹⁹F signal also around the tumour periphery but appears to occupy less area and may better represent the actual cell distribution since there is not the same sort of blooming artifact due to iron in ¹⁹F images. The presence of TAMs is confirmed in 2E&F (brown). Figure 3 shows PFC (red) and positive macrophages (green) along with the ¹⁹F MRI obtained at 3 weeks. Quantification of images is illustrated in Figure 4. Both cell tracking methods suggest that there is a higher density of TAMs present in the tumours at 4 days. No other cell tracking study has looked at TAMs at such an early time point during tumour progression. The use of ¹⁹F-based cell tracking may provide a more accurate representation of TAM infiltration when compared to iron-based cell tracking. This is achieved through both the ability to quantify ¹⁹F atoms as well as the lack of blooming artifact as seen with iron-labeled cells.