Introduction: The complex interplay among cancer cells, fibroblasts, endothelial cells, and diverse immune cells, drives tumor growth and metastasis. Visualizing these cells’ spatiotemporal distribution within individual mice, especially in immunocompetent models, can transform our understanding of tumor biology. These insights would also be highly valuable for understanding therapy response, especially with emerging immunotherapies.

Various reporter genes are used to dynamically track the fate of transplanted cells in-vivo. However, common optical reporters, including luciferase, are foreign to mice—altering normal immune responses or provoking rejection of the cells being tracked [1,2]. Previously, rat organic anion transporting polypeptide 1a1 (rOatp1a1) [3] and human OATP1B3 [4] have been developed as MRI reporters by promoting cellular uptake of the positive-contrast agent Gd-EOB-DTPA. These tools can enhance the conspicuity of viable cancer cells within tumors and sensitively track spontaneous metastases in immunodeficient mice [4, 5]. However, similar reporters for immunocompetent mice have not been described. Additionally, Flourine-19 (¹⁹F) MRI has been used to visualize tumor-associated macrophages (TAMs) following systemic administration of ¹⁹F perfluorocarbon emulsions [6]. In this work, a novel mouse-derived OATP1 MR reporter gene is used to sensitively visualize viable cancer cells within syngeneic tumors longitudinally, and ¹⁹F MRI is used to co-visualize TAM distribution during tumor development.

Methods and Results: Cell Engineering: 4T1 murine breast cancer cells were transduced with lentiviruses to stably express mOatp1a1 without immunogenic selection genes. Sulforhodamine-101 showed uptake into engineered cells, allowing sorting via FACS (Figure 2B&C).
In-vitro MRI: Naïve and engineered cells were incubated with and without Gd-EOB-DTPA. MR images were acquired using a fast spin-echo inversion-recovery pulse sequence to compare spin-lattice relaxation rates (R1), revealing engineered cells had significantly higher R1 rates than naive cells after incubation (p<0.01; Figure 2E).
In-vivo MRI: Naive and mOatp1a1-expressing 4T1 cells were implanted bilaterally into the 4th mammary fat pads of immunocompetent female Balb/c mice (n=3). Mice were imaged weekly before and 5 hours after Gd-EOB-DTPA administration. At day 26, mice were also imaged 24 hours after injection of a perfluorocarbon agent. mOatp1a1 tumors showed significantly increased Gd-EOB-DTPA-enhancement compared to naïve tumors (p<0.01; Figure 3B). As mOatp1a1 tumors developed, distinct enhancing (live cancer cells) and non-enhancing (necrosis) regions became evident (Figure 3A). At day 26, ¹⁹F signal was observed at the periphery of both naive and engineered tumors, and next to non-enhancing regions of mOatp1a1-engineered tumors (Figure 1A). Significant ¹⁹F signal in the spleen and lymph nodes were also present. Next, mice bearing singular mOatp1a1-expressing 4T1 tumors were imaged. Heterogeneous mOatp1a1-mediated intratumoral enhancement patterns were seen, which increased at later time points. At early time points, ¹⁹F signal was present in enlarged ipsilateral lymph nodes, whereas at later time points, contralateral lymph nodes also contained ¹⁹F signal and distinct regions of intratumoral ¹⁹F signal became notable (Figure 1B). At endpoint, tissues were excised and stained for macrophages. Macrophages were visualized in the spleen and tumor (Figure 3C).

Discussion: By developing the first mouse-derived MRI reporter, the ability to distinguish viable cancer cells from necrotic regions within individual syngeneic tumors was significantly enhanced. Additionally, ¹⁹F MRI provides complementary information about TAM biodistribution within tumors, potentially improving our understanding of treatment effectiveness in cancer models involving immune cells. Future work includes integrating this tool with other imaging technologies, like PET, to provide a deeper understanding of the cell interactions during tumorigenesis and metastasis. Given the scarcity of immunotolerant reporter genes in the cell-tracking toolbox, these findings provide a robust system for tracking cancer development in various immunocompetent mouse models. Importantly, beyond tracking cancer cells, the mOatp1a1 reporter can also be applied to monitor gene- or cell-based therapeutics for many diseases in immunocompetent mice.

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Figure 1. A) MR images of a Balb/c mouse 26 days after bilateral mammary fat pad injection of naïve or mOatp1a1-engineered murine breast cancer 4T1 cells. (Left) The 1H MR image 5 hours after IV injection of Gd-EOB-DTPA shows uptake in engineered tumors only, with distinct regions containing viable cancer cells as well as a necrotic core. (Right) Overlay of 1H and 19F MR images (axial view) reveal 19F perfluorocarbon agent uptake in the spleen and distinct regions within the tumor adjacent to the necrotic core. B) Similar imaging in a mouse implanted with a single mOatp1a1-engineered 4T1 mammary fat pad tumor over time. The development of necrotic regions is clear as well as the distinct intratumoral regions of 19F signal at later time points. 19F signal is also clearly seen in peritumoral and distant enlarged lymph nodes, even at day 12.

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