Potential of [11C](R)-PK11195 PET Imaging for Evaluating Tumor Inflammation: A Murine Mammary Tumor Model

Abstract

Background: Breast tumor inflammation is an immunological process that occurs mainly by mediation of Tumor-Associated Macrophages (TAM). Aiming for a specific measurement of tumor inflammation, the current study evaluated the potential of Positron Emission Tomography (PET) imaging with [¹¹C](R)-PK11195 to evaluate tumor inflammation in a mammary tumor animal model.

Methods: Female Balb/C mice were inoculated with 4T1 cells. The PET imaging with [¹¹C](R)-PK11195 and [¹⁸F]FDG was acquired 3 days, 1 week, and 2 weeks after cell inoculation.

Results: The [¹¹C](R)-PK11195 tumor uptake increased from 3 days to 1 week, and decreased at 2 weeks after cell inoculation, as opposed to the [¹⁸F]FDG uptake, which showed a slight decrease in uptake at 1 week and increased uptake at 2 weeks. In the control group, no significant differences occurred in tracer uptake over time. Tumor uptake of both radiopharmaceuticals is more expressed in tumor edge regions, with greater intensity at 2 weeks, as demonstrated by [¹¹C](R)-PK11195 autoradiography and immunofluorescence with TSPO antibodies and CD86 pro-inflammatory phenotype.

Conclusion: The [¹¹C](R)-PK11195 was able to identify heterogeneous tumor inflammation in a murine model of breast cancer and the uptake varied according to tumor size. Together with the glycolytic marker [¹⁸F]FDG, molecular imaging with [¹¹C](R)-PK11195 may provide a better characterization of inflammatory responses in cancer.

Keywords: 4T1 cells; TSPO; [11C](R)-PK11195; breast cancer; inflammation; macrophages; positron emission tomography.

Figure 1

Experimental study design and the divisions/sub-groups at each time point, illustrated by different colors. Note: As the main scientific question was related to the [¹¹C](R)-PK11195 uptake in the tumor, the sample size for this tracer was bigger than for [¹⁸F]FDG, as higher variations in the results were expected due to tumor growing heterogeneity.

Figure 2

Tumor volumes were measured at 3 days, 1 week, and 2 weeks after 4T1 cell inoculation (**** p < 0.0001, *** p = 0.0005, unpaired Student t-test). Note: The volume was considered zero at the 3-day time point due to the undetectable volume at the cell injection site.

Figure 3

Illustrative [¹¹C](R)-PK11195 and [¹⁸F]FDG PET images fused with anatomic reference (CT image). The white arrows indicate the tumor site.

Figure 4

Tumor volume and tracer uptake: (A) [¹¹C](R)-PK11195 uptake in tumor group (red) at 3 days (n = 4), 1 week (n = 20), and 2 weeks (n = 12), and in the control group (gray) at 3 days (n = 6), 1 week (n = 4), and 2 weeks (n = 4); and (B) [¹⁸F]FDG PET uptake in the tumor group (purple) at 3 days (n = 5), 1 week (n = 5), and 2 weeks (n = 4), and in the control group (gray) at 3 days (n = 7), 1 week (n = 6), and 2 weeks (n = 6). (**** p < 0.0001; decreased [¹¹C](R)-PK11195 uptake in 2 weeks compared to 1 week time point, Mann–Whitney U test).

Figure 5

Tumor/muscle uptake ratio: the [¹¹C](R)-PK11195 tumor/muscle uptake ratio at 3 days (n = 4), 1 week (n = 20), and 2 weeks (n = 12), and the [¹⁸F]FDG tumor/muscle uptake ratio at 3 days (n = 5), 1 week (n = 5), and 2 weeks (n = 4), (Mann–Whitney U test).

Figure 6

Illustrative [¹¹C](R)-PK11195 autoradiography images of mammary murine tumor at 1 and 2 weeks after 4T1 cell inoculation. The white arrows indicate tumor edge uptake.

Figure 7

Images of immunofluorescence reactions in tumor tissue with Hoechst dye (Nucleus), TSPO, CD11 (macrophages), and fusion of all three. The dashed line divides the tissue sections into an upper (edge) and lower (center) region.

Figure 8

Immunofluorescence images representing tumor staining for Hoechst uptake (Nucleus), CD206 (macrophage M2, anti-inflammatory), CD86 (macrophage M1, pro-inflammatory), and fusion of all three. The dashed line divides the tissue section, where the upper region corresponds to the tumor edge and the lower region corresponds to the tumor center.

Figure 9

Images of immunofluorescence reactions in tumor tissue with Hoechst dye (Nucleus) and OXPHOS (oxidative phosphorylation), and fusion of the two of them. The dashed line divides the tissue sections into an upper (edge) and lower (center) region.

Figure 10

Western blot of TSPO expression in three cell lines of breast cancer (4T1, MDA-MB231, and MCF7).