Nanoparticle-Based PET Imaging of T-cell Trafficking in Immuno-Competent Murine Tumor Models

1207 Objectives: Due to the expanding interest and development of immunotherapies in oncologic applications, there is an increased need for quantitative measures of immune trafficking and localization. Current methods of imaging inflammation have several serious limitations, such as the lack of specificity to the immune cell type involved (WBC-SPECT) or to metabolically active but non-inflamed tissues (FDG-PET). Previously (Yuan et al, PLOS One, 2017), we demonstrated that the FDA-approved nanoparticle drug Feraheme (FH) could be modified by click chemistry to include a cell-penetrating peptide (protamine) and radiolabeled to allow for ex vivo labeling of specific cell populations, allowing for quantitative imaging by PET. Here, we apply this to T-cell trafficking in an immunocompetent murine cancer model and in response to an immunomodulatory drug challenge. Methods: Immunocompetent female C57bl/6 mice were subcutaneously implanted with TC-1 cells. Educated T-cells were purified by negative magnetic selection from the homogenized spleens of tumor-bearing donor mice, incubated for 1h at 37C with 89Zr-Prot-FH, then washed and resuspended in dPBS. Experimental mice (N=5) received r.o. injections of T cells labeled with 89Zr-Prot-FH (11.2±3.25MBq, 8.4 ± 2.4*106 cells). Control mice (N=5) received 3.96±0.79MBq of free 89Zr-Prot-FH. Animals were imaged daily for 72h by PET. 72h after injection of radioactivity, all mice were administered an immune targeted small molecule intraperitoneally. All animals underwent additional imaging 1,3,5,12,24, and 72 hours after treatment. After the final imaging session, animals were euthanized and tissues were harvested for ex vivo biodistribution measurements. Results: Control animals receiving injections of free 89Zr-Prot-FH displayed rapid uptake in both spleen and liver with rapid clearance from the blood pool. Animals injected with T cells labeled ex vivo with 89Zr-Prot-FH showed slower accumulation of activity into the spleen, continuing over the first 48 hours, consistent with trafficking of the re-injected cells, in contrast to direct uptake of free 89Zr-Prot-FH by tissue resident immune cells. Post mortem studies showed significantly lower liver uptake in mice injected with T-cells (SUV=3.37) than in controls (SUV=8.39) (p=.005), consistent with clearance of free 89Zr-Prot-FH dominated by hepatic stellate macrophages. In both cohorts, response to immunomodulatory small molecule treatment was clearly visible by PET. Immediately following drug administration, mobilization of immune cells could be detected out of the spleen and bone marrow. In both cohorts, splenic mobilization peaked 3 hours after administration, and began to recover by 5 hours. In mice injected with T-cells, splenic mobilization was more pronounced, with a 15± 7% peak loss of activity vs.10± 5% in controls. Recovery of activity in the spleen after mobilization was also more pronounced in T-cell injected mice: 12 hours after drug treatment splenic activity in T-cell injected mice was 17±8% higher than pre-drug baseline vs. 8±8% in control animals. Similar results were seen for bone marrow mobilization. Differences in mobilization between cohorts did not reach statistical significance, however mobilization in control was expected as i.v. administration of 89Zr-Prot-FH would lead to in vivo labeling of the pan-leukocyte population. In both cohorts, low levels of activity localized to the tumors (SUV=0.1±0.06 for all animals). Conclusion:Ex vivo labeling with 89Zr-Prot-FH allows for long term quantitative measurement of in vivo T-cell localization and trafficking. While T-cells could not be seen migrating to the tumor mass, the localization and trafficking could be quantitatively tracked, allowing for the measurement of immunomodulatory effects of drug challenge. Therefore this is a promising tool to assist in drug development and in monitoring of treatment response. Research Support: P41EB022544, VIC Innovation Fund