Quantitative In Vivo & Ex Vivo Multimodality Cell Imaging of Antigen-Specific T-Cells in Murine Metastatic Ovarian Cancer

Cellular immunotherapies are an emerging treatment for many cancer types, including epithelial ovarian cancer. Adoptive transfer of antigen-specific T-cells has shown promise in pre-clinical and clinical studies, and has demonstrated efficacy in settings of low tumor burden. We are interested in the quantitative aspects of homing and cell-mediated destruction by antigen-specific T-cells. Specifically, how T-cell number influences the ability to detect and destroy individual metastatic lesions as a function of metastatic lesion growth rate, absolute size, and location, is poorly understood. First, we comprehensively characterized tumor kinetics and distribution in a metastatic ovarian cancer model. We used serial MRI imaging and optical in vivo imaging, ex vivo imaging, and ex vivo luminometry. Our in vivo MRI and in vivo bioluminescent data indicated that after intraperitoneal injection, ID8 ovarian tumor cells primarily home to the abdomen within four hours of transplantation (n = 10). Further, our data indicates that from day 0 (4-hour timepoint), abdominal signal decreased by over 90% by week 3, followed by linear growth until week 7 (n = 2). This data suggests that week 3 and week 5 tumor differ by a 15-fold increase in signal, indicating a large difference in tumor mass, confirmed by ex vivo bioluminescence and luminometry. Using this information, we developed a non-invasive, quantitative platform for imaging T-cells in vivo and in vitro in this well-defined model of metastatic ovarian cancer. First, OVA+ IE9-MP1 ovarian tumor cells were cultured and administered intraperitoneal in female C57BL6/J mice at a dose of 10 million each. Primary T-cells were then cultured from OT-1 TCR transgenic mice, in which the CD8+ T-cells express a TCR specific to ovalbumin (OVA), and employed to target the OVA+ IE9-MP1 cells. CD8+ T-cells were isolated, cultured, transduced with retrovirus (Fluc+ GFP+), and retroorbitally injected into lymph node-depleted mice. We hypothesized that following adoptive transfer, T-cells would traffic to major tumor locations (omentum), metastatic sites, and lymphatic organs. After 3 weeks of tumor growth, we administered luciferase-positive, OVA-specific T-cells at doses of 3 x 10⁶, 1 x 10⁶ and 3.33 x 10⁵ cells. Next, we performed serial in vivo and end point ex vivo bioluminescence. In vivo imaging of abdominal signal indicates increase in T-cell proliferation between days 2-6, and days 6-8 demonstrate at least a 10-fold signal increase compared to day 2 signals for all three doses (n = 3 per dose). Interestingly, in vivo imaging demonstrated that approximately 25-50% of cells home to primary tumor site (omentum), while the remaining T-cells home to other abdominal locations. Normalized data reveals a >50% shift in T-cell location from primary to metastatic tumor site between days 1-5 for all doses (n = 9). T-cell homing analysis of tumor-bearing mice after 5 weeks are currently underway. Our data indicates that tumor-bearing mice can be separated into 3 week (rare mets) and 5 week (early mets) models, and that T-cells distribute between primary and metastatic sites. Future studies will determine T-cell homing in the 5-week model and decipher mechanisms of T-cell distribution at metastatic sites.