Detailed temporal characterization of in vivo response to anti-tumor immunotherapies are limited by the frequency and volume of tissue that can be practically collected. We have successfully generated a novel double-transgenic murine model, which incorporates a nuclear factor-kappaB (NF-kB) reporter transgene (NGL) into the polyoma virus middle T oncogene (PyVT) model, a widely accepted transgenic mouse with spontaneous tumor formation and disease progression comparable to that of human breast cancer. The reporter transgene uses luciferase to quantitatively indicate NF-kB activation, which is central to immunomodulatory inflammation. In vivo imaging systems (IVIS) were used for luminescent imaging of luciferase activity as a spatial reporter of NF-kB activation that was carried out repeatedly on individual double transgenic (Py-NGL) mice. Magnetic resonance thermometry (MRT) was used to guide therapeutic, high intensity focused ultrasound (HIFU) for quantitatively repeatable, sub-ablative thermal dosing as an inflammatory stimulus.
Py-NGL mice were treated with the MRT-HIFU system at a sub-ablative level of 42 °C for up to 25 minutes. Mice were imaged with IVIS before treatment and every twelve hours after treatment until the collection of tissues. The untreated tumors, treated tumors, and spleens were collected, digested, and quantitatively analyzed with flow cytometry for immunophenotyping and cytokine expression.
Py-NGL mice have a peak in NF-kB activation, spatially correlated with the administration of MRT-guided HIFU, between 48-96 hours post treatment. Preliminary measures of inflammation by NF-kB activity demonstrate up to a 20-fold increase from baseline in the HIFU treated tissue. Following treatment, NF-kB activity increased from baseline as early as 24 hours and reached a peak between 48 and 96 hours, consistent with recruitment and activation of inflammatory cell phenotypes as a consequence of MRT-HIFU treatment. Elevated NF-kB activity remained localized in the HIFU-treated tissue throughout the measurement period, and smoothly declined towards, but remained above, baseline levels following the activation peak. Immunophenotyping data reveals changes in immune cell infiltrates following treatment, including the number and phenotypes of T cells and macrophages.
This novel murine model enabled real-time, quantitative live-animal imaging of spatially specific, time-dependent NF-kB activation. Measurement of NF-kB response to controlled-dose HIFU provides the first opportunity to correlate induced inflammation with immune cell and cytokine characterization of the tumors. The objective is to quantitatively elucidate the spatiotemporal immunologic response to inflammatory stimulus in tumors. In this way, therapeutic methods based on elevating inflammation in tumors can be assessed for reversal of the immunosuppressive microenvironment and the generation of functional anti-tumor immunotherapy.
Citation Format: Mary D. Dockery, Megan E. Poorman, Vandiver L. Chaplin, Ryan A. Spears, Charles F. Caskey, William A. Grissom, Todd D. Giorgio. Real-time in vivo characterization of spatiotemporal immunotherapeutic response to high intensity focused ultrasound with a novel NF-kB reporter model of human breast cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A086.