DEVELOPMENT OF IMAGING STRATEGIES FOR INVESTIGATION OF TCR WITH DEFINED ANTITUMOR REACTIVITY IN VIVO
Henrique Bianchi, Sabine Mall, Nicolas Beziere, Uwe Klemm, Christian Peschel, Vasilis Ntziachristos, Angela M. Krackhardt. (02-06-2016).
Medizinische Klinik III, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
Institute of Biological and Medical Imaging, Helmholtz Zentrum München, Germany
Research Area C
T-cell based immunotherapies are novel and promising therapeutic approaches for a variety of malignant diseases. However, diverse approaches including those using T-cell receptor (TCR)- and chimeric antigen receptor (CAR)-transgenic T cells show highly different characteristics in vitro and in vivo. Preclinical in vivo models providing high predictive value with respect to tumor reactivity and toxicity or treatment failure due to tumor evasion are currently missing. For surveillance of therapeutic efficacy of adoptive T cell transfer, nuclear imaging has been used as a non-invasive and sensitive cell tracking technology, although limits in spatial resolution are given. We aimed to develop optoacoustic imaging as an alternative non-invasive and novel method to track TCR-transgenic T-cell responses in vivo. Multispectral optoacoustical imaging (MSOT) operates in the near-infrared (NIR) spectral region and allows deep penetration in tissue with high resolution. Cell dyes and reporter genes were tested as suitable tracers for detecting T cells with MSOT. T cells labeled with DiR, a stable cell membrane dye, presented bright fluorescence and strong absorption in the NIR spectrum. As an alternative labeling method, T cell were stably transduced with variations of the reporter gene near-infrared fluorescent protein (iRFP), in which the variation iRFP720 showed a higher brightness and a detectable signal by MSOT due to its higher emission in the near-infrared spectrum. T cells labeled with DiR, T cells expressing iRFP720 and T cells harboring both tracers were compared with respect to the limit of detection by MSOT in agarose phantoms and in vivo. For the in vivo analysis, T cells mixed with matrigel were subcutaneously injected in the back of a mouse. T cells labeled with DiR presented the most sensitive detection by MSOT both in phantoms and in vivo. However, in case of DiR-labeled T cells simultaneously expressing iRFP720, the detection of the DiR signal by MSOT was highly impaired and the sensitivity of the method decreased around 10 times. For T cells expressing iRFP alone, up to 2,5x106 cells could be detected in vivo by MSOT. A xenogenic mouse model of myeloid sarcoma was used and human central memory T cells (TCM) transgenic for the leukemia-specific TCR2.5D6 and subsequently labeled with DiR were adoptively transferred. MSOT imaging was performed at different time points post TCM cell transfer in order to investigate TCM-distribution in vivo over time. Tumor rejection correlated with the infiltration of DiR-labeled TCM cells in the ML2 B7 tumor, which was observed by epi-fluorescence imaging and confirmed with fluorescence microscopy and immunohistochemistry (IHC). MSOT analysis of the tumor region alone allowed detection of specific DiR-labeled TCM cell signal in the ML2 B7 tumor only, which correlates with the oxygenated areas of this tumor. Further analyses are necessary to confirm the infiltration and concentration of TCM cells preferentially in tumor areas with high oxygenation.