In sum, in this work we developed a new hepatocellular carcinoma murine model that generates spontaneous fluorescent tumor masses and expresses nominal antigens (a protein of HBV and an oncogene), recognized by a population of lymphocytes (cells of the adaptive immunity that exert liver protection). First of all, we tried to understand whether our mouse model recapitulates the most important features of human hepatocellular carcinoma, like arterialization, sinusoidal capillarization and multi trabecular chords formation, typical of the human HCC, and we observed that we were able to summarize some of the common features used by clinicians to distinguish HCC from other liver cancers. So, we concluded that our mouse model generates well-differentiated HCC distributed in healthy liver parenchyma and the mouse disease that we obtain is mimicking sufficiently well the human disease. We then transferred activated lymphocytes able to recognize our nominal antigens expressed by transformed hepatocytes and by performing Magnetic resonance imaging scanning, we evaluated the volume of tumor masses. The reduction of tumor lesions that is around 10-15 days post cell transfer proved the effective activity of these cells in killing the transformed hepatocytes.
Using the multiphoton intravital microscopy (MPIVM) technique present in our BLS-3 animal facility, we could assessed the spatiotemporal dynamics of the tumor specific lymphocytes injected in HCC-bearing mice surgically prepared for liver MPIVM. We observed that the tumor specific lymphocytes displayed a more static and slow migratory behavior once they accumulate in the HCC lesions, compared to the control cells that maintain a motile phenotype. This behavior is possibly due to the cell recognition of the oncogene antigen expressed by the tumor mass since the tumor specific lymphocytes get activated, they produce IFN-γ and they increase their PD-1 and CD25 levels, indicating an activated state. Moreover they exert their cytotoxic function by inducing Caspase 3 in HCC lesions. We were able to obtain an imaging platform to study HCC in living animals and we presented our results in National and International Congresses and well as involving in the project 2 students for their master thesis.
