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Spatiotemporal dynamics of CD8+ T cell responses to hepatocellular carcinoma

Periodic Reporting for period 1 - Hepatocarcinoma IVM (Spatiotemporal dynamics of CD8+ T cell responses to hepatocellular carcinoma)

Reporting period: 2017-07-01 to 2019-06-30

The hepatocellular carcinoma (HCC) is one of the most common primary liver tumour, is derived from hepatocytes and it is the second cause of cancer-related death all over the world. HCC has an aggressive nature and a poor prognosis; indeed it is the second most common cause of cancer-related mortality but only the sixth most common cause of cancer worldwide. Chronic liver disease and cirrhosis are the most important risk factors to develop HCC (85-90% incidence). Liver cirrhosis often is a resultant of chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection and autoimmune hepatitis. Since CD8+ T cells are thought to play a critical role in controlling HCC, and since the protective capacity is mediated by antigen-experienced effector cells and depends on their ability to migrate to the liver, recognize tumor antigens and deploy effector functions, in this project we studied the role of CD8+ T cells in a novel murine model of HCC. While some of the rules that characterize CD8+ T cell behavior in the cancerous liver have been characterized at the population level, we have limited knowledge of the dynamics of CD8+ T cell interactions with tumor and hepatic cells at the single-cell level. Although recent advances in intravital microscopy may enable us to investigate the extent to which anatomical and hemodynamical cues that characterize HCC differently shape CD8+ T cell behavior and function, we still lack a proper mouse where to address these issues. In conclusion we developed a functional platform to study the immune system behaviour inside spontaneous HCC. This platform will be used to study cell activation and the therapeutic effect of anti-tumoral compounds.
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.
Our work showed that the tumor specific lymphocytes are able to reach the tumor and getting activated; we will next address the anatomical distribution of these cells inside the tumors: are the cells preferentially located in the parenchyma surrounding the HCC? Or is the distribution homogeneous accordingly to the HCC size? We are now developing a Matlab scripts to quantify the location of the injected cells in different tumor masses. Moreover, we are wondering whether the tumor specific lymphocytes recognize the antigen remaining in the sinusoidal wall or are they recognize the antigen being in the parenchyma? Finally, since human HCC mostly develops in a chronic liver disease, like HBV infection and cirrhosis, and these conditions are characterized by a strong inflammatory and suppressive response, we will also address the role of the innate cells, such as NK and NKT cells in exerting anti tumor activity. We will also address the mechanism of hepatocellular priming by T naïve cells in HCC context, in order to understand how these cells recognize tumor antigens, they get activated and deploy their functions towards tumor antigen recognition. In particular, Benechet et al. recently demonstrated that naïve CD8+ T cells primed by hepatocytes do not become full functional effector CD8+ T cells and these cells show the lack of IL-2 up-regulation. This dysfunctional state can be rescued by IL-2 treatment in a Kupffer cell dependent fashion (Nature, in press). For future experiments, we will address whether the administration of IL-2 can have a role in the antigen recognition of T cells in HCC context.
Our study can understand how the immune system control the HCC growth, where and how it fails to respond, opening new possible therapeutic approaches in a disease that has a poor survival rate. Our study can determine new diagnostic markers that could be used by clinicians to diagnose HCC in the early phase of the disease where could be a therapeutic window.