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Introduction: Current literature suggests that immunologic tolerance plays a major role in allowing hepatic tumor growth. However, the immune system is dynamic and its response to tumor antigens may very well be different at various stages of tumor growth. We have developed a reliable model of spontaneous HCC in which tumor progression can be accurately followed with MRI even at very small sizes. Using this model, we evaluated tumor specific immune responses at different stages of tumor growth.
Methods: C57BL/6 mice were inoculated with syngeneic HCC cells expressing Tag of the SV40 virus via splenic injections (ISPL). Tumor growth was evaluated with contrast enhanced magnetic resonance imaging (MRI) after ISPL. Basal immune responses were evaluated at 9, 14, 21, 28, 42 and 56 days after ISPL. Animals were vaccinated with WT-19 (cell line expressing Tag) at three time points 14, 28, and 56 days after ISPL. Tetramer analysis was used to quantify Tag-specific CD8+ T cell proliferation and T-cell function was assessed through Tag-specific γ-interferon production. Autopsies were performed for macroscopic evaluation of tumor. Immunohistochemistry (IHC) was used to confirm Tag expression in the tumor foci.
Results: All animals injected ISPL developed HCC. These tumors were first detectable by 28 days after injection with MRI. MRI performed at 14 days did not reveal evidence of tumor foci. At 28 days, MRI demonstrated small tumor foci, < 1mm. At 56 days, MRI demonstrated tumor foci, 7-10 mm in size. At all time points there was no basal tumor specific CD8+ T cell proliferation or activation. At 14 and 28 days after injection there was a robust tumor specific CD8+ T cell proliferation and activation after vaccination. At 56 days after injection there was no tumor specific CD8+ T cell proliferation or activation after vaccination. Necropsies confirmed findings of MRI examinations. IHC showed T ag expression in tumor foci.
Conclusion: Otherwise immunogenic tumor cells when delivered to the liver will grow. When this occurs, T cells are present and remain capable of producing a robust immune response when presented with the same tumor antigen. This response however, is not evident in animals with larger tumors. These results suggest that immunological ignorance is responsible for allowing tumor growth during early stages of tumorigenesis. Immunological tolerance appears to be important when tumors grow to a certain size. The exact size of tumor and mechanism responsible for this phenomenon is yet to be fully characterized.

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