Development of inCVAX, In situ Cancer Vaccine, and Its Immune Response in Mice with Hepatocellular Cancer.

Manipulation of immune system toward the rejection of established cancers has become the standard of care in some patients. Here we propose the development of an in situ autologous cancer vaccine, inCVAX, for the treatment of hepatocellular cancer (HCC). inCVAX is based on the induction of local immunogenic cancer cell death combined with local dendritic cell stimulation by intratumoral injection of the immune-activator N-dihydro-galacto-chitosan (GC). In a first set of experiments, cellular and molecular studies were performed to investigate the effect of inCVAX on immune activation in a murine model of HCC that we previously developed. Once large tumors were formed in mice, the tumor is surgically exposed and a laser fiber was inserted into the center of an individual tumor mass. Using a 10 mm diffuser tip, laser irradiation of 1.5 W was applied to heat the tumor at different durations (6-10 min) to assess tolerability of photothermal application at different temperatures. The laser application was followed by immediate injection of GC, and each mouse received one laser treatment and one GC injection. ELISA was used to assess the level of cytokines; immunohistochemical staining was conducted to analyze the effect of inCVAX on immune cell tumor-filtration and expression of tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). Results indicate that survival correlated to thermal exposure. At lower temperatures the photothermal effect was sufficient to induce tumor necrosis, but without obvious complication to the mice, although at these temperatures the treatment didn't alter the level of TSAs and TAAs, so further optimization is suggested. Nevertheless, in response to the inCVAX treatment, cytotoxic cytokine IFN-γ was significantly increased, but suppressive cytokine TGF-β was dramatically reduced. Furthermore, inCVAX prompted tumor infiltration of CD3+, CD4+, and CD8+ T cells; but modulated macrophage subsets differently. In conclusion, while the protocol needs further optimization, it would appear that inCVAX for the treatment of HCC activates an immune response in tumor-bearing mice, which in turn may have potential for the treatment of HCC.