A New Listeria Vaccine Against Hepatocellular Stem Cells

BackgroundHepatocellular carcinoma(HCC) is one of the most common cancer in China and often leads to poor prognosis. Current therapies for HCC including radiotherapy, chemotherapy and surgery. All the therapies did not decrease the risk of chemoresistance and metastasis of HCC. The new concept of cancer stem cells(CSCs) was put forward to elucidate the mechanism of HCC chemoresistance, metastasis and recurrence and brought a novel light for cacner immunotherapy. Attenuated Listeria monocytogene, which could effectively stimulate immune response and was safe after intravenous injection, was an eligible candidate vector to deliver tumor associated antigen. In this study, we constructed hepatic cancer stem cell vaccine Lmdd-CD24to elicit anti-tumor response and evaluate its safety and effectiveness in tumor-beared mice. Lmdd-CD24could be potentially applied in clinical practice.Methods1) Hepatic cancer stem cell biomarker CD24was chosen as a eligible tumor associated antigen to prevent HCC recurrence and metastasis2) CD24reduced apoptosis and enhanced migration of human HCC cells in vitro. After over-expression of on human HCC cell line HepG2, those cells were collected and stained with PI and Annexin V for flow cytometry analysis. By western blot, increased levels of cleaved caspase-3and caspase-9were observed. In the wound-healing test, HepG2-CD24cells displayed a higher migration rate.3) Recombinant Listeria strains secreting CD24proteins were constructed and proved to express CD24stably and effectively.4) By intravenous injection in mice, Lmdd-CD24vaccine was rather safe and mainly distributed in spleen. After phagocytosis by macrophages, the bacterium was rapidly cleared in three days and did not cause obvious inflammation and organ injuries.5) Lmdd-CD24could induce Thl and Th2cell-mediated immune responses by measuring the level of IFN-yand the number of IFN-yproducing CD8+T lymphocytes and IL-4,IL-10producing lymphocytes.6) Lmdd-CD24vaccine can cause regression of inoculated Hepal-6-CD24tumors in mice.7) A decreased level of Tregs in immunized mice were observed by flow cytometry and immunohistochemistry.8) ELISPOT and LDH-releasing assays showed that CD8+T lymphocytes in TILs could target the epitopes specifically.ResultsCD24, as a heptic cancer stem cell biomarker, could reduce the apoptosis and enhance the migration of HCC cells. A reliable Lmdd-CD24vaccine was constructed by genetic engineering. After intravenous injection, Lmdd-CD24were mainly distributed in spleen and liver and did not cause serious organ injuries. This vaccine could effectively elicit Thl-and Th2-cell mediated immuine response and decrease the level of Tregs. As well, this bacterium could cause the regression of inoculated Hepal-6-CD24cells.ConclusionThe promising attenuated Lmdd-CD24vaccine was safe and cause a specific anti-tumor immunity. It demonstrated a feasibility for potential clinical application.