RESIDUAL DORMANT CANCER STEM CELL FOCI ARE RESPONSIBLE FOR TUMOR RELAPSE AFTER ANGIOGENIC METRONOMIC THERAPY IN HEPATOCELLULAR CARCINOMA XENOGRAFTS

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related death. Current available chemotherapeutic options are not curative due in part to their resistance to conventional therapies. We have generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2rγ null mice by injection of AFP- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. Low dose metronomic administration of cyclophosphamide (LDM-CTX) causes in this model complete regression of the tumor mass with a significant increase in survival (p<0.0001), and reduction in aberrant angiogenesis, IL-6 and TNFα (but not VEGF) levels, hyperproliferation, and alteration of normal liver parenchyma, compared to untreated animals. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA- phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, off-therapy mice developed rapidly tumor growth, which was still sensitive to LDM-CTX therapy. The capacity of developing hepatoshperes in vitro was drastically reduced upon LDM-CTX treatment, which resulted in selection of CD13+ cells, showing that these cells are particularly resistant to therapy. Co-treatment of the CD13-targeting drug bestatin with LDM-CTX resulted in a dramatic reduction in tumor burden. Therefore, our results demonstrate the therapeutic efficacy of administering LDM-CTX and targeting the residual CD13+ CSC-like population in these novel orthotopic HCC models, and strongly suggests that this therapy could be implemented in clinical trials.