281 INTRAHEPATIC ULTRASOUND-MEDIATED GENE DELIVERY

Quiescence is a strategy that cancer stem cells (CSCs) adopt in stressful environments such are those triggered by antineoplastic treatments. CSCs form micrometastases that stay dormant for variable time periods and regain proliferation after unknown stimuli. cMYC induces pluripotency in differentiated cells and may have a role in dormant cells activation. Aim: To analyze cMYC role in the switch from dormant (generated by HCC line BCLC5) to growing tumors formed by BCLC5 cells transfected with cMYC (BCLC5-cMYC), compared to BCLC9 already expressing cMYC. Methods: Creation of a stable BCLC5 cell line expressing cMYC by transfection of pCMV6-cMYC, selection of cells expressing cMYC was done by G418 treatment. cMYC levels were confirmed by PCR/Western Blot (WB). Subcutaneous (s.c.) injection of cells into SCID mice: 10 BCLC9, BCLC5 and BCLC5-cMYC cells were s.c. injected. Stable cell lines derived from xenograft tumors: eight lines (t1 to t8) were derived from a BCLC5-cMYC tumor and 3 cell lines (c1, c2, c4) from tumors produced by injection of BCLC5-cMYC t7 cells. Gene expression of proangiogenic genes (VEGFA, CD13), epithelial– mesenchymal markers (CDH1, VIM), invasion markers (NM23H1, cMET, ITGB1, MMP2, MMP9) and genes associated with cell pluripotency (OCT4, SOX2, NANOG, CD133, EPCAM) were assessed by real-time PCR, immunohistochemistry and WB. Results: BCLC9 cells produced tumors in SCID mice while BCLC5 cells developed viable tumors <1–2mm in size that remained stable after 6 months of injection. BCLC5-cMYC cells generated tumors with high proliferation and cell lines showed increased expression of VEGFA, CD13 and CDH1 and decreased VIM, cMET and MMP2 compared to BCLC5. Tumor derived cell lines keep expressing pluripotency related genes and acquire EPCAM’s, which was originally expressed only by BCLC9. Second generation of tumors BCLC5-cMYC and their cell lines showed increased expression of proangiogenic markers, genes associated to epithelial phenotype and those related to CSC while markers associated with invasive behavior decreased. Conclusion: cMYC activation induces angiogenesis and epithelial markers while preserving pluripotent phenotype and reducing invasive potential. These changes consolidate along tumor evolution explaining the aggressive behavior of residual disease after antineoplastic treatment.