The Therapeutic Effect Of Oncolytic Adenovirus SG655-mGMP On Cancer Stem Cells

Cancer Stem Cells (CSCs) are small amount of cells that have infinite capability of self-renewal and are able to differentiate and generate heterogenotic tumor cell mass. According tonumerous studies, it is showed that CSCs is the potential source of tumor recurrence andmetastasis, and therefore has become a critical subject in target therapies. However, CSCsgenerally stay in quiescent stage, and are insensitive to micro molecule target drugs; infectionefficiency of some regular virus vectors (such as lentiviral vector, retroviral vector) towardsCSCs keeps low, which led to insufficient effects among gene therapy programs. Thus, newtherapeutic methods are in great need, so that treatment effect toward CSCs could be raised.Recently, immunotherapy has achieved superb success in clinical treatment of tumor,which had been listed as one of six most worth-concern fields of science in Science.Nevertheless, antigen presentation of CSCs is weak and could not stimulate effective immuneresponses. Hence, combining with other therapies, immunotherapy needs to be establishedand focus on CSCs, in order to increase sensitivity of immunocyte.Oncolytic Adenovirus could infect the cells in stationary phase effectively, mediating theeffective expression of exogenous theoretical gene; meanwhile, Oncolytic Adenovirus has thecapability of lysine and killing tumor cells, and the released antigen after cell lysis couldinduce strong immunological reaction. P53protein could boost apoptosis and cell senescence,and activating the immunological reaction indirectly; Granulocyte-macrophage colony-stimulating factor (GM-CSF) could induce differentiation and maturation of phagocytic cellsand Dendritic Cell(DC), and increasing immunological reaction. Therefore, if we couldcombine the oncolytic adenovirus, P53and GM-CSF together, a new effective therapeuticregimen that aims at CSCs could be established hopefully.Based on the above-mentioned concerns, we would like to use oncolytic adenoviruscarrying with11R-P53(P53protein fused with11R peptide) and GM-CSF gene, structuringrecombination adenovirus to express P53and GM-CSF, and observing the treatment effects inhuman hepatic carcinoma stem cells in vitro model and mice teratomas stem cells in vivomodel.The primary method and results are as below:I. Construction and preparation for SG655-mGMP and relevant control virusStructuring the oncolytic adenovirus shuttle plasmid P74-Tp-mGM-CSF via molecularcloning technology. The plasmid P74-Tp-mGM-CSF was co-transfected with pPE3-11R-P53 into293cells to produce the new recombinant adenovirus by homologous recombination,wecall it SG655-mGMP PCR proved that the virus packing is correct. Western Blotting studyshowed that the virus could express the theoretical gene P53effectively; ELISA showed thatthe virus could express the mGM-CSF protein.II. Study of in vitro effect of SG655-mGMP against tumor stem cells1.11R peptide could increase the in vitro infection efficiency of adenovirus towardstumor stem cellsBoth fluorescence microscope and flow cytometry indicated that oncolytic adenoviruswith11R peptide could increase the efficiency of infection towards Hep3B-C and ECCG5.2. Adenovirus effectively mediates the expression of theoretical gene in tumor stem cellsAfter virus infecting Hep3B-C (MOI=10), Western Blotting proved that P53proteincould express inside cell; also, ELISA indicated that the expression of mGM-CSF could befound in extracellular supernatant, and increasing with multipe of infection.3. SG655-mGMP inhibits the growth of tumor stem cells effectivelyAfter virus infecting Hep3B-C (MOI=10), CCK-8results showed that the virus couldinhibit the growth of cells; the inhibition ratio of oncolytic adenovirus with11R was higherthan that without11R.4. SG655-mGMP promotes the apoptosis of tumor stem cells effectivelyAfter virus infecting Hep3B-C (MOI=10), Annexin FITC/PI showed that the virus couldinduce the apoptosis of Hep3B-C effectively (apoptosis rate12.5%), the apoptosis rateobviously is higher than that of the blank control (0.2%), wild-type adenovirus (6.0%) andoncolytic adenovirus without11R (7.7%).III. SG655-mGMP activates immunity, inhibiting the growth of transplanted tumor withteratoma stem cellsECCG5tumor xenografts were established by a s.c. inoculation of5×106cells into theright flanks of C57BL/6mice.1. Oncolytic adenovirus SG655-mGMP could inhibit the growth of transplantable tumoras compared to control virus2. HE stain showed the increase of ecthyma gangrenosa in tumor after treatment;immunohistochemistry results (AIF1antibody) indicated an increase of infiltrating immunecells in tumor.3. Flow cytometry test showed that, after SG655-mGMP treatment, macrophage and DCcells (GM-CSF worked) increased significantly in tumor; granulocyte and NK cells showed no change; T cells increased slightly (P53worked).The results dedicated that SG655-mGMP could infect tumor stem cells effectively, andmediate expression of exogenous theoretical gene P53and GM-CSF; in vitro, promoting theapoptosis of tumor stem cells, inhibiting cell growth; in vivo, inhibiting the growth of tumorstem cells in transplantable tumor, and activating theimmunological reaction effectively. The new strategy could be provided while targetingtumor stem cells treatment.