Interestingly, the corresponding histological and immunohistochemical results showed that tumors derived from spheres exhibited a loss of E-cadherin and upregulation of fibronectin, appeared to be more aggressive, and had a mesenchymal-like appearance compared with tumors derived from parental cells. As mentioned earlier, the enriched spheres cultured from OSCC cell lines via a nonadhesive culture system may initially become suspended and detached from the parental cells, and form small clusters. Such spheres grown in a nonadhesive condition subsequently exhibit reduced cell–cell or cell–matrix interactions, lose their anchorage, and became homeless. This triggers a phenomenon called ‘‘anoikis,’’ presumably resulting in apoptotic response. Floating spheres in a state of anoikis in the culture medium are isolated and, although they attempt to adhere, are unable to attach to the underlying or surrounding plate which are expected to vanish in the end. How can these cancer cells survive and proliferate to overcome the threat of anoikis? What mechanism is involved in the acquisition of survival signals that offer the ability to survive and proliferate in a floating tumor population that lacks the normal solid-phase scaffolding, which constitutes a challenged microenvironment? Several studies have suggested that the adversity met by spheres in a nonadhesive, suspended condition can be stimulated by EMT and also encourage the enrollment of the potential of CSC properties. The literature also reveals that some signaling pathways mediate EMT and CSC properties, such as WNT, Sonic hedgehog, Snail/Slug, and NOTCH. There is increasing evidence suggesting that a link exists between EMT and CSCs that involves cell morphology alteration and motility. These concepts explain why our nonadhesive culture system can be used to enrich CSCs from cancer cell lines. In conclusion, using a modified nonadhesive culture system and a subsequent series of experiments, we not only validated the CSC properties of spheres isolated from OSCC cell lines, but also successfully established a rapid and economic method that can provide new insights and a newly applicable model for CSC research. Baculovirus expression system is one of the most ideal systems for routine production of recombinant eukaryotic proteins in insect cells, larvae and mammalian cells, which is widely-used in developing virus-like particles vaccine, displaying heterologous peptides or proteins, and transducing genes into mammalian cells. Compared with the AcMNPVSf9 system, the BmNPV-silkworm system provides enhanced expression level and pretty low cost in silkworm larvae or pupae, which shows promising industrialization future. Moreover, recent study has found that the N-acetyl glucosamine and galactose residues also exist in the N-glycan structures produced by silkworms, indicating silkworm larvae might be a useful host for producing human glycoproteins. Until today, great efforts have been made for efficiently constructing recombinant BmNPV, including the BmNPV-based Bac-to-Bac system, the mating-assisted genetically integrated cloning method and a method based on zerobackground Tn7-mediated transposition in E. coli. Other improvements relating to the baculovirus expression system also have been presented, such as utilizing cysteine protease and chitinase-deficient Bacmid to improve recombinant protein production and keep its stability, as well as a transfectantfree method by directly infecting insect cells or injecting silkworm larva with invasive E. coli containing recombinant Bacmid.