Therapies designed to target cancer stem cells (CSCs) in colorectal cancer (CRC) may improve treatment outcomes. based on GFP expression, and were traced by LUC (Physique ?(Figure2B).2B). The CXCR4+ cancer cells transduced with AAV-pCXCR4-LUC-RFP expressed both luciferase (LUC) and an RFP reporter. The transduced CXCR4+ cells (transduction efficiency of 85.5 6.5%) were purified by flow cytometry based on RFP expression, and were traced by LUC (Determine ?(Figure2C).2C). The Lgr5+/CXCR4+ cancer cells were generated by co-transduction with both AAVs. The transduced Lgr5+/CXCR4- cells, CXCR4+/Lgr5- cells, Lgr5+/CXCR4+ cells (transduction efficiency for double viruses was 72.2 6.1%) were purified by flow cytometry based on RFP and GFP co-expression, and were traced by LUC (Physique ?(Figure2D).2D). The purified Lgr5+/CXCR4- CRC cells appeared green in culture (Physique ?(Figure2E).2E). The purified CXCR4+/Lgr5- CRC cells appeared red in culture (Physique ?(Figure2F).2F). The purified Lgr5+/CXCR4+ CRC cells appeared yellow (both green Pluripotin and red) in culture (Physique ?(Figure2G).2G). Moreover, the mRNA levels of Lgr5 (Physique ?(Physique2H)2H) and CXCR4 (Physique ?(Physique2I)2I) confirmed the enrichment of Lgr5 and/or CXCR4 in these cells. Physique 2 Preparation of Lgr5+/CXCR4-, CXCR4+/Lgr5- and Pluripotin Lgr5+/CXCR4+ CRC cells Lgr5+/CXCR4+ cells generate the best cancer mass after s.c. transplantation Thus, the same number of control (unpurified, transduced with LUC), CXCR4+/Lgr5-, Lgr5+/CXCR4- and Lgr5+/CXCR4+ Caco-2 cells were s.c. implanted into NOD/SCID mice. We found that, compared to unsorted control cells, CXCR4+/Lgr5-, Lgr5+/CXCR4- and Lgr5+/CXCR4+ cells generated tumors with significantly increased mass 8 weeks after transplantation; likewise, the Lgr5+/CXCR4+ cells generated the best tumor mass among all, based on bioluminescence examination, shown by representative images (Physique ?(Figure3A),3A), and by quantification (Figure ?(Figure3B).3B). Next, we evaluated the survival of the mice that had received transplantation of unsorted control cells, CXCR4+/Lgr5-, Lgr5+/CXCR4- and Lgr5+/CXCR4+ cells. We found that the mice that received Lgr5+/CXCR4+ cells had the shortest survival (Physique ?(Physique3C3C). Physique 3 Lgr5+/CXCR4+ cells generate the best cancer mass after s.c Lgr5+/CXCR4+ cells generate more tumor spheres and and tracing of cells. GFP is usually a green fluorescent protein and RFP is usually a red fluorescent protein. The pLgr5 in the AAV-pLgr5-LUC-GFP plasmid and the pCXCR4 in the AAV-pCXCR4-LUC-RFP plasmid were prepared from a full-length human Lgr5 or CXCR4 promoter, respectively. The 5 and 3 homology regions for the Lgr5 promoter were 1.9 kb (between -1954 from human Lgr5 transcript start and -48 from human Lgr5 transcript start) and the 5 and 3 homology regions for the CXCR4 promoter were 2.6 kb (between C2760 from human CXCR4 transcript start and -85 from human CXCR4 transcript start). The pLgr5 and pCXCR4 were amplified by PCR with EcoRI-restriction-endonuclease-forward and NheI-restriction-endonuclease-reverse primers, using human genomic DNA as a template. The pLgr5 construct was then subcloned into the 50-EcoRI and 30-NheI sites of the pAAV-CMV-LUC-2A-GFP vector (Clontech, Mountain View, CA, USA) to replace the CMV promoter to generate pAAV-pLgr5-LUC-GFP. The pCXCR4 construct was then subcloned into the 50-EcoRI and 30-NheI sites of the pAAV-CMV-LUC-2A-RFP vector (Clontech, Mountain View, CA, USA) to replace the CMV promoter to generate pAAV-pCXCR4-LUC-RFP. Sequencing was performed to confirm the correct orientation of the prepared pAAV-pLgr5-LUC-GFP and pAAV-pCXCR4-LUC-RFP, which were then used to generate AAV, with a Pluripotin packaging plasmid carrying the serotype 6 rep and cap genes and a helper plasmid carrying the adenovirus helper functions (Applied Viromics, LLC. Fremont, CA, USA), using Lipofectamine 2000 reagent (Invitrogen). The control cells were transduced with AAV generated from pAAV-CMV-LUC-2A-GFP vector. The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single Rabbit polyclonal to APLP2 vector through a novel cleavage event within the 2A peptide sequence. The AAVs were purified using CsCl density centrifugation and then titration was decided by a quantitative densitometric dot-blot assay. For cell transduction by their expression of luciferase. Mouse manipulation Ten week-old male NOD/SCID mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China) were used for subcutaneous (s.c.) transplantation of tumor cells Pluripotin and serial adoptive transfer. Bioluminescence was monitored 4 weeks after s.c. transplantation. For s.c. transplantation of cancer cells into NOD/SCID mice, 500 cancer cells were implanted s.c. and Pluripotin tumor formation was examined after 8 weeks by bioluminescence. For serial adoptive transplantation of cancer.