Objective  To establish a green fluorescent protein (GFP) and firefly luciferase (Luc) double-labeled Epstein-Barr virus (EBV) infected B lymphoblastoid cell lines (B-LCL) and apply them to mouse models, then compare the advantages and disadvantages of models inoculated by intravenous (IV) or subcutaneous (SC).

  Methods  B lymphoblastoid cell lines double-tagged with GFP/Luc （B-LCL-GL ）were constructed through lentivirus transduction, puromycin intervention. Subcutaneous xenograft and hematogenous metastasis models were respectively established by subcutaneous or intravenous injection of B-LCL-GL cells at three concentrations in (NOD) /Prkdc^scid/IL-2Rγ^null(NPG) mice for in vivo bioluminescence imaging.

  Results  In the B-LCL-GL group, the ratio of the GFP-positive cell population was 92.5%, and the average luminescence intensity was as high as 4.80E+08 Photons/s, which was considerably higher than that of untreated B-LCLs. In the hematogenous metastasis models, tumor bioluminescence was initially located in the peritoneal area and then spread throughout the entire body between 7 and 28 days. In the subcutaneous xenograft models, strong central and weak peripheral tumor-related bioluminescence signal was detected on day 7 in the three groups, which then spread throughout the body on day 28 in the high-dose group. Taken together, there was no significant difference in tumor progression between the two routes of administration when using the same dose of B-LCL-GL cells. However, the survival analysis indicated that the IV injection group, in which all the mice ultimately died, had a shorter time frame for testing than that of the SC injection group, in which the mice survived until day 100 in the low-dose and medium-dose groups, thus allowing for long-term testing.

  Conclusions  GFP and Luc dual-positive B-LCLs were successfully established to generate hematogenous metastasis and subcutaneous xenograft models, which allow the monitoring of the location and size of lymphomas in vivo. It provide platform for the study of tumor characteristics and selecting anti-tumor drugs.