Tet-On regulating HSV-sr39tk suicide gene expressing mesenchymal stem cells with dual reporter system exert bystander effect on anaplastic thyroid cancer

Objectives Anaplastic thyroid cancer (ATC) is the most aggressive malignancy of thyroid gland and is the cause of death up to 40% from thyroid cancer by poor response to conventional therapies. Therefore, new therapeutic modality is urgently need for the cancer and gene-directed enzyme/prodrug therapy mediated by genetically engineered mesenchymal stromal cells (MSC) might be one of promising therapeutic modalities. Aim of the study was to develop therapeutic MSC having suicidal gene which can be induced by an artificial cue and to verify the therapeutic gene expression by the cue in vitro and in vivo models using optical molecular imaging. In addition, therapeutic effect of the MSC to the ATC was also assessed.

Methods We first designed the Tet on system in retroviral vector fused Herpes simplex virus thymidine kinase (HSV-sr-39tk) with dual reporters (GFP-Fluc2). Then mouse bone marrow derived MSC was transduced retrovirally. The transduced cells were screened and characterized. The engineered MSC were co-cultured with CAL-62 ATC cells in the presence of prodrug ganciclovir after stimulation with doxycycline. The transduced MSCs (MSC-Tet-TK/Fluc2) was injected into the ATC xenograft of nude mice and 1mg/kg of doxycycline (DOX) and ganciclovir (GCV) were IV injected. MSC-Tet-TK/Fluc2 was visualized by bioluminescence imaging (BLI). The treatment efficiency was periodically evaluated using the Rluc activity from Cal62/Rluc by BLI.

Results MSC-Tet-TK/Fluc2 was established and demonstrated enhanced therapeutic effect to Cal62/Rluc cells by DOX and GCV treatment. In vivo BLI successfully revealed expression of Tet-TK-Fluc2 in the transduced MSC cells in vitro and in vivo models. In addition, intratumoral MSC-Tet-TK/Fluc2 injection coupled with DOX and GCV administrations significantly decreased the tumor growth compared to without DOX as well as control tumor having naïve MSC injection.

Conclusions We successfully developed the MSC having TetOn gene-directed enzyme/prodrug (TK/GCV) therapeutic system and verified its therapeutic effect for ATC tumor both in vitro and in vivo models. The innovative therapeutic approach using the TetOn system can reduce adverse effects by artificial controlling of the suicidal TK gene expression by DOX.