Purpose: To develop a new interventional oncologic technique, namely “image-guided intratumoral radiofrequency hyperthermia (RFH)-enhanced local Herpes Simplex Virus Thymidine Kinase (HSV-TK)/ganciclovir (GCV)-mediated suicide gene therapy of lung cancers” and investigate its associated bio-molecular mechanisms.
Materials and Methods: Human lung cancer cells (A549) transduced with Luciferase/mCherry/lentivirus for in-vitro confirmation, and 24 nude rats with the same orthotopic lung cancers for in-vivo validation were divided into four study groups with different treatments of (i) combination therapy with intratumoral HSV-TK/GCV gene therapy followed by RFH at 41-42 ℃ for 30 minutes; (ii) HSV-TK/GCV gene therapy alone;(iii) RFH alone; (iv) phosphate-buffered solution (PBS) as a control. In in-vitro experiments, bioluminescence assay, confocal microscopy and flow cytometry were used to determine the viability and apoptosis of cells, while in the in-vivo experiments molecular optical/x-ray imaging was used to evaluate the changes of bioluminescent signals among different groups over 2 weeks. To investigate the potential mechanisms of apoptosis, immunohistochemistical (IHC) staining and western blotting (WB) were used for detecting the expression of Bcl-2/Bax, and Caspase-3. To determine mechanisms of immune-response, IHC and WB were used to exam the expression of HSP-70, IL-2 and CD94.
Results: Of in-vitro experiments, compared with gene therapy alone, RFH alone or PBS, combination therapy induced the lowest cell viability (30.56±9.12% vs 65.47±8.02% vs 89.95±6.33% vs 100%, P<0.01), the highest cell apoptosis (20.05±2.12% vs 9.87±2.02% vs 4.31±4.31% vs 3.12±1.03%, P<0.001), and a significant decrease of relative bioluminescence signal (16.54±0.12% vs 54.8±0.10% vs 85.01±0.08% vs 100%, P<0.01). Of in-vivo experiments, optical imaging detected a significantly decreased bioluminescence signal of the tumor with combination therapy (1.29±0.18 vs 1.76±0.13 vs 2.26±0.17 vs 2.94±0.15, P<0.05). Regarding to the mechanisms, both WB analysis and IHC staining displayed the significantly decreased expression of Bcl-2, as well as increased expression of Bax, Caspase-3, HSP-70, IL-2 and CD94 in cancer tissues of combination therapy, compare to other control treatments.
Conclusion: This study validated the feasibility of image-guided interventional RFH-enhanced direct suicide gene therapy of orthotopic lung cancers, which is activated through the mechanisms of augmenting Bax/Bcl-2/caspase-3-depended apoptosis and the HSP-70/IL-2 depended immune regulation pathway. This alternative technique may open new avenues for effective treatment of lung cancers via integrating image-guided interventional oncology, RF technology, and direct gene therapy.