Background: The control of lymph node metastasis (LNM) is one of the most important prognostic factors in head and neck squamous cell carcinoma (HNSCC) treatment. In clinical N0 cases, however, it is difficult to decide whether we should select elective neck dissection or watchful waiting even at present. In this situation, a novel less invasive treatment might allow head and neck surgeons to reduce the incidence of postoperative morbidities and improve the survival of HNSCC patients. We recently presented the successful gene transfer into HNSCC cells and anti-tumor effect by recombinant Sendai virus vector (rSeV) encoding a therapeutic gene (Tanaka et al, Gene Therapy, 2015), and oncolytic virus therapy against anaplastic thyroid cancer and HNSCC cells using rSeV that shows uPA-specific cell-killing activity via cell-cell fusion, which is named “BioKnife” (Miyagawa et al, 2014. Tanaka et al, 2015). The therapeutic use of rSeV is thought to be safer than that of DNA viruses, because it’s not pathogenic in humans and the RNA genome of rSeV does not go through a DNA phase. rSeV mediated sentinel lymph node (SLN) targeted gene therapy is expected to be safe and have a potential in protection of metastasis and suppression of micrometastasis in cN0 cases, enhancing the possibilities of its application in clinical settings. As a preliminary study of rSeV encoding a therapeutic gene in a micrometastasis animal model, we examined Sendai virus vector migration into SLN and expression of its gene in a HNSCC mouse model.
Method: We investigated Sendai virus vector migration into SLN and expression of its reporter gene in SLN after injection of the vector into primary tumor. rSeV encoding GFP reporter gene (rSeV-GFP) was injected into tongue tumor in an orthotopic nude mouse model of human highly metastatic tongue squamous cell carcinoma (HSC-3-M3). To demonstrate the vector migration into SLN, we performed real-time PCR using rSeV primer. To demonstrate the evidence of GFP expression in SLN, frozen sectioned SLN was stained with anti-GFP antibody, and real-time PCR was performed using GFP primer. Fluorescence intensity was measured using Image J.
Results: Figure.1&2 are representative confocal laser microscopic images of SLN of control group and rSeV-GFP group respectively (immunohistochemistry (IHC) of DAPI & anti-GFP antibody, x40). Figure.1 shows little GFP expression compared to Figure.2. Peripheral regions of SLN have a tendency to show a stronger GFP expression than the central region. In IHC, fluorescence intensity of rSeV-GFP group was significantly higher than that of control group in both tongue and SLN (p < 0.05). In real-time PCR, the relative mRNA expressions of rSeV and GFP were significantly increased in rSeV-GFP group when compared to control group in both tongue and SLN (p < 0.05).
Conclusions: In this study, we revealed that rSeV gene was able to be transferred into SLN in an orthotopic nude mouse model of HNSCC. These results show that rSeV has a potential to be a novel safe and less invasive option for LNM treatment in HNSCC using rSeV encoding a therapeutic gene or an oncolytic virus (BioKnife).