Aim: A large majority (~70%) of Nasopharyngeal Carcinoma (NPC) patients present with locally advanced disease at diagnosis carry a high risk of recurrence (~30%) following definitive cisplatin based chemoradiotherapy (CRT). Our locally endemic nasopharyngeal cancer (NPC) is ubiquitously associated with the Epstein Barr Virus (EBV) infection, and the presence of detectable post-treatment circulating EBV-DNA is well-documented to represent minimal residue disease (MRD), which portends a high risk of relapse following definitive treatment and thus a worse prognosis. Therefore, we aim to investigate if “off the shelf” Natural Killer (NK) cells can complement cisplatin chemotherapy in eradicating NPC in both in-vitro and in-vivo NPC models.
Methods: NK cells are expanded from PBMCs from healthy donors using an IL-2 expansion protocol, with iK562 cells as feeder cells. These expanded NK cells are characterized on flow cytometry to determine the NK cell composition and phenotype (CD3, CD4, CD8, CD16, CD56 and CD57). In addition, the status and amount of the residual iK562 cells in the expanded NK cells culture will be determined based on the presence of surface markers CD33, CD44, CD45, CD65, CD146 and SSEA-4. NK cell mediated cytotoxicity of NPC cells (C666-1) will also be determined to ensure the expanded NK cells’ functionality.
We have established a cell line derived NPC model using NOD/MrkBomTac-PrkdcSCID mice (4-6 weeks, male). In brief, 3x106 C666-1 cells suspended with Matrigel were grafted subcutaneously onto the right flank of the mice. After 2-3 weeks, 100-200 mm2 NPC tumour size was obtained. “Off the shelf” NK cells were purified from peripheral blood mononuclear cells (PBMC) of healthy donor. For each mouse, 0.5x106 activated NK cells (CD3- CD56+ CD16+) were injected intravenously three days before the first dose of cisplatin (5 mg/kg) treatment. A total of 3 cycles of NK cell treatment with cisplatin were administered.
Results: “Off the shelf” allogeneic NK cells demonstrate considerable cytotoxicity (77.6 ± 4.93%) against C-666 NPC cells at an effector to Target ratio of 5:1. Using our NPC rodent model, NK cells treatment alone significantly suppressed NPC growth from 85.7 ± 9.42 to 48.1 ± 8.47 mm3/day (p=0.0032). Notably, NK cell treatment suppressed tumor growth more effectively when the tumors are small (first 11 days) (34.2 ± 4.48 mm3/day) compared to larger tumors (next 11 days) (60.7 ± 12.69 mm3/day). NK cell treatment plus cisplatin treated tumors also demonstrated greater tumor suppression than cisplatin treated tumors ( mean tumor reduction of 15.2±1.97 mm3/day versus 31.7±2.54 mm3/day) (p=0.0014). This additive anti-NPC effect of combining NK cell treatment with cisplatin was not observed in larger tumors ( next 11 days).
Conclusion: “Off the shelf” allogeneic NK cells demonstrate anti-NPC effects in both in-vitro and in-vivo NPC preclinical models. Combining NK cells treatment with cisplatin chemotherapy was more effective during the early stage of tumor growth, rather than in larger established tumors. This observation opens up the rationale using NK cell treatment to eradicate MRD following definitive CRT in advanced NPC patients in order to reduce the risk of future NPC relapse.