Spontaneous HNSCC Model and Therapy

The Bmi1^CreER; Rosa^tdTomato mice received drinking water with 4NQO for 16 weeks to allow HNSCC to develop, followed by normal drinking water for 6 weeks, to form a spontaneous model of HNSCC. The mice were randomly divided into groups at 22 weeks. Before sacrificing the mice, they were given tamoxifen to label Bmi1⁺ CSCs. For the treatment assay, Bmi1^CreER; Rosa^tdTomato mice were divided randomly into the indicated groups, and injected with the indicated ASO PVT1 (10 nM, Integrated Biotech Solutions Co., Ltd, Shanghai, China) and ASO NC over the whole mouse tongue by twice-weekly subcutaneous injection for 4 weeks. For combination therapy, mice were intraperitoneally injected with anti-PD1 (BioXcell, Lebanon, NH, USA, 200 μg/mouse twice per week). After mice were sacrificed, the cervical lymph nodes and tongues were removed, and the lesion surface areas were calculated. For histological investigation and immunostaining, longitudinally cut tongues (dorsal/ventral) and intact lymph nodes were fixed overnight in 4% paraformaldehyde and paraffin-embedded. 10 sections of 5 mm thick tissue blocks were cut, and they were then stained with hematoxylin and eosin (HE). The SCC number was counted and regions were measured [8 (link)]. The following criteria were used to grade the invasiveness of the HNSCC: showing signs of normal or epithelial dysplasia appearance (grade 1); distinct invasion, unclearness of the basement membrane, drop and diffuse infiltration into the superficial portion of the muscle layer (grade 2); loss of the basement membrane; extensive invasion into deep muscle layer (grade 3). To examine cervical lymph node metastasis of HNSCC, the sections of cervical lymph nodes were immunostained with anti-PCK antibodies.