Single-Cell Analysis of Different Stages of Oral Cancer Carcinogenesis in a Mouse Model.

Ling-Yu Huang,Shih Sheng Jiang,Ko-Jiunn Liu,Wei-Fan Chiang,Yi-Ping Hsieh,Tze-Ta Huang,Wen-Tsung Huang,Daw-Yang Hwang,Yen-Yun Wang

doi:10.3390/ijms21218171

Abstract

Oral carcinogenesis involves the progression of the normal mucosa into potentially malignant disorders and finally into cancer. Tumors are heterogeneous, with different clusters of cells expressing different genes and exhibiting different behaviors. 4-nitroquinoline 1-oxide (4-NQO) and arecoline were used to induce oral cancer in mice, and the main factors for gene expression influencing carcinogenesis were identified through single-cell RNA sequencing analysis. Male C57BL/6J mice were divided into two groups: a control group (receiving normal drinking water) and treatment group (receiving drinking water containing 4-NQO (200 mg/L) and arecoline (500 mg/L)) to induce the malignant development of oral cancer. Mice were sacrificed at 8, 16, 20, and 29 weeks. Except for mice sacrificed at 8 weeks, all mice were treated for 16 weeks and then either sacrificed or given normal drinking water for the remaining weeks. Tongue lesions were excised, and all cells obtained from mice in the 29- and 16-week treatment groups were clustered into 17 groups by using the Louvain algorithm. Cells in subtypes 7 (stem cells) and 9 (keratinocytes) were analyzed through gene set enrichment analysis. Results indicated that their genes were associated with the MYC_targets_v1 pathway, and this finding was confirmed by the presence of cisplatin-resistant nasopharyngeal carcinoma cell lines. These cell subtype biomarkers can be applied for the detection of patients with precancerous lesions, the identification of high-risk populations, and as a treatment target.

Highlights

Oral cancer is currently one of the most common cancers worldwide, and its incidence and mortality are increasing annually [1]
4-nitroquinoline 1-oxide (4-NQO) (200 μg/mL) and arecoline (500 μg/mL) were given to mice in their drinking water every day to simulate the carcinogenic effects of cigarettes and betel nut in transforming healthy oral mucosa cells into precancerous lesions and eventually malignant lesions (Figure 1A)
The results indicated the poor differentiation of squamous cell carcinoma at 16 weeks and advanced squamous cell carcinoma at 29 weeks

Summary

Introduction

Oral cancer is currently one of the most common cancers worldwide, and its incidence and mortality are increasing annually [1]. Oral cancer is mainly treated through surgical resection, whereas radiotherapy, chemotherapy, and immunotherapy are used as postoperative adjunctive therapy for patients with more advanced cancer [3]. These treatments have limited therapeutic effects on patients with advanced cancer. Identifying key factors for tumor cell subtypes that predict the progression of precancerous lesions into cancer is essential for developing treatment for preventing the rapid carcinogenesis of tumor cells. The gene expression of an important subtype of cancer cells can be overlooked when differences in the expression of all cells are being averaged. Single-cell analysis can be used to distinguish between genetic and nongenetic mechanisms, identify characteristics of the tumor microenvironment and cell interactions, determine the likelihood of disease recurrence, identify minimal residual disease and rare tumor cell subpopulations, explore cell sources and influence pathways, and identify cell subpopulations that significantly affect disease progression [6]

Methods

Results

Conclusion