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

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.

Comprehensive Integrated Single-Cell Whole Transcriptome Analysis Revealed the p-EMT Tumor Cells—CAFs Communication in Oral Squamous Cell Carcinoma

“…In the total cell sample of OSCC tumors, we identified 15 main cell types that were confirmed previously in both human and mouse samples [ 1 , 5 ]. The main populations were composed of immune cells and non-immune cells, including cancer-associated cells that were similar to HNSCC data [ 12 , 18 ].…”

“…This leads to difficulties in prognosis and in the development of risk-stratified cancer screening, precision screening, and treatment strategies. Intra-tumoral heterogeneity represents a major challenge in oncology [ 5 ]. Among emerging technologies, single-cell RNA sequencing (scRNA-seq) has facilitated the identification of developmental hierarchies, drug resistance programs, and patterns of immune infiltration relevant to tumor biology, diagnosis, and therapy [ 6 ].…”

Characterization of normal and cancer stem-like cell populations in murine lingual epithelial organoids using single-cell RNA sequencing

“…To improve the treatment of heterogeneous malignancies, such as tongue carcinoma, tumor cells—particularly cell populations with stem cell properties—must be characterized at the single-cell level. To the best of our knowledge, there has been only one study on single-cell RNA-sequence analysis of 4-NQO-induced tongue cancer tissue in mice 13 . The authors reported that the most significantly enriched pathway in cancer-related clusters was the Myc targets regulatory pathway.…”

New Insights Into Oral Squamous Cell Carcinoma: From Clinical Aspects to Molecular Tumorigenesis

“…Accordingly, exploring the intricate immune network regulating tumor growth is a critical task in cancer management. By using a high-throughput sequencing platform, the single-cell RNA-seq (scRNA-seq) technique can be a powerful tool for analyzing genomics, epigenomics, and transcriptomics at cellular resolution [ 138 , 139 ]. This rapidly evolving technique can lead to innovative discoveries in tumor biology, immuno-oncology, and therapeutic resistance.…”

“…This rapidly evolving technique can lead to innovative discoveries in tumor biology, immuno-oncology, and therapeutic resistance. In a recent study examining our previously established oral SCC mouse model, the scRNA-seq technique demonstrated robust analytic value in determining the oncogenic signaling pathway and therapy resistance [ 139 ]. Enrichment analysis of gene expression profiling revealed that the MYC signaling pathway is predominant in clusters of stem cells and keratinocytes.…”