Joint Research Projects
Study In Malaysia
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Author/Institute
Introduction
According to the Ministry of Health and Welfare in Taiwan, oral cancer is the fifth cancer deaths in Taiwan. Oral cancers are mainly associated with the usage of tobacco, betel nuts and alcohol consumption. Other risk factors include poor oral hygiene, candidiasis, viral infection, and chronic oral mucosa trauma.
With the technologies of Single-cell RNA sequencing, tumor cell invasion and metastasis between different tumor cell groups can be detected in oral cancer carcinogenesis. The clinical mucosal specimens from normal oral mucosa, oral cancer and oral precancerous patients were collected and candida infection was detected. The differences in oral mucosa carcinogenesis and between candida infection or not were analyzed.
The total cells can be distinguished into 24 cell clusters, 6 cell clusters are cancer related cells, and 18 cell cluster are immune cell cluster. The gain or loss of the cell cluster, and gene expression difference can be observed in oral mucosa carcinogenesis, and between Candida infection. The above results can be utilized to investigate the carcinogenesis of oral tumors and subcluster cells concerning to Candida infection.
Methods
We obtained clinical oral mucosal specimens from normal oral mucosa, oral precancerous, and OSCC patients and candida infection were detected. There were 4 specimens obtained—candida infected and non-infected patients of OSCC; candida infected oral precancer; and normal oral mucosa tissue without candida infected.
Tissue dissociated with tissue specimens washed with PBS and mince into about 1-2 mm3 pieces. Treat with Collagenase type IV and dispase type II diluted in PRMI basal. The tissues were dissociated into single-cell suspension and collected through 40 µm filters. Single-Cell preparation with the 10x Genomics platform and the Single-Cell RNA sequencing were performed with NextSeq 2000. The Single-cell RNA analysis were reviewed and examined the differences in oral mucosa carcinogenesis and between candida infection or not.
Results
The total cells can be distinguished into 24 cell clusters, 6 cell clusters are cancer related cells, and 18 cell cluster are immune cell cluster. The gain or loss of the cell cluster, and gene expression difference can be observed in oral mucosa carcinogenesis, and between Candida infection. The MSigDB gene set database was utilized to perform Gene Set Variation Analysis (GSVA) and hallmark genes were sited to the target genes related to biological functions, and pathways with OSCC with and without candida infection. The cancer-related cell population Candida OSCC has significant differences in pathways including K-RAS Signaling Downregulation, Spermatogenesis, E2F targets, the pathway of Estrogen response late.
In the GSVA analysis, the K-ras signaling downregulation and E2F target pathways, and the expression of all pathway-related genes is shown in a dot plot. K-ras signal down-regulation is mainly due to gene down-regulation caused by K-ras activation while E2F targets represents genes encoding cell cycle related targets of E2F transcription factors. The results show several genes is overexpressed in the cancer group.
Discussion
The goal of this study is to examine the changes in the cell population during the malignant transformation from normal oral mucosa, oral precancerous lesions, to OSCC. The Myc targets K-RAS signaling and Hedgehog signaling are related to OSCC that are not infected by candida and oral normal mucosa tissue.
With the related mechanism of pro-inflammation cytokines. The difference between Candida and non-Candida OSCC were analyzed by GSVA to investigate the oncogenic pathway promoted by Candida. Angiogenesis, and epithelial mesenchymal transition (EMT), coagulation and other related pathways showed that Candida OSCC was significantly higher than non-Candida OSCC in immune cell clusters.
In the pathways related to cancer related cell clusters, the K-ras signaling downregulation pathway and E2F target pathway were detected among different cell clusters in each group. These over expression genes are related to metastasis, poor prognosis, or drug resistance. These genes are involved in the carcinogenesis of OSCC. The results can be utilized to investigate the carcinogenesis of oral tumors and subcluster cells concerning to Candida infection. These results can be utilized as treatment biomarker of precision medicine.