Abstract
The development of esophageal cancer accompanied by the presence of human papillomavirus (HPV) DNA into the host genome. By evaluating the expression of this virus for tumor cell origin and also their cell grows and migrations, we examined esophageal cancer clonality in the context of intra-tumor heterogeneity. In this research, we have checked the expression of HPV18 E6 and E7 in different single cell clones by the manual cell picking method in the HPV positive esophageal cancer (EC109), EC109 cell line used as a negative control, and Hela cell line used as the positive control. Quantitative real-time PCR (QRT-PCR) was run to detect the expression levels of HPV E6 and E7, Cell Counting Kit-8 (CCK-8) assay was used to examine cell proliferation, invasion assays performed using Costar chambers and wounding assay to study cell migrations in vitro. We investigated the intra-tumor heterogeneity of HPV E6 and E7 in esophageal cancer and the evaluation of the growth and migrations at the clonal level, using 10 single cell clones. In particular clones, C7 & C10 displayed a highly variable expression in both HPV E6 and E7 and weak in four clones (C1, C3, C4, and C9) consequently, the cell invasion, proliferation, and migration increase with increasing the level of HPV expression and inverse. In conclusion, the resulting based on single cell cloning showed the relationship between HPV and cell growth and migration in esophageal cancer. Future study in HPV DNA integration needed to explore the mains specific integration site of HPV DNA in esophageal cancer and molecular monitoring of the HPV for future prevention researches and also effective therapeutic strategies.
Highlights
The population of cancer cells have detected in different researches and explained heterogeneity in term of tumorigenicity, mutations, activation of metabolic and signaling pathway, grows and migrations, different ploidy, metastasis, alternation of copy number, and responding to the anticancer agent [1] [2] [3]
We have checked the expression of HPV18 E6 and E7 in different single cell clones by the manual cell picking method in the human papillomavirus (HPV) positive esophageal cancer (EC109), EC109 cell line used as a negative control, and Hela cell line used as the positive control
To analyze the expression of HPV18 E6 and E7 at the clonal level, 10 single cell clones were obtained from a primary culture of the human esophageal cancer (EC109)
Summary
The population of cancer cells have detected in different researches and explained heterogeneity in term of tumorigenicity, mutations, activation of metabolic and signaling pathway, grows and migrations, different ploidy, metastasis, alternation of copy number, and responding to the anticancer agent [1] [2] [3]. Tumor heterogeneity has observed in different kind of cancers like esophageal cell carcinoma [8]. Studies detected the HPV DNA in esophageal cancer base on intra-heterogeneity. The integration of HPV DNA may result in esophageal cancer intra-heterogeneity. Following, rising HPV DNA integrity results in increasing HPV DNA expressions and cell grows and proliferation [11]. In this research we are going to proof intra-tumour heterogeneity in esophageal cancer by detecting HPV E6 and E7 genes in 10 different single clones of esophageal cancer cells and clonal expansion by evaluating proliferation as well as invasions and migrations of each single clone to better understand HPV E6 and E7 roles in ESCC intra-heterogeneity and disease monitoring
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