Oral tongue squamous cell carcinoma (OTSCC) is one of the major causes of fatality in India owing to very high percentages of patients with smoking and chewing habits. Being highly heterogeneous in nature, every patient poses a different challenge clinically. To better understand disease progression, knowledge of cross talk between tumor stroma and the tumor cells becomes indispensable. Patient-derived in vitro cell line models are helpful to understand the complexity of diseases. However, they have very low efficiency of establishment from the tumor samples, especially the cancer associated fibroblasts (CAFs). In the present study, two novel autologous pairs have been immortalized spontaneously from non-habitual, HPV-positive patients, presented with tongue squamous cell carcinoma. The epithelial and fibroblast primary lines had typical polygonal and spindle shaped morphology, respectively. Positive staining with Pan-cytokeratin (PanCK) and Fibroblast Specific Protein (FSP-1) further confirmed their epithelial and fibroblast origin. Unique Short Tandem Repeat (STR) profile of the cultures confirmed their novelty, while the similarity of the STR profiles between the epithelial and fibroblast cells from the same patient, confirmed their autologous nature. DNA analysis revealed aneuploidy of the established cultures. Increase in the tumorigenic potential of the established epithelial cultures upon treatment with CAF-conditioned medium proved the CAF-ness of the established fibroblast cells. The established cultures are the first of their kind which would serve as an useful platform in understanding the cross talk between tumor-stroma and tumor, along with studying tongue cancer progression.
Background Quantitative real time PCR (qPCR) remains by far the most cost-effective, fast yet sensitive technique to check the gene expression levels in various systems. The traditionally used reference genes over the years were found to be regulated heavily based on sample sources and/or experimental conditions. This paper therefore presents a data science driven -omic approach for selection of reference genes from ~60,000 candidates from The Cancer Genome Atlas (TCGA) and Broad Institute Cancer Cell Line Encyclopaedia (CCLE) for gene expression studies in head and neck squamous cell carcinoma (HNSCC). mRNA-sequencing data of 500 patient samples and 33 cell lines from publicly available databases were analysed to assess stability of genes in terms of multiple statistical measures. A final set of 12 candidate genes were studied in the Indian set of data in Gene Expression Omnibus (GEO) and validated experimentally using qPCR in 35 different types of samples from platforms like drug sensitive and resistant cell lines, normal and tumor samples, fibroblast and epithelial primary culture derived from HNSCC patients from India. Result The study lead to the choice of five most stable reference genes –TYW5, RIC8B, PLEKHA3, CEP57L1 and GPR89B across three experimental platforms. Conclusion In addition to providing a set of five most stable reference genes for future gene expression analysis experiments across different types of samples in HNSCC, the study also presents an objective framework for assessing reference genes for other disease areas as well.
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