Establishment, molecular and biological characterization of HCB-514: a novel human cervical cancer cell line

Cervical cancer is the fourth most common cancer in women. Although cure rates are high for early stage disease, clinical outcomes for advanced, metastatic, or recurrent disease remain poor. To change this panorama, a deeper understanding of cervical cancer biology and novel study models are needed. Immortalized human cancer cell lines such as HeLa constitute crucial scientific tools, but there are few other cervical cancer cell lines available, limiting our understanding of a disease known for its molecular heterogeneity. This study aimed to establish novel cervical cancer cell lines derived from Brazilian patients. We successfully established one (HCB-514) out of 35 cervical tumors biopsied. We confirmed the phenotype of HCB-514 by verifying its' epithelial and tumor origin through cytokeratins, EpCAM and p16 staining. It was also HPV-16 positive. Whole-exome sequencing (WES) showed relevant somatic mutations in several genes including BRCA2, TGFBR1 and IRX2. A copy number variation (CNV) analysis by nanostring and WES revealed amplification of genes mainly related to kinases proteins involved in proliferation, migration and cell differentiation, such as EGFR, PIK3CA, and MAPK7. Overexpression of EGFR was confirmed by phospho RTK-array and validated by western blot analysis. Furthermore, the HCB-514 cell line was sensitive to cisplatin. In summary, this novel Brazilian cervical cancer cell line exhibits relevant key molecular features and constitutes a new biological model for pre-clinical studies.Barretos Cancer Hospital Research Support Department (NAP) for sample collection, Barretos Cancer Hospital Biobank for sample processing, Dr. Flávia de Paula and Gabriela Fernandes for technical support of STRs and BRCA2 Sanger validation, respectively, and Dr. Laura Musselwhite (Duke University) for revising the manuscript. This study was supported by grants from the FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII- BIOPLAT - Process number 01.13.0469.00) and Barretos Cancer Hospital. PhD scholarship from FINEP (Grant numbers 384088/2014-7 and 380434/2015-6) and Barretos Cancer Hospital to MNR

Mining RNA–Seq Data for Infections and Contaminations

RNA sequencing (RNA–seq) provides novel opportunities for transcriptomic studies at nucleotide resolution, including transcriptomics of viruses or microbes infecting a cell. However, standard approaches for mapping the resulting sequencing reads generally ignore alternative sources of expression other than the host cell and are little equipped to address the problems arising from redundancies and gaps among sequenced microbe and virus genomes. We show that screening of sequencing reads for contaminations and infections can be performed easily using ContextMap, our recently developed mapping software. Based on mapping–derived statistics, mapping confidence, similarities and misidentifications (e.g. due to missing genome sequences) of species/strains can be assessed. Performance of our approach is evaluated on three real–life sequencing data sets and compared to state–of–the–art metagenomics tools. In particular, ContextMap vastly outperformed GASiC and GRAMMy in terms of runtime. In contrast to MEGAN4, it was capable of providing individual read mappings to species and resolving non–unique mappings, thus allowing the identification of misalignments caused by sequence similarities between genomes and missing genome sequences. Our study illustrates the importance and potentials of routinely mining RNA–seq experiments for infections or contaminations by microbes and viruses. By using ContextMap, gene expression of infecting agents can be analyzed and novel insights in infection processes and tumorigenesis can be obtained