Isolation of single, intact chromosomes from single, selected ovarian cancer cells for in situ hybridization and sequencing

The first step towards effective therapy of cancer is to reveal molecular profiles of all cell clones propelling tumor growth. The specific aim of this project was to develop a technology helping us to isolate patient's single, living cells based upon their cancer-specific, cell surface biomarkers, to reveal their molecular profiles, and to isolate, from these selected cells, intact chromosomes for in situ hybridization (FISH) and for next generation sequencing (NGS). We attained this aim, while probing the cells from the ovarian cancer patients.Ovarian cancer is the most deadly of all gynecological cancers. In most of the patients with the advanced stages of this cancer, the gene for epidermal growth factors receptor (EGFR) is mutated, as the deletion variant III, resulting in the truncated transcripts and products. From these patients, we collected cells from peritoneal fluid, blood, lymph, and biopsies. We genetically engineered fluorescent and superparamagnetic single chain variable fragments (scFvs) targeting EGFRwt and EGFRvIII. Using these scFvs, we isolated single, living ovarian cancer cells and analyzed their transcripts and products. We further genetically engineered scFv targeting dsDNA. Using these scFvs, we isolated the entire single, intact chromosomes from the selected, single ovarian cancer cells for NGS and for liquid phase FISH.This novel work-flow opens new routes not only for molecular profiling of the entire spectrum of cancer cell clones in the diagnosed patient, one cell clone at a time, but also for manufacturing targeted contrast for in vivo imaging and for designing and guiding targeted delivery of therapeutic genes in cancer therapy.

► Human scFvs targeting human EGFR, EGFRvIII, dsDNA were genetically engineered.
► Single cells expressing EGFRvIII or EGFR were isolated from patients with ovarian cancers.
► Proteomes and transcriptomes from these cells were analyzed.
► Single, selected chromosomes were isolated and sequences validated by FISH and NGS.
► Integrated work flow was developed to study single cell genome, transcriptome, proteome.