Abstract
Cytological analysis of body fluids is currently used for detecting cancer. The objective of this study was to determine if the herpes virus carrying an enhanced green fluorescent protein (EGFP) could detect rare cancer cells in body fluids against millions of normal cells. Human cancer cells suspended with normal murine cells were infected with NV1066 at a multiplicity of infection (MOI) of 0.5 and 1.0 for 18 h. Fluorescent microscopy and flow cytometry were used for EGFP detection of cancer cells. EGFP-expressing cells were confirmed as cancer cells with specific markers by immunohistochemistry staining. Limits of detection of cancer cells in body fluid were measured by serial dilutions. Applicability of technique was confirmed with samples from patients with malignant pleural effusions. NV1066 expressed EGFP in 111 human cancer cell lines detected by fluorescent microscopy at an MOI of 0.5. NV1066 selectively infected cancer cells and spared normal cells as confirmed by immunohistochemistry. Sensitivity of detecting fluorescent green cells was 92% (confidence interval [CI] 83% to 97%) at a ratio of 1 cancer cell to 1 million normal cells. EGFP-positive cells were detected by fluorescent microscopy in patients' malignant pleural effusion samples. Our data show proof of the concept that NV1066-induced EGFP expression allows detection of a single cancer cell against a background of 1 million normal cells. This method was demonstrated to be a reliable screening tool for human cancer cells in a suspension of normal murine cells as well as clinical specimens of malignant pleural effusions.
Highlights
Detection of cancer before it has had a chance to metastasize remains the best strategy for reducing cancer mortality [1]
We present the technique of using recombinant herpes virus–expressing enhanced green fluorescent protein (EGFP) to detect rare cancer cells in body fluids
10 human lung (A549) and bladder (KU19-19) cancer cells were mixed with 1 × 106 normal cells from murine bronchoalveolar lavage or collected urine which was infected with NV1066 at an multiplicity of infection (MOI) of 0.5 or 1 for 18 h
Summary
Detection of cancer before it has had a chance to metastasize remains the best strategy for reducing cancer mortality [1]. One routinely used method for screening high-risk patients involves microscopic examination of body fluids (for example, sputum and urine) for the presence of tumor cells [5,6]. Such cytological tests are labor intensive and highly depend on the skill of the cytopathologist. The sensitivity of such sputum or urinary cytology studies is governed by technical limitations of identifying the few cancer cells in the background of many normal cells. We present the technique of using recombinant herpes virus–expressing enhanced green fluorescent protein (EGFP) to detect rare cancer cells in body fluids
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