Fluorescence-assisted cytological testing (FACT): Ex Vivo viral method for enhancing detection of rare cancer cells in body fluids

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.

Figure 1

EGFP-positive cancer cells can be identified against a background of millions of normal cells. (A–C) A rare cancer cell in a mixture of millions of normal cells is difficult to identify under bright-field microscopy and is time-consuming (A). Under fluorescent microscopy, EGFP-positive NV1066-infected cancer cells can be easily identified by means of green fluorescence (B). Overlap of fluorescent picture with bright-field identifies the cancer cell (C) for further studies. (Upper panel shows mixture of lung cancer cells and normal cells in bronchoalveolar lavage, and lower panel demonstrates a mixture of bladder cancer and normal cells in urine.) A rare cancer cell among millions of cells can be detected and separated out for further studies by flow cytometry. (D) NV1066 selective infection of cancer cells among a mixture of millions of normal cells is confirmed by counterstaining with immunohistochemistry. Human mesothelioma cancer cells were mixed with normal pleural cells (upper left panel ) and were incubated with NV1066 for 18 h. Examination under fluorescence microscope identified cancer cells by expression of strong green fluorescence (upper right panel ). These cancer cells express integrin (CD 51/61) surface antigen. Incubation with R-PE–conjugated mouse antihuman CD51/61 monoclonal antibody confirmed that EGFP expression is selective to only cancer cells (identified by red fluorescence, lower left panel). Overlap of fluorescent pictures with bright-field identifies cancer cells among normal cells (lower right panel). Live cells among the cell clumps were identified by nuclear Hoechst staining (blue). (E) A similar experiment repeated with human lung cancer cells mixed with normal bronchoalveolar cells and incubated with NV1066 confirms similar results as described in (D).

Figure 2

Detection of EGFP-positive cells by flow cytometry. (A) Two million cells were sorted out by their size and autofluorescence by flow cytometry. (B) Among the same cell population, cancer cells were identified by strong green fluorescence in the FL-1 channel. These rare cancer cells can be separated out for further histological studies by flow cytometric sorting. (C) Because of their strong expression of EGFP, NV1066-infected cancer cells in body fluids can be easily identified, even in a background of millions of cells or cell clumps. Lung cancer cells were mixed with normal cells from bronchoalveolar lavage in ratios from 1:10 to 1:1,000,000 and incubated with NV1066 for 18 h. Cancer cells mixed with NV1066 served as positive controls, and normal cells mixed with NV1066 served as negative controls. The mean fluorescence intensities of EGFP-expressing cells in each sample were plotted.

Figure 3

Detection of NV1066-infected cancer cells using EGFP fluorescence in human samples (magnification 30×). (A) Cytological examination showed benign mesothelial cells, as read by an attending pathologist. (B) These benign cells did not fluoresce when viewed under the green fluorescent protein (EGFP) filter. (C, E) Cytological examination showed malignant cells in samples from patients with non–small cell lung cancer. (D, F) Malignant cells expressed green fluorescence under the enhanced EGFP filter.