Charles A. Smith

Multiparameter analysis of human epithelial tumor cell lines by laser scanning cytometry

Laser scanning cytometry (LSC) is a relatively new slide-based technology developed for commercial use by CompuCyte (Cambridge, MA) for performing multiple fluorescence measurements on individual cells. Because techniques developed for performing four or more measurements on individual lymphoid cells based on light scatter as a triggering parameter for cell identification are not suitable for the identification of fixed epithelial tumor cells, an alternative approach is required for the analysis of such cells by LSC. Methods for sample preparation, event triggering, and the performance of multiple LSC measurements on disaggregated fixed human cells were developed using normal lymphocytes and two human breast cancer cell lines, JC-1939 and MCF-7, as test populations. Optimal conditions for individual cell identification by LSC were found to depend on several factors, including deposited cell density (cells per unit area), the dynamic range of probe fluorescence intensities, and intracellular distribution of the fluorescent probe. Sparsely deposited cells exhibited the least cell overlap and the brightest immunofluorescent staining. Major advantages of using DNA probes over a cytoplasmic immunofluorescent protein marker such as tubulin for event triggering are that the former exhibit greater fluorescence intensity within a relatively sharply demarcated nuclear region. The DNA-binding dye LDS-751 was found to be suboptimal for quantitative DNA measurements but useful as a triggering measurement that permits the performance of simultaneous fluorescein isothiocyanate-, phycoerythrin-, and indodicarbocyanine-based measurements on each cell. A major potential advantage of LSC over flow cytometry is the high yields of analyzable cells by LSC, permitting the performance of multiple panels of multicolor measurements on each tumor. In conclusion, we have developed and optimized a technique for performing multiple fluorescence measurements on fixed epithelial cells by LSC based on event triggering using the DNA-binding dye LDS 751. Although not ideal for quantitative measurements of cell DNA content, the large Stokes shift of this dye permits the performance of three or more additional fluorescence measurements on each cell.

A Suitable Method for Identifying Cell Aggregates in Laser Scanning Cytometry Listmode Data for Analyzing Disaggregated Cell Suspensions Obtained from Human Cancers

The presence of cell aggregates in cell suspensions obtained from human solid tumors can interfere with the measurement of cell DNA content of cell singlets, and can confound multiparameter analysis of other measurements on the same cells. Flow cytometric corrections for cell aggregates based on signal pulse shape have not proven to be reliable. Mathematical models have been developed to correct for cell aggregates in binned DNA histogram data, but they are not suitable for the correction of correlated non‐DNA measurements obtained on the same cells.

A simple correction for cell autofluorescence for multiparameter cell‐based analysis of human solid tumors

Corrections that have been proposed to minimize the unwanted contribution of cell autofluorescence to the total fluorescence signal often require either specialized instrumentation or the sacrifice of a data channel so as to perform a measurement that can be used to correct for autofluorescence in individual cells. Here we propose a simple cell by cell correction for autofluorescence that is suitable for multiparameter laser scanning cytometry (LSC) studies in human solid tumors that relies on the ratio of mean autofluorescence to mean total cell fluorescence (mean Flauto/mean Fltotal). This approach assumes a correlation between the autofluorescence component and the total signal in individual cells. This correction does not require specialized instrumentation, and does not sacrifice a data channel in multiparameter studies. A potential disadvantage is that errors may be introduced by the assumption of a correlation between the two components of the total fluorescence signal in individual cells in samples in which no such correlation exists.

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell‐based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell‐based immunofluorescence measurements.

Breast Cancer: Expression of HER-2 and Epidermal Growth Factor Receptor as Clinical Markers for Response to Targeted Therapy

In this chapter we consider the relationships that involve both HER-2 and epidermal growth factor receptor (EGFR), with a particular focus on the potential use of these two transmembrane growth factor receptors as biomarkers for predicting response to targeted therapeutic agents directed at either or both of them. Effective clinical use of these receptors as biomarkers depends both on an understanding of their complex biological interactions, and an awareness of the shortcomings of current methods of measuring these two cellular constituents in the clinical setting. Here we will review selected aspects of these two topics, and attempt to develop working hypotheses and organizing principles that might be useful in translating basic knowledge into clinical practice. CELL AND MOLECULAR BIOLOGY OF HER-2/EPIDERMAL GROWTH FACTOR RECEPTOR INTERACTIONS