Multiparameter analysis of progeny of individual cells by laser scanning cytometry

Background: Effectiveness of antitumor drugs to suppress unrestricted proli feration of cancer cells is commonly measured by cell clonogenicity assays. Assays of clonogenicity are also used in studies of stem/progenitor cells and in analysis of carcinogenic transformation. The conventional assays are limited to providing information about frequency of colonies (cloning effi ciency) and do not reveal the qualitative (phenotype) attributes of individ ual colonies that may yield clues on mechanisms by which cell proliferation was affected by the studied agent. Methods: Laser scanning cytometry (LSC) was adapted to identify and charact erize size and phenotype of colonies of MCF-7 cells growing in microscope s lide chambers, untreated and treated with the cytotoxic ribonuclease, oncon ase (Onc). Individual colonies were located and data representing each colo ny were segmented based on >650-nm fluorescence excited by a I-Ie-Ne laser of the cells whose protein was stained with BODIPY 630/G50-X. The DNA of th e cells was stained with propidium iodide (red fluorescence) whereas specif ic proteins (estrogen receptor [ER] or tumor suppressor p53) were detected immunocytochemically (green fluorescence), each excited by an Ar ion laser. Results: A plethora of attributes of individual colonies were measured, suc h as (a) morphometric features (area, circumference, area/circumference rat io, DNA or protein content per area ratio), (b) number of cells (nuclei), ( c) DNA content, (d) protein content and protein/DNA ratio, and (e) expressi on of ER or p53 per colony, per total protein, per nucleus or per DNA, with in a colony. Also cell cycle distribution within individual colonies and he terogeneity of colonies with respect to all the measured features could be assessed. The colonies growing in the presence of One had many of the above attributes different than the colonies from the untreated cultures. Conclusions: Analysis of the features of cell colonies by LSC provides a we alth of information about the progeny of individual cells. Changes in colon y size and phenotype, reflecting altered cell shape, cell size, colony prot ein/DNA ratio, and expression of individual proteins, may reveal mechanisms by which drugs suppress the proliferative capacity of the cells. This may include inducing growth imbalance and differentiation and modulating expres sion of the genes that may be associated with cell cycle, apoptosis, or dif ferentiation in a progeny of individual cells. Extensions of LSC may make i t applicable for automatic analysis of cloning efficiency and multiparamete r analysis of cell colonies in soft agar. Such analyses may be useful in st udies of the mechanisms and effectiveness of antitumor drugs, in the field of carcinogenesis, and for analyzing primary cultures and assessing tumor p rognosis and drug sensitivity. The assay can also be adapted to analysis of microbial colonies. (C) 2000 Wiley Liss, Inc.