Multiparameter analysis of progeny of individual cells by laser scanning cytometry.
BACKGROUND
Effectiveness of antitumor drugs to suppress unrestricted proliferation 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 efficiency) and do not reveal the qualitative (phenotype) attributes of individual 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 characterize size and phenotype of colonies of MCF-7 cells growing in microscope slide chambers, untreated and treated with the cytotoxic ribonuclease, onconase (Onc). Individual colonies were located and data representing each colony were segmented based on >650-nm fluorescence excited by a He-Ne laser of the cells whose protein was stained with BODIPY 630/650-X. The DNA of the cells was stained with propidium iodide (red fluorescence) whereas specific 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, such as (a) morphometric features (area, circumference, area/circumference ratio, DNA or protein content per area ratio), (b) number of cells (nuclei), (c) DNA content, (d) protein content and protein/DNA ratio, and (e) expression of ER or p53 per colony, per total protein, per nucleus or per DNA, within a colony. Also cell cycle distribution within individual colonies and heterogeneity of colonies with respect to all the measured features could be assessed. The colonies growing in the presence of Onc 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 wealth of information about the progeny of individual cells. Changes in colony size and phenotype, reflecting altered cell shape, cell size, colony protein/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 expression of the genes that may be associated with cell cycle, apoptosis, or differentiation in a progeny of individual cells. Extensions of LSC may make it applicable for automatic analysis of cloning efficiency and multiparameter analysis of cell colonies in soft agar. Such analyses may be useful in studies of the mechanisms and effectiveness of antitumor drugs, in the field of carcinogenesis, and for analyzing primary cultures and assessing tumor prognosis and drug sensitivity. The assay can also be adapted to analysis of microbial colonies.
Brander Cancer Research Institute, New York Medical College, Hawthorne 10532, USA.
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Bedner
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Ruan
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Chen
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Kamentsky
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Darzynkiewicz
D001896:Boron Compounds; D001943:Breast Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology*; D002352:Carrier Proteins / Q000032:analysis; D002453:Cell Cycle; D004273:DNA, Neoplasm / Q000032:analysis; D004527:Egg Proteins / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007150:Immunohistochemistry; D011419:Propidium; D011960:Receptors, Estrogen; D012260:Ribonucleases / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000473:pathology; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002352:Carrier Proteins; D004273:DNA, Neoplasm; D004527:Egg Proteins; D005456:Fluorescent Dyes; D011960:Receptors, Estrogen; D016159:Tumor Suppressor Protein p53; C010140:estrophilin; D011419:Propidium; D012260:Ribonucleases; C058703:ranpirnase