This study investigates the maturation of human neuroblastoma (NB) cells in vitro response to differentiation-inducing agents. Such agents may be useful in the therapy of NB by inducing irreversible maturation and/or a benign phenotype as a result of decreased oncogene expression, especially the N-myc oncogene. An objective assay for the differentiated state will be developed using Digital Image Microscopy (DIM), which allows quantitation of multicolor immunofluorescence, cell size, and cell shape on individual tumor cells. Variables that will be assessed include morphology, differentiation antigen expression, N-myc protein expression, and cell cycle distribution. Cell surface antigens increase or decrease, and the N-myc protein markedly decreases in morphologically differentiated cells. The response of well characterized neuroblastoma cell lines to a panel of differentiation inducers will be determined; because oncogene activation may be important in determining the state of differentiation, the panel of cell lines include neuroblastomas with single or multiple copies of the N-myc oncogene. The time course, uniformity, and irreversibility of the differentiation response for each agent and cell line will be correlated with N-myc gene copy number, N-myc protein expression, and/or differentiation antigen expression. If a uniform response to an agent is not obtained, the resistant subpopulation will be selected and cross-resistance to other inducers determined. The ability of combinations of inducers to overcome resistance will be investigated. Expression of N-myc protein and gene copy number will be compared in differentiation inducer sensitive and resistant cells. These studies should provide assays for the differentiated neuroblastoma phenotype, and in vitro models for neuroblastoma maturation, leading to understanding the mechanisms of such maturation, and to effective combinations of agents for therapeutic use. Methods developed for assessing neuroblastoma differentiation in vitro will be valuable in assessing the effects of differentiation inducers in vivo during clinical trials.
