Cellular heterogeneity is a hallmark of human neuroblastoma tumors and cell lines. Within a single neuroblastoma are cells from distinct neural crest lineages whose relative abundance is significant for prognosis. We postulate that a self-renewing multipotent tumor stem cell, which gives rise to diverse cell lineages, is the malignant progenitor of this cancer. To test this hypothesis, we have established 22 cloned, phenotypically homogeneous populations of the three major cell types from 17 neuroblastoma cell lines. In vitro, malignant neuroblastoma stem cells, termed I-type (intermediate type), have distinct morphologic, biochemical, differentiative, and tumorigenic properties. I-type cells express features of both neuroblastic (N) cells (scant cytoplasm, neuritic processes, neurofilaments, pseudoganglia, and granin and neurotransmitter enzyme expression) and substrate-adherent (S) cells (extensive cytoplasm and vimentin and CD44 expression). Moreover, they show bidirectional differentiation to either N or S cells when induced by specific agents. I-type cells are significantly more malignant than N- or S-type cells, with four- to five-fold greater plating efficiencies in soft agar and six-fold higher tumorigenicity in athymic mice. Differences in malignant potential are unrelated to N-myc amplification/overexpression or the ability to digest and migrate through the extracellular matrix. Immunocytochemical analyses of a small series of tumors reveal that frequency of cells coexpressing N and S cell markers correlates with poor prognosis. Thus, I-type stem cells may be instrumental in the genesis and growth of tumors in the patient. Their unique biology deserves attention and further investigation.

No abstract

Four cell lines, SK-N-SH, SK-N-MC, SK-N-BE(2), and IMR-32, established in vitro from tumor tissue of patients with neuroblastoma were analyzed by trypsin-Giemsa banding methods. In two of the lines a large, abnormally staining chromosome region was observed. This "homogeneously staining region" (HSR) was considerably longer than any of the bands present in normal human cells and, as revealed by both G- and Q-banding, stained with an intermediate intensity. It was located on chromosomes No 6, 10, 17, or 19 of the SK-N-BE(2) cell line and on chromosome No 1 of the IMR-32 line. In concurrent studies, long HSR's were also observed in Chinese hamster sublines that had been exposed to and had developed high levels of resistance to methotrexate or methasquin and high levels of activity of target enzyme dihydrofolate reductase. For several sublines with the highest levels of enzyme activity, approximately 2% of the total cell protein was dihydrofolate reductase. Of 13 independently derived sublines with acquired resistance to antifolate, only those 7 with greater than 100-fold increases in enzyme activity consistently exhibited HSR's. These regions comprised 2-5% of the total length of the chromosome complement and were specifically localized, as demonstrated by G-banding. Analysis of chromosome replication patterns of the HSR in human neuroblastoma and in drug-resistant Chinese hamster cells by tritiated thymidine radioautography indicated that the long, abnormally staining region replicated relatively rapidly and synchronously and terminated replication before the midpoint of the S phase. The HSR thus appeared to represent a novel chromosome abnormality that may be present in cells with specialized functions. Drug-resistant Chinese hamster cells were characterized by overproduction of target enzyme, whereas human neuroblastoma cells had phenotypes of normal neuronal cells. Whether the HSR is transcriptionally active was not elucidated.

Large, homogeneously staining chromosome regions which lack the longitudinal differentiation ordinarily revealed by cytogenetic "banding" methods have been found in antifolate-resistant Chinese hamster cells and also in human neuroblastoma cells established in vitro. The drug-resistant cells are characterized by excessive production of the target enzyme, dihydrofolate reducatase, while the human neuroblastoma cells have phenotypes of normal neuronal cells. The homogeneously staining region appears to represent a novel metaphase chromosome anaomaly which may have functional significance in cells with specialized properties.

No abstract