Characteristics of stem cells from human neuroblastoma cell lines and in tumors

Abstract

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.

Figure 1

Phase contrast photomicrographs of human neuroblastoma cell lines of the three different phenotypes, all at x135 magnification. (A) N-type SK-N-CH; (B) S-type SH-EP1; and I-types (C) SK-N-JD, (D) SK-N-LP, (E) SK-N-HM, and (F) SK-N-ER.

Figure 2

(A) Western blot analysis of N, I, and S cell lines stained for CgA, CD44, and actin. Lane, line, phenotype: (1) BE(2)-M17, N; (2) SK-N-JD, I; (3) SK-N-LP, I; (4) BE(2)-C, I; and (5) SH-EP1, S. (B) Bar graph showing relative expression of three N marker proteins [neuronal-specific RNA-binding protein Hu (Hu-IR), CgA, and neurofilament 68 (NF68)] and three S marker proteins (CD44, VIM, and ACTN) in phenotypically homogeneous populations of two N-type [BE(2)-M17 and SH-SY5Y], seven I-type [BE(2)-C, SH-IN, SK-N-HM, SK-N-LP, SK-N-JD, SK-N-ER, and CB-JMN], and two S-type [LA1-5s and SMS-KCNs] cell lines. Bar height represents the amount of protein (relative to actin) as a percentage ofmaximumamount of protein present in any of the lines.

Figure 3

RT-PCR analysis of relative expression levels ± SEM of CD133 and c-kit in three N-type (■), five I-type (), and two S-type (□) human neuroblastoma cell lines. Each bar represents the average of one to five separate determinations. Lane, line: (1) KCN-69n; (2) LA1-55n; (3) BE(2)-M17; (4) BE(2)-C; (5) SK-N-LP; (6) LAN-2; (7) CB-JMN; (8) SK-N-ER; (9) SH-EP1, and (10) LA1-5s.

Figure 4

Effects of 1 µM RA and 10 µM BUdR on the cell phenotype of: (1) BE(2)-C; (2) SK-N-ER; and (3) SK-N-HM I-type cells. Note that whereas RA induces a neuroblastic (N) phenotype, BUdR induces an S cell phenotype.

Figure 5

Colony-forming efficiencies ± SEM in soft agar for four N-type (■), six I-type (), and three S-type (□) cell lines. Lane, line: (1) BE(2)-M17; (2) SMS-KAN; (3) KCN-69n; (4) SH-SY5Y; (5) BE(2)-C; (6) CB-JMN; (7) SK-NER; (8) SK-N-JD; (9) SK-N-LP; (10) SH-IN; (11) SK-N-BE(2)s; (12) LA1-5s; and (13) SH-EP1.

Figure 6

Frequency ± SEM of I-like cells in human neuroblastoma tumor sections of patients who were progression-free (PF) (n = 10) or who relapsed with disease (R) (n = 5). **P < .001.