Surface marker profiling of SH-SY5Y cells enables small molecule screens identifying BMP4 as a modulator of neuroblastoma differentiation

Abstract

Neuroblastoma is the most common extra-cranial solid tumor in children. Its broad spectrum of clinical outcomes reflects the underlying inherent cellular heterogeneity. As current treatments often do not lead to tumor eradication, there is a need to better define therapy-resistant neuroblastoma and to identify new modulatory molecules. To this end, we performed the first comprehensive flow cytometric characterization of surface molecule expression in neuroblastoma cell lines. Exploiting an established clustering algorithm (SPADE) for unbiased visualization of cellular subsets, we conducted a multiwell screen for small molecule modulators of neuroblastoma phenotype. In addition to SH-SY5Y cells, the SH-EP, BE(2)-M17 and Kelly lines were included in follow-up analysis as in vitro models of neuroblastoma. A combinatorial detection of glycoprotein epitopes (CD15, CD24, CD44, CD57, TrkA) and the chemokine receptor CXCR4 (CD184) enabled the quantitative identification of SPADE-defined clusters differentially responding to small molecules. Exposure to bone morphogenetic protein (BMP)-4 was found to enhance a TrkAhigh/CD15−/CD184− neuroblastoma cellular subset, accompanied by a reduction in doublecortin-positive neuroblasts and of NMYC protein expression in SH-SY5Y cells. Beyond yielding novel marker candidates for studying neuroblastoma pathology, our approach may provide tools for improved pharmacological screens towards developing novel avenues of neuroblastoma diagnosis and treatment.

Wnt signalling drives context-dependent differentiation or proliferation in neuroblastoma

“…To further evaluate the growth and differentiation variance of these cell lines at the protein level, we examined whether induction of two recently characterised inducers of differentiation, BMP4 [29] and EPAS1 [28], corresponded with the propensity of cells to differentiate. As shown in Figure 4A…”

A Wnt-BMP4 signalling axis induces MSX and NOTCH proteins and promotes growth suppression and differentiation in neuroblastoma

“…Similarly, BMP2 was shown to induce growth arrest and neuronal differentiation of SH-SY5Y and RTBM cell-lines subsequent to accumulation of p27 (23). Although BMP2 has been shown to be a Wnt target gene in other cell types, our RNA-seq of Wnt3a/Rspo2 treated neuroblastoma cells highlighted BMP4 as the most prominent BMP gene downstream of Wnt/β-catenin signalling, and a recent study showed that BMP4 can trigger neurite-like extensions in SH-SY5Y cells, induce TrkA, and decrease MYCN and Ki-67 (24). Our study in part agrees with these findings, but with important differences and clearer mechanistic insights.…”

“…One of our most highly Wnt-induced genes following Wnt induction was BMP4. Previous studies have alluded to a role for BMPs in neuroblastoma growth and differentiation, including BMP2 in mouse neuroblastoma and SH-SY5Y cells (22,23) and BMP4, which also affected SH-SY5Y differentiation and decreased proliferation markers (24). However, the mechanisms and signalling crosstalk involved in BMP-mediated phenotypes and the significance to primary disease of BMPs is not known.…”

Comprehensive Cell Surface Antigen Analysis Identifies Transferrin Receptor Protein-1 (CD71) as a Negative Selection Marker for Human Neuronal Cells

“…Human neural cells obtained from proliferation and differentiation conditions were screened for the expression of cell surface molecules using human BD Lyoplate screening panels as recently reported for neuroblastoma cells . For flow cytometric analysis, single cell suspensions were dispensed into 96‐well plates at 200,000–500,000 cells per well.…”