Evaluating the RIST Molecular-Targeted Regimen in a Three-Dimensional Neuroblastoma Spheroid Cell Culture Model

Abstract

Background: The outcome for patients with high-risk neuroblastoma remains poor and novel treatment strategies are urgently needed. The RIST protocol represents a novel metronomic and multimodal treatment strategy for high-risk neuroblastoma combining molecular-targeted drugs as 'pre-treatment' with a conventional chemotherapy backbone, currently evaluated in a phase II clinical trial. For preclinical drug testing, cancer cell growth as spheroid compared to mo-nolayer cultures is of advantage since it reproduces a wide range of tumor characteristics, including the three-dimensional architecture and cancer stem cell (CSC) properties. The objective of this study was to establish a neuroblastoma spheroid model for the rigorous assessment of the RIST treatment protocol.

Methods: Evaluation of CSC marker expression was performed by mRNA and protein analysis and spheroid viability by luminescence-based assays. Aberrant expression of RNA-binding protein La in neuroblastoma was assessed by tissue microarray analysis and patients' data mining.

Results: Spheroid cultures showed increased expression of a subgroup of CSC-like markers (CXCR4, NANOG and BMI) and higher Thr389 phosphorylation of the neuroblastoma-associated RNA-binding protein La when compared to monolayer cultures. Molecular-targeted 'pre-treatment' of spheroids decreased neoplastic signaling and CSC marker expression.

Conclusions: The RIST treatment protocol efficiently reduced the viability of neuroblastoma spheroids characterized by advanced CSC properties.

Keywords: 3D model; CSC; La (LARP3; La autoantigen); La/SSB; RIST; RNA-binding protein; cancer stem cells; neuroblastoma; spheroids; three-dimensional.

Figure 1

Establishment of spheroid culture conditions. (a) Phase-contrast imaging of SK-N-SH spheroids on day 7 and SK-N-AS spheroids on day 10 after seeding increasing cell numbers on round-bottom 96-well ULA plates. Scale = 100 µm. (b) Analysis of the diameter of SK-N-SH (left) and SK-N-AS (right) spheroids seeded with increasing cell numbers and grown on round-bottom 96-well ULA plates for 3 days (d3), 7 days (d7), 10 days (d10) and 14 days (d14). Three independent experiments (n = 3) in triplicate were performed. Degrees of significance (unpaired student’s t-test): * < 0.05, ** < 0.01 and *** < 0.001.

Figure 2

Size of spheroids of five different neuroblastoma cell lines grown in 3D culture. Diameter of spheroids from neuroblastoma cell lines SK-N-SH (SH), SK-N-AS (AS), SK-N-BE(2) (BE(2)), IMR-32 and Kelly grown under established conditions (Table 1) on 96-well ULA plates for 3 days (d3) or 7 days (d7). MycN-non-amplified (MNN), MycN-amplified (MNA). Three independent experiments (n = 3) in triplicate were performed. Degrees of significance (unpaired student’s t-test): * < 0.05.

Figure 3

CSC-like marker expression in neuroblastoma spheroids compared to monolayer cell cultures. Fold change increase in cancer stem cell (CSC)-like marker (a) CXCR4, (b) NANOG and (c) BMI1 expression in spheroids compared to monolayer cultures of neuroblastoma cell lines SK-N-SH (SH), SK-N-AS (AS), SK-N-BE(2) (BE(2)), IMR-32 and Kelly determined by RTqPCR. Normalized to GAPDH expression. Statistical analysis of variance (ANOVA) was performed to determine the association between CSC-like marker expression in MNN and MNA cell lines. MycN-non-amplified (MNN), MycN-amplified (MNA). At least two independent experiments (n = 2) in triplicate were performed. Degrees of significance (unpaired student’s t-test): * < 0.05 and ** < 0.01.

Figure 4

Scheme of the experimental design for the in vitro RIST treatment protocol.

Figure 5

Viability of NB spheroids after R+D ‘pre-treatment’. To determine the impact of the Rapamycin plus Dasatinib (R+D) ‘pre-treatment’ on spheroid viability, spheroids were harvested on day 3 and analyzed applying the CellTiter-Glo 3D Cell Viability Assay. Viability of SK-N-SH (SH), SK-N-AS (AS), SK-N-BE(2) (BE(2)), IMR-32 and Kelly spheroids determined 3 days after treatment with R+D (+) or vehicle (−) DMSO treatment. MycN-non-amplified (MNN), MycN-amplified (MNA). Two independent experiments (n = 2) in triplicate were performed. Degrees of significance (unpaired student’s t-test): *** < 0.001.

Figure 6

Neoplastic signaling pathway response to R+D ‘pre-treatment’ comparing 2D and 3D cultures. Immunoblot (IB) analysis showing the phosphorylation status of ribosomal protein S6 kinase (p70S6K), translation initiation factor 4E-BP and tyrosine kinase Src under control (C)- and Rapamycin plus Dasatinib (R+D)-treated conditions in two-dimensional (2D) monolayer and three-dimensional (3D) spheroid cultures of neuroblastoma cell line SK-N-AS and SK-N-BE(2). Numbers below bands state phosphoprotein (P) normalized to total protein expression. GAPDH was applied as loading control. C: Vehicle-treated control cells. R+D: Rapamycin plus Dasatinib treatment as depicted in Table 2. Star (*): unknown band. MycN-non-amplified (MNN), MycN-amplified (MNA). IB: Immunoblot. Original immunoblot (IB) images (Figure S4).

Figure 7

The R+D ‘pre-treatment’ reduces CXCR4, BMI1 and NANOG protein expression in NB spheroid cultures. Immunoblot (IB) analysis showing (a) CXCR4, BMI1 and NANOG protein expression in four different neuroblastoma cell lines grown in 2D cultures with medium supplemented with 10% FBS, and (b) CXCR4, BMI1 and NANOG protein expression under control (C)- and Rapamycin plus Dasatinib (R+D)-treated conditions comparing 2D and 3D cultures grown in medium supplemented with 5% KOSR. GAPDH was applied as loading control. Numbers below bands state protein expression normalized to GAPDH. C: Vehicle-treated control cells. R+D: Rapamycin plus Dasatinib treatment as depicted in Table 2. MycN-non-amplified (MNN), MycN-amplified (MNA). IB: Immunoblot. Original immunoblot (IB) images (Figure S4).

Figure 8

Viability of NB spheroids after RIST treatment. To determine the impact of the multimodal RIST treatment (Figure 4) on spheroid viability, spheroids were harvested on day 7 (complete RIST) and analyzed applying the CellTiter-Glo 3D Cell Viability Assay. NB cell lines: SK-N-SH (SH), SK-N-AS (AS), SK-N-BE(2) (BE(2)), IMR-32 and Kelly. RIST (+) or DMSO (−) (vehicle) treatment. MycN-non-amplified (MNN), MycN-amplified (MNA). At least three independent experiments (n = 3) in triplicate were performed. Degrees of significance (unpaired student’s t-test): *** < 0.001.

Figure 9

High expression of the RNA-binding protein La correlates with low overall survival probability in neuroblastoma. (a,b) Immunohistochemical analysis of tissue microarray TMA MC809 demonstrates significant overexpression of the La protein in neuroblastoma, nephroblastoma and glioblastoma tissue compared to normal cerebrum, cerebellum and kidney tissue. (c) Kaplan–Meier curve indicating that elevated expression of La mRNA is an indicator for reduced overall survival probability in neuroblastoma. The neuroblastoma data set SEQC-498 consists of 498 cases including 398 patient samples with low (red) La and 100 with high (blue) La mRNA expression levels, leading to a significant difference. The datasets for the Kaplan–Meier curves were retrieved from the R2 platform (https://r2.amc.nl, dataset ID: GSE62564, accessed on 9 March 2023). Degrees of significance (unpaired student’s t-test): * < 0.05 and *** < 0.001.

Figure 10

R+D ‘pre-treatment’ reduced augmented Thr389 phosphorylation of RNA-binding protein La in NB spheroids. Immunoblot (IB) analysis indicates (a) the expression level of oncogene MycN and the La protein as well as the phosphorylation status of La at threonine 389 (Thr389) in various neuroblastoma cell lines (SK-N-AS, SK-N-SH, Kelly, IMR-32, SK-N-BE(2)) and SK-N-MC*. grown in 2D monolayer cultures in medium supplemented with 10% FBS. The star (*) indicates that SK-N-MC has been originally described as neuroblastoma but is now widely regarded as an Askin‘s tumor cell line related to Ewing‘s sarcoma [47]. In (b), the phosphorylation status of La at Thr389 is depicted, comparing 2D and 3D cultures under control (vehicle)- and Rapamycin plus Dasatinib (R+D)-treated conditions. Cells were grown in medium supplemented with 5% KOSR. Numbers below bands state phosphoprotein (P-La (Thr389)) normalized to total La protein expression. GAPDH was applied as loading control. C: Vehicle-treated control cells. R+D: Rapamycin plus Dasatinib treatment as depicted in Table 2. MycN-non-amplified (MNN), MycN-amplified (MNA). IB: Immunoblot. Original immunoblot (IB) images (Figure S4).