Analysis of Serial Neuroblastoma PDX Passages in Mice Allows the Identification of New Mediators of Neuroblastoma Aggressiveness

Abstract

Neuroblastoma is a neural crest cell-derived pediatric tumor characterized by high inter- and intra-tumor heterogeneity, and by a poor outcome in advanced stages. Patient-derived xenografts (PDXs) have been shown to be useful models for preserving and expanding original patient biopsies in vivo, and for studying neuroblastoma biology in a more physiological setting. The maintenance of genetic, histologic, and phenotypic characteristics of the original biopsy along serial PDX passages in mice is a major concern regarding this model. Here we analyze consecutive PDX passages in mice, at both transcriptomic and histological levels, in order to identify potential changes or highlight similarities to the primary sample. We studied temporal changes using mRNA and miRNA expression and correlate those with neuroblastoma aggressiveness using patient-derived databases. We observed a shortening of tumor onset and an increase in proliferative potential in the PDXs along serial passages. This behavior correlates with changes in the expression of genes related to cell proliferation and neuronal differentiation, including signaling pathways described as relevant for neuroblastoma malignancy. We also identified new genes and miRNAs that can be used to stratify patients according to survival, and which could be potential new players in neuroblastoma aggressiveness. Our results highlight the usefulness of the PDX neuroblastoma model and reflect phenotypic changes that might be occurring in the mouse environment. These findings could be useful for understanding the progression of tumor aggressiveness in this pathology.

Keywords: PDX; differentiation; gene expression; miRNA; neuroblastoma.

Figure 1

(a) Scheme showing PDX serial transplantation approach. (b) Percentage of mice alive at the indicated time points and tumor onset of different NB PDXs. Median survival is indicated. (c) Normalized summary heatmap with the mRNA gene expression changes in PDX NB27T from all passages and replicates. (d) Normalized summary heatmap with the miRNA expression changes in PDX NB27T from all passages and replicates.

Figure 2

(a) Gene expression profiles obtained from the STEM analysis of PDX NB27T. In each box, the number in the top left is number of genes, in the bottom left, p value. Only the ones with significant p values are shown. Profiles are grouped as Up, Down, or Mixed. (b) Volcano plots showing the most significant mRNA expression changes (left) or miRNA changes (right) from the PDX5 vs PDX1 comparison. In blue, the downregulated genes in PDX5, and in red, the upregulated genes in PDX5.

Figure 3

Heatmaps showing normalized gene expression changes on PDX passage 1 to 5 of PDX NB27T for genes on different published signatures. Ratio of the relative change in the different cell types is shown in each case: (a) signature from [6], (b) signature from [20], (c) signature from [7]. Lines delimitate genes decreasing or increasing their expression with time.

Figure 4

(a) miRNAs found differentially expressed among PDX passages affecting CD44 expression or MYCN expression. (b) miRNAs affecting CD44, PROM1, or KIT mRNAs. p values are shown. FC: fold change (c) expression of the miRNA99-a gene (MIR99AHG) in neuroblastoma patient tumor samples divided according to MYCN amplification or patient outcome. *: p < 0.005. (d) Kaplan– Meyer curves showing overall survival of patients according to miRNA-99a expression.

Figure 5

(a) Immunohistochemistry showing expression of the indicated markers in the original NB27T patient sample. Scale bar: 100 μm. (b) Immunohistochemistry showing expression of the indicated markers in PDX NB27T tissue from the indicated PDXs passages. Quantification of the expression is shown on the graphs. Scale bar: 100 μm. ***: p < 0.001 (c) Table showing genes differentially expressed between PDX5 and PDX1, whose expressions stratify neuroblastoma patients according to overall survival. Low: low expression, resulting in worse survival; high: high expression, resulting in worse survival.