Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma.

In this study chemotherapy response in neuroblastoma (NB) was assessed for the first time in a transplantation model comprising non-malignant human embryonic microenvironment of pluripotent stem cell teratoma (PSCT) derived from diploid bona fide hESC. Two NB cell lines with known high-risk phenotypes; the multi-resistant BE(2)-C and the drug sensitive IMR-32, were transplanted to the PSCT model and the tumour growth was exposed to single or repeated treatments with doxorubicin, and thereafter evaluated for cell death, apoptosis, and proliferation. Dose dependent cytotoxic effects were observed, this way corroborating the experimental platform for this type of analysis. Notably, analysis of doxorubicin-resilient BE(2)-C growth in the PSCT model revealed an unexpected 1,5-fold increase in Ki67-index (p3-fold increase in active cell cycle (i.e. cells positive staining for PH3 together with incorporation of EdU) (p<0.01). Considering the clinical challenge for treating high-risk NB, the discovery of a therapy-provoked growth-stimulating effect in the multi-resistant and p53-mutated BE(2)-C cell line, but not in the drug-sensitive p53wt IMR-32 cell line, warrants further studies concerning generality and clinical significance of this new observation

The effects of doxo on BE(2)-C and IMR-32 tumours in the PSCT model.

<p>Fraction of cells (%; mean±SD) expressing indicated markers following indicated treatments. mock = PBS; 4+4 = Repeated dose 4+4mg/kg doxo with 48h interval; 8 = single dose 8mg/kg doxo. Effects measured 96h after first administration of doxo. Data based on 5 PSCT per group. For statistical analysis, see text.</p

Micro colonisation of IMR-32 tumours in the PSCT model.

<p>(A) Schematic illustration; NB cells were injected into an arbitrary position centrally in the PSCT cellular mass, resulting in multiple micro-colonisations from migrating NB cells. (B) A representative FFPE section of a PSCT with four IMR-32 colonies indicated (red borders). (C) IMR-32 colony surrounded by loose mesenchyme. LM = loose mesenchyme; NE = neural epithelium; C = cartilage; M = muscle; Blue arrows = vessels. Size bars: B:5mm, C:500μm.</p

The effects of doxo on BE(2)-C tumours growing in the PSCT model.

<p>The effects of doxo on BE(2)-C tumours growing in the PSCT model.</p

Antibodies and kits used for immunohistochemistry.

<p>Antibodies and kits used for immunohistochemistry.</p

The effects of doxo on PSCT non-malignant embryonic tissues.

<p>Immunohistochemistry staining of formalin-fixed paraffin-embedded PSCT histological slides, following intra peritoneal injection of the host mouse with 8mg/kg doxo (A-D), or 4+4mg/kg doxo (E-H). High frequencies of positive staining for Ki67 in tissues compatible with neural epithelium, muscle and cartilage can be seen, indicative of extensive proliferation (A,C,E,G). Low frequencies of positive staining for cleaved caspase 3 can be seen in NE, muscle and cartilage, indicative of low frequencies of apoptosis (B,D,F,H). Size bars: 50μm.</p

Data set for BE(2)-C <i>in vitro</i>.

<p>Data set for BE(2)-C <i>in vitro</i>.</p

The effects of doxo in neural epithelium in the PSCT model.

<p>The effects of doxo in neural epithelium in the PSCT model.</p

Data set for IMR-32 <i>in vitro</i>.

<p>Data set for IMR-32 <i>in vitro</i>.</p

Percentage Ki67 positive cells in BE(2)-C tumour following doxo treatment in the PSCT model.

<p>Fraction of cells (%; mean±SD) expressing Ki67 96 hours after indicated treatments. Mock = diluent (1xPBS). Data based on 5 PSCT per group. For statistical analysis, see text.</p