These data suggest that some neuroblastomas acquire p53 mutations during therapy, which is associated with a loss of p53 function, and can confer high-level multidrug resistance.

Neuroblastoma, the second most common solid tumor in children, frequently metastasizes to the bone marrow and the bone. Neuroblastoma cells present in the bone marrow stimulate the expression of interleukin-6 (IL-6) by bone marrow stromal cells (BMSC) to activate osteoclasts. Here we have examined whether stromal-derived IL-6 also has a paracrine effect on neuroblastoma cells.

Ability to grow under anchorage-independent conditions is one of the major hallmarks of transformed cells. Key to this is the capacity of cells to suppress anoikis, or programmed cell death induced by detachment from the extracellular matrix. To model this phenomenon in vitro, we plated Ewing tumor cells under anchorage-independent conditions by transferring them to dishes coated with agar to prevent attachment to underlying plastic. This resulted in marked up-regulation of E-cadherin and rapid formation of multicellular spheroids in suspension. Addition of calcium chelators, antibodies to E-cadherin (but not to other cadherins or B 1 -integrin), or expression of dominant negative E-cadherin led to massive apoptosis of spheroid cultures whereas adherent cultures were unaffected. This correlated with reduced activation of the phosphatidylinositol 3-kinase-Akt pathway but not the Rasextracellular signal-regulated kinase 1/2 cascade. Furthermore, spheroid cultures showed profound chemoresistance to multiple cytotoxic agents compared with adherent cultures, which could be reversed by A-E-cadherin antibodies or dominant negative E-cadherin. In a screen for potential downstream effectors of spheroid cell survival, we detected E-cadherin-dependent activation of the ErbB4 receptor tyrosine kinase but not of other ErbB family members. Reduction of ErbB4 levels by RNA interference blocked Akt activation and spheroid cell survival and restored chemosensitivity to Ewing sarcoma spheroids. Our results indicate that anchorage-independent Ewing sarcoma cells suppress anoikis through a pathway involving E-cadherin cell-cell adhesion, which leads to ErbB4 activation of the phosphatidylinositol 3-kinase-Akt pathway, and that this is associated with increased resistance of cells to cytotoxic agents. [Cancer Res 2007;67(7):3094-105]

Background The National Cancer Institute (NCI) has established the Pediatric Preclinical Testing Program (PPTP) for testing drugs against in vitro and in vivo childhood cancer models to aid in the prioritization of drugs considered for early phase pediatric clinical trials. Procedures In vitro cytotoxicity testing employs a semi-automated fluorescence-based digital imaging cytotoxicity assay (DIMSCAN) that has a 4-log dynamic range of detection. Curve fitting of the fractional survival data of the cell lines in response to various concentrations of the agents was used to calculate relative IC50, absolute IC50, and Ymin values The panel of 23 pediatric cancer cell lines included leukemia (n=6), lymphoma (n=2), rhabdomyosarcoma (n=4), brain tumors (n=3), Ewing family of tumors (EFT, n=4), and neuroblastoma (n=4). The doubling times obtained using DIMSCAN were incorporated into data analyses to estimate the relationship between input cell numbers and final cell number. Results We report in vitro activity data for three drugs (vincristine, melphalan, and etoposide) that are commonly used for pediatric cancer and for the mTOR inhibitor rapamycin, an agent that is currently under preclinical investigation for cancer. To date, the PPTP has completed in vitro testing of 39 investigational and approved agents for single drug activity and two investigational agents in combination with various “standard” chemotherapy drugs. Conclusions This robust in vitro cytotoxicity testing system for pediatric cancers will enable comparisons to response data for novel agents obtained from xenograft studies and from clinical trials.