J. S. Laue1, C. H. Quinn1, J. R. Julson1, N. Nazam1, S. Butey1, R. L. Stinson1, L. Markert1, J. E. Stewart1, C. A. Martin1, E. A. Beierle1 1University Of Alabama at Birmingham, Pediatric Surgery, Birmingham, Alabama, USA
Introduction:
Children living in lower socioeconomic conditions are subject to higher levels of stress. It is well documented that children from lower socioeconomic backgrounds have worse survival rates for neuroblastoma. We propose that higher levels of stress may impact neuroblastoma tumor biology and may partially explain these disparities in survival. Our previous research demonstrated that cortisol, the human stress hormone, increased neuroblastoma proliferation and motility, suggesting a more aggressive phenotype. Cancer cell stemness is another factor known to contribute to a more aggressive neuroblastoma phenotype. We hypothesized that cortisol might alter the stem cell phenotype of neuroblastoma accounting for the more aggressive tumor properties seen after cortisol treatment.
Methods:
Two established human neuroblastoma cell lines, SK-N-AS (MYCN non-amplified) and SK-N-BE(2) (MYCNamplified), and the human patient derived xenograft (PDX) COA6 (MYCN amplified), were utilized. Cells were treated with cortisol (0-1 μM) for 72 hours. Two methods were employed to evaluate stemness. q-RT-PCR was utilized to assess the mRNA abundance of established stem cell markers, NANOG, SOX2, and OCT4. An extreme limiting dilution assay (ELDA) was performed to assess tumorsphere formation in the COA6 PDX cells. Data were reported as mean ± standard error of the mean, compared with two-tailed Student’s t-test or ANOVA as appropriate, and p≤0.05 considered significant.
Results:
The mRNA abundance of neuroblastoma stem cell markers NANOG, SOX2, and OCT4, was significantly increased in SK-N-BE(2) and COA6 cells and trended toward an increase in SK-N-AS cells following cortisol treatment (Figure). Using an ELDA, we found that cortisol treatment significantly increased tumorsphere forming capacity in COA6 cells, indicating that the cells were more stem-like.
Conclusion:
Cortisol treatment resulted in an increase in neuroblastoma stemness as demonstrated by increased mRNA abundance of stem cell markers and increased tumorsphere forming capacity. These data suggest that increased levels of stress may play an important role in neuroblastoma biology. Further research is necessary to translate these findings to begin to address and eliminate the effects of health disparities in neuroblastoma.
