A. L. Alvarez1, B. T. Craig1, E. J. Rellinger1, J. Qiao1, D. H. Chung1 1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA
Introduction: Neuroblastoma is responsible for nearly 15% of all pediatric cancer-related deaths and remains a difficult pediatric solid tumor to cure. Acute tumor hypoxia in neuroblastoma is known to promote dedifferentiation and drug resistance in part by stabilizing hypoxia-inducible factors (HIF-1α and HIF-2α ) which function as key drivers of glycolysis, and has been well characterized. Chronic tumor cell hypoxia is also likely to exist in aggressive solid tumors such as neuroblastoma, yet its exact role in tumorigenicity remains poorly understood. We hypothesize that chronic hypoxia is a microenvironmental stress stimulus that plays a critical role in driving neuroblastoma progression. The aim of this study was to establish an in vitro model of chronic hypoxia to assess neuroblastoma cell growth.
Methods: Human neuroblastoma cell lines, BE(2)-C (MYCN-amplified) and SK-N-SH (MYCN-single copy), were cultured in 1% ambient oxygen for 1, 3, 7, or 10 d. Post-hypoxia cell viability was assessed over a time course (0 to 96 h) using CCK-8 assay. Soft agar colony formation after 2 weeks incubation in 0.4%/0.8% agarose gel was used to assess anchorage-independent growth. SDS-PAGE was used to assess protein expression. Two-tailed unpaired Student’s t-test was used for statistical analysis and a p value of <0.05 was considered significant.
Results: Ten days of 1% O2 increased the proliferative capacity of both cell lines compared to controls cultured at ambient O2 (p <0.01 for each individual comparison). Specifically, MYCN–single copy SK-N-SH cells demonstrated a two-fold increase in proliferation following 10 d exposure to 1% O2 compared to controls cultured in 21% O2. A more modest increase (1.5-fold) was observed in the MYCN-amplified BE(2)-C cells. Anchorage-independent growth was also observed in both BE(2)-C and SK-N-SH cells cultured for 10 d at 1% O2. The well-established mediators of the acute hypoxic response HIF-1α and HIF-2α were both upregulated by 1 d as expected, validating the hypoxia culture chamber. Interestingly, expression levels of both factors returned to basal levels by 10 d of continuous hypoxic exposure. Hexokinase-1 and lactate dehydrogenase-A, two downstream markers of glycolytic flux, were similarly decreased at 10 d of continuous exposure to 1% O2.
Conclusion: Neuroblastoma is a pediatric solid tumor with significant intra-tumoral heterogeneity featuring areas of necrosis and hemorrhage that may implicate chronic hypoxia as a key microenvironmental regulator of tumor progression. Our model shows that prolonged continuous exposure to subnormal oxygen levels induces a phenotype that enhances both cellular proliferation and anchorage independent growth. This behavioral change appears to be both a HIF- and glycolysis-independent phenomenon.