Abstract 1279: Sigma-2 receptor-mediated cell death is altered by cell density in human SK-N-SH neuroblastoma 2-D and 3-D cultures

Sigma-2 receptors are highly expressed in tumor cell lines compared to normal cells and are more highly upregulated in proliferative than in quiescent cells. Sigma-2 receptor agonists induce apoptotic cell death. Here, we investigate the effect of cell confluence at the time of harvest, cell plating density, and 3-D cell aggregation on the cytotoxic potency of the irreversible sigma-2 receptor partial agonist CM572. Sensitivity to sigma-2 receptor agonists has in practice appeared to vary with cell confluency, an effect likely due to proliferative state. To examine 2-D SK-N-SH cell culture sensitivity to CM572, we varied two parameters: 1) cell confluence at the time of harvest (cells harvested upon reaching 60% confluency or cells harvested after remaining at 100% confluency for 3 days) and 2) cell plating density (low: 7,000, medium: 15,000, or high: 30,000 cells/well). Cells were treated with CM572 24 h after plating. Using MTT assay, CM572 EC50 values for cells harvested at 60% confluence were similar regardless of cell plating densities (μM): 4.92 ± 2.24, 7.6 ± 1.68, and 6.06 ± 1.30 for cells plated at low, medium, and high densities, respectively. In contrast, CM572 EC50 values for cells harvested at 100% confluence for 3 days were significantly dependent on plating density (μM): 17.62 ±7.62, 35.33 ± 35.3, and >50 for cells plated at low, medium, and high densities, respectively. [3H]DTG Scatchard analysis of cells harvested at 60% confluence vs. 100% confluence for 3 days revealed no significant difference in sigma-2 receptor number: Bmax = 212.3 ± 34.22 and 173.0 ± 4.31 fmol bound/mg protein, respectively. The effect of CM572 on cell aggregation and cytotoxicity was additionally studied in 3-D SK-N-SH cultures. Cells were seeded into rod-shaped agarose wells, to which they could not adhere but only self-aggregate over a period of 24 h. Subsequent treatment with CM572 over 24-72 h significantly reduced 3-D aggregation, which was defined as an increase in the length of the 3-D culture rod after aggregation. However, treatment of these 3-D cultures with up to 70 μM CM572 for 24-72 h resulted in cytotoxicity levels of less than 10%, as measured by the LDH assay. This indicates a significant decrease in CM572 potency compared to the 2-D EC50. Therefore, sensitivity to CM572 decreases both after cells are confluent for 3 days at 100% in 2-D culture and in cells that form aggregates in 3-D culture. These results show that sensitivity of SK-N-SH cell cultures to CM572 is dependent on cell density, suggesting that CM572 may cause cell death in a cell-cycle dependent manner. This may be related to SK-N-SH cells transitioning from a proliferative to quiescent state when they are confluent in 2-D culture or form aggregates in 3-D culture. Taken together, these results suggest that cytotoxic sigma-2 receptor ligands such as CM572 may be more potent in proliferating cancer cells and/or cells leaving tumor masses in the process of metastasis. Citation Format: Cheri Z. Liu, Vira Behnam, Wayne D. Bowen. Sigma-2 receptor-mediated cell death is altered by cell density in human SK-N-SH neuroblastoma 2-D and 3-D cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1279.