Investigating the effects of MAD-28 on BT474 spheroids
Abstract
Two-dimensional (2D) cell cultures are primarily used in breast cancer research, yet the results do not correspond with in vivo studies. Tumors often have complex microenvironments with interactions which are difficult to mimic in vitro. With the use of three-dimensional (3D) spheroids, a more accurate environment is generated. In this thesis, we established a protocol that allows culture of BT474 under 3D conditions and evaluated the sensitivity of the cell line to MAD-28 under both 2D and 3D growth conditions. Different numbers of BT474 cells were grown in a 96-well non tissue culture treated plate for up to 96h. After 72h of culture, 80% of the spheroids had sizes up to 200μm, when starting with 5,000 or 10,000 cells per well. Drug sensitivity tests were performed using 10,000 BT474 cells per well after 72h of growth under 2D and 3D conditions. Western blot analysis showed a pronounced increase in the expression levels of E-cadherin at the 3D conditions. Upon treatment with different concentrations of MAD-28, HER-2, a known client of Hsp90, degraded quickly, while Hsp70 was slightly upregulated. Treatment with 2.0µM of MAD-28 efficiently activated the apoptotic cascade, as indicated with the presence of cleaved PARP. Interestingly, a more detailed analysis of viability under both 2D and 3D conditions, using an ATP luminescence assay, showed that the spheroids were more susceptible to MAD-28 compared to the 2D growing BT474 cells. Future experiments are necessary to address potential initiation of alternative cell death pathways along with apoptosis.
Investigating the effects of MAD-28 on BT474 spheroids
Two-dimensional (2D) cell cultures are primarily used in breast cancer research, yet the results do not correspond with in vivo studies. Tumors often have complex microenvironments with interactions which are difficult to mimic in vitro. With the use of three-dimensional (3D) spheroids, a more accurate environment is generated. In this thesis, we established a protocol that allows culture of BT474 under 3D conditions and evaluated the sensitivity of the cell line to MAD-28 under both 2D and 3D growth conditions. Different numbers of BT474 cells were grown in a 96-well non tissue culture treated plate for up to 96h. After 72h of culture, 80% of the spheroids had sizes up to 200μm, when starting with 5,000 or 10,000 cells per well. Drug sensitivity tests were performed using 10,000 BT474 cells per well after 72h of growth under 2D and 3D conditions. Western blot analysis showed a pronounced increase in the expression levels of E-cadherin at the 3D conditions. Upon treatment with different concentrations of MAD-28, HER-2, a known client of Hsp90, degraded quickly, while Hsp70 was slightly upregulated. Treatment with 2.0µM of MAD-28 efficiently activated the apoptotic cascade, as indicated with the presence of cleaved PARP. Interestingly, a more detailed analysis of viability under both 2D and 3D conditions, using an ATP luminescence assay, showed that the spheroids were more susceptible to MAD-28 compared to the 2D growing BT474 cells. Future experiments are necessary to address potential initiation of alternative cell death pathways along with apoptosis.