Figure 1.
Simplified PI3K/AKT pathway map.
In this study we examine the levels of PI3K, mTOR and phosphorylated Protein S6 as indicators of cellular response to PI3K/mTOR inhibition.
Figure 2.
U2OS cell proliferation is unaffected by seeding density.
U2OS cells were seeded at various densities in 300 µL collagen gels. Cells were imaged immediately after seeding and 96 hours later. Data is presented as a ratio of the cell population after 96 hours to the cell population at initial seeding.
Figure 3.
Proliferation of U2OS in 3D collagen gels with different concentration of live cell dye CMRA.
Cells were grown in 3 mg/ml collagen gels. Cells were counted immediately after seeding and 96 h post-seeding. Data is presented as a ratio of 96 h data to 0 h data. No significant difference in cell proliferation was observed.
Figure 4.
Proliferation of U2OS on standard 2D tissue culture plastic compared to 3D collagen gels.
Cells were plated and immediately imaged. 96 hours later the cells were imaged again and the cell population was compared to the initial numbers. Cells in 3D show a dramatic reduction in proliferation, with approximately 1 cell division occurring over the 96 h experiment. Cells in 2D showed much more aggressive growth, with cell doubling occurring every 24–30 hours. (p<.05 between 2D and 3D samples).
Figure 5.
Proliferation of MCF7 cells on standard 2D tissue culture plastic compared to 3D collagen gels.
Cells were plated and immediately imaged. 96 hours later the cells were imaged again and the cell population was compared to the initial numbers. Cells in 3D show a dramatic reduction in proliferation. Cells in 2D showed much more aggressive growth, with cell doubling occurring every 30–36 hours. (p<.05 between 2D and 3D samples).
Figure 6.
Migration of U2OS cells on standard 2D tissue culture plastic compared to 3D collagen gels.
Cells were plated, and then immediately underwent timelapse imaging for 12 hours. Data here is the average speed of the cells over the course of the imaging sequence. Cell tracking was done using Imaris® software suite (Bitplane Inc., St. Paul, MN), with additional data processing done in MatLab®. Cells on tissue culture plastic moved 2.5 times fasters than cells grown in 3D collagen gels (p<.01).
Figure 7.
Protein expression blots from U20S cells grown on tissue culture plastic or in 3D collagen gels.
Cells were plated, and then harvested 24 hours later. Cell lysates were kept at −80°C if not immediately used. Cells in 3D had undetectable amount of pAkt, indicating a low level of overall pathway activity. This is consistent with the reduction in cell proliferation seen in earlier experiments.
Figure 8.
Ratio of U20S cell number in samples treated with PI103 to samples left untreated.
Data here is presented as a ratio of cell count in drug treated sample to untreated controls. Cells in 4 mg/ml collagen gels had a 90% cell survival rate after 8 days of drug treatment. Cells were more sensitive to the effects of the drug on 2D tissue culture plastic and in 3 mg/ml gels. Interestingly, cells showed similar behavior in 3 mg/ml and 4 mg/ml gels when untreated, but had a markedly different response to PI103. All Day 8 data points are significantly different (p<.05).
Figure 9.
Ratio of MCF7 cell number in samples treated with PI103 to samples left untreated.
Data here is presented as a ratio of cell count in drug treated sample to untreated controls. As in figure 8, data is presented as a ratio of cell counts in the drug treated and untreated samples. Similar to U2OS cells, MCF7 cell survived very well in 4 mg/ml collagen gels when compared to cells in 2D. Unlike U2OS cells, MCF7 cells in 3 mg/ml collagen gels survived equally as well as cells in stiffer gels. Both of these conditions yielded cells with higher drug resistance than cells grown in monolayer. Although this behavior is not as complex as observed in U2OS cells, it highlights the difference between 2D and 3D culture platforms.
Figure 10.
Migration of U2OS cells over 6 hours in different culture conditions.
Cells were stained with CMRA whole cell stain prior to being embedded in collagen. Cells were tracked by imaging the gels every 15 minutes for 6 hours. Cell speed was calculated using a 3D displacement algorithm performed on cell centroid location data from Imaris®.
Figure 11.
Western Blots for protein S6, PRAS, Akt and GAPDH in U2OS cells on different substrates with and without drug treatment.
Cells were plated, and then harvested 24 hours later. Cell lysates were kept at −80°C if not immediately used. Drug treatment reduced levels of pS6 in all samples, consistent with the observed increase in cell doubling time. Paradoxically, this reduction was most pronounced in cells grown in 4 mg/ml despite these cells showing the highest proliferative resistance to the effects of PI103.