Fig 1.
Optimization of co-culture system.
A) Optimization of the tumor cell: fibroblast ratio. Cancer cells and fibroblasts (MRC5) were cultured in 96 well-well plates as either monocultures or co-cultures as described in the cell viability assay in the Materials and Methods section. Different ratios of tumor cells to fibroblasts were used as indicated. Cell viability was measured on day 5. We observed that the cell viability on day 5 was the highest at tumor cell: fibroblasts ratio of 1:1.5) on day 5. B) Optimization of the co-culture duration. Cancer cells and fibroblasts (MRC5) were cultured in 96-well plates as monocultures or co- cultures as described in the cell viability assay in the Materials and Methods section. The tumor cells and fibroblasts were co-cultured at a ratio of (1:1.5), and cell viability was measured on days 3, 5 and 7. The viability in of the co- cultured cells increased from day 3 to day 5 and then decreased slightly on day 7.
Fig 2.
Comparison of the Boyden chamber, 2D co-culture and 3D co-culture systems.
To compare the 3D co-culture system to the 2D co-culture and trans- well co-culture systems, tumor cells and fibroblasts were cultured as either as mono-cultures or co-cultures for 5 days as described in the Materials and Methods section. Cell viability was measured on day 5. We observed that 3D co-culture of the tumor cells with fibroblasts induced differential proliferation in co-cultures compared to the Boyden chamber or the 2D co-culture system.
Table 1.
Cell line panel.
Fig 3.
Co- culturing the tumor cells with MRC5 fibroblasts influences cell survival.
Tumor cells and MRC5 fibroblasts were cultured as either co- cultures or monocultures as described. Cell viability was measured based on the total ATP content on day 5 after cell seeding using CellTiterGlo. A) Seven of the 9 pancreatic cancer cell lines showed exhibited a significant increase in cell survival upon co- culturing with MRC5 cells. Bxpc3 cells exhibited the greatest fold-change in proliferation among these cell lines upon co-culturing. B) Two out of the 7 of the lung cancer cell lines exhibited a significant increase in cell survival upon co- culturing with MRC5 cells; out of which the H596 cells exhibited the greatest fold-change in proliferation upon co-culturing. C. Of the two breast cancer cell lines that exhibited an increase in proliferation upon co-culturing with MRC5 fibroblasts, only the BT20 cells exhibited a significant increase in cell survival.
Fig 4.
Co- culturing the tumor cells with primary tumor associated fibroblasts (TAFs) influences cell survival similar to MRC5 fibroblasts.
One tumor cell line that exhibited the greatest fold-change in cell survival due to co-culturing (Bxpc3, H596 and BT20) and one cell line that did not exhibit an increase in survival upon co-culture with MRC5 cells from each cancer type (Suit2, H1993 and SKBR3) were co-cultured with corresponding primary TAFs (129A, lung TAFs and or 161A, breast TAFs) or organs-specific fibroblasts (LT2, pancreatic fibroblasts) for 5 days followed by measurement of cell viability on day 5 using CellTiterGlo. All three cell lines that exhibited a significant increase in cell survival upon co-culturing with MRC5 fibroblasts (Bxpc3, H596 and BT20) also exhibited increased survival in co-culture with TAFs, whereas the cell lines that did not exhibit increased survival in co-culture with MRC5 (Suit2, H1993 and SKBR3) retained their proliferative properties even upon co-culturing with TAFs.
Fig 5.
Co- culturing the tumor cells with MRC5 fibroblasts induces differential secretion of growth factors and cytokines.
Tumor cells and fibroblasts were co-cultured for 5 days as described, and supernatants were collected. The levels of EGF, HGF and IL6 secreted by the mono- or co-cultures in the supernatants were measured using Luminex multiplex technology. The relative levels of these secreted factors are plotted in the graphs as the mean fluorescence intensities (MFI). The error bars represent the standard deviation of three replicates. A) The pancreatic and breast cancer co-culture supernatants contained increased EGF levels compared to the lung cancer co-culture supernatants. B) Increased HGF levels were detected in the supernatants from the lung cancer co-cultures but not in those from the corresponding mono-cultures. The Lung fibroblast cell line MRC5 and the TAFs, 129A, also secreted high levels of HGF into the supernatants in monoculture. C) High levels of IL6 were detected in the supernatants from BT20 cells co-cultured with MRC5 or 161A primary breast TAFs and in the supernatants from the H596 cell line mono- and co-cultures. The fibroblasts cell lines MRC5, and LT2 and the primary TAFs, 129A also produced IL6 in mono-culture.
Fig 6.
Co-culturing the tumor cells with fibroblasts influences their response to therapeutic agents.
Cancer cells and fibroblasts (MRC5 or primary TAFs) were co-cultured as described in the cell viability assay for 5 days in the presence of inhibitory antibodies against EGFR, cMet, IL6 and or IGF1R. Cell viability was measured on day 5 using the CellTiterGlo as described for the cell viability assay. The percentage of surviving cells (% survival) was calculated relative to the respective IgG control. A) The mono-cultured BxPc3 cells treated with Erbitux exhibited a significant reduction in cell survival, whereas in the BxPc3 cells co-cultured with MRC5 or LT2 cells were not strongly affected by Erbitux (approximately 20%). Upon treatment with the IGF1R antibody, the survival of Bxpc3 cells was moderately reduced in the co-cultures compared to the monocultures. Treatment with the IL6 and or cMet antibodies induced a slight reduction in cell survival. B) Upon treatment with the anti-cMet antibody lung cancer cell line, H596, did not exhibit a significant reduction in cell survival in monoculture. In co-culture with MRC5 or 129A TAFs, treatment with the anti-cMet antibody induced a significant reduction of in cell survival. C) The breast cancer cell line, BT20 responded to treatment with the anti-IL6 antibody in co-culture exhibiting a significant reduction of cell survival. The mono-cultured BT20 cells treated with the anti- IL6 antibody exhibited no reduction in cell survival.