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Quantitative analysis of the EGF receptor autocrine system reveals cryptic regulation of cell response by ligand capture

¹ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
² Newborn Medicine, Children’s Hospital, Boston, MA 02115, USA
³ Fundamental Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA
⁴ Division of Bioengineering and Environmental Health and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

View larger version (14K): [in a new window]   Fig. 1. EGF accumulation in the bulk medium in the presence and absence of EGFR-blocking antibodies. Monolayers of cells were induced to produce EGF at a rate of approximately 1000 moleculesxcell-1xmin-1 using tetracycline. Fresh medium with () or without () 5.0 µg/ml 225 mAb was added to the cells and, at the indicated time, the medium was collected and assayed for EGF using an ELISA. The fraction of EGF captured, as calculated from the ratio of EGF concentrations without and without 225 mAb, remained constant over 12 hours (insert). The error bars represent the s.d. of one experiment in duplicate.

View larger version (14K): [in a new window]   Fig. 2. The fraction of ligand that is captured decreases as ligand release increases. Cells that had been induced to release ligand at different rates using tetracycline and a ligand-release inhibitor were allowed to condition their medium for 8-14 hours. The medium was collected and assayed for EGF by ELISA. The concentration of EGF was then converted to a ligand secretion rate. The total ligand secretion rate, VLT, was calculated from samples containing 5.0 µg/ml 225 mAb during cell medium conditioning. Fraction ligand captured is determined by comparing ligand secretion rates calculated from samples without 5.0 µg/ml 225 mAb to those with 5.0 µg/ml 225 mAb during cell medium conditioning. The error bars represent the s.e.m. of a set of data points taken and analyzed in duplicate.

View larger version (10K): [in a new window]   Fig. 3. As the concentration of anti-EGFR blocking antibody increases, the concentration of EGF in the bulk medium increases. Cells induced to secrete ligand at a rate of approximately 600 moleculesxcell-1xminute-1using tetracycline were allowed to condition their medium in the presence of 0.0, 0.05, 0.1, 0.5 or 5.0 µg/ml 225 mAb for 12 hours. The medium was collected and assayed for EGF by ELISA. The error bars represent the s.d. of one experiment in duplicate.

View larger version (19K): [in a new window]   Fig. 4. The relationship between EGF production, EGFR expression and fraction of EGF captured. (A) Cells were induced to release ligand at the rate of 90 (), 490 (), 780 () and 1700 () moleculesxcell-1xminute-1using tetracycline and a ligand-release inhibitor. Fresh medium with 0.0, 0.05, 0.1, 0.5 and 5.0 µg/ml 225 mAb was then added, and the cells were allowed to condition their medium for 8-14 hours. The medium was collected and assayed for EGF by ELISA. Fraction ligand captured was calculated as described in Fig. 2. The concentration of 225 mAb was converted to number of accessible surface receptors as described in Appendix A. The y error bars represent the s.e.m. of a set of data points taken and analyzed in duplicate. The x error bars represent propagated error as described in Materials and Methods. (B) The data presented in Figs 2 () and 4A were used to calculate the effective receptor production rate in moleculesxcell-1xminute-1(VR) at a certain total ligand secretion rate (VLT) for a given concentration of 225 mAb as outlined in Appendix A. The error bars represent propagated error as described in Materials and Methods. The line through the data is a Gaussian distribution fit by least squares and weighed by the estimated errors.

View larger version (22K): [in a new window]   Fig. 5. ECAR increases in response to increasing endogenous and exogenous EGF. (A) Cells lacking EGF expression were grown in transwells and placed in a cytosensor. Once a baseline ECAR was established, the cells were challenged with an addition of exogenous EGF, 7 nM () or 0.1 nM (). The reported ECAR is normalized to the baseline signal that was set to 0 for clarity. (B) Cells were induced to secrete ligand at a steady state rate of 0 (), 45 (), 400 (), 700 (), 1300 (), 3600 () moleculesxcell-1xminute-1 using tetracycline and a ligand-release inhibitor. The cells were placed on the cytosensor and once a baseline was established, they were challenged with 40, 20, 5, 2.5, 1.2, 0.6, 0.3, 0.15, 0.08 ng/ml EGF. The maximal change in ECAR, normalized to baseline, was recorded as a function of the challenge concentration. The error bars represent the s.e.m. of three experiments in duplicate.

View larger version (12K): [in a new window]   Fig. 6. The effective autocrine ligand concentration increases as ligand secretion increases. The effective autocrine ligand concentration was calculated by using the cytosensor data shown in Fig. 5B in conjunction with the whole cell model outlined in Appendix B. The error bars represent propagated error propagated from the s.e.m. of receptor synthesis rate, the s.e.m. of the fraction of ligand degraded, the s.e.m. of the ligand production rate, and the standard error of the estimate associated with the ECAR-Max curve fits.

View larger version (17K): [in a new window]   Fig. 7. Fraction of occupied receptors increases as ligand secretion increases. The fraction of occupied receptors was calculated by using the cytosensor data shown in Fig. 6 with the whole cell model outlined in Appendix B and can be calculated based on the total number of occupied receptors () or the number of occupied surface receptors (). The error bars represent propagated error from the s.e.m. of receptor synthesis rate, the s.e.m. of the fraction of ligand degraded, the s.e.m. of the ligand production rate, and the standard error of the estimate associated with the ECAR-Max curve fits. The dashed line (···) represents the data from Fig. 4B.

View larger version (13K): [in a new window]   Fig. 8. Autocrine signaling in human mammary epithelial cells. (A) Cells were grown in 12-well dishes for 6 days with medium alone (control), mAb 225 (10 µg/ml), BB-2116 (10 µM) or EGF (2 nM). The medium was changed every other day and the cells were counted on day 6. (B) Cells were grown as described above with concentrations of BB-2116 ranging from 0.01 to 10 µM. Cell numbers were determined on day 6. Parallel cultures of cells in 60 mm dishes were treated with the same concentrations of BB-2116 and 20 µg/ml of mAb 225 for 18 hours. The medium was then collected and the TGF- levels were determined by RIA. Cell number was 2x106 per dish. Error bars represent the SD of triplicate samples.

View larger version (13K): [in a new window]   Fig. 9. Receptor de-sensitization is not responsible for the shift in ECAR-Max as cells release more ligand. Cells releasing ligand at 1300 () and 830 () moleculesxcell-1xminute-1 as controlled by tetracycline concentration and a ligand-release inhibitor were placed on the cytosensor with () or without () 0.1 µg/ml 225 mAb in the medium. This resulted in both cells with essentially the same number of surface accessible receptors (7500 per cell). The cells were challenged with 40, 20, 5, 2.5, 1.2, 0.6, 0.3, 0.15 ng/ml EGF and the ECAR-Max was determined as described in Fig. 5. The error bars represent the s.e.m. of three experiments in duplicate.

View larger version (13K): [in a new window]   Fig. 10. Whole-cell modeling, with endogenous ligand concentration determined from microphysiometer experiments, predicts the occupancy of surface receptors on autocrine cells. Nearly confluent monolayers of autocrine cells secreting different levels of ligand as controlled by tetracycline and a ligand-release inhibitor were incubated with 0.6 µg/ml 125I-13A9 mAb, an EGFR non-blocking antibody, for 3 hours at 37°C. 125I-13A9 mAb was removed by acid stripping as described in Materials and Methods to determine the number of surface receptors (). Using whole-cell kinetic modeling as described in Appendix B, the predicted total number of surface receptors was determined (). The error bars for the radiolabeling results represent the s.e.m. of two to five experiments in triplicate. The error bars for the modeling results represent propagated error from the s.e.m. of receptor synthesis rate, the s.e.m. of the fraction of ligand degraded, the s.e.m. of the ligand production rate, and the standard error of the estimate associated with the ECAR-Max curve fits.