Proc. Natl. Acad. Sci. USA Vol. 95, pp. 15368–15373, December 1998 Cell Biology Real-time quantitative measurement of autocrine ligand binding indicates that autocrine loops are spatially localized DOUGLAS A. LAUFFENBURGER*†‡,GREGORY T. OEHRTMAN†,LAURA WALKER†, AND H. STEVEN WILEY§ *Division of Bioengineering & Environmental Health and Center for Biomedical Engineering and †Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; and §Department of Pathology, University of Utah Medical Center, Salt Lake City, UT 84132 Communicated by Edwin N. Lightfoot, Jr., University of Wisconsin, Madison, WI, October 15, 1998 (received for review July 21, 1998) ABSTRACT Autocrine ligands are important regulators rapid (,30 sec) and small (,0.1 unit) changes in solution pH of many normal tissues and have been implicated in a number in the cellular microenvironment in an '1 ml chamber above of disease states, including cancer. However, because by the sensor. These pH changes [termed ‘‘extracellular acidifi- definition autocrine ligands are synthesized, secreted, and cation rate’’ (ECAR)] can arise from both metabolic and bound to cell receptors within an intrinsically self-contained regulatory events and have been shown to be quantitatively ‘‘loop,’’ standard pharmacological approaches cannot be used related to specific activation of many types of cell receptors, to investigate relationships between ligandyreceptor binding including tyrosine kinase receptors, G protein receptors, and and consequent cellular responses. We demonstrate here a ion channel receptors (16) with EC50 values similar to those new approach for measurement of autocrine ligand binding to derived from direct-labeled ligand binding (17). Hence, for cells, using a microphysiometer assay originally developed for exogenous ligands the microphysiometer can be used to obtain investigating cell responses to exogenous ligands. This tech- real-time, kinetic measurements of receptor binding once a nique permits quantitative measurements of autocrine re- calibration curve has been generated relating ECAR data to sponses on the time scale of receptor binding and internal- labeled ligandyreceptor-binding data. This has proven of spe- ization, thus allowing investigation of the role of receptor cial value for high-throughput screening of pharmacological trafficking and dynamics in cellular responses. We used this compounds. technique to investigate autocrine signaling through the epi- Because the quantitative relationship between ECAR and dermal growth factor receptor by transforming growth factor ligandyreceptor binding should be identical regardless of alpha (TGFa) and found that anti-receptor antibodies are far whether the particular ligand is added exogenously or is more effective than anti-ligand antibodies in inhibiting auto- self-produced in an autocrine fashion, we reasoned that we crine signaling. This result indicates that autocrine-based could adapt the microphysiometer to permit real-time quan- signals can operate in a spatially restricted, local manner and titative determination of autocrine ligand binding in an anal- thus provide cells with information on their local microenvi- ogous manner. We demonstrate here this new methodology by ronment. calibrating ligandyreceptor binding to ECAR and establishing key dose–response relationships. As an example of the utility Awareness of the crucial role that autocrine ligands play in of this approach, we test a theoretical prediction of the tissue physiology and pathology is increasing across a wide comparative effectiveness of anti-receptor (‘‘blocker’’) versus spectrum of fields including embryonic and tissue develop- anti-ligand (‘‘decoy’’) antibodies in interrupting autocrine ment, immunology, cancer, angiogenesis, dermatology, neu- signaling. roscience, and biotechnology (1–8). The concept of autocrine ligandyreceptor cell signaling was introduced almost two de- MATERIALS AND METHODS cades ago (9), and a range of physiological and pathological Materials. Parental B82 mouse fibroblasts lacking epider- situations are now known to be regulated by self-secreted mal growth factor receptor (EGFR) and EGFR-expressing factors (10). Unfortunately, understanding how autocrine sys- B82 cells were a gift from Gordon Gill (University of Cali- tems work is severely limited by an inability to construct fornia, San Diego). Use of the tetracycline-controlled two- something as simple and fundamental to receptor biology as a plasmid system (20) to create the transforming growth factor dose–response curve for ligandyreceptor binding. This is due alpha (TGFa) autocrine cell system has been described by to the recursive nature of autocrine signaling and the difficulty Oehrtman et al. (18). The constructed cell lines relevant to the of selectively labeling autocrine ligands. Without being able to present work are denoted as R1yL2 (B82 cells with EGFR) quantify relationships between ligand production and receptor and R1yL1 (B82 cells with EGFR and TGFa). The R1yL1 binding, interpreting cell behavioral changes after a molecular cells can express TGFa at a range of levels depending on the intervention remains ambiguous. Current methodologies for medium concentration of the suppresser tetracycline (18). analyzing autocrine signaling are indirect, but whether they are Dialyzed bovine calf serum (10,000 M -cutoff dialyzed measuring end-point cell functions such as migration, prolif- r against 0.15 M NaCl), DMEM, methotrexate, geneticin sulfate eration, or differentiation (e.g., see refs. 11–13) or short-term (G418), histidinol, BSA fraction IV (RIA grade), glutamine, receptor activation events such as phosphorylation (e.g., see penicillin, and streptomycin were purchased from Sigma. refs. 14, 15), they are laborious and time-consuming— Hybridoma producing mAb 225 were obtained from the therefore not ‘‘real-time’’—and are only poorly quantitative at American Type Culture Collection and the antibodies pro- best. duced as described by Opresko et al. (34). Goat anti-TGFa The Cytosensor microphysiometer (Molecular Devices) (16, antibody was obtained fromR&DSystems. Rabbit IgG was 17) uses a light-addressable potentiometric sensor to measure obtained from Sigma. Both anti-EGFR blocking antibody 225 and anti-TGFa antibody bind to EGFR and TGFa, respec- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: EGFR, epidermal growth factor receptor; ECAR, accordance with 18 U.S.C. §1734 solely to indicate this fact. extracellular acidification rate. © 1998 by The National Academy of Sciences 0027-8424y98y9515368-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: lauffen@ PNAS is available online at www.pnas.org. mit.edu. 15368 Downloaded by guest on September 27, 2021 Cell Biology: Lauffenburger et al. Proc. Natl. Acad. Sci. USA 95 (1998) 15369 ay y tively, with affinity comparable with TGF EGFR binding mM CaCl2 20 mM Hepes). Bound ligand was determined by 5 (Kd 2 nM) (27). lysing cells with 1 M NaOH for 10 min and counting in a R1yL2 cells were grown in DMEM with 1 mM glutamine, gamma spectrophotometer. Free ligand concentration was 100 unitsyml penicillin, and 2.5 mgyml streptomycin along with determined by counting an aliquot of the binding medium. 10% dialyzed bovine calf serum and 1 mM methotrexate to maintain selection on the EGFR expression plasmid pXER RESULTS AND DISCUSSION (19). R1yL1 cells were grown in DMEM with 10% dialyzed bovine calf serum, 1 mM glutamine, 100 unitsyml penicillin, For our experimental autocrine cell system, we used a B82 and 2.5 mgyml streptomycin along with maintaining plasmid mouse fibroblast cell line in which we have created an artificial selection with 600 mgyml G418 (pUHD15.1), 2,400 mM his- autocrine loop consisting of TGFa and EGFR (18). These cells tidinol (pUHD10.3 and pR8), and 1 mM methotrexate do not normally express EGFR or its ligands (TGFa or EGF). (pXER). After expression of EGFR in B82 cells by transfection, there Microphysiometer Assay. Cells (250,000) were seeded into is no significant activation of the receptor in the absence of each Cytosensor transwell (Corning Transwell #3402; 3 mm exogenous EGF ligands (19). TGFa expression is under con- pore size, 12 mm diameter) in the appropriate growth medium. trol of the ‘‘tetracycline-off’’ two-plasmid system (20) permit- Twenty-four hours later, cell-transwell assemblies were pre- ting quantitative manipulation of the level of autocrine ligand pared for placement on the Cytosensor by inserting a spacer production. We have previously shown that in these cells, (50 mm high, 6 mm inner diameter; #R7026B, Molecular TGFa is expressed in its normal manner as a transmembrane Devices) into the transwell medium directly over the cells precursor, and is efficiently processed at the plasma membrane followed by a transwell insert (3 mm pore size; #R7025, to yield almost exclusively the 5.5-kDa soluble form (18). Molecular Devices), fitting flush inside the original transwell Clones transfected with both EGFR and TGFa (therefore, and the addition of 1 ml of running buffer to each insert cup autocrine clones) were designated R1yL1. They were fully to seat the insert into the transwell. The cell-transwell assem- induced for ligand production in the absence of tetracycline blies were then placed in Cytosensor silicon sensor chambers and uninduced in the presence of 1 mgyml tetracycline. Control and equilibrated for 2–3