[CANCER RESEARCH 61, 4740 – 4743, June 15, 2001] The Cell Transmembrane pH Gradient in Tumors Enhances Cytotoxicity of Specific Weak Acid Chemotherapeutics 1 Sergey V. Kozin, 2 Pavel Shkarin, and Leo E. Gerweck Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 [S. V. K., L. E. G.], and Magnetic Resonance Center, Yale University School of Medicine, New Haven, Connecticut 06520 [P. S.] ABSTRACT The extracellular pH is lower in tumor than in normal tissue, whereas their intracellular pH is similar. In this study, we show that the tumor- specific pH gradient may be exploited for the treatment of cancer by weak acid chemotherapeutics. i.v.-injected glucose substantially decreased the electrode estimated extracellular pH in a xenografted human tumor while its intracellular pH, evaluated by 31 P magnetic resonance spectroscopy, remained virtually unchanged. The resulting increase in the average cell pH gradient caused a parallel increase in tumor growth delay by the weak acid chlorambucil (CHL). Regardless of glucose administration, the effect of CHL was significantly greater in tumors preirradiated with a large dose of ionizing radiation. This suggests that CHL was especially pronounced in radioresistant hypoxic cells possessing a larger transmembrane pH gradient. These results indicate that the naturally occurring cell pH gradient difference between tumor and normal tissue is a major and exploitable determinant of the uptake of weak acids in the complex tumor microenvironment. The use of such drugs may be especially effective in combination with radiation. INTRODUCTION It has long been established that the electrode-estimated pH is on average lower in tumor than in normal tissue (1). However, various attempts to exploit this difference for the treatment of cancer have largely been unsuccessful. This is likely because of, in large part, the lack of distinction between extracellular and intracellular tissue pH, which substantially differ. Prior to the application of 31 P-MRS 3 to living tissue, it was not recognized that its pH is compartmentalized into an intracellular component (pHi), which is similar in tumor and normal tissues, and an extracellular component (pHe), which is rela- tively acidic in tumors (2). This gives rise to a cellular transmembrane pH gradient difference between these tissues, which in principle may be exploited for the treatment of cancer by drugs that are weak electrolytes with the appropriate pKa (3, 4). The passage of noncarrier-mediated weak electrolytes through the plasma membrane to their intracellular target(s) is strongly influenced by the ionization status of the compounds, with penetration occurring when the molecule is in its uncharged (lipophilic) form. As a result, at equilibrium such drugs predominantly concentrate on that side of the barrier where their ionized fraction is larger (5). Thus, under physi- ological pH conditions, the concentration of weak acids with pKa  6.5 is expected to be substantially greater in a more basic compartment, intracellularly in tumors and in the extracellular space in normal tissue. The central role of the pH gradient in governing the intracellular uptake and cytotoxicity of weak electrolytes, such as CHL, doxoru- bicin, and mitoxantrone, has been demonstrated clearly in cells under defined in vitro conditions (6 – 8). The importance of the cellular pH gradient on the efficacy of such drugs in vivo is less certain, and its analysis is complicated by a number of potential difficulties. Tumors exhibit considerable spatial and perhaps temporal heterogeneity in blood flow, pO 2 , and pH, which may modulate the delivery and cytotoxicity of chemotherapeutics. Furthermore, if the method used to modify the pH gradient concurrently alters tumor blood flow, both drug delivery and the tumor microenvironment will be affected, thus additionally obscuring the impact of any transmembrane drug redis- tribution on tumor treatment response. Here we report the results of two approaches for evaluating the role of the intra-extracellular pH gradient on the cytotoxicity of a weak acid, CHL, in a human tumor xenograft. In the first approach, this gradient was increased by the use of glucose (without undesirable changes of perfusion), and the resultant impact on CHL-induced tumor growth delay was determined. In the second, the effect of CHL was compared in tumors with and without preirradiation. Although exceptions may exist at particular loci, both tissue pHe and pO 2 decrease with increasing distance from supplying tumor vessels (9, 10). Therefore, the sterilization of radiosensitive oxygenated tumor cells by radiation permits the selective evaluation of CHL cytotoxicity in the remaining subpopulation of cells residing in a relatively acid environment, and presumably possessing the largest transmembrane pH gradient. The results of both approaches demonstrate that the cell pH gradient can significantly enhance the toxicity of certain weak acid drugs in tumors. We thus identify both a tumor-specific microenvi- ronmental property and the type of damaging molecules that may be used to exploit it for the treatment of cancer. MATERIALS AND METHODS Animals and Tumors. Tumors were transplanted into athymic NCr/Sed nude (nu/nu) male mice, 8 –10 weeks of age, bred and maintained in our defined flora and specific pathogen-free colony (11). The human small cell lung carcinoma 54A (12) was implanted into the mice 24 h after further immunosuppression by whole-body irradiation ( 137 Cs, 0.7 Gy/min) to a dose of 5 Gy. Third to sixth generation source tumors were excised, cleaned of necrotic tissue, cut into small chunks, and transplanted s.c. into the right hind leg. The tumors were used for experiments when they reached an average diameter of 8 mm. Measurements of Tumor pHe, pHi, and Blood Flow. Tumor pHe and perfusion were continuously measured before and after glucose administration in mice anesthetized with sodium pentobarbital and gently restrained by tape. An initial i.p. pentobarbital dose of 50 mg/kg was supplemented with up to an additional 30 mg/kg for long duration monitoring of these parameters. A 0.65-mm diameter, steel-sheathed needle, glass pH electrode (type MI 408B) was inserted into a central part of the tumors through a puncture made by a 23-gauge needle. The micro-reference electrode with flexible barrel (type MI 402) was placed into the subcutis nearby. The electrodes (Microelectrodes, Inc., Londonderry, NH) were connected to the Chemical Microsensor II (Diamond General, Ann Arbor, MI). Concurrently with pHe measurements, changes in blood flow (RBC flux) were also assessed in half of the tumors, using the laser Doppler technique. A 0.8-mm diameter needle probe connected to the LASERFLOW Blood Perfusion Monitor 403A (TSI, Inc., St. Paul, MN) was used as described previously (13, 14). For insertion of the probe, the skin was pierced with a 23-gauge needle, and the probe was inserted to a point Received 7/13/00; accepted 4/30/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Cancer Institute Grant CA-22860 (to L. E. G.) and National Cancer Institute Merit Award CA-13311 (to H. D. S.). 2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Cox 7, Massachusetts General Hospital, Boston, MA 02114. 3 The abbreviations used are: MRS, magnetic resonance spectroscopy; pHe, extracel- lular pH; pHi, intracellular pH; pKa, dissociation constant of a weak electrolyte; pO 2 , oxygen partial pressure; CHL, chlorambucil. 4740 Research. on September 23, 2015. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from