Intra-tumoural extra-cellular pH: a useful parameter of response to chemotherapy in syngeneic tumour lines

Abstract

Reliable surrogate markers of response to anticancer therapy remain a desirable tool for preclinical modelling and clinical practice in oncology. Clinical evaluation is relatively unreliable when attempting to assess rapidly and prospectively the outcome of treatment. Fluxes in released or circulating tumour marker levels are a useful but inconsistent marker of cytotoxic response. Serial measurement of circulating tumour cells appears to have some utility as a surrogate marker, but assay systems are expensive, and many cancers are not associated with the presence of circulating tumour cells. Because tissue breakdown is associated with release of nucleic acids and other cellular products, we reasoned that serial measurement of intra-tumoural pH may correlate with the extent of tumour lysis, and thus with outcomes of cytotoxic chemotherapy. Doxorubicin-sensitive and doxorubicin-resistant sublines of P388 murine monocytic leukaemia in C57BL/6 mice were treated with increasing concentrations of doxorubicin. Tumours were serially measured by conventional bi-dimensional methods and pH was sampled using a bevelled tip electrode. Mean and median pH changes were statistically different in responsive and resistant tumours, and amplitude of change correlated with long-term responses to doxorubicin. Serial sampling of pH in tumour masses may provide a useful surrogate of long-term response to chemotherapy.

Figure 1

Measurement of extra-cellular pH in P388 murine monocytic leukaemia tumours and normal tissues in DBA/2 mice. Extra-cellular pH measurements of established (11-day-old) subcutaneous tumours and the subcutaneous tissues of the posterior neck were obtained using a micro-electrode. Mice had received PBS or cyclophosphamide (Cy) 2 days previously (n=24 each group).

Figure 2

P388 tumour volume in DBA/2 mice. Mice bearing established s.c. P388 tumours received 100 mg kg⁻¹ Cy on day 0. Intra-tumoural pH was assessed on day 2 (indicated by arrow), as above.

Figure 3

Survival of mice bearing P388 tumours. Mice from Figure 2 were monitored for survival following single-dose of Cy or vehicle.

Figure 4

Antiproliferative effects of doxorubicin (ADR) against B16-BL6 murine melanoma. Doxorubicin sensitive (Sen) and resistant (Res) sublines of B16-BL6 were treated with doxorubicin in vitro and growth was assessed after 4 days. Negative values on the y-axis indicate death of initially plated cells (n=8 each dose).

Figure 5

Measurement of intra-tumoural pH in B16-BL6 tumours. Extra-cellular pH measurements of sensitive and resistant B16-BL6 tumours were obtained following doxorubicin (ADR, 2 mg kg⁻¹ × 6 days) or PBS treatment. SP (sensitive, PBS), RP (resistant, PBS), SA (sensitive, ADR), RA (resistant, ADR) designations indicate cell subline and treatment (n=16 mice each group).

Figure 6

Survival of C57Bl6 mice bearing B16-BL6 tumours and kinetics of pH response. (A) Mice implanted with tumours as in Figure 5 were monitored for survival following treatment with ADR or PBS (n=16). Intra-tumoural pH was measured at a single time point (day 12, indicated by arrow). Cell subline and treatment designations as in Figure 5. (B) A second series of C57Bl6 mice (n=6 per group) bearing B16-BL6 tumours with indwelling intra-tumoural guide tubes received ADR or PBS treatment (days 6–12) and underwent repetitive pH measurement on days 6 (pre- and post-treatment), 7, 10, 13, and 16.