Figure 1.

Effect of NaHCO₃ on metastases and survival. MDA-MB-231 were obtained from American Type Culture Collection and maintained in growth media (DMEM/F-12 supplemented with 10% FBS) at 37°C with 5% CO₂ in a humidified atmosphere. These cells were stably transfected with expression vectors for hygromycin-resistant pcDNA3.1/LacZ (Invitrogen). These β-gal–labeled MDA-MB-231 cells (10⁷), suspended in 0.2 mL of 0.8% sterile saline, were injected s.c. into the left inguinal mammary fat pads of 6-wk-old female SCID mice. Mice (n = 8) were started on drinking water (ad libitum) supplemented with 200 mmol/L NaHCO₃ at 6 d postinjection and maintained along with untreated animals (n = 8). After 30 d of primary tumor growth, the animals were sacrificed and the β-gal–positive lung lesions were counted and sized after staining, as shown in A. Mean lesion diameters (P < 0.0001) and frequencies (P = 0.0342) were significantly different between the two groups as determined by two-tailed unpaired t test with Welch's correction for unequal variances. In a repeat of this experiment, 10⁶ β-gal-MDA-MB-231 cells were injected into inguinal mammary fat pads, and control (n = 9) and NaHCO₃-treated (n = 15) animals were maintained for 60 d before sacrifice. In this experiment, lung images were analyzed using ImagePro Plus to determine the metastatic tumor burden by counting the number of β-gal–positive pixels per animal. B, numbers of lung lesions per animal following 60 d of growth in the presence of NaHCO₃ in drinking water. The frequency of lesions per animal in the NaHCO₃-treated mice was compared with that in untreated controls by unpaired t test (P = 0.0004). In a third experiment, MDA-MB-231 cells were stably transfected to express neomycin-resistant pcDNA3/EGFP (a gift from Peter Ratcliffe, Oxford University, Oxford, United Kingdom). MDA-MB-231/eGFP cells (6.5 × 10⁶) were injected into inguinal mammary fat pads of animals that were randomized into bicarbonate and control groups (n = 12 per group) 6 d postinoculation. Tumors were allowed to grow for 5 to 6 wk (to a volume of ∼600 mm³), at which time they were surgically removed. If the primary regrew (as was the case in 9 of 24 animals), it was resected again. Animals were monitored biweekly and maintained on bicarbonate or water until they evidenced a lymph node lesion >300 mm³ in size, at which time they were sacrificed and necropsied by examination with a fluorescence dissecting scope. Data from this experiment are plotted as a Kaplan-Meier survival curve (C). The difference in the survival curve for the bicarbonate versus control animals was tested using the log-rank test (P = 0.027).