Specifically, we seek to employ the natural tendency of tumors to convert glucose to lactate as a method for selective intracellular tumor acidification, which has been reported to potentiate tumor response to radiation (1), hyperthermia (2) as well as to chemotherapy with N-mustards (3, 4), doxorubicin (5) and alkylating agents. This study monitors intracellular pH (pHi), extracellular pH (pHe), bioenergetics (βNTP/Pi) and lactate in androgen-independent (PC3) and androgen-dependent (LNCaP) prostate cancer xenografts by ³¹P and ¹H magnetic resonance spectroscopy (MRS) following administration of lonidamine (LND), a putative inhibitor of transmembrane monocarboxylate transporters (MCTs) (unpublished data), the mitochondrial pyruvate carrier (unpublished data) and complex II of electron transport chain (6). These findings point to the potential utility of radiation, hyperthermia and chemotherapeutic drugs in combination with LND in the treatment of disseminated prostate cancer. PC3 prostate cancer cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 1% penicillin-streptomycin. 7×10⁶ PC3 cells were inoculated subcutaneously (s.c.) in each mouse (n=5) as a 0.1 mL suspension. LNCaP cells were grown in RPMI 1640 with 10% fetal bovine serum and 0.5% penicillin/streptomycin. 5×10⁶ cells in a mixture of 75 μl matrigel and 75 μl of RPMI 1640 medium were inoculated s.c. into the right flank of the nude mouse (n=5). Prostate cancer xenografts were allowed to grow until they reached 7-10 mm in diameter along the longest axis of the tumor. The pHi (n=5), extracellular pH (pHe) (n=5), βNTP/Pi (n=5) and steady-state levels of tumor lactate (n=5) were measured in PC3 and LNCaP xenografts by ³¹P MRS (pH and bioenergetics) and ¹H MRS (Hadamard-selective multiple quantum coherence transfer pulse sequence), respectively, as described in our previous publications (3-5). Analysis of variance with Tukey multiple comparisons was used for statistical analysis (SPSS 16). The data on pHi, pHe, bioenergetics and lactate at various time points following LND administration were compared by ANOVA and t test analysis. In vivo ³¹P MRS (Fig. 1) demonstrates that PC3 and LNCaP prostate cancer xenografts in immunosuppressed mice treated with the MCT inhibitor LND exhibit a sustained and tumor-selective decrease in pHi from 6.94 ± 0.02 to 6.49 ± 0.05 (p = 0.02), pHe from 7.06 ± 0.03 to 6.72 ± 0.08 (p = 0.08) (Fig. 2 A) and 6.90 ± 0.03 to 6.45 ± 0.05 (p = 0.05), pHe from 7.0 ± 0.03 to 6.69 ± 0.06 (p = 1.0) (Fig. 2 B), respectively. Tumor bioenergetics (βNTP/Pi) decreased by 75.0 ± 0.12% (p = 0.01) and 79.0 ± 0.18% (p = 0.00) in PC3 and LNCaP (Fig. 2C), respectively, relative to the baseline level immediately prior to LND administration. Steady-state levels of tumor lactate (intracellular plus extracellular) were monitored by ¹H MRS with the Hadamard-selective multiple quantum coherence transfer pulse sequence in PC3 (Fig. 3A, C) and LNCaP (Fig. 3B, C) tumors following LND administration at time zero. The lactate intensity, which peaked at around 40 min in both cell type, PC3 (p = 0.03) and LNCaP (p = 0.02) remained stable for 120 min (p = 0.03) in PC3 and 100 min (p = 0.02) in LNCaP post- LND administration, and then decreased monotonically (Figs 3 A, B, C). The ³¹P MR spectra clearly show that LND leads to intracellular acidification and depression of the bioenergetics of the prostate cancer xenograft in vivo; these parameters are critical indices for tumor thermosensitization and/or for improving tumor response to antineoplastic agents. LND appears to inhibit the MCT on the plasma membrane and may also block the mitochondrial pyruvate carrier thereby impeding the delivery of pyruvate to produce acetyl-CoA for the TCA (tricarboxylic acid) cycle (unpublished data). Thus, LND is very attractive because it may simultaneously cause selective tumor acidification and tumor de-energization. While LND clearly inhibits export of lactate from human DB-1 melanoma and MCF-7 breast cancer and human 9L gliomas in rats, it is not clear if it is inhibiting transport of pyruvate into mitochondria as α-cyano-4-hydroxycinnamate (CHC) does (7-9). However, the similar effect of CHC and LND on the bioenergetics of DB-1 melanomas (3, 10) and prostate cancer here strongly suggest that it is. Therefore, the decline of bioenergetics that was evident both from the decrease in βNTP/Pi and from direct monitoring of NTP by ³¹P MRS vs. time in each animal could be explained by a profound decrease in mitochondrial metabolism following LND administration.