2014 Intracellular acidification of human melanoma xenografts by MIBG and hyperglycemia

Proceedings

of the 39th

Annual

247

Meeting

ASTRO

2013 The Impact of Hypoxia JM Bean’, DD Bignd, ‘Department ‘Department

and Oxygenation

GE Archer’, MT Mu&y’, MW Dewbirst,’ of Radiation of Pathology,

Modification

E Ong’,

on the Rodirtion

SA Snyder’,

Oncology, Duke University DUMC, ‘Gray Laboratory,

ZA Haroo!?,

Medical Middlesex,

Center UK

Response of on Intracranial RE McL.wdon,3

(DUMC),

LB Marks’,

2Department

Rat Gliomr MRL

Stratford

of Surgery,

Division

4, DJ Chaplin4,

DM Brizel’,

of Neurosurgery,

DUMC,

puroosq: The median survival for a patient treated for GBM is 1 year Reasons for the poor long term benefit of conventional therapy, which includes radiation therapy, are poorly understood. Gliomas have areas of hypoxia which may predict for radioresistance. The purpose of this project was to test the hypothesis that hyperbaric oxygen was superior to nicotinamide plus carbogen in decreasing the extent of hypoxia and in increasing survival followingfracrionnredirradiation in an intracranial glioma tumor model. Materials and Methods: RG-2 tumor cells were transplanted intracranially into female Fischer 344 rats. Hypoxia was studied using immunohistochemical staining of drug-protein adducts in tumor following removal of the brain after i.p. injection of pimonidazole hydrochloride under conditions of room air, 3 atmospheres oxygen (HBO), or niwtinamide (200 mg/kg i.p.) with carbogen. Serum nicotinamide and metabolites were measured by HPLC on samples taken 20 minutes after giving nicotinamide (200 mg/kg, i.p.). In a survival study animals were randomized on day I4 post-transplant to 1 of 4 treatment groups: 1) sham - anesthesia only; 2) XRT - 4 Gy x 5; 3) HBO - XRT after breathing for 5 minutes once 3 atmospheres of lOO?h oxygen was obtained; and 4) Carbogetiicotinamide - XRT with i.p. nicotinamide 20 minutes prior to irradiation and carbogen breathing for 5 minutes after saturation of the chamber. Following treatment, animals were followed until death. M: Immunohistochemical evaluation of the tumors from animals breathing room air revealed pimonidazole adducts indicating the presence of hypoxia. Those tumors evaluated from animals treated with HBO did not show evidence of hypoxia marker binding. The median serum nicotinamide level obtained was 258.2 &ml. Median survivals post-transplant were: 1) Sham - 22 days, 2) XRT - 28 days, 3) HBO - 28 days, and 4) Carbogen/Nicotinamide - 26 days. Only XRT and HBO were statistically better than Sham (p = 0.002 and p i 0.001 respectively). XRT and HBO showed a trend towards improved survival over CarbogenMicotinamide (p = 0.067 and p = 0.066 respectively). There was no difference between XRT and HBO (p = 0.976) or between Sham and Carbogeflicotinamide (p = 0.154). Conclusions: The results demonstrated both the presence of hypoxia in the tumor model under air breathing conditions and the absence of hypoxia under HBO conditions. However, there was no improvement in survival with the addition of either HBO or CarbogenMicotinamide. This might be explained by one of the following: 1) the tumors were not hypoxic to begin with; 2) the degree of hypoxia was not radiobiologically significant; 3) not all animals had tumors that were hypoxic; 4) the treatments designed to decrease hypoxia were ineffective; or 5) there was reoxygenation between the radiation treatments in air breathing animals. The results indicate that the tumors were hypoxic and that HBO was effective in reducing the hypoxia. It is possible that not aIl of the animals had tumors that were hypoxic which would mask the benefit seen for those animals with hypoxic tumors. The degree of hypoxia was not quantitated, but even minimal hypoxia can have a significant impact on survival for a radiosensitive tumor in the absence of reoxygenation. Thus, it is our conclusion that this tumor was relatively radioresistant and that there was significant reoxygenation between fractions of radiation in air breathing animals which resulted in a response that was indistinguishable from radiation treatment with HBO.

2014 INTRACELLULAR Dennis

B. Leeper,

Depts. of Radiation

Purpose/Objective:

ACIDIFICATION Ph.D.,

Marea

Oncology

D. Pollard,

& Medicine],

OF HUMAN L.A.T.,

David

Thomas

Jefferson

MELANOMA Berd,

M.D.1

University,

XENOGRAFTS and Jerry and Dept.

D. Glickson, of Radiologyz,

BY MIBG

AND

HYPERGLYCEMIA.

Ph.D.2 University

of Pennsylvania,

Philadelphia,

PA

Stimulation of the intrinsically higher glycolytic capacity of tumors by extra glucose supply is a strategy to selectively acidify tumors by increased lactic acid production. However, the average decrease in tumor extracellular pH (p&) in 87 patients was 0.17M.02 unit, and only 17% of tumors exhibited a decrease in pH, a.2 pH unit. Therefore, additional efforts are needed to enhance acute tumor acidification beyond that of hyperglycemia alone to achieve significant sensitization to therapeutic agents. Kuin et al. (Cancer Res. 54:3785-92, 1994) showed that combining the mimchondrial respiratory inhibitor meta-iodobenzylguanidine (MIBG) with hyperglycemia greatly enhanced extracellular acidification in rodent tumors with only transient and modest effects on normal tissues. The following experiments were performed to determine whether MIBG combined wirh hyperglycemia would enhance intracellular acidification (pHi) by increased lactate production in F2 human melanoma xenografts. Materials & Methods: Second generation human melanoma xenografts were grown subcutaneously in the thighs of male ICR SCID mice 8-12 wk of age weighing 33-38 gm obtained from Taconic Farms. Xenograft bearing animals were fasted for 5 br before administration of 2 g/kg oral glucose which prevents the osmotic effects of i.p. administration. Prior to oral glucose animals were anesthetized with 25 mgikg Ketamine + 2.5 mg/kg Diazepam + 10 mg/kg Nembutal, a cocktail which does not produce hyperglycemia. Rectal temperature was kept at 36.5* 0.5” C. Glucose concentration was measured by Chemstrips in a glucometer (Accu-Chek) every 20-30 min in blood drawn from the orbital sinus. MIBG (Sigma, 15 mg/kg, i.p.) was administered SO-65 min post-glucose. Tumor pHe was measured with a 20 g needle pH combination microelectrode (S. Agulian, New Haven, CT) advanced with a micromanipulator. Tumor pHI was monitored by 3LP NMR spectra obtained with a G.E. CSYAccustar spectrometer equipped with a 4.7 T/40 cm bore magnet and actively shielded gradient coils (E. Aboagye, Johns Hopkins Univ). Lactate production was monitored by proton NMR spectra measured by selective multiple quantum coherence transfer (He et al., J. Magn. Reson. Med. 106:203-l 1, 1995) on a Broker Biospec 4.7 T/30 cm bore magnet equipped with SMIS gradients and a SMIS console (Q. He, Univ. of Corm., Yale Univ.). The spectrum was taken over the entire tumor and spectral acquisition required 8 min. Results: MIBG at 15 mg/kg could bc administered daily without effect; the LDlo was -40 mg/kg for fasted. hyperglycemic non-tumor bearing ICR mice. In a 1.2 mm3 xenograft there was no intracellular acidification during hyperglycemia alone, however, when 15 mg/kg MIBG was injected i.p., the tumor pHi fell within minutes by 0.24 pH unit from 7.07 to 6.83. Blwd glucose concentration was 231 mg/dL. If [H+]mR is associated with [H+]i, then [H+]i was completely unaffected by induction of hyperglycemia, but increased by 73% upon inhibition of respiration with MIBG (Fig. 1). While the change in pHi was ~0.25 pH unit, it reflects substantial acidification if the buffer capacity of the tumor ~25-30 mmole/pH/L. This would correspond to about 5-6 ~molelml of acid generated by the tumor in response to hyperglycemia plus MIBG. Five days later the same xenograft was studied for extracellular 0acidification. The pHe of the xenograft was 6.91. The extracellular acidification during hyperglycemia alone was 0.06 pH unit; and when 15 mg/kg MIBG was injected, the pH, decreased by another 0.31 pH unit to 6.54. Blood glucose Mlnum was 260 mg/dL. Lactate was detected by ‘H NMR spectroscopy in another melanoma xenograft 0.73 Fig. 1. Hydrogen ion concenrrarion in a concentration human mefonomo xenogrofr @?frer oral mm3. No lactate was detected under normoglycemic or hyperglycemic conditions without MIBG. However, the &cose and&r i.~. injection @MlBG. lactate resonance at 1.3 ppm was detected immediately after administration of 15 mg/kg MIBG during hyperglycemia. Conclusions: It is likely the melanoma xenografts exhibit low levels of lactate production and relatively high levels of oxidative metabolism under normoglycemic and hyperglycemic conditions; however, upon administration of MIBG, oxidative phosphorylation is blocked, resulting in increased production of ADP and stimulation of sufficiently high levels of glycolytic flux to produce detectable levels of lactate. Therefore, combining MIBG with hyperglycemia may be useful for sensitizing melanomas to hyperthermia or pH sensitive chemotherapeutic agents. Furthermore, MIBG by virtue of inhibiting oxidative phosphorylation may increase the oxygen distribution in tumors prior to irradiation. (Supported by NIH Grant PO1 CA56690.)