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ORIGINAL ARTICLES |
From the Departments of Surgery (RJC, SBK, DLF), Radiology (RZ, JDG), and Biostatistics & Epidemiology (YZ, DFH), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and Department of Radiation Oncology (DBL), Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania.
Correspondence: Address correspondence and reprint requests to: Douglas L. Fraker, MD, Division of Surgical Oncology, Department of Surgery, University of Pennsylvania Medical Center, 3400 Spruce Street, 4 Silverstein, Philadelphia, PA 19104; Fax: 215-662-3629; E-mail: frakerd{at}uphs.upenn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methods: Human melanoma xenografts were implanted into the hind limbs of athymic rats. When tumors reached 12 to 15 mm in diameter, animals were randomized to ILP with or without melphalan, with or without systemic MIBG, and hyperglycemia of 485 ± 35 mg/dL. Intratumoral pH was measured during MIBG and glucose treatment by using magnetic resonance spectroscopy.
Results: MIBG at 30 mg/kg plus hyperglycemia decreased intracellular pH by .6 units and extracellular pH by .8 units. MIBG at 22.5 mg/kg plus hyperglycemia decreased intracellular and extracellular pH by .4 and .5 units, respectively. Tumor growth was unaffected by systemic MIBG and hyperglycemia alone. When MIBG at 30 mg/kg and hyperglycemia were combined with ILP, tumor growth was delayed for 33 days after control ILP and for 44 days after melphalan ILP. However, this dose of MIBG was complicated by a 40% mortality rate after ILP. MIBG at 22.5 mg/kg, in combination with MIBG in the perfusate, did not cause mortality and delayed tumor growth by 51 days after melphalan ILP.
Conclusions: MIBG and hyperglycemia improve tumor response rates after ILP in a rodent model of human melanoma. Selective tumor acidification with MIBG and hyperglycemia may offer added benefit to current regional perfusion strategies.
Key Words: Isolated limb perfusion • Acidification • MIBG • Human melanoma xenografts
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, for in-transit extremity melanoma have shown overall response rates ranging from 80% to 100% and complete response rates ranging from 53% to 75%. Furthermore, approximately one third of patients experience a sustained and durable complete response after ILP, underscoring the value of this technique for an otherwise refractory condition. Nevertheless, despite these generally favorable results, methods to optimize the response to ILP could benefit a substantial number of patients.
We and others have shown that ILP is feasible in rodent models, and results with conventional agents (i.e., melphalan and tumor necrosis factor-) reproduce those seen clinically.2,3 We have also demonstrated in rodents that direct perfusate acidification with dilute hydrochloric acid, targeting a venous pH of 6.8, markedly augments tumor response rates to melphalan ILP.4 However, the significant therapeutic benefit of direct perfusate acidification is offset by increased regional toxicity (i.e., ischemic necrosis of the toes) in a small proportion of animals (Bauer et al., unpublished data, 2003). Consequently, we have sought methods to selectively acidify tumors to sensitize them to the effects of ILP without increasing regional toxicity.
Tumor cells exhibit enhanced glucose transport and glycolytic metabolism as an adaptation to hypoxia. Consequently, systemic hyperglycemia is capable of acutely acidifying tumors through glycolysis-mediated lactic acid production. Investigators have repeatedly used hyperglycemia-induced acidification in experimental tumor models to augment the effect of various antineoplastic modalities, including hyperthermia, radiation, and chemotherapy.5–7 Metaiodobenzylguanidine (MIBG), an inhibitor of mitochondrial function, also shunts cellular glucose metabolism to lactic acid production.8 In studies in both mice and immunocompetent rats, MIBG has been shown to increase hyperglycemia-induced tumor acidification.9–12 This study was performed to evaluate the effect of selective tumor acidification with hyperglycemia and MIBG on response rates to ILP in a rodent model of human melanoma.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Line
The human melanoma cell line NIH 1286 was obtained from Dr. Steven Rosenberg at the National Cancer Institute (Bethesda, MD). Cells were maintained in tissue culture with RPMI medium (Gibco BRL, Green Island, NY) supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, penicillin G, streptomycin, amphotericin, and gentamicin.
Animals and Tumor Cell Implantation
Female homozygous nude rats, aged 5 to 7 weeks, were obtained from the National Cancer Institute (Frederick, MD) and housed under standard light and accommodation conditions. To optimize tumor cell implantation, animals were subjected to 5 Gy of total body irradiation with a cesium-137 irradiator (Atomic Energy of Canada Ltd., Ontario, Canada) 1 to 2 days before tumor inoculation. For tumor inoculation, animals were anesthetized with a combination of IP ketamine 62.5 mg/kg (Abbott Laboratories, Chicago, IL) and IP xylazine 5 mg/kg (Webster Veterinary Supply, Sterling, MA), and 5 x 106 cells were injected subcutaneously in the lateral aspect of the distal hind limb. This regimen reliably produced 12- to 15-mm elliptical subcutaneous tumors approximately 3 weeks after inoculation. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
Glucose Infusion
A stock solution of D-glucose (750 mM) was delivered by a syringe pump (Harvard Apparatus, Holliston, MA) through a 22-gauge Angiocath (Abbott Laboratories) inserted by cutdown into the left common femoral vein. An infusion protocol of 45 µL/min for 5 minutes followed by 5 µL/min was developed in age- and weight-matched animals that reproducibly yielded a blood glucose concentration of 485 ± 35 mg/dL (mean ± SEM). Blood samples were obtained from the tail and analyzed with an Accu-Chek Advantage diabetes monitoring kit (Boehringer Mannheim, Indianapolis, IN). Glucose infusions were performed for 45 minutes for MR spectroscopy and 45 to 65 minutes for ILP experiments, depending on the amount of time required to cannulate the rodent’s vessels and begin isolation perfusion.
MR Analysis of Intratumoral pH
Animals were anesthetized with 125 mg/kg of IP ketamine and 10 mg/kg of IP xylazine. A left common femoral vein catheter was placed for intravenous infusion of glucose. Two IP catheters were placed so that additional anesthetics and MIBG could be delivered during the MR experiments without removing the animals from the magnet. The animals were then placed on a water-circulating blanket (42°C) to maintain core temperature at 37°C and were positioned inside the magnet with the surface coil secured immediately over the tumor to minimize interference from other tissues.
The MR studies were performed on a GE (Fairfield, CT) 9.4-T/8.9-cm vertical bore Omega system. In vivo phosphorus-31 spectra were acquired with a homemade single-turn solenoidal surface coil by using the following parameters: 128 scans with an interpulse delay of 2 seconds; a radiofrequency pulse width of 15 to 20 µs, corresponding approximately to a 90° flip angle; and 2048 data points. Data were processed on a Sun computer (Sun Microsystems, Santa Clara, CA) by using 25-Hz line broadening to increase the signal:noise ratio and a convolution difference routine to minimize the broad phospholipid peaks underneath the spectrum. A Lorentzian line-fitting routine provided by the manufacturer was used to resolve peaks and measure their areas. No corrections were applied for partial saturation of resonances. Intracellular and extracellular pH (pHi and pHe, respectively) were determined as described by Zhou et al.12 by using the Henderson-Hasselbach equation to calculate the chemical shifts of inorganic phosphate and 3-APP (for pHi and pHe, respectively) referenced to the -nucleoside triphosphate resonance.
ILP Technique and Measurement of Tumor Volume
When tumors reached 12 to 15 mm in diameter, animals were randomized to the following treatment groups: (1) no treatment; (2) treatment with systemic MIBG and hyperglycemia; (3) control ILP with hetastarch solution; (4) melphalan ILP; (5) MIBG, hyperglycemia, and control ILP; and (6) MIBG, hyperglycemia, and melphalan ILP.
ILP was performed as described by Kelley et al.4 In brief, animals were anesthetized with IP ketamine (125 mg/kg) and xylazine (10 mg/kg), and the right groin was cleansed with povidone-iodine solution. A 3-cm incision was made, and the femoral artery and vein were exposed. A tourniquet was then loosely placed inferior to the inguinal ligament, and 80 U of heparin (Abbott Laboratories) was injected IP. With an operating microscope, 6-0 silk ties were placed to obtain proximal and distal control of the vessels. Perfusate—consisting of 6 mL of 6% hetastarch in .9% sodium chloride (Abbott Laboratories) plus 80 U of heparin with or without melphalan—was then added to a glass oxygenating reservoir (Radnoti Glass Technology Inc., Monrovia, CA), and the system was primed. Veterinary silastic tubing was used to cannulate the vessels (arterial: .30-mm inner diameter, .64-mm outer diameter; venous: .64-mm inner diameter, 1.19-mm outer diameter; Konigsberg Instruments, Pasadena, CA), and after cannulation, the tourniquet was tightened. Venous drainage flowed by gravity into the reservoir, and arterial inflow was infused by a rotary pump (Masterflex 7524-00; Barnant Corp., Barrington, IL) at 1.5 mL/min.
Normothermic ILP was then performed for 10 minutes, followed by a 2-minute washout perfusion with hetastarch solution. The pump was then stopped, the tourniquet was loosened, and the femoral cannulas were removed. The vessels were then ligated with 6-0 silk ties, the incision was sutured with 4-0 Vicryl (Ethicon Inc., Somerville, NJ), and the animals allowed to recover from anesthesia.
The response to treatment was followed up for 60 days unless large tumors (i.e., diameter >30 mm) were present, necessitating killing the animals in accordance with animal care protocols. Tumors were measured with calipers every 3 days, and tumor volume was calculated by using a formula (length x width2 x .52) that approximates the volume of an ellipse. The volume of full-thickness eschar or ulceration of tumors was similarly measured and subtracted from the overall volume to estimate viable tumor volume. The percentage change in tumor volume was determined by normalizing to the starting volume at the time of ILP.
Statistical Analysis
Absolute tumor volume measurements were transformed to a logarithmic scale to improve the normality and homogeneity of the error variances between measurements. Logarithmic transformation also enabled the slopes of the data sets to be interpreted as growth rates because they achieved an approximately linear pattern.13 Tumor growth was then modeled with mixed linear models that assumed that the animals were independent but that serial tumor volumes for each individual rodent were correlated.14 Analysis of the data in this way suggested that in each treatment group, there was an initial decline in tumor volume that lasted 9 to 15 days, followed by a return to a steady pattern of growth. Therefore, we modeled the tumor growth curves on the basis of a linear spline with a single knot at day 9.15 This assumed that growth progressed with one slope until day 9, at which point the slope changed to another value that remained constant for the remainder of the observation period. Differences in tumor volume were thus analyzed by comparing declining slopes in the early period (before day 9) and increasing slopes in the later period (after day 9). Analyses were performed with SAS MIXED (SAS Institute, Cary, NC), and a P value of 
.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast to the tumor pH changes, pH monitoring of both muscle and the venous effluent from the ILP circuit revealed only slight differences. Figure 1C depicts changes in pHi and pHe in thigh muscle after treatment with systemic hyperglycemia and MIBG at a dose of 22.5 mg/kg. Baseline pHi and pHe were both maintained at approximately 7.2 and declined by a maximum of .1 ± .03 units over 80 minutes of treatment. When systemic MIBG at a dose of 30 mg/kg and hyperglycemia were administered concurrently with ILP (n = 9), the venous pH (mean ± SD) from the ILP circuit at the conclusion of the procedure was 7.19 ± .09. When MIBG at a dose of 22.5 mg/kg and hyperglycemia (n = 7) were administered with ILP, venous pH (mean ± SD) was 7.24 ± .08. For the animals undergoing ILP under standard conditions, i.e., hetastarch perfusion with or without melphalan (n = 11), the venous pH (mean ± SD) was 7.25 ± .03. Although there was a slight trend toward decreased serum pH with the highest dose of MIBG (7.19 vs. 7.25), this difference was negligible when compared with the .4- to .6-unit and .5- to .8-unit reductions for pHi and pHe, respectively, after MIBG and hyperglycemia treatment. This suggests that the acidification produced in the tumor microenvironment by MIBG and hyperglycemia either did not reach the serum or was adequately buffered by it.
Comparison of Treatment Groups Using High-Dose MIBG
On the basis of our MR results and reports in the literature demonstrating selective tumor acidification with systemic hyperglycemia of 450 to 500 mg/dL and MIBG at a dose of 30 mg/kg, we chose to test those doses on response rates to ILP. Hyperglycemia and MIBG alone did not significantly affect tumor growth when compared with no treatment (Fig. 2). However, when combined with melphalan and even control ILP, MIBG and hyperglycemia were associated with increased tumor regression. The maximal reduction (mean ± SEM) in viable tumor volume relative to starting tumor volume was 0% without treatment; 0% for MIBG and hyperglycemia alone; 25% ± 15% for control ILP alone; 57% ± 9% for melphalan ILP; 86% ± 8% for MIBG, hyperglycemia, and control ILP; and 75% ± 16% for MIBG, hyperglycemia, and melphalan ILP.
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However, the combination of hyperglycemia, MIBG at a dose of 30 mg/kg, and ILP (independent of the use of melphalan) was lethal to approximately 40% of the animals. No mortality occurred when MIBG and hyperglycemia were used alone, but in the setting of MIBG, hyperglycemia, and ILP, death typically occurred 6 to 8 hours after surgery, before the animals could successfully recover from the effects of anesthesia. Given this toxicity, we modified our treatment protocol to include reduced systemic MIBG (22.5 mg/kg) and regionally delivered MIBG in the perfusate solution, because during the ILP procedure the concentration of MIBG within the tumor tissue was likely decreasing below the threshold for activity. In addition, because of the prolonged general anesthetic required to both deliver intravenous glucose and perform the ILP procedure, in addition to concern about hyperglycemia-induced hypovolemia, we began to use boluses of intravenous crystalloid (10 mL/kg) at the conclusion of the ILP procedure. These protocol modifications successfully prevented further animal deaths. In addition, there was no evidence of regional toxicity from systemic hyperglycemia and MIBG at any dose.
Comparison of Treatment Groups Using Intermediate-Dose MIBG
Figure 3 depicts tumor response rates after ILP among the various treatment groups with the modified MIBG protocol. At a dose of 22.5 mg/kg, MIBG and hyperglycemia augmented the response to melphalan, but not to control ILP. The maximal reduction (mean ± SEM) in viable tumor volume relative to the starting tumor volume was 25% ± 15% for control ILP; 52% ± 5% for MIBG, glucose, and control ILP; 57% ± 9% for melphalan ILP; and 82% ± 9% for MIBG, glucose, and melphalan ILP. Thirty days after treatment, the relative viable tumor volume (mean ± SEM) for the groups was 382% ± 273% for MIBG, glucose, and control ILP; 320% ± 62% for control ILP; 157% ± 26% for melphalan ILP; and 36% ± 6% for MIBG, glucose, and melphalan ILP. The time required to return to 100% of the starting tumor volume was 9 days for MIBG, glucose, and control ILP; 11 days for control ILP; 21 days for melphalan ILP; and 51 days for MIBG, glucose, and melphalan ILP. Statistical analysis revealed that the differences in early tumor regression for days 0 to 9 were not statistically significant between the treatment groups, but the growth rate after day 9 was significantly reduced in the group that received MIBG, hyperglycemia, and melphalan ILP (P = .021). Figure 4 shows pictures of representative tumors 14 days after treatment for each of the groups. The improved response from the addition of systemic MIBG and hyperglycemia to a standard melphalan ILP can be seen.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have observed in an experimental model that direct perfusate acidification with dilute hydrochloric acid dramatically augments tumor response rates to both control and melphalan ILP.4 However, this marked increase in tumor response (100% complete response rate for acid plus melphalan–treated tumors at 60 days) is complicated by an approximately 20% incidence of ischemic necrosis of the distal phalanges, in a pattern of injury that resembles compartment syndrome (Bauer et al., unpublished data, 2003).
Enhanced glucose transport and glycolysis, with a concomitant increase in lactate production, are critical adaptations of tumor cells to hypoxia. This metabolic phenomenon was initially described by Warburg in 1930,11 and recent molecular studies have confirmed the central role of hypoxia-inducible factor-1 in this response.16,17 The high glycolytic potential of tumor cells, which enables them to produce energy in the presence of hypoxia, also presumably explains the repeated observation that systemic hyperglycemia is capable of acutely acidifying tumors.5–7 This phenomenon has also been demonstrated clinically in patients with superficial tumor deposits.18 However, the relatively small degree of acidification observed in patients with hyperglycemia alone (.15–.20 pH units, on average) has prompted investigators to test additional agents in an attempt to increase the extent of acidification.
MIBG, an inhibitor of mitochondrial oxidative phosphorylation, also shunts tumor cells to lactic acid production. When combined with hyperglycemia in several experimental models, MIBG has increased tumor acidification by an additional .3 to .6 pH units while decreasing pH in healthy tissue by .1 units.9,10,12 Moreover, because MIBG is an analog of norepinephrine, it concentrates in tissues of neural crest origin, which actively transport MIBG by a catecholamine receptor pathway.8 As a result, MIBG has been used with success in the diagnosis and treatment of neuroblastoma, pheochromocytoma, and metastatic carcinoid syndrome,19–22 and melanoma seems to be an appropriate target for this agent as well.23
In our nude rat model of human melanoma, MIBG at 30 mg/kg with hyperglycemia of 450 to 500 mg/dL decreased pHi by .6 pH units and pHe by .8 pH units, and MIBG at 22.5 mg/kg with hyperglycemia decreased pHi by .4 pH units and pHe by .5 pH units. Neither of these doses significantly affected muscle pH in the limb or venous pH in the ILP circuit. At 30 mg/kg, MIBG and hyperglycemia produced selective tumor acidification, which translated into improved tumor response rates for both melphalan ILP and, interestingly, control ILP, which used no drug. This suggests that the acidification from MIBG and hyperglycemia, in the absence of melphalan, sensitized the tumors to an antineoplastic effect of the ILP procedure itself. After control ILP alone, we have typically observed a 25% reduction in tumor volume that takes approximately 10 days to reverse. The precise mechanism for this initial minimal response is unclear, although we have hypothesized that it is secondary to ischemia, tissue edema, or both. Acidification with systemic MIBG at 30 mg/kg seems to render the tumors more susceptible to this nonspecific effect from control ILP, as evidenced by the 86% reduction in starting tumor volume and the 33-day delay in time required to return to 100% of the starting tumor volume. However, when systemic MIBG at 22.5 mg/kg is used in combination with hyperglycemia and control ILP, this increased antitumor effect is not observed, and tumor growth resembles that of control ILP.
This steep dose-response relationship for MIBG suggests that a threshold of intratumoral acidification may be necessary before a therapeutic effect occurs. Because tumor cells exist in a chronic low-pH environment, they may have limited ability to adapt to additional acidification.24 Once these buffering mechanisms are overwhelmed and intratumoral pH begins to decrease, tumor cells presumably are susceptible to further insults, which may then signal them to undergo apoptosis.25,26 Some data have suggested that this threshold is reached when pHi begins to decrease (which is maintained at more physiologic levels despite significant extracellular acidity), but further work is needed to clarify this.23,27,28
Although systemic MIBG at 30 mg/kg and hyperglycemia by themselves were not toxic to the animals, when MIBG at 30 mg/kg and hyperglycemia were combined with ILP, there was an approximately 40% mortality rate. Other investigators have also reported lethal effects of MIBG to rodents at doses of 
40 mg/kg,10 presumably secondary to uptake of the compound in nonneoplastic tissues and subsequent inhibition of cellular adenosine triphosphate production. This effect in combination with the prolonged general anesthetic necessary to perform the ILP likely explains the high animal mortality in our model. It is interesting to note that in this study, there were no deaths at MIBG 22.5 mg/kg combined with ILP, suggesting a steep dose-toxicity relationship of the agent.
Systemic MIBG at 30 mg/kg with hyperglycemia was effective at augmenting tumor response rates to melphalan ILP. At this dose of MIBG, tumors regressed by 75%, and regrowth was delayed by 45 days. Systemic MIBG at 22.5 mg/kg and hyperglycemia, in conjunction with MIBG in the ILP perfusate, was also effective in increasing response rates to melphalan ILP. This combination of systemic and regional MIBG with hyperglycemia, which had no effect on response rates to control ILP, caused tumors to regress by 82% and delayed regrowth by 51 days in the setting of melphalan ILP. Although our results with this dose of systemic MIBG are somewhat confounded by the addition of regional MIBG to the perfusate, this empirically derived protocol demonstrates that systemic and regional MIBG can be combined successfully without significant systemic or regional toxicity. The precise dose-response relationship of regionally delivered MIBG to the efficacy of melphalan ILP is an important subject for further research when considering the possible clinical application of this therapy, because ILP in patients lasts for 60 to 90 minutes. This period could potentially allow MIBG to be delivered exclusively as a regional agent.
Because melphalan itself is an acid with an equilibrium constant of approximately 2.5, increased activity of the drug is expected in acidic conditions. This has been demonstrated in vitro,29,30 and our data show that selective tumor acidification with MIBG and hyperglycemia is an effective technique for increasing the activity of melphalan in vivo as well. Given that ILP permits the delivery of high-dose melphalan to its target tissue, acidification strategies may be well suited to increasing its activity if concerns regarding potential toxicity can be answered. Moreover, although questions regarding the relative contribution of regional versus systemic MIBG in augmenting the effect of melphalan require further study, our data demonstrate that intermediate-dose systemic MIBG can be combined successfully with regional MIBG in a rodent model of ILP to enhance the therapeutic effect of melphalan without prohibitive toxicity.
In summary, systemic MIBG and hyperglycemia seem to improve tumor response rates after ILP in a rodent model of human melanoma. Selective tumor acidification with MIBG and hyperglycemia may augment response rates to current regional perfusion in patients without increasing regional toxicity.
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ACKNOWLEDGMENTS |
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Supported by National Institutes of Health grant 5PO1 CA56690 (RZ, JDG, DBL) and the Georgene S. Harmelin Endowment Fund.
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FOOTNOTES |
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Metaiodobenzylguanidine (MIBG) and hyperglycemia selectively acidify tumors and thereby sensitize them to the effects of radiotherapy and hyperthermia. In a rodent model of human melanoma, MIBG and hyperglycemia increase tumor response rates after isolated limb perfusion with melphalan.
Received for publication May 16, 2003. Accepted for publication October 27, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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