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. Author manuscript; available in PMC: 2015 Aug 15.
Published in final edited form as: Mol Imaging Biol. 2013 Dec;15(6):768–775. doi: 10.1007/s11307-013-0655-6

Evaluation of Arginine Deiminase Treatment in Melanoma Xenografts Using 18F-FLT PET

Lars Stelter 1,2, Simon Fuchs 1, Achim A Jungbluth 3, Gerd Ritter 3, Valerie A Longo 2, Pat Zanzonico 2, Nathanael Raschzok 4, Igor M Sauer 4, John S Bomalaski 5, Steven M Larson 2,3,6
PMCID: PMC4537614  NIHMSID: NIHMS707717  PMID: 23722880

Abstract

Purpose

This study aims to develop a molecular imaging strategy for response assessment of arginine deiminase (ADI) treatment in melanoma xenografts using 3′-[18F]fluoro-3′-deoxythymidine ([18F]-FLT) positron emission tomography (PET).

Procedures

F-FLT response to ADI therapy was studied in preclinical models of melanoma in vitro and in vivo. The molecular mechanism of response to ADI therapy was investigated, with a particular emphasis on biological pathways known to regulate 18F-FLT metabolism.

Results

Proliferation of SK-MEL-28 melanoma tumors was potently inhibited by ADI treatment. However, no metabolic response was observed in FLT PET, presumably based on the known ADI-induced degradation of PTEN, followed by instability of the tumor suppressor p53 and a relative overexpression of thymidine kinase 1, the enzyme mainly responsible for intracellular FLT processing.

Conclusion

The specific pharmacological properties of ADI preclude using 18F-FLT to evaluate clinical response in melanoma and argue for further studies to explore the use of other clinically applicable PET tracers in ADI treatment.

Keywords: Arginine deiminase, Melanoma xenograft, Response evaluation, FLT PET

Introduction

Arginine deiminase (ADI) has been recently introduced into clinic as an anticancer treatment. By depleting cancer cells of the semi-essential amino acid l-arginine (Arg), it has been shown in multicenter phase I/II trials to have beneficial effects in patients suffering from irresectable hepatocellular carcinoma or metastasized malignant melanoma [1–4]. Due to the mutual characteristic of lacking the essential enzyme for intrinsic Arg synthesis, argininosuccinate synthetase (ASS), these cancer entities face nutrient starvation and apoptotic cell death [5, 6]. More recent studies indicate that ADI could not only be an interesting therapeutic option in ASS-negative lung cancers but also describe that the present or absent expression of ASS in cancer cells can function as a predictive biomarker for the development of, e.g., pulmonary metastasis, and thus implies a potential target for pharmacologic intervention [7, 8]. Using quantitative molecular imaging modalities to assess and follow ADI treatment, however, has so far failed. The use of 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG PET), which has been well established as a biomarker in imaging treatment response in oncology, failed to show the ADI-associated clinical improvement due to its cross-reactivity with the PI3K signaling axis [9–11]. Still, finding a noninvasive way to follow and evaluate ADI treatment would be desirable for its further translation into clinic, as the early identification of responders and non-responders is particularly crucial for the patients' outcome and management. Based on this premise and the fact that FDG PET did not reflect the effective therapeutic intervention with ADI, we choose the clinically established tracer 3′-[18F]fluoro-3′-deoxythymidine (FLT PET), which objects proliferation by representing DNA synthesis [12, 13]. Relating to preliminary findings [10], we used an analogous experimental set up and, in a proof-of-principle approach, aimed to evaluate FLT PET in treatment response of melanoma xenograft mice undergoing ADI therapy.

Material and Methods

Materials

The melanoma cell lines SK-MEL-10 and SK-MEL-28 were obtained from Memorial Sloan-Kettering Cancer Center and maintained in glucose-containing RPMI and DMEM, respectively, supplemented with 10 % fetal calf serum, l-glutamine, and 100 μg/ml penicillin-streptomycin at 37 °C and 5 % CO2. ADI-EG 20 was provided by DesigneRx Pharmaceuticals Inc., Vacaville, CA, USA, a subsidiary of Polaris Group, San Diego, CA, USA. For immunoblot analysis of protein expression levels, α-ASS (BD Biosciences, Franklin Lakes, NJ, USA), α-β-actin (AC-15, GeneTex, Irvine, CA, USA), α-PTEN (Cell Signaling Technology, Danvers, MA, USA), α-p53 (Cell Signaling Technology), α-mouse, and α-rabbit IgG-HRP secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used according to manufacturer's instructions. Statistical analysis of all data (student t test) was performed with Prism 5.0 (GraphPad, La Jolla, CA, USA) and Microsoft Excel 2008 for Mac Version 12.0. A p value <0.05 was regarded as significant. Error bars represent standard error of the mean (SEM).

ADI Dosing Studies in Xenograft Models

All animal studies were conducted in accordance with the institutional guidelines established at the Memorial Sloan-Kettering Cancer Center (MSKCC). Subcutaneous SK-MEL-28 xenografts were established by injecting 4×106 cells in Matrigel™ (BD, Franklin Lakes, NJ, USA) in the shoulder region of 6–8-week-old female NOD-SCID mice (NOD.CB17-Prkdcscid/J, Jackson Laboratory, Bar Harbor, ME, USA). The mice were randomized into control (PBS, n=3 each) and ADI groups (n=5 each) for PET imaging and further weekly histopathological work-up. Seven days post inoculation (average tumor volume=∼500 mm3), the mice received weekly intramuscular injections of ADI (160 U/m2, equal to approx. 17.8 mg/m2) or PBS. Body weight and tumor volume (caliper measurements: calculation: 2/3 π×((diameter 1+diameter 2)/2)3) were measured once a week for 4 weeks.

FLT PET Imaging of Xenograft Models

PET imaging was performed once a week prior to each treatment. All mice were injected with about 0.3 mCi (11.1 MBq) of 18F-FLT via a lateral tail vein. After 1 h of tracer uptake, the animals were anesthetized with 1–2 % isoflurane (Baxter Healthcare, Deerfield, IL, USA), and PET imaging was performed on an R4 microPET (Concorde Microsystems Inc., Knoxville, TN, USA), a dedicated 3-dimensional small-animal PET scanner. An energy window of 350–750 keV and a coincidence timing window of 6 ns were used, with a minimum of 20 million events, typically acquired over 5 min. The resulting list-mode data were sorted into 2-dimensional histograms by Fourier rebinning, and transverse images were reconstructed by filtered back projection into a 128×128×63 (0.72×0.72×1.3 mm) matrix. The reconstructed spatial resolution for 18F is 2.2-mm full width half maximum (FWHM) at the center of the field of view to 3.2-mm FWHM offset 5 cm from the center of the field of view. The image data were corrected for nonuniformity of response of the microPET, dead-time count losses, and physical decay to the time of injection, but no attenuation or scatter correction was applied. An empirically determined system calibration factor (i.e., in millicurie per milliliter per centipoise per voxel) for mice was used to convert voxel counting rates to activity concentrations, and the resulting image data were then normalized to the administered activity to determine, by region-of-interest analysis, the maximum percentage of injected dose per gram (%ID/g) of tumor corrected for radioactive decay to the time of injection in the tumors, using the ASIPro VM software (Concorde Microsystems Inc.). Finally, the received values were corrected to the partial volume effect to preclude an effect of the tumor sizes on PET quantification, applying an empirically determined equation (measured activity concentration×{1−e−0.80×(source diameter−2.90)}).

Immunohistochemistry

Immunohistochemistry was performed on sections of formalin-fixed paraffin-embedded human melanoma cell line SK-MEL-28 murine xenografts. As primary reagents, α-Ki-67 rabbit antihuman polyclonal antibody (Chemicon, EMD Millipore, Billerica, MA, USA) and a murine monoclonal antibody to thymidine kinase 1 (TK1, Abcam, Cambridge, UK) were used. After an initial blocking step, primary antibodies were applied overnight at 5 °C. Both, a negative (no primary antibody) and a positive control (human tonsil tissue for Ki-67 and TK1; both not shown) were employed. Detection of the primaries was done with a biotinylated rabbit-anti-sheep and horse-anti-mouse secondary antibody, respectively (Vector Labs, Burlingame, CA, USA). Diaminobenzidine (Liquid DAB, Biogenex, San Ramon, CA, USA) served as a chromogen. The Ki-67 index was estimated by counting the percentage of Ki-67-positive cell nuclei per 500–1,000 tumor cells in the tumor region with the greatest density of staining. A pathologist who was unaware of the clinical data and PET findings examined the immunohistochemistry (IHC) slides.

Immunoblot Analysis of Protein Expression Levels

SK-MEL-10 and SK-MEL-28 cells were harvested for protein analysis using M-PER® (Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Calbiochem, EMD Millipore). Protein concentration was determined with a Bradford protein assay (Bio Rad, Hercules, CA, USA) and bovine serum albumin standards. Samples were prepared in LDS sample buffer (Invitrogen, Carlsbad, CA, USA), and cell lysates (25–75 μg/lane) were resolved by gel electrophoresis. Lysate samples were transferred, blocked (SuperBlock® Blocking Buffer, Thermo Scientific, Logan, UT, USA), and probed for expression with primary antibodies for 12 h at 4 °C. After incubation with the appropriate secondary antibody (1 h at room temperature), proteins were visualized with chemoluminescence (ECL, SuperSignal® West Pico, Thermo Scientific).

18F-FLT Uptake in Vitro

A 2×106 of SK-MEL-28 cells were plated in culture flasks and assayed in triplicate. Cells were treated with PBS or 4.3 mU/ml ADI daily for 4 weeks, followed by an incubation period of 10 ml of culture medium containing 0.1 mCi (3.7 MBq) of FLT. After 1 h of incubation, the cells were harvested, and a liquid scintillation counter was used to assay the radioactivity in the medium and in the cell pellets. Percentage uptakes of FLT in cells were determined using the following formula: (net counts in cell pellet)/(net counts in cell pellet + net counts in medium) × 100 %. A sample of cells from each flask was withdrawn after harvest to determine the number of viable cells in each group. Finally, the activity measured in each cell sample was normalized to the number of viable cells.

Results

ADI Treatment of Melanoma Xenografts

ASS-negative SK-MEL-28 cells were inoculated s.c. into the shoulder region of SCID mice. After a tumor size of approximately 500 mm3 (usually within 1 week) was reached, each mouse was assigned to an either weekly ADI or PBS treatment for a 4-week observation/treatment period. ADI-treated mice showed a decline in tumor volumes by more than 50 % of the initial value, whereas the tumors of the PBS-treated mice continued to grow and almost doubled their size (Fig. 1). The differences between the treatment groups reached statistical significance within the second week of treatment (p=0.03). None of the animals showed signs of an adverse reaction towards the treatment or a significant change in body weight.

Fig. 1.

Fig. 1

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SK-MEL-28 xenografts were established in female SCID mice and animals were treated weekly with intramuscular injections of ADI (160 U/m2) or vehicle (PBS). Tumor volume declined continuously from 1 to 4 weeks of ADI treatment. Bars represent SEM (*p<0.05, **p<0.01).

18F-FLT Positron Emission Tomography

To see if the observed response to ADI treatment could be represented or predicted by metabolic changes in the tumors, a weekly FLT PET was performed on each mouse. FLT PET showed a distinct tracer uptake in the tumor region of both groups before initiation of treatment (week 0, Fig. 2a), which visually seemed to be stable during the first 2 weeks of treatment (week 2, Fig. 2a) and even increased at the end of the observation period (week 4, Fig. 2a). Quantification of the tumor volume-corrected tracer uptake confirmed this observation, showing a basically unchanged FLT accumulation in the tumors of both groups for the first 3 weeks at around 3.3 %ID, followed by an increase to 5.4 %ID on week 4 (Fig. 2b).

Fig. 2.

Fig. 2

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a Subcutaneous SK-MEL-28 xenografts were imaged immediately prior (week 0) to ADI therapy (160 U/m2) and once weekly for 4 weeks. Representative PET images before the onset of treatment, week 2, and from the end of the study are shown. Arrowheads indicate the region of FLT uptake associated with the xenograft. b ROI analysis of intratumoral FLT uptake did not show a metabolic response in proportion to the tumor volume reductions associated with ADI therapy (see Fig. 1). Bars represent SEM.

Immunohistochemistry—Ki-67

To elucidate the above-described discrepancy of a clinical tumor response without concomitant changes in FLT accumulation, we analyzed the Ki-67 proliferation index of all treated and untreated xenograft tumors. Ki-67 immunostaining clearly revealed a loss of tumor proliferation after 1 week of ADI treatment (Fig. 3). The Ki-67 index dropped statistically significant from a mean proliferation fraction of 62.7±6.2 % before the onset of treatment to 19.7±0.3 % (p= 0.002) within the first week and stayed low for the whole observation period (Figs. 3 and 4a, b). IHC revealed no significant changes in Ki-67 expression in the control group. The observed increase in FLT retention in week 4 of the treatment was not represented in the in vitro or ex vivo data. A longer observation period needs to confirm and hopefully explain this finding.

Fig. 3.

Fig. 3

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Immunohistochemistry of excised tumors revealed a constant decline in Ki-67-expression in the treatment group compared to that in the PBS group. Representative sections of excised tumors before the onset of treatment, week 1, and from the end of the study are shown.

Fig. 4.

Fig. 4

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A direct comparison of the quantified volume-corrected FLT uptake (%ID/g) and Ki-67 proliferation index (in percentage) in numbers (a) and visualized in the graph (b) reveals the disconnection of an unchanged FLT retention despite a significant drop in cell proliferation. Bars represent SEM (**p<0.01).

Immunoblotting—p53 and IHC–TK1

We previously showed that ADI treatment affects the PTEN/PI3K/Akt pathway, mainly by downregulating PTEN, promoting downstream activation of the PI3K/Akt axis [10]. There is a well-known association between PTEN and the tumor suppressor p53 as well as to the expression of thymidine kinase 1 (TK1) [14, 15], the enzyme that mainly contributes to cellular FLT phosphorylation. Consequently, we analyzed the expression of both PTEN and TK1 and observed a loss of PTEN expression in ADI-sensitive melanoma cells within the first 2 weeks of treatment, reaching basically undetectable levels after 3 weeks of treatment. Mock treatment with PBS as well as treatment of ADI-insensitive cells did not result in any alteration of cellular PTEN expression (Fig. 5a, b). Accompanied with PTEN downregulation, we also observed a decrease of p53 in ADI-susceptible cells (Fig. 5b). Regarding the expression of TK1 in excised xenograft specimen, no clear dynamic in the expression levels could be observed within the ADI treatment period or the PBS group, using IHC (Fig. 6).

Fig. 5.

Fig. 5

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ADI treatment (4.3 mU/ml) causes loss of PTEN expression accompanied by a decrease in p53 in ASS-negative and ADI-sensitive SK-MEL-28 cells, whereas no changes were observed in the ADI-insensitive cell line SK-MEL-10. HT29 colon carcinoma cells were used as a positive control.

Fig. 6.

Fig. 6

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Immunohistochemistry of excised tumors revealed a basically unchanged expression of TK1 upon ADI treatment and PBS. Representative sections of excised tumors before the onset of treatment, week 1, and from the end of the study are shown.

18F-FLT Uptake in Vitro

Incubation of ADI-sensitive and ADI-insensitive cells with FLT revealed no major differences regarding the tracers' uptake between both cell lines. Also, no significant changes, i.e., loss, of cellular FLT accumulation could be observed during the course of ADI treatment in viable SK-MEL-28 cells (Fig. 7).

Fig. 7.

Fig. 7

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In the context of an unchanged TK1 expression, no significant changes of FLT uptake could be observed in ADI-sensitive SK-MEL-28 and -insensitive SK-MEL-10 cells upon ADI treatment. Bars represent SEM.

Discussion

The metabolic strategy to deprive tumors of the semi-essential amino acid l-arginine (Arg) by enzymatic degradation using pegylated ADI has been promising and successful in numerous preclinical and clinical studies [1–3, 6, 7, 16, 17]. Arg auxotrophy of these tumor entities is mediated by ASS promoter methylation and the absence of the ASS protein, which functions as the rate-limiting enzyme in Arg synthesis [4, 18]. ADI proved to be effective as a single treatment and in combination with other antiproliferative drugs and is currently evaluated in clinical trials of advanced hepatocellular carcinoma, refractory small cell lung cancer, malignant pleural mesothelioma, and prostate cancer [17–19, http://clinicaltrials.gov].

Using functional imaging modalities to evaluate anticancer treatment is a constantly evolving field and has been the gold standard for response assessment in numerous cancer entities for many years [20–22]. PET in ADI-guided therapy, however, has only been evaluated in very few cases. Especially the use of FDG PET has been hampered for response evaluation due to ADI-mediated modulations associated with the PTEN/PI3K/Akt pathway [10, 11, 23, 24]. In our study, we used FLT PET for treatment evaluation and, despite a good clinical response and a significant drop in the Ki-67 proliferation index in IHC, failed to observe the expected metabolic response in FLT PET. A comparable discordance of tumoral FLT retention and changes in Ki-67 activity has been described for other cancer entities in different kinds of treatment. Possible sample mismatch or treatment-associated changes in TK1 activity, the known key determinant for FLT uptake, has been discussed as the underlying cause in these cases [15, 25–28]. In our study, however, IHC revealed no changes in tumoral TK1 expression but a constantly high protein expression for the whole observation period. This apparent disconnection of FLT retention/TK1 activity and Ki-67 activity has also been observed by others and proved to be indicative for high levels of cells in S-phase and DNA repair [15, 29]. ADI is generally known to cause G1 arrest in tumor cells, and we repeatedly observed loss in cell proliferation and apoptotic cell death, followed by tumor volume decrease in ADI-treated melanomas [10, 23, 30]. As a next step, we probed for PTEN expression in our melanoma cells and confirmed the loss of PTEN expression within 2 weeks of ADI treatment. Interestingly, we further observed an indication for an accompanied attenuation of cellular p53 protein levels. There is a known interaction of these two tumor suppressors, which has most commonly been described as a functional cooperation in a protective role of PTEN, anticipating p53 degradation. In case of PTEN loss, however, PI3K is activated, leading to the phosphorylation of Mdm2 and subsequent p53 degradation [10, 31]. Further on, there have been a few reports that link expression of p53 to TK1 activity [32]. P53 is described to play a pivotal role in maintaining the relationship of TK1 activity and S-phase of the cell cycle. If p53 gets activated in cancers, e.g., under cytotoxic therapies, TK1 activity and cell proliferation get downregulated leading to a G1 arrest [33, 34]. As there is a presumably PTEN-influenced downregulation of p53 in our study, this regulatory mechanism might be altered, resulting in an unchanged high TK1 activity and an unchanged tracer uptake. Consequently, and despite a clear treatment-associated decrease in tumoral proliferation, PTEN loss seems to result in a disconnection of the cells' proliferative status and p53/TK1 activity that masks the ADI effect in FLT PET. There are other known activities that can modify cellular FLT uptake and retention, such as the equilibrative nucleoside transporter hENT1 or nucleotide efflux pumps [35– 37]. Additional studies are under way to further examine the biological effect of these mechanisms on PET imaging. As two of the most well-known metabolic markers for treatment response, based on ADI-related modulations of the tumor suppressor PTEN, failed in our experimental setting, there is a strong need to further continue the effort to find an imaging tool for evaluating the therapeutic proceeding of ADI. In that respect, targeting apoptosis could be a promising approach, especially the recently introduced 18F-labeled 5-fluoropentyl-2-methyl-malonic acid (18F-ML-10) seems to imply favorable characteristics to be evaluated in preclinical and clinical settings [38, 39].

Acknowledgments

The authors would like to thank Dr. Marcus Kelly for his informative discussions and Megan Holz for her invaluable assistance. We also thank the staff of the Radiochemistry/Cyclotron Core at MSKCC. Technical services provided by the MSKCC Small-Animal Imaging Core Facility were supported in part by NIH grants R24 CA83084 and P30 CA08748. L.S. was supported by a grant (Ste 1837/1-1) of the Deutsche Forschungsgemeinschaft.

Footnotes

Conflict of Interest. J.S.B. is the Executive Vice President of Polaris Group that provided ADI-PEG 20 for this study. All other authors declare that they have no conflict of interest.

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