Journal of Drug Targeting, 2009; 17(4): 268–277

R E S E A R C H A R T I C L E

Targeting malignant glioma cells in vitro using platelet-derived growth factor AA-based conjugates

Maria Dahlström Wester, Åke Wasteson, and Annelie Lindström

Division of Cell Biology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden

Address for Correspondence: Maria Dahlström Wester, Division of Cell Biology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden. E-mail: maria.wester@liu.se

(Received 03 December 2008; revised 02 January 2009; accepted 04 January 2009)

Introduction

The use of cell-toxic substances, directed against specific structures on cancer cells, is of great potential impor-tance. The aim of the targeting approach is specific exposure of the tumor to cell-damaging agents inducing cell growth inhibition or apoptosis in the tumor cells, but sparing normal cells.

Glioblastoma multiforme (GBM) is an unusu-ally aggressive brain tumor; it is also highly hetero-geneous (Kleihues & Ohgaki, 1999; Lassman, 2004; Sathornsumetee & Rich, 2006; Chandana et al., 2008; Colman & Aldape, 2008). GBM presents a poor progno-sis and median survival is less than 1 year using conven-tional treatment, surgery, radiotherapy, and chemother-apy, calling for development of new treatment strategies. GBM shows incorrect control of essential growth regu-lating functions such as growth factor receptors, PI3K/Akt, Ras/MAPK, and PLC (Rich & Bigner, 2004; Alper Arslan, Kutuk, & Basaga, 2006; Sathornsumetee & Rich,

2006; Wong, Kaye, & Hovens, 2007). It is a challenging possibility to utilize such aberrant characteristics in molecular therapies.

Earlier studies have shown that platelet-derived growth factor receptors (PDGFRs) are overexpressed or amplified in certain cancers, for example, prostate, colon, breast, ovarian, and malignant brain tumors (Nistér et al., 1988; Westermark, 1993; Westermark, Heldin, & Nistér, 1995; Hermansson et al., 1996; George, 2003; Hunter et al., 2003; Lassman, 2004; Board & Jayson, 2005). PDGFRs might therefore act as potential targets for therapy. The PDGF receptors exist in two forms, alpha () and beta (), that can form three types of dimers (:, :, :). Overexpressed PDGFR is localized on malignant glioma tumor cells, whereas PDGFR has been found in high amounts on newly formed, normal, endothelial cells in the vicinity of such tumors (Hermansson et al., 1992; Kirsch, Wilson, & Black, 1997). The PDGF ligands have, until now, been shown to exist in five isoforms, AA, BB, AB, CC, or DD, where A, B, C, and D are different

ISSN 1061-186X print/ISSN 1029-2330 online © 2009 Informa UK LtdDOI: 10.1080/10611860902718698

AbstractGlioblastoma multiforme (GBM) is an unusually aggressive brain tumor; it is also highly heterogeneous. Poor prognosis and a median survival of less than 1 year, using conventional treatment, calls for develop-ment of new treatment strategies. Overexpression and/or amplification of platelet-derived growth factor alpha receptors (PDGFαRs) in GBM might act as potential targets for a novel therapeutic approach. In this study, conjugates based on PDGFAA-ligand and dextran, of different sizes (10 and 40 kDa dextran), were prepared and investigated regarding targeting properties in vitro. Three human malignant glioma cell lines, U343MGa31L, U343MGaCl2:6, and U563MG, were used because of their previously reported differ-ences in receptor expression and behavior. PDGFAA-based 10 kDa dextran iodine-125 radiolabeled conju-gates showed the most favorable properties according to results achieved in accumulation, retention, and localization of cell-associated radioactivity. In comparison with dextran-125I-tyrosine delivered radioactivity, the PDGFAA-based dextran conjugates confirm the potential of receptor targeting.

Keywords: Conjugate; dextran; glioblastoma multiforme; PDGFAA; PDGF receptors; targeting

http://www.informapharmascience.com/drt

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Targeting PDGFAA-based conjugates to glioma cells 269

polypeptide chains. The protein isoforms have different affinity for the - and -receptors (Heldin & Westermark, 1999; Board & Jayson, 2005). Coexpression of a PDGF-ligand and a responsive PDGF-receptor, which has been described for PDGFAA in a number of cell types, would enable autocrine/paracrine stimulation of cancer cells (Hermansson et al., 1992; Kleihues & Ohgaki, 1999; Dai et al., 2001; Lokker et al., 2002; Lassman, 2004). It would also offer a novel therapeutic approach.

It is therefore of interest to synthesize substances on basis of the PDGFAA-ligand to target PDGFR-carrying cells. Relatively low molecular weight ligands often show short half-lives in the circulation whereas conjugation to a water-soluble polymer may have a stabilizing effect (Andersson et al., 1991; Zalipsky & Lee, 1992; Francis et al., 1996; Sjöström et al., 1997; Zhao et al., 1997; Mehvar, 2000; Hägg et al., 2002). Conjugation of PDGF-ligand to a polymer, for example, dextran or polyethyleneglycol (PEG), can improve its handling properties influencing internalization and retention of the protein in vitro and in vivo. Dextran is a water-soluble polysaccharide which is considered to be biologically inert (Molteni, 1979). It is possible to chemically activate dextran, that is, introduce reactive groups that readily react with proteins and/or other substances resulting in covalent bonding between these moieties. Different chemical reagents are avail-able for the activation of dextran, resulting in different reactive groups (Axén, Porath, & Ernback, 1967; Molteni, 1979). CDAP (1-cyano-4-dimethyl amino pyridinium tetra-fluoroborate), a nonhazardous alternative to cyanogen bromide, together with triethylamine (TEA) activates hydroxyl groups on dextran. Thereby cyanate esters, carbamates, imidocarbonates, and pyridinium-isourea derivatives are formed. The cyanate esters and pyridinium-isourea derivatives readily react with primary amino groups in proteins forming secondary stable carbamates. During the conjugation reaction, the pyridinium-isourea derivatives are released in the form of inert 4-dimethylamino pyridinium salt (Kohn & Wilchek, 1983, 1984). It is crucial to optimize the conjugation procedure, that is, the degree of activation in relation to protein concentration, to avoid cross-linking in the polysaccharide resin (Molteni, 1979). The conjugate can be loaded with toxic substances, such as chemotherapeutic agents or stable/radioactive isotopes. Delivered to a target cell such substances may cause cell damage and induce cell death if allowed to be retained for a sufficient period of time.

The aim of this study was to prepare PDGFAA conju-gates, on the basis of the dextran of different sizes, and investigate their receptor-binding properties in vitro. No such study has been reported previously. For trac-ing the conjugates, the radioactive isotope 125I was used. Previous studies show the use of dextran to increase the biological durability of low molecular weight ligands in

vitro and in vivo (Molteni, 1979; Andersson et al., 1991). For example, epidermal growth factor (EGF) conjugated to dextran with a molecular weight of 19.5 kDa showed extensively prolonged half-life when tested on GBM cells with overexpression of EGF receptors (Andersson et al., 1991). When using EGF-dextran-tyrosine-131I con-jugates on GBM in vitro, a strong anticlonogenic effect was achieved as a result of the prolonged cellular reten-tion of the radioactive isotope (Andersson, Capala, & Carlsson, 1992).

Considering the molecular size of dextran and that of the ligand, there might be an optimal mass relation between the two. It is reasonably possible that the length of the dextran molecule may determine the in vitro and in vivo properties of the conjugate. Although a longer dextran molecule would allow the delivery of a higher number of cell-damaging agents, it may structurally hinder specific receptor binding. Therefore, dextrans of two different molecular sizes, 10 and 40 kDa, were used for conjugation. Experimental settings were performed to reveal the impact of different dextran molecular sizes on the targeting quality of PDGFAA-dextran conjugates. It is of significance for a future drug to possess optimal in vivo properties for maximal exposure to target tissue. Therefore, the conjugates were investigated regarding cellular accumulation as a function of time, retention, and localization (membrane-associated or internalized radioactivity over time) of cell-associated radioactivity in vitro.

Facing the choice of an in vitro model system, the selection of a target cell is significant for mimicking the tumor in vivo properties. In this case, the studied material should be of human origin, well established, consistent, and representative for GBM in vivo. The three human malignant glioma cell lines, U343MGa31L, U343MGaCl2:6, and U563MG, seem to fulfill these cri-teria and therefore were used in this study. They have extensively been used as in vitro model systems for GBM. This includes their characteristics in receptor expression and behavior (Nistér et al., 1987, 1991; Claesson-Welsh et al., 1989; Dahlström et al., 2004).

Materials and methods

Cells and cell culture

The human malignant GBM cell lines, U343MGa31L, U343MGaCl2:6, and U563MG, were gifts from Dr. B. Westermark (Uppsala, Sweden). U343MGa31L and U563MG were grown in MEM-Earles media and U343MGaCl2:6 in D-MEM/F-12 medium. The different media were all supplemented with 10% heat-inactivated fetal bovine serum (FBS), l-glutamine (2 mM), strep-tomycin (45 µg/mL), and penicillin (45 IU/mL). Media

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270 Maria Dahlström Wester, Åke Wasteson, and Annelie Lindström

were routinely changed three times a week, and cells were subcultivated once a week. Cells were maintained in humidified air with 5% CO

2 (g) at 37ºC. Experiments

were repeated in triplicates, and individual cell cultures were visually inspected using a microscope (Olympus CK2, Japan). Cell culture media and supplements were from Invitrogen Life Technologies (Stockholm, Sweden).

The number of cells per Petri dish (TC-dish 60 mm TCT) during experiments was 0.76–4.05· × 106 for U343MGa31L (corresponding to 2.71–14.5· × 104 cells/cm2), 1.56–7.55· × 106 for U343MGaCl2:6 (5.58–27.0· × 104 cells/cm2), and 0.98–4.85· × 106 for U563MG (3.50–17.3· × 104 cells/cm2).

Radiolabeling

Human recombinant PDGFAA (Cat. No. 100-13A, Lot. No. 111CY31; Peprotech EC, London, UK) was used in all experiments. PDGFAA (5 µg in 50 µL of 0.1 M NaHCO

3,

pH 8.2) and l-tyrosine (5 µg in 40 µL of 0.1 M NaHCO3)

were radiolabeled with 125I (18.5 MBq in 0.01 M NaOH, pH 8–12, IMS30; Amersham Biosciences) using the chloramine-T method (Greenwood, Hunter, & Glover, 1963). Briefly, after the addition of 125I (4.7–11.3 µL) to PDGFAA, 10 µL chloramine-T (2 mg chloramine-T/mL of 0.5 M potassium phosphate buffer, pH 7.5) was added. The solution was incubated for 1 min at room tempera-ture before termination with 25 µL sodium metabisulfite (2 mg sodium metabisulfite/mL of 0.5 M potassium phosphate buffer). 125I-PDGFAA and 125I-tyrosine prepa-rations were used directly for conjugation (see below). Nonconjugated ligand, 125I-PDGFAA, preparation was directly separated from low molecular weight reagents by gel chromatography (see below).

Conjugation procedure

Activation of dextranDextran (dx) with an average molecular weight of 10 kDa (8.5–11.5 kDa) or 40 kDa (35–45 kDa) was used. Dextran (10 mg in 1 mL of 0.1 M NaHCO

3, 0ºC) was mixed under

constant stirring with 10 mg 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP). After 10 sec, 6 µL triethylamine (TEA) (equal number of moles as CDAP) was added dropwise for 45 sec. Total activation time was approximately 120 sec.

Preparation of 125I-labeled conjugates (10 or 40 kDa dx)125I-tyrosine (80 µL) and PDGFAA (5 µg in 50 µL of 0.1 M NaHCO

3) was added to activated dextran (250 µL) to

yield PDGFAA-dextran-125I-tyrosine. To obtain dextran-125I-tyrosine, radiolabeled tyrosine (80 µL) was added to activated dextran (250 µL). The solutions were mixed

under constant stirring for at least 180 min at room temperature before dilution with 0.1 M NaHCO

3 to a

final volume of 500 µL. 125I-PDGFAA-dextran (10 kDa dx) was obtained by mixing 125I-PDGFAA, directly after radiolabeling, with activated dextran according to the procedure described earlier. Thereafter, high molecular weight molecules were separated from low molecular weight reagents on NAP-5 columns (Sephadex G-25). The columns were equilibrated with 1 M HAc (5 mg BSA/mL) (125I-PDGFAA and 125I-PDGFAA-dextran, 10 kDa dx) or 0.1 M NaHCO

3 (1 mg BSA/mL) [PDGFAA-dextran-125I-

tyrosine (10 and 40 kDa dx) and dextran-125I-tyrosine (10 and 40 kDa dx)]. The high molecular weight fractions were eluted in 1 mL of corresponding equilibrium solu-tion. A small sample (5 µL) from each high molecular weight fraction was measured in a gamma counter (1470 Automatic gamma counter; Perkin Elmer Wallac), and data were used for the calculation of specific activity in MBq/µg PDGFAA or tyrosine. The high molecular weight fractions obtained from gel chromatography were diluted in supplemented cell culture media for the respective cell line to a concentration of 14.3–18.5 kBq/mL.

Accumulation of cell-associated radioactivity

Cells were incubated with 1 mL of 18.5 kBq/mL 125I-PDGFAA, 125I-PDGFAA-dextran (10 kDa dx), or PDGFAA-dextran-125I-tyrosine (10 and 40 kDa dx) incubation solutions, respectively, for different time periods (10, 20, 40, 60, 90, 120, 240, 360 min). The cells were washed three times with 1 mL phosphate-buffered saline (PBS; pH 7.4–7.5, room temperature) before treatment with 1 mL trypsin (0.25% trypsin). The cell suspension was collected and combined with a 1 mL wash solution and thereafter measured due to radioactive content using a gamma counter. Cell count measurements (Z2 Coulter particle count and size analyzer; Beckman Coulter) were performed at each time point in parallel cultures. Incubation time, for the respective cell line in this experimental setting, was determined as the time point with the highest registered amount of accumulated cell-associated radioactivity.

Retention of cell-associated radioactivity

Cells were incubated with 1 mL of 14.3–18.5 kBq/mL of respective solution of nonconjugated ligand and conju-gates for the corresponding incubation time (Table 1). The incubation medium was then replaced with 1 mL nonradioactive cell culture medium and cells were fur-ther cultured for 0, 10, 20, 40, 60, 120, and 1440 min. Each medium was collected and combined with the first wash out of three with PBS; thereafter, the cells were treated with trypsin as described earlier. Cell suspensions were collected; the Petri dishes were washed once with 1 mL

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Targeting PDGFAA-based conjugates to glioma cells 271

PBS and the wash solution combined with the cell sus-pension and measured.

Localization of cell-associated radioactivity

Cells were incubated with 1 mL 18.3–18.5 kBq/mL of respective solution of nonconjugated ligand or conju-gates for the corresponding incubation time (Table 1). Incubation was carried out as described earlier for the retention of cell-associated radioactivity and was ter-minated by treatment with 1 mL sodium acetate (0.5 M NaCl in 0.2 M NaAc, diluted with 17.5 M HAc to pH 2.5) per Petri dish for 6 min at 4ºC (Haigler et al., 1980; Andersson et al., 1991). The acetate fractions were col-lected and combined with 1 mL wash solution per Petri dish. Cell remnants were thereafter treated with 1 mL of 1 M NaOH for 60 min at 37ºC and suspensions were collected. Washing with 1 mL NaOH was combined with corresponding suspension and measured.

Statistical analysis

Standard errors for the different measurements are presented by error bars in the figures. Each data point represents three individual Petri dishes for all experi-ments. Student’s t-test, two sample unequal variance test (Microsoft Excel), was used to calculate differences in retention. Retained radioactivity within each experi-mental setting (cell line and type of administered radio-activity) was compared. A p-value of ≤0.05 (two-tail) was considered to be statistically significant.

Results

Preparation of nonconjugated radiolabeled PDGFAA and PDGFAA-based dextran conjugate incubation solutions

The preparation procedure resulted in incubation solutions of the respective constructs at a radioactive concentration of 14.3–18.5 kBq/mL. Specific activity was determined to be 125I-PDGFAA: 0.43–1.47; 125I-PDG-FAA-dx: 0.81; PDGFAA-dx-125I-tyr: 10 kDa dx 2.11, 40 kDa dx 4.39; dx-125I-tyr: 10 kDa dx 2.75 and 40 kDa dx 4.81 MBq/µg PDGFAA or tyrosine.

Accumulation of cell-associated radioactivity

Incubation of U343MGa31L, U343MGaCl2:6, and U563MG with 125I-PDGFAA, 125I-PDGFAA-dextran (10 kDa dx), and PDGFAA-dextran-125I-tyrosine (10 and 40 kDa dx) lead to time-dependent accumulation of cell-associated radioactivity. Incubation time, for respective cell line and conjugate and nonconjugated ligand, respectively, was determined from Figure 1 as the time where the highest amount of cell-associated radioactivity was attained. U563MG (Figure 1C) showed a uniform cell-associated radioactivity, delivered by 125I-PDGFAA, at the incubation times tested. For con-venience, the incubation time was decided to be 60 min (equal to incubation times estimated for U343MGa31L and U343MGaCl2:6). Table 1 summarizes the incubation time for respective cell line, conjugated, and nonconju-gated ligand. For comparison, the same incubation times, as for the respective conjugates, were used for dextran-125I-tyrosine, 10 kDa and 40 kDa dextran (Table 1).

Effect of dextran on the accumulation of cell-associated radioactivity

Conjugation of PDGFAA with dextran effected the required incubation time to reach the highest level of cell-associated radioactivity. A longer incubation time was needed for PDGFAA-based dextran conjugates com-pared with nonconjugated ligand (Table 1). Although the 40 kDa dextran-based conjugate had a higher specific activity, the results show a slow accumulation rate and a very low amount of total cell-associated radioactivity in the cell lines tested.

Effect of dextran on retention of cell-associated radioactivity

Retention of cell-associated radioactivity delivered by 125I-PDGFAA, 125I-PDGFAA-dextran, PDGFAA-dextran-125I-tyrosine (10 kDa dextran), and dextran-125I-tyrosine (10 kDa dextran) is presented in Figure 2. Cell-associated radioactivity, Bq/106 cells, was related to the cell-associated radioactivity at incubation time zero (i.e., the amount of cell-associated radioactivity at the time at which the radioactive incubation medium was replaced with nonradioactive cell culture medium,

Table 1. Maximal accumulation of cell-associated radioactivity after incubation with 125I-labeled substances as a function of time.

125I-PDGFAA

(min)

125I-PDGFAA-dx (10 kDa dx)

(min)

PDGFAA-dx-125I-tyrosine (10 kDa

dx) (min)

PDGFAA-dx-125I-tyrosine (40 kDa

dx) (min)

dx-125I-tyrosine (10 kDa dx)

(min)

dx-125I-tyrosine

(40 kDa dx) (min)

U343MGa31L 60 240 360 360 360 360

U343MGaCl2:6 60 240 360 60 360 60

U563MG 60 240 360 360 360 360

Data recorded from Figure 1. dx: dextran.

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272 Maria Dahlström Wester, Åke Wasteson, and Annelie Lindström

set to 100%) and given in % (Figure 2). Table 2 summa-rizes data from Figure 2 at time points 10 min and 24 h (1440 min). The results indicate differences between the cell lines. For U343MGa31L (Figure 2A), the 125I-PDGFAA-dextran (10 kDa dx) showed an unchanged amount of cell-associated radioactivity (95% at 10 min) during a long time (0–120 min). Although a decrease in detected amount, 74% at 24 h, a statistical difference

could not be seen (p > 0.05 at all time points). PDGFAA-dextran-125I-tyrosine (10 kDa dx) showed a decrease to 71% during the first 10 min in U343MGa31L, but there-after results were in the same range till 120 min (p > 0.05 at 10–120 min). At 24 h, 50% of the cell-associated radio-activity remained in the cells (half-life time 24 h, p < 0.05 at 24 h). Radioactivity delivered as 125I-PDGFAA, was in comparison, rapidly released from the U343MGa31L

125I-PDGFAA

(A) (D)

(B) (E)

(C) (F)

(G) (J)

(H) (K)

(I) (L)

U343MGa31L U343MGa31L

U343MGaC12:6 U343MGaC12:6

U563MG U563MG

U343MGa31L U343MGa31L

U343MGaC12:6 U343MGaC12:6

U563MG U563MG

15001000

Bq/

106 c

ells

5000

0 100 200Time (min)

300 400

600400

Bq/

106 c

ells

2000

0 100 200Time (min)

300 400

15001000

Bq/

106 c

ells

5000

0 100 200Time (min)

300 400

600400

Bq/

106 c

ells

2000

0 100 200Time (min)

300 400

125I-PDGFAA-dx(10 kDa dx)

15001000

Bq/

106 c

ells

5000

0 100 200Time (min)

300 400

600400

Bq/

106 c

ells

2000

0 100 200Time (min)

300 400

150100

Bq/

106 c

ells

500

0 100 200Time (min)

300 400

6040

Bq/

106 c

ells

200

0 100 200Time (min)

300 400

6040

Bq/

106 c

ells

200

0 100 200Time (min)

300 400

150100

Bq/

106 c

ells

500

0 100 200Time (min)

300 400

6040

Bq/

106 c

ells

200

0 100 200Time (min)

300 400

150100

Bq/

106 c

ells

500

0 100 200Time (min)

300 400

PDGFAA-dx-125 I-tyrosine(10 kDa dx)

PDGFAA-dx-125 I-tyrosine(40 kDa dx)

Figure 1. (A–L) Accumulation of cell-associated radioactivity (Bq/106 cells) after incubation with 125I-labeled substances as a function of time. Cells were incubated for 10–360 min before harvest. Each data point represents three individual experiments ± SD. dx: dextran.

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Targeting PDGFAA-based conjugates to glioma cells 273

cells as shown in Table 2, 68% at 10 min and only 16% left at 24 h (p < 0.05 at all time points). Even though the amount of cell-associated radioactivity delivered as dextran-125I-tyrosine (10 kDa dx) showed a rapid release at 10 min, it was thereafter more or less unchanged at the following incubation times analyzed (49% at 10 min, 45% at 24 h, p > 0.05 at all time points). Results for U343MGaCl2:6, treated with conjugates and non-conjugated ligand are shown in Figure 2B. Differences were seen for 125I-PDGFAA at 24 h in comparison with all tested substances for this cell line (p < 0.05 at all tested time points). Only 35% of 125I-PDGFAA was retained at 24 h while the corresponding value was 71–78% (p > 0.05 at all tested time points) for the conju-gates (Table 2). At 10 min, cell-associated radioactivity delivered as dextran-125I-tyrosine to U343MGa31L was decreased to 60% (and thereafter increased to 78% at 24 h, p < 0.05 only at 24 h). The corresponding value at 10 min for 125I-PDGFAA-dextran and PDGFAA-dextran-125I-tyrosine was 78% and 90% (p > 0.05 at all tested time points), respectively (Table 2). In U343MGaCl2:6 cells, the half-life for nonconjugated ligand was esti-mated to approximately 12 h but exceeded 24 h for the PDGFAA-based conjugates. For U563MG, 97% of the cell-associated radioactivity delivered by 125I-PDGFAA remained at 10 min in comparison with 68% and 90% for U343MGa31L and U343MGaCl2:6, respectively. U563MG showed, after the first 10 min, a rapid decrease in cell-associated radioactivity delivered as 125I-PDG-FAA resulting in a half-life of 50 min (28% remained at 24 h, Table 2, p < 0.05 at all time points), in comparison with 95 min for U343MGa31L and approximately 12 h for U343MGaCl2:6. Half-life of dextran-based conju-gates exceeded 24 h except for a 10 min half-life for dextran-125I-tyrosine and U343MGa31L. In U563MG cells, the pattern for PDGFAA-dextran-125I-tyrosine and dextran-125I-tyrosine (10 kDa dx) were mutually similar at the tested time points (Figure 2C, p > 0.05 at all time points). In this cell line, 125I-PDGFAA-dextran showed a curve with several phases but ends at the same level

Table 2. Retention of cell-associated radioactivity, incorporated after exposure of cells to 125I-PDGFAA and 125I-radiolabeled dextran conjugates.

125I-PDGFAA (%) 125I-PDGFAA-dx (%) PDGFAA-dx-125I-tyrosine (%) dx-125I-tyrosine (%)

U343MGa31L

10 min 68 95 71 49

24 h 16 74 50 45

U343MGaCl2:6

10 min 90 78 90 60

24 h 35 72 71 78

U563MG

10 min 97 74 65 60

24 h 28 59 63 59

Cells were incubated with nonconjugated ligand and conjugates before replacement by nonradioactive cell culture media. After replacement (Table 1) with nonradioactive cell culture media, cell cultures were further incubated for 0–1440 min before harvesting. Retained radioactivity is given in % of total cell-associated radioactivity. Data recorded from Figure 2. dx: dextran, 10 kDa.

180(A)

(B)

160140120100

% 80604020

010 20 40

Time (min)

U343MGa31L

60 120 1440

180160140120100%

80604020

010 20 40

Time (min)

U343MGaC12:6

60 120 1440

(C)180160140120100%

80604020

010

125-PGDFAA 125I-PDGFAA-dx

PDGFAA-dx-I125I-tyr dx-125tyr

20 40Time (min)

U563MG

60 120 1440

Figure 2. (A–C) Retention of cell-associated radioactivity, incor-porated after exposure of cells to 125I-PDGFAA and 125I-radiolabeled dextran conjugates, given in %, as a function of time. Cell cultures were incubated with conjugates and nonconjugated ligand before replacement with nonradioactive cell culture media. Cell cultures were harvested 0–1440 min after replacement. Each data point repre-sents three individual experiments ± SD. dx: dextran, 10 kDa.

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274 Maria Dahlström Wester, Åke Wasteson, and Annelie Lindström

as the other two dextran substances, PDGFAA-dextran-125I-tyrosine and dextran-125I-tyrosine, at 24 h (59–63%, Table 2, p > 0.05). The corresponding value at 10 min was 74% for 125I-PDGFAA-dextran, 65% for PDGFAA-dextran-125I-tyrosine, and 60% for dextran-125I-tyrosine. In comparison with nonconjugated ligand, 125I-PDGFAA, the 10 kDa dextran-based conjugates showed extended retention properties, especially 125I-PDGFAA-dextran.

Conjugates based on 40 kDa dextran gave a poor retention in all cell lines tested. Radioactivity was not retained cell-associated at a sufficient amount, not even at the shortest incubation time (10 min). This indicates, in this experiment, less favorable targeting properties of conjugates based on 40 kDa dextran. Consequently, further experiments with these conjugates, PDGFAA-dx-125I-tyrosine (40 kDa dx) and dx-125I-tyrosine (40 kDa dx), were not performed, and 125I-PDGFAA-dextran based on this dextran size was not prepared.

Effect of dextran on localization of cell-associated radioactivity

Table 3 summarizes the distribution, in %, of mem-brane-associated and internalized radioactivity at 0 and 24 h. At 0 h, 125I-PDGFAA showed an approximately equal distribution between membrane-associated and internalized radioactivity for the cell lines tested. At 24 h, a higher membrane-associated fraction was seen for U343MGa31L (60%) and U343MGaCl2:6 (76%) while U563MG showed approximately a uniform distribu-tion (55% membrane-associated, 45% internalized). Cell-associated radioactivity delivered as 125I-PDGFAA-dextran was to the greatest part internalized at all ana-lyzed times and for all cell lines. At 24 h, the amount of internalized radioactivity delivered by this conjugate was increased for all cell lines. Radioactivity delivered as PDGFAA-dextran-125I-tyrosine was to the greatest part membrane-associated at all time points and for all cell lines (85–88% at 0 h, 65–85% at 24 h). Over time, the membrane-associated radioactivity declined in a

similar pattern for all cell lines. Consequently, the frac-tion of internalized radioactivity in relation to total cell-associated radioactivity increased at 24 h (12–15% at 0 h, 29–35% at 24 h). Dextran-125I-tyrosine was mainly associ-ated with the membrane at all analyzed times and for all cell lines (86–90% at 0 h, 63–86% at 24 h). U343MGaCl2:6 indicated an increase in internalized radioactivity deliv-ered by this conjugate at 24 h (14% internalized at 0 h, 37% at 24 h). The relation between membrane-associ-ated and internalized radioactivity at 24 h was similar for U343MGa31L and U563MG (membrane-associated 86%, internalized 14%; Table 3). In summary, the char-acter of the conjugates seems to influence the distribu-tion of cell-associated radioactivity.

Visual inspection of cell cultures

The visual inspection, using a microscope, of all cell cul-tures incubated with the radioactive conjugate incuba-tion solution showed no differences in morphology or cell density in comparison with cell cultures used for cell number determination. In addition, the culture media showed no cloudiness or change in pH.

Discussion

In cancer cells, activation of PDGFR results in signaling events inducing various cellular responses, including cell proliferation, survival, and migration (Pietras et al., 2003). Among other changes, GBM has been shown to overexpress and/or have an amplified PDGFR (Pontén & Westermark, 1978; Shapiro et al., 1991) and coexpres-sion with PDGFAA suggests autocrine/paracrine stimu-lation of these cancer cells (Hermansson et al., 1992; Kleihues & Ohgaki, 1999; Dai et al., 2001; Lokker et al., 2002; Lassman, 2004). This indicates a potential route for PDGFAA to target PDGFR-carrying cells. PDGFAA conjugates carrying toxic substances, i.e., radioactive/stable isotopes or chemotherapeutic substances, may

Table 3. Localization of accumulated cell-associated radioactivity.

125I-PDGFAA (%) 125I-PDGFAA-dx (%) PDGFAA-dx-125I-tyrosine (%) dx-125I-tyrosine (%)

Membrane Internal. Membrane Internal. Membrane Internal. Membrane Internal.

U343MGa31L

0 h 44 56 29 71 88 12 90 10

24 h 60 40 13 87 65 35 86 14

U343MGaCl2:6

0 h 54 46 27 73 86 14 86 14

24 h 76 24 16 84 60 40 63 37

U563MG

0 h 52 48 31 69 85 15 90 10

24 h 55 45 23 77 71 29 86 14

Cell cultures were treated as described in Table 2; membrane-associated (Membrane) and internalized (Internal.) radioactivity was determined as described in Material and methods section and given in % of total cell-associated radioactivity. dx: dextran, 10 kDa.

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Targeting PDGFAA-based conjugates to glioma cells 275

therefore be of interest to specifically reach cancer cells. To understand the cellular handling of such con-structs, the accumulation, retention, and localization of PDGFAA-based dextran conjugates were studied in GBM cell cultures.

The radioactive PDGFAA-based dextran conjugates were investigated regarding retention and localization of cell-associated radioactivity. Consideration was taken to the differences in cell number per Petri dish and in the specific activity of the conjugates and the nonconju-gated ligand, respectively. Since 125I was used only in low amounts to track the different molecules, it was not likely to induce cell damage potentially perturbing the results. The visual inspection, using a microscope, showed no indication of cell damage induced by these radioactive conjugate incubation solutions in vitro.

The experimental setting was designed to show the impact of dextran on the different conjugates. As noted earlier, a prolonged retention in cells could be of importance for the therapeutic effect on tumor cells. PDGFAA-based conjugates with 10 kDa dextran showed the most favorable targeting properties. In comparison with nonconjugated radioactive ligand, the conjugates gave a prolonged retention of cell-associated radioac-tivity. This is probably due to the properties of dextran since dextran itself, in the form of dextran-125I-tyrosine, showed a prolonged retention of cell-associated radio-activity in comparison with nonconjugated ligand at 24 h (Figure 2). Moreover, the potential of receptor targeting is confirmed by the results obtained with PDGFAA-based dextran conjugates, in comparison with dextran-125I-tyrosine. Even though the specific activity of PDGFAA-based dextran conjugates (0.81 MBq/µg; 2.11 MBq/µg) was lower than that of dextran-125I-tyrosine (2.75 MBq/µg), a higher total amount of cell-associated radioactivity delivered by PDGFAA-based conjugates was registered. Although 40 kDa dextran conjugates had a higher specific activity in comparison with the 10 kDa dextran conjugates, the amount of cell-associated radio-activity was very low. The longer dextran chain seems, in this case, to negatively change the behavior of the conjugates regarding uptake and internalization. In the 40 kDa conjugate, dextran constitutes 57% of the final conjugate while the corresponding quota for the 10 kDa conjugate is 25%. It is likely that the relation between the size of the dextran and protein as well as the total size of the conjugate may influence the characteristics of the conjugates in a negative way.

The localization of cell-associated radioactivity was carried out to discriminate between membrane-associ-ated and internalized radioactivity (Haigler et al., 1980; Andersson et al., 1991). For nonconjugated radioactive ligand, a higher membrane-associated fraction at 24 h, in comparison with 0 h, was observed in all cell lines and indicates release of internalized radioactivity. This trend

was not seen for the conjugates and reveals the impact of dextran on retention (Table 3). All the conjugates show an increase in internalized fraction at 24 h, in compari-son with 0 h, which may reflect a lower internalization rate induced by dextran. Once, the dextran-based con-jugates are cell-associated they are retained for a longer period of time compared with nonconjugated ligand. The extended retention observed for these dextran-based conjugates is shown by statistical analysis.

Radioactivity measurements form the basis of the results achieved. However, such measurements do not mirror the metabolic fate of the conjugate molecule retrieved in/at the membrane or inside the cell. The conjugates are structurally different and their character may influence the properties regarding receptor bind-ing and internalization. Evaluation of the localization of cell-associated radioactivity shows differences where PDGFAA-dextran-125I-tyrosine and dextran-125I-tyrosine distinguish substantially from 125I-PDGFAA-dextran regarding distribution of cell-associated radioactivity (Table 3). This may indicate that radiolabeling of the dextran with 125I-tyrosine has an impact on the distribu-tion toward higher extent of membrane association.

The three cell lines used, U343MGa31L, U343MGaCl2:6, and U563MG, originate from GBM biop-sies. Even though the quantity and affinity of PDGF receptors on these cells are described in the literature (Nistér et al., 1987; Claesson-Welsh et al., 1989), their biological activity is not well known. U343MGa31L has, in previous studies, been shown to have 47,000 recep-tors/cell (Nistér et al., 1987; Claesson-Welsh et al., 1989). An earlier study showed U343MGaCl2:6 to be negative for PDGFR mRNA (Claesson-Welsh et al., 1989), but in experiments performed by us, shown to be PDGFR mRNA positive (data not shown). U343MGaCl2:6 has been used in previous studies exploring EGFR in vitro targeting. The cell line has been shown to have a high amount of functional and amplified EGF receptors; 180,000 per cell (Westermark, Magnusson, & Heldin, 1982). U563MG was included because it is regarded to have a low expression of PDGF and EGF receptors (Dr. B. Westermark, personal communication, Uppsala University, Sweden; Claesson-Welsh et al., 1989). The results obtained with U343MGa31L presented in this study agree with earlier work regarding U343MGaCl2:6 and EGF-based dextran conjugates (Andersson et al., 1991). Analogous experiments, with EGF-based dextran conjugates and U343MGa31L and U563MG, have not been performed.

Chemotherapeutic agents of potential interest for use in PDGFAA-based conjugates are radioactive and stable isotopes as well as cytotoxins. The use of stable isotopes, which are activated at their target, is a way to circumvent the negative effects of radioactive isotopes on the circu-latory system and other organs. Boron neutron capture

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276 Maria Dahlström Wester, Åke Wasteson, and Annelie Lindström

therapy (BNCT), for example, utilizes the stable isotope boron-10 (10B) (Dahlström et al., 2004). When activated by neutrons, the 10B deposits short-ranged high-LET fis-sion products, an -particle, and a Li-ion, with poten-tial cell-damaging properties. Radioactive isotopes, for example, iodine and other halogens may be used for therapeutic or diagnostic purpose.

Knowledge of the properties of the different subpopu-lations in the solid, heterogeneous GBM tumor may argue for the use of a combinatorial drug approach to specifi-cally target different tumoral compartments (Debinski, 2008). This might be of importance in a therapeutic per-spective. The present study extends differences between the two cell lines, U343MGa31L and U343MGaCl2:6, that originate from the same tumor material (Nistér et al., 1987), regarding their handling, in vitro, of PDGFAA-based dextran conjugates. As described previously, these two cell lines illustrate remarkable heterogeneity in terms of accumulation of a boronated phenylalanine derivative, boronophenylalanine (BPA), for the purpose of BNCT (Dahlström et al., 2004).

Considering the results regarding the extended retention of cell-associated radioactivity obtained with PDGFAA-based dextran conjugates in this in vitro study, it is of interest to further investigate the properties and potential of such conjugates, especially 125I-PDGFAA-dextran (10 kDa). Dextran conjugates carrying toxic substances, for therapeutic purpose, should be prepared and evaluated.

Acknowledgments

The authors thank Dr B. Westermark, Uppsala University, Sweden, for kindly providing the GBM cell lines. This work was supported by the Swedish Cancer Foundation (No. 4430-B00-01XAB)

Declaration of interest: The authors report no conflicts of interest.

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