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Keywords:

  • glioma;
  • interleukin 6 receptor;
  • magnetic resonance imaging;
  • tumor targeting

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

The interleukin 6 receptor (IL-6R) and its ligand interleukin 6 (IL-6) play a crucial role in glioma growth and development accomplished by autocrine growth promotion and induction of angiogenesis via activation of vascular epithelial growth factor A (VEGF-A). Therefore, IL-6R represents a target for both therapy (preventing VEGF-A activation by blocking the receptor) and imaging (higher receptor density on tumor cells). A short heptapeptide that selectively binds to IL-6R and which inhibits the effect of IL-6 was coupled to the magnetic resonance imaging (MRI) contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and the fluorescent dye rhodamine. MRI, confocal laser scanning microscopy, and flow cytometry showed that our IL-6-DOTA-rhodamine conjugate was taken up into the cytoplasm of human U373 glioma cells without any cytotoxic effects. Competition experiments indicate that this uptake was receptor-mediated. This conjugate might be used for future MRI studies of brain tumors after systemic or intraoperative local application. The cytoplasm specificity of the conjugate also makes it a potential building block for the design of future cytoplasm-directed imaging and therapeutic conjugates.

Interleukin-6 (IL-6) is a cytokine that is involved in the immune response, inflammation, hematopoiesis, and physiology of virtually every organ system. It also plays a major role in the response to injury and infection.

However, disregulation of IL-6 and its receptor has been found in diverse human illnesses (e.g. autoimmune diseases, neurologic diseases, and cancer) (1). IL-6 plays a pivotal role in angiogenesis by strongly inducing vascular endothelial growth factor A (VEGF-A) (2).

Autocrine growth promotion via the IL-6 and the IL-6 receptor (IL-6R) was found for several human glioblastoma cell lines including U373 (3). IL-6 as well as IL-6R have been found in human pituitary tumors (4). In a syngeneic mouse model, IL-6 was required for astrocytoma development (5). However, healthy astrocytes did not express IL-6R (3).

Considering all this, IL-6R presents an attractive target for both therapy (preventing VEGF-A activation by blocking the receptor) and imaging (higher receptor density on tumor cells).

Using the phage display method, a short heptapeptide (LSLITRL) was identified that selectively binds to IL-6R and which inhibits the effect of IL-6 (2).

To construct a compound usable as an imaging marker for tumors with high IL-6R density as well as a therapeutic agent, we coupled the extracellular magnetic resonance imaging (MRI) contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and the fluorescent dye rhodamine to the IL-6R-binding heptapeptide via an additional N-terminal lysine carrying both markers. Gd-DOTA is a commonly used MRI contrast agent in clinical routine examinations and also a very stable complex with a half-life of over 4000 h (6). We used MRI experiments, confocal laser scanning microscopy (CLSM), and flow cytometry to evaluate the binding characteristics of our conjugate to U373 glioma cells and whether binding could be influenced by competition with unmarked peptide.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Synthesis of gadolinium-DOTA-Lys(rhodamine)-Leu-Ser-Leu-Ile-Thr-Arg-Leu

Conjugate synthesis was performed on 25-μmol scale by solid phase peptide synthesis on a Syro II automatic synthesizer (MutiSynTech, Witten, Germany) using the fmoc strategy. TCP-resin (PepChem, Reutlingen, Germany) was used as carrier. O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU) was used as coupling agent and diisopropyl-ethylamine (DIPEA) as adjuvant base for amino acid coupling in sequential peptide elongation. The fmoc protective group was cleaved off with 25% piperidine in dimethylformamide. The side chain of the N-terminal lysine was protected with 1-(4,4-dimethyl-2,6-dioxycyclohex-1-ylidine)-ethyl (Dde). After fmoc deprotection of the N-terminus, 1,4,7,10 tetraazacyclododecane-1,4,7 tri-tert-butyl acetate-10-acetic acid (DOTA) was coupled to the lysine with TBTU/DIPEA. The Dde side chain protective group was cleaved off by using 2.5% hydrazine in dimethylformamide over the course of 1 h. Rhodamine isothiocyanate (RITC) was coupled to the free side-chain amino group using three equivalents of RITC and an equal amount of DIPEA in dimethylsulfoxide at room temperature over night. The peptide sequences synthesized were analyzed and separated by RP-HPLC (Merck Hitachi, L-4000 A UV detector; Merck Hitachi, Darmstadt, Germany) using a C8 column (150 × 10 mm; Reprosil 100; Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) with the following solvent systems: (A) 0.055% v/v trifluoroacetic acid in water. (B) 0.05% v/v trifluoroacetic acid in 80% v/v acetonitrile in water. Elution was performed using a linear gradient from 20% to 100% B within 40 min at the flow rate of 2.5 mL/min and at 214 nm absorbance. The purified conjugate (>97% purity) was then lyophilized.

The resulting substance was stirred for 5 h at 50 °C with one equivalent of Gd(III)chloride hexahydrate in water at pH 5.6 (adjusted with NaOH). After acidification with diluted acetic acid, the substance was relyophilized.

The product was verified by matrix assisted laser desorption/ionization–time of flight mass spectrometry on a Bruker Daltonics reflex IV (Bruker Daltonics Inc., Billerica, MA, USA) (theoretic mass: 1985.9, measured mass: 1984.0).

CLSM

U373 human glioma cells were grown to 70% confluency at 37 °C, 5% CO2 (vol/vol) in RPMI-1640 Ready Mix Medium containing L-glutamine and 10% fetal bovine serum (FBS)-Gold (PAA laboratories, Pasching, Austria), in 4-well plates (NUNC, Wiesbaden, Germany) with about 250 000 cells per well.

The conjugate was dissolved at 130 μm in medium. Cells were incubated at 37 °C in an atmosphere of 5% CO2 for 60 min with the conjugate. After this, the cells were rinsed once with medium and then incubated with medium containing Annexin-Fluos (Roche Diagnostics, Mannheim, Germany). Annexin-Fluos binds to phosphatidylserine that is only accessible in the outer membrane leaflet of apoptotic or necrotic cells but not in intact cells where phosphatidylserine is exclusively found in the inner membrane leaflet.

CLSM was performed on an inverted LSM510 laser scanning microscope (Carl Zeiss, Jena, Germany) (objective: LD Achroplan 40 × 0.6). For Annexin-Fluos and rhodamine fluorescence excitation, the 488-nm line of an Argon laser and the 543-nm line of a helium–neon laser with appropriate beam splitters and barrier filters were used. All measurements were performed at least three times on living, non-fixed cells.

MR-relaxometry

For MR relaxometry, U373 human glioma cell lines were grown in 75-cm2 culture flasks (Corning Costar, Bodenheim Germany) (80% confluency). Accutase™ (PAA laboratories, Pasching, Austria) was added to achieve detachment of the cells, which were harvested and subsequently aliquoted into Eppendorf tubes (6 × 106 cells per tube). The cells in the tubes were incubated with the conjugate (5.2 μm, 65 μm, 130 μm and 520 μm). After a 60-min incubation period at 37 °C in an atmosphere of 5% CO2, the cells were washed three times in phosphate buffered saline (PBS) and centrifuged at 800 rpm for 5 min.

Competition samples were preincubated with 20-fold excess of unmarked peptide for 10 min. Then the unmarked peptide solution was replaced with solutions containing 5.2, 65, 130, and 520 μm conjugate concentration and 20-fold excess of unmarked peptide and then incubated for 60 min. Control samples were only incubated with medium. Washing and centrifugation were performed in line with the samples containing conjugate alone.

In vitro imaging was performed with a 3-tesla whole body MRI-system (Trio, Siemens, circular polarized wrist coil, Siemens, Erlangen, Germany).

Sagittal T1-weighted MR images were obtained using the following spin echo sequence: repetition time (TR): 200 mseconds, TE (echo time): 7.4 mseconds, flip angle 90°, averages: 1, concatenations: 2, measurements: 1, number of slices: 19, distance factor: 30%, slice thickness: 3 mm, field of view read: 180 mm, field of view phase: 100%, base resolution: 256, phase resolution: 100%, voxel size: 0.7 × 0.7 × 3.0 mm, scan time: 1:48 min. T1 relaxation times were evaluated from signal intensities obtained by multiple spin echo measurements: TR: 20–8000 mseconds (50 different TR values), TE: 6.4 mseconds, flip angle 90°, averages: 1, measurements: 1, number of slices: 1, slice thickness: 1 mm, field of view read: 120 mm, field of view phase: 87.5, base resolution 128, phase resolution: 100%, voxel size: 0.9 × 0.9 × 1 mm.

Analyses and calculations were performed using a Matlab program (Math Works, Natick, MA, USA). T1 values were approximated by a three-parameter fit procedure. All signal curves were examined and found to be monoexponential. The relaxivity value obtained as the reciprocal value of the T1 relaxation time correlates directly to the conjugate uptake. The investigations were performed in triplicate.

Flow cytometry

For fluorescence activated cell sorting (FACS), cells were grown in 75-cm2 culture flasks (Corning Costar, Bodenheim Germany) (80% confluency) under the same conditions as described under CLSM. Accutase™ (PAA laboratories, Pasching, Austria) was added to achieve detachment of the cells, which were harvested and subsequently aliquoted into Eppendorf tubes (Eppendorf, Hamburg, Germany) (1 × 106 cells per tube). The cells were incubated for 60 min with the conjugate (5.2, 65, 130 and 520 μm). As described before, competition samples were first preincubated with unmarked peptide for 10 min and then incubated for another 60 min with 20:1 peptide:conjugate mix.

Afterwards, the cells were washed three times with PBS and centrifuged at 800 rpm for 5 min. Then, 300 μl of FACS buffer (D-PBS containing 1% paraformaldehyde) was added. The samples were measured immediately. Approximately 20 000 events were recorded per sample. Fluorescence excitation was achieved by an argon laser (488 nm). Rhodamine fluorescence was detected using a 580-610-nm bandpass filter. All investigations were performed in triplicate.

Mean rhodamine fluorescence values of the samples were acquired using the WinMDI software (Joseph Trotter, Scripps Research Institute, San Diego, CA, USA) and then statistically evaluated.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

CLSM

Many if not all cells were stained. Staining was cytoplasmic and the cell nuclei appeared to be free of conjugate. The cell cytoplasm showed dot-like staining pattern (Figure 1). Lack of green fluorescence from the Annexin-Fluos vitality test (Roche Diagnostics) showed that the cells remained viable.

image

Figure 1.  Confocal laser scanning microscopy images of living adherent U373 glioma cells incubated with the conjugate at 130 μm concentration showed dot-like cytoplasmic staining. Cell nuclei remained free of conjugate. The majority if not all cells were stained. The Annexin-Fluos vitality test showed no green fluorescence, i.e. no cell death.

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MRI

The mean relaxivity value of the 520-μm samples was highest (0.00380) and the values decreased with the lower concentrations (130 μm: 0.00124; 65 μm: 0.00095; 5.2 μm: 0.00052). Relaxivity values of samples in which 20-fold excess of unmarked peptide was added to the conjugate solution were lower than the values of the respective samples without competition at all concentrations (520 μm: 0.00278; 130 μm: 0.00110; 65 μm: 0.00085; 5.2 μm: 0.00048). Finally, the native U373 glioma cell sample showed the lowest relaxivity value of only 0.00038 (Figure 2A, B).

image

Figure 2.  Magnetic resonance (MR) relaxometry experiments. (A) The relaxivity values (1/T1 relaxation time) of U373 human glioma cell samples incubated solely with the conjugate and with conjugate in competition with 20-fold excess of unmarked peptide at 520, 130, 65, and 5.2 μm conjugate concentration and with medium alone (native sample). Mean values and standard deviation from three investigations were depicted. The relaxivity value of the native sample was used as arbitrary origin. The scale of the y-axis was adjusted for each of the four different concentrations used to better illustrate the effect of the competition. Decrease of conjugate uptake through competition was found for all concentrations with the decrease ranging from 16% to 30%. (B) Relaxivity diagram using the y-axis scale of the 520-μm sample for all samples. Conjugate uptake was strongest at the highest conjugate concentration (520 μm) and decreased with lower concentrations. Competition was observed at all concentrations. (C) Axial T1 magnetic resonance imaging (MRI)-images of 0.5-mL tubes containing U373 human glioma cell samples incubated at 520 μm (left) and 5.2 μm (right) conjugate concentration with and without competition with unmarked peptide were taken at different repetition times (TR). The MRI signals of the samples without competition are stronger and can be seen at a lower TR at both concentrations.

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Using the native sample value as arbitrary origin, the effect of the competition was found highest at 520 μm concentration with a reduction of conjugate uptake by 30%. The reduction in conjugate uptake for the other concentrations was between 16% and 28%, but no specific order was found. The effect of the competition could already be observed qualitatively in the axial T1 MRI images (Figure 2C). The MRI signal of the sample without competition could be first discerned at a lower TR than the sample with competition. At higher TR values, the sample without competition was stronger than the competition sample. Only at TR values high enough for all samples to show full relaxation the signal of the competition sample matches the signal of the sample without competition (Figure 2C).

Flow cytometry

Mean rhodamine fluorescence intensity was strongest in the samples incubated with 520 μm of conjugate alone and decreasing with lower conjugate concentrations (520 μm: 277.7; 130 μm: 98.4; 65 μm: 52.8; 5.2 μm: 19.5). The samples with 20-fold excess of unmarked peptide all showed reduced mean fluorescence intensity if compared to the respective samples without competition (520 μm: 234.4; 130 μm: 89.4; 65 μm: 46.3; 5.2 μm: 16.1) (Figure 3). The decrease in mean rhodamine fluorescence achieved through competition with the unmarked peptide was found to be between 10% and 18%.

image

Figure 3.  Fluorescence activated cell sorting analysis of U373 human glioma cells after incubation with the conjugate alone (520, 130, 65 and 5.2 μm) and the conjugate in competition with 20-fold excess of unmarked peptide. The mean rhodamine fluorescence intensity of the samples without competition was highest for the 520-μm sample and decreased with the lower concentrations. Competition with 20-fold excess of unmarked peptide led to reduced mean rhodamine fluorescence at all concentrations if compared to the samples without competition. The decrease in mean rhodamine fluorescence caused by the competition ranged from 10% to 18% in the four different conjugate concentrations examined. Mean and standard deviation of three measurements were depicted.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Interleukin receptors have already been used as targets for cancer therapy and diagnostics using different approaches: Fusion proteins of diphtheria toxin with interleukin 2 (7) and interleukin 3 (8), interleukin 2 antibodies coupled to radioactive bismuth-DOTA (9) and a conjugate consisting of a cyclic interleukin 11 nonapeptide labeled with both a near infrared dye and a radioactive indium-diethylene triamine pentaacetic acid (10). However, no compounds targeting the interleukin 6 receptor (IL-6R) have been used so far.

Interleukin-6 (IL-6) and IL-6R have been found in autocrine growth promotion of brain tumors [e.g. glioblastoma (3), pituitary tumors (4)]. To enable IL-6R-based CLSM and MRI, we coupled rhodamine (fluorescent dye) and gadolinium (Gd)-DOTA (MRI contrast agent) to a heptapeptide that specifically binds to IL-6R [LSLITRL; (2)].

MRI with contrast agents is a common method in brain tumor imaging. In tumors of WHO grade higher than II, the blood–brain barrier is disrupted and the contrast agent accumulates in the interstitial space. The advantage of magnetic resonance imaging compared to other methods like positron emission tomography is the high anatomic resolution.

The conjugate was taken up into the cytoplasm of U373 glioma cells while the cell nuclei were omitted. This and the dot-like staining pattern of cells (Figure 1) indicate receptor binding with subsequent receptor internalization.

Binding of the conjugate to IL-6R could be inhibited with 20-fold excess of unmarked LSLITRL peptide. This inhibitory effect was observed at all four examined concentrations in magnetic resonance (MR) relaxometry as well as flow cytometry experiments. However, the inhibition resulted in no more than a 30% reduction in gadolinium uptake in MR relaxometry and at most 18% reduction in mean rhodamine fluorescence in flow cytometry (Figures 2 and 3). This was remarkably low considering the 20:1 ratio of unmarked peptide to conjugate that the cells were incubated with.

An explanation for this effect could be that the conjugate is already competing with autocrinely secreted IL-6 so the additional competition has lower effect on the uptake than if the unmarked peptide was the only source of competition. On the other hand, an effect of the imaging markers cannot be excluded resulting in different binding affinities of the conjugate and the unmarked peptide to IL-6R.

We attributed the overlapping error bars in the MRI evaluation to statistical deviation that plays a stronger role at the low MRI signals we received for the low conjugate concentrations. At the 520-μM concentration, the error bars are far from overlapping with the effect of the competition being a reduction of gadolinium uptake and MRI signal by 30%, while at the 5.2-μm concentration, the error bars overlap at a signal reduction of 28%.

Keeping in mind that the relaxivity values of the competition samples were consistently lower than those of the samples without competition in every single experiment and that the error bars at the high conjugate concentration were clearly not overlapping, we did not give any significance to the overlapping of the error bars at the low conjugate concentrations.

Vitality testing showed that blocking the IL-6R with our IL-receptor specific conjugate did not result in cell death. Therefore, its future antitumor therapeutic use seems to be restricted. The conjugate’s cytoplasmic localization and lack of cytotoxic effects makes it of potential value for the use as a contrast agent. A possible application could be intraoperative MRI during brain tumor surgery. When the tumor tissue is dissected, the currently used extracellular contrast agent flows out along the interstitial space into the healthy tissue (11). This new conjugate would remain within the cells resulting in no blurred section margins in MRI.

The exclusive cytoplasmic accumulation of the conjugate is a welcome finding for future design of cytoplasm-directed conjugates. Some targets for glioma therapy (e.g. inhibitor of apoptosis proteins) are located only in the cytoplasm. The regularly used cationic membrane transport sequences like HIV-tat or polyarginines resemble nuclear localization sequences (NLS) (12) and are thus also directed to the cell nucleus (13). Even though conjugates of sizes comparable to our IL-6-DOTA-rhodamine conjugate do not contain any NLS, they should still be able to pass the nuclear pore complex by passive diffusion (cut-off size < 10kDa, (14)).

Further experiments will show whether this conjugate can distinguish between healthy and tumorous astrocytes based upon their different IL-6R expression and whether the in vitro results can be confirmed in vivo, e.g. on mice bearing human U373 glioma xenografts.

In summary, the novel IL-6-DOTA-rhodamine conjugate was taken up into the cytoplasm of human U373 glioma cells without any cytotoxic effects. Competition experiments indicate that this uptake was receptor-mediated. The conjugate might be used for future MRI studies of brain tumors by systemic or intraoperative local application.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

This study is supported by the Hertie Foundation for Brain Research and the Interdisciplinary Center for Clinical Research, University of Tübingen.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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