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- Materials and Methods
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.
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- Materials and Methods
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.