Lysyl Oxidases as Targets for Cancer Therapy and Diagnostic Imaging.

The understanding of the contribution of the tumour microenvironment to cancer progression and metastasis, in particular the interplay between tumour cells, fibroblasts and the extracellular matrix has grown tremendously over the last years. Lysyl oxidases are increasingly recognised as key players in this context, in addition to their function as drivers of fibrotic diseases. These insights have considerably stimulated drug discovery efforts towards lysyl oxidases as targets over the last decade. This review article summarises the biochemical and structural properties of theses enzymes. Their involvement in tumour progression and metastasis is highlighted from a biochemical point of view, taking into consideration both the extracellular and intracellular action of lysyl oxidases. More recently reported inhibitor compounds are discussed with an emphasis on their discovery, structure-activity relationships and the results of their biological characterisation. Molecular probes developed for imaging of lysyl oxidase activity are reviewed from the perspective of their detection principles, performance and biomedical applications.

Specific and safe targeting of glioblastoma using switchable and logic-gated RevCAR T cells.

Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo.

CRISPR/Cas9 Mediated Knockout of Cyclooxygenase-2 Gene Inhibits Invasiveness in A2058 Melanoma Cells.

The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tumor microenvironment, tumor growth, apoptosis resistance, angiogenesis and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the functional knockout effect of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by Western blot and flow cytometry analysis. When comparing COX-2 knockout cells to controls, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor development in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic activity of cells under normoxia and experimental hypoxia as demonstrated by using the radiotracers [18F]FDG and [18F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.

The Pyrazolo[3,4-d]pyrimidine-Based Kinase Inhibitor NVP-BHG712: Effects of Regioisomers on Tumor Growth, Perfusion, and Hypoxia in EphB4-Positive A375 Melanoma Xenografts.

In a previous study, EphB4 was demonstrated to be a positive regulator of A375-melanoma growth but a negative regulator of tumor vascularization and perfusion. To distinguish between EphB4 forward and ephrinB2 reverse signaling, we used the commercially available EphB4 kinase inhibitor NVP-BHG712 (NVP), which was later identified as its regioisomer NVPiso. Since there have been reported significant differences between the inhibition profiles of NVP and NVPiso, we compared the influence of NVP and NVPiso on tumor characteristics under the same experimental conditions. Despite the different inhibitory profiles of NVP and NVPiso, the comparative study conducted here showed the same EphB4-induced effects in vivo as in the previous investigation. This confirmed the conclusion that EphB4-ephrinB2 reverse signaling is responsible for increased tumor growth as well as decreased tumor vascularization and perfusion. These results are further substantiated by microarrays showing differences between mock-transfected and EphB4-transfected (A375-EphB4) cells with respect to at least 9 angiogenesis-related proteins. Decreased expression of vascular endothelial growth factor (VEGF), angiotensin 1 (Ang-1), and protein kinase B (Akt/PKB), together with the increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta-2 (TGF-ß2), is consistent with the impaired vascularization of A375-EphB4 xenografts. Functional overexpression of EphB4 in A375-EphB4 cells was confirmed by activation of a variety of signaling pathways, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT), rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen activated protein kinase kinase (Ras/Raf/MEK), and nuclear factor kappa-B (NFkB) pathways.

Radiopharmacologist's and Radiochemist's View on Targeting the Eph/Ephrin Receptor Tyrosine Kinase System.

In the past decade, there have been extensive efforts to open up the Eph/ephrin subfamily of the receptor tyrosine kinase family for diagnostic and therapeutic applications. Besides classical pharmaceutical developments, which focus either on drugs targeting the extracellular ligand binding domains or on the intracellular tyrosine kinase domains of these receptors, there also have been first radiopharmaceutical approaches. Here the focus is on the development of specific and selective probes for molecular imaging, particularly by means of positron emission tomography, and the functional characterization of the Eph/ephrin subfamily in certain target tissues. The aim of this mini-review is to summarize the different approaches toward Eph-targeting radiotracers by using antibodies, peptides, and small molecules and to discuss their radiopharmacological characterization. With regard to the small molecules, further considerations will focus on the design and synthesis of nonradioactive reference compounds and precursors as well as on radiolabeling strategies.

Synthesis, radiolabelling and initial biological characterisation of 18F-labelled xanthine derivatives for PET imaging of Eph receptors.

Eph receptor tyrosine kinases, particularly EphA2 and EphB4, represent promising candidates for molecular imaging due to their essential role in cancer progression and therapy resistance. Xanthine derivatives were identified to be potent Eph receptor inhibitors with IC50 values in the low nanomolar range (1-40 nm). These compounds occupy the hydrophobic pocket of the ATP-binding site in the kinase domain. Based on lead compound 1, we designed two fluorine-18-labelled receptor tyrosine kinase inhibitors ([18F]2/3) as potential tracers for positron emission tomography (PET). Docking into the ATP-binding site allowed us to find the best position for radiolabelling. The replacement of the methyl group at the uracil residue ([18F]3) rather than the methyl group of the phenoxy moiety ([18F]2) by a fluoropropyl group was predicted to preserve the affinity of the lead compound 1. Herein, we point out a synthesis route to [18F]2 and [18F]3 and the respective tosylate precursors as well as a labelling procedure to insert fluorine-18. After radiolabelling, both radiotracers were obtained in approximately 5% radiochemical yield with high radiochemical purity (>98%) and a molar activity of >10 GBq µmol-1. In line with the docking studies, first cell experiments revealed specific, time-dependent binding and uptake of [18F]3 to EphA2 and EphB4-overexpressing A375 human melanoma cells, whereas [18F]2 did not accumulate at these cells. Since both tracers [18F]3 and [18F]2 are stable in rat blood, the novel radiotracers might be suitable for in vivo molecular imaging of Eph receptors with PET.

Synthesis and preliminary radiopharmacological characterisation of an 11 C-labelled azadipeptide nitrile as potential PET tracer for imaging of cysteine cathepsins.

An O-methyltyrosine-containing azadipeptide nitrile was synthesised and investigated for its inhibitory activity towards cathepsins L, S, K, and B. Labelling with carbon-11 was accomplished by reaction of the corresponding phenolic precursor with [11 C]methyl iodide starting from cyclotron-produced [11 C]methane. Radiopharmacological evaluation of the resulting radiotracer in a mouse xenograft model derived from a mammary tumour cell line by small animal PET imaging indicates tumour targeting with complex pharmacokinetics. Radiotracer uptake in the tumour region was considerably lower under treatment with the nonradioactive reference compound and the epoxide-based irreversible cysteine cathepsin inhibitor E64. The in vivo behaviour observed for this radiotracer largely confirms that of the corresponding 18 F-fluoroethylated analogue and suggests the limited suitability of azadipeptide nitriles for the imaging of tumour-associated cysteine cathepsins despite target-mediated uptake is evidenced.

Radiochemical and radiopharmacological characterization of a 64 Cu-labeled α-MSH analog conjugated with different chelators.

Radiolabeled α-melanocyte-stimulating hormone (α-MSH) derivatives have a high potential for diagnosis and treatment of melanoma, because of high specificity and binding affinity to the melanocortin-1 receptor (MC1R). Hence, the α-MSH-derived peptide NAP-NS1 with a ß-Ala linker (ε-Ahx-ß-Ala-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 ) was conjugated to different chelators: either to NOTA (p-SCN-Bn-1,4,7-triazacyclononane-1,4,7-triacetic acid), to a hexadentate bispidine carbonate derivative (dimethyl-9-(((4-nitrophenoxy)carbonyl)oxy)-2,4-di(pyridin-2-yl)-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate), or to DMPTACN (p-SCN-Ph-bis(2-pyridyl-methyl)-1,4,7-triaza-cyclononane), labeled with 64 Cu, and investigated in terms of radiochemical and radiopharmacological properties. For the three 64 Cu-labeled conjugates negligible transchelation, suitable buffer and serum stability, as well as appropriate water solubility, was determined. The three conjugates exhibited high binding affinity (low nanomolar range) in murine B16F10, human MeWo, and human TXM13 cells. The Bmax values of [64 Cu]Cu-bispidine-NAP-NS1 ([64 Cu]Cu-2) and [64 Cu]Cu-DMPTACN-NAP-NS1 ([64 Cu]Cu-3) were higher than those of [64 Cu]Cu-NOTA-NAP-NS1 ([64 Cu]Cu-1), implying that different charged chelate units might have an impact on binding capacity. Preliminary in vivo biodistribution studies suggested the main excretion pathway of [64 Cu]Cu-1 and [64 Cu]Cu-3 to be renal, while that of [64 Cu]Cu-2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60 minutes. All three 64 Cu-labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.

Diverse effects of phospholipase A2 receptor expression on LNCaP and PC-3 prostate cancer cell growth in vitro and in vivo.

Physiological and pathophysiological functions of the phospholipase A2 receptor 1 (PLA2R1) are still not completely understood. To elucidate PLA2R1's function in prostate carcinoma, the receptor was ectopically overexpressed in LNCaP with silenced PLA2R1, and diminished in PC-3 cells with constitutively increased PLA2R1 expression relative to normal prostate epithelial cells. LNCaP cells were transfected to overexpress PLA2R1 (LNCaP-PLA2R1) and compared to control vector transfected cells (LNCaP-Ctrl). Alternatively, a CRISPR/Cas9-knockdown of PLA2R1 was achieved in PC-3 cells (PC-3 KD) and compared to the corresponding control-transfected cells (PC-3 Ctrl). The impact of PLA2R1 expression on proliferative and metastatic parameters was analysed in vitro. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP and PC-3 cells. Cell viability/proliferation and motility were significantly increased in LNCaP-PLA2R1 and PC-3 Ctrl compared to LNCaP-Ctrl and PC-3 KD cells, respectively. However, levels of apoptosis, clonogenicity and cell invasion were reduced in LNCaP-PLA2R1 and PC-3 Ctrl cells. Gene expression analysis revealed an up-regulation of fibronectin 1 (FN1), TWIST homolog 1 (TWIST1), and cyclin-dependent kinase 6 (CDK6) in LNCaP-PLA2R1. In LNCaP xenografts, PLA2R1-dependent regulation of clonogenicity appeared to outweigh the receptor's pro-oncogenic properties, resulting in decreased tumour growth, supporting the tumour-suppressive role of PLA2R1. Alternatively, PC-3 Ctrl xenografts exhibited faster tumour growth compared to PC-3 KD cells, suggesting a pro-oncogenic effect of endogenous PLA2R1 expression. The differential growth-regulatory effects of PLA2R1 may be mediated by FN1, TWIST1, and CDK6 expression, although further investigation is required.

Evaluation of Fluorine-18-Labeled α1(I)-N-Telopeptide Analogs as Substrate-Based Radiotracers for PET Imaging of Melanoma-Associated Lysyl Oxidase.

Accumulating evidence suggests an unequivocal role of lysyl oxidases as key players of tumor progression and metastasis, which renders this enzyme family highly attractive for targeted non-invasive functional imaging of tumors. Considering their function in matrix remodeling, malignant melanoma appears as particularly interesting neoplasia in this respect. For the development of radiotracers that enable PET imaging of the melanoma-associated lysyl oxidase activity, substrates derived from the type I collagen α1 N-telopeptide were labeled with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) as prosthetic reagent. With regards to potential crosslinking to tumor-associated collagen in vivo, their interaction with triple-helical type I collagen was studied by SPR. A mouse model of human melanoma was established on the basis of the A375 cell line, for which the expression of the oncologically relevant lysyl oxidase isoforms LOX and LOXL2 was demonstrated in Western blot and immunohistochemical experiments. The radiopharmacological profiles of the peptidic radiotracers were evaluated in normal rats and A375 melanoma-bearing mice by ex vivo metabolite analysis, whole-body biodistribution studies and dynamic PET imaging. Out of three 18F-labeled telopeptide analogs, the one with the most favorable substrate properties has shown favorable tumor uptake and tumor-to-muscle ratio. Lysyl oxidase-mediated tumor uptake was proven by pharmacological inhibition using ß-aminopropionitrile and by employing negative-control analogs of impeded or abolished targeting capability. The latter were obtained by substituting the lysine residue by ornithine and norleucine, respectively. Comparing the tumor uptake of the lysine-containing peptide with that of the non-functional analogs indicate the feasibility of lysyl oxidase imaging in melanoma using substrate-based radiotracers.

Synthesis, Characterization, and Initial Biological Evaluation of [99m Tc]Tc-Tricarbonyl-labeled DPA-α-MSH Peptide Derivatives for Potential Melanoma Imaging.

α-Melanocyte stimulating hormone (α-MSH) derivatives target the melanocortin-1 receptor (MC1R) specifically and selectively. In this study, the α-MSH-derived peptide NAP-NS1 (Nle-Asp-His-d-Phe-Arg-Trp-Gly-NH2 ) with and without linkers was conjugated with 5-(bis(pyridin-2-ylmethyl)amino)pentanoic acid (DPA-COOH) and labeled with [99m Tc]Tc-tricarbonyl by two methods. With the one-pot method the labeling was faster than with the two-pot method, while obtaining similarly high yields. Negligible trans-chelation and high stability in physiological solutions was determined for the [99m Tc]Tc-tricarbonyl-peptide conjugates. Coupling an ethylene glycol (EG)-based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [99m Tc]Tc-tricarbonyl-peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the 99m Tc-labeled peptides for in vivo imaging.

Bridging from Brain to Tumor Imaging: (S)-(-)- and (R)-(+)-[18F]Fluspidine for Investigation of Sigma-1 Receptors in Tumor-Bearing Mice.

Sigma-1 receptors (Sig1R) are highly expressed in various human cancer cells and hence imaging of this target with positron emission tomography (PET) can contribute to a better understanding of tumor pathophysiology and support the development of antineoplastic drugs. Two Sig1R-specific radiolabeled enantiomers (S)-(-)- and (R)-(+)-[18F]fluspidine were investigated in several tumor cell lines including melanoma, squamous cell/epidermoid carcinoma, prostate carcinoma, and glioblastoma. Dynamic PET scans were performed in mice to investigate the suitability of both radiotracers for tumor imaging. The Sig1R expression in the respective tumors was confirmed by Western blot. Rather low radiotracer uptake was found in heterotopically (subcutaneously) implanted tumors. Therefore, a brain tumor model (U87-MG) with orthotopic implantation was chosen to investigate the suitability of the two Sig1R radiotracers for brain tumor imaging. High tumor uptake as well as a favorable tumor-to-background ratio was found. These results suggest that Sig1R PET imaging of brain tumors with [18F]fluspidine could be possible. Further studies with this tumor model will be performed to confirm specific binding and the integrity of the blood-brain barrier (BBB).

Overexpression of Receptor Tyrosine Kinase EphB4 Triggers Tumor Growth and Hypoxia in A375 Melanoma Xenografts: Insights from Multitracer Small Animal Imaging Experiments.

Experimental evidence has associated receptor tyrosine kinase EphB4 with tumor angiogenesis also in malignant melanoma. Considering the limited in vivo data available, we have conducted a systematic multitracer and multimodal imaging investigation in EphB4-overexpressing and mock-transfected A375 melanoma xenografts. Tumor growth, perfusion, and hypoxia were investigated by positron emission tomography. Vascularization was investigated by fluorescence imaging in vivo and ex vivo. The approach was completed by magnetic resonance imaging, radioluminography ex vivo, and immunohistochemical staining for blood and lymph vessel markers. Results revealed EphB4 to be a positive regulator of A375 melanoma growth, but a negative regulator of tumor vascularization. Resulting in increased hypoxia, this physiological characteristic is considered as highly unfavorable for melanoma prognosis and therapy outcome. Lymphangiogenesis, by contrast, was not influenced by EphB4 overexpression. In order to distinguish between EphB4 forward and EphrinB2, the natural EphB4 ligand, reverse signaling a specific EphB4 kinase inhibitor was applied. Blocking experiments show EphrinB2 reverse signaling rather than EphB4 forward signaling to be responsible for the observed effects. In conclusion, functional expression of EphB4 is considered a promising differentiating characteristic, preferentially determined by non-invasive in vivo imaging, which may improve personalized theranostics of malignant melanoma.

Extracellular S100A4 affects endothelial cell integrity and stimulates transmigration of A375 melanoma cells.

High extracellular S100A4 level proves a specific characteristic of some cancer cases, including malignant melanoma. Concerning the latter, extracellular S100A4 in an autocrine manner was shown to promote prometastatic activation of A375 cells by interaction with the receptor for advanced glycation endproducts (RAGE). We hypothesized that interaction of extracellular S100A4 with RAGE in a paracrine manner will affect endothelial cell (EC) integrity thus further promoting melanoma metastasis. We investigated the influence of recombinant and cell (A375)-derived S100A4 on junction protein expression and EC (hCMEC/D3) integrity by measuring transendothelial electrical resistance (TEER). Decrease of TEER and diminished expression of both occludin and VE-cadherin revealed the loss of EC integrity. Transmigration of transgenic A375 cells (A375-hS100A4/A375-hRAGE) through the EC monolayer was significantly higher compared to wild-type A375 cells, and was substantially decreased by sRAGE. A pilot study in mice, intracardially injected with A375-hS100A4 or A375-hRAGE cells, showed lower survival rates and a higher incidence of metastases compared to wild-type A375 cells. Tumor development was mostly located in the brain, bones, and ovaries. These findings provide further evidence on extracellular S100A4 as paracrine mediator of prometastatic endothelial dysfunction involving its interaction with RAGE.

Interaction of extracellular S100A4 with RAGE prompts prometastatic activation of A375 melanoma cells.

S100A4, a member of the S100 protein family of EF-hand calcium-binding proteins, is overexpressed in various tumour entities, including melanoma, and plays an important role in tumour progression. Several studies in epithelial and mesenchymal tumours revealed a correlation between extracellular S100A4 and metastasis. However, exact mechanisms how S100A4 stimulates metastasis in melanoma are still unknown. From a pilot experiment on baseline synthesis and secretion of S100A4 in human melanoma cell lines, which are in broad laboratory use, A375 wild-type cells and, additionally, newly generated A375 cell lines stably transfected with human S100A4 (A375-hS100A4) or human receptor for advanced glycation endproducts (A375-hRAGE), were selected to investigate the influence of extracellular S100A4 on cell motility, adhesion, migration and invasion in more detail. We demonstrated that A375 cells actively secrete S100A4 in the extracellular space via an endoplasmic reticulum-Golgi-dependent pathway. S100A4 overexpression and secretion resulted in prometastatic activation of A375 cells. Moreover, we determined the influence of S100A4-RAGE interaction and its blockade on A375, A375-hS100A4, A375-hRAGE cells, and showed that interaction of RAGE with extracellular S100A4 contributes to the observed activation of A375 cells. This investigation reveals additional molecular targets for therapeutic approaches aiming at blockade of ligand binding to RAGE or RAGE signalling to inhibit melanoma metastasis.