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Upregulation of prostate-specific membrane antigen (PSMA) in neovasculature has been described in glioblastoma multiforme (GBM), whereas vasculature in nonaffected brain shows hardly any expression of PSMA. It is unclear whether PSMA-targeting tracer uptake on PET is based on PSMA-specific binding to neovasculature or aspecific uptake in tumor. Here, we quantified uptake of various PSMA-targeting tracers in GBM and correlated this with PSMA expression in tumor biopsy samples from the same patients. Methods: Fourteen patients diagnosed with de novo (n = 8) or recurrent (n = 6) GBM underwent a preoperative PET scan after injection of 1.5 MBq/kg [68Ga]Ga-PSMA-11 (n = 7), 200 MBq of [18F]DCFpyl (n = 3), or 200 MBq of [18F]PSMA-1007 (n = 4). Uptake in tumor and tumor-to-background ratios, with contralateral nonaffected brain as background, were determined. In a subset of patients, PSMA expression levels from different regions in the tumor tissue samples (n = 40), determined using immunohistochemistry (n = 35) or RNA sequencing (n = 13), were correlated with tracer uptake on PET. Results: Moderate to high (SUVmax, 1.3-20.0) heterogeneous uptake was found in all tumors irrespective of the tracer type used. Uptake in nonaffected brain was low, resulting in high tumor-to-background ratios (6.1-359.0) calculated by dividing SUVmax of tumor by SUVmax of background. Immunohistochemistry showed variable PSMA expression on endothelial cells of tumor microvasculature, as well as on dispersed individual cells (of unknown origin), and granular staining of the neuropil. No correlation was found between in vivo uptake and PSMA expression levels (for immunohistochemistry, r = -0.173, P = 0.320; for RNA, r = -0.033, P = 0.915). Conclusion: Our results indicate the potential use of various PSMA-targeting tracers in GBM. However, we found no correlation between PSMA expression levels on immunohistochemistry and uptake intensity on PET. Whether this may be explained by methodologic reasons, such as the inability to measure functionally active PSMA with immunohistochemistry, tracer pharmacokinetics, or the contribution of a disturbed blood-brain barrier to tracer retention, should still be investigated.
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Glioblastoma , Neoplasias de la Próstata , Masculino , Humanos , Glioblastoma/diagnóstico por imagen , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radioisótopos de Galio , Células Endoteliales/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología , Tomografía de Emisión de PositronesRESUMEN
Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein's dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.
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Fibroblastos Asociados al Cáncer , Neoplasias Pancreáticas , Fotoquimioterapia , Animales , Ratones , Serina Endopeptidasas/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Distribución Tisular , Proteínas de la Membrana/metabolismo , Neoplasias Pancreáticas/patología , Fibroblastos/metabolismo , Ácido Pentético/metabolismoRESUMEN
Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model (CKP). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.
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Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Fotoquimioterapia , Ratones , Animales , Serina Endopeptidasas/metabolismo , Caspasa 3/metabolismo , Distribución Tisular , Modelos Animales de Enfermedad , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/patología , Fibroblastos/metabolismo , Anticuerpos/metabolismo , Línea Celular Tumoral , Neoplasias PancreáticasRESUMEN
PURPOSE: Incomplete resection of prostate cancer (PCa) results in increased risk of disease recurrence. Combined fluorescence-guided surgery with tumor-targeted photodynamic therapy (tPDT) may help to achieve complete tumor eradication. We developed a prostate-specific membrane antigen (PSMA) ligand consisting of a DOTA chelator for 111In labeling and a fluorophore/photosensitizer IRDye700DX (PSMA-N064). We evaluated the efficacy of PSMA-tPDT using PSMA-N064 in cell viability assays, a mouse xenograft model and in an ex vivo incubation study on fresh human PCa tissue. METHODS: In vitro, therapeutic efficacy of PSMA-N064 was evaluated using PSMA-positive LS174T cells and LS174T wild-type cells. In vivo, PSMA-N064-mediated tPDT was tested in immunodeficient BALB/c mice-bearing PSMA-positive LS174T xenografts. Tumor growth and survival were compared to control mice that received either NIR light or ligand injection only. Ex vivo tPDT efficacy was evaluated in excised fresh human PCa tissue incubated with PSMA-N064. RESULTS: In vitro, tPDT led to a PSMA-specific light- and ligand dose-dependent loss in cell viability. In vivo, tPDT-induced tumor cell apoptosis, delayed tumor growth, and significantly improved survival (p = 0.004) of the treated PSMA-positive tumor-bearing mice compared with the controls. In fresh ex vivo human PCa tissue, apoptosis was significantly increased in PSMA-tPDT-treated samples compared to non-treated control samples (p = 0.037). CONCLUSION: This study showed the feasibility of PSMA-N064-mediated tPDT in cell assays, a xenograft model and excised fresh human PCa tissue. This paves the way to investigate the impact of in vivo PSMA-tPDT on surgical outcome in PCa patients.
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Fotoquimioterapia , Neoplasias de la Próstata , Masculino , Humanos , Animales , Ratones , Medicina de Precisión , Ligandos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Glutamato Carboxipeptidasa II , Antígenos de Superficie , Fotoquimioterapia/métodos , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/tratamiento farmacológico , Línea Celular TumoralRESUMEN
INTRODUCTION: Fibroblast activation protein (FAP) is highly overexpressed in stromal tissue of various cancers. While FAP has been recognized as a potential diagnostic or therapeutic cancer target for decades, the surge of radiolabeled FAP-targeting molecules has the potential to revolutionize its perspective. It is presently hypothesized that FAP targeted radioligand therapy (TRT) may become a novel treatment for various types of cancer. To date, several preclinical and case series have been reported on FAP TRT using varying compounds and showing effective and tolerant results in advanced cancer patients. Here, we review the current (pre)clinical data on FAP TRT and discuss its perspective towards broader clinical implementation. METHODS: A PubMed search was performed to identify all FAP tracers used for TRT. Both preclinical and clinical studies were included if they reported on dosimetry, treatment response or adverse events. The last search was performed on July 22 2022. In addition, a database search was performed on clinical trial registries (date 15th of July 2022) to search for prospective trials on FAP TRT. RESULTS: In total, 35 papers were identified that were related to FAP TRT. This resulted in the inclusion of the following tracers for review: FAPI-04, FAPI-46, FAP-2286, SA.FAP, ND-bisFAPI, PNT6555, TEFAPI-06/07, FAPI-C12/C16, and FSDD. CONCLUSION: To date, data was reported on more than 100 patients that were treated with different FAP targeted radionuclide therapies such as [177Lu]Lu-FAPI-04, [90Y]Y-FAPI-46, [177Lu]Lu-FAP-2286, [177Lu]Lu-DOTA.SA.FAPI and [177Lu]Lu-DOTAGA.(SA.FAPi)2. In these studies, FAP targeted radionuclide therapy has resulted in objective responses in difficult to treat end stage cancer patients with manageable adverse events. Although no prospective data is yet available, these early data encourages further research.
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Proteínas de la Membrana , Radioisótopos , Humanos , Línea Celular Tumoral , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Radioisótopos/uso terapéutico , Fibroblastos/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radioisótopos de GalioRESUMEN
PURPOSE: Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. METHODS: We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. RESULTS: Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). CONCLUSION: We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.
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Péptidos de Penetración Celular , Insulinoma , Neoplasias Pancreáticas , Humanos , Exenatida/metabolismo , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/metabolismo , Distribución Tisular , Insulinoma/metabolismo , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Ponzoñas/farmacología , Ponzoñas/química , Ponzoñas/metabolismoRESUMEN
Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.
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Anticuerpos Monoclonales , Neoplasias de Cabeza y Cuello , Albúminas/metabolismo , Animales , Anticuerpos Monoclonales/química , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX/metabolismo , Línea Celular Tumoral , Semivida , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Humanos , Hipoxia , Ratones , Ácido Pentético , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
INTRODUCTION: The first generation ligands for prostate-specific membrane antigen (PSMA)-targeted radio- and fluorescence-guided surgery followed by adjuvant photodynamic therapy (PDT) have already shown the potential of this approach. Here, we developed three new photosensitizer-based dual-labeled PSMA ligands by crucial modification of existing PSMA ligand backbone structures (PSMA-1007/PSMA-617) for multimodal imaging and targeted PDT of PCa. METHODS: Various new PSMA ligands were synthesized using solid-phase chemistry and provided with a DOTA chelator for 111In labeling and the fluorophore/photosensitizer IRDye700DX. The performance of three new dual-labeled ligands was compared with a previously published first-generation ligand (PSMA-N064) and a control ligand with an incomplete PSMA-binding motif. PSMA specificity, affinity, and PDT efficacy of these ligands were determined in LS174T-PSMA cells and control LS174T wildtype cells. Tumor targeting properties were evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wildtype tumors using µSPECT/CT imaging, fluorescence imaging, and biodistribution studies after dissection. RESULTS: In order to synthesize the new dual-labeled ligands, we modified the PSMA peptide linker by substitution of a glutamic acid into a lysine residue, providing a handle for conjugation of multiple functional moieties. Ligand optimization showed that the new backbone structure leads to high-affinity PSMA ligands (all IC50 < 50 nM). Moreover, ligand-mediated PDT led to a PSMA-specific decrease in cell viability in vitro (P < 0.001). Linker modification significantly improved tumor targeting compared to the previously developed PSMA-N064 ligand (≥ 20 ± 3%ID/g vs 14 ± 2%ID/g, P < 0.01) and enabled specific visualization of PMSA-positive tumors using both radionuclide and fluorescence imaging in mice. CONCLUSION: The new high-affinity dual-labeled PSMA-targeting ligands with optimized backbone compositions showed increased tumor targeting and enabled multimodal image-guided PCa surgery combined with targeted photodynamic therapy.
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Fotoquimioterapia , Neoplasias de la Próstata , Animales , Antígenos de Superficie/metabolismo , Línea Celular Tumoral , Glutamato Carboxipeptidasa II/metabolismo , Humanos , Ligandos , Masculino , Ratones , Ratones Desnudos , Imagen Multimodal , Fármacos Fotosensibilizantes/uso terapéutico , Medicina de Precisión , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/terapia , Distribución TisularRESUMEN
Nanobodies are well-established targeting ligands for molecular imaging and therapy. Their short circulation time enables early imaging and reduces systemic radiation exposure. However, shorter circulation time leads to lower tracer accumulation in the target tissue. Cell-penetrating peptides (CPPs) improve cellular uptake of various cargoes, including nanobodies. CPPs could enhance tissue retention without compromising rapid clearance. However, systematic investigations on how the functionalities of nanobody and CPP combine with each other at the level of 2D and 3D cell cultures and in vivo are lacking. Here, we demonstrate that conjugates of the epidermal growth factor receptor (EGFR)-binding nanobody 7D12 with different CPPs (nonaarginine, penetratin, Tat and hLF) differ with respect to cell binding and induction of endocytosis. For nonaarginine and penetratin we compared the competition of EGF binding and performance of L- and D-peptide stereoisomers, and tested the D-peptide conjugates in tumor cell spheroids and in vivo. The D-peptide conjugates showed better penetration into spheroids than the unconjugated 7D12. Both in vivo and in vitro, the behavior of the agent reflects the combination of both functionalities. Although CPPs cause promising increases in in vitro uptake and 3D penetration, the dominant effect of the CPP in the control of biodistribution warrants further investigation.
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Variable domains of heavy chain only antibodies (VHHs) are valuable agents for application in tumor theranostics upon conjugation to both a diagnostic probe and a therapeutic compound. Here, we optimized site-specific conjugation of the chelator DTPA and the photosensitizer IRDye700DX to anti-epidermal growth factor receptor (EGFR) VHH 7D12, for applications in nuclear imaging and photodynamic therapy. 7D12 was site-specifically equipped with bimodal probe DTPA-tetrazine-IRDye700DX using the dichlorotetrazine conjugation platform. Binding, internalization and light-induced toxicity of DTPA-IRDye700DX-7D12 were determined using EGFR-overexpressing A431 cells. Finally, ex vivo biodistribution of DTPA-IRDye700DX-7D12 in A431 tumor-bearing mice was performed, and tumor homing was visualized with SPECT and fluorescence imaging. DTPA-IRDye700DX-7D12 was retrieved with a protein recovery of 43%, and a degree of labeling of 0.56. Spectral properties of the IRDye700DX were retained upon conjugation. 111In-labeled DTPA-IRDye700DX-7D12 bound specifically to A431 cells, and they were effectively killed upon illumination. DTPA-IRDye700DX-7D12 homed to A431 xenografts in vivo, and this could be visualized with both SPECT and fluorescence imaging. In conclusion, the dichlorotetrazine platform offers a feasible method for site-specific dual-labeling of VHH 7D12, retaining binding affinity and therapeutic efficacy. The flexibility of the described approach makes it easy to vary the nature of the probes for other combinations of diagnostic and therapeutic compounds.
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Ovarian cancer is the most lethal gynecological malignancy due to late detection associated with dissemination throughout the abdominal cavity. Targeted photodynamic therapy (tPDT) aimed at epithelial cell adhesion molecule (EpCAM), overexpressed in over 90% of ovarian cancer metastatic lesions, is a promising novel therapeutic modality. Here, we tested the specificity and activity of conjugates of EpCAM-directed designed ankyrin repeat proteins (DARPins) with the photosensitizer IRDye 700DX in in vitro and in vivo ovarian cancer models. EpCAM-binding DARPins (Ec1: Kd = 68 pM; Ac2: Kd = 130 nM) and a control DARPin were site-specifically functionalized with fluorophores or IRDye 700DX. Conjugation of anti-EpCAM DARPins with fluorophores maintained EpCAM-specific binding in cell lines and patient-derived ovarian cancer explants. Penetration of DARPin Ec1 into tumor spheroids was slower than that of Ac2, indicative of a binding site barrier effect for Ec1. DARPin-IRDye 700DX conjugates killed EpCAM-expressing cells in a highly specific and illumination-dependent fashion in 2D and 3D cultures. Furthermore, they effectively homed to EpCAM-expressing subcutaneous OV90 xenografts in mice. In conclusion, the high activity and specificity observed in preclinical ovarian cancer models, combined with a high specificity in patient material, warrant a further investigation of EpCAM-targeted PDT for ovarian cancer.
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Treatment of hyperinsulinemic hypoglycemia is challenging. Surgical treatment of insulinomas and focal lesions in congenital hyperinsulinism is invasive and carries major risks of morbidity. Medication to treat nesidioblastosis and diffuse congenital hyperinsulinism has varying efficacy and causes significant side effects. Here, we describe a novel method for therapy of hyperinsulinemic hyperglycemia, highly selectively killing ß-cells by receptor-targeted photodynamic therapy (rtPDT) with exendin-4-IRDye700DX, targeting the glucagon-like peptide 1 receptor (GLP-1R). Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with the GLP-1R. The efficacy and specificity of rtPDT with exendin-4-IRDye700DX were examined in vitro in cells with different levels of GLP-1R expression. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. Induction of cellular damage and the effect on tumor growth were analyzed to determine treatment efficacy. Results: Exendin-4-IRDye700DX has a high affinity for the GLP-1R, with a half-maximal inhibitory concentration of 6.3 nM. rtPDT caused significant specific phototoxicity in GLP-1R-positive cells (2.3% ± 0.8% and 2.7% ± 0.3% remaining cell viability in CHL-GLP-1R and INS-1 cells, respectively). The tracer accumulates dose-dependently in GLP-1R-positive tumors. In vivo, rtPDT induces cellular damage in tumors, shown by strong expression of cleaved caspase-3, and leads to a prolonged median survival of the mice (36.5 vs. 22.5 d, respectively; P < 0.05). Conclusion: These data show in vitro as well as in vivo evidence of the potency of rtPDT using exendin-4-IRDye700DX. This approach might in the future provide a new, minimally invasive, highly specific treatment method for hyperinsulinemic hypoglycemia.
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Hiperinsulinismo Congénito/tratamiento farmacológico , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Fotoquimioterapia/métodos , Animales , Línea Celular Tumoral , Cricetinae , Cricetulus , Exenatida/metabolismo , Exenatida/uso terapéutico , Femenino , Humanos , Indoles/metabolismo , Indoles/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Nesidioblastosis/tratamiento farmacológico , Compuestos de Organosilicio/metabolismo , Compuestos de Organosilicio/uso terapéutico , RatasRESUMEN
BACKGROUND: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in various types of cancer and induce metabolic alterations resulting from the neomorphic activity that causes production of D-2-hydroxyglutarate (D-2-HG) at the expense of α-ketoglutarate (α-KG) and NADPH. To overcome metabolic stress induced by these alterations, IDH-mutated (IDH mut ) cancers utilize rescue mechanisms comprising pathways in which glutaminase and glutamate dehydrogenase (GLUD) are involved. We hypothesized that inhibition of glutamate processing with the pleiotropic GLUD-inhibitor epigallocatechin-3-gallate (EGCG) would not only hamper D-2-HG production, but also decrease NAD(P)H and α-KG synthesis in IDH mut cancers, resulting in increased metabolic stress and increased sensitivity to radiotherapy. METHODS: We performed 13C-tracing studies to show that HCT116 colorectal cancer cells with an IDH1 R132H knock-in allele depend more on glutaminolysis than on glycolysis for the production of D-2-HG. We treated HCT116 cells, HCT116-IDH1 R132H cells, and HT1080 cells (carrying an IDH1 R132C mutation) with EGCG and evaluated D-2-HG production, cell proliferation rates, and sensitivity to radiotherapy. RESULTS: Significant amounts of 13C from glutamate accumulate in D-2-HG in HCT116-IDH1 wt/R132H but not in HCT116-IDH1 wt/wt . Preventing glutamate processing in HCT116-IDH1 wt/R132H cells with EGCG resulted in reduction of D-2-HG production. In addition, EGCG treatment decreased proliferation rates of IDH1 mut cells and at high doses sensitized cancer cells to ionizing radiation. Effects of EGCG in IDH-mutated cell lines were diminished by treatment with the IDH1mut inhibitor AGI-5198. CONCLUSIONS: This work shows that glutamate can be directly processed into D-2-HG and that reduction of glutamatolysis may be an effective and promising new treatment option for IDH mut cancers.
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Insulinomas, neuroendocrine tumors arising from pancreatic beta cells, often show overexpression of the glucagon-like peptide-1 receptor. Therefore, imaging with glucagon-like peptide analog exendin-4 can be used for diagnosis and preoperative localization. This review presents an overview of the development and clinical implementation of exendin-based tracers for nuclear imaging, and the potential use of exendin-4 based tracers for optical imaging and therapeutic applications such as peptide receptor radionuclide therapy or targeted photodynamic therapy.
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Diagnóstico por Imagen/métodos , Exenatida/química , Exenatida/uso terapéutico , Insulinoma/diagnóstico por imagen , Insulinoma/terapia , Animales , Humanos , Insulinoma/tratamiento farmacológico , FotoquimioterapiaRESUMEN
Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1mut), resulting in metabolic stress. Although IDHmut gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDHmut gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1R132H mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1mut- and IDHwt-glioma xenografts. Gene set enrichment analyses of clinical IDH1mut gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDHwt gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDHmut glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDHmut gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.
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Neoplasias Encefálicas/patología , Glioma/patología , Ácido Glutámico/metabolismo , Isocitrato Deshidrogenasa/genética , Ácido Láctico/metabolismo , Mutación , Estrés Fisiológico , 4-Aminobutirato Transaminasa/genética , 4-Aminobutirato Transaminasa/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/metabolismo , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Glutaminasa/genética , Glutaminasa/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Succionato-Semialdehído Deshidrogenasa/genética , Succionato-Semialdehído Deshidrogenasa/metabolismo , Transcriptoma , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this issue of Diabetologia, Alavi and Werner ( https://doi.org/10.1007/s00125-018-4676-1 ) criticise the attempts to use positron emission tomography (PET) for in vivo imaging of pancreatic beta cells, which they consider as 'futile'. In support of this strong statement, they point out the limitations of PET imaging, which they believe render beta cell mass impossible to estimate using this method. In our view, the Alavi and Werner presentation of the technical limitations of PET imaging does not reflect the current state of the art, which leads them to questionable conclusions towards the feasibility of beta cell imaging using this approach. Here, we put forward arguments in favour of continuing the development of innovative technologies enabling in vivo imaging of pancreatic beta cells and concisely present the current state of the art regarding putative technical limitations of PET imaging. Indeed, far from being a 'futile' effort, we demonstrate that beta cell imaging is now closer than ever to becoming a long-awaited clinical reality.
Asunto(s)
Células Secretoras de Insulina , Inutilidad Médica , Páncreas , Tomografía de Emisión de PositronesRESUMEN
Photodynamic therapy (PDT) is an emerging method to treat light-accessible malignancies. To increase specificity and allow dose reduction, conjugates of photosensitizers (PS) with antibodies against tumor-associated antigens have been developed for photoimmunotherapy (PIT). However, so far it is unclear whether cellular internalization of these conjugates after binding affects PIT efficacy. The use of low molecular weight llama single domain antibodies (VHHs, nanobodies) for PIT is preferred above full size antibodies because of better tumor penetration. Therefore, we functionalized the VHH 7D12, directed against the epidermal growth factor receptor (EGFR), with a PS (IRDye700DX). To assess the impact of cellular internalization on activity, the VHHs were additionally conjugated to a cell-penetrating peptide (VHH[PS]-CPP). Here we show that upon illumination with near-infrared (NIR) light, both VHH[PS] and VHH[PS]-CPP conjugates specifically induce cell death of EGFR expressing cancer cell lines and of EGFR-expressing cells derived from surgically obtained ascites from patients with high-grade serous ovarian cancer. However, VHH[PS] conjugates were significantly more effective compared to internalizing VHH[PS]-CPP suggesting that cell surface association is required for optimal therapeutic activity.
Asunto(s)
Receptores ErbB/metabolismo , Inmunoconjugados/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Anticuerpos de Dominio Único/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Endocitosis , Receptores ErbB/inmunología , Femenino , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Nanomedicina/métodos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Tecnología Farmacéutica/métodosRESUMEN
Overexpression of (mutated) receptor tyrosine kinases is a characteristic of many aggressive tumors, and induction of receptor uptake has long been recognized as a therapeutic modality. A conjugate of a synthetically produced cell-penetrating peptide (CPP), corresponding to amino acids 38-59 of human lactoferrin, and the recombinant llama single-domain antibody (VHH) 7D12, which binds the human epidermal growth factor receptor (EGFR), was generated by sortaseâ A mediated transpeptidation. The conjugate blocks EGF-mediated EGFR activation with higher efficacy than that of both modalities alone; a phenomenon that is caused by both effective receptor blockade and internalization. Thus, the VHH-CPP conjugate shows a combination of activities that implement a highly powerful new design principle to block receptor activation by its clearance from the cell surface.
Asunto(s)
Péptidos de Penetración Celular/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Inmunoconjugados/farmacología , Péptidos de Penetración Celular/inmunología , Endocitosis , Humanos , Inmunoconjugados/uso terapéutico , Lactoferrina/inmunología , Fragmentos de Péptidos/inmunologíaRESUMEN
Recombinant llama heavy-chain antibody fragments (VHHs) are promising tools in the field of targeted nanomedicine. 7D12, a VHH against the epidermal growth factor receptor (EGFR) that is overexpressed in various cancers, has been evaluated as an effective cancer-targeting VHH in multiple studies. The small size of VHHs (15-20 kDa) results in a low circulation half-life, which can be disadvantageous for certain applications. A solution to this problem is to attach VHHs to the surface of nanoparticles to increase the hydrodynamic radius of the conjugate. This approach simultaneously allows the incorporation of different VHHs and other targeting moieties and therapeutic components into one structure, creating multispecificity and versatility for therapy and diagnosis. Here, we present the construction of highly defined 7D12-containing nanoparticles by utilizing thermoresponsive diblock elastin-like peptides that reversibly self-assemble into micellar structures. The resulting particles have a hydrodynamic radius of 24.3 ± 0.9 nm and retain full EGFR-binding capacity. We present proof of concept of the usability of such particles by controlled incorporation of a photosensitizer and show that the resulting nanoparticles induce EGFR-specific light-induced cell killing. This approach is easily extended to the controlled incorporation of various functional modules, improving therapy and diagnosis with targeted nanomedicine.
Asunto(s)
Elastina/química , Nanopartículas/química , Péptidos/química , Fármacos Fotosensibilizantes/química , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Dominio Único/química , Animales , Camélidos del Nuevo Mundo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Estabilidad de Medicamentos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Escherichia coli/genética , Humanos , Luz , Nanomedicina , Fotoquimioterapia , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu+-independent strain-promoted alkyne-azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.