Subject(s)
Lead , Prostatic Neoplasms, Castration-Resistant , Humans , Male , Prostate-Specific Antigen , Prostatic Neoplasms, Castration-Resistant/diagnostic imaging , Prostatic Neoplasms, Castration-Resistant/radiotherapy , Prostatic Neoplasms, Castration-Resistant/pathology , Radiopharmaceuticals , Single Photon Emission Computed Tomography Computed TomographyABSTRACT
The aim of this prospective clinical study was to evaluate the potential of the prostate specific membrane antigen (PSMA) targeting ligand, [68Ga]-PSMA-Glu-NH-CO-NH-Lys-2-naphthyl-L-Ala-cyclohexane-DOTA ([68Ga]Ga-PSMA-617) as a positron emission tomography (PET) imaging biomarker in recurrent glioblastoma patients. Patients underwent [68Ga]Ga-PSMA-617 and O-(2-[18F]-fluoroethyl)-L-tyrosine ([18F]FET) PET scans on two separate days. [68Ga]Ga-PSMA-617 tumour selectivity was assessed by comparing tumour volume delineation and by assessing the intra-patient correlation between tumour uptake on [68Ga]Ga-PSMA-617 and [18F]FET PET images. [68Ga]Ga-PSMA-617 tumour specificity was evaluated by comparing its tumour-to-brain ratio (TBR) with [18F]FET TBR and its tumour volume with the magnetic resonance imaging (MRI) contrast-enhancing (CE) tumour volume. Ten patients were recruited in this study. [68Ga]Ga-PSMA-617-avid tumour volume was larger than the [18F]FET tumour volume (p = 0.063). There was a positive intra-patient correlation (median Pearson r = 0.51; p < 0.0001) between [68Ga]Ga-PSMA-617 and [18F]FET in the tumour volume. [68Ga]Ga-PSMA-617 had significantly higher TBR (p = 0.002) than [18F]FET. The [68Ga]Ga-PSMA-617-avid tumour volume was larger than the CE tumour volume (p = 0.0039). Overall, accumulation of [68Ga]-Ga-PSMA-617 beyond [18F]FET-avid tumour regions suggests the presence of neoangiogenesis in tumour regions that are not overly metabolically active yet. Higher tumour specificity suggests that [68Ga]-Ga-PSMA-617 could be a better imaging biomarker for recurrent tumour delineation and secondary treatment planning than [18F]FET and CE MRI.
Subject(s)
Brain Neoplasms , Glioblastoma , Prostatic Neoplasms , Male , Humans , Adult , Glioblastoma/diagnostic imaging , Glioblastoma/pathology , Gallium Radioisotopes , Prospective Studies , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/pathology , Positron-Emission Tomography/methods , Contrast Media , Magnetic Resonance Imaging , Chronic Disease , Prostatic Neoplasms/pathologyABSTRACT
Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.
Subject(s)
SARS-CoV-2 , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Indicators and Reagents , SARS-CoV-2/geneticsABSTRACT
Rationale: An antibody-drug conjugate (ADC) is a targeted therapy consisting of a cytotoxic payload that is linked to an antibody which targets a protein enriched on malignant cells. Multiple ADCs are currently used clinically as anti-cancer agents significantly improving patient survival. Herein, we evaluated the rationale of targeting the cell surface oncoreceptor CUB domain-containing protein 1 (CDCP1) using ADCs and assessed the efficacy of CDCP1-directed ADCs against a range of malignant tumors. Methods: CDCP1 mRNA expression was evaluated using large transcriptomic datasets of normal/tumor samples for 23 types of cancer and 15 other normal organs, and CDCP1 protein expression was examined in 34 normal tissues, >300 samples from six types of cancer, and in 49 cancer cell lines. A recombinant human/mouse chimeric anti-CDCP1 antibody (ch10D7) was labelled with 89Zirconium or monomethyl auristatin E (MMAE) and tested in multiple pre-clinical cancer models including 36 cancer cell lines and three mouse xenograft models. Results: Analysis of CDCP1 expression indicates elevated CDCP1 expression in the majority of the cancers and restricted expression in normal human tissues. Antibody ch10D7 demonstrates a high affinity and specificity for CDCP1 inducing cell signalling via Src accompanied by rapid internalization of ch10D7/CDCP1 complexes in cancer cells. 89Zirconium-labelled ch10D7 accumulates in CDCP1 expressing cells enabling detection of pancreatic cancer xenografts in mice by PET imaging. Cytotoxicity of MMAE-labelled ch10D7 against kidney, colorectal, lung, ovarian, pancreatic and prostate cancer cells in vitro, correlates with the level of CDCP1 on the plasma membrane. ch10D7-MMAE displays robust anti-tumor effects against mouse xenograft models of pancreatic, colorectal and ovarian cancer. Conclusion: CDCP1 directed imaging agents will be useful for selecting cancer patients for personalized treatment with cytotoxin-loaded CDCP1 targeting agents including antibody-drug conjugates.
Subject(s)
Antineoplastic Agents , Colorectal Neoplasms , Immunoconjugates , Male , Female , Humans , Animals , Mice , Immunoconjugates/pharmacology , Zirconium , Cell Line, Tumor , Xenograft Model Antitumor Assays , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cytotoxins , RNA, Messenger , Antigens, Neoplasm , Cell Adhesion MoleculesABSTRACT
Survival outcomes for patients with glioblastoma multiforme (GBM) have remained poor for the past 15 years, reflecting a clear challenge in the development of more effective treatment strategies. The efficacy of systemic therapies for GBM is greatly limited by the presence of the blood-brain barrier (BBB), which prevents drug penetration and accumulation in regions of infiltrative tumour, as represented in a consistent portion of GBM lesions. Focused ultrasound (FUS) - a technique that uses low-frequency ultrasound waves to induce targeted temporary disruption of the BBB - promises to improve survival outcomes by enhancing drug delivery and accumulation to infiltrating tumour regions. In this review we discuss the current state of preclinical investigations using FUS to enhance delivery of systemic therapies to intracranial neoplasms. We highlight critical methodological inconsistencies that are hampering clinical translation of FUS and we provide guiding principles for future preclinical studies. Particularly, we focus our attention on the importance of the selection of clinically relevant animal models and to the standardization of methods for FUS delivery, which will be paramount to the successful clinical translation of this promising technology for treatment in GBM patients. We also discuss how preclinical FUS research can benefit the development of GBM immunotherapies.
Subject(s)
Brain Neoplasms , Glioblastoma , Glioma , Animals , Blood-Brain Barrier/pathology , Brain Neoplasms/drug therapy , Brain Neoplasms/therapy , Drug Delivery Systems , Glioblastoma/drug therapy , Glioma/diagnostic imaging , Glioma/drug therapy , Humans , MicrobubblesABSTRACT
BACKGROUND: Multicentre clinical trials evaluating the role of 18F-Fluoroethyl-L-tyrosine (18F-FET) PET as a diagnostic biomarker in glioma management have highlighted a need for standardised methods of data analysis. 18F-FET uptake normalised against background in the contralateral brain is a standard imaging technique to delineate the biological tumour volume (BTV). Quantitative analysis of 18F-FET PET images requires a consistent and robust background activity. Currently, defining background activity involves the manual selection of an arbitrary region of interest, a process that is subject to large variability. This study aims to eliminate methodological errors in background activity definition through the introduction of a semiautomated method for region of interest selection. A new method for background activity definition, involving the semiautomated generation of mirror-image (MI) reference regions, was compared with the current state-of-the-art method, involving manually drawing crescent-shape (gCS) reference regions. The MI and gCS methods were tested by measuring values of background activity and resulting BTV of 18F-FET PET scans of ten patients with recurrent glioblastoma multiforme generated from inputs provided by seven readers. To assess intra-reader variability, each scan was evaluated six times by each reader. Intra- and inter-reader variability in background activity and BTV definition was assessed by means of coefficient of variation. RESULTS: Compared to the gCS method, the MI method showed significantly lower intra- and inter-reader variability both in background activity and in BTV definition. CONCLUSIONS: The proposed semiautomated MI method minimises intra- and inter-reader variability, providing a valuable approach for standardisation of 18F-FET PET quantitative parameters. Trial registration ANZCTR, ACTRN12618001346268. Registered 9 August 2018, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=374253.
ABSTRACT
The efficient encapsulation of small molecule active ingredients has been a challenge for many decades across many commercial applications. Recently, successful attempts to address this issue have included deposition of thin metal shells onto liquid filled polymer microcapsules or emulsion droplets to provide an impermeable barrier to diffusion. In this work we have developed a novel method to protect small molecule active ingredients by deposition of thin mineral shells. Platinum nanoparticles are used to catalyse and direct growth of a calcium phosphate shell onto liquid filled polymer microcapsules under various reaction conditions. Findings indicate that a non-porous protective shell is formed on the majority of the microcapsule population, with small concentrations of the core material being released only from those microcapsules with defects, over a 7 days period, when conducting forced release studies into a solvent for the core oil. The resulting microcapsules show no significant cell toxicity when exposed to HEK 293 cells for 72 h.
Subject(s)
Metal Nanoparticles , Calcium Phosphates , Capsules , HEK293 Cells , Humans , PlatinumABSTRACT
Colorectal cancer (CRC) is the third most common malignancy in the world, with 22% of patients presenting with metastatic disease and a further 50% destined to develop metastasis. Molecular imaging uses antigen-specific ligands conjugated to radionuclides to detect and characterise primary cancer and metastases. Expression of the cell surface protein CDCP1 is increased in CRC, and here we sought to assess whether it is a suitable molecular imaging target for the detection of this cancer. CDCP1 expression was assessed in CRC cell lines and a patient-derived xenograft to identify models suitable for evaluation of radio-labelled 10D7, a CDCP1-targeted, high-affinity monoclonal antibody, for preclinical molecular imaging. Positron emission tomography-computed tomography was used to compare zirconium-89 (89Zr)-10D7 avidity to a nonspecific, isotype control 89Zr-labelled IgGκ1 antibody. The specificity of CDCP1-avidity was further confirmed using CDCP1 silencing and blocking models. Our data indicate high avidity and specificity for of 89Zr-10D7 in CDCP1 expressing tumors at. Significantly higher levels than normal organs and blood, with greatest tumor avidity observed at late imaging time points. Furthermore, relatively high avidity is detected in high CDCP1 expressing tumors, with reduced avidity where CDCP1 expression was knocked down or blocked. The study supports CDCP1 as a molecular imaging target for CRC in preclinical PET-CT models using the radioligand 89Zr-10D7.
Subject(s)
Antigens, Neoplasm/genetics , Cell Adhesion Molecules/genetics , Colorectal Neoplasms/genetics , Positron Emission Tomography Computed Tomography , Radioisotopes/pharmacology , Zirconium/pharmacology , Animals , Antigens, Neoplasm/isolation & purification , Cell Adhesion Molecules/isolation & purification , Colorectal Neoplasms/diagnostic imaging , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , Heterografts , Humans , Ligands , MiceABSTRACT
Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Cell Adhesion Molecules/antagonists & inhibitors , Fluorescent Dyes/administration & dosage , Optical Imaging/methods , Ovarian Neoplasms/diagnosis , Animals , Antibodies, Monoclonal/chemistry , Antigens, Neoplasm , Cell Line, Tumor , Female , Fluorescent Dyes/chemistry , Humans , Indocyanine Green/administration & dosage , Indocyanine Green/chemistry , Injections, Intravenous , Mice , Ovarian Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
The implementation of accurate and sensitive molecular detection for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is paramount to effectively control the ongoing coronavirus disease 2019 (COVID-19) pandemic. In this regard, we herein propose the specific and highly sensitive SARS-CoV-2 detection based on nanoyeast single-chain-variable fragment (scFv) and ultrasensitive plasmonic nanobox-integrated nanomixing microassay. Importantly, this designed platform showcases the utility of nanoyeast-scFvs as specific capture reagents targeting the receptor-binding domain (RBD) of the virus and as monoclonal antibody alternatives suitable for cost-effective mass production and frequent testing. By capitalizing on single-particle active nanoboxes as plasmonic nanostructures for surface-enhanced Raman scattering (SERS), the microassay utilizes highly sensitive Raman signals to indicate virus infection. The developed microassay further integrated nanomixing for accelerating molecular collisions. Through the synergistic working of nanoyeast-scFv, plasmonic nanoboxes, and nanomixing, the highly specific and sensitive SARS-CoV-2 detection is achieved as low as 17 virus/µL without any molecular amplification. We successfully demonstrate SARS-CoV-2 detection in saliva samples of simulated patients at clinically relevant viral loads, suggesting the possibility of this platform for accurate and noninvasive patient screening.
Subject(s)
COVID-19 , Single-Chain Antibodies , Humans , SARS-CoV-2 , Saliva , Spectrum Analysis, RamanABSTRACT
BACKGROUND: Novel targeted therapies for children diagnosed with medulloblastoma (MB), the most common malignant pediatric brain tumor, are urgently required. A major hurdle in the development of effective therapies is the impaired delivery of systemic therapies to tumor cells due to a specialized endothelial blood-brain barrier (BBB). Accordingly, the integrity of the BBB is an essential consideration in any preclinical model used for assessing novel therapeutics. This study sought to assess the functional integrity of the BBB in several preclinical mouse models of MB. METHODS: Dynamic contrast enhancement magnetic resonance imaging (MRI) was used to evaluate blood-brain-tumor barrier (BBTB) permeability in a murine genetically engineered mouse model (GEMM) of Sonic Hedgehog (SHH) MB, patient-derived orthotopic xenograft models of MB (SHH and Gp3), and orthotopic transplantation of GEMM tumor cells, enabling a comparison of the direct effects of transplantation on the integrity of the BBTB. Immunofluorescence analysis was performed to compare the structural and subcellular features of tumor-associated vasculature in all models. RESULTS: Contrast enhancement was observed in all transplantation models of MB. No contrast enhancement was observed in the GEMM despite significant tumor burden. Cellular analysis of BBTB integrity revealed aberrancies in all transplantation models, correlating to the varying levels of BBTB permeability observed by MRI in these models. CONCLUSIONS: These results highlight functional differences in the integrity of the BBTB and tumor vessel phenotype between commonly utilized preclinical models of MB, with important implications for the preclinical evaluation of novel therapeutic agents for MB.
Subject(s)
Cerebellar Neoplasms , Medulloblastoma , Animals , Blood-Brain Barrier , Cell Line, Tumor , Child , Hedgehog Proteins , Heterografts , Humans , MiceABSTRACT
BACKGROUND: High-grade glioma (HGG) remains a recalcitrant clinical problem despite many decades of research. A major challenge in improving prognosis is the inability of current therapeutic strategies to address a clinically significant burden of infiltrating tumor cells that extend beyond the margins of the primary tumor mass. Such cells cannot be surgically excised nor efficiently targeted by radiation therapy. Therapeutic targeting of this tumor cell population is significantly hampered by the presence of an intact blood-brain barrier (BBB). In this study, we performed a preclinical investigation of the efficiency of MR-guided Focused Ultrasound (FUS) to temporarily disrupt the BBB to allow selective delivery of a tumor-targeting antibody to infiltrating tumor. METHODS: Structural MRI, dynamic-contrast enhancement MRI, and histology were used to fully characterize the MR-enhancing properties of a patient-derived xenograft (PDX) orthotopic mouse model of HGG and to develop a reproducible, robust model of nonenhancing HGG. PET-CT imaging techniques were then used to evaluate the efficacy of FUS to increase 89Zr-radiolabeled antibody concentration in nonenhancing HGG regions and adjacent non-targeted tumor tissue. RESULTS: The PDX mouse model of HGG has a significant tumor burden lying behind an intact BBB. Increased antibody uptake in nonenhancing tumor regions is directly proportional to the FUS-targeted volume. FUS locally increased antibody uptake in FUS-targeted regions of the tumor with an intact BBB, while leaving untargeted regions unaffected. CONCLUSIONS: FUS exposure successfully allowed temporary BBB disruption, localized to specifically targeted, nonenhancing, infiltrating tumor regions and delivery of a systemically administered antibody was significantly increased.
ABSTRACT
Increasing accumulation and retention of nanomedicines within tumor tissue is a significant challenge, particularly in the case of brain tumors where access to the tumor through the vasculature is restricted by the blood-brain barrier (BBB). This makes the application of nanomedicines in neuro-oncology often considered unfeasible, with efficacy limited to regions of significant disease progression and compromised BBB. However, little is understood about how the evolving tumor-brain physiology during disease progression affects the permeability and retention of designer nanomedicines. We report here the development of a modular nanomedicine platform that, when used in conjunction with a unique model of how tumorigenesis affects BBB integrity, allows investigation of how nanomaterial properties affect uptake and retention in brain tissue. By combining different in vivo longitudinal imaging techniques (including positron emission tomography and magnetic resonance imaging), we have evaluated the retention of nanomedicines with predefined physicochemical properties (size and surface functionality) and established a relationship between structure and tissue accumulation as a function of a new parameter that measures BBB leakiness; this offers significant advancements in our ability to relate tumor accumulation of nanomedicines to more physiologically relevant parameters. Our data show that accumulation of nanomedicines in brain tumor tissue is better correlated with the leakiness of the BBB than actual tumor volume. This was evaluated by establishing brain tumors using a spontaneous and endogenously derived glioblastoma model providing a unique opportunity to assess these parameters individually and compare the results across multiple mice. We also quantitatively demonstrate that smaller nanomedicines (20 nm) can indeed cross the BBB and accumulate in tumors at earlier stages of the disease than larger analogues, therefore opening the possibility of developing patient-specific nanoparticle treatment interventions in earlier stages of the disease. Importantly, these results provide a more predictive approach for designing efficacious personalized nanomedicines based on a particular patient's condition.
ABSTRACT
The clinical translation of new nanoparticle-based therapies for high-grade glioma (HGG) remains extremely poor. This has partly been due to the lack of suitable preclinical mouse models capable of replicating the complex characteristics of recurrent HGG (rHGG), namely the heterogeneous structural and functional characteristics of the blood-brain barrier (BBB). The goal of this study is to compare the characteristics of the tumor BBB of rHGG with two different mouse models of HGG, the ubiquitously used U87 cell line xenograft model and a patient-derived cell line WK1 xenograft model, in order to assess their suitability for nanomedicine research. Method: Structural MRI was used to assess the extent of BBB opening in mouse models with a fully developed tumor, and dynamic contrast enhanced MRI was used to obtain values of BBB permeability in contrast enhancing tumor. H&E and immunofluorescence staining were used to validate results obtained from the in vivo imaging studies. Results: The extent of BBB disruption and permeability in the contrast enhancing tumor was significantly higher in the U87 model than in rHGG. These values in the WK1 model are similar to those of rHGG. The U87 model is not infiltrative, has an entirely abnormal and leaky vasculature and it is not of glial origin. The WK1 model infiltrates into the non-neoplastic brain parenchyma, it has both regions with intact BBB and regions with leaky BBB and remains of glial origin. Conclusion: The WK1 mouse model more accurately reproduces the extent of BBB disruption, the level of BBB permeability and the histopathological characteristics found in rHGG patients than the U87 mouse model, and is therefore a more clinically relevant model for preclinical evaluations of emerging nanoparticle-based therapies for HGG.
Subject(s)
Blood-Brain Barrier/physiopathology , Glioma/pathology , Nanomedicine/methods , Nanoparticles/administration & dosage , Animals , Blood-Brain Barrier/chemistry , Blood-Brain Barrier/drug effects , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Capillary Permeability , Cell Line, Tumor , Drug Delivery Systems/methods , Drug Evaluation, Preclinical/methods , Female , Glioma/drug therapy , Glioma/metabolism , Humans , Magnetic Resonance Imaging/methods , Mice , Mice, Inbred NOD , Mice, SCID , Nanoparticles/chemistry , Xenograft Model Antitumor AssaysABSTRACT
At diagnosis, 22% of colorectal cancer (CRC) patients have metastases, and 50% later develop metastasis. Peptide receptor radionuclide therapy (PRRT), such as 177Lu-PSMA-617, is used to treat metastatic prostate cancer. 177Lu-PSMA-617 targets prostate-specific membrane antigen (PSMA), a cell-surface protein enriched in prostate cancer and the neovasculature of other solid tumors, including CRC. We performed 68Ga-PSMA-11 PET/CT imaging of 10 patients with metastatic CRC to assess metastasis avidity. Eight patients had lesions lacking avidity, and 2 had solitary metastases exhibiting very low avidity. Despite expression of PSMA in CRC neovasculature, none of the patients exhibited tumor avidity sufficient to be considered for 177Lu-PSMA-617 PRRT.
Subject(s)
Colorectal Neoplasms/diagnostic imaging , Edetic Acid/analogs & derivatives , Oligopeptides/pharmacokinetics , Positron-Emission Tomography/methods , Radiopharmaceuticals/pharmacokinetics , Colorectal Neoplasms/pathology , Colorectal Neoplasms/radiotherapy , Dipeptides/therapeutic use , Edetic Acid/pharmacokinetics , Gallium Isotopes , Gallium Radioisotopes , Heterocyclic Compounds, 1-Ring/therapeutic use , Humans , Lutetium , Neoplasm Metastasis , Prostate-Specific Antigen , RadioisotopesABSTRACT
Background: CUB domain-containing protein 1 (CDCP1) is a cell surface receptor regulating key signalling pathways in malignant cells. CDCP1 has been proposed as a molecular target to abrogate oncogenic signalling pathways and specifically deliver anti-cancer agents to tumors. However, the development of CDCP1-targeting agents has been questioned by its frequent proteolytic processing which was thought to result in shedding of the CDCP1 extracellular domain limiting its targetability. In this study, we investigated the relevance of targeting CDCP1 in the context of pancreatic ductal adenocarcinoma (PDAC) and assess the impact of CDCP1 proteolysis on the effectiveness of CDCP1 targeting agents. Methods: The involvement of CDCP1 in PDAC progression was assessed by association analysis in several PDAC cohorts and the proteolytic processing of CDCP1 was evaluated in PDAC cell lines and patient-derived cells. The consequences of CDCP1 proteolysis on its targetability in PDAC cells was assessed using immunoprecipitation, immunostaining and biochemical assays. The involvement of CDCP1 in PDAC progression was examined by loss-of-function in vitro and in vivo experiments employing PDAC cells expressing intact or cleaved CDCP1. Finally, we generated antibody-based imaging and therapeutic agents targeting CDCP1 to demonstrate the feasibility of targeting this receptor for detection and treatment of PDAC tumors. Results: High CDCP1 expression in PDAC is significantly associated with poorer patient survival. In PDAC cells proteolysis of CDCP1 does not always result in the shedding of CDCP1-extracellular domain which can interact with membrane-bound CDCP1 allowing signal transduction between the different CDCP1-fragments. Targeting CDCP1 impairs PDAC cell functions and PDAC tumor growth independently of CDCP1 cleavage status. A CDCP1-targeting antibody is highly effective at delivering imaging radionuclides and cytotoxins to PDAC cells allowing specific detection of tumors by PET/CT imaging and superior anti-tumor effects compared to gemcitabine in in vivo models. Conclusion: Independent of its cleavage status, CDCP1 exerts oncogenic functions in PDAC and has significant potential to be targeted for improved radiological staging and treatment of this cancer. Its elevated expression by most PDAC tumors and lack of expression by normal pancreas and other major organs, suggest that targeting CDCP1 could benefit a significant proportion of PDAC patients. These data support the further development of CDCP1-targeting agents as personalizable tools for effective imaging and treatment of PDAC.
Subject(s)
Antigens, Neoplasm/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Cell Adhesion Molecules/metabolism , Pancreatic Neoplasms/metabolism , Animals , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/therapy , Cell Line, Tumor , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Mice , Pancreatic Neoplasms/therapy , Precision Medicine , ProteolysisABSTRACT
INTRODUCTION: Pretargeting strategies that do not rely on the expression of molecular targets have expanded imaging and therapy options for cancer patients. Nanostars with designed multivalency and which highly accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect may therefore be the ideal vectors for the development of a passive pretargeting approach. METHODS: Nanostars were synthesized, consisting of 7-8 center-cross-linked arms that were modified with trans-cyclooctene (TCO) using poly(ethylene glycol) (PEG) linkers of 12 or 106 monomer units or without linker. The bioorthogonal click reaction with radiofluorinated 2,2'-(7-(2-(tetrazine-poly(ethyleneglycol)11-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-PEG11-NODA) or 2,2'-(7-(2-(tetrazine-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-NODA) was measured by ex vivo biodistribution studies and positron emission tomography (PET) in mice bearing tumors with high EPR characteristics. Bioorthogonal masking was performed using a tetrazine-functionalized dextran polymer (Tz-DP). RESULTS: Highest tumor accumulation of [18F]F-Tz-PEG11-NODA was observed for nanostars functionalized with TCO without linker, with a tumor uptake of 3.2 ± 0.4%ID/g and a tumor-to-muscle ratio of 12.8 ± 4.2, tumor-to-large intestine ratio of 0.5 ± 0.3 and tumor-to-kidney ratio of 2.0 ± 0.3, being significantly higher than for nanostars functionalized with TCO-PEG12 (P < 0.05) or TCO-PEG106 (P < 0.05). Tumor uptake and tumor-to-tissue ratios did not improve upon bioorthogonal masking with Tz-DP or when using a smaller, more lipophilic tetrazine([18F]F-Tz-NODA). CONCLUSIONS: A pretargeting strategy was developed based on the passive delivery of TCO-functionalized nanostars. Such a strategy would allow for the imaging and treatment of tumors with apparent EPR characteristics, with high radioactive tumor doses and minimal doses to off-target tissues.
Subject(s)
Drug Design , Nanomedicine/methods , Nanostructures , Positron-Emission Tomography/methods , Radiopharmaceuticals/chemistry , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic , Cyclooctanes/chemistry , Female , Isotope Labeling , Mice , Mice, Inbred BALB C , Polyethylene Glycols/chemistry , Radiopharmaceuticals/pharmacokinetics , Tissue DistributionABSTRACT
CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.
Subject(s)
Cell Adhesion Molecules/antagonists & inhibitors , Immunoconjugates/immunology , Membrane Proteins/metabolism , Ovarian Neoplasms/metabolism , Surface Plasmon Resonance/methods , Animals , Antigens, Neoplasm/immunology , Cell Adhesion Molecules/immunology , Cell Line, Tumor , Cell Membrane/metabolism , Cell Movement/immunology , Female , Mice , Models, Animal , Positron-Emission Tomography/methods , Radioisotopes/chemistry , Radioisotopes/metabolism , Transplantation, Heterologous/methods , Zirconium/chemistry , Zirconium/metabolism , src-Family Kinases/metabolismABSTRACT
Expression levels of biomarkers are generally unknown at initial diagnosis. The development of theranostic probes that do not rely on biomarker availability would expand therapy options for cancer patients, improve patient selection for nanomedicine and facilitate treatment of inoperable patients or patients with acquired therapy resistance. Herein, we report the development of star polymers, also known as nanostars, that allow for molecular imaging and/or endoradiotherapy based on passive targeting via the enhanced permeability and retention (EPR) effect. Methods: We synthesised a star copolymer, consisting of 7-8 centre-cross-linked arms that were modified with Gd3+ for magnetic resonance imaging (MRI), and functionalised either with 89Zr for in vivo quantification and positron emission tomography (PET) imaging, or with 177Lu for endoradiotherapy. 1H longitudinal relaxivities were determined over a continuum of magnetic field strengths ranging from 0.24 mT - 0.94 T at 37 °C (nuclear magnetic relaxation dispersion (NMRD) profile) and T1-weighted MRI contrast enhancement was visualized at 3 T and 7 T. PET imaging and ex vivo biodistribution studies were performed in mice bearing tumours with high EPR (CT26) or low EPR (BxPC3) characteristics. Therapy studies were performed in mice with high EPR tumours and mean absorbed organ doses were estimated for a standard human model. Results: The star copolymer with Gd3+ displayed a significantly superior contrast enhancement ability (T1 = 0.60 s) compared to the standard clinical contrast agent Gadovist (T1 = 1.0 s). Quantification of tumour accumulation using the radiolabelled nanostars in tumour-bearing mice demonstrated an exceptionally high uptake in tumours with high EPR characteristics (14.8 - 21.7 %ID/g). Uptake of the star polymers in tumours with low EPR characteristics was significantly lower (P<0.001), suggesting passive tumour accumulation of the nanostars via the EPR effect. Survival of mice treated with high dose 177Lu-labelled star polymers was significantly higher than survival of mice treated with lower therapy doses or control mice (P=0.001), demonstrating the utility of the 177Lu-labelled star polymers as platforms for endoradiotherapy. Conclusion: Our work highlights the potential of star polymers as probes for the molecular imaging of cancer tissue or for the passive delivery of radionuclides for endoradiotherapy. Their high functionalisability and high tumour accumulation emphasises their versatility as powerful tools for nanomedicine.