RESUMO
Triple-negative breast cancer (TNBC) has the worst prognosis among the subtypes of breast cancer, with no targeted therapy available. Immunotherapy targeting programmed cell death protein-1 (PD-1) and its ligand (PD-L1) has resulted in some promising outcomes in cancer patients. The common treatments are monoclonal antibodies (mAbs). Despite novel methodologies in developing mAbs, there are several drawbacks with these medications. Immunological reactions, expensive and time-consuming production and requiring refrigeration are some of the challenging characteristics of mAbs that are addressed with using aptamers. Aptamers are nucleotide-based structures with high selectivity and specificity for target. Their small size helps aptamers penetrate the tissue better. In this review, we have discussed the nature of PD-1/PD-L1 interaction and summarised the available mAbs and aptamers specific for these two targets. This review highlights the role of aptamers as a future pathway for PD-1/PD-L1 modulation.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antígeno B7-H1/imunologia , Feminino , Humanos , Receptor de Morte Celular Programada 1/imunologia , Neoplasias de Mama Triplo Negativas/imunologiaRESUMO
The development of an aptamer-based therapeutic has rapidly progressed following the first two reports in the 1990s, underscoring the advantages of aptamer drugs associated with their unique binding properties. In 2004, the US Food and Drug Administration (FDA) approved the first therapeutic aptamer for the treatment of neovascular age-related macular degeneration, Macugen developed by NeXstar. Since then, eleven aptamers have successfully entered clinical trials for various therapeutic indications. Despite some of the pre-clinical and clinical successes of aptamers as therapeutics, no aptamer has been approved by the FDA for the treatment of cancer. This review highlights the most recent and cutting-edge approaches in the development of aptamers for the treatment of cancer types most refractory to conventional therapies. Herein, we will review (1) the development of aptamers to enhance anti-cancer immunity and as delivery tools for inducing the expression of immunogenic neoantigens; (2) the development of the most promising therapeutic aptamers designed to target the hard-to-treat cancers such as brain tumors; and (3) the development of "carrier" aptamers able to target and penetrate tumors and metastasis, delivering RNA therapeutics to the cytosol and nucleus.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Neoplasias/tratamento farmacológico , Aptâmeros de Nucleotídeos/imunologia , Portadores de Fármacos , Desenvolvimento de Medicamentos , Humanos , Neoplasias/imunologia , Evasão Tumoral/efeitos dos fármacosRESUMO
Breast cancer is one of the most commonly occurring cancers in women globally and is the primary cause of cancer mortality in females. BC is highly heterogeneous with various phenotypic expressions. The overexpression of HER2 is responsible for 15-30% of all invasive BC and is strongly associated with malignant behaviours, poor prognosis and decline in overall survival. Molecular imaging offers advantages over conventional imaging modalities, as it provides more sensitive and specific detection of tumours, as these techniques measure the biological and physiological processes at the cellular level to visualise the disease. Early detection and diagnosis of BC is crucial to improving clinical outcomes and prognosis. While HER2-specific antibodies and nanobodies may improve the sensitivity and specificity of molecular imaging, the radioisotope conjugation process may interfere with and may compromise their binding functionalities. Aptamers are single-stranded oligonucleotides capable of targeting biomarkers with remarkable binding specificity and affinity. Aptamers can be functionalised with radioisotopes without compromising target specificity. The attachment of different radioisotopes can determine the aptamer's functionality in the treatment of HER2(+) BC. Several HER2 aptamers and investigations of them have been described and evaluated in this paper. We also provide recommendations for future studies with HER2 aptamers to target HER2(+) BC.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Anticorpos de Domínio Único/uso terapêutico , Feminino , Humanos , PrognósticoRESUMO
Hospital-acquired infections (HAIs) are a growing concern around the world. They contribute to increasing mortality and morbidity rates and are an economic threat. All hospital patients have the potential to contract an HAI, but those with weakened or inferior immune systems are at highest risk. Most hospital patients will contract at least one HAI, but many will contract multiple ones. Bacteria are the most common cause of HAIs and contribute to 80-90% of all HAIs, with Staphylococcus aureus, Clostridium difficile, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae accounting for the majority. Each of these bacteria are highly resistant to antibiotics and can produce a protective film, known as a biofilm, to further prevent their eradication. It has been shown that by detecting and eradicating bacteria in the environment, infection rates can be reduced. The current methods for detecting bacteria are time consuming, non-specific, and prone to false negatives or false positives. Aptamer-based biosensors have demonstrated specific, time-efficient and simple detection, highlighting the likelihood that they could be used in a similar way to detect HAI-causing bacteria.
Assuntos
Infecção Hospitalar/diagnóstico , Infecção Hospitalar/microbiologia , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/prevenção & controle , Gerenciamento Clínico , Suscetibilidade a Doenças , Farmacorresistência Bacteriana , Humanos , Técnicas Microbiológicas , Técnicas de Diagnóstico Molecular , Sensibilidade e EspecificidadeRESUMO
This planet is home to countless species, some more well-known than the others. While we have developed many techniques to be able to interrogate some of the "omics", proteomics is becoming recognized as a very important part of the puzzle, given how important the protein is as a functional part of the cell. Within human health, the proteome is fairly well-established, with numerous reagents being available to decipher cellular pathways. Recent research advancements have assisted in characterizing the proteomes of some model (non-human) species, however, in many other species, we are only just touching the surface. This review considers three main reagent classes-antibodies, aptamers, and nanobodies-as a means of continuing to investigate the proteomes of non-model species without the complications of understanding the full protein signature of a species. Considerations of ease of production, potential applications, and the necessity for producing a new reagent depending on homology are presented.
Assuntos
Anticorpos/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Proteoma/análise , Anticorpos de Domínio Único/metabolismo , Animais , Humanos , Ligação Proteica , Proteoma/química , Proteômica/métodosRESUMO
The mainstay of clinical diagnostics is the use of specialised ligands that can recognise specific biomarkers relating to pathological changes. While protein antibodies have been utilised in these assays for the last 40 years, they have proven to be unreliable due to a number of reasons. The search for the 'perfect' targeting ligand or molecular probe has been slow, though the description of chemical antibodies, also known as aptamers, nearly 30 years ago suggested a replacement reagent. However, uptake has been slow to progress into the clinical environment. In this review, we discuss the issues associated with antibodies and describe some of the applications of aptamers that have relevancy to the clinical diagnostic environment.
Assuntos
Anticorpos , Aptâmeros de Nucleotídeos , Técnicas de Diagnóstico Molecular , Técnica de Seleção de Aptâmeros , Anticorpos/química , Biomarcadores , Humanos , Imuno-Histoquímica , Imunofenotipagem , Ligantes , Sondas MolecularesRESUMO
Glioblastoma is a highly aggressive primary brain tumour, renowned for its infiltrative growth and varied genetic profiles. The current treatment options are insufficient, and their off-target effects greatly reduce patient quality of life. The major challenge in improving glioblastoma diagnosis and treatment involves the development of a targeted imaging and drug delivery platform, capable of circumventing the blood brain barrier and specifically targeting glioblastoma tumours. The unique properties of aptamers demonstrate their capability of bridging the gap to the development of successful diagnosis and treatment options, where antibodies have previously failed. Aptamers possess many characteristics that make them an ideal novel imaging and therapeutic agent for the treatment of glioblastoma and other brain malignancies, and are likely to provide patients with a better standard of care and improved quality of life. Their target sensitivity, selective nature, ease of modification and low immunogenicity make them an ideal drug-delivery platform. This review article summarises the aptamers previously generated against glioblastoma cells or its identified biomarkers, and their potential application in diagnosis and therapeutic targeting of glioblastoma tumours.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/terapia , Glioblastoma/diagnóstico por imagem , Glioblastoma/terapia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Humanos , Técnica de Seleção de AptâmerosRESUMO
Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros/métodos , Bioensaio/métodos , Humanos , Ligação ProteicaRESUMO
INTRODUCTION: Prostate cancer (CaP) is the second leading malignancy in older men in Western countries. The role of CD44 variant 6 (CD44v6) in CaP progression and therapeutic resistance is still uncertain. Here, we investigated the roles of CD44v6 in CaP metastasis and chemo/radioresistance. Expression of CD44v6 in metastatic CaP cell lines, human primary CaP tissues and lymph node metastases was assessed using immunofluorescence and immunohistochemistry, respectively. METHODS: Knock down (KD) of CD44v6 was performed in PC-3M, DU145, and LNCaP cells using small interfering RNA (siRNA), and confirmed by confocal microscope, Western blot and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The adhesive ability and invasive potential were assessed using a hyaluronic acid (HA) adhesion and a matrigel chamber assay, respectively. Tumorigenesis potential and chemo-/radiosensitivity were measured by a sphere formation assay and a colony assay, respectively. RESULTS: Over-expression of CD44v6 was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of CD44v6 suppressed CaP proliferative, invasive and adhesive abilities, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated epithelial-mesenchymal transition (EMT), PI3K/Akt/mTOR, and Wnt/ß-catenin signaling pathway proteins in vitro. CONCLUSIONS: Our findings demonstrate that CD44v6 is an important cancer stem cell-like marker associated with CaP proliferation, invasion, adhesion, metastasis, chemo-/radioresistance, and the induction of EMT as well as the activation PI3K/Akt/mTOR and Wnt signaling pathways, suggesting that CD44v6 is a novel therapeutic target to sensitize CaP cells to chemo/radiotherapy.
Assuntos
Receptores de Hialuronatos/metabolismo , Metástase Linfática/genética , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Receptores de Hialuronatos/genética , Metástase Linfática/patologia , Masculino , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Aptamers are functional oligonucleotide ligands used for the molecular recognition of various targets. The natural characteristics of aptamers make them an excellent alternative to antibodies in diagnostics, therapeutics, and biosensing. DNA aptamers are mainly single-stranded oligonucleotides (ssDNA) that possess a definite binding to targets. However, the application of aptamers to the fields of brain health and neurodegenerative diseases has been limited to date. Herein, a DNA aptamer against the brain-derived neurotrophic factor (BDNF) protein was obtained by in vitro selection. BDNF is a potential biomarker of brain health and neurodegenerative diseases and has functions in the synaptic plasticity and survival of neurons. We identified eight aptamers that have binding affinity for BDNF from a 50-nucleotide library. Among these aptamers, NV_B12 showed the highest sensitivity and selectivity for detecting BDNF. In an aptamer-linked immobilized sorbent assay (ALISA), the NV_B12 aptamer strongly bound to BDNF protein, in a dose-dependent manner. The dissociation constant (Kd) for NV_B12 was 0.5 nM (95% CI: 0.4-0.6 nM). These findings suggest that BDNF-specific aptamers could be used as an alternative to antibodies in diagnostic and detection assays for BDNF.
Assuntos
Aptâmeros de Nucleotídeos , Doenças Neurodegenerativas , Humanos , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/química , Fator Neurotrófico Derivado do Encéfalo/genética , DNA de Cadeia Simples , Biblioteca GênicaRESUMO
Despite significant advances in surgery, radiotherapy and chemotherapy to treat prostate cancer (CaP), many patients die of secondary disease (metastases). Current therapeutic approaches are limited, and there is no cure for metastatic castration-resistant prostate cancer (CRPC). Epithelial cell adhesion molecule (EpCAM, also known as CD326) is a transmembrane glycoprotein that is highly expressed in rapidly proliferating carcinomas and plays an important role in the prevention of cell-cell adhesion, cell signalling, migration, proliferation and differentiation. Stably and highly expressed EpCAM has been found in primary CaP tissues, effusions and CaP metastases, making it an ideal candidate of tumour-associated antigen to detect metastasis of CaP cells in the circulation as well as a promising therapeutic target to control metastatic CRPC disease. In this review, we discuss the implications of the newly identified roles of EpCAM in terms of its diagnostic and metastatic relevance to CaP. We also summarize EpCAM expression in human CaP and EpCAM-mediated signalling pathways in cancer metastasis. Finally, emerging and innovative approaches to the management of the disease and expanding potential therapeutic applications of EpCAM for targeted strategies in future CaP therapy will be explored.
Assuntos
Antígenos de Neoplasias/fisiologia , Moléculas de Adesão Celular/fisiologia , Neoplasias da Próstata/patologia , Animais , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/química , Biomarcadores Tumorais/análise , Adesão Celular , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/química , Progressão da Doença , Molécula de Adesão da Célula Epitelial , Humanos , Masculino , Metástase Neoplásica , Células Neoplásicas Circulantes , Neoplasias da Próstata/tratamento farmacológicoRESUMO
Aptamers, and the selection process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) used to generate them, were first described more than twenty years ago. Since then, there have been numerous modifications to the selection procedures. This review discusses the use of modified bases as a means of enhancing serum stability and producing effective therapeutic tools, as well as functionalising these nucleic acids to be used as potential diagnostic agents.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Aptâmeros de Peptídeos/uso terapêutico , Técnicas Biossensoriais/métodos , Técnica de Seleção de Aptâmeros/instrumentação , Técnica de Seleção de Aptâmeros/métodos , Animais , HumanosRESUMO
Despite medical advances in treatment strategies over the past 30-years, epithelial ovarian cancer (EOC) continues to be defined by poor patient survival rates and aggressive, drug resistant relapse. Traditional approaches to cancer chemotherapy are typically limited by severe off-target effects on healthy tissue and aggressive drug-resistant recurrence. Recent shifts towards targeted therapies offer the possibility of circumventing the obstacles experienced by these treatments. While antibodies are the pioneering agents in such targeted therapies, several intrinsic characteristics of antibodies limits their clinical translation and efficacy. In contrast, oligonucleotide chemical antibodies, known as aptamers, are ideal for this application given their small size and lack of immunogenicity. This study explored the efficacy of a DNA aptamer, designed to target a well-established cancer biomarker, EpCAM, to deliver a chemotherapeutic drug. The results from this study support evidence that EpCAM aptamers can bind to epithelial ovarian cancer; and offers a valid alternative as a targeting ligand with tuneable specificity and sensitivity. It also supports the growing body of evidence that aptamers show great potential for application-specific, post-SELEX engineering through rational modifications. Through in vitro assays, these aptamers demonstrated cytotoxicity in both monolayer and tumoursphere assays, as well as in tumourigenic enriching assays. Further experimentation based on the results achieved in this project might aid in the development of novel cancer therapeutics and guide the novel designs of drugs for targeted drug delivery.
Assuntos
Antineoplásicos , Aptâmeros de Nucleotídeos , Neoplasias Ovarianas , Humanos , Feminino , Molécula de Adesão da Célula Epitelial , Carcinoma Epitelial do Ovário/tratamento farmacológico , Antineoplásicos/uso terapêutico , Doxorrubicina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Preparações Farmacêuticas , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/uso terapêutico , Linhagem Celular Tumoral , Técnica de Seleção de AptâmerosRESUMO
The key challenges to treating glioblastoma multiforme (GBM) are the heterogeneous and complex nature of the GBM tumour microenvironment (TME) and difficulty of drug delivery across the blood-brain barrier (BBB). The TME is composed of various neuronal and immune cells, as well as non-cellular components, including metabolic products, cellular interactions, and chemical compositions, all of which play a critical role in GBM development and therapeutic resistance. In this review, we aim to unravel the complexity of the GBM TME, evaluate current therapeutics targeting this microenvironment, and lastly identify potential targets and therapeutic delivery vehicles for the treatment of GBM. Specifically, we explore the potential of aptamer-targeted delivery as a successful approach to treating brain cancers. Aptamers have emerged as promising therapeutic drug delivery vehicles with the potential to cross the BBB and deliver payloads to GBM and brain metastases. By targeting specific ligands within the TME, aptamers could potentially improve treatment outcomes and overcome the challenges associated with larger therapies such as antibodies.
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Dengue infection, caused by the dengue virus, is a global threat which requires immediate attention and appropriate disease management. The current diagnosis of dengue infection is largely based on viral isolation, RT-PCR and serology-based detection, which are time-consuming and expensive, and require trained personnel. For early diagnosis of dengue, the direct detection of a dengue antigenic target is efficacious, and one such target is NS1. NS1-based detection is primarily antibody-centric and is beset by drawbacks pertaining to antibodies such as the high cost of synthesis and large batch-to-batch variation. Aptamers are potential surrogates of antibodies and are much cheaper, without exhibiting batch-to-batch variation. Given these advantages, we sought to isolate RNA aptamers against the NS1 protein of dengue virus serotype 2. A total of 11 cycles of SELEX were carried out, resulting in two potent aptamers, DENV-3 and DENV-6, with dissociation constant values estimated at 37.57 ± 10.34 nM and 41.40 ± 9.29 nM, respectively. These aptamers can be further miniaturized to TDENV-3 and TDENV-6a with an increased LOD upon their usage in direct ELASA. Moreover, these truncated aptamers are highly specific against the dengue NS1 while showing no cross-reactivity against the NS1 of the Zika virus, the E2 protein of the Chikungunya virus or the LipL32 protein of Leptospira, with target selectivity retained even in human serum. The usage of TDENV-3 as the capturing probe and TDENV-6a as the detection probe underpinned the development of an aptamer-based sandwich ELASA for the detection of dengue NS1. The sensitivity of the sandwich ELASA was further improved with the stabilization of the truncated aptamers and the repeated incubation strategy, which enabled a LOD of 2 nM when used with the target NS1 spiked in human serum diluted at 1:2000.
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The limited treatment options for triple-negative breast cancer with brain metastasis (TNBC-BM) have left the door of further drug development for these patients wide open. Although immunotherapy via monoclonal antibodies has shown some promising results in several cancers including TNBC, it cannot be considered the most effective treatment for brain metastasis. This is due to the protective role of the blood-brain barrier (BBB) which limits the entrance of most drugs, especially the bulky ones such as antibodies, to the brain. For a drug to traverse the BBB via passive diffusion, various physicochemical properties should be considered. Since natural medicine has been a key inspiration for the development of the majority of current medicines, in this paper, we review several naturally-derived molecules which have the potential for immunotherapy via blocking the interaction of programmed cell death protein-1 (PD-1) and its ligand, PD-L1. The mechanism of action, physicochemical properties and pharmacokinetics of these molecules and their theoretical potential to be used for the treatment of TNBC-BM are discussed.
RESUMO
The key challenges with the treatment of triple-negative breast cancer brain metastasis (TNBC-BM) are the lack of any targeted therapy and difficulties associated with drug delivery to the brain. These add to the high toxicity profile of existing treatments and the poor outcomes for patient. In this review, we introduce current drugs based on their molecular targets and look to improve brain drug delivery using more efficient and promising drug delivery systems. We describe ongoing clinical trials on druggable targets in TNBC-BM for a more targeted treatment and introduce the obstacles hindering drug delivery to the brain, bringing strategies and advancing knowledge for future steps in the treatment of patients with TNBC-BM.
Assuntos
Neoplasias Encefálicas , Neoplasias de Mama Triplo Negativas , Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Humanos , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The blood-brain barrier (BBB) is a strong barrier against the entrance of drugs, which has made brain cancer treatment a major challenge. We have previously shown that targeting transferrin receptors using aptamers increased brain drug delivery. To get a better understanding of this phenomenon, in the present article, a mathematical model based on the finite element method was developed accounting for the fluid flow and mass transport of the aptamer molecule inside an 8 µm capillary vessel across a 14 µm blood-brain barrier domain. The fluid flow and mass transport equations were coupled to calculate the blood velocity and aptamer concentration profiles across the BBB. It was identified that the thickness of the astrocyte and endothelial cell layers are key parameters affecting the concentration of the aptamer delivered to the last neuron dendrites in the BBB. The predicted efficacy of the drug delivery (Capt/Cin) of 10.9% to 13.8% was calculated at a porosity of 0.5 to 0.9, respectively, at a blood velocity of 0.38 mm/s, which was independent of the inlet concentration of the aptamer. This low efficacy was attributed to the mass transfer resistance across endothelial cells, astrocyte and pericyte layers, which decreased the concentration by 6.7%. It was also identified that the main mechanism of drug delivery is switched from convective mass transport in the capillary layer (with Peclet number > 50) to mixed convection mass transport (1 < Peclet number < 5) in the porous layers and to diffusion only once aptamer reached the brain parenchyma (Peclet number < 1).
Assuntos
Barreira Hematoencefálica , Neoplasias Encefálicas , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Células Endoteliais/metabolismo , Humanos , Receptores da Transferrina/metabolismo , Receptores da Transferrina/uso terapêuticoRESUMO
A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias , Anticorpos Monoclonais/metabolismo , Aptâmeros de Nucleotídeos/química , Molécula de Adesão da Célula Epitelial/metabolismo , Citometria de Fluxo , Humanos , Ligantes , Proteínas de Membrana/metabolismo , Ligação Proteica , Técnica de Seleção de AptâmerosRESUMO
The lack of a specific targeting strategy against cancer stem cells in current cancer treatment regimens is at least partly responsible for life-threatening cytotoxicity for patients undergoing traditional chemotherapy. An effective cancer stem cell targeting system is urgently required for the next generation of cancer medicine. Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and it has recently been identified as a cancer stem cell marker. In this study, we isolated a 40-base RNA aptamer that binds to EpCAM from a random oligonucleotide library using systematic evolution of ligands by exponential enrichment. The aptamer was further truncated to 19 bases. This 19-nt RNA aptamer interacts specifically with a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM, but not with those not expressing EpCAM, as analyzed using flow cytometry and confocal microscopy. The binding affinity of the EpCAM RNA aptamer to human cancer cells is approximately 55 nM. Importantly, this EpCAM RNA aptamer is efficiently internalized after binding to cell surface EpCAM. To our knowledge, this is the first RNA aptamer against a cancer stem cell surface marker being developed. Such cancer stem cell aptamers will greatly facilitate the development of novel targeted nanomedicine and molecular imaging agents for cancer theranostics.