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1.
J Biomed Sci ; 31(1): 72, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-39010070

RESUMEN

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) has been widely studied as a tumor antigen due to its expression in varieties of solid tumors. Moreover, the glycoprotein contributes to critical cancer-associated cellular functionalities via its extracellular (EpEX) and intracellular (EpICD) domains. In colorectal cancer (CRC), EpCAM has been implicated in the Wnt signaling pathway, as EpICD and ß-Catenin are coordinately translocated to the nucleus. Once in the nucleus, EpICD transcriptionally regulates EpCAM target genes that; however, remains unclear whether Wnt signaling is modulated by EpICD activity. METHODS: Patient-derived organoids (PDOs), patient-derived xenografts (PDXs), and various CRC cell lines were used to study the roles of EpCAM and EpICD in Wnt receptor expression. Fluorescence and confocal microscopy were used to analyze tumors isolated from PDX and other xenograft models as well as CRC cell lines. EpCAM signaling was intervened with our humanized form of EpCAM neutralizing antibody, hEpAb2-6. Wnt receptor promoters under luciferase reporters were constructed to examine the effects of EpICD. Luciferase reporter assays were performed to evaluate promoter, γ-secretase and Wnt activity. Functional assays including in vivo tumor formation, organoid formation, spheroid and colony formation experiments were performed to study Wnt related phenomena. The therapeutic potential of EpCAM suppression by hEpAb2-6 was evaluated in xenograft and orthotopic models of human CRC. RESULTS: EpICD interacted with the promoters of Wnt receptors (FZD6 and LRP5/6) thus upregulated their transcriptional activity inducing Wnt signaling. Furthermore, activation of Wnt-pathway-associated kinases in the ß-Catenin destruction complex (GSK3ß and CK1) induced γ-secretase activity to augment EpICD shedding, establishing a positive-feedback loop. Our hEpAb2-6 antibody blocked EpICD-mediated upregulation of Wnt receptor expressions and conferred therapeutic benefits in both PDX and orthotopic models of human CRC. CONCLUSIONS: This study uncovers relevant functions of EpCAM where Wnt receptors are upregulated via the transcriptional co-factor activity of EpICD. The resultant enhancement of Wnt signaling induces γ-secretase activity further stimulating EpICD cleavage and its nuclear translocation. Our humanized anti-EpCAM antibody hEpAb2-6 blocks these mechanisms and may thereby provide therapeutic benefit in CRC.


Asunto(s)
Neoplasias Colorrectales , Molécula de Adhesión Celular Epitelial , Vía de Señalización Wnt , Humanos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Molécula de Adhesión Celular Epitelial/genética , Ratones , Animales , Línea Celular Tumoral , Progresión de la Enfermedad
2.
Nat Commun ; 15(1): 5888, 2024 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-39003254

RESUMEN

Archived patient-derived tissue specimens play a central role in understanding disease and developing therapies. To address specificity and sensitivity shortcomings of existing single-cell resolution proteoform analysis tools, we introduce a hybrid microfluidic platform (DropBlot) designed for proteoform analyses in chemically fixed single cells. DropBlot serially integrates droplet-based encapsulation and lysis of single fixed cells, with on-chip microwell-based antigen retrieval, with single-cell western blotting of target antigens. A water-in-oil droplet formulation withstands the harsh chemical (SDS, 6 M urea) and thermal conditions (98 °C, 1-2 hr) required for effective antigen retrieval, and supports analysis of retrieved protein targets by single-cell electrophoresis. We demonstrate protein-target retrieval from unfixed, paraformaldehyde-fixed (PFA), and methanol-fixed cells. Key protein targets (HER2, GAPDH, EpCAM, Vimentin) retrieved from PFA-fixed cells were resolved and immunoreactive. Relevant to biorepositories, DropBlot profiled targets retrieved from human-derived breast tumor specimens archived for six years, offering a workflow for single-cell protein-biomarker analysis of sparing biospecimens.


Asunto(s)
Western Blotting , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , Línea Celular Tumoral , Formaldehído/química , Femenino , Receptor ErbB-2/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Fijación del Tejido/métodos , Proteómica/métodos , Vimentina/metabolismo , Microfluídica/métodos , Microfluídica/instrumentación , Polímeros
3.
J Mater Chem B ; 12(29): 7203-7214, 2024 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-38952178

RESUMEN

Fluorescence-based LB (liquid biopsy) offers a rapid means of detecting cancer non-invasively. However, the widespread issue of sample loss during purification steps will diminish the accuracy of detection results. Therefore, in this study, we introduce a magnetic lanthanide sensor (MLS) designed for sensitive detection of the characteristic protein, epithelial cell adhesion molecule (EpCAM), on epithelial tumor exosomes. By leveraging the inherent multi-peak emission and time-resolved properties of the sole-component lanthanide element, combined with the self-ratiometric strategy, MLS can overcome limitations imposed by manual operation and/or sample complexity, thereby providing more stable and reliable output results. Specifically, terbium-doped NaYF4 nanoparticles (NaYF4:Tb) and deformable aptamers terminated with BHQ1 were sequentially introduced onto superparamagnetic silica-decorated Fe3O4 nanoparticles. Prior to target binding, emission from NaYF4:Tb at 543 nm was partially quenched due to the fluorescence resonance energy transfer (FRET) from NaYF4:Tb to BHQ1. Upon target binding, changes in the secondary structure of aptamers led to the fluorescence intensity increasing since the deconfinement of distance-dependent FRET effect. The characteristic emission of NaYF4:Tb at 543 nm was then utilized as the detection signal (I1), while the less changed emission at 583 nm served as the reference signal (I2), further reporting the self-ratiometric values of I1 and I2 (I1/I2) to illustrate the epithelial cancerous features of exosomes while ignoring possible sample loss. Consequently, over a wide range of exosome concentrations (2.28 × 102-2.28 × 108 particles per mL), the I1/I2 ratio exhibited a linear increase with exosome concentration [Y(I1/I2) = 0.166 lg (Nexosomes) + 3.0269, R2 = 0.9915], achieving a theoretical detection limit as low as 24 particles per mL. Additionally, MLS effectively distinguished epithelial cancer samples from healthy samples, showcasing significant potential for clinical diagnosis.


Asunto(s)
Exosomas , Exosomas/química , Exosomas/metabolismo , Humanos , Elementos de la Serie de los Lantanoides/química , Transferencia Resonante de Energía de Fluorescencia , Terbio/química , Molécula de Adhesión Celular Epitelial/metabolismo , Luminiscencia , Nanopartículas de Magnetita/química , Tamaño de la Partícula , Itrio/química , Técnicas Biosensibles/métodos , Fluoruros
4.
PeerJ ; 12: e17602, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38952968

RESUMEN

Background: Peritoneal metastasis (PM) is the most prevalent type of metastasis in patients with gastric cancer (GC) and has an extremely poor prognosis. The detection of free cancer cells (FCCs) in the peritoneal cavity has been demonstrated to be one of the worst prognostic factors for GC. However, there is a lack of sensitive detection methods for FCCs in the peritoneal cavity. This study aimed to use a new peritoneal lavage fluid cytology examination to detect FCCs in patients with GC, and to explore its clinical significance on diagnosing of occult peritoneal metastasis (OPM) and prognosis. Methods: Peritoneal lavage fluid from 50 patients with GC was obtained and processed via the isolation by size of epithelial tumor cells (ISET) method. Immunofluorescence and fluorescence in situ hybridization (FISH) were used to identify FCCs expressing chromosome 8 (CEP8), chromosome 17 (CEP17), and epithelial cell adhesion molecule (EpCAM). Results: Using a combination of the ISET platform and immunofluorescence-FISH, the detection of FCCs was higher than that by light microscopy (24.0% vs. 2.0%). Samples were categorized into positive and negative groups, based on the expressions of CEP8, CEP17, and EpCAM. Statistically significant relationships were demonstrated between age (P = 0.029), sex (P = 0.002), lymphatic invasion (P = 0.001), pTNM stage (P = 0.001), and positivity for FCCs. After adjusting for covariates, patients with positive FCCs had lower progression-free survival than patients with negative FCCs. Conclusion: The ISET platform highly enriched nucleated cells from peritoneal lavage fluid, and indicators comprising EpCAM, CEP8, and CEP17 confirmed the diagnosis of FCCs. As a potential detection method, it offers an opportunity for early intervention of OPM and an extension of patient survival.


Asunto(s)
Hibridación Fluorescente in Situ , Lavado Peritoneal , Neoplasias Peritoneales , Neoplasias Gástricas , Humanos , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/patología , Neoplasias Peritoneales/diagnóstico , Masculino , Femenino , Persona de Mediana Edad , Neoplasias Gástricas/patología , Neoplasias Gástricas/diagnóstico , Anciano , Líquido Ascítico/patología , Líquido Ascítico/citología , Pronóstico , Molécula de Adhesión Celular Epitelial/metabolismo , Molécula de Adhesión Celular Epitelial/genética , Adulto , Citodiagnóstico/métodos , Células Neoplásicas Circulantes/patología , Células Neoplásicas Circulantes/metabolismo , Citología
5.
Reprod Biol Endocrinol ; 22(1): 92, 2024 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-39085882

RESUMEN

BACKGROUND: Endometriosis is a gynecological disease characterized by the presence of endometrial tissue in abnormal locations, leading to severe symptoms, inflammation, pain, organ dysfunction, and infertility. Surgical removal of endometriosis lesions is crucial for improving pain and fertility outcomes, with the goal of complete lesion removal. This study aimed to analyze the location and expression patterns of poly (ADP-ribose) polymerase 1 (PARP-1), epithelial cell adhesion molecule (EpCAM), and folate receptor alpha (FRα) in endometriosis lesions and evaluate their potential for targeted imaging. METHODS: Gene expression analysis was performed using the Turku endometriosis database (EndometDB). By immunohistochemistry, we investigated the presence and distribution of PARP-1, EpCAM, and FRα in endometriosis foci and adjacent tissue. We also applied an ad hoc platform for the analysis of images to perform a quantitative immunolocalization analysis. Double immunofluorescence analysis was carried out for PARP-1 and EpCAM, as well as for PARP-1 and FRα, to explore the expression of these combined markers within endometriosis foci and their potential simultaneous utilization in surgical treatment. RESULTS: Gene expression analysis revealed that PARP-1, EpCAM, and FOLR1 (FRα gene) are more highly expressed in endometriotic lesions than in the peritoneum, which served as the control tissue. The results of the immunohistochemical study revealed a significant increase in the expression levels of all three biomarkers inside the endometriosis foci compared to the adjacent tissues. Additionally, the double immunofluorescence analysis consistently demonstrated the presence of PARP-1 in the nucleus and the expression of EpCAM and FRα in the cell membrane and cytoplasm. CONCLUSION: Overall, these three markers demonstrate significant potential for effective imaging of endometriosis. In particular, the results emphasize the importance of PARP-1 expression as a possible indicator for distinguishing endometriotic lesions from adjacent tissue. PARP-1, as a potential biomarker for endometriosis, offers promising avenues for further investigation in terms of both pathophysiology and diagnostic-therapeutic approaches.


Asunto(s)
Endometriosis , Molécula de Adhesión Celular Epitelial , Receptor 1 de Folato , Poli(ADP-Ribosa) Polimerasa-1 , Endometriosis/metabolismo , Endometriosis/cirugía , Endometriosis/genética , Endometriosis/diagnóstico , Endometriosis/patología , Femenino , Humanos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Adulto , Biomarcadores/metabolismo , Inmunohistoquímica , Endometrio/metabolismo , Endometrio/patología , Endometrio/cirugía
6.
Anal Chim Acta ; 1312: 342778, 2024 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-38834257

RESUMEN

BACKGROUND: The technology of capturing circulating tumor cells (CTCs) plays a crucial role in the diagnosis, evaluation of therapeutic efficacy, and prediction of prognosis in lung cancer. However, the presence of complex blood environment often results in severe nonspecific protein adsorption and interferences from blood cells, which negatively impacts the specificity of CTCs capture. There is a great need for development of novel nanomaterials for CTCs capture with prominent anti-nonspecific adsorptions from proteins or blood cells. RESULTS: We present a novel immune magnetic probe Fe3O4@(PEI/AA)4@Apt. The surface of Fe3O4 particles was modified with four layers of PEI/AA composite by layer-by-layer assembly. Furthermore, aptamers targeting epithelial marker EpCAM (SYL3C) and mesenchymal marker CSV (ZY5C) were simultaneously connected on Fe3O4@(PEI/AA)4 to improve the detection of different phenotypic CTCs and reduce false negatives. The results demonstrated that the (PEI/AA)4 coatings not only minimized non-specific protein adsorptions, but also significantly reduced the adsorption rate of red blood cells to a mere 1 %, as a result of which, the Fe3O4@(PEI/AA)4@Apt probe achieved a remarkably high capture efficiency toward CTCs (95.9 %). In the subsequent validation of clinical samples, the probe was also effective in capturing rare CTCs from lung cancer patients. SIGNIFICANCE AND NOVELTY: A (PEI/AA) polymerized composite with controllable layers was fabricated by layer-by-layer self-assembly technique, which displayed remarkable anti-nonspecific adsorption capabilities toward proteins and cells. Importantly, Fe3O4@(PEI/AA)4@Apt probe significantly improved CTCs capture purity in lung cancer patients to 89.36 %. For the first time, this study combined controllable (PEI/AA) layers with magnetic separation to innovatively build a resistant interface that significantly improves the specific capture performances of CTCs, broadening the application of this polymerized composite.


Asunto(s)
Alginatos , Células Neoplásicas Circulantes , Polietileneimina , Humanos , Células Neoplásicas Circulantes/patología , Polietileneimina/química , Alginatos/química , Nanopartículas de Magnetita/química , Neoplasias Pulmonares/patología , Aptámeros de Nucleótidos/química , Adsorción , Propiedades de Superficie , Molécula de Adhesión Celular Epitelial/inmunología
7.
Sci Rep ; 14(1): 14273, 2024 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-38902362

RESUMEN

Tumor-derived extracellular vesicles (EVs) show great potential as biomarkers for several diseases, including pancreatic cancer, due to their roles in cancer development and progression. However, the challenge of utilizing EVs as biomarkers lies in their inherent heterogeneity in terms of size and concentration, making accurate quantification difficult, which is highly dependent on the isolation and quantification methods used. In our study, we compared three EV isolation techniques and two EV quantification methods. We observed variations in EV concentration, with approximately 1.5-fold differences depending on the quantification method used. Interestingly, all EV isolation techniques consistently yielded similar EV quantities, overall size distribution, and modal sizes. In contrast, we found a notable increase in total EV amounts in samples from pancreatic cancer cell lines, mouse models, and patient plasma, compared to non-cancerous conditions. Moreover, individual tumor-derived EVs exhibited at least a 3-fold increase in several EV biomarkers. Our data, obtained from EVs isolated using various techniques and quantified through different methods, as well as originating from various pancreatic cancer models, suggests that EV profiling holds promise for the identification of unique and cancer-specific biomarkers in pancreatic cancer.


Asunto(s)
Biomarcadores de Tumor , Molécula de Adhesión Celular Epitelial , Vesículas Extracelulares , Glipicanos , Neoplasias Pancreáticas , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Vesículas Extracelulares/metabolismo , Humanos , Biomarcadores de Tumor/metabolismo , Animales , Ratones , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial/metabolismo , Glipicanos/metabolismo , Integrina alfaV/metabolismo
8.
Int J Mol Sci ; 25(12)2024 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-38928484

RESUMEN

Platinum-resistant high-grade serous carcinoma (HGSC) is an incurable disease, so biomarkers that could help with timely treatment adjustments and personalized approach are extensively being sought. Tumor-derived extracellular vesicles (EVs) that can be isolated from ascites and blood of HGSC patients are such promising biomarkers. Epithelial cell adhesion molecule (EpCAM) expression is upregulated in most epithelium-derived tumors; however, studies on prognostic value of EpCAM overexpression in ovarian carcinoma have shown contradictory results. The aim of our study was to evaluate the potential of total and EpCAM-positive EVs as prognostic and predictive biomarkers for advanced HGSC. Flow cytometry was used to determine the concentration of total and EpCAM-positive EVs in paired pretreatment ascites and plasma samples of 37 patients with advanced HGSC who underwent different first-line therapy. We found that higher EpCAM-positive EVs concentration in ascites is associated with shorter progression-free survival (PFS) regardless of treatment strategy. We also found a strong correlation of EpCAM-positive EVs concentration between ascites and plasma. Our findings indicate that EpCAM-positive EVs in ascites of patients with advanced HGSC have the potential to serve as prognostic biomarkers for predicting early recurrence and thereby likelihood of more aggressive tumor biology and development of chemoresistance.


Asunto(s)
Ascitis , Biomarcadores de Tumor , Cistadenocarcinoma Seroso , Molécula de Adhesión Celular Epitelial , Vesículas Extracelulares , Neoplasias Ováricas , Supervivencia sin Progresión , Humanos , Molécula de Adhesión Celular Epitelial/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Ascitis/metabolismo , Ascitis/patología , Persona de Mediana Edad , Anciano , Biomarcadores de Tumor/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Cistadenocarcinoma Seroso/mortalidad , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/mortalidad , Pronóstico , Adulto , Clasificación del Tumor
9.
Biosens Bioelectron ; 261: 116492, 2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-38870828

RESUMEN

Exosomes have been considered as promising biomarkers for cancer diagnosis due to their abundant information from originating cells. However, sensitive and reliable detection of exosomes is still facing technically challenges due to the lack of a sensing platform with high sensitivity and reproducibility. To address the challenges, here we propose a portable surface plasmon resonance (SPR) sensing of exosomes with a three-layer Au mirror/SiO2 spacer/Au nanohole sensor, fabricated by an economical polystyrene nanosphere self-assembly method. The SiO2 spacer can act as an optical cavity and induce mode hybridization, leading to excellent optimization of both sensitivity and full width at half maximum compared with normal single layer Au nanohole sensors. When modified with CD63 or EpCAM aptamers, a detection of limit (LOD) of as low as 600 particles/µL was achieved. The sensors showed good capability to distinguish between non-tumor derived L02 exosomes and tumor derived HepG2 exosomes. Additionally, high reproducibility was also achieved in detection of artificial serum samples with RSD as low as 2%, making it feasible for clinical applications. This mode hybridization plasmonic sensor provides an effective approach to optimize the detection sensitivity of exosomes, pushing SPR sensing one step further towards cancer diagnosis.


Asunto(s)
Exosomas , Oro , Límite de Detección , Dióxido de Silicio , Resonancia por Plasmón de Superficie , Exosomas/química , Humanos , Oro/química , Dióxido de Silicio/química , Aptámeros de Nucleótidos/química , Molécula de Adhesión Celular Epitelial , Tetraspanina 30 , Células Hep G2 , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , Reproducibilidad de los Resultados , Diseño de Equipo , Nanosferas/química , Hibridación de Ácido Nucleico , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/análisis
10.
Anal Chem ; 96(26): 10800-10808, 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38904228

RESUMEN

Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 105-107 particles/mL, along with a detection limit of 1.1 × 104 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.


Asunto(s)
Aptámeros de Nucleótidos , Vesículas Extracelulares , Oro , Espectrometría de Masas , Nanopartículas del Metal , Técnicas de Amplificación de Ácido Nucleico , Humanos , Aptámeros de Nucleótidos/química , Vesículas Extracelulares/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Nanopartículas del Metal/química , Oro/química , Espectrometría de Masas/métodos , Neoplasias , Molécula de Adhesión Celular Epitelial/metabolismo , Límite de Detección
11.
Int Immunopharmacol ; 137: 112424, 2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-38878486

RESUMEN

Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.


Asunto(s)
Complejo CD3 , Neoplasias Colorrectales , Molécula de Adhesión Celular Epitelial , Animales , Humanos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/terapia , Molécula de Adhesión Celular Epitelial/metabolismo , Complejo CD3/inmunología , Ratones , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Células HCT116 , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Femenino , Línea Celular Tumoral , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Ratones Endogámicos BALB C , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Inmunoterapia/métodos
12.
Mikrochim Acta ; 191(7): 424, 2024 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-38922365

RESUMEN

The enumeration of circulating tumor cells (CTCs) in peripheral blood plays a crucial role in the early diagnosis, recurrence monitoring, and prognosis assessment of cancer patients. There is a compelling need to develop an efficient technique for the capture and identification of these rare CTCs. However, the exclusive reliance on a single criterion, such as the epithelial cell adhesion molecule (EpCAM) antibody or aptamer, for the specific recognition of epithelial CTCs is not universally suitable for clinical applications, as it usually falls short in identifying EpCAM-negative CTCs. To address this limitation, we propose a straightforward and cost-effective method involving triplex fluorescently labelled aptamers (FAM-EpCAM, Cy5-PTK7, and Texas Red-CSV) to modify Fe3O4-loaded dendritic SiO2 nanocomposite (dmSiO2@Fe3O4/Apt). This multi-recognition-based strategy not only enhanced the efficiency in capturing heterogeneous CTCs, but also facilitated the rapid and accurate identification of CTCs. The capture efficiency of heterogenous CTCs reached up to 93.33%, with a detection limit as low as 5 cells/mL. Notably, the developed dmSiO2@Fe3O4/Apt nanoprobe enabled the swift identification of captured cells in just 30 min, relying solely on the fluorescently modified aptamers, which reduced the identification time by approximately 90% compared with the conventional immunocytochemistry (ICC) technique. Finally, these nanoprobe characteristics were validated using blood samples from patients with various types of cancers.


Asunto(s)
Aptámeros de Nucleótidos , Colorantes Fluorescentes , Nanocompuestos , Células Neoplásicas Circulantes , Dióxido de Silicio , Humanos , Células Neoplásicas Circulantes/patología , Dióxido de Silicio/química , Aptámeros de Nucleótidos/química , Nanocompuestos/química , Colorantes Fluorescentes/química , Separación Inmunomagnética/métodos , Molécula de Adhesión Celular Epitelial/inmunología , Límite de Detección , Línea Celular Tumoral , Óxido Ferrosoférrico/química
13.
Medicina (Kaunas) ; 60(6)2024 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-38929532

RESUMEN

Background and Objectives: Hepatocellular carcinoma (HCC) is a prevalent form of malignancy that is characterized by high mortality rates and prognosis that remain suboptimal, largely due to treatment resistance mechanisms. Recent studies have implicated cancer stem cells (CSCs), particularly those expressing epithelial cell adhesion molecule (EpCAM), in HCC progression and resistance. In the present study, we sought to assess EpCAM expression in HCC patients and its correlation with various clinicopathological parameters. Materials and Methods: Tissue samples from 42 HCC patients were subjected to immunohistochemical staining to evaluate EpCAM expression. Clinicopathological data were obtained including the size, grade and stage of tumors, vascular invasion status, alpha-fetoprotein levels, and cirrhosis status. The Chi square and Fisher's exact tests were employed to assess the association between categorical groups. Independent Student-t test or Mann-Whitney U test was used to investigate the association between continuous patient characteristics and survival. Results: Immunohistochemical analysis revealed EpCAM expression in 52.5% of HCC cases. EpCAM-positive tumors exhibited characteristics indicative of aggressive disease, including larger tumor sizes (p = 0.006), greater tumor multiplicity (p = 0.004), higher grades (p = 0.002), more advanced stages (p = 0.003), vascular invasion (p = 0.023), elevated alpha-fetoprotein levels (p = 0.013), and cirrhosis (p = 0.052). Survival analysis demonstrated that EpCAM expression was significantly associated with lower overall rates of survival and higher rates of recurrence in HCC patients. Conclusions: Our findings suggest that EpCAM expression may serve as a prognostic biomarker for HCC with a potential role in patient management. Targeting EpCAM-positive CSCs may represent a promising approach to overcome treatment resistance and improve clinical outcomes in HCC. However, further investigation into the molecular mechanisms underlying EpCAM's role in HCC progression is warranted to facilitate the development of personalized therapeutic interventions.


Asunto(s)
Biomarcadores de Tumor , Carcinoma Hepatocelular , Molécula de Adhesión Celular Epitelial , Neoplasias Hepáticas , Células Madre Neoplásicas , Humanos , Carcinoma Hepatocelular/patología , Molécula de Adhesión Celular Epitelial/análisis , Neoplasias Hepáticas/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Biomarcadores de Tumor/análisis , Anciano , Adulto , Inmunohistoquímica , Pronóstico , alfa-Fetoproteínas/análisis , alfa-Fetoproteínas/metabolismo
14.
ACS Appl Mater Interfaces ; 16(23): 29760-29769, 2024 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-38813974

RESUMEN

Multivalent receptor-ligand interactions (RLIs) exhibit excellent affinity for binding when targeting cell membrane receptors with low expression. However, existing strategies only allow for limited control of the valency and spacing of ligands for a certain receptor, lacking recognition patterns for multiple interested receptors with complex spatial distributions. Here, we developed flexible DNA nanoclaws with multivalent aptamers to achieve powerful cell recognition by controlling the spacing of aptamers to match the spatial patterns of receptors. The DNA nanoclaw with spacing-controllable binding sites was constructed via hybrid chain reaction (HCR), enabling dual targeting of HER2 and EpCAM molecules. The results demonstrate that the binding affinity of multivalent DNA nanoclaws to tumor cells is enhanced. We speculate that the flexible structure may conform better to irregularly shaped membrane surfaces, increasing the probability of intermolecular contact. The capture efficiency of circulating tumor cells successfully verified the high affinity and selectivity of this spatial pattern. This strategy will further promote the potential application of DNA frameworks in future disease diagnosis and treatment.


Asunto(s)
Aptámeros de Nucleótidos , ADN , Molécula de Adhesión Celular Epitelial , Receptor ErbB-2 , Humanos , Aptámeros de Nucleótidos/química , Molécula de Adhesión Celular Epitelial/metabolismo , Receptor ErbB-2/metabolismo , ADN/química , Línea Celular Tumoral , Nanoestructuras/química , Células Neoplásicas Circulantes/patología , Células Neoplásicas Circulantes/metabolismo
15.
Biosens Bioelectron ; 259: 116382, 2024 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-38749284

RESUMEN

Small extracellular vesicles (sEVs) reflect the genotype and phenotype of original cells and are biomarkers for early diagnosis and treatment monitoring of tumors. Yet, their small size and low density make them difficult to isolate and detect in body fluid samples. This study proposes a novel acDEP-Exo chip filled with transparent micro-beads, which formed a non-uniform electrical field, and finally achieved rapid, sensitive, and tunable sEVs capture and detection. The method requires only 20-50 µL of sample, achieved a limit of detection (LOD) of 161 particles/µL, and can detect biomarkers within 13 min. We applied the chip to analyze the two markers of sEV's EpCAM and MUC1 in clinical plasma samples from breast cancer (BC) patients and healthy volunteers and found that the combined evaluation of sEV's biomarkers has extremely high sensitivity, specificity and accuracy. The present study introduces an alternative approach to sEVs isolation and detection, has a great potential in real-time sEVs-based liquid biopsy.


Asunto(s)
Biomarcadores de Tumor , Técnicas Biosensibles , Neoplasias de la Mama , Molécula de Adhesión Celular Epitelial , Vesículas Extracelulares , Dispositivos Laboratorio en un Chip , Mucina-1 , Humanos , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/sangre , Vesículas Extracelulares/química , Femenino , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Mucina-1/sangre , Mucina-1/análisis , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/aislamiento & purificación , Límite de Detección , Diseño de Equipo , Electroforesis/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Biopsia Líquida/métodos , Biopsia Líquida/instrumentación
16.
Sci Rep ; 14(1): 12245, 2024 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-38806508

RESUMEN

Following the discovery of circulating tumor cells (CTCs) in the peripheral blood of cancer patients, CTCs were initially postulated to hold promise as a valuable prognostic tool through liquid biopsy. However, a decade and a half of accumulated data have revealed significant complexities in the investigation of CTCs. A challenging aspect lies in the reduced expression or complete loss of key epithelial markers during the epithelial-mesenchymal transition (EMT). This likely hampers the identification of a pathogenetically significant subset of CTCs. Nevertheless, there is a growing body of evidence regarding the prognostic value of such molecules as CD24 expressing in the primary breast tumor. Herewith, the exact relevance of CD24 expression on CTCs remains unclear. We used two epithelial markers (EpCAM and cytokeratin 7/8) to assess the count of CTCs in 57 breast cancer patients, both with (M0mts) and without metastasis (M0) during the follow-up period, as well as in M1 breast cancer patients. However, the investigation of these epithelial markers proved ineffective in identifying cell population expressing different combinations of EpCAM and cytokeratin 7/8 with prognostic significance for breast cancer metastases. Surprisingly, we found CD24+ circulating cells (CCs) in peripheral blood of breast cancer patients which have no epithelial markers (EpCAM and cytokeratin 7/8) but was strongly associated with distant metastasis. Namely, the count of CD45-EpCAM-CK7/8-CD24+ N-cadherin-CCs was elevated in both groups of patients, those with existing metastasis and those who developed metastases during the follow-up period. Simultaneously, an elevation in these cell counts beyond the established threshold of 218.3 cells per 1 mL of blood in patients prior to any treatment predicted a 12-fold risk of metastases, along with a threefold decrease in distant metastasis-free survival over a 90-month follow-up period. The origin of CD45-EpCAM-CK7/8-CD24+ N-cadherin-CCs remains unclear. In our opinion their existence can be explained by two most probable hypotheses. These cells could exhibit a terminal EMT phenotype, or it might be immature cells originating from the bone marrow. Nonetheless, if this hypothesis holds true, it's worth noting that the mentioned CCs do not align with any of the recognized stages of monocyte or neutrophil maturation, primarily due to the presence of CD45 expression in the myeloid cells. The results suggest the presence in the peripheral blood of patients with metastasis (both during the follow-up period and prior to inclusion in the study) of a cell population with a currently unspecified origin, possibly arising from both myeloid and tumor sources, as confirmed by the presence of aneuploidy.


Asunto(s)
Biomarcadores de Tumor , Neoplasias de la Mama , Antígeno CD24 , Molécula de Adhesión Celular Epitelial , Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Molécula de Adhesión Celular Epitelial/metabolismo , Antígeno CD24/metabolismo , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/sangre , Neoplasias de la Mama/mortalidad , Pronóstico , Persona de Mediana Edad , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/metabolismo , Anciano , Adulto , Transición Epitelial-Mesenquimal , Queratina-7/metabolismo , Queratina-8/metabolismo
17.
J Nanobiotechnology ; 22(1): 231, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38720360

RESUMEN

BACKGROUND: Circulating tumor cells (CTCs) are considered as a useful biomarker for early cancer diagnosis, which play a crucial role in metastatic process. Unfortunately, the tumor heterogeneity and extremely rare occurrence rate of CTCs among billions of interfering leukocytes seriously hamper the sensitivity and purity of CTCs isolation. METHODS: To address these, we firstly used microfluidic chips to detect the broad-spectrum of triple target combination biomarkers in CTCs of 10 types of cancer patients, including EpCAM, EGFR and Her2. Then, we constructed hybrid engineered cell membrane-camouflaged magnetic nanoparticles (HE-CM-MNs) for efficient capture of heterogeneous CTCs with high-purity, which was enabled by inheriting the recognition ability of HE-CM for various CTCs and reducing homologous cell interaction with leukocytes. Compared with single E-CM-MNs, HE-CM-MNs showed a significant improvement in the capture efficiency for a cell mixture, with an efficiency of 90%. And the capture efficiency of HE-CM-MNs toward 12 subpopulations of tumor cells was ranged from 70 to 85%. Furthermore, by using HE-CM-MNs, we successfully isolated heterogeneous CTCs with high purity from clinical blood samples. Finally, the captured CTCs by HE-CM-MNs could be used for gene mutation analysis. CONCLUSIONS: This study demonstrated the promising potential of HE-CM-MNs for heterogeneous CTCs detection and downstream analysis.


Asunto(s)
Biomarcadores de Tumor , Membrana Celular , Separación Celular , Nanopartículas de Magnetita , Células Neoplásicas Circulantes , Células Neoplásicas Circulantes/patología , Células Neoplásicas Circulantes/metabolismo , Humanos , Nanopartículas de Magnetita/química , Separación Celular/métodos , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/química , Biomarcadores de Tumor/sangre , Receptor ErbB-2 , Molécula de Adhesión Celular Epitelial/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias
18.
Biosens Bioelectron ; 259: 116380, 2024 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-38754193

RESUMEN

Exosomes, as novel biomarker for liquid biopsy, exhibit huge important potential value for cancer diagnosis. However, various proteins show different expression levels on exosomal membrane, and the absolute concentration of exosomes in clinical samples is easily influenced by a number of factors. Here, we developed a CRISPR/Cas12a and aptamer-chemiluminescence based analysis (CACBA) for the relative abundance determination of tumor-related protein positive exosomes in plasma for breast cancer diagnosis. The total concentration of exosomes was determined through captured CD63 using a CRISPR/Cas12a-based method with the LoD of 8.97 × 103 particles/µl. Meanwhile, EpCAM and MUC1 positive exosomes were quantitatively detected by aptamer-chemiluminescence (ACL) based method with the LoD of 1.45 × 102 and 3.73 × 102 particles/µl, respectively. It showed that the percentages of EpCAM and MUC1 positive exosomes offered an excellent capability to differentiate breast cancer patients and healthy donors. The high sensitivity, strong specificity, outstanding anti-interference capability, and steady recovery rate of this approach offered higher accuracy and robustness than the commercialized method in clinical trial. In addition with good stability, easy preparation and low cost, this method not only provides a new approach to rapid analysis of exosome proteins, it may be quickly extended to the diagnoses of various cancers.


Asunto(s)
Aptámeros de Nucleótidos , Biomarcadores de Tumor , Técnicas Biosensibles , Neoplasias de la Mama , Sistemas CRISPR-Cas , Molécula de Adhesión Celular Epitelial , Exosomas , Mucina-1 , Humanos , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Exosomas/química , Exosomas/genética , Femenino , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Mucina-1/sangre , Mucina-1/genética , Mucina-1/análisis , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Molécula de Adhesión Celular Epitelial/genética , Mediciones Luminiscentes/métodos , Tetraspanina 30 , Límite de Detección
19.
Anal Chem ; 96(19): 7747-7755, 2024 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-38691774

RESUMEN

Accurate classification of tumor cells is of importance for cancer diagnosis and further therapy. In this study, we develop multimolecular marker-activated transmembrane DNA computing systems (MTD). Employing the cell membrane as a native gate, the MTD system enables direct signal output following simple spatial events of "transmembrane" and "in-cell target encounter", bypassing the need of multistep signal conversion. The MTD system comprises two intelligent nanorobots capable of independently sensing three molecular markers (MUC1, EpCAM, and miR-21), resulting in comprehensive analysis. Our AND-AND logic-gated system (MTDAND-AND) demonstrates exceptional specificity, allowing targeted release of drug-DNA specifically in MCF-7 cells. Furthermore, the transformed OR-AND logic-gated system (MTDOR-AND) exhibits broader adaptability, facilitating the release of drug-DNA in three positive cancer cell lines (MCF-7, HeLa, and HepG2). Importantly, MTDAND-AND and MTDOR-AND, while possessing distinct personalized therapeutic potential, share the ability of outputting three imaging signals without any intermediate conversion steps. This feature ensures precise classification cross diverse cells (MCF-7, HeLa, HepG2, and MCF-10A), even in mixed populations. This study provides a straightforward yet effective solution to augment the versatility and precision of DNA computing systems, advancing their potential applications in biomedical diagnostic and therapeutic research.


Asunto(s)
ADN , Molécula de Adhesión Celular Epitelial , MicroARNs , Humanos , Molécula de Adhesión Celular Epitelial/metabolismo , ADN/química , MicroARNs/análisis , MicroARNs/metabolismo , Mucina-1/metabolismo , Mucina-1/análisis , Computadores Moleculares , Células MCF-7 , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/análisis , Membrana Celular/metabolismo , Membrana Celular/química , Células Hep G2
20.
Anal Chem ; 96(23): 9585-9592, 2024 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-38816678

RESUMEN

The PD-L1 protein on extracellular vesicles (EVs) is a promising biomarker for tumor immunotherapy. However, PD-L1+ EVs have various cell origins, so further analysis of the subpopulations is essential to help understand better their relationship with tumor immunotherapy. Different from the previous work which focus on the level of total PD-L1+ EVs expression, we, herein, report a dual-recognition mediated autocatalytic amplification (DRMAA) assay to detect the PD-L1 derived from tumors (EpCAM+), immune T cells (CD3+), and total (Lipids) EVs, respectively. The DRMAA assay employed proximity hybridization to construct a complete trigger sequence and then catalyzed the cross-hybridization of three hairpin probes, producing a three-way DNA junction (3-WJ) structure carrying the newly exposed trigger sequence. The 3-WJ complex subsequently initiated an autocatalytic amplification reaction and higher sensitivity than the traditional catalytic hairpin assembly assay was obtained. It was found that the EpCAM+ and PD-L1+ EVs were more effective than others in distinguishing lung cancer patients from healthy people. Surprisingly, the CD3+ and PD-L1+ EVs in lung cancer patients were also upregulated, indicating that immune cell-derived PD-L1+ EVs are also non-negligible marker in a tumor microenvironment. Our results suggested that the DRMAA assay would improve the study of subpopulations of PD-L1+ EVs to provide new insights for cancer immunotherapies.


Asunto(s)
Antígeno B7-H1 , Vesículas Extracelulares , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Humanos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Catálisis , Molécula de Adhesión Celular Epitelial/metabolismo , Técnicas de Amplificación de Ácido Nucleico , Biomarcadores de Tumor , Hibridación de Ácido Nucleico
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