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1.
Cancer Sci ; 111(2): 467-476, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31845453

RESUMO

Collective invasion of cancer cells is the key process of circulating tumor cell (CTC) cluster formation, and greatly contributes to metastasis. Cancer stem-like cells (CSC) have a distinct advantage of motility for metastatic dissemination. To verify the role of CSC in the collective invasion, we performed 3D assays to investigate the collective invasion from cancer cell spheroids. The results demonstrated that CSC can significantly promote both collective and single-cell invasion. Further study showed that CSC prefer to move outside and lead the collective invasion. More interestingly, approximately 60% of the leader CSC in collective invasion co-expressed both epithelial and mesenchymal genes, while only 4% co-expressed in single invasive CSC, indicating that CSC with hybrid epithelial/mesenchymal phenotype play a key role in cancer cell collective invasion.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Invasividade Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
2.
Chem Asian J ; 14(24): 4837-4846, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31756283

RESUMO

Three-dimensional (3D) scaffolds formed from natural biopolymers gelatin and chitosan that are chemically modified by galactose have shown improved hepatocyte adhesion, spheroid geometry and functions of the hepatocytes. Galactose specifically binds to the hepatocytes via the asialoglycoprotein receptor (ASGPR) and an increase in galactose density further improves the hepatocyte proliferation and functions. In this work, we aimed to increase the galactose density within the biopolymeric scaffold by physically blending the biopolymers chitosan and gelatin with an amphiphlic ß-galactose polypeptide (PPO-GP). PPO-GP, is a di-block copolymer with PPO and ß-galactose polypeptide, exhibits lower critical solution temperature and is entrapped within the scaffold through hydrophobic interactions. The uniform distribution of PPO-GP within the scaffold was confirmed by fluorescence microscopy. SEM and mechanical testing of the hybrid scaffolds indicated pore size, inter connectivity and compression modulus similar to the scaffolds made from 100 % biopolymer. The presence of the PPO-GP on the surface of the scaffold was tested monitoring the interaction of an analogous mannose containing PPO-GP scaffold and the mannose binding lectin Con-A. In vitro cell culture experiments with HepG2 cells were performed on GLN-GP and CTS-GP and their cellular response was compared with GLN and CTS scaffolds for a period of seven days. Within three days of culture the Hep G2 cells formed multicellular spheroids on GLN-GP and CTS-GP more efficiently than on the GLN and CTS scaffolds. The multicellular spheroids were also found to infiltrate more in GLN-GP and CTS-GP scaffolds and able to maintain their round morphology as observed by live/dead and SEM imaging.


Assuntos
Quitosana/química , Gelatina/química , Glicoproteínas/química , Hepatócitos/metabolismo , Polímeros/química , Propilenoglicóis/química , Tecidos Suporte/química , Animais , Módulo de Elasticidade , Galactosídeos/química , Células Hep G2 , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Esferoides Celulares/metabolismo , Suínos , Engenharia Tecidual/métodos
3.
Int J Nanomedicine ; 14: 7339-7352, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31686810

RESUMO

Purpose: To deliver the chemotherapeutics through the nanoparticles, the delivery system should accumulate at the tumor site first and then penetrate through the interstitium into the interior. The specific tumor-targeting pathway mediated via the receptor-ligand binding could achieve the desirable accumulation of nanoparticles, and the nanoparticles with smaller sizes were required for penetration. Methods and materials: We constructed a size-shrinkable nanocluster modified with a tumor-targeting motif IF-7 (IF-7-MNC) based on a pH-sensitive framework which could be disintegrated in an acid environment to release the micelles aggregated inside. The micelles were constructed by amphiphilic block copolymers PEG-PLA to encapsulate paclitaxel (PTX), while the cross-linked framework consisting of TPGS-PEI was used as a net to gather and release micelles. This nanoplatform could specifically bind with the tumor receptor Annexin A1 through the ligand IF-7 and then shrunk into small micelles with a desirable size for penetration. Conclusion: IF-7-MNC of 112.27±6.81 nm could shrink into micelles in PBS (0.01 M, pH 5.0) with sizes of 14.89±0.32 nm. The cellular-uptake results showed that IF-7-MNC could be significantly internalized by A549 cells and HUVEC cells, while the penetration of IF-7-MNC could be more prominent into the 3D-tumor spheroids compared with that of MNC. The biodistribution results displayed that the fluorescence of IF-7-MNC in the tumor site at 24 hrs was 4.5-fold stronger than that of MNC. The results of anti-tumor growth demonstrated that IF-7-MNC was more favorable for the tumor therapy than MNC, where the inhibitory rate of tumor growth was 88.29% in the PTX-loaded IF-7-MNC (IF-7-PMNC) treated group, significantly greater than PMNC treatment group (p<0.05).


Assuntos
Antineoplásicos/farmacologia , Carboidratos/química , Sistemas de Liberação de Medicamentos , Micelas , Nanopartículas/química , Neoplasias/tratamento farmacológico , Tamanho da Partícula , Peptídeos/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Endocitose , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/ultraestrutura , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Polietilenoglicóis , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Eletricidade Estática , Distribuição Tecidual
4.
Invest Ophthalmol Vis Sci ; 60(14): 4759-4773, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31738824

RESUMO

Purpose: Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods: Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results: After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions: This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.


Assuntos
Diferenciação Celular/fisiologia , Colinérgicos/farmacologia , Células Ependimogliais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/embriologia , Transdução de Sinais/fisiologia , Esferoides Celulares/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Embrião de Galinha , Colina O-Acetiltransferase/metabolismo , Crioultramicrotomia , Ácido Glutâmico/farmacologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neurônios Retinianos/citologia , Esferoides Celulares/metabolismo , Fixação de Tecidos , Vimentina/metabolismo
5.
Nat Commun ; 10(1): 4862, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649238

RESUMO

Hypoxia is known to be detrimental in cancer and contributes to its development. In this work, we present an approach to fate-map hypoxic cells in vivo in order to determine their cellular response to physiological O2 gradients as well as to quantify their contribution to metastatic spread. We demonstrate the ability of the system to fate-map hypoxic cells in 2D, and in 3D spheroids and organoids. We identify distinct gene expression patterns in cells that experienced intratumoral hypoxia in vivo compared to cells exposed to hypoxia in vitro. The intratumoral hypoxia gene-signature is a better prognostic indicator for distant metastasis-free survival. Post-hypoxic tumor cells have an ROS-resistant phenotype that provides a survival advantage in the bloodstream and promotes their ability to establish overt metastasis. Post-hypoxic cells retain an increase in the expression of a subset of hypoxia-inducible genes at the metastatic site, suggesting the possibility of a 'hypoxic memory.'


Assuntos
Espécies Reativas de Oxigênio/metabolismo , Esferoides Celulares/metabolismo , Transcriptoma , Hipóxia Tumoral/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Transgênicos , Metástase Neoplásica/genética , Imagem Óptica , Fenótipo , Prognóstico , Microambiente Tumoral
6.
Nature ; 574(7776): 112-116, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554966

RESUMO

Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1-7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.


Assuntos
Sistema Biliar/embriologia , Intestinos/embriologia , Fígado/embriologia , Modelos Biológicos , Morfogênese , Pâncreas/embriologia , Animais , Sistema Biliar/citologia , Biomarcadores/análise , Biomarcadores/metabolismo , Padronização Corporal , Endoderma/citologia , Endoderma/embriologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/citologia , Fígado/citologia , Masculino , Camundongos , Organoides/citologia , Organoides/embriologia , Pâncreas/citologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Fatores de Transcrição HES-1/análise , Fatores de Transcrição HES-1/metabolismo
7.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510100

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, and thus better understanding of its molecular pathology is crucial for us to devise more effective treatment of this deadly disease. As cancer cell line remains a convenient starting point for discovery and proof-of-concept studies, here we report the miRNA expression characteristics of two cell lines, MIA PaCa-2 and PANC-1, and discovered three miRNAs (miR-7-5p, let-7d, and miR-135b-5p) that are involved in cancer stem cells (CSCs) suppression. After transfection of each miRNA's mimic into PANC-1 cells which exhibits higher stemness feature than MIA-PaCa-2 cells, partial reduction of CSC surface markers and inhibition of tumor sphere formation were observed. These results enlighten us to consider miRNAs as potential therapeutic agents for pancreatic cancer patients via specific and effective inhibition of CSCs.


Assuntos
Carcinoma Ductal Pancreático/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Neoplasias Pancreáticas/patologia , Esferoides Celulares/metabolismo
8.
Int J Mol Sci ; 20(17)2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484286

RESUMO

Cerebral small vessel diseases (SVD) have been causally correlated with ischemic strokes, leading to cognitive decline and vascular dementia. Neuroimaging and molecular genetic tests could improve diagnostic accuracy in patients with potential SVD. Several types of monogenic, hereditary cerebral SVD have been identified: cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cathepsin A-related arteriopathy with strokes and leukoencephalopathy (CARASAL), hereditary diffuse leukoencephalopathy with spheroids (HDLS), COL4A1/2-related disorders, and Fabry disease. These disorders can be distinguished based on their genetics, pathological and imaging findings, clinical manifestation, and diagnosis. Genetic studies of sporadic cerebral SVD have demonstrated a high degree of heritability, particularly among patients with young-onset stroke. Common genetic variants in monogenic disease may contribute to pathological progress in several cerebral SVD subtypes, revealing distinct genetic mechanisms in different subtype of SVD. Hence, genetic molecular analysis should be used as the final gold standard of diagnosis. The purpose of this review was to summarize the recent discoveries made surrounding the genetics of cerebral SVD and their clinical significance, to provide new insights into the pathogenesis of cerebral SVD, and to highlight the possible convergence of disease mechanisms in monogenic and sporadic cerebral SVD.


Assuntos
Doenças de Pequenos Vasos Cerebrais/genética , Leucoencefalopatias/genética , Acidente Vascular Cerebral/genética , Animais , Demência Vascular/metabolismo , Humanos , Esferoides Celulares/metabolismo
9.
BMC Cancer ; 19(1): 864, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470802

RESUMO

BACKGROUND: Bronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Studies of tumor initiating cell (TIC) biology in bronchial carcinoids has been hindered by the lack of appropriate in-vitro and xenograft models representing the bronchial carcinoid phenotype and behavior. METHODS: Bronchial carcinoid cell lines (H727, TC and H720, AC) were cultured in serum-free growth factor supplemented medium to form 3D spheroids and serially passaged up to the 3rd generation permitting expansion of the TIC population as verified by expression of stemness markers, clonogenicity in-vitro and tumorigenicity in both subcutaneous and orthotopic (lung) models. Acetazolamide (AZ), sulforaphane (SFN) and the AZ + SFN combination were evaluated for targeting TIC in bronchial carcinoids. RESULTS: Data demonstrate that bronchial carcinoid cell line 3rd generation spheroid cells show increased drug resistance, clonogenicity, and tumorigenic potential compared with the parental cells, suggesting selection and expansion of a TIC fraction. Gene expression and immunolabeling studies demonstrated that the TIC expressed stemness factors Oct-4, Sox-2 and Nanog. In a lung orthotopic model bronchial carcinoid, cell line derived spheroids, and patient tumor derived 3rd generation spheroids when supported by a stroma, showed robust tumor formation. SFN and especially the AZ + SFN combination were effective in inhibiting tumor cell growth, spheroid formation and in reducing tumor formation in immunocompromised mice. CONCLUSIONS: Human bronchial carcinoid tumor cells serially passaged as spheroids contain a higher fraction of TIC exhibiting a stemness phenotype. This TIC population can be effectively targeted by the combination of AZ + SFN. Our work portends clinical relevance and supports the therapeutic use of the novel AZ+ SFN combination that may target the TIC population of bronchial carcinoids.


Assuntos
Acetazolamida/administração & dosagem , Anticarcinógenos/administração & dosagem , Neoplasias Brônquicas/tratamento farmacológico , Tumor Carcinoide/tratamento farmacológico , Isotiocianatos/administração & dosagem , Células-Tronco Neoplásicas/efeitos dos fármacos , Acetazolamida/farmacologia , Animais , Anticarcinógenos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Brônquicas/genética , Neoplasias Brônquicas/metabolismo , Tumor Carcinoide/genética , Tumor Carcinoide/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isotiocianatos/farmacologia , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Anal Bioanal Chem ; 411(27): 7087-7094, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31471684

RESUMO

Accurate measurement and understanding of therapeutic uptake and metabolism is key in the drug development process. This work examines the amount of doxorubicin that can penetrate into spheroids after being encapsulated in a liposomal configuration in comparison with free drug. Through a process known as serial trypsinization, three distinct cellular populations of a spheroid were successfully separated and a small molecule extraction was used to isolate the chemotherapeutic. Doxorubicin showed a time-dependent permeability into spheroids with the most drug accumulating in the core at 24 h of treatment. Entrapment of the chemotherapeutic delayed the permeability of the drug and resulted in reduced amounts quantified at the earlier time points. These findings validate the claim that liposomal therapeutics have the ability to alter the pharmacokinetics and pharmacodynamics profiles of a drug while also demonstrating the combined power of mass spectrometry and three-dimensional cell cultures to evaluate drug penetration and metabolism. Graphical abstract.


Assuntos
Antibióticos Antineoplásicos/metabolismo , Doxorrubicina/análogos & derivados , Esferoides Celulares/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/metabolismo , Doxorrubicina/farmacocinética , Células HCT116 , Humanos , Espectrometria de Massas , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacocinética , Tripsina/metabolismo
11.
Biomed Pharmacother ; 118: 109371, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31545281

RESUMO

BACKGROUND: Disulfiram (DSF) is a drug used for treatment of alcoholism that has also displayed promising anti-cancer activity. It unfolds its effects by inhibiting the enzyme activity of aldehyde dehydrogenase (ALDH) isoforms. METHODS: MTT assay, spheroid formation, clonogenicity assay, qRT-PCR, and ALDH enzyme activity analysis were performed using ovarian cancer cell lines IGROV1, SKOV3 and SKOV3IP1. Cell cycle analyses and measurement of intracellular reactive oxygen species (ROS) were carried out by flow cytometry. ALDH+ and ALDH- cells were isolated by FACS sorting. RESULTS: ALDH activity was inhibited in ovarian cancer stem cells (the proportion of ALDH+ cells was reduced from 21.7% to 0.391%, 8.4% to 0%, 6.88% to 0.05% in cell lines IGROV1, SKOV3, and SKOV3IP1, respectively). DSF with or without the cofactor copper (Cu2+) exhibited cytotoxicity dose- and time-dependent and enhanced cisplatin-induced apoptosis. DSF + Cu2+ increased intracellular ROS levels triggering apoptosis of ovarian cancer stem cells (CSC). Significantly more colony and spheroid formation was observed in ALDH+ compared with ALDH- cells (P < 0.01). Moreover, ALDH+ cells were more resistant to cisplatin treatment compared with ALDH-cells (P < 0.05) and also exhibited a lower basal level of ROS. However, no significant difference in ROS accumulation nor in cellular viability was observed in ALDH + cells in comparison to ALDH- cells after pre-treatment with DSF (0.08 µM). CONCLUSION: Our findings provide evidence that DSF might be employed as a novel adjuvant chemotherapeutic agent in combination with cisplatin for treatment of ovarian cancer.


Assuntos
Aldeído Desidrogenase/metabolismo , Cobre/farmacologia , Dissulfiram/farmacologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Humanos , Concentração Inibidora 50 , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
12.
Tissue Cell ; 59: 39-43, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383287

RESUMO

Major Glioblastoma's hallmarks include proliferation, invasion and heterogeneity. Biological 3D tumor spheroid models can serve as intermediate systems between traditional 2D cell culture and complex in vivo models. Tumor spheroids have been shown to more accurately reproduce the spatial organization and microenvironmental factors of in vivo micro-tumors, such as relevant gradients of nutrients and other molecular agents, while they maintain cell-to-cell and cell-to-matrix interactions. In vitro 3D assays are useful to monitor these properties. Here, we test the suitability of the well-known T98 G Glioblastoma cell line in such a 3D assay. The doubling time and death rate parameters of T98 G are estimated, as well as their spheroidal growth-expansion curves with and without the presence of basement membrane substrate. The T98 G invasive profile is characterized by collective morphology and proliferation-associated invasion. We show that the T98 G secondary GB cell line exhibits both invasive and proliferative capabilities in 3D and thus, can serve as control cell line for the 3D in vitro study of primary GB cell cultures.


Assuntos
Glioblastoma , Modelos Biológicos , Esferoides Celulares , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
13.
Anal Chim Acta ; 1080: 104-115, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31409459

RESUMO

We have implemented a linear ion trap (LIT)-based SIM-stitching method for ultra-high-resolution Fourier transform mass spectrometry (FTMS) that increases the S/N over a wide m/z range compared to non-segmented wide full-scan (WFS) spectra. Here we described an improved segmented spectral scan stitching method that was based on quadrupole mass filter (QMF)-SIM, which overcame previous limitations of ion signal loss in LIT. This allowed for accurate representation of isotopologue distributions, both at natural abundance and in stable isotope-resolved metabolomics (SIRM)-based experiments. We also introduced a new spectral binning method that provided more precise and resolution-independent bins for irreversibly noise-suppressed FTMS spectra. We demonstrated a substantial improvement in S/N and sensitivity (typically > 10-fold) for 13C labeled lipid extracts of human macrophages grown as three-dimensional (3D) cell culture, with detection of an increased number of 13C isotopologue ions. The method also enabled analysis of extracts from very limited biological samples.


Assuntos
Lipídeos/análise , Macrófagos/química , Isótopos de Carbono/química , Análise de Fourier , Glucose/química , Glucose/metabolismo , Humanos , Marcação por Isótopo , Macrófagos/metabolismo , Espectrometria de Massas/métodos , Metabolômica/métodos , Esferoides Celulares/química , Esferoides Celulares/metabolismo
14.
Nat Commun ; 10(1): 3838, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444335

RESUMO

Developing biomimetic nanoparticles without loss of the integrity of proteins remains a major challenge in cancer chemotherapy. Here, we develop a biocompatible tumor-cell-exocytosed exosome-biomimetic porous silicon nanoparticles (PSiNPs) as drug carrier for targeted cancer chemotherapy. Exosome-sheathed doxorubicin-loaded PSiNPs (DOX@E-PSiNPs), generated by exocytosis of the endocytosed DOX-loaded PSiNPs from tumor cells, exhibit enhanced tumor accumulation, extravasation from blood vessels and penetration into deep tumor parenchyma following intravenous administration. In addition, DOX@E-PSiNPs, regardless of their origin, possess significant cellular uptake and cytotoxicity in both bulk cancer cells and cancer stem cells (CSCs). These properties endow DOX@E-PSiNPs with great in vivo enrichment in total tumor cells and side population cells with features of CSCs, resulting in anticancer activity and CSCs reduction in subcutaneous, orthotopic and metastatic tumor models. These results provide a proof-of-concept for the use of exosome-biomimetic nanoparticles exocytosed from tumor cells as a promising drug carrier for efficient cancer chemotherapy.


Assuntos
Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Exossomos/química , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Exocitose , Feminino , Humanos , Masculino , Camundongos , Nanopartículas/química , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Porosidade , Estudo de Prova de Conceito , Silício/química , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Chem Commun (Camb) ; 55(73): 10952-10955, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31441915

RESUMO

Triggering antibody-mediated innate immune mechanisms to kill cancer cells is an attractive therapeutic avenue. In this context, recruitment of endogenous antibodies to the cancer cell surface could be a viable alternative to the use of monoclonal antibodies. We report on antibody-recruiting polymers containing multiple antibody-binding hapten motifs and cyclooctynes that can covalently conjugate to azides introduced onto the glycocalyx of cancer cells by metabolic labeling with azido sugars.


Assuntos
Resinas Acrílicas/química , Anticorpos/imunologia , Azidas/metabolismo , Dinitrobenzenos/imunologia , Hexosaminas/metabolismo , Resinas Acrílicas/síntese química , Animais , Azidas/química , Linhagem Celular Tumoral , Química Click , Reação de Cicloadição , Ciclo-Octanos/síntese química , Ciclo-Octanos/química , Dinitrobenzenos/síntese química , Dinitrobenzenos/química , Fluorescência , Corantes Fluorescentes/química , Glicocálix/metabolismo , Hexosaminas/química , Humanos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Estudo de Prova de Conceito , Esferoides Celulares/metabolismo
16.
Nanoscale ; 11(35): 16488-16498, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31453605

RESUMO

Magnetic nanoparticles (MNPs) internalized within stem cells have paved the way for remote magnetic cell manipulation and imaging in regenerative medicine. A full understanding of their interactions with stem cells and of their fate in the intracellular environment is then required, in particular with respect to their surface coatings. Here, we investigated the biological interactions of MNPs composed of an identical magnetic core but coated with different molecules: phosphonoacetic acid, polyethylene glycol phosphonic carboxylic acid, caffeic acid, citric acid, and polyacrylic acid. These coatings vary in the nature of the chelating function, the number of binding sites, and the presence or absence of a polymer. The nanoparticle magnetism was systematically used as an indicator of their internalization within human stem cells and of their structural long-term biodegradation in a 3D stem cell spheroid model. Overall, we evidence that the coating impacts the aggregation status of the nanoparticles and subsequently their uptake within stem cells, but it has little effect on their intracellular degradation. Only a high number of chelating functions (polyacrylic acid) had a significant protective effect. Interestingly, when the nanoparticles aggregated prior to cellular internalization, less degradation was also observed. Finally, for all coatings, a robust dose-dependent intracellular degradation rate was demonstrated, with higher doses of internalized nanoparticles leading to a lower degradation extent.


Assuntos
Materiais Revestidos Biocompatíveis , Nanopartículas de Magnetita , Células-Tronco Mesenquimais , Esferoides Celulares , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacocinética , Materiais Revestidos Biocompatíveis/farmacologia , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/uso terapêutico , Nanopartículas de Magnetita/ultraestrutura , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Esferoides Celulares/metabolismo , Esferoides Celulares/ultraestrutura
17.
Eur Phys J E Soft Matter ; 42(8): 112, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31456065

RESUMO

Computational models aiming at the spatio-temporal description of cancer evolution are a suitable framework for testing biological hypotheses from experimental data, and generating new ones. Building on our recent work (J. Theor. Biol. 389, 146 (2016)) we develop a 3D agent-based model, capable of tracking hundreds of thousands of interacting cells, over time scales ranging from seconds to years. Cell dynamics is driven by a Monte Carlo solver, incorporating partial differential equations to describe chemical pathways and the activation/repression of "genes", leading to the up- or down-regulation of specific cell markers. Each cell-agent of different kind (stem, cancer, stromal etc.) runs through its cycle, undergoes division, can exit to a dormant, senescent, necrotic state, or apoptosis, according to the inputs from its systemic network. The basic network at this stage describes glucose/oxygen/ATP cycling, and can be readily extended to cancer-cell specific markers. Eventual accumulation of chemical/radiation damage to each cell's DNA is described by a Markov chain of internal states, and by a damage-repair network, whose evolution is linked to the cell systemic network. Aimed at a direct comparison with experiments of tumorsphere growth from stem cells, the present model will allow to quantitatively study the role of transcription factors involved in the reprogramming and variable radio-resistance of simulated cancer-stem cells, evolving in a realistic computer simulation of a growing multicellular tumorsphere.


Assuntos
Carcinogênese/metabolismo , Evolução Clonal , Modelos Teóricos , Esferoides Celulares/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Dano ao DNA , Glucose/metabolismo , Humanos , Cadeias de Markov , Oxigênio/metabolismo , Esferoides Celulares/patologia , Células Tumorais Cultivadas
18.
Oncol Rep ; 42(5): 2029-2038, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31432145

RESUMO

In vitro culture of patient­derived tumor cells offers many advantages in the development of novel therapies for colorectal cancer. Although various culture systems have been developed, the long­term expansion of patient­derived tumor cells remains challenging. The present results suggested that tumor cells isolated from colorectal cancer patient­derived xenografts can be efficiently immortalized in conditioned medium from irradiated feeder cells containing Y­27632, a rho­associated coiled­coil containing protein kinase (ROCK) inhibitor. Patient­derived tumor cells proliferated rapidly, reaching 90­95% confluence in ~6 days. Short tandem repeat analysis suggested that these tumor tissues and cultured cells presented 13 identical short tandem repeat loci, including Amelogenin, Penta E, Penta D, D2S1338 and D19S433. Their epithelial phenotype was confirmed by staining for epithelial cell adhesion molecule and cytokeratin 20, whereas vimentin was used as a mesenchymal marker. When cells were transferred to 3D cultures, they continued to proliferate, forming well­defined tumor spheroids. Expression levels of human telomerase reverse transcriptase and C­Myc mRNA were increased in cultured cells. Finally, immortalized cells were used for the screening of 65 anticancer drugs approved by the Food and Drug Administration, allowing the identification of gene­drug associations. In the present study, primary culture models of colorectal cancer were efficiently established using a ROCK inhibitor and feeder cells, and this approach could be used for personalized treatment strategies for patients with colorectal cancer.


Assuntos
Amidas/farmacologia , Neoplasias Colorretais/patologia , Células Alimentadoras/citologia , Cultura Primária de Células/métodos , Piridinas/farmacologia , Células Tumorais Cultivadas/citologia , Idoso , Animais , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Meios de Cultivo Condicionados/química , Feminino , Humanos , Masculino , Camundongos , Repetições de Microssatélites , Pessoa de Meia-Idade , Transplante de Neoplasias , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos
19.
Mol Biol Rep ; 46(5): 5103-5112, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31290055

RESUMO

The therapeutic application of recombinant proteins is limited due to their inherent structural complexity. Additionally, screening of therapeutic potential of protein products requires an appropriate testing platform to achieve biological relevance. Fabrication of three dimensional cultures bridges the gap between in vitro based monolayer cultures and clinical applications. In this perspective, glioblastoma U-87 MG and breast cancer MCF7 spheroids were generated to assess the therapeutic prospect of recombinant PTEN protein. PTEN bound to silver nanoclusters was encapsulated within PEG coating, which resulted in fabrication of spherical nanocarriers named as PTEN-nanocomposites. Internalization of PTEN-nanocomposites in the spheroids was confirmed by confocal microscopy. Upon uptake, PTEN-nanocomposites led to modulation of cyclins and apoptosis gene regulators culminating in cell cycle arrest and reduced cell viability as confirmed by calcein-AM/PI dual staining and alamar blue assay. Further, combination of tamoxifen and PTEN-nanocomposites on U-87 MG spheroids resulted in two-fold reduction of drug dosage. The study revealed that the monolayer culture results translated to the 3D culture as well, however higher dose of the recombinant PTEN was required for the spheroid system. The anti-proliferative role of PTEN-nanocomposites in a complex 3D environment augments its biological implication and paves the way for recombinant PTEN based therapeutic applications.


Assuntos
PTEN Fosfo-Hidrolase/farmacologia , Polietilenoglicóis/química , Esferoides Celulares/citologia , Tamoxifeno/farmacologia , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Nanopartículas Metálicas , Microscopia Confocal , Nanocompostos , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo
20.
J Agric Food Chem ; 67(28): 8007-8019, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31268702

RESUMO

Cow and human milk have been reported to contain dioxins ranging from 0.023 to 26.46 and 0.88 to 19 pg/g of fat, respectively. However, the toxic effects of the dioxins in the milk in this range of concentrations were not explored. Therefore, considering the outbred livestock tissues as better models than inbred laboratory animals, the present study targeted to study the effect of dioxins present in the milk on three-dimensionally (3D) cultured buffalo primary hepatocyte spheroids. The spheroids were treated with a model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), directly and also through milk fat at different concentrations (i.e, 0.02-20 pg/mL) for 24 h. Among the liver-cell-specific (ALB, HNF4α, and AFP) genes, a similar ALB and upregulated HNF4α expression at all treatments indicated the functional and transcriptionally active hepatocyte spheroids. Supportingly, no significant difference in the antiapoptotic gene expression between the treatments of milk fat and milk fat containing dioxins indicated the survivability of the spheroids during dioxin treatments. Among the selected TCDD responsive (CYP1A1, CYP1A2, AHR, CYP1B1, and TIPARP) genes, a nonsignificant increasing trend of the CYP1A1 expression was observed from 0.2 to 10 pg/mL of TCDD concentration through milk fat. This pattern was similar to the reported insensitive response of human primary hepatocytes toward dioxins than that of rat primary hepatocytes. This may indicate that the buffalo hepatocyte spheroids could be better models than rats for TCDD hepatotoxic studies. Further, TCDD in the milk in the range of 0.02-20 pg/mL concentration may not be very hepatotoxic.


Assuntos
Dioxinas/farmacologia , Hepatócitos/efeitos dos fármacos , Leite/química , Animais , Búfalos , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Dioxinas/análise , Contaminação de Alimentos/análise , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Modelos Animais , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo
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