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1.
Langmuir ; 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32216370

RESUMO

Maintaining the integrity of cell membranes is indispensable for cellular viability. Poloxamer 188 (P188), a poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer with a number-average molecular weight of 8700 g/mol and containing 80% by mass PEO, protects cell membranes from various external injuries and has the potential to be used as a therapeutic agent in diverse applications. The membrane protection mechanism associated with P188 is intimately connected with how this block copolymer interacts with the lipid bilayer, the main component of a cell membrane. Here, we report the distribution of P188 in a model lipid bilayer comprising 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) using neutron reflectivity (NR) and atomic force microscopy (AFM). We also investigated the association of a PEO homopolymer (PEO8.4K; Mn = 8400 g/mol) that does not protect living cell membranes. These experiments were conducted following incubation of a 4.5 mmol/L polymer solution in a buffer that mimics physiological conditions with supported POPC bilayer membranes followed by washing with the aqueous medium. In contrast to previous reports, which dealt with P188 and PEO in salt-free solutions, both P188 and PEO8.4K penetrate into the inner portion of the lipid bilayer as revealed by NR, with approximately 30% by volume occupancy across the membrane without loss of bilayer structural integrity. These results indicate that PEO is the chemical moiety that principally drives P188 binding to bilayer membranes. No defects or phase-separated domains were observed in either P188- or PEO8.4K-incubated lipid bilayers when examined by AFM, indicating that polymer chains mingle homogeneously with lipid molecules in the bilayer. Remarkably, the breakthrough force required for penetration of the AFM tip through the bilayer membrane is unaffected by the presence of the large amount of P188 and PEO8.4K.

2.
Ann Biomed Eng ; 48(4): 1230-1240, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31916125

RESUMO

Irreversible electroporation (IRE) is an emerging technology for non-thermal ablation of solid tumors. This study sought to integrate electrodes into microporous poly(caprolactone) (PCL) scaffolds previously shown to recruit metastasizing cancer cells in vivo in order to facilitate application of IRE to disseminating cancer cells. As the ideal parallel plate geometry would render much of the porous scaffold surface inaccessible to infiltrating cells, numerical modeling was utilized to predict the spatial profile of electric field strength within the scaffold for alternative electrode designs. Metal mesh electrodes with 0.35 mm aperture and 0.16 mm wire diameter established electric fields with similar spatial uniformity as the parallel plate geometry. Composite PCL-IRE scaffolds were fabricated by placing cylindrical porous PCL scaffolds between two PCL dip-coated stainless steel wire meshes. PCL-IRE scaffolds exhibited no difference in cell infiltration in vivo compared to PCL scaffolds. In addition, upon application of IRE in vivo, cells infiltrating the PCL-IRE scaffolds were successfully ablated, as determined by histological analysis 3 days post-treatment. The ability to establish homogeneous electric fields within a biomaterial that can recruit metastatic cancer cells, especially when combined with immunotherapy, may further advance IRE technology beyond solid tumors to the treatment of systemic cancer.

3.
Biomicrofluidics ; 13(6): 064119, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31768205

RESUMO

The blood-brain barrier (BBB) is composed of brain microvascular endothelial cells (BMECs) that regulate brain homeostasis, and astrocytes within the brain are involved in the maintenance of the BBB or modulation of its integrity in disease states via secreted factors. A major challenge in modeling the normal or diseased BBB is that conventional in vitro models lack either the physiological complexity of the BBB or key functional features such as formation of a sufficiently tight barrier. In this study, we utilized human induced pluripotent stem cell (hiPSC)-derived BMECs in a BBB-on-a-chip device that supports flow and coculture with an astrocyte-laden 3D hydrogel. The BMECs are separated from the hydrogel by a porous membrane with either 0.4 or 8.0 µm pore size, making the device suitable for studying the transport of molecules or cells, respectively, across the BBB. In addition, all cells seeded in the device are differentiated from the same hiPSC line, which could enable genetic and rare disease modeling. Formation of a confluent BMEC barrier was confirmed by immunocytochemistry of tight junction proteins and measurement of fluorescein permeability. Integrity of the barrier was further assessed by performing impedance spectroscopy in the device. Finally, the ability of this device to recapitulate a disease model of BBB disruption was demonstrated, with apical addition of TGF-ß1 leading to transendothelial electrical resistance reduction and indicators of astrocyte activation. These results demonstrate the utility of the fabricated device for a broad range of applications such as drug screening and mechanistic studies of BBB disruption.

4.
Biotechnol Bioeng ; 116(9): 2353-2363, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31038193

RESUMO

Local delivery of viral vectors can enhance the efficacy of therapies by selectively affecting necessary tissues and reducing the required vector dose. Pluronic F127 is a thermosensitive polymer that undergoes a solution-gelation (sol-gel) transition as temperature increases and can deliver vectors without damaging them. While pluronics can be spread over large areas, such as the surface of an organ, before gelation, they lack sufficient adhesivity to remain attached to some tissues, such as the surface of the heart or mucosal surfaces. Here, we utilized blends of pluronic F127 and polycarbophil (PCB), a mucoadhesive agent, to provide the necessary adhesivity for local delivery of viral vectors to the cardiac muscle. The effects of PCB concentration on adhesive properties, sol-gel temperature transition and cytocompatibility were evaluated. Rheological studies showed that PCB decreased the sol-gel transition temperature at concentrations >1% and increased the adhesive properties of the gel. Furthermore, these gels were able to deliver viral vectors and transduce cells in vitro and in vivo in a neonatal mouse apical resection model. These gels could be a useful platform for delivering viral vectors over the surface of organs where increased adhesivity is required.

5.
Int J Hyperthermia ; 36(1): 130-138, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30676126

RESUMO

Focal thermal therapy (Heat), cryosurgery (Cryo) and irreversible electroporation (IRE) are increasingly used to treat cancer. However, local recurrence and systemic spread are persistent negative outcomes. Nevertheless, emerging work with immunotherapies (i.e., checkpoint blockade or dendritic cell (DC) vaccination) in concert with focal therapies may improve outcomes. To understand the role of focal therapy in priming the immune system for immunotherapy, an in vitro model of T cell response after exposure to B16 melanoma cell lysates after lethal exposures was designed. Exposure included: Heat (50 °C, 30 min), Cryo (-80 °C, 30 min) and IRE (1250 V/cm, 99 pulses, 50 µs pulses with 1 Hz intervals). After viability assessment (CCK-8 assay), cell lysates were collected and assessed for protein release (BCA assay), protein denaturation (FTIR-spectroscopy), TRP-2 antigen release (western blot), and T cell activation (antigen-specific CD8 T cell proliferation). Results showed IRE released the most protein and antigen (TRP-2), followed by Cryo and Heat. In contrast, Cryo released the most native (not denatured) protein, compared to IRE and Heat. Finally, IRE dramatically outperformed both Cryo and Heat in T cell activation while Cryo modestly outperformed Heat. This study demonstrates that despite all focal therapies ability to destroy cells, the 'quantity' (i.e., amount) and 'quality' (i.e., molecular state) of tumor protein (including antigen) released can dramatically change the ensuing priming of the immune system. This suggests protein-based metrics whereby focal therapies can be designed to prime the immune system in concert with immunotherapies to eventually achieve improved and durable cancer treatment in vivo.


Assuntos
Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Neoplasias/imunologia , Animais , Humanos , Camundongos , Camundongos Transgênicos
6.
J Biol Eng ; 12: 12, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30127847

RESUMO

Background: While hypoxia has been well-studied in various tumor microenvironments, its role in cancer cell dormancy is poorly understood, in part due to a lack of well-established in vitro and in vivo models. Hypoxic conditions under conventional hypoxia chambers are relatively unstable and cannot be maintained during characterization outside the chamber since normoxic response is quickly established. To address this challenge, we report a robust in vitro cancer dormancy model under a hypoxia-mimicking microenvironment using cobalt chloride (CoCl2), a hypoxia-mimetic agent, which stabilizes hypoxia inducible factor 1-alpha (HIF1α), a major regulator of hypoxia signaling. Methods: We compared cellular responses to CoCl2 and true hypoxia (0.1% O2) in different breast cancer cell lines (MCF-7 and MDA-MB-231) to investigate whether hypoxic regulation of breast cancer dormancy could be mimicked by CoCl2. To this end, expression levels of hypoxia markers HIF1α and GLUT1 and proliferation marker Ki67, cell growth, cell cycle distribution, and protein and gene expression were evaluated under both CoCl2 and true hypoxia. To further validate our platform, the ovarian cancer cell line OVCAR-3 was also tested. Results: Our results demonstrate that CoCl2 can mimic hypoxic regulation of cancer dormancy in MCF-7 and MDA-MB-231 breast cancer cell lines, recapitulating the differential responses of these cell lines to true hypoxia in 2D and 3D. Moreover, distinct gene expression profiles in MCF-7 and MDA-MB-231 cells under CoCl2 treatment suggest that key cell cycle components are differentially regulated by the same hypoxic stress. In addition, the induction of dormancy in MCF-7 cells under CoCl2 treatment is HIF1α-dependent, as evidenced by the inability of HIF1α-suppressed MCF-7 cells to exhibit dormant behavior upon CoCl2 treatment. Furthermore, CoCl2 also induces and stably maintains dormancy in OVCAR-3 ovarian cancer cells. Conclusions: These results demonstrate that this CoCl2-based model could provide a widely applicable in vitro platform for understanding induction of cancer cell dormancy under hypoxic stress.

7.
Fluids Barriers CNS ; 15(1): 9, 2018 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-29615068

RESUMO

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene. 40% of X-ALD patients will convert to the deadly childhood cerebral form (ccALD) characterized by increased permeability of the brain endothelium that constitutes the blood-brain barrier (BBB). Mutation information and molecular markers investigated to date are not predictive of conversion. Prior reports have focused on toxic metabolic byproducts and reactive oxygen species as instigators of cerebral inflammation and subsequent immune cell invasion leading to BBB breakdown. This study focuses on the BBB itself and evaluates differences in brain endothelium integrity using cells from ccALD patients and wild-type (WT) controls. METHODS: The blood-brain barrier of ccALD patients and WT controls was modeled using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO-PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. RESULTS: iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 Ω cm2) compared to WT controls (5001 ± 172 Ω cm2). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO-PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. CONCLUSIONS: The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD.


Assuntos
Adrenoleucodistrofia/metabolismo , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Microvasos/metabolismo , Modelos Biológicos , Adrenoleucodistrofia/tratamento farmacológico , Adrenoleucodistrofia/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Permeabilidade Capilar , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Microvasos/efeitos dos fármacos , Microvasos/patologia , Fármacos Neuroprotetores/farmacologia , Polímeros/farmacologia , Transcriptoma
8.
Adv Healthc Mater ; 7(10): e1700903, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29521008

RESUMO

Primary tumor (PT) immune cells and pre-metastatic niche (PMN) sites are critical to metastasis. Recently, synthetic biomaterial scaffolds used as PMN mimics are shown to capture both immune and metastatic tumor cells. Herein, studies are performed to investigate whether the scaffold-mediated redirection of immune and tumor cells would alter the primary tumor microenvironment (TME). Transcriptomic analysis of PT cells from scaffold-implanted and mock-surgery mice identifies differentially regulated pathways relevant to invasion and metastasis progression. Transcriptomic differences are hypothesized to result from scaffold-mediated modulations of immune cell trafficking and phenotype in the TME. Culturing tumor cells with conditioned media generated from PT immune cells of scaffold-implanted mice decrease invasion in vitro more than two-fold relative to mock surgery controls and reduce activity of invasion-promoting transcription factors. Secretomic characterization of the conditioned media delineates interactions between immune cells in the TME and tumor cells, showing an increase in the pan-metastasis inhibitor decorin and a concomitant decrease in invasion-promoting chemokine (C-C motif) ligand 2 (CCL2) in scaffold-implanted mice. Flow cytometric and transcriptomic profiling of PT immune cells identify phenotypically distinct tumor-associated macrophages (TAMs) in scaffold-implanted mice, which may contribute to an invasion-suppressive TME. Taken together, this study demonstrates biomaterial scaffolds systemically influence metastatic progression through manipulation of the TME.


Assuntos
Materiais Biocompatíveis , Materiais Biomiméticos , Neoplasias da Mama/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Tecidos Suporte/química , Microambiente Tumoral , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Neoplasias da Mama/patologia , Rastreamento de Células , Quimiocina CCL2/metabolismo , Decorina/metabolismo , Feminino , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Transcriptoma
9.
Biomaterials ; 166: 27-37, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29533788

RESUMO

Currently, there are very few therapeutic options for treatment of metastatic disease, as it often remains undetected until the burden of disease is too high. Microporous poly(ε-caprolactone) biomaterials have been shown to attract metastasizing breast cancer cells in vivo early in tumor progression. In order to enhance the therapeutic potential of these scaffolds, they were modified such that infiltrating cells could be eliminated with non-invasive focal hyperthermia. Metal disks were incorporated into poly(ε-caprolactone) scaffolds to generate heat through electromagnetic induction by an oscillating magnetic field within a radiofrequency coil. Heat generation was modulated by varying the size of the metal disk, the strength of the magnetic field (at a fixed frequency), or the type of metal. When implanted subcutaneously in mice, the modified scaffolds were biocompatible and became properly integrated with the host tissue. Optimal parameters for in vivo heating were identified through a combination of computational modeling and ex vivo characterization to both predict and verify heat transfer dynamics and cell death kinetics during inductive heating. In vivo inductive heating of implanted, tissue-laden composite scaffolds led to tissue necrosis as seen by histological analysis. The ability to thermally ablate captured cells non-invasively using biomaterial scaffolds has the potential to extend the application of focal thermal therapies to disseminated cancers.


Assuntos
Materiais Biocompatíveis , Hipertermia Induzida , Tecidos Suporte , Animais , Humanos , Hipertermia Induzida/instrumentação , Hipertermia Induzida/métodos , Camundongos , Metástase Neoplásica/terapia , Neoplasias/patologia , Neoplasias/terapia , Engenharia Tecidual/métodos
10.
Tissue Eng Part C Methods ; 24(5): 289-299, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29478388

RESUMO

Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.


Assuntos
Criopreservação , Dimetil Sulfóxido/farmacologia , Congelamento , Células-Tronco Pluripotentes Induzidas/citologia , Análise de Célula Única , Amidas/química , Apoptose , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Gelo , Análise Espectral Raman
11.
Phys Med Biol ; 61(19): 6892-6904, 2016 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-27618507

RESUMO

In cancer biology, there has been a recent effort to understand tumor formation in the context of the tissue microenvironment. In particular, recent progress has explored the mechanisms behind how changes in the cell-extracellular matrix ensemble influence progression of the disease. The extensive use of in vitro tissue culture models in simulant matrix has proven effective at studying such interactions, but modalities for non-invasively quantifying aspects of these systems are scant. We present the novel application of an imaging technique, Inverse Spectroscopic Optical Coherence Tomography, for the non-destructive measurement of in vitro biological samples during matrix remodeling. Our findings indicate that the nanoscale-sensitive mass density correlation shape factor D of cancer cells increases in response to a more crosslinked matrix. We present a facile technique for the non-invasive, quantitative study of the micro- and nano-scale structure of the extracellular matrix and its host cells.


Assuntos
Proliferação de Células , Colágeno/metabolismo , Neoplasias do Colo/patologia , Reagentes para Ligações Cruzadas/metabolismo , Matriz Extracelular/patologia , Tomografia de Coerência Óptica/métodos , Neoplasias do Colo/metabolismo , Matriz Extracelular/metabolismo , Humanos , Células Tumorais Cultivadas
12.
Cancer Res ; 76(18): 5209-18, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27635043

RESUMO

The onset of distant organ metastasis from primary breast cancer marks the transition to a stage IV diagnosis. Standard imaging modalities often detect distant metastasis when the burden of disease is high, underscoring the need for improved methods of detection to allow for interventions that would impede disease progression. Here, microporous poly(ε-caprolactone) scaffolds were developed that capture early metastatic cells and thus serve as a sentinel for early detection. These scaffolds were used to characterize the dynamic immune response to the implant spanning the acute and chronic foreign body response. The immune cell composition had stabilized at the scaffold after approximately 1 month and changed dramatically within days to weeks after tumor inoculation, with CD11b(+)Gr1(hi)Ly6C(-) cells having the greatest increase in abundance. Implanted scaffolds recruited metastatic cancer cells that were inoculated into the mammary fat pad in vivo, which also significantly reduced tumor burden in the liver and brain. Additionally, cancer cells could be detected using a label-free imaging modality termed inverse spectroscopic optical coherence tomography, and we tested the hypothesis that subsequent removal of the primary tumor after early detection would enhance survival. Surgical removal of the primary tumor following cancer cell detection in the scaffold significantly improved disease-specific survival. The enhanced disease-specific survival was associated with a systemic reduction in the CD11b(+)Gr1(hi)Ly6C(-) cells as a consequence of the implant, which was further supported by Gr-1 depletion studies. Implementation of the scaffold may provide diagnostic and therapeutic options for cancer patients in both the high-risk and adjuvant treatment settings. Cancer Res; 76(18); 5209-18. ©2016 AACR.


Assuntos
Neoplasias da Mama/patologia , Metástase Neoplásica/patologia , Tecidos Suporte , Animais , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Poliésteres , Tomografia de Coerência Óptica
13.
Acta Biomater ; 33: 13-24, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26844426

RESUMO

UNLABELLED: Metastatic tumor cells colonize the pre-metastatic niche, which is a complex microenvironment consisting partially of extracellular matrix (ECM) proteins. We sought to identify and validate novel contributors to tumor cell colonization using ECM-coated poly(ε-caprolactone) (PCL) scaffolds as mimics of the pre-metastatic niche. Utilizing orthotopic breast cancer mouse models, fibronectin and collagen IV-coated scaffolds implanted in the subcutaneous space captured colonizing tumor cells, showing a greater than 2-fold increase in tumor cell accumulation at the implant site compared to uncoated scaffolds. As a strategy to identify additional ECM colonization contributors, decellularized matrix (DCM) from lungs and livers containing metastatic tumors were characterized. In vitro, metastatic cell adhesion was increased on DCM coatings from diseased organs relative to healthy DCM. Furthermore, in vivo implantations of diseased DCM-coated scaffolds had increased tumor cell colonization relative to healthy DCM coatings. Mass-spectrometry proteomics was performed on healthy and diseased DCM to identify candidates associated with colonization. Myeloperoxidase was identified as abundantly present in diseased organs and validated as a contributor to colonization using myeloperoxidase-coated scaffold implants. This work identified novel ECM proteins associated with colonization using decellularization and proteomics techniques and validated candidates using a scaffold to mimic the pre-metastatic niche. STATEMENT OF SIGNIFICANCE: The pre-metastatic niche consists partially of ECM proteins that promote metastatic cell colonization to a target organ. We present a biomaterials-based approach to mimic this niche and identify ECM mediators of colonization. Using murine breast cancer models, we implanted microporous PCL scaffolds to recruit colonizing tumor cells in vivo. As a strategy to modulate colonization, we coated scaffolds with various ECM proteins, including decellularized lung and liver matrix from tumor-bearing mice. After characterizing the organ matrices using proteomics, myeloperoxidase was identified as an ECM protein contributing to colonization and validated using our scaffold. Our scaffold provides a platform to identify novel contributors to colonization and allows for the capture of colonizing tumor cells for a variety of downstream clinical applications.


Assuntos
Matriz Extracelular/metabolismo , Metástase Neoplásica/patologia , Tecidos Suporte/química , Ensaio Tumoral de Célula-Tronco/métodos , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Materiais Revestidos Biocompatíveis/farmacologia , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Peroxidase/metabolismo , Poliésteres/farmacologia , Proteômica , Reprodutibilidade dos Testes , Sus scrofa
14.
Sci Rep ; 5: 17566, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26634905

RESUMO

Metastatic cell homing is a complex process mediated in part by diffusible factors secreted from immune cells found at a pre-metastatic niche. We report on connecting secretomics and TRanscriptional Activity CEll aRray (TRACER) data to identify functional paracrine interactions between immune cells and metastatic cells as novel mediators of homing. Metastatic breast cancer mouse models were used to generate a diseased splenocyte conditioned media (D-SCM) containing immune cell secreted factors. MDA-MB-231 metastatic cell activity including cell invasion, migration, transendothelial migration, and proliferation were increased in D-SCM relative to control media. Our D-SCM secretome analysis yielded 144 secreted factor candidates that contribute to increased metastatic cell activity. The functional mediators of homing were identified using MetaCore software to determine interactions between the immune cell secretome and the TRACER-identified active transcription factors within metastatic cells. Among the 5 candidate homing factors identified, haptoglobin was selected and validated in vitro and in vivo as a key mediator of homing. Our studies demonstrate a novel systems biology approach to identify functional signaling factors associated with a cellular phenotype, which provides an enabling tool that complements large-scale protein identification provided by proteomics.


Assuntos
Neoplasias da Mama/patologia , Fatores Imunológicos/metabolismo , Comunicação Parácrina/genética , Proteômica , Biologia de Sistemas , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Proliferação de Células/genética , Feminino , Humanos , Fatores Imunológicos/genética , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/imunologia , Metástase Neoplásica , Comunicação Parácrina/imunologia , Transdução de Sinais/genética
15.
Nat Commun ; 6: 8094, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26348915

RESUMO

Breast cancer is a leading cause of death for women, with mortality resulting from metastasis. Metastases are often detected once tumour cells affect the function of solid organs, with a high disease burden limiting effective treatment. Here we report a method for the early detection of metastasis using an implanted scaffold to recruit and capture metastatic cells in vivo, which achieves high cell densities and reduces the tumour burden within solid organs 10-fold. Recruitment is associated with infiltration of immune cells, which include Gr1(hi)CD11b(+) cells. We identify metastatic cells in the scaffold through a label-free detection system using inverse spectroscopic optical coherence tomography, which identifies changes to nanoscale tissue architecture associated with the presence of tumour cells. For patients at risk of recurrence, scaffold implantation following completion of primary therapy has the potential to identify metastatic disease at the earliest stage, enabling initiation of therapy while the disease burden is low.


Assuntos
Adenocarcinoma/diagnóstico , Materiais Biocompatíveis , Neoplasias da Mama/diagnóstico , Neoplasias Hepáticas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Células Neoplásicas Circulantes , Tecidos Suporte , Adenocarcinoma/secundário , Animais , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Detecção Precoce de Câncer , Feminino , Humanos , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Próteses e Implantes , Tomografia de Coerência Óptica , Carga Tumoral
16.
Sci Rep ; 4: 4160, 2014 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-24561821

RESUMO

Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction, and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm(2)). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.


Assuntos
Barreira Hematoencefálica/metabolismo , Modelos Biológicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Tretinoína/farmacologia , Astrócitos/citologia , Barreira Hematoencefálica/química , Encéfalo/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Condutividade Elétrica , Endotélio Vascular/citologia , Humanos , Células-Tronco Neurais/citologia , Neurônios/citologia , Pericitos/citologia , Células-Tronco Pluripotentes/citologia
17.
Biomaterials ; 35(6): 2024-31, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24309498

RESUMO

Biomaterial scaffolds are central to many tissue engineering strategies as they create a space for tissue growth and provide a support for cell adhesion and migration. However, biomaterial implantation results in unavoidable injury resulting in an inflammatory response, which can impair integration with the host and tissue regeneration. Toward the goal of reducing inflammation, we investigated the hypothesis that a lentiviral gene therapy-based approach to localized and sustained IL-10 expression at a scaffold could modulate the number, relative proportions, and cytokine production of infiltrating leukocyte populations. Flow cytometry was used to quantify infiltration of six leukocyte populations for 21 days following implantation of PLG scaffolds into intraperitoneal fat. Leukocytes with innate immune functions (i.e., macrophages, dendritic cells, neutrophils) were most prevalent at early time points, while T lymphocytes became prevalent by day 14. Reporter gene delivery indicated that transgene expression persisted at the scaffold for up to 28 days and macrophages were the most common leukocyte transduced, while transduced dendritic cells expressed the greatest levels of transgene. IL-10 delivery decreased leukocyte infiltration by 50% relative to controls, increased macrophage IL-10 expression, and decreased macrophage, dendritic cell, and CD4 T cell IFN-γ expression. Thus, IL-10 gene delivery significantly decreased inflammation following scaffold implant into the intraperitoneal fat, in part by modulating cytokine expression of infiltrating leukocytes.


Assuntos
Regulação da Expressão Gênica , Interleucina-10/metabolismo , Leucócitos/citologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Citometria de Fluxo , Terapia Genética , Imunomodulação/fisiologia , Masculino , Camundongos
18.
Nat Protoc ; 8(1): 162-75, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23257984

RESUMO

The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of ß-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening and cell-based therapeutic applications.


Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Via de Sinalização Wnt , Citometria de Fluxo , Humanos , Lentivirus/metabolismo , RNA Interferente Pequeno/metabolismo , beta Catenina/metabolismo
19.
Nat Biotechnol ; 30(8): 783-91, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22729031

RESUMO

The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover, because of its barrier properties, this endothelial interface restricts uptake of neurotherapeutics. Thus, a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues, including those involved in Wnt/ß-catenin signaling. The resulting endothelial cells have many BBB attributes, including well-organized tight junctions, appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably, they respond to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2), and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients.


Assuntos
Barreira Hematoencefálica/citologia , Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Endotélio Vascular/citologia , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Separação Celular/métodos , Técnicas de Cocultura/métodos , Humanos , Neurônios/citologia , Fenótipo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
20.
Proc Natl Acad Sci U S A ; 109(27): E1848-57, 2012 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-22645348

RESUMO

Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of ß-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of ß-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.


Assuntos
Técnicas de Cultura de Células/métodos , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Via de Sinalização Wnt/fisiologia , Diferenciação Celular/fisiologia , Meios de Cultura/farmacologia , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , RNA Interferente Pequeno/genética , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética
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