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1.
Altern Lab Anim ; 50(6): 414-422, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36263982

RESUMO

The use of in vitro 3-D cell culture models in cancer research has yielded substantial gains in knowledge on various aspects of tumour biology. Such cell culture models could be useful in the study of head and neck squamous cell carcinoma (HNSCC), where mimicking intratumoral and intertumoral heterogeneity is especially challenging. Our research aims to establish 3-D spheroid models for HNSCC that reproduce in vitro the connections between tumour cells and the surrounding microenvironment. The aims of this study were to determine the optimal conditions for the culture and use of spheroids from HNSCC cell lines and optimal timepoint for using the spheroids obtained, to evaluate the effects of coculture with tumour-specific fibroblasts on spheroid formation, and to investigate spheroid responses to cisplatin treatment. Four HNSCC cell lines (UMSCC-11A, UMSCC-11B, UMSCC-22B and UD-SCC-01) were seeded in flat or round bottom well ultra-low attachment spheroid plates, and spheroid formation was evaluated. The HNSCC cell lines were then cocultured with stromal cells of the tumour microenvironment, producing an accelerated formation of dense spheroids. The viability of cells within the spheroids was assessed during cell culture by using a fluorescent dye. Our results suggest that: three out of the four cell lines tested could form usable spheroids with acceptable viability; the addition of stromal cells did not improve the number of viable cells; and the use of round bottom well plates supported the formation of a single spheroid, whereas flat bottom well plates led to the formation of multiple spheroids of different sizes.


Assuntos
Neoplasias de Cabeça e Pescoço , Animais , Carcinoma de Células Escamosas de Cabeça e Pescoço , Esferoides Celulares , Técnicas de Cultura de Células/métodos , Linhagem Celular , Microambiente Tumoral
2.
Laryngorhinootologie ; 100(1): 23-29, 2021 01.
Artigo em Alemão | MEDLINE | ID: mdl-33401320

RESUMO

An increasing amount of evidence suggests the existence of a stem cell-like population in head and neck squamous cell carcinoma (HNSCC). These cells have been termed cancer stem cells (CSC) due to the shared properties with somatic stem cells, such as the ability to self-renew and differentiate. Furthermore, the CSC are thought to be resistant to antineoplastic treatments and are therefore clinically relevant. As with somatic stem cells, CSC are thought to reside in a specialized supportive microenvironment, called the stem cell niche. One possible strategy to target the CSC could be through affecting functions of the stem cell niche.Stromal cell-derived factor-1 (SDF-1) is a multifunctional cytokine, which is secreted by e. g. stromal cells within the niche. SDF-1 is known to be the major regulator of stem cell trafficking between the niche and the peripheral vascular system. It elicits the chemotactic activity through interaction with a transmembrane receptor CXCR4, expressed by CSC. The SDF-1-CXCR4-axis is thought to play a crucial role in the interaction between CSC and their supportive cells in the tumor niche. A better understanding of these interactions could help in gaining further insight into the pathophysiology of progression/recurrence of malignant diseases and aid in finding new strategies for therapy.Specialized cell culture models are of advantage for deciphering the mechanisms of interaction between CSC and their niche. We anticipate that the recent technological advancements in bioprinting and the development of complex 3D cell culture model systems will contribute to our understanding of these mechanisms and to the establishment of individualized therapies.Here were provide an overview of the current knowledge on the CSC-tumor stem cell niche interactions in HNSCC with a focus on the SDF-1-CXCR4 axis.


Assuntos
Neoplasias de Cabeça e Pescoço , Nicho de Células-Tronco , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Recidiva Local de Neoplasia , Células-Tronco Neoplásicas , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Microambiente Tumoral
3.
Exp Cell Res ; 338(2): 162-9, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26410556

RESUMO

BACKGROUND: Fibrotic diseases encompass numerous systemic and organ-specific disorders characterized by the development and persistence of myofibroblasts. TGFß1 is considered the key inducer of fibrosis and drives myofibroblast differentiation in cells of diverse histological origin by a pro-oxidant shift in redox homeostasis associated with decreased nitric oxide (NO)/cGMP signaling. Thus, enhancement of NO/cGMP represents a potential therapeutic strategy to target myofibroblast activation and therefore fibrosis. METHODS: Myofibroblast differentiation was induced by TGFß1 in human primary prostatic (PrSCs) and normal dermal stromal cells (NDSCs) and monitored by α smooth muscle cell actin (SMA) and IGF binding protein 3 (IGFBP3) mRNA and protein levels. The potential of enhanced cGMP production by the sGC stimulator BAY 41-2272 or the sGC activator BAY 60-2770 to inhibit and revert myofibroblast differentiation in vitro was analyzed. Moreover, potential synergisms of BAY 41-2272 or BAY 60-2770 and inhibition of cGMP degradation by the PDE5 inhibitor vardenafil were investigated. RESULTS: BAY 41-2272 and BAY 60-2770 at doses of 30µM significantly inhibited induction of SMA and IGFBP3 levels in PrSCs and reduced myofibroblast marker levels in TGFß1-predifferentiated cells. At lower concentrations (3 and 10µM) only BAY 41-2272 but not BAY 60-2770 significantly inhibited and reverted myofibroblast differentiation. In NDSCs both substances significantly inhibited differentiation at all concentrations tested. Attenuation of SMA expression was more pronounced in NDSCs whereas reduction of IGFBP3 levels by BAY 41-2272 appeared more efficient in PrSCs. Moreover, administration of BAY 41-2272 or BAY 60-2770 enhanced the efficiency of the PDE5 inhibitor vardenafil to inhibit and revert myofibroblast differentiation in vitro. CONCLUSIONS: Increase of cGMP by sGC stimulation/activation significantly inhibited and reverted myofibroblast differentiation. This effect was even more pronounced when a combination treatment with a PDE5 inhibitor was applied. Thus, enhancement of NO/cGMP-signaling by sGC stimulation/activation is a promising strategy for the treatment of fibrotic diseases. Whereas, in NDSCs BAY 60-2770 and BAY 41-2272 exerted similar effects on myofibroblast differentiation, higher potency of BAY 41-2272 was observed in PrSCs, indicating phenotypical differences between fibroblasts form different organs that should be taken into account in the search for antifibrotic therapies.


Assuntos
Diferenciação Celular/fisiologia , Guanilato Ciclase/metabolismo , Miofibroblastos/metabolismo , Próstata/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Estromais/metabolismo , Actinas/metabolismo , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibrose/metabolismo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miofibroblastos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Próstata/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Guanilil Ciclase Solúvel , Células Estromais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
4.
Nanomedicine ; 12(3): 823-833, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26654993

RESUMO

Biofunctionalized scaffold facilitates complete healing of large defects. Biological constraints are induction and ingrowth of vessels. Angiogenic growth factors such as vascular endothelial growth factor or angiopoietin-1 can be bound to nano-scaled diamond particles. Corresponding bioactivities need to be examined after biofunctionalization. We therefore determined the physisorptive capacity of distinctly manufactured, differently sized nDP and the corresponding activities of bound factors. The properties of biofunctionalized nDPs were investigated on cultivated human mesenchymal stem cells and on the developing chicken embryo chorio-allantoic membrane. Eventually porous bone substitution material was coated with nDP to generate an interface that allows biofactor physisorption. Angiopoietin-1 was applied shortly before scaffold implantation into an osseous defect in sheep calvaria. Biofunctionalized scaffolds exhibited significantly increased rates of angiogenesis already one month after implantation. Conclusively, nDP can be used to ease functionalization of synthetic biomaterials. FROM THE CLINICAL EDITOR: With the advances in nanotechnology, many nano-sized materials have been used in the biomedical field. This is also true for nano-diamond particles (nDP). In this article, the authors investigated the physical properties of functionalized nano-diamond particles in both in-vitro and in-vivo settings. The positive findings would help improve understanding of these nanomaterials in regenerative medicine.


Assuntos
Indutores da Angiogênese/farmacologia , Angiopoietina-1/farmacologia , Diamante/química , Nanoestruturas/química , Neovascularização Fisiológica , Alicerces Teciduais/química , Fator A de Crescimento do Endotélio Vascular/farmacologia , Adsorção , Indutores da Angiogênese/química , Angiopoietina-1/química , Animais , Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Embrião de Galinha , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Nanoestruturas/ultraestrutura , Neovascularização Fisiológica/efeitos dos fármacos , Ovinos , Engenharia Tecidual , Fator A de Crescimento do Endotélio Vascular/química
5.
BMC Cancer ; 15: 738, 2015 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-26483043

RESUMO

BACKGROUND: Novel synthesized analogs of Aplidin, PM01215 and PM02781, were tested for antiangiogenic effects on primary human endothelial cells in vitro and for inhibition of angiogenesis and tumor growth in vivo. METHODS: Antiangiogenic activity of both derivatives was evaluated by real-time cell proliferation, capillary tube formation and vascular endothelial growth factor (VEGF)-induced spheroid sprouting assays. Distribution of endothelial cells in the different phases of the cell cycle was analyzed by flow cytometry. Aplidin analogs were tested in vivo in chicken chorioallantoic membrane (CAM) assays. RESULTS: Both derivatives inhibited angiogenic capacities of human endothelial cells (HUVECs) in vitro at low nanomolar concentrations. Antiangiogenic effects of both analogs were observed in the CAM. In addition, growth of human multiple myeloma xenografts in vivo in CAM was significantly reduced after application of both analogs. On the molecular level, both derivatives induced cell cycle arrest in G1 phase. This growth arrest of endothelial cells correlated with induction of the cell cycle inhibitor p16(INK4A) and increased senescence-associated beta galactosidase activity. In addition, Aplidin analogs induced oxidative stress and decreased production of the vascular maturation factors Vasohibin-1 and Dickkopf-3. CONCLUSIONS: From these findings we conclude that both analogs are promising agents for the development of antiangiogenic drugs acting independent on classical inhibition of VEGF signaling.


Assuntos
Bortezomib/farmacologia , Depsipeptídeos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Antineoplásicos/farmacologia , Western Blotting , Ciclo Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Microscopia Confocal , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Estresse Oxidativo , Peptídeos Cíclicos , Gravidez , Células Tumorais Cultivadas
6.
Cells ; 13(2)2024 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-38247833

RESUMO

Tissue engineering (TE) techniques offer solutions for tissue regeneration but require large quantities of cells. For microtia patients, TE methods represent a unique opportunity for therapies with low donor-site morbidity and reliance on the surgeon's individual expertise. Microtia-derived chondrocytes and perichondrocytes are considered a valuable cell source for autologous reconstruction of the pinna. The aim of this study was to investigate the suitability of perichondrocytes from microtia patients for autologous reconstruction in comparison to healthy perichondrocytes and microtia chondrocytes. Perichondrocytes were isolated via two different methods: explant culture and enzymatic digestion. The isolated cells were analyzed in vitro for their chondrogenic cell properties. We examined migration activity, colony-forming ability, expression of mesenchymal stem cell markers, and gene expression profile. We found that microtic perichondrocytes exhibit similar chondrogenic properties compared to chondrocytes in vitro. We investigated the behavior in three-dimensional cell cultures (spheroids and scaffold-based 3D cell cultures) and assessed the expression of cartilage-specific proteins via immunohistochemistry, e.g., collagen II, which was detected in all samples. Our results show that perichondrocytes from microtia patients are comparable to healthy perichondrocytes and chondrocytes in terms of chondrogenic cell properties and could therefore be a promising cell source for auricular reconstruction.


Assuntos
Microtia Congênita , Células-Tronco Mesenquimais , Humanos , Condrócitos , Condrogênese , Nível de Saúde
7.
Biomedicines ; 11(9)2023 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-37761029

RESUMO

Microtia is a congenital condition of abnormal development of the outer ear. Tissue engineering of the ear is an alternative treatment option for microtia patients. However, for this approach, the identification of high regenerative cartilage progenitor cells is of vital importance. Raman analysis provides a novel, non-invasive, label-free diagnostic tool to detect distinctive biochemical features of single cells or tissues. Using micro-Raman spectroscopy, we were able to distinguish and characterize the particular molecular fingerprints of differentiated chondrocytes and perichondrocytes and their respective progenitors isolated from healthy individuals and microtia patients. We found that microtia chondrocytes exhibited lower lipid concentrations in comparison to healthy cells, thus indicating the importance of fat storage. Moreover, we suggest that collagen is a useful biomarker for distinguishing between populations obtained from the cartilage and perichondrium because of the higher spectral contributions of collagen in the chondrocytes compared to perichondrocytes from healthy individuals and microtia patients. Our results represent a contribution to the identification of cell markers that may allow the selection of specific cell populations for cartilage tissue engineering. Moreover, the observed differences between microtia and healthy cells are essential for gaining better knowledge of the cause of microtia. It can be useful for designing novel treatment options based on further investigations of the discovered biochemical substrate alterations.

8.
Int J Oncol ; 63(3)2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37503786

RESUMO

Although checkpoint inhibitors (CPI) have recently extended the treatment options and improved clinical response of advanced stage head and neck squamous cell carcinoma (HNSCC), treatment success remains unpredictable. Programmed cell death ligand­1 (PD­L1) is a key player in immunotherapy. Tumor cells, and exosomes derived therefrom, are carriers of PD­L1 and efficiently suppress immune responses. The aim of the present study was to analyze the influence of established therapies on PD­L1 expression of HNSCC cell lines and their exosomes. The HNSCC cell lines, UM­SCC­11B, UM­SCC­14C and UM­SCC­22C were treated with fractionated radiotherapy (RT; 5x2 Gy), cisplatin (CT) and cetuximab (Cetux) as monotherapy, or combined therapy, chemoradiotherapy (CRT; RT and CT) or radioimmunotherapy (RT and Cetux). The expression of PD­L1 and phosphorylated (p)ERK1/2 as a mediator of radioresistance were assessed using western blotting, immunohistochemistry and an ex vivo vital tissue culture model. Additionally, exosomes were isolated from concentrated supernatants of the (un­)treated HNSCC cell lines by size exclusion chromatography. Exosomal protein expression levels of PD­L1 were detected using western blotting and semi­quantitative levels were calculated. The functional impact of exosomes from the (un­)treated HNSCC cell lines on the proliferation (MTS assay) and apoptosis (Caspase 3/7 assay) of the untreated HNSCC cell lines were measured and compared. The HNSCC cell lines UM­SCC­11B and UM­SCC­22B showed strong expression of pERK1/2 and PD­L1, respectively. RT upregulated the PD­L1 expression in UM­SCC­11B and UM­SCC­14C and in exosomes from all three cell lines. CT alone induced PD­L1 expression in all cell lines. CRT induced the expression of PD­L1 in all HNSCC cell lines and exosomes from UM­SCC­14C and UM­SCC­22B. The data indicated a potential co­regulation of PD­L1 and activated ERK1/2, most evident in UM­SCC­14C. Exosomes from irradiated UM­SCC­14C cells protected the unirradiated cells from apoptosis by Caspase 3/7 downregulation. The present study suggested a tumor cell­mediated regulation of PD­L1 upon platinum­based CRT in HNSCC and in exosomes. A co­regulation of PD­L1 and MAPK signaling response was hypothesized.


Assuntos
Exossomos , Neoplasias de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Caspase 3/metabolismo , Exossomos/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Cetuximab/farmacologia , Cisplatino/farmacologia , Linhagem Celular Tumoral
9.
Tissue Eng Part B Rev ; 28(3): 531-541, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-33966486

RESUMO

The clinical relevance of perichondrium was recognized more than a century ago. In children and adolescents, perichondrium is essential for the formation and growth of the cartilaginous part of craniofacial features and must be considered during reconstructive surgery in the head and neck area. Also in adults, perichondrium must be preserved during surgical intervention for adequate postoperative healing and cartilage maintenance. Furthermore, the regenerative function of perichondrium in the ribs enables the harvesting of the rib cartilage tissue for reconstruction of craniofacial features. With the advancement of tissue engineering, renewed attention has been focused on the perichondrium, because without this crucial tissue, the function of cartilage engineered for craniofacial reconstruction is incomplete and may not be suitable for long-term reconstructive goals. Furthermore, interest in the perichondrium was revived owing to its possible role as a microenvironment containing stem and progenitor cells. Here we will revisit seminal studies on the perichondrium and review the current literature to provide a holistic perspective on the importance of this tissue in the context of regenerative medicine. We will also highlight the functional significance of perichondrium for cartilage tissue engineering. Impact statement All adult cartilage tissues, with the exception of articular and fibrocartilage, are lined by a stratified tissue called the perichondrium. The perichondrium contributes to growth, structural stability, and regeneration and maintenance of the organ, but the cellular mechanisms underlying these processes are not well understood. This review provides a comprehensive summary of past and recent studies on perichondrium from the vantage point of tissue engineering and regenerative medicine. Of particular relevance is the evidence that perichondrium might contain chondrogenic progenitor cells. Cartilage tissue engineering holds great promise for novel treatments of craniofacial defects, and a better understanding of the function and structure of the perichondrium could contribute to improved therapies for head and neck reconstructive surgery and beyond.


Assuntos
Cartilagem , Engenharia Tecidual , Adolescente , Adulto , Criança , Condrogênese , Humanos , Medicina Regenerativa , Células-Tronco
10.
Int J Oncol ; 61(1)2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35642667

RESUMO

Immunotherapy has evolved into a powerful tool in the fight against a number of types of cancer, including head and neck squamous cell carcinomas (HNSCC). Although checkpoint inhibition (CPI) has definitely enriched the treatment options for advanced stage HNSCC during the past decade, the percentage of patients responding to treatment is widely varying between 14­32% in second­line setting in recurrent or metastatic HNSCC with a sporadic durability. Clinical response and, consecutively, treatment success remain unpredictable in most of the cases. One potential factor is the expression of target molecules of the tumor allowing cancer cells to acquire therapy resistance mechanisms. Accordingly, analyzing and modeling the complexity of the tumor microenvironment (TME) is key to i) stratify subgroups of patients most likely to respond to CPI and ii) to define new combinatorial treatment regimens. Particularly in a heterogeneous disease such as HNSCC, thoroughly studying the interactions and crosstalking between tumor and TME cells is one of the biggest challenges. Sophisticated 3D models are therefore urgently needed to be able to validate such basic science hypotheses and to test novel immuno­oncologic treatment regimens in consideration of the individual biology of each tumor. The present review will first summarize recent findings on immunotherapy, predictive biomarkers, the role of the TME and signaling cascades eliciting during CPI. Second, it will highlight the significance of current promising approaches to establish HNSCC 3D models for new immunotherapies. The results are encouraging and indicate that data obtained from patient­specific tumors in a dish might be finally translated into personalized immuno­oncology.


Assuntos
Neoplasias de Cabeça e Pescoço , Inibidores de Checkpoint Imunológico , Biomarcadores , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Microambiente Tumoral
11.
Oncol Lett ; 23(6): 177, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35464304

RESUMO

Epidermal growth factor receptor (EGFR) upregulation is a typical characteristic of head and neck squamous cell carcinoma (HNSCC). However, tyrosine kinase inhibitors have not yet been able to achieve enough therapeutic benefit in clinical trials to justify their use in standard therapy regimens. At present, little is known about the reasons for this treatment failure. In the present study, the HNSCC cell lines UM-SCC-11B and UM-SCC-22B were tested for their response to tyrosine kinase inhibitors (TKI) under 2D and 3D cell culture conditions. Absorption and luciferase-based viability assays were used for this, as well as optical evaluation via fluorescence microscopy. In addition, EGFR and HER3 expression as well as the downstream signalling pathways PI3K/AKT/mTOR and RAS/RAF/MEK/ERK were investigated using western blotting. Cell line UM-SCC-11B revealed a strong resistance to lapatinib under 3D cell culture conditions, while a good response to TKI therapy was observed under 2D cell culture conditions. An associated overexpression of phosphorylated HER3 under 3D cell culture conditions offered a plausible explanation for the altered treatment response. The results of the present study represent an idea of how signalling mechanisms of cancer cells can be changed using different cell culture methods. Overall, 3D cell culture could be an important component in the analysis of resistance mechanisms in cancer therapy.

12.
J Biomed Mater Res A ; 110(5): 1021-1035, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34967101

RESUMO

Decellularized extracellular matrices (DECM) are among the most common types of materials used in tissue engineering due to their cell instructive properties, biodegradability, and accessibility. Particularly in cartilage, a natural collagen type II matrix can be a promising means to provide the necessary cues and support for chondrogenic stem and progenitor cells (CSPCs). However, efficient remodeling of the transplanted DECM is largely dependent on the host immune response, with macrophages playing the central role in orchestrating both inflammatory and regenerative processes. Here we assessed the reaction of human primary macrophages to the cartilage DECM. Our findings show that the xenogeneic collagen matrix can elicit a mixed response in human macrophages, whereby the inflammatory response (M1) and the activation of remodeling (M2) type of macrophages are both present. Additionally, we demonstrate the inhibitory effect of macrophage response on the migratory capacity of human CSPCs. We further show that the inflammatory reaction of macrophages to the cartilage DECM, as well as the resulting inhibitory effects on CSPC migration, can be attenuated by interleukin-4 (IL-4). Finally, we demonstrate that IL-4 can effectively bind the matrix, thereby modulating macrophage response by reducing the inflammatory reaction and inducing the M2 phenotype.


Assuntos
Matriz Extracelular , Interleucina-4 , Cartilagem/fisiologia , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-4/metabolismo , Regeneração , Engenharia Tecidual/métodos
13.
J Tissue Eng ; 13: 20417314221114423, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36158899

RESUMO

Nasal septum defects can currently only be reconstructed using autologous cartilage grafts. In this study, we examine the reconstruction of septal cartilage defects in a rabbit model using porcine decellularized nasal septal cartilage (DNSC) functionalized with recombinant platelet-derived growth factor-BB (PDFG-BB). The supportive function of the transplanted DNSC was estimated by the degree of septum deviation and shrinkage using magnetic resonance imaging (MRI). The biocompatibility of the transplanted scaffolds was evaluated by histology according to international standards. A study group with an autologous septal transplant was used as a reference. In situ regeneration of cartilage defects was assessed by histological evaluation 4 and 16 weeks following DNSC transplantation. A study group with non-functionalized DNSC was introduced for estimation of the effects of PDFG-BB functionalization. DNSC scaffolds provided sufficient structural support to the nasal septum, with no significant shrinkage or septal deviations as evaluated by the MRI. Biocompatibility analysis after 4 weeks revealed an increased inflammatory reaction of the surrounding tissue in response to DNSC as compared to the autologous transplants. The inflammatory reaction was, however, significantly attenuated after 16 weeks in the PDGF-BB group whereas only a slight improvement of the biocompatibility score was observed in the untreated group. In situ regeneration of septal cartilage, as evidenced by the degradation of the DNSC matrix and production of neocartilage, was observed in both experimental groups after 16 weeks but was more pronounced in the PDFG-BB group. Overall, DNSC provided structural support to the nasal septum and stimulated in situ regeneration of the cartilage tissue. Furthermore, PDFG-BB augmented the regenerative potential of DNSC and enhanced the healing process, as demonstrated by reduced inflammation after 16 weeks.

14.
Oncol Rep ; 48(3)2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35856431

RESUMO

Increased submaxillary gland androgen­regulated protein 3A (SMR3A) expression was previously shown to serve as an independent risk factor for oropharyngeal squamous cell carcinoma (OPSCC) and as a surrogate biomarker for active estrogen receptor 2 signaling in radioresistant tumor cells. In the present study, it was aimed to unravel the expression and clinical significance of another member of the opiorphin family, opiorphin prepropeptide (OPRPN), in the radiotherapy for head and neck squamous cell carcinoma (HNSCC). Expression of SMR3A and OPRPN were analyzed for the prior and post fractionated irradiation (4x2 Gy) by double immunofluorescence staining in established HNSCC cell lines as well as by immunohistochemical (IHC) staining in ex vivo tumor tissues. Next, in a retrospective experimental cohort study, primary tumor samples from OPSCC patients (n=96), who received definitive surgery and adjuvant radiotherapy were reviewed, and expression levels of OPRPN protein were detected by IHC. Immunoreactivity scores (IRS) were associated with pathological and clinical risk factors by Chi­square analysis. Survival analysis was performed by using the Kaplan­Meier plot, log­rank test and Cox regression analysis. The expression levels of OPRPN and SMR3A protein were both induced by fractionated irradiation in vitro and ex vivo. In primary tumor samples, IRS of OPRPN was significantly higher than scores of SMR3A expression and positively correlated with expression patterns of SMR3A. SMR3A was confirmed to serve as an unfavorable factor, while OPRPN protein had no significant association with the clinical outcome of patients with OPSCC. A combinational analysis revealed that the subgroup with SMR3AhighOPRPNlow staining pattern had the worst clinical outcome among the various subgroups. Multivariate Cox regression analyses indicated that high expression of SMR3A serves as an independent unfavorable biomarker, while increased expression of OPRPN appears to exert protective function. In summary, the present study indicated that SMR3A and OPRPN serve as potential prognostic markers for HNSCC after radiotherapy.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Androgênios , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Estudos de Coortes , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Prognóstico , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Glândula Submandibular/química , Glândula Submandibular/metabolismo , Glândula Submandibular/patologia
15.
J Tissue Eng Regen Med ; 16(1): 36-50, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34687154

RESUMO

Lesions of aural, nasal and tracheal cartilage are frequently reconstructed by complex surgeries which are based on harvesting autologous cartilage from other locations such as the rib. Cartilage tissue engineering (CTE) is regarded as a promising alternative to attain vital cartilage. Nevertheless, CTE with nearly natural properties poses a significant challenge to research due to the complex reciprocal interactions between cells and extracellular matrix which have to be imitated and which are still not fully understood. Thus, we used a custom-made glass bioreactor to enhance cell migration into decellularized porcine cartilage scaffolds (DECM) and mimic physiological conditions. The DECM seeded with human nasal chondrocytes (HPCH) were cultured in the glass reactor for 6 weeks and examined by histological and immunohistochemical staining, biochemical analyses and real time-PCR at 14, 28 and 42 days. The migration depth and the number of migrated cells were quantified by computational analysis. Compared to the static cultivation, the dynamic culture (DC) fostered migration of HPCH into deeper tissue layers. Furthermore, cultivation in the bioreactor enhanced differentiation of the cells during the first 14 days, but differentiation diminished in the course of further cultivation. We consider the DC in the presented bioreactor as a promising tool to facilitate CTE and to help to better understand the complex physiological processes during cartilage regeneration. Maintaining differentiation of chondrocytes and improving cellular migration by further optimizing culture conditions is an important prerequisite for future clinical application.


Assuntos
Condrogênese , Alicerces Teciduais , Animais , Cartilagem , Movimento Celular , Condrócitos , Matriz Extracelular , Suínos , Engenharia Tecidual , Alicerces Teciduais/química
16.
Blood ; 114(18): 3960-7, 2009 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-19713465

RESUMO

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.


Assuntos
Inibidores da Angiogênese/farmacologia , Ácidos Borônicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Pirazinas/farmacologia , Animais , Bortezomib , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Chaperona BiP do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Front Cell Dev Biol ; 9: 666515, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34307351

RESUMO

Despite the current progress in the development of new concepts of precision medicine for head and neck squamous cell carcinoma (HNSCC), in particular targeted therapies and immune checkpoint inhibition (CPI), overall survival rates have not improved during the last decades. This is, on the one hand, caused by the fact that a significant number of patients presents with late stage disease at the time of diagnosis, on the other hand HNSCC frequently develop therapeutic resistance. Distinct intratumoral and intertumoral heterogeneity is one of the strongest features in HNSCC and has hindered both the identification of specific biomarkers and the establishment of targeted therapies for this disease so far. To date, there is a paucity of reliable preclinical models, particularly those that can predict responses to immune CPI, as these models require an intact tumor microenvironment (TME). The "ideal" preclinical cancer model is supposed to take both the TME as well as tumor heterogeneity into account. Although HNSCC patients are frequently studied in clinical trials, there is a lack of reliable prognostic biomarkers allowing a better stratification of individuals who might benefit from new concepts of targeted or immunotherapeutic strategies. Emerging evidence indicates that cancer stem cells (CSCs) are highly tumorigenic. Through the process of stemness, epithelial cells acquire an invasive phenotype contributing to metastasis and recurrence. Specific markers for CSC such as CD133 and CD44 expression and ALDH activity help to identify CSC in HNSCC. For the majority of patients, allocation of treatment regimens is simply based on histological diagnosis and on tumor location and disease staging (clinical risk assessments) rather than on specific or individual tumor biology. Hence there is an urgent need for tools to stratify HNSCC patients and pave the way for personalized therapeutic options. This work reviews the current literature on novel approaches in implementing three-dimensional (3D) HNSCC in vitro and in vivo tumor models in the clinical daily routine. Stem-cell based assays will be particularly discussed. Those models are highly anticipated to serve as a preclinical prediction platform for the evaluation of stable biomarkers and for therapeutic efficacy testing.

18.
BMC Cancer ; 9: 284, 2009 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-19682397

RESUMO

BACKGROUND: The murine homologue of human vasohibin (mVASH1), a putative antiangiogenic protein, was investigated for its effects on in vitro and in vivo angiogenesis. METHODS: Cell growth and migration were analyzed in murine fibroblasts, smooth muscle cells and endothelial cells. Angiogenic sprouting was studied in human umbilical vein endothelial cells (HUVECs) in the spheroid sprouting assay. In vivo effects on blood vessel formation were investigated in the chorioallantoic membrane (CAM) assay and in the C57BL/6 melanoma xenograft model. RESULTS: Purified murine and human VASH1 protein induced apoptosis of murine fibroblasts in vitro, but not of vascular aortic smooth muscle cells (AoSMC) or endothelial cells. Adenoviral overexpression of murine and human VASH1 inhibited capillary sprouting of HUVECs in the spheroid assay. Administration of recombinant murine and human VASH1 inhibited growth of large vessels in the CAM assay and promoted the formation of a dense, fine vascular network. Murine VASH1-overexpressing B16F10 melanomas displayed a reduction in large vessels and vascular area. Moreover, tumors showed more microvessels that stained positive for the mural cell markers alpha-smooth muscle cell actin (ASMA) and proteoglycan (NG2). CONCLUSION: Our data imply that murine VASH1 causes angiogenic remodelling by inhibiting angiogenic sprouting and large vessel growth, thereby supporting the formation of a vascular bed consisting predominantly of mature microvessels.


Assuntos
Inibidores da Angiogênese/metabolismo , Vasos Sanguíneos/crescimento & desenvolvimento , Proteínas de Ciclo Celular/metabolismo , Membrana Corioalantoide/irrigação sanguínea , Regulação para Baixo , Neovascularização Fisiológica , Inibidores da Angiogênese/genética , Animais , Vasos Sanguíneos/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica , Esferoides Celulares , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Arterioscler Thromb Vasc Biol ; 28(3): 478-84, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18187668

RESUMO

OBJECTIVE: In this study, the alternative splicing product of vasohibin 1 (VASH1B) was analyzed in direct comparison to the major isoform (VASH1A) for antiangiogenic effects on endothelial colony forming cells (ECFCs) from peripheral blood and on human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Expression studies in primary human endothelial cells revealed that both vasohibin proteins, hVASH1A and hVASH1B, localized in the nucleus and cytoplasm. Adenoviruses carrying the cDNA for VASH1A/B and purified recombinant proteins were used to study the function of both molecules in ECFCs and HUVECs. Recombinant VASH1A protein did not inhibit cell proliferation, tube formation, or vessel growth in vivo in the chick chorioallantoic membrane (CAM) assay, but promoted endothelial cell migration in vitro. The VASH1B protein had an inhibitory effect on cell proliferation, migration, tube formation, and inhibited blood vessel formation in the CAM assay. Adenoviral overexpression of VASH1B, but not of VASH1A, resulted in inhibition of endothelial cell growth, migration, and capillary formation. Interestingly, overexpression of VASH1A and B induced apoptosis in proliferating human fibroblasts, but did not affect cell growth of keratinocytes. CONCLUSIONS: Our data point out that alternative splicing of the VASH1 pre-mRNA transcript generates a potent antiangiogenic protein.


Assuntos
Processamento Alternativo , Inibidores da Angiogênese/genética , Capilares/citologia , Proteínas de Ciclo Celular/genética , Células Endoteliais/citologia , Neovascularização Fisiológica/genética , Adulto , Inibidores da Angiogênese/farmacologia , Western Blotting , Capilares/fisiologia , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Neovascularização Fisiológica/efeitos dos fármacos , Probabilidade , RNA Mensageiro/análise , Proteínas Recombinantes , Veias Umbilicais/citologia
20.
Front Oncol ; 9: 1530, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32039016

RESUMO

Background: Mechanisms mediating resistance against the proteasome inhibition by bortezomib (BTZ) in multiple myeloma (MM) cells are still unclear. We analyzed the activation of the unfolded protein response (UPR), induction of prosurvival, and apoptotic pathways after proteasome inhibition in BTZ-sensitive and -resistant cells. Thereafter, these findings from tissue culture were proofed on MM cells of BTZ-sensitive and BTZ-refractory patients. Methods: Proteasomal and ABC transporter activities were measured in sensitive and resistant cell lines by the use of the respective substrates. TP53 gene loss and mutations were determined by cytogenetics and targeted NGS. UPR pathways, proteasome subunit levels and protein secretion were studied by Western Blot analysis, and apoptosis was determined by flow cytometry. MM cell lines were stably transfected with inducible GRP78 expression to study unfolded protein expression. Transient knock-down of GRP78 was done by RNA interference. Splicing of XBP1 and expression of GRP78 was studied by real-time PCR in CD138-enriched MM primary cells of BTZ-refractory and -sensitive patients. Results: BTZ-sensitive cells displayed lower basal proteasomal activities. Similar activities of all three major ABC transporter proteins were detected in BTZ-sensitive and resistant cells. Sensitive cells showed deficiencies in triggering canonical prosurvival UPR provoked by endoplasmic reticulum (ER) stress induction. BTZ treatment did not increase unfolded protein levels or induced GRP78-mediated UPR. BTZ-resistant cells and BTZ-refractory patients exhibited lower sXBP1 levels. Apoptosis of BTZ-sensitive cells was correlating with induction of p53 and NOXA. Tumor cytogenetics and NGS analysis revealed more frequent TP53 deletions and mutations in BTZ-refractory MM patients. Conclusions: We identified low sXBP1 levels and TP53 abnormalities as factors correlating with bortezomib resistance in MM. Therefore, determination of sXBP1 levels and TP53 status prior to BTZ treatment in MM may be beneficial to predict BTZ resistance.

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