Simulating tumor microenvironment: changes in protein expression in an in vitro co-culture system.

BACKGROUND: The role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance, both for the enhanced understanding of fundamental cancer biology as well as for improving molecular diagnostics and therapeutics. The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells. METHODS: The HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24 h to 7 days. Cell morphology and organization were studied using phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloprotease 1 (MMP1) was investigated by Real Time PCR and Western Blotting. RESULTS: The results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured. CONCLUSIONS: We concluded that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity.

Novel PLA microspheres with hydrophilic and bioadhesive surfaces for the controlled delivery of fenretinide.

Novel polylactide (PLA) microspheres endowed with hydrophilic and bioadhesive surfaces as injectable formulations for the controlled release of fenretinide were prepared, using a novel technique based on the co-precipitation of PLA with gelatin, at the interface of a liquid dispersion formed by the addition of N-methylpyrrolidone containing PLA and dextrin (DX), towards an aqueous solution of gelatin (G). The resulting PLA-G-DX microspheres were compared with others prepared by the same technique using polylactide-co-glycolide (PLGA), with or without DX, and with or without phosphatidylcholine. Of the different systems, the PLA-G-DX microspheres had the best morphological, dimensional and functional characteristics. They had the highest drug loading, and their drug release was the most efficient over time without any burst effect. Their in vitro anti-tumoural activity was strongly enhanced with respect to the pure fenretinide. This paralleled the increased drug concentration inside the cells due to their marked bioadhesion to the tumour cell membranes as indicated by scanning electron microscope images.

The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload.

Background: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) failure. While current PAH therapies improve patient outlook, they show limited benefit in attenuating RV dysfunction. Recent investigations demonstrated that the thromboxane (TX) A2 receptor (TP) antagonist NTP42 attenuates experimental PAH across key hemodynamic parameters in the lungs and heart. This study aimed to validate the efficacy of NTP42:KVA4, a novel oral formulation of NTP42 in clinical development, in preclinical models of PAH while also, critically, investigating its direct effects on RV dysfunction. Methods: The effects of NTP42:KVA4 were evaluated in the monocrotaline (MCT) and pulmonary artery banding (PAB) models of PAH and RV dysfunction, respectively, and when compared with leading standard-of-care (SOC) PAH drugs. In addition, the expression of the TP, the target for NTP42, was investigated in cardiac tissue from several other related disease models, and from subjects with PAH and dilated cardiomyopathy (DCM). Results: In the MCT-PAH model, NTP42:KVA4 alleviated disease-induced changes in cardiopulmonary hemodynamics, pulmonary vascular remodeling, inflammation, and fibrosis, to a similar or greater extent than the PAH SOCs tested. In the PAB model, NTP42:KVA4 improved RV geometries and contractility, normalized RV stiffness, and significantly increased RV ejection fraction. In both models, NTP42:KVA4 promoted beneficial RV adaptation, decreasing cellular hypertrophy, and increasing vascularization. Notably, elevated expression of the TP target was observed both in RV tissue from these and related disease models, and in clinical RV specimens of PAH and DCM. Conclusion: This study shows that, through antagonism of TP signaling, NTP42:KVA4 attenuates experimental PAH pathophysiology, not only alleviating pulmonary pathologies but also reducing RV remodeling, promoting beneficial hypertrophy, and improving cardiac function. The findings suggest a direct cardioprotective effect for NTP42:KVA4, and its potential to be a disease-modifying therapy in PAH and other cardiac conditions.

The Hypoxia-Mimetic Agent Cobalt Chloride Differently Affects Human Mesenchymal Stem Cells in Their Chondrogenic Potential.

Adult stem cells are a promising cell source for cartilage regeneration. They resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Cobalt chloride (CoCl2) imitates hypoxia in vitro by stabilizing hypoxia-inducible factor-alpha (HIF-1α), which is the master regulator in the cellular adaptive response to hypoxia. In this study, the influence of CoCl2 on the chondrogenic potential of human MSCs, isolated from dental pulp, umbilical cord, and adipose tissue, was investigated. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 µM. Cell viability, HIF-1α protein synthesis, and the expression of the chondrogenic markers were analyzed. The results showed that the CoCl2 supplementation had no effect on cell viability, while the upregulation of chondrogenic markers such as SOX9, COL2A1, VCAN, and ACAN was dependent on the cellular source. This study shows that hypoxia, induced by CoCl2 treatment, can differently influence the behavior of MSCs, isolated from different sources, in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies.

Wharton's Jelly Derived Mesenchymal Stem Cells: Comparing Human and Horse.

Wharton's jelly (WJ) is an important source of mesenchymal stem cells (MSCs) both in human and other animals. The aim of this study was to compare human and equine WJMSCs. Human and equine WJMSCs were isolated and cultured using the same protocols and culture media. Cells were characterized by analysing morphology, growth rate, migration and adhesion capability, immunophenotype, differentiation potential and ultrastructure. Results showed that human and equine WJMSCs have similar ultrastructural details connected with intense synthetic and metabolic activity, but differ in growth, migration, adhesion capability and differentiation potential. In fact, at the scratch assay and transwell migration assay, the migration ability of human WJMSCs was higher (P < 0.05) than that of equine cells, while the volume of spheroids obtained after 48 h of culture in hanging drop was larger than the volume of equine ones (P < 0.05), demonstrating a lower cell adhesion ability. This can also revealed in the lower doubling time of equine cells (3.5 ± 2.4 days) as compared to human (6.5 ± 4.3 days) (P < 0.05), and subsequently in the higher number of cell doubling after 44 days of culture observed for the equine (20.3 ± 1.7) as compared to human cells (8.7 ± 2.4) (P < 0.05), and to the higher (P < 0.05) ability to form fibroblast colonies at P3. Even if in both species tri-lineage differentiation was achieved, equine cells showed an higher chondrogenic and osteogenic differentiation ability (P < 0.05). Our findings indicate that, although the ultrastructure demonstrated a staminal phenotype in human and equine WJMSCs, they showed different properties reflecting the different sources of MSCs.

The tumor microenvironment promotes cancer progression and cell migration.

The tumor microenvironment contributes to cancer progression, in part through interactions between tumor and normal stromal cells. This study analyzed morphological and molecular changes induced in co-cultured human fibroblasts (HFs) and the MG-63 osteosarcoma cell line. Co-cultured cell monolayers were morphologically analyzed using high resolution scanning electron microscopy (HR-SEM), and trans-well assays were performed to assess cell migration and invasion. Proteins involved in inflammatory responses, cancer cell invasion, and angiogenesis were assessed using western blotting. HR-SEM showed progressive spatial orientation loss by fibroblasts in contact with MG-63s, while MG-63s proliferated rapidly and invaded HF space. Trans-well assays showed enhanced MG-63 migration in the presence of HFs. IL-6 expression was increased in co-cultured HFs, possibly stimulated by TNF-α. HFs do not normally express YKL-40 but did so in co-culture. Band densitometric analyses showed that increasing YKL-40 expression was followed by VEGF overexpression, especially in MG-63s. Finally, our results confirmed fibroblasts as the main matrix metalloproteinase source in this tumor microenvironment. Our study sheds new light on how tumor-stroma interactions promote tumor development and progression, and may support identification of novel anti-cancer therapeutics.

Calcium/Cobalt Alginate Beads as Functional Scaffolds for Cartilage Tissue Engineering.

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes the in vitro behavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co(+2) ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-ßs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes.

Changes in the gene expression of co-cultured human fibroblast cells and osteosarcoma cells: the role of microenvironment.

BACKGROUND: The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work, we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells. METHODS: In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL-40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP-9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown. RESULTS: In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knockdown of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs. CONCLUSIONS: These findings demonstrated that the tumor microenvironment has an influence on the gene expression of healthy surrounding tissues and on the process of tumorigenicity and it is emerging as attractive targets for therapeutic strategies.

Stem cell origin differently affects bone tissue engineering strategies.

Bone tissue engineering approaches are encouraging for the improvement of conventional bone grafting technique drawbacks. Thanks to their self-renewal and multi-lineage differentiation ability, stem cells are one of the major actors in tissue engineering approaches, and among these adult mesenchymal stem cells (MSCs) hold a great promise for regenerative medicine strategies. Bone marrow MSCs (BM-MSCs) are the first- identified and well-recognized stem cell population used in bone tissue engineering. Nevertheless, several factors hamper BM-MSC clinical application and subsequently, new stem cell sources have been investigated for these purposes. The fruitful selection and combination of tissue engineered scaffold, progenitor cells, and physiologic signaling molecules allowed the surgeon to reconstruct the missing natural tissue. On the basis of these considerations, we analyzed the capability of two different scaffolds, planned for osteochondral tissue regeneration, to modulate differentiation of adult stem cells of dissimilar local sources (i.e., periodontal ligament, maxillary periosteum) as well as adipose-derived stem cells (ASCs), in view of possible craniofacial tissue engineering strategies. We demonstrated that cells are differently committed toward the osteoblastic phenotype and therefore, taking into account their specific features, they could be intriguing cell sources in different stem cell-based bone/periodontal tissue regeneration approaches.

In vitro osteogenic and odontogenic differentiation of human dental pulp stem cells seeded on carboxymethyl cellulose-hydroxyapatite hybrid hydrogel.

Stem cells from human dental pulp have been considered as an alternative source of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Recently, polysaccharide based hydrogels have become especially attractive as matrices for the repair and regeneration of a wide variety of tissues and organs. The incorporation of inorganic minerals as hydroxyapatite nanoparticles can modulate the performance of the scaffolds with potential applications in tissue engineering. The aim of this study was to verify the osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) cultured on a carboxymethyl cellulose-hydroxyapatite hybrid hydrogel. Human DPSCs were seeded on carboxymethyl cellulose-hydroxyapatite hybrid hydrogel and on carboxymethyl cellulose hydrogel for 1, 3, 5, 7, 14, and 21 days. Cell viability assay and ultramorphological analysis were carried out to evaluate biocompatibility and cell adhesion. Real Time PCR was carried out to demonstrate the expression of osteogenic and odontogenic markers. Results showed a good adhesion and viability in cells cultured on carboxymethyl cellulose-hydroxyapatite hybrid hydrogel, while a low adhesion and viability was observed in cells cultured on carboxymethyl cellulose hydrogel. Real Time PCR data demonstrated a temporal up-regulation of osteogenic and odontogenic markers in dental pulp stem cells cultured on carboxymethyl cellulose-hydroxyapatite hybrid hydrogel. In conclusion, our in vitro data confirms the ability of DPSCs to differentiate toward osteogenic and odontogenic lineages in presence of a carboxymethyl cellulose-hydroxyapatite hybrid hydrogel. Taken together, our results provide evidence that DPSCs and carboxymethyl cellulose-hydroxyapatite hybrid hydrogel could be considered promising candidates for dental pulp complex and periodontal tissue engineering.

HEMA but not TEGDMA induces autophagy in human gingival fibroblasts.

Polymerized resin-based materials are successfully used in restorative dentistry. Despite their growing popularity, one drawback is the release of monomers from the polymerized matrix due to an incomplete polymerization or degradation processes. Released monomers are responsible for several adverse effects in the surrounding biological tissues, inducing high levels of oxidative stress. Reactive oxygen species are important signaling molecules that regulate many signal-trasduction pathways and play critical roles in cell survival, death, and immune defenses. Reactive oxygen species were recently shown to activate autophagy as a mechanism of cell survival and cell death. Although the toxicity induced by dental resin monomers is widely studied, the cellular mechanisms underlying these phenomena are still unknown. The aim of the study was to investigate the behavior of human gingival cells exposed to 2-hydroxy-ethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) to better elucidate the mechanisms of cell survival and cell death induced by resin monomers. Primary culture of human gingival cells were exposed to 3 mmol/L of HEMA or 3 mmol/L of TEGDMA for 24, 48, and 72 h. Morphological investigations were performed by transmission electron microscopy to analyze the ultrastructure of cells exposed to the monomers. The expression of protein markers for apoptosis (caspase - 3 and PARP) and autophagy (beclin - 1 and LC3B I/II) were analyzed by western blot to investigate the influence of dental resin monomers on mechanisms underlying cell death. Results showed that HEMA treatment clearly induced autophagy followed by apoptosis while the lack of any sign of autophagy activation is observed in HGFs exposed to TEGDMA. These data indicate that cells respond to monomer-induced stress by the differential induction of adaptive mechanisms to maintain cellular homeostasis.

Anti-tumor activity of fenretinide complexed with human serum albumin in lung cancer xenograft mouse model.

Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and ACSVL3 mainly connected with tumor growth. Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and ACSVL3 suggested a reduction of tumor growth. In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.

Ultrastructural changes in human gingival fibroblasts after exposure to 2-hydroxy-ethyl methacrylate.

Polymerized resin-based materials are successfully utilized in medical applications. One draw- back is the release of monomers from the matrix due to an incomplete polymerization or degradation processes. Released monomers can diffuse in the systemic circulation and induceadverse effects to biological tissues. Although there are many hypotheses about the induction of cell death by resin monomers, the underlying mechanisms are still under discussion. The aim of the study was to investigate the morphological modifications in human gingival fibroblasts exposed to 2-hydroxy-ethyl methacrylate (HEMA) to better elucidate the mechanism of cell death induced by resin monomers. Primary cultures of gingival fibroblasts were exposed to 3mM HEMA for 24 h, 72 h, 96 h. Morphological investigations were performed by scanning and transmission electron microscopy, while western blot for caspase-3 was carried out to ver- ify apoptosis. Electron microscopy images showed deep changes in the cell surface and cyto- plasm after 72 h and 96 h of HEMA treatment. Autophagic vesicles were easily observed just after 24 h. Cleaved caspase-3 was detected after 72 h of treatment. These findings suggest that resin based materials induced cell death by the cooperation of apoptosis and autophagy mecha- nisms. The understanding of these mechanisms will lead to the development of smart biomate- rials without or with low adverse effects.

Chondrogenic differentiation of human adipose mesenchimal stem cells: influence of a biomimetic gelatin genipin crosslinked porous scaffold.

Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system.