Use of extracellular matrix components for cell culture

Extracellular matrix components when used as a substratum in vitro can greatly influence cell behavior. The response observed is dependent on the type of cell and matrix used. Cells in vitro usually respond best to the matrix components with which they are normally in contact in vivo. More differentiated phenotypes are observed and cells generally survive longer on such matrices. In some cases, the presence of such matrices allows cells to be cultured in the absence of serum and growth factors. As more investigators try the matrices and matrix components described, as well as new components and combinations of them, it is anticipated that improvement in the culture of many cells can be expected.     

Alterations in cell surface carbohydrate composition of a human colon carcinoma cell line affect adhesion to extracellular matrix components.

The adhesion of HT29 human colon adenocarcinoma cells to different extracellular matrix components was studied. While treatment of the cells with sialidase had no detectable effect on binding to laminin and fibronectin, attachment to collagen IV was decreased. However, additional removal of beta-(1-4)-bound galactose led to significantly reduced binding to all of the substrates, including fibronectin and laminin. Tunicamycin treatment, monitored by lectin-induced aggregation, drastically diminished cell adhesion to laminin and fibronectin, whereas cell binding to collagen IV was not affected. Arg-Gly-Asp (RGD)-related peptides were used to study the adhesion to collagen IV. The results show that a serine-containing RGD-related peptide GRGDSP has virtually no effect on colon carcinoma cell adhesion to type IV collagen. In contrast, when serine was substituted for threonine (GRGDTP) adhesion to collagen IV was strongly inhibited. After incubation of sialidase-treated cells with the threonine-containing peptide adhesion was almost totally blocked. These results demonstrate the existence of both RGD-dependent and carbohydrate-based mechanisms for metastatic human HT29 cell binding to collagen IV.     

Fibroblasts: The arbiters of extracellular matrix remodeling

Extracellular matrix (ECM) is the foundation on which all cells and organs converge to orchestrate normal physiological functions. In the setting of pathology, the ECM is modified to incorporate additional roles, with modifications including turnover of existing ECM and deposition of new ECM. The fibroblast is center stage in coordinating both normal tissue homeostasis and response to disease. Understanding how fibroblasts work under normal conditions and are activated in response to injury or stress will provide mechanistic insight that triggers discovery of new therapeutic treatments for a wide range of disease. We highlight here fibroblast roles in the cancer, lung, and heart as example systems where fibroblasts are major contributors to homeostasis and pathology.     

The matrix reorganized: extracellular matrix remodeling and integrin signaling

Via integrins, cells can sense dimensionality and other physical and biochemical properties of the extracellular matrix (ECM). Cells respond differently to two-dimensional substrates and three-dimensional environments, activating distinct signaling pathways for each. Direct integrin signaling and indirect integrin modulation of growth factor and other intracellular signaling pathways regulate ECM remodeling and control subsequent cell behavior and tissue organization. ECM remodeling is critical for many developmental processes, and remodeled ECM contributes to tumorigenesis. These recent advances in the field provide new insights and raise new questions about the mechanisms of ECM synthesis and proteolytic degradation, as well as the roles of integrins and tension in ECM remodeling.     

MiR-26a modulates extracellular matrix homeostasis in cartilage

MicroRNAs (miRNAs) may represent new therapeutic targets for bone and joint diseases. We hypothesized that several cartilage-specific proteins are targeted by a single miRNA and used bioinformatics to identify a miRNA that can modulate extracellular matrix (ECM) homeostasis in cartilage.Bioinformatic analysis of miRNA binding sequences in the 3′-untranslated region (3′-UTR) of target genes was performed to identify a miRNA that could bind to the 3′-UTR of cartilage matrix-related genes. MiRNA expression was studied by quantitative PCR of microdissected growth plate cartilage and binding to the 3′-UTR sequences was analyzed by luciferase interaction studies. Levels of proteins encoded by target genes in cultures of miR-26a mimic- or inhibitor-transfected chondrocytes were determined by FACS or immunoblot analysis.The complementary binding sequence of miR-26a and miR-26b was found in the 3′-UTR of the prehypertrophic/hypertrophic-specific genes Cd200, Col10a1 as well as Col9a1 and Ctgf. Both miRNAs were expressed in cartilage and only miR-26a was downregulated in hypertrophic growth plate cartilage. MiR-26a could interact with the 3′-UTR of Cd200 and Col10a1 in luciferase binding studies, but not with Col9a1 and Ctgf. However, protein expression of target genes and the ECM adaptor genes matrilin-3 and COMP was significantly altered in miR-26a mimic- or inhibitor-transfected chondrocytes, whereas the abundance of the cell surface receptor for insulin was not changed. In conclusion, miR-26a suppresses hypertrophic and ECM adaptor protein production. Dysregulation of miR-26a expression could contribute to ECM changes in cartilage diseases and this miRNA may therefore act as a therapeutic target.     

The extracellular matrix viscoelasticity as a regulator of cell and tissue dynamics

The extracellular matrix mechanical properties regulate processes in development, cancer, and fibrosis. Among the distinct mechanical properties, the vast majority of research has focused on the extracellular matrix's elasticity as the primary determinant of cell and tissue behavior. However, both cells and the extracellular matrix are not only elastic but also viscous. Despite viscoelasticity being a universal feature of living tissues, our knowledge of the influence of the extracellular matrix's viscoelasticity in cell behavior is limited. This mini-review describes some of the recent findings that have highlighted the role of the extracellular matrix's viscoelasticity in cell and tissue dynamics.     

Cytologic diagnosis of laryngeal and hypopharyngeal squamous cell carcinoma in sputum

A prospective study of the value of sputum cytology in the diagnosis of squamous cell carcinoma of the larynx and hypopharynx is reported. Sputum cytology established the diagnosis in 63.5% of the patients with laryngeal lesions and in 77.4% of the patients with hypopharyngeal lesions. In laryngeal cancer, a positive diagnosis by sputum cytology was related to clinical T factors (according to the TNM classification): while only 29.4% of T1 lesions were positively detected by sputum cytology, 63.3% of T2 lesions, 69.7% of T3 lesions and 79.2% of T4 lesions were so detected. In hypopharyngeal cancer, there was no discernible relationship between sputum cytodiagnosis and clinical T factors. Generally, there was only a small number of cancer cells present in the sputum in these cases. Some of the squamous cancer cells were not very conspicuous and would require careful screening of the sputum specimens to be detected.     

Proteomics-based identification of autoantibody against CDC25B as a novel serum marker in esophageal squamous cell carcinoma

This study was aimed to identify tumor proteins that elicit a humoral response in patients with esophageal squamous cell carcinoma (ESCC). Autologous sera of 15 newly diagnosed patients with ESCC and age- and gender-matched 15 healthy controls were analyzed individually for antibody-based reactivity against proteins from 15 homogenized ESCC tissue mixture resolved by two-dimensional PAGE. One protein spot, which reacted with sera from ESCC patients but not with those from controls, was identified to be CDC25B by mass spectrometry and Western blotting. High expression of CDC25B was detected in ESCC cell lines and primary tumor tissues, but not in normal esophageal tissues. In addition, CDC25B expression was significantly higher in tumor tissue of patients with sera positive CDC25B-Abs than that of patients without CDC25B-Abs. Finally, anti-CDC25B antibodies were readily detectable in sera from 45 of 124 (36.29%) patients with ESCC, 13 of 150 (8.67%) patients with other types of cancer and 0 of 102 (0%) of healthy individuals. Thus, CDC25B autoantibodies may have clinical utility in ESCC screening and diagnosis.     

Matrix proteoglycans are markedly affected in advanced laryngeal squamous cell carcinoma

Proteoglycans (PGs) are implicated in the growth and progression of malignant tumors. In this study, we examined the concentration and localization of PGs in advanced (stage IV) laryngeal squamous cell carcinoma (LSCC) and compared with human normal larynx (HNL). LSCC and HNL sections were examined immunohistochemically with a panel of antibodies, and tissues extracts were analyzed by biochemical methods including immunoblotting and high performance liquid chromatography (HPLC). The results demonstrated significant destruction of cartilage in LSCC, which was followed by marked decrease of aggrecan and link protein. In contrast to the loss of aggrecan in LSCC, accumulation of versican and decorin was observed in the tumor-associated stroma. Biochemical analyses indicated that aggrecan, versican, decorin and biglycan comprise the vast majority of total PGs in both healthy and cancerous tissue. In LSCC the absolute amounts of KS/CS/DS-containing PGs were dramatically decreased about 18-fold in comparison to HNL. This decrease is due to the loss of aggrecan. Disaccharide analysis of CS/DSPGs from LSCC showed a significant reduction of 6-sulfated Delta-disaccharides (Deltadi-6S) with a parallel increase of 4-sulfated Delta-disaccharides (Deltadi-4S) as compared to HNL. The obtained data clearly demonstrate that tumor progression is closely related to specific alteration of matrix PGs in LSCC. The altered composition of PGs in cartilage, as well as in tumor-associated stroma, is crucial for the biological behaviour of cancer cells in the diseased tissue.     

Immunogenicity of xenogeneic cartilage matrix components in a rabbit model

Purified xenogeneic cartilage matrix components, including proteoglycan subunits, chondroitin 4 sulfate, and chondroitin 6 sulfate, were inoculated into the knee joint of rabbits, and local as well as systemic responses were evaluated. proteoglycan was associated with synovial hyperplasia and infiltrates of eosinophils and lymphocytes and with rising titers of antiproteoglycan antibodies in a tanned sheep rbc hemagglutination assay over a six-week period of weekly intra-articular injections and observations. Chondroitin sulfates failed to evoke detectable changes in the joint or serum. Immunogenicity of cartilage matrix components may play a role in allogeneic and xenogeneic osteochondral grafts, and it is also possible that autogenous matrix immunogens exist and contribute to progression of degenerative joint disease. The immunogenicity of allogeneic and autogenous cartilage matrix components remains undefined.     

In situ localization of cartilage extracellular matrix components by immunoelectron microscopy after cryotechnical tissue processing

Localization and distribution of proteoglycans within rat growth plate cartilage were investigated by immunoelectron microscopy. By use of a mixture of three monoclonal antibodies directed against chondroitin sulfate chains and of post-embedding staining by protein A-gold, the immunosensitivity and resolution achieved by electron microscopy within tissue processed by high-pressure freezing, freeze-substitution, and low-temperature embedding were compared with those in tissue preserved by three alternative procedures (i.e., mild chemical fixation in combination with either low-temperature embedding or conventional embedding, and high-pressure freezing and freeze-substitution followed by conventional embedding). The loss of matrix components incurred during each stage of high-pressure freezing, freeze-substitution, and low temperature embedding was also determined by measuring the loss of [35S]-proteoglycans from tissue labeled in vivo, and the results compared with previously determined estimates for tissue processed using conventional techniques. Immunosensitivity, determined as the number of gold particles per unit area, was highest in tissue processed by high-pressure freezing, freeze substitution, and low-temperature embedding. Comparable results (with a reduction of only 3-7%) were achieved within tissue preserved by mild chemical fixation followed by low-temperature embedding. In both procedures where conventional embedding was adopted, sensitivity was considerably reduced (by 51% for high-pressure freezing and freeze substitution and by 74% for mild chemical fixation). Loss of matrix components was negligible during all stages of high-pressure freezing, freeze-substitution, and low-temperature embedding. Such information, and that derived from morphological inspection of the various matrix compartments in cartilage processed by high-pressure freezing, freeze-substitution, and low-temperature embedding (J Cell Biol 98:277, 1984), together demonstrate that application of this technique results in successful immobilization of proteoglycans in situ within cartilage matrix. Although loss of proteoglycans from mildly fixed cartilage embedded under low-temperature conditions is minor, morphological examination of this tissue reveals considerable shifting of proteoglycans within matrix compartments. Hence, even though immunosensitivity may be high, resolution is poor. The beauty of the high-pressure freezing, freeze-substitution, and low-temperature embedding technique is that it combines high immunosensitivity with precise localization of matrix components at the molecular level.     

Hyaluronidase and CD44 hyaluronan receptor expression in squamous cell laryngeal carcinoma

Squamous cell laryngeal carcinoma undergoes significant structural-related modifications of the extracellular matrix components (ECM), the most characteristics being the presence of degraded collagen, aggrecan and hyaluronan. We examined the presence of hyaluronidase and of the cellular hyaluronan receptor CD44 during the various stages of cancer. ECM components were extracted by using PBS, 4 M GdnHCl and 4 M GdnHCl-0.1% Triton-X 100 sequentially and hyaluronidase and CD44 analyzed by zymography and immunochemistry techniques. Total RNA was also extracted and the mRNA of the various hyaluronidases and of CD44 was analyzed after amplification with RT-PCR. Hyaluronidase was detected as a double band of 45 and 55 kDa molecular mass, only in cancer samples. The analysis of mRNA indicated an aberrant expression of PH-20, the testicular-type hyaluronidase, at late stages of cancer and an overexpression of HYAL1 only at stage IV. In addition, CD44 was identified in two protein bands of 80 and 64 kDa in cancer samples. The analysis of mRNA showed that hyaluronan receptor was expressed in a stage-related order. Thus, it could be suggested that in laryngeal squamous cell carcinoma, cancer cells migrated and proliferated under the influence of small molecular mass hyaluronan, by expressing increased amounts of its receptor.     

Aspects of extracellular matrix remodeling in development and disease

The extracellular matrix is the major constituent of organic matter in both plants and animals, where it provides the interface between individual cells. In most tissues, with some notable exceptions such as bone marrow, the volume of extracellular matrix equals or exceeds the volume of intracellular space and organelles, making matrix an abundant constituent through which cells exert their functions and receive cues. The matrix may therefore be considered the basic structural entity that supports the function of an organ, and in connective tissues the matrix is the organ itself to which function is tied throughout the life of its resident cells. In this review, a select number of proteinases involved in some of the more conspicuous matrix remodeling events of the mammalian organism are explored. Evidence from both animal models and human diseases is discussed in relation to normal physiological processes, including instances in which aberrant matrix remodeling leads to disease states.     

Neural cell adhesion molecule (NCAM) as a quantitative marker in synaptic remodeling

The neural cell adhesion molecule (NCAM) participates in adhesion and neuritic outgrowth during nervous system development. In the adult brain, NCAM is considered to be involved in neuronal sprouting and synaptic remodeling. the NCAM concentration of brain tissue has proved to be a useful marker of these processes, especially when viewed in comparison with the concentration of a marker of mature synapses, e.g. D3-protein (SNAP-25) or synaptophysin. The present review focusses on studies of adult brain in which NCAM concentration estimates and NCAM/D3 ratios have been used to evaluate the rate of synaptic remodeling in brain damage and degenerative diseases.Special issue dedicated to Dr. Robert Balázs.     

Integrative analysis of mRNA and microRNA expression profiles in laryngeal squamous cell carcinoma

Larynx cancer is a therapeutically challenging disease. Rapidly evolving experimentally validated data have significantly improved our understanding of the complex role of numerous RNA, DNA, and proteins that play a role in the development and progression of cancer. Based on the insights from approximately two decades of research, it seems clear that microRNAs (miRNAs) have revolutionized our concepts related to the main role of noncoding RNAs in different cancers’ progression, development, and metastasis. Mechanistically, miRNAs have been reported to regulate different RNAs and finally protein-coding genes. The expression profiling of miRNAs and messenger RNA (mRNAs) was conducted for a deeper analysis of the miRNAs and mRNAs which play an essential role in larynx cancer. Downregulation or upregulation over twofolds in the miRNAs was considered to be significant, and that of sixfolds or below was considered to be significant for the mRNAs. In accordance with this approach, the expression levels of 43 miRNAs were increased in this study, whereas the expression levels of 129 were decreased. Accordingly, all the genomic expression studies provided evidence of upregulation of 97 genes, whereas 128 genes were found to be downregulated. Among these miRNAs, hsa-miR-20a-3p and hsa-miR-1972 were noted to be important in the etiology of larynx cancer.     

Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling

Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.     

Cdc42 regulates extracellular matrix remodeling in three dimensions

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.