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Results for “Cellular Microenvironment”

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PubMedDate not supplied

Spheroids: relation between tumour and endothelial cells.

It has recently been established that the microenvironment plays a major role in many physiological and pathological events. Indeed cell-cell and cell-extracellular matrix contacts are necessary for much cellular function such as differentiation, proliferation, cell death, apoptosis and angiogenesis. For growth, proliferating tumour cells need to be fed by nutrients and oxygen brought by new vessels. In this context, scientists seek a new model that allows for the investigation of both angiogenesis and the influence of the microenvironment on this phenomenon. The purpose of this paper is to review the literature on the relation between tumour and endothelial cells grown as spheroids, a technique that allows us to study in three-dimensions the influence of cell contact on this growth. For the purpose of clarification, this review has recategorised the different studies on spheroids into three classes: (1) spheroids grown in vitro and then reimplanted in animals to follow endothelial cell infiltration; (2) spheroids grown in vitro and then cultured on endothelial cell monolayers; (3) tumours grown in vitro such as organotypic culture. This review attempts to demonstrate that spheroid cell cultures are useful for studying the relation between tumour and endothelial cells and to analyse physiological phenomena such as wound healing, extravasation and intravasation.

Animals↗

PubMed2003

Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor.

The human coxsackievirus and adenovirus receptor (CAR) represents the primary cellular site of adenovirus attachment during infection. An understanding of the mechanisms regulating its expression could contribute to improving efficacy and safety of adenovirus-based therapies. We characterized regulation of CAR expression in a 3D cell culture model of human breast cancer progression, which mimics aspects of the physiological tissue context in vitro. Phenotypically normal breast epithelial cells (S1) and their malignant derivative (T4-2 cells) were grown either on tissue culture plastic (2D) or 3D cultures in basement membrane matrix. S1 cells grown in 3D showed low levels of CAR, which was expressed mainly at cell-cell junctions. In contrast, T4-2 cells expressed high levels of CAR, which was mainly in the cytoplasm. When signaling through the epidermal growth factor receptor was inhibited in T4-2 cells, cells reverted to a normal phenotype, CAR protein expression was significantly reduced, and the protein relocalized to cell-cell junctions. Growth of S1 cells as 2D cultures or in 3D in collagen-I, a nonphysiological microenvironment for these cells, led to up-regulation of CAR to levels similar to those in T4-2 cells, independently of cellular growth rates. Thus, expression of CAR depends on the integrity and polarity of the 3D organization of epithelial cells. Disruption of this organization by changes in the microenvironment, including malignant transformation, leads to up-regulation of CAR, thus enhancing the cell's susceptibility to adenovirus infection.

Base Sequence↗

PubMed2025

Understanding proneural-mesenchymal transition using patient-derived glioma stem-like cell (GSC) organoids and engineered extracellular matrix.

Glioblastoma multiforme (GBM) is a highly aggressive, angiogenic WHO grade IV glioma marked by rapid progression, therapeutic resistance, and poor prognosis. A defining feature of GBM is the presence of glioma stem-like cells (GSCs), which reside in specialized perivascular niches and drive tumor progression, recurrence, and therapeutic resistance. The blood-brain barrier, coupled with the complex and dynamic tumor microenvironment, poses significant challenges for both treatment and mechanistic investigation. Current in vitro GBM models inadequately recapitulate the structural and biochemical cues of the native perivascular niche due to the absence of functional vasculature and brain-mimetic extracellular matrix (ECM), limiting their physiological relevance and predictive power. To address the limitations of existing in vitro GBM models, we developed a patient-derived glioma stem cells (GSC) derived Matrigel spheroid system that transitions into organoids and enables integration into engineered microenvironments. Our model incorporates GSC organoids representing proneural and mesenchymal GBM subtypes, a synthetic engineered extracellular matrix (eECM), and endothelial cells (ECs) seeded on the matrix surface. We evaluated the expression of subtype-specific, pro-angiogenic, stemness, and differentiation markers under increasingly complex co-culture conditions. Our results show that Matrigel-derived GSC spheroids progressively differentiate into organoids over two weeks, with significantly enhanced expression of cell-specific markers in the presence of ECs. Encapsulation of these organoids within eECM, combined with EC co-culture, further promoted cellular invasion and induction of GBM associated genes. This in situ encapsulation strategy enables real-time observation of GSC behavior in a tunable microenvironment that mimics key features of the native tumor niche. Together, this platform provides a physiologically relevant and modular in vitro system for investigating GBM pathophysiology. It holds promise for uncovering tumor-specific cellular dependencies, studying GSC-vascular interactions, and conducting high-throughput drug screening under controlled, biomimetic conditions.

Engineered extracellular matrix↗

PubMed1995

Interaction between ionizing radiation, estrogens and antiestrogens in the modification of tumor microenvironment in estrogen dependent multicellular spheroids.

MCF7 human breast cancer cells growing as multicellular spheroids were examined as a model of three-dimensional cellular organization. Estrogen-free medium inhibited spheroid formation. In medium containing estrogens, the antiestrogen hydroxytamoxifen decreased the spheroid growth rate. Analyses with the recursion formula after Gompertz fitting showed that the rate of exponential decrease in growth rate (alpha) was alpha 0.099 +/- 0.013 d-1, and the decrease in alpha' was 0.061 +/- 0.015 d-1 for 0.1 microM hydroxytamoxifen and control spheroids respectively. MCF7 cells which had been growth arrested in an estrogen-free medium showed a significant decrease in radiosensitivity (surviving fraction at 2 Gy, SF2 = 63%) when compared with 0.1 nM 17 beta-estradiol-treated cells (SF2 = 38%). No differences in radiosensitivity were seen in MCF7 spheroids in estrogen-supplemented medium (radiation dose necessary to control 50% of spheroids (SCD50) was 5.51 Gy; derived alpha, beta and SF2 were 0.301 +/- 0.110 Gy-1, 0.018 +/- 0.005 Gy-2, and 51% respectively) when compared with monolayer cultures in the same medium (alpha = 0.316 +/- 0.059 Gy-1, beta = 0.023 +/- 0.006 Gy-2 and SF2 = 50%). In the spheroid model, manipulating the cellular environment, i.e., with estrogen treatment, modulates sensitivity to ionizing radiation.

Breast Neoplasms↗

PubMed2005

Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy.

BACKGROUND: The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. RESULTS: Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. CONCLUSION: Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

Animals↗

PubMed2003

Polyvinylalcohol three-dimensional matrices for improved long-term dynamic culture of hepatocytes.

Rat hepatocytes were seeded on three-dimensional highly porous polyvinylalcohol (PVA) and aminoethyl-modified polyvinylalcohol (AE-PVA) matrices. Hepatocytes were cultured under static and dynamic conditions. The three-dimensional matrices offered an improved extracellular microenvironment for long-term (5 days) maintenance of hepatocytes, compared to reference monolayer cultures on collagen. Cellular adhesion exceeded 80% with a viability superior to 70%. The preservation of albumin secretion after 5 days of culture was two times higher for static cultures on three-dimensional matrices (18% on PVA, 13% on AE-PVA) and three times higher for dynamic three-dimensional cultures (25% PVA and AE-PVA), compared to the static two-dimensional culture on collagen film (8%). The biotransformation of ammonia into urea was also maintained throughout the culture period. The addition of the aminoethyl function demonstrated no toxicity for the hepatocyte cultures. This function could be suitable eventually to further improve the hepatocyte culture system by linking more specific adhesion molecules on the polymer surface. This study demonstrated the efficiency of polyvinylalcohol as a three-dimensional matrix coupled to a perfusion culture system, which improves extracellular conditions for hepatocyte survival and promotes preservation of long-term hepatospecific functions.

Animals↗

PubMed2006

What is a stem cell niche?

Niche has become the most important issue in stem cell biology, but it is still a hypothetical notion that cannot be defined in a better way than the microenvironment surrounding stem cells. Using a melanocyte stem cell system as a model, we have analyzed the cellular and molecular requirements for differentiation of quiescent stem cells. Our results demonstrate the multiple subsets within the stem cell compartment and thus suggests the complexity of niche.

Animals↗

PubMed2000

High glucose modulates P2X7 receptor-mediated function in human primary fibroblasts.

AIMS/HYPOTHESIS: Purinergic receptors are a family of newly characterized plasma membrane molecules involved in several and as yet only partially known cellular functions such as vascular reactivity, apoptosis and cytokine secretion. Little is known about the effect extracellular microenvironment has on their function. Fibroblasts share several features with smooth muscle cells and are an important constituent of the atherosclerotic plaque. Our aim was to evaluate the effect of high glucose concentration on ATP-mediated responses in human fibroblasts. METHODS: Fibroblasts were obtained by skin biopsies and grown at two different glucose concentrations. We evaluated receptor expression by RT-PCR and immunoblotting and receptor localization by immunofluorescence. Plasma membrane potential and calcium changes were measured by fluorescent indicators. Apoptosis was determined by ethidium bromide staining and caspase-3 activation. RESULTS: We show that cells grown in a medium with high glucose concentration underwent great ATP-mediated morphological changes, enhanced apoptosis, caspase 3 activation and interleukin-6 release. We identified P2X7 as the main purinergic receptor involved in these responses. Furthermore, high glucose concentration triggered the assembly of P2X7 into ring-like structures located at the periphery of the cells. CONCLUSION/INTERPRETATION: Given that ATP is frequently released into the extracellular milieu upon cell and tissue damage, secretory exocytosis or activation of plasma membrane transporters, we hypothesize that ATP receptors participate in the pathogenesis of vascular complications of diabetes.

Adenosine Triphosphate↗

PubMed1988

DNA repair in lymphocytes from humans and rats with chronic iron overload.

A marked reduction of the proliferative capability after a mitogenic stimulus and a dramatic decrease of the capacity to repair DNA damages were found in lymphocytes from iron overloaded rats. These immunological parameters were not significantly different from controls in peripheral blood lymphocytes from patients with primary iron overload: hereditary hemochromatosis and porphyria cutanea tarda. This discrepancy could be due to the accelerated modality of iron overload in the rat model and to the fact that rat lymphocytes were obtained from an highly iron repleted microenvironment (i.e. spleen). Our data indicate that iron overload can affect the structure and/or the function of cellular DNA thus offering new insights on the close association of iron overload conditions and cancer.

Animals↗

PubMed1994

Localization of c-fos, c-jun, and hsp70 mRNA expression in brain after neonatal hypoxia-ischemia.

The sites of expression of early response mRNAs were determined in the brains of 7-day-old rat pups exposed to unilateral carotid artery ligation followed by 3 h of hypoxia. Pups were sacrificed after recovery periods ranging from 10 min to 24 h. In agreement with our previous northern blot analysis, in situ hybridization of coronal brain sections to probes for c-fos, c-jun, and heat-inducible hsp70 revealed a marked induction and subsequent disappearance of all three mRNAs during this time period. We observed co-localization of the 2 immediate early gene (IEG) mRNAs, c-fos and c-jun, which encode proteins that act in combination to regulate subsequent gene expression. These mRNAs were expressed in all regions known to be vulnerable to permanent injury in this model, such as the cortex, hippocampus, and striatum, as well as in other regions that are spared from permanent damage, such as contralateral cortex and lateral ventricular neuroepithelium. The temporal and regional co-localization of c-fos and c-jun suggests that the transcriptional regulatory activity of their protein products could play a role in plasticity associated with death or recovery from injury in the immature brain. Hsp70 mRNA expression was induced in nearly all of the animals that were positive for IEG mRNAs. Although the most frequent site of expression for all three mRNAs was the ipsilateral cerebral cortex, hsp70 expression was restricted to the ipsilateral hemisphere and absent from a number of structures that were positive for c-fos and c-jun. In addition, the patterns of expression of hsp70 within specific structures frequently differed from those of the IEGs, implying that although both cellular early response systems are activated in this model, their specific functions are carried out within different microenvironments.

Animals↗

PubMed2004

Thymus cell-cell interactions.

The recent advances in molecular biology and genetics, as well as the progress of in vitro techniques, have provided a more coherent image of the thymic function on the molecular level. But they have shifted the attention away from studies on the cellular level, which are necessary to clarify the biological roles of different cell types of the thymic microenvironment. The structure and function of the normal thymus depend on mutual interactions between thymocytes and nonlymphocyte cells. In this review a detailed description of morphological and phenotypic features of both maturing thymocytes and nonlymphocyte cells is given. The recent genetic and biochemical data are presented in conjunction with cytological results to enlighten the thymus cell-cell interactions during thymopoiesis and organization of thymic microstructure. Special emphasis is put on the experimental approaches, which may be used to study the interactions between thymocytes and nonlymphocyte cells in vivo.

Animals↗

PubMed2006

Lymphatic tissue fibrosis is associated with reduced numbers of naive CD4+ T cells in human immunodeficiency virus type 1 infection.

The organized structure of lymphatic tissues (LTs) constitutes a microenvironment referred to as a niche that plays a critical role in immune system homeostasis by promoting cellular interactions and providing access to cytokines and growth factors on which cells are dependent for survival, proliferation, and differentiation. In chronic human immunodeficiency virus type 1 (HIV-1) infection, immune activation and inflammation result in collagen deposition and disruption of this LT niche. We have previously shown that these fibrotic changes correlate with a reduction in the size of the total population of CD4+ T cells. We now show that this reduction is most substantial within the naïve CD4+ T-cell population and is in proportion to the extent of LT collagen deposition in HIV-1 infection. Thus, the previously documented depletion of naïve CD4+ T cells in LTs in HIV-1 infection may be a consequence not only of a decreased supply of thymic emigrants or chronic immune activation but also of the decreased ability of those cells to survive in a scarred LT niche. We speculate that LT collagen deposition might therefore limit repopulation of naïve CD4+ T cells with highly active antiretroviral therapy, and thus, additional treatments directed to limiting or reversing inflammatory damage to the LT niche could potentially improve immune reconstitution.

Adult↗

PubMed1997

Thymic nurse cells forming a dynamic microenvironment in spontaneous thymoma BUF/Mna rats.

Thymic nurse cells (TNCs) were studied using an animal model, BUF/Mna rats, which spontaneously develop benign thymomas of epithelial origin with age. The unusual increment and high availability of TNCs in this thymus enabled us to analyze TNCs directly either in tissue sections or on smears after enzymatic isolation. No structural or phenotypical abnormality in these TNCs was detected as assessed by electron microscopy and immunohistochemistry. Typical TNCs were widely distributed in the cortical areas but not in the medullary areas. They showed characteristic euchromatic bright nuclei and enclosed intra-TNC cells with an investment of relatively light cytoplasm with abundant small vesicles and rough-endoplasmic reticulum. The intra-TNC cells were mostly double positive (CD4+CD8+) cortical thymocytes, though macrophage populations could also be distinguished by their content of membrane-bounded phagosomes, multivesicular bodies and other inclusion bodies, and by their lack of cytoskeletal keratin filaments. High voltage electron microscopy revealed that intra-TNC cells were separated into several compartments by extremely thin internal veils of the TNC processes. The outer veils of the TNCs were continuous with occasional small gaps through which intra-TNC cells could migrate in and out of the compartments. Immunohistochemical analyses revealed that the TNCs per se were positive for MHC class I and class II, keratin and thymulin, but lacked both lymphocyte and macrophage markers. Among all adhesion molecules tested, ICAM-1 was strongly expressed on almost all TNCs. A minority of TNCs also contained either LFA-1 alpha or LFA-1 beta positive cells. These results suggest that TNCs may form a rather dynamic microenvironment for T cell development where either nursing or clearance of thymocytes take place, depending on the cellular components of intra-TNC cells. Macrophage populations may also play crucial roles as the third component within TNCs.

Age Factors↗

PubMed2005

Integrin function and signaling as pharmacological targets in cardiovascular diseases and in cancer.

The microenvironment is now considered as an important source of potential therapeutic targets in diverse pathologies. In cardiovascular diseases and in cancer, common processes involving stromal remodeling, cell invasion, and angiogenesis can promote progression of the pathology. At each step of the pathogenesis, cell adhesion needs to be modulated to allow adaptation of cell survival/motility/proliferation functions to the microenvironment. Among adhesion receptors, integrins, responsible for cell/matrix or cell/cell interactions, play a key role in the cellular responses. Moreover, their engagement conditions the sensitivity to apoptosis induced by therapeutic drugs. Targeting of the extracellular side of integrins in order to modulate their adhesive functions is under development and has reached clinical indications. However, improvement of oral availability and of cell signaling control is required in the future. Targeting of the extracellular or the intracellular key proteins involved in integrin-dependent signaling pathway seems promising. Yet, although some common key enzyme inhibitors are under development, a better knowledge of the specificity of integrin activation and interaction with partners upon pathogenesis is of major importance in envisaging the antagonism of integrin-linked signals as a therapeutic tool alone or in association with other therapies.

Cardiovascular Diseases↗

PubMed2005

Cellular signaling in neural stem cells: implications for restorative neurosurgery.

Over the course of the past few decades, it has become apparent that in contrast to previously held beliefs, the adult central nervous system (CNS) may have the capability of regeneration and repair. This greatly expands the possibilities for the future treatment of CNS disorders, with the potential strategies of treatment targeting the entire scope of neurological diseases. Indeed, there is now ample evidence that stem cells exist in the CNS throughout life, and the progeny of these stem cells may have the ability to assume the functional role of neural cells that have been lost. The existence of stem cells is no longer in dispute. In addition, once transplanted, stem cells have been shown to survive, migrate, and differentiate. Nevertheless, the clinical utility of stem cell therapy for neurorestoration remains elusive. Without question, the control of the behavior of stem cells for therapeutic advantage poses considerable challenges. In this paper, the authors discuss the cellular signaling processes that influence the behavior of stem cells. These signaling processes take place in the microenvironment of the stem cell known as the niche. Also considered are the implications attending the replication and manipulation of elements of the stem cell niche to restore function in the CNS by using stem cell therapy.

Animals↗

PubMed2002

Expression of alpha-smooth muscle actin in the stromal cells of bone marrow in fetuses in different stages of development, in multiple myeloma and monoclonal gammopathy of unknown significance.

Several disorders are associated with a monoclonal immunoglobulin detected by serum or urine electrophoresis, the most common being a monoclonal gammopathy of unknown significance, multiple myeloma, Waldenstrom's macroglobulinemia, and amyloidosis. Plasma cells, the immunoglobulin secretory cells of the immune system, are normal constituents of bone marrow (BM). Plasma cells are seen in small numbers in the stroma, surrounding blood vessels in the marrow. Their perivascular disposition is consistent with their secreting capacity. The hematopoietic microenvironment has a crucial role homing and regulating precursor cell growth both in physiologic and pathologic conditions. Cellular components such as branched adventitial reticular cells, macrophages, endothelial cells and fat cells constitute the supporting framework (stroma) for hematopoiesis, which takes place in the extravascular compartment. The presence of myiod cells (MCs) in human bone marrow has been observed during hematopoiesis in embryonic life, whereas during adult life, it is strictly related to various pathologic conditions. The aim of this study was to examine in the stroma of BM the presence, distribution and quantitation of cells expressing a-smooth muscle actin (MCs) from patients with monoclonal gammopathy of unknown significance, those with plasma cell myeloma and embryos (gestational age 15 to 25 weeks). For this reason, a series of 20 trephine bone marrow biopsies from adult patients and ten fetal specimens of the spine and femur were examined for the presence of stromal myoid cells using a monoclonal recognising alpha-smooth muscle actin, a contractile microfilament expressed solely by smooth muscle cells, myofibroblasts and related cells. Our results suggest that the appearance of MCs and subsequent fibrosis is not a feature of malignant BM disorders such as MM but it is also seen to a lesser degree in the BM stroma of individuals with monoclonal gammopathy of unknown significance (MGUS). Stromal cells with phenotypic smooth muscle features appear in bone marrow during pathological situations in a manner reminiscent of what occurs during normal development.

Actins↗

PubMedDate not supplied

Arising podosomal structures are associated with neoplastic cell morphological phenotype induced by the microenvironment.

Increased numbers of rosettes of podosomes were observed in overgrown rat Rous sarcoma RsK4 cells. A possible role of these structures in nutrient uptake in tumour cell survival was investigated by exposure to acute starvation. A single cell suspension of RsK4 cells in Hanks balanced salt solution was allowed to interact with either clean uncoated or serum-coated for bait coverglasses. Confocal microscopy revealed contrasting 3D cell morphologies that were associated with conspicuous patterns of podosomal structures, which on the coated coverglasses resembled the sealing zones of osteoclasts, while on the uncoated coverglasses they resembled the marginal podosomes of migrating monocyte-derived cells. Thus, the arising podosomal structures, the involvement of which in an uptake of nutrients appeared feasible morphologically, were associated with the emerging 3D cell shapes guided by the microenvironment. Such phenotypic plasticity of neoplastic RsK4 cells in response to microenvironmental challenge suggested that uniqueness in cellular attributes within the neoplastic cell population could be crucial for the malignant potential.

Actins↗

PubMed2005

An extracellular matrix microarray for probing cellular differentiation.

We present an extracellular matrix (ECM) microarray platform for the culture of patterned cells atop combinatorial matrix mixtures. This platform enables the study of differentiation in response to a multitude of microenvironments in parallel. The fabrication process required only access to a standard robotic DNA spotter, off-the-shelf materials and 1,000 times less protein than conventional means of investigating cell-ECM interactions. To demonstrate its utility, we applied this platform to study the effects of 32 different combinations of five extracellular matrix molecules (collagen I, collagen III, collagen IV, laminin and fibronectin) on cellular differentiation in two contexts: maintenance of primary rat hepatocyte phenotype indicated by intracellular albumin staining and differentiation of mouse embryonic stem (ES) cells toward an early hepatic fate, indicated by expression of a beta-galactosidase reporter fused to the fetal liver-specific gene, Ankrd17 (also known as gtar). Using this technique, we identified combinations of ECM that synergistically impacted both hepatocyte function and ES cell differentiation. This versatile technique can be easily adapted to other applications, as it is amenable to studying almost any insoluble microenvironmental cue in a combinatorial fashion and is compatible with several cell types.

Animals↗