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1.

Background

Compressive mechanical stress produced during growth in a confining matrix limits the size of tumor spheroids, but little is known about the dynamics of stress accumulation, how the stress affects cancer cell phenotype, or the molecular pathways involved.

Methodology/Principal Findings

We co-embedded single cancer cells with fluorescent micro-beads in agarose gels and, using confocal microscopy, recorded the 3D distribution of micro-beads surrounding growing spheroids. The change in micro-bead density was then converted to strain in the gel, from which we estimated the spatial distribution of compressive stress around the spheroids. We found a strong correlation between the peri-spheroid solid stress distribution and spheroid shape, a result of the suppression of cell proliferation and induction of apoptotic cell death in regions of high mechanical stress. By compressing spheroids consisting of cancer cells overexpressing anti-apoptotic genes, we demonstrate that mechanical stress-induced apoptosis occurs via the mitochondrial pathway.

Conclusions/Significance

Our results provide detailed, quantitative insight into the role of micro-environmental mechanical stress in tumor spheroid growth dynamics, and suggest how tumors grow in confined locations where the level of solid stress becomes high. An important implication is that apoptosis via the mitochondrial pathway, induced by compressive stress, may be involved in tumor dormancy, in which tumor growth is held in check by a balance of apoptosis and proliferation.  相似文献   

2.
Tse HT  Weaver WM  Di Carlo D 《PloS one》2012,7(6):e38986
As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 μm) to low (7 μm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.  相似文献   

3.
Multicellular tumour spheroids (MCTS) are three-dimensional cell culture systems which are widely used in cancer research. They are characterized by an outer zone of proliferating cells, an inner region of differentiating quiescent cells and an area of so-called necrotic cell death in their centre. The exact cause of this cell death, a controversy for many years, was the aim of the present study. Our data show that cell death in the centre of MCTS of three colorectal adenocarcinoma cell lines (HRT-18, HT-29 and CX-2) was induced by apoptosis. Apoptotic cells were initially distributed at random but accumulated very quickly in the quiescent and central area at day 4-5, suggesting a time- rather than size-dependent synchronization of apoptosis parallel to the formation of the proliferation gradient in MCTS. To study mechanisms inducing apoptosis, the Fas-pathway was investigated. A cell-cell contact-dependent expression of CD95 was found in all MCTS. FasL was not detected in monolayer cultures, but was expressed in spheroids of HRT-18 and CX-2. We found that TNF alpha and TGF beta 1 activated the CD95 pathway in all three cell lines. Since both TNF-alpha and TGF-beta are known to be inducible by hypoxia in a variety of cell types, we suggest that these hypoxia-induced factors sensitize the CD95 pathway in the quiescent area of MCTS. Furthermore, a loss of the heat shock proteins 27, 32, 60, 73 and 90 was observed in the quiescent area of spheroids. This suggests that tumour cell differentiation in the inner region of MCTS may be an additional factor inducing apoptosis.  相似文献   

4.
Tumor spheroids constitute an effective in vitro tool to investigate the avascular stage of tumor growth. These three-dimensional cell aggregates reproduce the nutrient and proliferation gradients found in the early stages of cancer and can be grown with a strict control of their environmental conditions. In the last years, new experimental techniques have been developed to determine the effect of mechanical stress on the growth of tumor spheroids. These studies report a reduction in cell proliferation as a function of increasingly applied stress on the surface of the spheroids. This work presents a specialization for tumor spheroid growth of a previous more general multiphase model. The equations of the model are derived in the framework of porous media theory, and constitutive relations for the mass transfer terms and the stress are formulated on the basis of experimental observations. A set of experiments is performed, investigating the growth of U-87MG spheroids both freely growing in the culture medium and subjected to an external mechanical pressure induced by a Dextran solution. The growth curves of the model are compared to the experimental data, with good agreement for both the experimental settings. A new mathematical law regulating the inhibitory effect of mechanical compression on cancer cell proliferation is presented at the end of the paper. This new law is validated against experimental data and provides better results compared to other expressions in the literature.  相似文献   

5.
Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.  相似文献   

6.
We consider how cell proliferation and death generate residual stresses within a multi-cell tumour spheroid (MCTS). Previous work by Jones and co-workers [8] has shown that isotropic growth in a purely elastic MCTS produces growth induced stresses which eventually become unbounded, and hence are physically unrealistic. Since viscoelastic materials show stress relaxation under a fixed deformation we consider the effect of the addition of a small amount of viscosity to the elastic system by examining formation of equilibrium stress profiles within a Maxwell type viscoelastic MCTS. A model of necrosis formation based upon that proposed by Please and co-workers (see [16] [17] [18]) is then presented in which necrosis forms under conditions of adverse mechanical stress rather than in regions of extreme chemical stress as is usually assumed. The influence of rheology on necrosis formation is then investigated, and it is shown that the excessive stress generated in the purely elastic tumour can be relieved either by the addition of some viscosity to the system or by accounting for an inner necrotic interface with an appropriate stress boundary condition.  相似文献   

7.
Experiments on tumor spheroids have shown that compressive stress from their environment can reversibly decrease tumor expansion rates and final sizes. Stress release experiments show that nonuniform anisotropic elastic stresses can be distributed throughout. The elastic stresses are maintained by structural proteins and adhesive molecules, and can be actively relaxed by a variety of biophysical processes. In this paper, we present a new continuum model to investigate how the growth-induced elastic stresses and active stress relaxation, in conjunction with cell size control feedback machinery, regulate the cell density and stress distributions within growing tumors as well as the tumor sizes in the presence of external physical confinement and gradients of growth-promoting chemical fields. We introduce an adaptive reference map that relates the current position with the reference position but adapts to the current position in the Eulerian frame (lab coordinates) via relaxation. This type of stress relaxation is similar to but simpler than the classical Maxwell model of viscoelasticity in its formulation. By fitting the model to experimental data from two independent studies of tumor spheroid growth and their cell density distributions, treating the tumors as incompressible, neo-Hookean elastic materials, we find that the rates of stress relaxation of tumor tissues can be comparable to volumetric growth rates. Our study provides insight on how the biophysical properties of the tumor and host microenvironment, mechanical feedback control and diffusion-limited differential growth act in concert to regulate spatial patterns of stress and growth. When the tumor is stiffer than the host, our model predicts tumors are more able to change their size and mechanical state autonomously, which may help to explain why increased tumor stiffness is an established hallmark of malignant tumors.  相似文献   

8.
Understanding the mechanical behavior of multicellular monolayers and spheroids is fundamental to tissue culture, organism development, and the early stages of tumor growth. Proliferating cells in monolayers and spheroids experience mechanical forces as they grow and divide and local inhomogeneities in the mechanical microenvironment can cause individual cells within the multicellular system to grow and divide at different rates. This differential growth, combined with cell division and reorganization, leads to residual stress. Multiple different modeling approaches have been taken to understand and predict the residual stresses that arise in growing multicellular systems, particularly tumor spheroids. Here, we show that by using a mechanically robust agent-based model constructed with the peridynamic framework, we gain a better understanding of residual stresses in multicellular systems as they grow from a single cell. In particular, we focus on small populations of cells (1–100 s) where population behavior is highly stochastic and prior investigation has been limited. We compare the average strain energy density of cells in monolayers and spheroids using different growth and division rules and find that, on average, cells in spheroids have a higher strain energy density than cells in monolayers. We also find that cells in the interior of a growing spheroid are, on average, in compression. Finally, we demonstrate the importance of accounting for stochastic fluctuations in the mechanical environment, particularly when the cellular response to mechanical cues is nonlinear. The results presented here serve as a starting point for both further investigation with agent-based models, and for the incorporation of major findings from agent-based models into continuum scale models when explicit representation of individual cells is not computationally feasible.  相似文献   

9.
Multicellular tumour spheroids (MCTS) are three-dimensional cell culture systems which are widely used in cancer research. They are characterized by an outer zone of proliferating cells, an inner region of differentiating quiescent cells and an area of so-called necrotic cell death in their centre. The exact cause of this cell death, a controversy for many years, was the aim of the present study. Our data show that cell death in the centre of MCTS of three colorectal adenocarcinoma cell lines (HRT-18, HT-29 and CX-2) was induced by apoptosis. Apoptotic cells were initially distributed at random but accumulated very quickly in the quiescent and central area at day 4–5, suggesting a time- rather than size-dependent synchronization of apoptosis parallel to the formation of the proliferation gradient in MCTS. To study mechanisms inducing apoptosis, the Fas-pathway was investigated. A cell--cell contact-dependent expression of CD95 was found in all MCTS. FasL was not detected in monolayer cultures, but was expressed in spheroids of HRT-18 and CX-2. We found that TNF and TGF1 activated the CD95 pathway in all three cell lines. Since both TNF- and TGF- are known to be inducible by hypoxia in a variety of cell types, we suggest that these hypoxia-induced factors sensitize the CD95 pathway in the quiescent area of MCTS. Furthermore, a loss of the heat shock proteins 27, 32, 60, 73 and 90 was observed in the quiescent area of spheroids. This suggests that tumour cell differentiation in the inner region of MCTS may be an additional factor inducing apoptosis.  相似文献   

10.
Multicellular tumor spheroids (MCTS) are three dimensional cell culture systems induced by suspension culture. MCTS are widely used in cancer research because of their similarity to solid tumors. CaSki cells are derived from a metastatic cervical cancer containing human papillomavirus 16 (HPV16). Cell death of CaSki cells in MCTS has been previously reported, and our model is used to better characterize the mechanisms of cell death of HPV16-positive keratinocytes. In this study, we found that apoptosis of CaSki cells was induced by suspension culture along with the formation of MCTS after 24 h of incubation. In suspended CaSki cells, monoclonal antibodies blocking E-cadherin function inhibited MCTS formation and suppressed suspension-induced apoptosis in a dose-dependent manner. Western blot for E-cadherin detected upregulation of the authentic 120 kDa band from MCTS of CaSki cells as well as a shorter 100 kDa band. Addition of EGF, whose receptor is known to form a complex with E-cadherin, abrogated apoptosis of suspended CaSki cells in a dose-dependent manner. These findings suggest that E-cadherin-dependent cell–cell contact, directly or indirectly, mediates the signal to undergo apoptosis of CaSki cells during MCTS formation, and thus provides new information on the role of E-cadherin in cervical cancer cell apoptosis.  相似文献   

11.
This study addresses establishment of an "in vitro" melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRT-PCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A1 and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor microenvironment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis.  相似文献   

12.
Multicellular tumor spheroids (MCTS) are a well established 3-D in vitro model system that reflects the pathophysiological in vivo situation in tumor microregions and of avascular micrometastatic sites. Because monocytes and other immune cells infiltrate into MCTS of different origin, such spheroid co-cultures are a valuable, still underestimated tool to systematically study heterologous interactions between tumor and immune cells. The present article gives a brief overview on work that has been published on tumor - immune cell interactions in MCTS and also summarizes mechanisms of immune suppression in the tumor milieu focussing on myeloid cells. Using the co-culture model, we recently demonstrated that tumor-derived lactic acid is a potent modulator of human monocyte as lactic acid inhibited the differentiation of monocytes (MO) into dendritic cells (DC) and also impaired antigen presentation. We show herein, that the capacity of various tumor cells in MCTS to secrete lactic acid differs up to tenfold, suggesting that this capacity is dependent on the tumor cell type. It is further demonstrated that lactic acid disturbs the migration of MO into MCTS as infiltration could be increased by blocking lactic acid production. We therefore discuss lactic acid which accumulates in many tumors and tumor microregions as a potent immune suppressor for MO/DC in the tumor milieu and conclude that these data are highly relevant for adoptive immunotherapy protocols with DC.  相似文献   

13.
Multicellular tumour spheroid (MCTS) cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR). This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA) against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci.  相似文献   

14.
15.
Multicellular tumor spheroids (MCTS) are routinely employed as three-dimensional in vitro models to study tumor biology. Cultivation of MCTS in spinner flasks provides better growing conditions, especially with regard to the availability of nutrients and oxygen, when compared with microtiter plates. The main endpoint of drug response experiments is spheroid size. It is common practice to analyze spheroid size manually with a microscope and an ocular micrometer. This requires removal of some spheroids from the flask, which entails major limitations such as loss of MCTS and the risk of contamination. With this new approach, the authors present an efficient and highly reproducible method to analyze the size of complete MCTS populations in culture containers with transparent, flat bottoms. MCTS sediments are digitally scanned and spheroid volumes are calculated by computerized image analysis. The equipment includes regular office hardware (personal computer, flatbed scanner) and software (Adobe Photoshop, Microsoft Excel, ImageJ). The accuracy and precision of the method were tested using industrial precision steel beads with known diameter. In summary, in comparison with other methods, this approach provides benefits in terms of semiautomation, noninvasiveness, and low costs.  相似文献   

16.
We develop a quantitative single cell-based mathematical model for multi-cellular tumor spheroids (MCTS) of SK-MES-1 cells, a non-small cell lung cancer (NSCLC) cell line, growing under various nutrient conditions: we confront the simulations performed with this model with data on the growth kinetics and spatial labeling patterns for cell proliferation, extracellular matrix (ECM), cell distribution and cell death. We start with a simple model capturing part of the experimental observations. We then show, by performing a sensitivity analysis at each development stage of the model that its complexity needs to be stepwise increased to account for further experimental growth conditions. We thus ultimately arrive at a model that mimics the MCTS growth under multiple conditions to a great extent. Interestingly, the final model, is a minimal model capable of explaining all data simultaneously in the sense, that the number of mechanisms it contains is sufficient to explain the data and missing out any of its mechanisms did not permit fit between all data and the model within physiological parameter ranges. Nevertheless, compared to earlier models it is quite complex i.e., it includes a wide range of mechanisms discussed in biological literature. In this model, the cells lacking oxygen switch from aerobe to anaerobe glycolysis and produce lactate. Too high concentrations of lactate or too low concentrations of ATP promote cell death. Only if the extracellular matrix density overcomes a certain threshold, cells are able to enter the cell cycle. Dying cells produce a diffusive growth inhibitor. Missing out the spatial information would not permit to infer the mechanisms at work. Our findings suggest that this iterative data integration together with intermediate model sensitivity analysis at each model development stage, provide a promising strategy to infer predictive yet minimal (in the above sense) quantitative models of tumor growth, as prospectively of other tissue organization processes. Importantly, calibrating the model with two nutriment-rich growth conditions, the outcome for two nutriment-poor growth conditions could be predicted. As the final model is however quite complex, incorporating many mechanisms, space, time, and stochastic processes, parameter identification is a challenge. This calls for more efficient strategies of imaging and image analysis, as well as of parameter identification in stochastic agent-based simulations.  相似文献   

17.
Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.  相似文献   

18.
Napolitano AP  Dean DM  Man AJ  Youssef J  Ho DN  Rago AP  Lech MP  Morgan JR 《BioTechniques》2007,43(4):494, 496-494, 500
Techniques that allow cells to self-assemble into three-dimensional (3-D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular--especially in fields such as stem cell research, tissue engineering, and cancer biology. Unfortunately, caveats involving scale, expense, geometry, and practicality have hindered the widespread adoption of these techniques. We present an easy-to-use, inexpensive, and scalable technology for production of complex-shaped, 3-D microtissues. Various primary cells and immortal cell lines were utilized to demonstrate that this technique is applicable to many cell types and highlight differences in their self-assembly phenomena. When seeded onto micromolded, nonadhesive agarose gels, cells settle into recesses, the architectures of which optimize the requisite cell-to-cell interactions for spontaneous self-assembly. With one pipeting step, we were able to create hundreds of uniform spheroids whose size was determined by seeding density. Multicellular tumor spheroids (MCTS) were assembled or grown from single cells, and their proliferation was quantified using a modified 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. Complex-shaped (e.g., honeycomb) microtissues of homogeneous or mixed cell populations can be easily produced, opening new possibilities for 3-D tissue culture.  相似文献   

19.
In most instances, the growth of solid tumors occurs in constrained environments and requires a competition for space. A mechanical crosstalk can arise from this competition. In this article, we dissect the biomechanical sequence caused by a controlled compressive stress on multicellular spheroids (MCSs) used as a tumor model system. On timescales of minutes, we show that a compressive stress causes a reduction of the MCS volume, linked to a reduction of the cell volume in the core of the MCS. On timescales of hours, we observe a reversible induction of the proliferation inhibitor, p27Kip1, from the center to the periphery of the spheroid. On timescales of days, we observe that cells are blocked in the cell cycle at the late G1 checkpoint, the restriction point. We show that the effect of pressure on the proliferation can be antagonized by silencing p27Kip1. Finally, we quantify a clear correlation between the pressure-induced volume change and the growth rate of the spheroid. The compression-induced proliferation arrest that we studied is conserved for five cell lines, and is completely reversible. It demonstrates a generic crosstalk between mechanical stresses and the key players of cell cycle regulation. Our results suggest a role of volume change in the sensitivity to pressure, and that p27Kip1 is strongly influenced by this change.  相似文献   

20.
In most instances, the growth of solid tumors occurs in constrained environments and requires a competition for space. A mechanical crosstalk can arise from this competition. In this article, we dissect the biomechanical sequence caused by a controlled compressive stress on multicellular spheroids (MCSs) used as a tumor model system. On timescales of minutes, we show that a compressive stress causes a reduction of the MCS volume, linked to a reduction of the cell volume in the core of the MCS. On timescales of hours, we observe a reversible induction of the proliferation inhibitor, p27Kip1, from the center to the periphery of the spheroid. On timescales of days, we observe that cells are blocked in the cell cycle at the late G1 checkpoint, the restriction point. We show that the effect of pressure on the proliferation can be antagonized by silencing p27Kip1. Finally, we quantify a clear correlation between the pressure-induced volume change and the growth rate of the spheroid. The compression-induced proliferation arrest that we studied is conserved for five cell lines, and is completely reversible. It demonstrates a generic crosstalk between mechanical stresses and the key players of cell cycle regulation. Our results suggest a role of volume change in the sensitivity to pressure, and that p27Kip1 is strongly influenced by this change.  相似文献   

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