Cancer cell invasion is enhanced by applied mechanical stimulation

Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor.     

Cell behavior observation and gene expression analysis of melanoma associated with stromal fibroblasts in a three‐dimensional magnetic cell culture array

A three‐dimensional (3D) multicellular tumor spheroid culture array has been fabricated using a magnetic force‐based cell patterning method, analyzing the effect of stromal fibroblast on the invasive capacity of melanoma. Formation of spheroids was observed when array‐like multicellular patterns of melanoma were developed using a pin‐holder device made of magnetic soft iron and an external magnet, which enables the assembly of the magnetically labeled cells on the collagen gel‐coated surface as array‐like cell patterns. The interaction of fibroblast on the invasion of melanoma was investigated using three types of cell interaction models: (i) fibroblasts were magnetically labeled and patterned together in array with melanoma spheroids (direct‐interaction model), (ii) fibroblasts coexisting in the upper collagen gel (indirect‐interaction model) of melanoma spheroids, and (iii) fibroblast‐sheets coexisting under melanoma spheroids (fibroblast‐sheet model). The fibroblast‐sheet model has largely increased the invasive capacity of melanoma, and the promotion of adhesion, migration, and invasion were also observed. In the fibroblast‐sheet model, the expression of IL‐8 and MMP‐2 increased by 24‐fold and 2‐fold, respectively, in real time RT‐PCR compared to the absence of fibroblasts. The results presented in this study demonstrate the importance of fibroblast interaction to invasive capacity of melanoma in the 3D in vitro bioengineered tumor microenvironment. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Quantitative analysis of glioma cell invasion by confocal laser scanning microscopy in a novel brain slice model

To quantitatively analyze the spatial extent of glioma cell migration in an organotypic brain slice culture, we developed a new invasion model with the aid of confocal laser scanning microscopy (CLSM). CLSM allowed not only for three-dimensional visualization of the invasive pattern of human T98G glioma cells in the living brain slice but also for serial analysis of the invasive process over several weeks. Twenty-four hours after the T98G glioma spheroid was initiated to coculture with a brain slice, the glioma cells detached themselves from the spheroid and spontaneously continued to migrate on the surface of the brain slice, while they diffusely invaded into the slice by migrating to a deeper site. Immunohistochemical analysis revealed that these migrating glioma cells much more strongly immunostained for matrix metalloproteinase (MMP)-2 and -9 than the tumor spheroid which remained at the implanted site. Treatment of the T98G glioma spheroid with 1,10-phenanthroline, a specific inhibitor of MMPs, significantly inhibited not only the cell migration on the surface of the brain slice but also the invasion of the glioma cells into the slice. The present version of the glioma invasion model using CLSM makes it possible to spatially and serially analyze the extent of glioma cell invasion in the living brain slice for several weeks, making it a very useful tool for investigating the cellular and molecular mechanisms of glioma invasion under conditions most analogous to those of normal brains in vivo.     

A mathematical model of glioblastoma tumor spheroid invasion in a three-dimensional in vitro experiment

Glioblastoma, the most malignant form of brain cancer, is responsible for 23% of primary brain tumors and has extremely poor outcome. Confounding the clinical management of glioblastomas is the extreme local invasiveness of these cancer cells. The mechanisms that govern invasion are poorly understood. To gain insight into glioblastoma invasion, we conducted experiments on the patterns of growth and dispersion of U87 glioblastoma tumor spheroids in a three-dimensional collagen gel. We studied two different cell lines, one with a mutation to the EGFR (U87DeltaEGFR) that is associated with increased malignancy, and one with an endogenous (wild-type) receptor (U87WT). We developed a continuum mathematical model of the dispersion behaviors with the aim of identifying and characterizing discrete cellular mechanisms underlying invasive cell motility. The mathematical model quantitatively reproduces the experimental data, and indicates that the U87WT invasive cells have a stronger directional motility bias away from the spheroid center as well as a faster rate of cell shedding compared to the U87DeltaEGFR cells. The model suggests that differences in tumor cell dispersion may be due to differences in the chemical factors produced by cells, differences in how the two cell lines remodel the gel, or different cell-cell adhesion characteristics.     

Evidence for a secreted chemorepellent that directs glioma cell invasion

Secreted chemotropic cues guide the migration of neuronal and glial cell precursors during neural development. It is not known if chemotropism contributes to directing the invasion of brain tissue by glioma cells. A model system has been developed that allows quantification of invasive behavior using gliomas spheroids embedded in collagen gels. Here we provide evidence that glioma spheroids secrete a chemorepellent factor(s) that directs cells away from the spheroid and into the collagen matrix. The relationship between total invasion, cell number, and implantation distance suggests that glioma cells respond to a gradient of the chemorepellent cue(s) that is well established at 48 h. C6 astrocytoma cells normally invade the collagen at an angle perpendicular to the spheroid edge. In contrast, an adjacent spheroid causes cells to turn away from their normal trajectory and slow their rate of invasion. Astrocytoma cells are repelled by an adjacent glioma spheroid but rapidly infiltrate astrocyte aggregates, indicating that astrocytes do not express the repellent cue. Uniform concentrations of repellent factor(s) in spheroid conditioned medium overwhelm endogenous gradients and render glioma cells less able to exhibit this chemotropic response. Concentration gradients of spheroid conditioned medium in cell migration assays also demonstrate the chemorepellent cue(s)'s tropic effect. Our findings indicate that glioma spheroids produce a secreted diffusible cue(s) that promotes glioma cell invasion. Identification of this factor(s) may advance current therapies that aim to limit tumor cell invasion.     

A 50 Hz sinusoidal magnetic field does not damage MG-63 three-dimensional tumor spheroids but induces changes in their invasive properties

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 1 mT can damage MG-63 osteosarcoma spheroids and induce variations in the invasive properties of these three-dimensional model systems after 2 days of exposure was investigated. Specifically, possible damage induced by these fields was examined by determining changes in spheroid surface morphology (light microscopy), growth (spheroid diameter and protein content determination), lactate dehydrogenase release, and reduced glutathione amount. Possible changes in the invasive properties were studied by invasion chambers. The results show no induction of cell damage by ELF fields while invasion chamber assays demonstrate a significant increase in the invasive potential of exposed spheroids. In order to determine if the fibronectin or hyaluronan receptors are involved, Western blot analysis was conducted on these two proteins. No significant variations were observed in either receptor in MG-63 multicellular tumor spheroids.     

Breast Cancer Cell Line Aggregate Morphology Does Not Predict Invasive Capacity

To invade and metastasize to distant loci, breast cancer cells must breach the layer of basement membrane surrounding the tumor and then invade through the dense collagen I-rich extracellular environment of breast tissue. Previous studies have shown that breast cancer cell aggregate morphology in basement membrane extract correlated with cell invasive capacity in some contexts. Moreover, cell lines from the same aggregate morphological class exhibited similarities in gene expression patterns. To further assess the capacity of cell and aggregate morphology to predict invasive capacity in physiologically relevant environments, six cell lines with varied cell aggregate morphologies were assessed in a variety of assays including a 3D multicellular invasion assay that recapitulates cell-cell and cell-environment contacts as they exist in vivo in the context of the primary breast tumor. Migratory and invasive capacities as measured through a 2D gap assay and a 3D spheroid invasion assay reveal that breast cancer cell aggregate morphology alone is insufficient to predict migratory speed in 2D or invasive capacity in 3D. Correlations between the 3D spheroid invasion assay and gene expression profiles suggest this assay as an inexpensive functional method to predict breast cancer invasive capacity.     

Three-Dimensional Quantification of Spheroid Degradation-Dependent Invasion and Invadopodia Formation

Invadopodia are actin-rich, proteolytic structures that enable cancer cell to invade into the surrounding tissues. Several in vitro invasion assays have been used in the literature ranging from directional quantitative assays to complex three-dimensional (3D) analyses. One of the main limitations of these assays is the lack of quantifiable degradation-dependent invasion in a three-dimensional (3D) environment that mimics the tumor microenvironment. In this article, we describe a new invasion and degradation assay based on the currently available tumor spheroid model that allows long-term high-resolution imaging of the tumor, precise quantification, and visualization of matrix degradation and multichannel immunocytochemistry. By incorporating a degradation marker (DQ-Green BSA) into a basement-membrane matrix, we demonstrate the ability to quantitate cancer cell-induced matrix degradation in 3D. Also, we describe a technique to generate histological sections of the tumor spheroid allowing the detection of invadopodia formation in the 3D tumor spheroid. This new technique provides a clear advantage for studying cancer in vitro and will help address critical questions regarding the dynamics of cancer cell invasion.     

Regulation of invadopodia by the tumor microenvironment

The tumor microenvironment consists of stromal cells, extracellular matrix (ECM), and signaling molecules that communicate with cancer cells. As tumors grow and develop, the tumor microenvironment changes. In addition, the tumor microenvironment is not only influenced by signals from tumor cells, but also stromal components contribute to tumor progression and metastasis by affecting cancer cell function. One of the mechanisms that cancer cells use to invade and metastasize is mediated by actin-rich, proteolytic structures called invadopodia. Here, we discuss how signals from the tumor environment, including growth factors, hypoxia, pH, metabolism, and stromal cell interactions, affect the formation and function of invadopodia to regulate cancer cell invasion and metastasis. Understanding how the tumor microenvironment affects invadopodia biology could aid in the development of effective therapeutics to target cancer cell invasion and metastasis.     

Regulation of invadopodia by the tumor microenvironment

The tumor microenvironment consists of stromal cells, extracellular matrix (ECM), and signaling molecules that communicate with cancer cells. As tumors grow and develop, the tumor microenvironment changes. In addition, the tumor microenvironment is not only influenced by signals from tumor cells, but also stromal components contribute to tumor progression and metastasis by affecting cancer cell function. One of the mechanisms that cancer cells use to invade and metastasize is mediated by actin-rich, proteolytic structures called invadopodia. Here, we discuss how signals from the tumor environment, including growth factors, hypoxia, pH, metabolism, and stromal cell interactions, affect the formation and function of invadopodia to regulate cancer cell invasion and metastasis. Understanding how the tumor microenvironment affects invadopodia biology could aid in the development of effective therapeutics to target cancer cell invasion and metastasis.     

The functional cross talk between cancer cells and cancer associated fibroblasts from a cancer mechanics perspective

The function of biological tissues in health and disease is regulated at cellular level and is highly influenced by the physical microenvironment, through the interaction of forces between cells and ECM, which are perceived through mechanosensing pathways. In cancer, both chemical and physical signaling cascades and their interactions are involved during cell-cell and cell-ECM communications to meet requirements of tumor growth. Among stroma cells, cancer associated fibroblasts (CAFs) play key role in tumor growth and pave the way for cancer cells to initiate metastasis and invasion to other tissues, and without recruitment of CAFs, the process of cancer invasion is dysfunctional. This is through an intense chemical and physical cross talks with tumor cells, and interactive remodeling of ECM. During such interaction CAFs apply traction forces and depending on the mechanical properties, deform ECM and in return receive physical signals from the micromechanical environment. Such interaction leads to ECM remodeling by manipulating ECM structure and its mechanical properties. The results are in form of deposition of extra fibers, stiffening, rearrangement and reorganization of fibrous structure, and degradation which are due to a complex secretion and expression of different markers triggered by mechanosensing of tumor cells, specially CAFs. Such events define cancer progress and invasion of cancer cells.A systemic knowledge of chemical and physical factors provides a holistic view of how cancer process and enhances the current treatment methods to provide more diversity among targets that involves tumor cells and ECM structure.     

Encapsulated human hepatocellular carcinoma cells by alginate gel beads as an in vitro metastasis model

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and often forms metastases, which are the most important prognostic factors. For further elucidation of the mechanism underlying the progression and metastasis of HCC, a culture system mimicking the in vivo tumor microenvironment is needed. In this study, we investigated the metastatic ability of HCC cells cultured within alginate gel (ALG) beads. In the culture system, HCC cells formed spheroids by proliferation and maintained in nuclear abnormalities. The gene and protein expression of metastasis-related molecules was increased in ALG beads, compared with the traditional adhesion culture. Furthermore, several gene expression levels in ALG bead culture system were even closer to liver cancer tissues. More importantly, in vitro invasion assay showed that the invasion cells derived from ALG beads was 7.8-fold higher than adhesion cells. Our results indicated that the in vitro three-dimensional (3D) model based on ALG beads increased metastatic ability compared with adhesion culture, even partly mimicked the in vivo tumor tissues. Moreover, due to the controllable preparation conditions, steady characteristics and production at large-scale, the 3D ALG bead model would become an important tool used in the high-throughput screening of anti-metastasis drugs and the metastatic mechanism research.     

Tumor morphology and phenotypic evolution driven by selective pressure from the microenvironment

Emergence of invasive behavior in cancer is life-threatening, yet ill-defined due to its multifactorial nature. We present a multiscale mathematical model of cancer invasion, which considers cellular and microenvironmental factors simultaneously and interactively. Unexpectedly, the model simulations predict that harsh tumor microenvironment conditions (e.g., hypoxia, heterogenous extracellular matrix) exert a dramatic selective force on the tumor, which grows as an invasive mass with fingering margins, dominated by a few clones with aggressive traits. In contrast, mild microenvironment conditions (e.g., normoxia, homogeneous matrix) allow clones with similar aggressive traits to coexist with less aggressive phenotypes in a heterogeneous tumor mass with smooth, noninvasive margins. Thus, the genetic make-up of a cancer cell may realize its invasive potential through a clonal evolution process driven by definable microenvironmental selective forces. Our mathematical model provides a theoretical/experimental framework to quantitatively characterize this selective pressure for invasion and test ways to eliminate it.     

Emergent behaviors from a cellular automaton model for invasive tumor growth in heterogeneous microenvironments

Understanding tumor invasion and metastasis is of crucial importance for both fundamental cancer research and clinical practice. In vitro experiments have established that the invasive growth of malignant tumors is characterized by the dendritic invasive branches composed of chains of tumor cells emanating from the primary tumor mass. The preponderance of previous tumor simulations focused on non-invasive (or proliferative) growth. The formation of the invasive cell chains and their interactions with the primary tumor mass and host microenvironment are not well understood. Here, we present a novel cellular automaton (CA) model that enables one to efficiently simulate invasive tumor growth in a heterogeneous host microenvironment. By taking into account a variety of microscopic-scale tumor-host interactions, including the short-range mechanical interactions between tumor cells and tumor stroma, degradation of the extracellular matrix by the invasive cells and oxygen/nutrient gradient driven cell motions, our CA model predicts a rich spectrum of growth dynamics and emergent behaviors of invasive tumors. Besides robustly reproducing the salient features of dendritic invasive growth, such as least-resistance paths of cells and intrabranch homotype attraction, we also predict nontrivial coupling between the growth dynamics of the primary tumor mass and the invasive cells. In addition, we show that the properties of the host microenvironment can significantly affect tumor morphology and growth dynamics, emphasizing the importance of understanding the tumor-host interaction. The capability of our CA model suggests that sophisticated in silico tools could eventually be utilized in clinical situations to predict neoplastic progression and propose individualized optimal treatment strategies.     

Small-molecule agonists of mammalian Diaphanous–related (mDia) formins reveal an effective glioblastoma anti-invasion strategy

The extensive invasive capacity of glioblastoma (GBM) makes it resistant to surgery, radiotherapy, and chemotherapy and thus makes it lethal. In vivo, GBM invasion is mediated by Rho GTPases through unidentified downstream effectors. Mammalian Diaphanous (mDia) family formins are Rho-directed effectors that regulate the F-actin cytoskeleton to support tumor cell motility. Historically, anti-invasion strategies focused upon mDia inhibition, whereas activation remained unexplored. The recent development of small molecules directly inhibiting or activating mDia-driven F-actin assembly that supports motility allows for exploration of their role in GBM. We used the formin inhibitor SMIFH2 and mDia agonists IMM-01/-02 and mDia2-DAD peptides, which disrupt autoinhibition, to examine the roles of mDia inactivation versus activation in GBM cell migration and invasion in vitro and in an ex vivo brain slice invasion model. Inhibiting mDia suppressed directional migration and spheroid invasion while preserving intrinsic random migration. mDia agonism abrogated both random intrinsic and directional migration and halted U87 spheroid invasion in ex vivo brain slices. Thus mDia agonism is a superior GBM anti-invasion strategy. We conclude that formin agonism impedes the most dangerous GBM component—tumor spread into surrounding healthy tissue. Formin activation impairs novel aspects of transformed cells and informs the development of anti-GBM invasion strategies.     

靶向纤溶酶原激活物抑制剂1的肿瘤治疗研究进展

肿瘤是严重危害人类健康的疾病.研究表明,实体瘤周围环境中的胞外基质蛋白、浸润性免疫细胞和间充质细胞分泌的蛋白质组等均与肿瘤的发生、发展以及肿瘤治疗的耐受性等密切相关.肿瘤微环境中一个重要调控因子,纤溶酶原激活物抑制剂1 (plasminogen activator inhibitor-1,PAI-1),不仅与组织型纤溶酶原激活物(tissue-type plasminogen activators,tPA)构成调节纤溶活性的一对关键物质,而且参与肿瘤的侵袭、浸润和转移等多个环节并扮演重要角色.本文针对近年来PAI-1的结构和功能方面研究新进展及其与肿瘤微环境的相关性进行综述,并提出PAI-1可作为抗肿瘤治疗的重要靶点.同时,本文也分析了PAI-1抑制剂对肿瘤干预的研究现状,指出PAI-1抑制剂对肿瘤治疗的潜在应用价值.     

成纤维细胞激活蛋白α与肿瘤发展进程的研究

肿瘤微环境对肿瘤的发生、发展具有重要的意义。选择性表达于肿瘤微环境重要组成部分——肿瘤相关成纤维细胞（carcinoma associated fibroblasts,CAFs）表面的成纤维细胞激活蛋白α（fibroblast activation protein-α,FAPα）广泛参与了肿瘤的生长、侵袭、转移以及肿瘤细胞外基质重建、血管生成、免疫逃逸等过程,从而促进了肿瘤的发展进程。FAPα具有蛋白水解酶活性,并作用于细胞信号通路,但FAPα在肿瘤微环境中发挥功能的具体分子机制还有待进一步研究。由于FAPα的表达具有肿瘤组织特异性,因此,以FAPα作为肿瘤基质标志物,对肿瘤进行病理诊断和免疫治疗将成为新兴的研究靶点。对FAPα的主要生物学性状进行概述,并综述了其对肿瘤细胞的生长、侵袭、转移以及肿瘤细胞外基质重建、血管生成、免疫逃逸等方面的重要影响。     

Air-liquid interface promotes invasive growth of laryngeal squamous cell carcinoma with or without hypoxia

Air-liquid interface (ALI) is a microenvironment of aerodigestive tract. In our previous study, ALI promoted invasive growth of laryngeal squamous cell carcinoma (SCC); but its mechanism was unclear. Hypoxia is also related to cancer spread. Here we show that ALI with or without hypoxia accelerated invasive growth of laryngeal SCC cells, using collagen gel invasion assay. Submerged condition (SMC) without ALI did not induce the invasion with or without hypoxia. ALI enhanced expression of the following growth-, invasion-, and motility-related molecules in the cells with or without hypoxia more greatly than SMC: c-Met, Ras, mitogen-activated protein kinase cascade proteins (Raf-1, MEK-1, and ERK-1/2), matrix metalloproteinase-1, and filamin A. The data indicate that ALI promotes invasive growth of SCC cells by enhancement of the invasive growth-related molecules above, through mechanisms that differ from hypoxia, suggesting that ALI microenvironment should be taken into account for the study of cancer biology.     

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.     

炎症-肿瘤转化在眼部相关疾病中的研究进展

炎症向癌症转化的机制一直是癌症研究中的重点。作为炎症-肿瘤转化起始时所处的环境，炎性微环境是一个多种调控因子、细胞的大集合，其中包含的肿瘤干细胞、肿瘤相关巨噬细胞以及细胞因子（如趋化因子、生长因子）等在常见的眼部肿瘤中对肿瘤的起始、发生、演进乃至恶性转化和转移的过程起到了至关重要的调控作用。基于此，主要讨论了在炎性微环境中的肿瘤相关细胞、细胞因子以及细胞外基质等对肿瘤细胞的增殖、转移、浸润、侵袭过程的影响，着重探讨了眼部炎症-肿瘤转化相关的分子机制；并综述了视网膜母细胞瘤、腺样囊性癌等常见眼部肿瘤的特征及其由炎症到肿瘤发生过程中起重要调控作用的分子；最后，针对这些眼部肿瘤普遍存在的信号通路和分子靶点做出了对未来诊断及治疗方法的展望，以期在今后对眼科肿瘤的诊治过程中，能够针对提及的炎性成分设计思路，最大化防止炎症-肿瘤转化和恶性转归出现。