Agent-based computational modeling of wounded epithelial cell monolayers

Computational modeling of biological systems, or ‘in silico biology’ is an emerging tool for understanding structure and order in biological tissues. Computational models of the behavior of epithelial cells in monolayer cell culture have been developed and used to predict the healing characteristics of scratch wounds made to urothelial cell cultures maintained in low and physiological [Ca2+] environments. Both computational models and in vitro experiments demonstrated that in low exogenous [Ca2+], the closure of 500mm scratch wounds was achieved primarily by cell migration into the denuded area. The wound healing rate in low (0.09mM) [Ca2+] was approximately twice as rapid as in physiological (2mM) [Ca2+]. Computational modeling predicted that in cell cultures that are actively proliferating, no increase in the fraction of cells in S-phase would be expected, and this conclusion was supported experimentally in vitro by BrdU incorporation assay. We have demonstrated that a simple rule-based model of cell behavior, incorporating rules relating to contact inhibition of proliferation and migration, is sufficient to qualitatively predict the calcium-dependent pattern of wound closure observed in vitro. Differences between the in vitro and in silico models suggest a role for wound-induced signaling events in urothelial cell cultures

Quantifying cancer epithelial-mesenchymal plasticity and its association with stemness and immune response

Cancer cells can acquire a spectrum of stable hybrid epithelial/mesenchymal (E/M) states during epithelial-mesenchymal transition (EMT). Cells in these hybrid E/M phenotypes often combine epithelial and mesenchymal features and tend to migrate collectively commonly as small clusters. Such collectively migrating cancer cells play a pivotal role in seeding metastases and their presence in cancer patients indicates an adverse prognostic factor. Moreover, cancer cells in hybrid E/M phenotypes tend to be more associated with stemness which endows them with tumor-initiation ability and therapy resistance. Most recently, cells undergoing EMT have been shown to promote immune suppression for better survival. A systematic understanding of the emergence of hybrid E/M phenotypes and the connection of EMT with stemness and immune suppression would contribute to more effective therapeutic strategies. In this review, we first discuss recent efforts combining theoretical and experimental approaches to elucidate mechanisms underlying EMT multi-stability (i.e. the existence of multiple stable phenotypes during EMT) and the properties of hybrid E/M phenotypes. Following we discuss non-cell-autonomous regulation of EMT by cell cooperation and extracellular matrix. Afterwards, we discuss various metrics that can be used to quantify EMT spectrum. We further describe possible mechanisms underlying the formation of clusters of circulating tumor cells. Last but not least, we summarize recent systems biology analysis of the role of EMT in the acquisition of stemness and immune suppression.Comment: 50 pages, 6 figure

Colorectal Cancer Through Simulation and Experiment

Colorectal cancer has continued to generate a huge amount of research interest over several decades, forming a canonical example of tumourigenesis since its use in Fearon and Vogelstein’s linear model of genetic mutation. Over time, the field has witnessed a transition from solely experimental work to the inclusion of mathematical biology and computer-based modelling. The fusion of these disciplines has the potential to provide valuable insights into oncologic processes, but also presents the challenge of uniting many diverse perspectives. Furthermore, the cancer cell phenotype defined by the ‘Hallmarks of Cancer’ has been extended in recent times and provides an excellent basis for future research. We present a timely summary of the literature relating to colorectal cancer, addressing the traditional experimental findings, summarising the key mathematical and computational approaches, and emphasising the role of the Hallmarks in current and future developments. We conclude with a discussion of interdisciplinary work, outlining areas of experimental interest which would benefit from the insight that mathematical and computational modelling can provide

Neoplastic transformation of porcine mammary epithelial cells in vitro and tumor formation in vivo.

BackgroundThe mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo.MethodsWe utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire -Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl) /SzJ (NSG) mice.ResultsWe demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo.ConclusionsHerein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers

Exploring hypotheses of the actions of TGF-beta 1 in epidermal wound healing using a 3D computational multiscale model of the human epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-beta 1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-beta 1 literature-derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units ( keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-beta 1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged ( by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-beta 1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-beta 1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing

The receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis

PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here we demonstrate that PTPRB negatively regulates branching morphogenesis in the mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in estrogen receptor positive luminal cells. During mammary development Ptprb expression is down-regulated during puberty, a period of extensive of ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of FGFR activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology

Agent based modelling helps in understanding the rules by which fibroblasts support keratinocyte colony formation

Background: Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings: A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions: A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine serum

25 years of epidermal stem cell research.

This is a chronicle of concepts in the field of epidermal stem cell biology and a historic look at their development over time. The past 25 years have seen the evolution of epidermal stem cell science, from first fundamental studies to a sophisticated science. The study of epithelial stem cell biology was aided by the ability to visualize the distribution of stem cells and their progeny through lineage analysis studies. The excellent progress we have made in understanding epidermal stem cell biology is discussed in this article. The challenges we still face in understanding epidermal stem cells include defining molecular markers for stem and progenitor sub-populations, determining the locations and contributions of the different stem cell niches, and mapping regulatory pathways of epidermal stem cell proliferation and differentiation. However, our rapidly evolving understanding of epidermal stem cells has many potential uses that promise to translate into improved patient therapy

Biofilm-stimulated epithelium modulates the inflammatory responses in co-cultured immune cells

The gingival epithelium is a physical and immunological barrier to the microbiota of the oral cavity, which interact through soluble mediators with the immune cells that patrol the tissue at the gingival epithelium. We sought to develop a three-dimensional gingivae-biofilm interface model using a commercially available gingival epithelium to study the tissue inflammatory response to oral biofilms associated with “health”, “gingivitis” and “periodontitis”. These biofilms were developed by sequential addition of microorganisms to mimic the formation of supra- and sub-gingival plaque in vivo. Secondly, to mimic the interactions between gingival epithelium and immune cells in vivo, we integrated peripheral blood mononuclear cells and CD14+ monocytes into our three-dimensional model and were able to assess the inflammatory response in the immune cells cultured with and without gingival epithelium. We describe a differential inflammatory response in immune cells cultured with epithelial tissue, and more so following incubation with epithelium stimulated by “gingivitis-associated” biofilm. These results suggest that gingival epithelium-derived soluble mediators may control the inflammatory status of immune cells in vitro, and therefore targeting of the epithelial response may offer novel therapies. This multi-cellular interface model, both of microbial and host origin, offers a robust in vitro platform to investigate host-pathogens at the epithelial surface