Growth of confined cancer spheroids : a combined experimental marker, critical modelling approach

A critical step in the dissemination of ovarian cancer is the formation of multicellular spheroids from cells shed from the primary tumour. The objectives of this study were to apply bioengineered three-dimensional (3D) microenvironments for culturing ovarian cancer spheroids in vitro and simultaneously to build on a mathematical model describing the growth of multicellular spheroids in these biomimetic matrices. Cancer cells derived from human epithelial ovarian carcinoma were embedded within biomimetic hydrogels of varying stiffness and grown for up to 4 weeks. Immunohistochemistry, imaging and growth analyses were used to quantify the dependence of cell proliferation and apoptosis on matrix stiffness, long-term culture and treatment with the anti-cancer drug paclitaxel. The mathematical model was formulated as a free boundary problem in which each spheroid was treated as an incompressible porous medium. The functional forms used to describe the rates of cell proliferation and apoptosis were motivated by the experimental work and predictions of the mathematical model compared with the experimental output. This work aimed to establish whether it is possible to simulate solid tumour growth on the basis of data on spheroid size, cell proliferation and cell death within these spheroids. The mathematical model predictions were in agreement with the experimental data set and simulated how the growth of cancer spheroids was influenced by mechanical and biochemical stimuli including matrix stiffness, culture duration and administration of a chemotherapeutic drug. Our computational model provides new perspectives on experimental results and has informed the design of new 3D studies of chemoresistance of multicellular cancer spheroids.

Gelatine methacrylamide-based hydrogels : an alternative three-dimensional cancer cell culture system

Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5-7% w/v) strongly influenced hydrogel stiffness (0.5±0.2kPa to 9.0±1.8kPa) but had little effect on solute diffusion. The diffusion coefficient of 70kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33-37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.

Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.

A multiscale road map of cancer spheroids : incorporating experimental and mathematical modelling to understand cancer progression

Computational models represent a highly suitable framework, not only for testing biological hypotheses and generating new ones but also for optimising experimental strategies. As one surveys the literature devoted to cancer modelling, it is obvious that immense progress has been made in applying simulation techniques to the study of cancer biology, although the full impact has yet to be realised. For example, there are excellent models to describe cancer incidence rates or factors for early disease detection, but these predictions are unable to explain the functional and molecular changes that are associated with tumour progression. In addition, it is crucial that interactions between mechanical effects, and intracellular and intercellular signalling are incorporated in order to understand cancer growth, its interaction with the extracellular microenvironment and invasion of secondary sites. There is a compelling need to tailor new, physiologically relevant in silico models that are specialised for particular types of cancer, such as ovarian cancer owing to its unique route of metastasis, which are capable of investigating anti-cancer therapies, and generating both qualitative and quantitative predictions. This Commentary will focus on how computational simulation approaches can advance our understanding of ovarian cancer progression and treatment, in particular, with the help of multicellular cancer spheroids, and thus, can inform biological hypothesis and experimental design.

Paclitaxel resistance and multicellular spheroid formation are induced by kallikrein-related peptidase 4 in serous ovarian cancer cells in an ascites mimicking microenvironment

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease. © 2011 Elsevier Ltd. All rights reserved.

The effect of nitroxides on swarming motility and biofilms, multicellular behaviors in Pseudomonas aeruginosa

The ability of NO to induce biofilm dispersion has been well established. Here we investigated the effect of nitroxides (sterically hindered nitric oxide analogues) on biofilm formation and swarming motility in Pseudomonas aeruginosa. A transposon mutant unable to produce nitric oxide endogenously (nirS) was deficient in swarming motility relative to wild type and the complemented strain. Moreover, expression of the nirS gene was up-regulated by 9.65-fold in wild type swarming cells when compared to planktonic cells. Wild type swarming levels were substantially restored upon exogenous addition of nitroxide containing compounds, consistent with the hypothesis that NO is necessary for swarming motility. Here, we showed that nitroxides not only mimicked the dispersal activity of NO, but also prevented biofilms from forming in flow cell chambers. In addition, a nirS transposon mutant was deficient in biofilm formation relative to wild type and the complemented strain, thus implicating NO in the formation of biofilms. Intriguingly despite its stand alone action in inhibiting biofilm formation and promoting dispersal, a nitroxide partially restored the ability of a nirS mutant to form biofilms.

The rapid manufacture of uniform composite multicellular-biomaterial micropellets, their assembly into macroscopic organized tissues, and potential applications in cartilage tissue engineering

We and others have published on the rapid manufacture of micropellet tissues, typically formed from 100-500 cells each. The micropellet geometry enhances cellular biological properties, and in many cases the micropellets can subsequently be utilized as building blocks to assemble complex macrotissues. Generally, micropellets are formed from cells alone, however when replicating matrix-rich tissues such as cartilage it would be ideal if matrix or biomaterials supplements could be incorporated directly into the micropellet during the manufacturing process. Herein we describe a method to efficiently incorporate donor cartilage matrix into tissue engineered cartilage micropellets. We lyophilized bovine cartilage matrix, and then shattered it into microscopic pieces having average dimensions < 10 μm diameter; we termed this microscopic donor matrix "cartilage dust (CD)". Using a microwell platform, we show that ~0.83 μg CD can be rapidly and efficiently incorporated into single multicellular aggregates formed from 180 bone marrow mesenchymal stem/stromal cells (MSC) each. The microwell platform enabled the rapid manufacture of thousands of replica composite micropellets, with each micropellet having a material/CD core and a cellular surface. This micropellet organization enabled the rapid bulking up of the micropellet core matrix content, and left an adhesive cellular outer surface. This morphological organization enabled the ready assembly of the composite micropellets into macroscopic tissues. Generically, this is a versatile method that enables the rapid and uniform integration of biomaterials into multicellular micropellets that can then be used as tissue building blocks. In this study, the addition of CD resulted in an approximate 8-fold volume increase in the micropellets, with the donor matrix functioning to contribute to an increase in total cartilage matrix content. Composite micropellets were readily assembled into macroscopic cartilage tissues; the incorporation of CD enhanced tissue size and matrix content, but did not enhance chondrogenic gene expression.

A hybrid cellular automata model of multicellular tumour spheroid growth in hypoxic microenvironment

A three-dimensional hybrid cellular automata (CA) model is developed to study the dynamic process of multicellular tumour spheroid (MTS) growth by introducing hypoxia as an important microenvironment factor which influences cell migration and cell phenotype expression. The model enables us to examine the effects of different hypoxic environments on the growth history of the MTS and to study the dynamic interactions between MTS growth and chemical environments. The results include the spatial distribution of different phenotypes of tumour cells and associated oxygen concentration distributions under hypoxic conditions. The discussion of the model system responses to the varied hypoxic conditions reveals that the improvement of the resistance of tumour cells to a hypoxic environment may be important in the tumour normalization therapy.

Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

Engineering an Ecosystem : Taking Cues from Nature’s Paradigm to Build Tissue in the Lab and the Body

This manuscript took a 'top down' approach to understanding survival of inhabitant cells in the ecosystem bone, working from higher to lower length and time scales through the hierarchical ecosystem of bone. Our working hypothesis is that nature “engineered” the skeleton using a 'bottom up' approach,where mechanical properties of cells emerge from their adaptation to their local me-chanical milieu. Cell aggregation and formation of higher order anisotropic struc- ture results in emergent architectures through cell differentiation and extracellular matrix secretion. These emergent properties, including mechanical properties and architecture, result in mechanical adaptation at length scales and longer time scales which are most relevant for the survival of the vertebrate organism [Knothe Tate and von Recum 2009]. We are currently using insights from this approach to har-ness nature’s regeneration potential and to engineer novel mechanoactive materials [Knothe Tate et al. 2007, Knothe Tate et al. 2009]. In addition to potential applications of these exciting insights, these studies may provide important clues to evolution and development of vertebrate animals. For instance, one might ask why mesenchymal stem cells condense at all? There is a putative advantage to self-assembly and cooperation, but this advantage is somewhat outweighed by the need for infrastructural complexity (e.g., circulatory systems comprised of specific differentiated cell types which in turn form conduits and pumps to overcome limitations of mass transport via diffusion, for example; dif-fusion is untenable for multicellular organisms larger than 250 microns in diameter. A better question might be: Why do cells build skeletal tissue? Once cooperatingcells in tissues begin to deplete local sources of food in their aquatic environment, those that have evolved a means to locomote likely have an evolutionary advantage. Once the environment becomes less aquarian and more terrestrial, self-assembled organisms with the ability to move on land might have conferred evolutionary ad-vantages as well. So did the cytoskeleton evolve several length scales, enabling the emergence of skeletal architecture for vertebrate animals? Did the evolutionary advantage of motility over noncompliant terrestrial substrates (walking on land) favor adaptations including emergence of intracellular architecture (changes in the cytoskeleton and upregulation of structural protein manufacture), inter-cellular con- densation, mineralization of tissues, and emergence of higher order architectures?How far does evolutionary Darwinism extend and how can we exploit this knowl- edge to engineer smart materials and architectures on Earth and new, exploratory environments?[Knothe Tate et al. 2008]. We are limited only by our ability to imagine. Ultimately, we aim to understand nature, mimic nature, guide nature and/or exploit nature’s engineering paradigms without engineer-ing ourselves out of existence.

Generalized binomial Tau-leap method for biochemical kinetics incorporating both delay and intrinsic noise

The delay stochastic simulation algorithm (DSSA) by Barrio et al. [Plos Comput. Biol.2, 117–E (2006)] was developed to simulate delayed processes in cell biology in the presence of intrinsic noise, that is, when there are small-to-moderate numbers of certain key molecules present in a chemical reaction system. These delayed processes can faithfully represent complex interactions and mechanisms that imply a number of spatiotemporal processes often not explicitly modeled such as transcription and translation, basic in the modeling of cell signaling pathways. However, for systems with widely varying reaction rate constants or large numbers of molecules, the simulation time steps of both the stochastic simulation algorithm (SSA) and the DSSA can become very small causing considerable computational overheads. In order to overcome the limit of small step sizes, various τ-leap strategies have been suggested for improving computational performance of the SSA. In this paper, we present a binomial τ- DSSA method that extends the τ-leap idea to the delay setting and avoids drawing insufficient numbers of reactions, a common shortcoming of existing binomial τ-leap methods that becomes evident when dealing with complex chemical interactions. The resulting inaccuracies are most evident in the delayed case, even when considering reaction products as potential reactants within the same time step in which they are produced. Moreover, we extend the framework to account for multicellular systems with different degrees of intercellular communication. We apply these ideas to two important genetic regulatory models, namely, the hes1 gene, implicated as a molecular clock, and a Her1/Her 7 model for coupled oscillating cells.