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High endothelial venules (HEV) are specialized post capillary venules that recruit naïve T cells and B cells into secondary lymphoid organs (SLO) such as lymph nodes (LN). Expansion of HEV networks in SLOs occurs following immune activation to support development of an effective immune response. In this study, we used a carcinogen-induced model of fibrosarcoma to examine HEV remodeling after depletion of regulatory T cells (Treg). We used light sheet fluorescence microscopy imaging to visualize entire HEV networks, subsequently applying computational tools to enable topological mapping and extraction of numerical descriptors of the networks. While these analyses revealed profound cancer- and immune-driven alterations to HEV networks within LNs, these changes did not identify successful responses to treatment. The presence of HEV networks within tumors did however clearly distinguish responders from nonresponders. Finally, we show that a successful treatment response is dependent on coupling tumor-associated HEV (TA-HEV) development to T-cell activation implying that T-cell activation acts as the trigger for development of TA-HEVs which subsequently serve to amplify the immune response by facilitating extravasation of T cells into the tumor mass. Significance: We used three-dimensional imaging methods with computational tools to analyze networks of specialized blood vessels called HEVs in LNs and tumors. By applying these techniques in a mouse model of carcinogen-induced tumors, we could identify network changes after depletion of Tregs.

Naïve LN HEV Characterisation.

High endothelial venules (HEV) are specialized post capillary venules that recruit naïve T cells and B cells into secondary lymphoid organs (SLO) such as lymph nodes (LN). Expansion of HEV networks in SLOs occurs following immune activation to support development of an effective immune response. In this study, we used a carcinogen-induced model of fibrosarcoma to examine HEV remodeling after depletion of regulatory T cells (Treg). We used light sheet fluorescence microscopy imaging to visualize entire HEV networks, subsequently applying computational tools to enable topological mapping and extraction of numerical descriptors of the networks. While these analyses revealed profound cancer- and immune-driven alterations to HEV networks within LNs, these changes did not identify successful responses to treatment. The presence of HEV networks within tumors did however clearly distinguish responders from nonresponders. Finally, we show that a successful treatment response is dependent on coupling tumor-associated HEV (TA-HEV) development to T-cell activation implying that T-cell activation acts as the trigger for development of TA-HEVs which subsequently serve to amplify the immune response by facilitating extravasation of T cells into the tumor mass.Significance:We used three-dimensional imaging methods with computational tools to analyze networks of specialized blood vessels called HEVs in LNs and tumors. By applying these techniques in a mouse model of carcinogen-induced tumors, we could identify network changes after depletion of Tregs.

Global overview of intra-tumoral HEVs in a 4T1 regressor tumour following PI-3065 treatment.

Global overview of intra-tumoral HEVs in a 4T1 regressor tumour following PI-3065 treatment.

Global overview of intra-tumoral HEVs in a 4T1 non-regressor tumour following PI-3065 treatment.

Background and purpose Inhibitors of DNA-dependent protein kinase (DNA-PK) are effective radiation sensitisers in preclinical tumours, but little is known about risks of normal tissue radiosensitisation. Here, we evaluate radiosensitisation of head and neck squamous cell carcinoma (HNSCC) cells by DNA-PK inhibitor AZD7648 under oxia and anoxia in vitro, and tumour (SCCVII), oral mucosa and small intestine in mice. Materials and methods Radiosensitisation of human (UT-SCC-54C) and murine (SCCVII) HNSCC cells by AZD7648 under oxia and anoxia was evaluated by clonogenic assay. Radiosensitisation of SCCVII tumours in C3H mice by oral AZD7648 (75 mg/kg) was determined by ex vivo clonogenic assay 3.5 days post-irradiation, with evaluation of normal tissue surrogate endpoints using 5-ethynyl-2’-deoxyuridine to facilitate detection of regenerating crypts in the ileum and repopulating S-phase cells in the ileum and oral mucosa of the same animals. Results AZD7648 potently radiosensitised both cell lines, with similar sensitiser enhancement ratios for 10% survival (SER10) under oxia and anoxia. AZD7648 diffused rapidly through multicellular layers, suggesting rapid equilibration between plasma and hypoxic zones in tumours. SCCVII tumours were radiosensitised by AZD7648 (SER10 2.5). AZD7648 also enhanced radiation-induced body weight loss and suppressed regenerating intestinal crypts and repopulating S-phase cells in the ileum and tongue epithelium with SER values similar to SCCVII tumours. Conclusion AZD7648 is a potent radiation sensitiser of both oxic and anoxic tumour cells, but also markedly radiosensitises stem cells in the small intestine and oral mucosa.

Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates for any diffusion limitations of the prodrug in tumour tissue. Metabolism and uptake of the nitroCBI in oxic and anoxic cells, and diffusion through multicellular layer cultures, was characterised by LC–MS/MS. To quantify bystander effects, clonogenic cell killing of HCT116 cells was assessed in multicellular spheroid co-cultures comprising cells transfected with cytochrome P450 oxidoreductase (POR) or E. coli nitroreductase NfsA. Spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) models, parameterised by the above measurements, were developed for spheroids and tumours using agent-based and Green’s function modelling, respectively. NitroCBI was reduced to aminoCBI by POR under anoxia and by NfsA under oxia, and was the only significant cytotoxic metabolite in both cases. In spheroid co-cultures comprising 30% NfsA-expressing cells, non-metabolising cells were as sensitive as the NfsA cells, demonstrating a marked bystander effect. Agent-based PK/PD models provided good prediction of cytotoxicity in spheroids, while use of the same parameters in a Green’s function model for a tumour microregion demonstrated that local diffusion of aminoCBI overcomes the penetration limitation of the prodrug. The nitroCBI HAP SN30548 generates a highly efficient bystander effect through local diffusion of its active metabolite in tumour tissue.

The ability of the baby to receive nutrients and oxygen in utero depends on the healthy development of the placenta. For maternal blood to adequately perfuse the placenta, it dramatically alters the arteries in the uterus that supply it with nutrient-rich blood right from the start of pregnancy. Placental cells (trophoblasts) invade both into the tissue of the uterus and into the maternal blood vessels nearest to the site of implantation (the spiral arteries (SAs)) and transform these allowing a relatively high and steady flow of nutrient-rich blood to perfuse the placenta. Trophoblasts also form plugs that occlude SAs, preventing maternal blood flow to the placenta until the late first trimester, at which point these plugs dislodge or disintegrate. Here we present an agent-based model of trophoblast migration within plugged SAs to tease apart the impact of chemical signals and mechanical factors on trophoblast behaviour. The model supports our previous in vitro hypothesis that plugging of the maternal arteries in early pregnancy can act to promote trophoblast invasion by providing a ‘low flow’ environment and extends our understanding by suggesting ‘weak spots’ in plug structure can lead to plug degeneration, allowing increased blood flow through the materno-fetal circulation.

Supplementary figures illustrating time courses of cell migration as predicted by the agent based model described in this study.

Multicellular tumour spheroids capture many characteristics of human tumour microenvironments, including hypoxia, and represent an experimentally tractable in vitro model for studying interactions between radiotherapy and anticancer drugs. However, interpreting spheroid data is challenging because of limited ability to observe cell fate within spheroids dynamically. To overcome this limitation, we have developed a hybrid continuum/agent-based model (ABM) for HCT116 tumour spheroids, parameterised using experimental models (monolayers and multilayers) in which reaction and diffusion can be measured directly. In the ABM, cell fate is simulated as a function of local oxygen, glucose and drug concentrations, determined by solving diffusion equations and intracellular reactions. The model is lattice-based, with cells occupying discrete locations on a 3D grid embedded within a coarser grid that encompasses the culture medium; separate solvers are employed for each grid. The generated concentration fields account for depletion in the medium and specify concentration-time profiles within the spheroid. Cell growth and survival are determined by intracellular oxygen and glucose concentrations, the latter based on direct measurement of glucose diffusion/reaction (in multilayers) for the first time. The ABM reproduces known features of spheroids including overall growth rate, its oxygen and glucose dependence, peripheral cell proliferation, central hypoxia and necrosis. We extended the ABM to describe in detail the hypoxia-dependent interaction between ionising radiation and a hypoxia-activated prodrug (SN30000), again using experimentally determined parameters; the model accurately simulated clonogenic cell killing in spheroids, while inclusion of reversible cell cycle delay was required to account for the marked spheroid growth delay after combined radiation and SN30000. This ABM of spheroid growth and response exemplifies the utility of integrating computational and experimental tools for investigating radiation/drug interactions, and highlights the critical importance of understanding oxygen, glucose and drug concentration gradients in interpreting activity of therapeutic agents in spheroid models., Author summary Studies in 3D cultures, notably multicellular tumour spheroids that mimic many features of solid tumours, have great potential for speeding up anticancer drug development. However the increased complexity of 3D cultures makes interpretation of experiments more difficult. We have developed a hybrid continuum/agent-based mathematical model, validated by experiments, to aid interpretation of spheroid experiments in developing drugs designed to eliminate radiation-resistant hypoxic cells. This model includes key features of the tumour microenvironment including oxygen and glucose transport and regions of hypoxia where the cells are resistant to radiation, but sensitive to hypoxia-activated prodrugs such as SN30000. This enables us to predict the growth and cell response in untreated spheroids and compare the results to spheroids treated with radiation and SN30000. We demonstrate good prediction of cellular responses in spheroids treated with radiation and SN30000 and good agreement with spheroid regrowth after treatment when additional effects of cellular growth delay are added. This demonstrates that the modelling approach has potential to improve interpretation of experimental investigations of drug and radiation combinations.

International audience; The initiation of T-cell responses in lymph nodes requires T cells to integrate signals delivered by dendritic cells (DCs) during long-lasting contacts (synapses) or more transient interactions (kinapses). However, it remains extremely challenging to understand how a specific sequence of contacts established by T cells ultimately dictates T-cell fate. Here, we have coupled a computational model of T-cell migration and interactions with DCs with a real-time, flow cytometry-like representation of T-cell activation. In this model, low-affinity peptides trigger T-cell proliferation through kinapses but we show that this process is only effective under conditions of high DC densities and prolonged antigen availability. By contrast, high-affinity peptides favor synapse formation and a vigorous proliferation under a wide range of antigen presentation conditions. In line with the predictions, decreasing the DC density in vivo selectively abolished proliferation induced by the low-affinity peptide. Finally, our results suggest that T cells possess a biochemical memory of previous stimulations of at least 1-2 days. We propose that the stability of T-cell-DC interactions, apart from their signaling potency, profoundly influences the robustness of T-cell activation. By offering the ability to control parameters that are difficult to manipulate experimentally, the virtual lymph node model provides new possibilities to tackle the fundamental mechanisms that regulate T-cell responses elicited by infections or vaccines.

Understanding of the microvasculature has previously been limited by the lack of methods capable of capturing and modelling complete vascular networks. We used novel imaging and computational techniques to establish the topology of the entire blood vessel network of a murine lymph node, combining 63706 confocal images at 2 μm pixel resolution to cover a volume of 3.88 mm3. Detailed measurements including the distribution of vessel diameters, branch counts, and identification of voids were subsequently re-visualised in 3D revealing regional specialisation within the network. By focussing on critical immune microenvironments we quantified differences in their vascular topology. We further developed a morphology-based approach to identify High Endothelial Venules, key sites for lymphocyte extravasation. These data represent a comprehensive and continuous blood vessel network of an entire organ and provide benchmark measurements that will inform modelling of blood vessel networks as well as enable comparison of vascular topology in different organs.

Background: Hypoxia in solid tumors acts as the major barrier to anti-cancer therapies and promotes tumor progression. Hypoxia-activated prodrugs (HAPs) are designed to not only eliminate these resistant cells, but also to exploit hypoxia as a basis of tumor selectivity. A key concept in exploiting hypoxia with HAPs is that active drug metabolite should be able to diffuse from hypoxic zones to maximize effect in surrounding aerobic tumor tissue. There is some evidence that these ‘bystander effects’ contribute to the antitumor activity of HAPs, but currently there is a lack of robust methods for their detection. Objectives: To investigate bystander effects from HAPs in a novel spheroid co-culture system using PR104A which releases a diffusible DNA crosslinking mustard and the tirapazamine analogue SN30000 which is converted to a short lived radical that is not expected to diffuse out of cells. Methods: A new method for measuring the bystander effect with multicellular spheroids under hypoxic conditions was developed using co-cultures of HCT116 colon cancer cells with high metabolic capacity for HAP metabolism (activators, transfected to overexpress cytochrome P450 reductase [POR]) and cells that cannot activate HAPs efficiently (targets, with POR knocked out by zinc finger nuclease gene targeting) along with differential antibiotic resistance and fluorescent protein markers to enable their quantitation in mixed cultures. Sensitivity of activators and targets to PR104A and SN30000 was determined in spheroid co-cultures by dissociation and clonogenic cell survival assay. Results were compared to output of a novel agent-based mathematical model (ABM) of spheroid co-cultures. Results: In monolayer cultures, activator cells showed markedly higher sensitivity to the HAPs than did targets. Tagging of activators and targets with fluorescent proteins allowed visualization of their spatial distribution in spheroid co-cultures. When spheroids were treated with PR104A, killing of targets markedly increased with increasing proportion of activators, confirming a bystander effect. However, increasing proportions of activators reduced cell killing of targets after SN30000 treatment, suggesting that rapid metabolism by activators inhibits its penetration, which was confirmed by flux experiments in multicellular layer co-cultures. ABM simulations of cell killing by PR104A and SN30000 in spheroids using our published transport parameters agreed with experimental results indicating diffusion of active metabolites of PR104A but not SN30000. Discussion: Using this novel spheroid co-culture model, a bystander effect of PR104A was demonstrated while SN30000 showed no bystander effect. This is the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the utility of the novel spheroid ABM in simulating active metabolite diffusion in 3D co-cultures. Citation Format: Cho Rong Hong, Gib Bogle, William R. Wilson, Kevin O. Hicks. Investigation of bystander effects of hypoxia activated prodrugs using three dimensional cell cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4513. doi:10.1158/1538-7445.AM2017-4513

Introduction: Hypoxia-activated prodrugs (HAPs) are a promising strategy to specifically target radioresistant hypoxic cells in tumors. We have used multicellular layers (MCLs) and steady state Green’s function pharmacokinetic/pharmacodynamic (PK/PD) modelling in microvascular networks to select SN30000 as a tirapazamine analogue with optimal spatial complementarity with radiation. However these models have several limitations such as lack of time dependent PK. Multicellular tumor spheroids are a more tractable model than MCLs for investigating PD parameters. However, the challenges of interpreting spheroid experiments in terms of spatial complementarity limits their application. We have developed an in silico agent-based (AB) tumor spheroid model, in which cell fate can be tracked as a function of time and is determined by spatially-varying concentrations of oxygen, glucose and therapeutic agents (radiation and HAP). The hypothesis is that our AB model can predict tumor spheroid response to radiation and SN30000, both alone and in combination. Methods: In the AB model, spheroid growth is simulated as a function of oxygen, glucose and drug concentrations determined by solving diffusion equations using oxygen consumption measured in HCT116 monolayers and glucose and SN30000 diffusion and metabolism measured in HCT116 MCLs and stirred cell suspensions, linked with intracellular reaction equations. Cell fate is determined by oxygen, glucose and drug concentrations. Spheroid diameter, cell survival and hypoxic fraction of HCT116 spheroids grown at a range of oxygen and initial glucose concentrations were compared to model predictions, as was glucose depletion from the culture medium. Radiation (cobalt-60) and SN30000 induced clonogenic cell killing under oxia and anoxia was determined using HCT116 monolayers. Model simulations of HCT116 spheroid response to radiation and SN30000 were compared to experimental results. Results: The model reproduced known features of spheroids including oxygen and glucose gradients, rapid cell proliferation at the periphery and central hypoxia and necrosis. Measured HCT116 spheroid diameters increased linearly with time, at rates that decreased at lower ambient O2 concentrations. Spheroid growth and glucose depletion was well-fitted by the AB model. Good agreement was found between simulated and measured clonogenic cell killing by radiation and SN30000, while the model underestimated spheroid growth delay by radiation and SN30000 than experimental observation. Discussion: An oxygen and glucose dependent AB model for growth and PD response (cell killing and growth delay) of HCT116 spheroids has been developed and calibrated using experimentally determined parameters. Further development of this model will link cell fates (including cell cycle arrest) explicitly to DNA damage responses and O2 and glucose-dependence of cell proliferation and survival. Citation Format: Xinjian Mao, Gib Bogle, Sarah McManaway, William Wilson, Kevin Hicks. Agent-based models for prediction of tumor spheroid response to radiation and the hypoxia-activated prodrug SN30000 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4523. doi:10.1158/1538-7445.AM2017-4523

We present a theoretical description of the propagation of composition waves along a strip of electrochemically-active conducting polymer, upon electrochemical stimulation. We develop an efficient solution of the electro-neutral Nernst–Plank equations in 2-D for electromigration and diffusional transport in the solution based on an extension of the methods of Scharfetter and Gummel [D. L. Scharfetter and H. K. Gummel, IEEE Trans. Electron Devices, 1969, ED16, 64–77.] and of Cohen and Cooley [H. Cohen and J. W. Cooley, Biophys. J., 1965, 5, 145–162.], and demonstrate important effects of the geometry of the cell. Under some circumstances, waves reflecting back from the end of the strip are predicted. We then demonstrate theoretically how such waves, associated as they are with expansion of the polymer, could be employed to enhance mixing or induce pumping in microfluidic systems.

Electrochemically-active conducting polymers (ECP) swell or shrink in response to ion and solvent incorporation or ejection as a result of electrochemical reaction of the polymer. As a consequence, they are, in principle, attractive materials to consider for inducing fluid motion of electrolytes in microfluidic systems. When anodic potential is applied to an electrode attached to one end of ECP strip, the oxidation process starts from the electrode and proceeds along the polymer, propagating as a wave. This wave is driven as a consequence of the electrochemical reactions and would be coupled to a propagating front of compositional change. This property of the ECP can be used to design pumps and mixers for microfluidic systems. We in this paper set up a 2-D transport model to explain this wave phenomenon that includes both diffusion and electro-migration, that is coupled to the reaction at the polymer-solution interface, and that also includes the effects of change of polymer conductivity on the charge transport in the polymer layer. We explore the design for a microfluidic pump that uses this process and its efficiency to pump electrolytes.

Activation of T cells by antigen-presenting cells (APCs) in lymph nodes (LNs) is a key initiating event in many immune responses. Our understanding of this process has been both improved and complicated in recent years by evidence from techniques such as intravital microscopy that has revealed new levels of dynamism in the interaction of T cells and APCs. In particular, the complex motility of T cells within LNs, and their serial interactions with many APCs, imply that earlier static models of T cell activation need to be updated. Here we review the first attempts to model T cell interactions with APCs in LNs that incorporate simulations of T cell motility, based on experimental observations. We show that lattice-based modeling approaches are the dominant trend in these models, and then chart a possible course for development of these models toward spatially-resolved models of immune responses within LNs.

Agent-based simulation modelling of T-cell trafficking, activation and proliferation in the lymph node paracortex requires a model for cell motility. Such a model must be able to reproduce the observed random-walk behaviour of T cells, while accommodating large numbers of tightly packed cells, and must be computationally efficient. We report the development of a motility model, based on a three-dimensional lattice geometry, that meets these objectives. Cells make discrete jumps between neighbouring lattice sites in directions that are randomly determined from specified discrete probability distributions, which are defined by a small number of parameters. It is shown that the main characteristics of the random motion of T cells as typically observed in vivo can be reproduced by suitable specification of model parameters. The model is computationally highly efficient and provides a suitable engine for a model capable of simulating the full T-cell population of the paracortex.

Agent-based simulation is a powerful method for investigating the complex interplay of the processes occurring in a lymph node during an adaptive immune response. We have previously established an agent-based modeling framework for the interactions between T cells and dendritic cells within the paracortex of lymph nodes. This model simulates in three dimensions the "random-walk" T cell motility observed in vivo, so that cells interact in space and time as they process signals and commit to action such as proliferation. On-lattice treatment of cell motility allows large numbers of densely packed cells to be simulated, so that the low frequency of T cells capable of responding to a single antigen can be dealt with realistically. In this paper we build on this model by incorporating new numerical methods to address the crucial processes of T cell ingress and egress, and chemotaxis, within the lymph node. These methods enable simulation of the dramatic expansion and contraction of the T cell population in the lymph node paracortex during an immune response. They also provide a novel probabilistic method to simulate chemotaxis that will be generally useful in simulating other biological processes in which chemotaxis is an important feature.