Global sensitivity analysis and model-based reactive scheduling of targeted cancer immunotherapy

Intra-patient variability is a key challenge in cancer treatment. This makes it necessary to find the factors affecting tumor growth and accordingly schedule therapies over the treatment horizon for the patient. In this work, model-based studies are performed to investigate these issues for optimal immunotherapeutic intervention. Dendritic cell therapy is a targeted immunotherapy where the dendritic cells and its activating agents such as interleukin are engineered, stimulated to recognize and specifically eradicate tumors. A mathematical model that integrates tumor dynamics and dendritic cell therapy is used to perform the analysis. Global sensitivity analysis of the model is done using high dimensional model reduction (HDMR) technique and the key parameters altering the tumor growth are identified. The variations in these key parameters are deemed to result in intra-patient variability during the treatment phase. Then, reactive scheduling is used to schedule dendritic cell interventions with and without interleukin interventions under the varying conditions of the patient. Moreover, the key parameters obtained from HDMR are verified using the reactive scheduling and nominal scheduling approaches. Besides saving costs, the in silico analysis done in this paper may be useful to the oncology community in designing experiments to clinically measure the influential parameters. It can also be used as a decision making tool to determine the required intervention dosage during the treatment.     

Effect of airway and left atrial pressures on microcirculation of newborn lungs

To determine the effect of lung inflation and left atrial pressure on the hydrostatic pressure gradient for fluid flux across 20- to 60-microns-diam venules, we isolated and perfused the lungs from newborn rabbits, 7-14 days old. We used the micropuncture technique to measure venular pressures in some lungs and perivenular interstitial pressures in other lungs. For all lungs, we first measured venular or interstitial pressures at a constant airway pressure of 5 or 15 cmH2O with left atrial pressure greater than airway pressure (zone 3). For most lungs, we continued to measure venular or interstitial pressures as we lowered left atrial pressure below airway pressure (zone 2). Next, we inflated some lungs to whichever airway pressure had not been previously used, either 5 or 15 cmH2O, and repeated venular or interstitial pressures under one or both zonal conditions. We found that at constant blood flow a reduction of left atrial pressure below airway pressure always resulted in a reduction in venular pressure at both 5 and 15 cmH2O airway pressures. This suggests that the site of flow limitation in zone 2 was located upstream of venules. When left atrial pressure was constant relative to airway pressure, the transvascular gradient (venular-interstitial pressures) was greater at 15 cmH2O airway pressure than at 5 cmH2O airway pressure. These findings suggest that in newborn lungs edema formation would increase at high airway pressures only if left atrial pressure is elevated above airway pressure to maintain zone 3 conditions.     

Glucuronoarabinoxylan structure in the walls of Aechmea leaf chlorenchyma cells is related to wall strength

In CAM-plants rising levels of malic acid in the early morning cause elevated turgor pressures in leaf chlorenchyma cells. Under specific conditions this process is lethal for sensitive plants resulting in chlorenchyma cell burst while other species can cope with these high pressures and do not show cell burst under comparable conditions. The non-cellulosic polysaccharide composition of chlorenchyma cell walls was investigated and compared in three cultivars of Aechmea with high sensitivity for chlorenchyma cell burst and three cultivars with low sensitivity. Chlorenchyma layers were cut from the leaf and the non-cellulosic carbohydrate fraction of the cell wall fraction was analyzed by gas-liquid chromatography. Glucuronoarabinoxylans (GAXs) were the major non-cellulosic polysaccharides in Aechmea. The fine structure of these GAXs was strongly related to chlorenchyma wall strength. Chlorenchyma cell walls from cultivars with low sensitivity to cell burst were characterized by an A/X ratio of ca. 0.13 while those from cultivars with high sensitivity showed an A/X ratio of ca. 0.23. Xylose chains from cultivars with high cell burst sensitivity were ca. 40% more substituted with arabinose compared to cultivars with low sensitivity for cell burst. The results indicate a relationship in vivo between glucuronoarabinoxylan fine structure and chlorenchyma cell wall strength in Aechmea. The evidence obtained supports the hypothesis that GAXs with low degrees of substitution cross-link cellulose microfibrils, while GAXs with high degrees of substitution do not. A lower degree of arabinose substitution on the xylose backbone implies stronger cell walls and the possibility of withstanding higher internal turgor pressures without cell bursting.     

A nonlinear characteristic regime of biomembrane force probe

A linear relation between stiffness and aspiration pressure is the basis for biomembrane force probe (BFP), a widely used technique to measure minuscule forces. Here we perform finite element simulations and semi-analytical modeling of the BFP operation to show that, at low aspiration pressures, there exists a characteristic regime in which the relation between stiffness and aspiration pressure is actually nonlinear. We find that this nonlinear characteristic regime arises from a transition in configuration of a partially aspirated biomembrane force probe under increasing aspiration pressure. We discuss both the conditions for the transition and the characteristics of the nonlinear characteristic regime, as well as its potential applications.     

Interdependence of flow between lobes reduces maximal emptying postresection in dogs.

The effect of pulmonary resection on the maximal emptying of the remaining lobes was examined in an open-chest preparation in normal canine lungs and in a unilobar papain emphysema model. The objectives were to determine whether, compared with when both lungs were deflated (BL), maximal emptying of the normal lower lobes or the emphysematous right lower lobe would be altered 1) when acute pneumonectomy of the contralateral lung was performed (OL) and 2) when the lower lobe deflated alone (LA). The alveolar capsule technique was used to measure alveolar pressures (Palv) at 75, 50, and 30% lobar vital capacity (VC). During forced deflation, the maximal rates of deflation (dPalv/dt) and flows (lobarV(max)) of the lower lobes were determined under the three different conditions. The Pitot-static tube technique was used to measure intrabronchial pressures and to estimate bronchial area and compliance in which values were obtained at the same central airway during the conditions studied. The results showed that, compared with BL and OL, dPalv/dt and lobar V(max) decreased during LA (P < 0.05). These findings were due to a reduction in bronchial area during LA that limited flow at a lower maximal value compared with BL. This decrease in area appeared to be due to a change in bronchial pressure area behavior that resulted in a smaller bronchial area during LA for similar transmural pressures between conditions. There were no differences in findings between normal and emphysematous lobes. This study suggested that removal of lobes may alter the pressure area behavior of central airways. Possible mechanisms considered were differences in axial tension between conditions, negative effort dependence, or parenchymal-bronchial interdependence that may be relevant to understanding the dynamic collapsibility of central as well as intraparenchymal airways.     

Automated selective dissociation of cells from different regions of multicellular spheroids

Summary In this report we describe a new apparatus which has been developed for the automated selective dissociation of multicellular spheroids into fractions of viable cells from different locations in the spheroid. This device is based on the exposure of spheroids to a 0.25% solution of trypsin under carefully controlled conditions, such that the cells are released from the outer spheroid surface in successive layers. Study of the spheroid size, number of cells per spheroid, and sections through the spheroid with increasing exposure to trypsin demonstrate the effectiveness of this technique. The technique has been successfully used on spheroids from five different cell lines over a wide range of spheroid diameters. We also present data detailing the effect of varying the dissociation temperature, the mixing speed, the trypsin concentration, and the number of spheroids being dissociated. The new apparatus has several advantages over previous selective dissociation methods and other techniques for isolating cells from different regions in spheroids, including: a) precise control over dissociation conditions, improving reproducibility; b) short time to recover cell fractions; c) ability to isolate large numbers of cells from many different spheroid locations; d) use of common, inexpensive laboratory equipment; and e) easy adaptability to new cell lines or various spheroid sizes. Applications of this method are demonstrated, including the measurement of nutrient consumption rates, regrowth kinetics, and radiation survivals of cells from different spheroid regions. This work was supported by grants CA-36535, CA-22585, and RR-02845 from the National Institutes of Health, Bethesda, MD, the National Flow Cytometry Resource (NIH grant RR-01315), and by the Department of Energy, Washington, DC.     

Microarray analysis of genes differentially expressed in hepG2 cells cultured in simulated microgravity: Preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.     

“Cancer Reviews 2021” series: Cell competition in intratumoral and tumor microenvironment interactions

Tumors are complex cellular and acellular environments within which cancer clones are under continuous selection pressures. Cancer cells are in a permanent mode of interaction and competition with each other as well as with the immediate microenvironment. In the course of these competitive interactions, cells share information regarding their general state of fitness, with less‐fit cells being typically eliminated via apoptosis at the hands of those cells with greater cellular fitness. Competitive interactions involving exchange of cell fitness information have implications for tumor growth, metastasis, and therapy outcomes. Recent research has highlighted sophisticated pathways such as Flower, Hippo, Myc, and p53 signaling, which are employed by cancer cells and the surrounding microenvironment cells to achieve their evolutionary goals by means of cell competition mechanisms. In this review, we discuss these recent findings and explain their importance and role in evolution, growth, and treatment of cancer. We further consider potential physiological conditions, such as hypoxia and chemotherapy, that can function as selective pressures under which cell competition mechanisms may evolve differently or synergistically to confer oncogenic advantages to cancer.     

Role of endothelial cells in the proliferative response of cultured pulmonary vascular smooth muscle cells to reduced oxygen tension

Summary The development of pulmonary hypertension in a wide variety of human disease states and experimental animal models characterized by chronic alveolar hypoxia is mediated by two pathologic vascular processes, a) vasoconstriction and b) vasoconstruction (structural remodeling). The anatomic changes seen within the pulmonary circulation include a) increased deposition of collagen and elastin in the adventitial layer and b) aberrant pulmonary vascular smooth muscle cell proliferation and maturation in the medial segments. Despite the demonstrated ability of pharmacologic manipulation in the experimental animal to ameliorate both the structural and hemodynamic changes, the actual etiologic mechanisms are only beginning to be explored. Using the cell culture technique of co-cultivation, we have investigated the potential role of bovine pulmonary arterial endothelial cell-derived factors in mediating abnormal bovine smooth muscle cell growth under conditions of reduced oxygen tension. We have demonstrated that these cultured endothelial cells exposed in vitro to reduced levels of atmospheric oxygen concentrations of 5.0% and 2.5% O₂ for durations of 24 to 72 h produce and secrete soluble growth factor(s) which stimulate smooth muscle cell proliferation when compared to cells maintained under standard tissue culture oxygen conditions of 95% room air. This growth-stimulatory effect required the concomitant presence of serum factors (0.5% fetal bovine serum), was inhibited by heparin, was distinct from platelet-derived growth factor, and seemed to have a molecular weight greater than 14 000 Da. We conclude that reduced levels of oxygen tension in vitro can selectively induce pulmonary arterial endothelial cells to release mitogen(s) which can stimulate vascular smooth muscle replication. Furthermore, we speculate that this in vitro finding may be of importance as an etiologic mechanism to explain the accelerated smooth muscle cell growth characteristic of hypoxic pulmonary arteriopathy.     

Expansion of mesenchymal stem cells under atmospheric carbon dioxide

Stem cells are needed for an increasing number of scientific applications, including both fundamental research and clinical disease treatment. To meet this rising demand, improved expansion methods to generate high quantities of high quality stem cells must be developed. Unfortunately, the bicarbonate buffering system – which relies upon an elevated CO₂ environment – typically used to maintain pH in stem cell cultures introduces several unnecessary limitations in bioreactor systems. In addition to artificially high dissolved CO₂ levels negatively affecting cell growth, but more importantly, the need to sparge CO₂ into the system complicates the ability to control culture parameters. This control is especially important for stem cells, whose behavior and phenotype is highly sensitive to changes in culture conditions such as dissolved oxygen and pH. As a first step, this study developed a buffer to support expansion of mesenchymal stem cells (MSC) under an atmospheric CO₂ environment in static cultures. MSC expanded under atmospheric CO₂ with this buffer achieved equivalent growth rates without adaptation compared to those grown in standard conditions and also maintained a stem cell phenotype, self‐renewal properties, and the ability to differentiate into multiple lineages after expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1298–1306, 2013     

A novel population balance model to investigate the kinetics of in vitro cell proliferation: part II. Numerical solution, parameters' determination, and model outcomes

Based on the general theoretical model developed in Part I of this work, a series of numerical simulations related to the in vitro proliferation kinetics of adherent cells is here presented. First the complex task of assigning a specific value to all the parameters of the proposed population balance (PB) model is addressed, by also highlighting the difficulties arising when performing proper comparisons with experimental data. Then, a parametric sensitivity analysis is performed, thus identifying the more relevant parameters from a kinetics perspective. The proposed PB model can be adapted to describe cell growth under various conditions, by properly changing the value of the adjustable parameters. For this reason, model parameters able to mimic cell culture behavior under microgravity conditions are identified by means of a suitable parametric sensitivity analysis. Specifically, it is found that, as the volume growth parameter is reduced, proliferation slows down while cells arrest in G0/G1 or G2/M depending on the initial distribution of cell population. On the basis of this result, model capabilities have been tested by means of a proper comparison with literature experimental data related to the behavior of synchronized and not-synchronized cells under micro- and standard gravity levels.     

Root cell-wall properties are proposed to contribute to phosphorus (P) mobilization by groundnut and pigeonpea

Groundnuts showed a superior ability to take up phosphorus (P) from two soils of extremely low fertility, where sorghum and soybean died of P deficiency. This ability could not be attributed to differences in root development, to P uptake parameters such as C_min, or to the excretion of root exudates capable of solubilizing iron- (Fe-P) and aluminum-bound P (Al-P), the sparingly soluble P forms in soils. A new P solubilizing mechanism (called `contact reaction') which occurs at the interface between root surface and soil particles, is therefore proposed. Isolated cell walls from groundnut roots solubilized more P from P-fixing minerals than those from sorghum and soybean roots. The P-solubilizing activity of groundnut root cell-walls might therefore be related to the superior growth of this crop under P-deficient conditions. The P-solubilizing active sites in groundnut root cell walls were located at the root surface and could act as chelating agent with Fe(III). This P-solubilizing active component in the cell walls could be extracted by NaOH, but not by HCl, and was identified as a small molecule through column chromatography with Sephadex LH-20. The P-solubilizing ability of pigeonpea root cell-walls was examined and found to be as high as that of groundnut. As pigeonpea plants excrete significant amount of root exudates with Fe-P solubilizing ability only after they flower, the P-solubilizing ability of root cell-walls may partially explain the high P efficiency of this species before it flowers.     

Water stress effects on guard cell anatomy and the mechanical advantage of the epidermal cells

Abstract Vicia faba plants grown under water deficit were found to have guard cells considerably smaller than those of plants grown under well-watered conditions. Stomala of plants adapted to drought conditions have been observed in past studies to maintain opening at plant water potentials lower than those of plants not so adapted. By employing the geometric interpretation of the mechanical advantage (Wu, Sharpe & Spence, 1985), an anatomical/mechanical basis was found that helps explain how such opening in drought conditions can occur. The geometry and resulting mechanical properties of small stomata, in contrast to larger stomata, give them the capability of opening or maintaining open pores with lower guard cell turgor pressures, relative to the turgor of the surrounding epidermal cells.     

Mechanical Response of Single Plant Cells to Cell Poking： A Numerical Simulation Model

Cell poking is an experimental technique that is widely used to study the mechanical properties of plant cells. A full understanding of the mechanical responses of plant cells to poking force Is helpful for experimental work. The aim of this study was to numerically investigate the stress distribution of the cell wall, cell turgor, and deformation of plant cells in response to applied poking force. Furthermore, the locations damaged during poking were analyzed. The model simulates cell poking, with the cell treated as a spherical, homogeneous, isotropic elastic membrane, filled with incompressible, highly viscous liquid. Equilibrium equations for the contact region and the non-contact regions were determined by using membrane theory. The boundary conditions and continuity conditions for the solution of the problem were found. The forcedeformation curve, turgor pressure and tension of the cell wall under cell poking conditions were obtained. The tension of the cell wall circumference was larger than that of the meridian. In general, maximal stress occurred at the equator around. When cell deformation increased to a certain level, the tension at the poker tip exceeded that of the equator. Breakage of the cell wall may start from the equator or the poker tip, depending on the deformation. A nonlinear model is suitable for estimating turgor, stress, and stiffness, and numerical simulation is a powerful method for determining plant cell mechanical properties.     

RAPID ALDOSTERONE-INDUCED CELL VOLUME INCREASE OF ENDOTHELIAL CELLS MEASURED BY THE ATOMIC FORCE MICROSCOPE

Atomic force microscopy (AFM) is a useful technique for imaging the surface of living cells in three dimensions. The authors applied AFM to obtain morphological information of individual cultured endothelial cells of bovine aorta under stationary and strain conditions and to simultaneously measure changes in cell volume in response to aldosterone. This mineralocorticoid hormone is known to have acute, non-genomic effects on intracellular pH, intracellular electrolytes and inositol-1,4,5-triphosphate production. In this study whether endothelial cells under tension change their volume in response to aldosterone was tested. Such changes were already shown in human leukocytes measured by Coulter counter. In contrast to leukocytes that are more or less spherical and live in suspension, endothelial cells exhibit a complex morphology and adhere to a substrate. Thus, measurements of discrete cell volume changes in endothelial cells under physiological condition is only feasible with more sophisticated techniques. By using AFM we could precisely measure the absolute cell volume of individual living endothelial cells. Before the addition of aldosterone the cell volume of mechanically stressed endothelial cells mimicking arterial blood pressure was 1827±172fl. Cell volume was found to increase by 28% 5min after hormone exposure. Twenty-five minutes later cell volume was back to normal despite the continuous presence of aldosterone in the medium. Amiloride, a blocker of the plasma membrane Na⁺/H⁺exchanger prevented the initial aldosterone-induced volume increase. Taken together, AFM disclosed a transient swelling of endothelial cells induced by the activation of an aldosterone sensitive plasma membrane Na⁺/H⁺exchanger.     

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.     

基于图像分析技术的红细胞膜力学特性多参数动态测定的研究

探索以图像分析技术,在无扰、在位、实时的情况下,对单个活态红细胞的多个力学参量：弯曲模量KC、剪切模量μ及切向与弯曲模量之比ε等进行非侵入性连续动态测定的新方法。以该技术对红细胞在不同外部条件（温度、氧分压、渗透压）下的力学参量进行动态监测,不但揭示出有关变量条件对细胞各个力学参量的影响。还证明了本技术适于对细胞的各种生理和病理过程进行连续监测。     

Within and between Population Variation in Epidermal Club Cell Investment in a Freshwater Prey Fish: A Cautionary Tale for Evolutionary Ecologists

Many prey fishes possess large club cells in their epidermis. The role of these cells has garnered considerable attention from evolutionary ecologists. These cells likely form part of the innate immune system of fishes, however, they also have an alarm function, releasing chemical cues that serve to warn nearby conspecifics of danger. Experiments aimed at understanding the selection pressures leading to the evolution of these cells have been hampered by a surprisingly large intraspecific variation in epidermal club cell (ECC) investment. The goal of our current work was to explore the magnitude and nature of this variation in ECC investment. In a field survey, we documented large differences in ECC investment both within and between several populations of minnows. We then tested whether we could experimentally reduce variation in mean ECC number by raising fish under standard laboratory conditions for 4 weeks. Fish from different populations responded very differently to being held under standard laboratory conditions; some populations showed an increase in ECC investment while others remained unchanged. More importantly, we found some evidence that we could reduce within population variation in ECC investment through time, but could not reduce among-population variation in mean ECC investment. Given the large variation we observed in wild fish and our limited ability to converge mean cell number by holding the fish under standard conditions, we caution that future studies may be hard pressed to find subtle effects of various experimental manipulations; this will make elucidating the selection pressures leading to the evolution of the cells challenging.     

Assessment of metabolic activity of single bacterial cells — new developments in microcolony and dehydrogenase assays

Abstract: Some bacteria lose culturability in natural environments but retain measurable metabolic activity and are thus considered viable. Several techniques have been proposed to determine the activity of nonculturable cells. Due to the considerable physiological heterogeneity of bacterial populations in the environment, it is imperative to apply methods which measure cellular activity at the single cell level. This review focuses on two promising methods: the microcolony assay and the respiration assay based on reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). In the microcolony assay, viable cells are identified by their ability to perform a limited number of cell divisions and this approach is thus related to conventional culture techniques. Some recent methodological developments of the technique aiming at improving the incubation conditions and the detection of microcolonies are presented. Results obtained by the microcolony technique are used to introduce its advantages and limitations. The CTC-reduction assay determines a central cellular metabolic activity, but does not measure cell growth. Results of studies using this assay are presented, and it is emphasized that great care should be taken to optimize assay conditions for the studied organisms. Finally, the results obtained by different viability assays are compared. For a specific bacterium, several assays, addressing different aspects of cell metabolism, can provide comparable results suggesting that they provide meaningful viability estimates. On the other hand, the use of viability assays on complex indigenous populations may be ambiguous.     

Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.