Differential effects of risuteganib and bevacizumab on AMD cybrid cells.

PURPOSE: Intravitreal injections of anti-vascular endothelial growth factor (VEGF) treatments are currently used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, and macular edema. Chronic, repetitive treatments with anti-VEGF may have unintended consequences beyond the inhibition of angiogenesis. Most recently, clinical trials have been conducted with risuteganib (RSG, Luminate®), which is anti-angiogenic and has neuroprotective and anti-inflammatory properties. Mitochondrial damage and dysfunction play a major role in development of AMD. Transmitochondrial cybrids are cell lines established by fusing human retinal pigment epithelial (RPE) cells that are Rho0 (lacking mtDNA) with platelets isolated from AMD subjects or age-matched normal subjects. Cybrid cell lines have identical nuclei but mitochondria from different subjects, enabling investigation of the functional consequences of damaged AMD mitochondria. The present study compares the responses of AMD cybrids treated with bevacizumab (Bmab, Avastin®) versus risuteganib (RSG, Luminate®). METHODS: Cybrids were created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 3) cybrids were treated for 48 h with or without 1x clinical dose of 1.25 mg/50 µl (25,000 µg/ml) of Bmab or 1.0 mg/50 µl (20,000 µg/ml) of RSG. Cultures were analyzed for levels of cleaved caspase 3/7 and NucLight Rapid Red staining (IncuCyte® Live Cell Imager), mitochondrial membrane potential (ΔΨm, JC1 assay) or reactive oxygen species (ROS, H2DCFDA assay). Expression levels of genes related to the following pathways were analyzed with qRT-PCR: Apoptosis (BAX, BCL2L13, CASP-3, -7, -9); angiogenesis (VEGFA, HIF1α, PDGF); integrins (ITGB-1, -3, -5, ITGA-3, -5, -V); mitochondrial biogenesis (PGC1α, POLG); oxidative stress (SOD2, GPX3, NOX4); inflammation (IL-6, -18, -1ß, IFN-ß1); and signaling (P3KCA, PI3KR1). Statistical analyses were performed using GraphPad Prism software. RESULTS: The untreated AMD cybrids had significantly higher levels of cleaved caspase 3/7 compared to the untreated normal cybrids. The Bmab-treated AMD cybrids showed elevated levels of cleaved caspase 3/7 compared to untreated AMD or RSG-treated AMD cybrids. The Bmab-treated cybrids had lower ΔΨm compared to untreated AMD or RSG-treated AMD cybrids. The ROS levels were not changed with Bmab or RSG treatment. Results showed that Bmab-treated cybrids had higher expression levels of inflammatory (IL-6, IL1-ß), oxidative stress (NOX4) and angiogenesis (VEGFA) genes compared to untreated AMD, while RSG-treated cybrids had lower expression levels of apoptosis (BAX), angiogenesis (VEGFA) and integrin (ITGB1) genes. CONCLUSIONS: These data suggest that the mechanism(s) of action of RSG, an integrin regulator, and Bmab, a recombinant monoclonal antibody, affect the AMD RPE cybrid cells differently, with the former having more anti-apoptosis properties, which may be desirable in treating degenerative ocular diseases.

Sequenced Somatic Cell Reprogramming and Differentiation Inside Nested Hydrogel Droplets.

The efficient genesis of pluripotent cells or therapeutic cells for regenerative medicine involves several external manipulations and conditioning protocols, which drives down clinical applicability. Automated programming of the genesis by microscale physical forces and chronological biochemistry can increase clinical success. The design and fabrication of nested polysaccharide droplets (millimeter-sized) with cell sustaining properties of natural tissues and intrinsic properties for time and space evolution of cell transformation signals between somatic cells, pluripotent cells and differentiated therapeutic cells in a swift and efficient manner without the need for laborious external manipulation are reported. Cells transform between phenotypic states by having single and double nested droplets constituted with extracellular matrix proteins and reprogramming, and differentiation factors infused chronologically across the droplet space. The cell transformation into germ layer cells and bone cells is successfully tested in vitro and in vivo and promotes the formation of new bone tissues. Thus, nested droplets with BMP-2 loaded guests synthesize mineralized bone tissue plates along the length of a cranial non-union bone defect at 4 weeks. The advantages of sequenced somatic cell reprogramming and differentiation inside an individual hydrogel module without external manipulation, promoted by formulating tissue mimetic physical, mechanical, and chemical microenvironments are shown.

Pioglitazone and Deoxyribonucleoside Combination Treatment Increases Mitochondrial Respiratory Capacity in m.3243A>G MELAS Cybrid Cells.

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease.

Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes.

The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization.

Involvement of Actin Cytoskeletal Components in Breast Cancer Cell Fusion with Human Mesenchymal Stroma/Stem-Like Cells.

Cell fusion as a rare event was observed following the co-culture of human MDA-MB-231cherry breast cancer cells or benign neoplastic MCF10Acherry breast epithelial cells together with different mesenchymal stroma/stem-like cells (MSCGFP) cultures, respectively, resulting in the generation of double-fluorescing hybrid cells. Analysis of potential molecular mechanisms for the formation of cancer hybrid cells revealed cytoskeletal components, including F-actin. Thus, a sub-lethal concentration of cytochalasin D, which blocks elongation of actin filaments, was able to significantly reduce cancer hybrid cell formation. Simultaneously, cell cycle progression of the different co-cultures remained unaffected following treatment with cytochalasin D, indicating continued proliferation. Moreover, exposure to 50 nM cytochalasin D revealed little if any effect on the expression of various integrins and cell adhesion molecules in the different co-cultures. However, LC-MS proteome analysis of the different control co-cultures compared to corresponding cytochalasin-treated co-cultures demonstrated predominant differences in the expression of actin-associated cytoskeletal proteins. In addition, the requirement of structured actin to provide an appropriate cytoskeletal network for enabling subsequent fusion processes was also substantiated by the actin filament disrupting latrunculin B, which inhibits the fusion process between the breast cancer populations and mesenchymal stroma/stem-like cells (MSC). Together, these findings suggest an important role of distinct actin structures and associated cytoskeletal components during cell fusion and the formation of breast cancer hybrid cells.

Amplification of arsenic genotoxicity by TiO2 nanoparticles in mammalian cells: new insights from physicochemical interactions and mitochondria.

Titanium dioxide nanoparticles (TiO2 NPs) have shown great adsorption capacity for arsenic (As); however, the potential impact of TiO2 NPs on the behavior and toxic responses of As remains largely unexplored. In the present study, we focused on the physicochemical interaction between TiO2 NPs and As(III) to clarify the underlying mechanisms involved in their synergistic genotoxic effect on mammalian cells. Our data showed that As(III) mainly interacted with TiO2 NPs by competitively occupying the sites of hydroxyl groups on the surface of TiO2 NP aggregates, resulting in more aggregation of TiO2 NPs. Although TiO2 NPs at concentrations used here had no cytotoxic or genotoxic effects on cells, they efficiently increased the genotoxicity of As(III) in human-hamster hybrid (AL) cells. The synergistic genotoxicity of TiO2 NPs and As(III) was partially inhibited by various endocytosis pathway inhibitors while it was completely blocked by an As(III)-specific chelator. Using a mitochondrial membrane potential fluorescence probe, a reactive oxygen species (ROS) probe together with mitochondrial DNA-depleted ρ0 AL cells, we discovered that mitochondria were essential for mediating the synergistic DNA-damaging effects of TiO2 NPs and As(III). These data provide novel mechanistic proof that TiO2 NPs enhanced the genotoxicity of As(III) via physicochemical interactions, which were mediated by mitochondria-dependent ROS.

Cisplatin selects short forms of the mitochondrial DNA OriB variant (16184-16193 poly-cytosine tract), which confer resistance to cisplatin.

A number of alternations in mitochondrial DNA (mtDNA) have been reported in different types of cancers, and the role of mtDNA in cancer has been attracting increasing interest. In order to investigate the relationship between mtDNA alternations and chemosensitivity, we constructed cybrid (trans-mitochondrial hybrid) cell lines carrying a HeLa nucleus and the mtDNA of healthy individuals because of the presence of somatic alternations in the mtDNA of many cancer cells. After a treatment with 1.0 µg/mL cisplatin for 10 days, we isolated 100 cisplatin-resistant clones, 70 of which carried the shorter mtDNA OriB variant (16184-16193 poly-cytosine tract), which was located in the control region of mtDNA. Whole mtDNA sequencing of 10 clones revealed no additional alternations. Re-construction of the HeLa nucleus and mtDNA from cisplatin-resistant cells showed that cisplatin resistance was only acquired by mtDNA alternations in the control region, and not by possible alternation(s) in the nuclear genome.

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids.

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψm) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψm in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.

Chemotherapy promotes tumour cell hybridization in vivo.

Spontaneous cell-cell fusion has been recognized to be an important mechanism for tissue and organ development and repair. In cancer, cell fusion is critically involved in tumourigenesis, metastasis and drug resistance, as illustrated by in vitro experiments. However, there has been no direct detection of tumour cell fusion or hybridization in an in vivo tumour environment, and the features of hybridized cells under selective pressures, such as chemotherapy, are unknown. Here, we expressed two fluorescent marker proteins in the human breast cancer cell line SKBR3 to detect tumour cell hybridization in vivo and performed a xenograft chemotherapy experiment in mice to evaluate the chemotherapeutic response of the hybrids. The mice treated by epirubicin showed that chemotherapy promoted tumour cell hybridization in vivo, which elicited the production of more hybrids in the outer section of the tumour. These results provide the first in vivo evidence of tumour cell fusion and indicate that chemotherapy may contribute to a poor prognosis by enriching for fused cells, which are more malignant. It is therefore necessary to reassess chemotherapy strategies.

Nesfatin-1 antagonized rotenone-induced neurotoxicity in MES23.5 dopaminergic cells.

Nesfatin-1 is a recently identified brain-gut peptide involved in feeding and energy homeostasis. Recently, it has been proved that nesfatin-1 could exert its neuroprotective effect against subarachnoid hemorrhage-induced injury via its anti-apoptotic and anti-inflammatory properties. However, whether it has neuroprotective effect on dopamine neurons is largely unknown. In the present study, we investigated the neuroprotective effect of nesfatin-1 on rotenone-treated MES23.5 dopaminergic cells and illustrated the underlying mechanisms. Our results showed that nesfatin-1 pretreatment could significantly attenuate rotenone-induced cell loss. Further studies showed that the neuroprotective effect of nesfatin-1 against rotenone was mediated by reversing rotenone-induced mitochondrial dysfunction. Nesfatin-1 could rescue rotenone-induced mitochondrial transmembrane potential collapse and restore the function of mitochondrial respiratory chain complex I. In addition, rotenone-induced release of cytochrome C from mitochondria, ROS production and the subsequent caspase-3 activation were also attenuated by nesfatin-1 pretreatment. Our data suggested that nesfatin-1 exerted its neuroprotective effect on dopaminergic cells against rotenone by ameliorating mitochondrial dysfunction and its anti-apoptotic property. This suggested that nesfatin-1 had the potential to be considered as an aid for prevention of Parkinson's disease.

Fusion in cancer: an explanatory model for aneuploidy, metastasis formation, and drug resistance.

Aneuploidy, metastasis formation, and drug resistance are major issues to overcome in most cancers. If there exists common underlying proceedings for the formation of these phenomena is still unknown. The searching and thereby better understanding of causal mechanisms could promote the generation of drugs targeting the ultimate cause of these cancer promoting events. The merging of a cancer cell with another cancer cell or normal cell could be one explanation how cancer cells could gain advantageous properties and escape eliminating cell fates thereby foster cancer progression. This chapter summarizes how cell-cell fusion could directly be involved in the pathogenesis of cancer and which often cancer associated mechanisms, like viral infections or chronic inflammation, are hitherto proposed to trigger cell fusion in cancer context.

Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential.

Most previous studies have linked cancer-macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression.

Deformability-based microfluidic cell pairing and fusion.

We present a microfluidic cell pairing device capable of sequential trapping and pairing of hundreds of cells using passive hydrodynamics and flow-induced deformation. We describe the design and operation principles of our device and show its applicability for cell fusion. Using our device, we achieved both homotypic and heterotypic cell pairing, demonstrating efficiencies up to 80%. The platform is compatible with fusion protocols based on biological, chemical and physical stimuli with fusion yields up to 95%. Our device further permits its disconnection from the fluidic hardware enabling its transportation for imaging and culture while maintaining cell registration on chip. Our design principles and cell trapping technique can readily be applied for different cell types and can be extended to trap and fuse multiple (>2) cell partners as demonstrated by our preliminary experiments.

Pro-apoptotic properties of morphine in neuroblastoma × glioma NG108-15 hybrid cells: modulation by yohimbine.

Short-term incubation with pharmacologically relevant concentrations of morphine has been shown to transiently affect the metabolism and redox status of NG108-15 cells through δ-opioid receptor stimulation, but apparently did not provoke cell death. The present work tries to determine if incubation with morphine at longer time intervals (24 h) provokes apoptosis and/or necrosis, as it has been described in other cell lines. We have also checked the potential modulatory role of yohimbine on these effects, on the basis of the previously described interactions between this drug and opioid receptor ligands. Incubation with morphine 0.1 and 10 µM provoked the appearance of images compatible with apoptosis (bebbling, pyknotic cells with cytoplasmic and nuclear condensation) and necrosis (cells swollen with vacuolated cytoplasm lacking cell processes) that could be observed directly and/or after staining with methylene blue, crystal violet and propidium iodide/4',6-diamidino-2-phenylindole (IP/DAPI). Quantification of apoptosis by activation of caspases 3 and 7 and DNA fragmentation with the Tunel assay revealed a modest but significant increase after incubation with the two concentrations of morphine used. Co-incubation with 10 µM yohimbine prevented all these effects of the opioid. The results extend previous findings of a yohimbine-sensitive, neurotoxic effect of morphine on NG108-15 cells.

Pluripotent hybrid stem cells from transgenic Huntington's disease monkey.

Huntington's disease (HD) is a devastating disease that currently has no cure. Transgenic HD monkeys have developed key neuropathological and cognitive behavioral impairments similar to HD patients. Thus, pluripotent stem cells derived from transgenic HD monkeys could be a useful comparative model for clarifying HD pathogenesis and developing novel therapeutic approaches, which could be validated in HD monkeys. In order to create personal pluripotent stem cells from HD monkeys, here we present a tetraploid technique for deriving pluripotent hybrid HD monkey stem cells.

Crosstalk from non-cancerous mitochondria can inhibit tumor properties of metastatic cells by suppressing oncogenic pathways.

Mitochondrial-nucleus cross talks and mitochondrial retrograde regulation can play a significant role in cellular properties. Transmitochondrial cybrid systems (cybrids) are an excellent tool to study specific effects of altered mitochondria under a defined nuclear background. The majority of the studies using the cybrid model focused on the significance of specific mitochondrial DNA variations in mitochondrial function or tumor properties. However, most of these variants are benign polymorphisms without known functional significance. From an objective of rectifying mitochondrial defects in cancer cells and to establish mitochondria as a potential anticancer drug target, understanding the role of functional mitochondria in reversing oncogenic properties under a cancer nuclear background is very important. Here we analyzed the potential reversal of oncogenic properties of a highly metastatic cell line with the introduction of non-cancerous mitochondria. Cybrids were established by fusing the mitochondria DNA depleted 143B TK- ρ0 cells from an aggressive osteosarcoma cell line with mitochondria from benign breast epithelial cell line MCF10A, moderately metastatic breast cancer cell line MDA-MB-468 and 143B cells. In spite of the uniform cancerous nuclear background, as observed with the mitochondria donor cells, cybrids with benign mitochondria showed high mitochondrial functional properties including increased ATP synthesis, oxygen consumption and respiratory chain activities compared to cybrids with cancerous mitochondria. Interestingly, benign mitochondria could reverse different oncogenic characteristics of 143B TK(-) cell including cell proliferation, viability under hypoxic condition, anti-apoptotic properties, resistance to anti-cancer drug, invasion, and colony formation in soft agar, and in vivo tumor growth in nude mice. Microarray analysis suggested that several oncogenic pathways observed in cybrids with cancer mitochondria are inhibited in cybrids with non-cancerous mitochondria. These results suggest the critical oncogenic regulation by mitochondrial-nuclear cross talk and highlights rectifying mitochondrial functional properties as a promising target in cancer therapy.

Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

Assessing cell fusion and cytokinesis failure as mechanisms of clone 9 hepatocyte multinucleation in vitro.

In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion.

Modelling the effects of cell-cycle heterogeneity on the response of a solid tumour to chemotherapy: biological insights from a hybrid multiscale cellular automaton model.

The therapeutic control of a solid tumour depends critically on the responses of the individual cells that constitute the entire tumour mass. A particular cell's spatial location within the tumour and intracellular interactions, including the evolution of the cell-cycle within each cell, has an impact on their decision to grow and divide. They are also influenced by external signals from other cells as well as oxygen and nutrient concentrations. Hence, it is important to take these into account when modelling tumour growth and the response to various treatment regimes ('cell-kill therapies'), including chemotherapy. In order to address this multiscale nature of solid tumour growth and its response to treatment, we propose a hybrid, individual-based approach that analyses spatio-temporal dynamics at the level of cells, linking individual cell behaviour with the macroscopic behaviour of cell organisation and the microenvironment. The individual tumour cells are modelled by using a cellular automaton (CA) approach, where each cell has its own internal cell-cycle, modelled using a system of ODEs. The internal cell-cycle dynamics determine the growth strategy in the CA model, making it more predictive and biologically relevant. It also helps to classify the cells according to their cell-cycle states and to analyse the effect of various cell-cycle dependent cytotoxic drugs. Moreover, we have incorporated the evolution of oxygen dynamics within this hybrid model in order to study the effects of the microenvironment in cell-cycle regulation and tumour treatments. An important factor from the treatment point of view is that the low concentration of oxygen can result in a hypoxia-induced quiescence (G0/G1 arrest) of the cancer cells, making them resistant to key cytotoxic drugs. Using this multiscale model, we investigate the impact of oxygen heterogeneity on the spatio-temporal patterning of the cell distribution and their cell-cycle status. We demonstrate that oxygen transport limitations result in significant heterogeneity in HIF-1 α signalling and cell-cycle status, and when these are combined with drug transport limitations, the efficacy of the therapy is significantly impaired.

Increasing glucose in KSOMaa basal medium on culture Day 2 improves in vitro development of cloned caprine blastocysts produced via intraspecies and interspecies somatic cell nuclear transfer.

This study was conducted to evaluate the efficiency of potassium simplex optimization medium with amino acids (KSOMaa) as a basal culture medium for caprine intraspecies somatic cell nuclear transfer (SCNT) and caprine-bovine interspecies somatic cell nuclear transfer (iSCNT) embryos. The effect of increased glucose as an energy substrate for late stage development of cloned caprine embryos in vitro was also evaluated. Enucleated caprine and bovine in vitro matured oocytes at metaphase II were reconstructed with caprine ear skin fibroblast cells for the SCNT and iSCNT studies. The cloned caprine and parthenogenetic embryos were cultured in either KSOMaa with 0.2 mM glucose for 8 days (Treatment 1) or KSOMaa for 2 days followed by KSOMaa with additional glucose at a final concentration of 2.78 mM for the last 6 days (Treatment 2). There were no significant differences in the cleavage rates of SCNT (80.7%) and iSCNT (78.0%) embryos cultured in KSOMaa medium. Both Treatment 1 and Treatment 2 could support in vitro development of SCNT and iSCNT embryos to the blastocyst stage. However, the blastocyst development rate of SCNT embryos was significantly higher (P < 0.05) in Treatment 2 compared to Treatment 1. Increasing glucose for later stage embryo development (8-cell stage onwards) during in vitro culture (IVC) in Treatment 2 also improved both caprine SCNT and iSCNT embryo development to the hatched blastocyst stage. In conclusion, this study shows that cloned caprine embryos derived from SCNT and iSCNT could develop to the blastocyst stage in KSOMaa medium supplemented with additional glucose (2.78 mM, final concentration) and this medium also supported hatching of caprine cloned blastocysts.