Reducing Tumour Hypoxia via Oral Administration of Oxygen Nanobubbles

Hypoxia has been shown to be a key factor inhibiting the successful treatment of solid tumours. Existing strategies for reducing hypoxia, however, have shown limited efficacy and/or adverse side effects. The aim of this study was to investigate the potential for reducing tumour hypoxia using an orally delivered suspension of surfactant-stabilised oxygen nanobubbles. Experiments were carried out in a mouse xenograft tumour model for human pancreatic cancer (BxPc-3 cells in male SCID mice). A single dose of 100 μL of oxygen saturated water, oxygen nanobubbles or argon nanobubbles was administered via gavage. Animals were sacrificed 30 minutes post-treatment (3 per group) and expression of hypoxia-inducible-factor-1α (HIF1α) protein measured by real time quantitative polymerase chain reaction and Western blot analysis of the excised tumour tissue. Neither the oxygen saturated water nor argon nanobubbles produced a statistically significant change in HIF1α expression at the transcriptional level. In contrast, a reduction of 75% and 25% in the transcriptional and translational expression of HIF1α respectively (p<0.001) was found for the animals receiving the oxygen nanobubbles. This magnitude of reduction has been shown in previous studies to be commensurate with an improvement in outcome with both radiation and drug-based treatments. In addition, there was a significant reduction in the expression of vascular endothelial growth factor (VEGF) in this group and corresponding increase in the expression of arrest-defective protein 1 homolog A (ARD1A)

Three-dimensional recapitulation of neuroblastoma: from model to drug screening platform

Neuroblastoma is one of the most common yet fatal paediatric cancer. Nevertheless, few drug treatments are available in the market. One of the difficulties is the drug responses reported in 2-D in vitro tests does not always accurately reflect those observed in 3-D in vivo models. As the process consumes time, money and animal lives, a 3-D in vitro drug testing bioreactor recapitulating neuroblastoma tumours are needed to screen out any unsuccessful drug candidates before they enter in vivo tests. In this thesis, a 3-D in vitro model comprised neural stem cells (NSCs) or neurob- lastoma cells (SH-SY5Ys) and human umbilical vein endothelial cells (HUVECs) were initially optimised and developed in the laminin-rich hydrogel matrix to recapitulate neuroblastoma tumours. Next, a modified polydimethylsiloxane bioreactor (TissueFlex®) was developed to extend the viability of the 3-D in vitro model by enhancing the mass transport of nutrients and oxygen. The bioreactor was continuously perfused with a supplemented endothelial basal medium (EGM-2) co-treated with cisplatin, retinoic acid (RA), both cisplatin and RA, or EGM-2 as a control for three weeks. Cell morphology, cell viability and expressions of markers (TuJ1, Nestin and CD31) were measured weekly. Results were compared to parallel cultures of 2-D monolayer and 3DFlo® automatic cell culture systems. Our results show that hydrogel fragmentation allowed the formation of concentrated laminin and cellular networks along the gel granule boundaries with neurite extensions observed. Besides, cell surface marker expressions suggested an optimal NSC-HUVEC seeding density ratio of 3:1, cultured in 100% EGM-2 medium. The incorporation of perfusion culture to the model gave rise to significant increases in the number of cells and the expression of Nestin compared to static culture. After 3 weeks, only 2-D cultures showed a significant decrease in marker expressions upon treatment with cisplatin or cisplatin and RA in combination. Also, drug treatments were significantly less effective in 3-D bioreactors than in 2-D cultures. This suggests that the 3-D interactions between cells and hydrogel have created a microenvironment that recapitulates phenomena present in neuroblastoma and provides resistance to cancer therapeutics. Our 3-D in vitro model and perfusion bioreactor have implications for a more efficient drug discovery process by closing the gap between cell culture and physiological tissue investigations. These can be applied towards personalised medicine, where a patientâs biopsy specimen can be tested in the system against a series of drugs to identify ones giving rise to optimal therapeutic outcomes.</p

Morphological analysis of human umbilical vein endothelial cells co-cultured with ovarian cancer cells in 3D: An oncogenic angiogenesis assay.

Antiangiogenic therapy for cancer is a strategy targeted at tumour vasculature, often in combination with conventional cytotoxicity treatments. Animal testing is still the most common method used for evaluating the efficacy of new drugs but tissue-engineered in vitro models are becoming more acceptable for replacing and reducing the use of animals in anti-cancer drug screening. In this study, a 3D co-culture model of human endothelial cells and ovarian cancer cells was developed. This model has the potential to mimic the interactions between endothelial cells and ovarian cancer cells. The feasibility of applying this model in drug testing was explored here. The complex morphology of the co-culture system, which features development of both endothelial tubule-like structures and tumour structures, was analysed quantitatively by an image analysis method. The co-culture morphology integrity was maintained for 10 days and the potential of the model for anti-cancer drug testing was evaluated using Paclitaxel and Cisplatin, two common anti-tumour drugs with different mechanisms of action. Both traditional cell viability assays and quantitative morphological analyses were applied in the drug testing. Cisplatin proved a good example showing the advantages of morphological analysis of the co-culture model when compared with mono-culture of endothelial cells, which did not reveal an inhibitory effect of Cisplatin on the tubule-like endothelial structures. Thus, the tubule areas of the co-culture reflected the anti-angiogenesis potential of Cisplatin. In summary, in vitro cancer models can be developed using a tissue engineering approach to more closely mimic the characteristics of tumours in vivo. Combined with the image analysis technique, this developed 3D co-culture angiogenesis model will provide more reproducible and reliably quantified results and reveal further information of the drug's effects on both tumour cell growth and tumour angiogenesis

Semi-automated image analysis comparing morphologies of different <i>in vitro</i> models.

<p>(A) Comparison of 3D on-top and 3D sandwich co-culture of OVCAR8 and HUVECs with regard to: (i) Total spheroid areas in μm² and (ii) Mean tubule areas. (B) Comparison of 3D mono-culture of OVCAR8 and 3D co-culture of HUVECs and OVCAR8 in terms of: (i) Total spheroid areas in μm² and (ii) Mean spheroid areas in μm². (C) Angiogenesis parameter comparisons for 3D on-top and 3D sandwich co-culture of OVCAR8 and HUVECs over 10 days for: (i) Total tubule areas and (ii) Branching points. Results are presented as (mean ± SD), and are considered significantly different when p < 0.05 based on Student’s t-test (marked as ‘*’ in the figures showing significant difference between the compared models on the same day). Note that since HUVECs mono-culture lost their tubule network at day 2 and eventually lost signs of living cells over longer term culture (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180296#pone.0180296.g002" target="_blank">Fig 2(A))</a>, only mono-culture of cancer cell OVCAR8 and co-culture of OVCAR8 and HUVECs were compared here over 10 days of culture.</p

Schematic procedure for 3D co-culture of HUVECs and OVCAR8 in Matrigel sandwich.

<p>(A) HUVECs seeded on polymerised Matrigel; (B) After 4 hours, HUVECs started to form tubules; (C) OVCAR8 cell suspension in medium containing 10% Matrigel was added; (D) 24 hours later, Matrigel sandwich structure formed and co-culture stabilised, ready for longer term culture or further drug testing.</p

Apoptosis assay comparing co-culture of OVCAR8 with HUVECs and mono-culture of OVCAR8 at day 10.

<p>(A) Fluorescence images of co-culture. It is noticeable that nearly no apoptotic signal was observed with thinner tubule structures, while more apoptosis was detected with the structures with larger dimensions. (B) Fluorescent images of mono-culture. Cleaved caspase 3 (magenta); DAPI (blue), HUVECs (red), and OVCAR8 (green). Scale bar: 100μm. (C) Zen localisation coefficient comparison between co-culture and mono-culture. No significant difference was observed.</p

Morphological analysis of human umbilical vein endothelial cells co-cultured with ovarian cancer cells in 3D: An oncogenic angiogenesis assay

<div><p>Antiangiogenic therapy for cancer is a strategy targeted at tumour vasculature, often in combination with conventional cytotoxicity treatments. Animal testing is still the most common method used for evaluating the efficacy of new drugs but tissue-engineered <i>in vitro</i> models are becoming more acceptable for replacing and reducing the use of animals in anti-cancer drug screening. In this study, a 3D co-culture model of human endothelial cells and ovarian cancer cells was developed. This model has the potential to mimic the interactions between endothelial cells and ovarian cancer cells. The feasibility of applying this model in drug testing was explored here. The complex morphology of the co-culture system, which features development of both endothelial tubule-like structures and tumour structures, was analysed quantitatively by an image analysis method. The co-culture morphology integrity was maintained for 10 days and the potential of the model for anti-cancer drug testing was evaluated using Paclitaxel and Cisplatin, two common anti-tumour drugs with different mechanisms of action. Both traditional cell viability assays and quantitative morphological analyses were applied in the drug testing. Cisplatin proved a good example showing the advantages of morphological analysis of the co-culture model when compared with mono-culture of endothelial cells, which did not reveal an inhibitory effect of Cisplatin on the tubule-like endothelial structures. Thus, the tubule areas of the co-culture reflected the anti-angiogenesis potential of Cisplatin. In summary, <i>in vitro</i> cancer models can be developed using a tissue engineering approach to more closely mimic the characteristics of tumours <i>in vivo</i>. Combined with the image analysis technique, this developed 3D co-culture angiogenesis model will provide more reproducible and reliably quantified results and reveal further information of the drug’s effects on both tumour cell growth and tumour angiogenesis.</p></div

Drug response comparisons between 3D mono-cultures and co-cultures.

<p>(A) Dose-dependent responses comparing monocultures and co-culture, measured by cell viability inhibition percentages in a Matrigel sandwich treated with: (i) Cisplatin; (ii) Paclitaxel. (B) Tubule area reduction comparing monocultures and co-culture, after treatment by: (i) Cisplatin and (ii) Paclitaxel. *p<0.05 **p<0.01 compared with control using Student’s t-test.</p