On the Evaluation of a Novel Hypoxic 3D Pancreatic Cancer Model as a Tool for Radiotherapy Treatment Screening

Tissue engineering is evolving to mimic intricate ecosystems of tumour microenvironments (TME) to more readily map realistic in vivo niches of cancerous tissues. Such advanced cancer tissue models enable more accurate preclinical assessment of treatment strategies. Pancreatic cancer is a dangerous disease with high treatment resistance that is directly associated with a highly complex TME. More specifically, the pancreatic cancer TME includes (i) complex structure and complex extracellular matrix (ECM) protein composition; (ii) diverse cell populations (e.g., stellate cells), cancer associated fibroblasts, endothelial cells, which interact with the cancer cells and promote resistance to treatment and metastasis; (iii) accumulation of high amounts of (ECM), which leads to the creation of a fibrotic/desmoplastic reaction around the tumour; and (iv) heterogeneous environmental gradients such as hypoxia, which result from vessel collapse and stiffness increase in the fibrotic/desmoplastic area of the TME. These unique hallmarks are not effectively recapitulated in traditional preclinical research despite radiotherapeutic resistance being largely connected to them. Herein, we investigate, for the first time, the impact of in vitro hypoxia (5% O2) on the radiotherapy treatment response of pancreatic cancer cells (PANC-1) in a novel polymer (polyurethane) based highly macroporous scaffold that was surface modified with proteins (fibronectin) for ECM mimicry. More specifically, PANC-1 cells were seeded in fibronectin coated macroporous scaffolds and were cultured for four weeks in in vitro normoxia (21% O2), followed by a two day exposure to either in vitro hypoxia (5% O2) or maintenance in in vitro normoxia. Thereafter, in situ post-radiation monitoring (one day, three days, seven days post-irradiation) of the 3D cell cultures took place via quantification of (i) live/dead and apoptotic profiles and (ii) ECM (collagen-I) and HIF-1a secretion by the cancer cells. Our results showed increased post-radiation viability, reduced apoptosis, and increased collagen-I and HIF-1a secretion in in vitro hypoxia compared to normoxic cultures, revealing hypoxia-induced radioprotection. Overall, this study employed a low cost, animal free model enabling (i) the possibility of long-term in vitro hypoxic 3D cell culture for pancreatic cancer, and (ii) in vitro hypoxia associated PDAC radio-protection development. Our novel platform for radiation treatment screening can be used for long-term in vitro post-treatment observations as well as for fractionated radiotherapy treatment

Novel anticancer and treatment sensitizing compounds against pancreatic cancer

The isolation of chemical compounds from natural origins for medical application has played an important role in modern medicine with a range of novel treatments having emerged from various natural forms over the past decades. Natural compounds have been exploited for their antioxidant, antimicrobial and antitumor capabilities. Specifically, 60% of today’s anticancer drugs originate from natural sources. Moreover, the combination of synthetic and natural treatments has shown applications for (i) reduced side effects, (ii) treatment sensitization and (iii) reduction in treatment resistance. This review aims to collate novel and natural compounds that are being explored for their preclinical anticancer, chemosensitizing and radiosensitizing effects on Pancreatic Ductal Adenocarcinoma (PDAC), which is a lethal disease with current treatments being inefficient and causing serve side effects. Two key points are highlighted by this work: (i) the availability of a range of natural compounds for potentially new therapeutic approaches for PDAC, (ii) potential synergetic impact of natural compounds with advanced chemo-and radio-therapeutic modalities for PDAC

A novel 3D polymeric tool for accurate screening of the performance of immunodiagnostic polymeric microneedles

INTRODUCTION: Melanoma is the most lethal skin cancer, having a rapid increase of occurrenceover the past 30 years1. To date, the most effective treatment for melanoma is the early diagnosis, whichis followed by surgical resection. Therefore, in order to improve these disappointing statistical figures,it is essential to develop efficient diagnostic tools for rapid detection of the disease’s specific markers.Minimally invasive microneedles (MNs) are promising candidates, as they enable rapid and pain-freeprotein biomarker detection in situ. However, validating the developed microneedle (MN) systemsremains a bottleneck. To date, the most commonly used systems for in vitro microneedle validation areeither homogeneous solutions that contain the target antigen to be detected by the MNs or excisedanimal skin. Animal skin strikes many similarities with the human skin, however the animal skinproperties, such as stiffness, elasticity, porosity, which vary between different patients cannot be easilytuned/ tailored2. Furthermore, antigen solutions can be informative for a preliminary evaluation of theMN arrays, but they are not representative models of in vivo skin structure and biomarker concentration.Biomaterial based 3D structures can simulate important skin tissue features, such as stiffness, elasticity,porosity, structure, extracellular matrix presence that can vary between different patients, different skintypes and with ageing. Moreover, they can provide a realistic structural environment for the penetrationand action of MN. Therefore, these biomaterial based 3D structures have great potential as screeningtools for MN evaluation. The aim of this work was to validate the S100 expression, a marker that isupregulated in melanoma, on a microporous polymer based 3D melanoma model. S100 expression inthe model was confirmed using a novel immunodiagnostic microneedle device.METHODS: 3D polymer (PU) based microporous scaffolds (5x5x2.5mm3) were developed using theThermally Induced Phase Separation (TIPS) method, as described previously3. The porosity was 80%and the pore size 100-120 μm. Thereafter, the metastatic melanoma cell line A-375 was injected andcultured in those scaffolds for 5 weeks. Evaluation of cell distribution within the PU matrix wasconducted with Scanning Electron Microscopy (SEM). Viable (live) cells were visualised in situ withconfocal laser scanning microscopy (CLSM) of several sections of each scaffold. Furthermore, thedetection of the S100 marker was carried out with PLA microneedles both on the 3D scaffold and forthe cell culture supernatants. The PLA microneedle device was produced, surface modified and coatedwith the S100 antibody as previously described, followed by the detection of the antigen viaimmunoassay analysis on the microneedle surface4.RESULTS: The MN device was able to detect the S100 secretion from the melanoma cells in the scaffold after 35 days of a viable culture, producing a clear and visible detection signal similar to theone detected for the positive control samples. However, S100 gradients were not detected in the cellculture supernatants, suggesting that this versatile scaffolding tool can be an advantageous low costanimal free tool to be use as a surrogate for the in vitro evaluations of the MNs.REFERENCES: 1American Cancer Society, 2018. 2K. Moronkeji and R. Akhtar, in Mechanical Propertiesof Aging Soft Tissues, eds. B. Derby and R. Akhtar, Springer International Publishing, Cham, 2015, pp. 237-263. 3S. Totti, M. Allenby, S. B. d. Santos, A. Mantalaris and E. Velliou, RSC adv, 2018, 8, 20928-40 4 K. W. Ng, W. M. Lau and A. C. Williams, Drug Deliv Transl Res, 2015, 5, 387-396

Novel in situ multi-level analysis of structural-mechanical relations in a bioinspired polyurethane-based tissue model

In this manuscript, we elucidated, for the first time, the substructural mechanisms present in our recently developed bioinspired polyurethane-based pancreatic tissue models. Different protein coatings of the model, i.e., collagen and fibronectin were examined. More specifically, analysis took place by combined real-time synchrotron X-ray scattering techniques and confocal laser scanning microscopy, to quantify the structural alteration of uncoated-polyurethane (PU) and protein-coated PU as well as the time-resolved structural reorganisation occurring at the micro-, nano- and lattice length scales during in situ micromechanical testing. We demonstrate that a clear increase of stiffness at the lamellar level following the fibronectin-PU modification, which is linked to the changes in the mechanics of the lamellae and interlamellar cohesion. This multi-level analysis of structural-mechanical relations in this polyurethane-based pancreatic cancer tissue model opens an opportunity in designing mechanically robust cost-effective tissue models not only for fundamental research but also for treatment screening

Multi-scale structural and mechanical characterisation in bioinspired polyurethane-based pancreatic cancer model

In this work, novel bioinspired polyurethane (PU) scaffolds were fabricated via freeze casting for PU-based Pancreatic Ductal Adenocarcinoma (PDAC) model. In order to reproduce the tumour micro-environment that facilitates cellular kinetics, the PU scaffolds were surface modified with extracellular matrix (ECM) proteins including collagen and fibronectin (Col and FN). Synchrotron-based small- and wide-angle X-ray scattering (SAXS/WAXS) techniques were applied to probe structural evolution during in situ mechanical testing. Strains at macroscopic, nano-, and lattice scales were obtained to investigate the effects of ECM proteins and pancreatic cell activities to PU scaffolds. Significant mechanical strengthening across length scales of PU scaffolds was observed in specimens surface modified by FN. A model of stiffness modulation via enhanced interlamellar recruitment is proposed to explain the multi-scale strengthening mechanisms. Understanding multi-scale deformation mechanisms of a series of PU scaffolds opens an opportunity in developing a novel pancreatic cancer model for studying cancer evolution and predicting outcomes of drug/treatments

Mesoporous Phosphate-Based Glasses Prepared via Sol-Gel

In the present study, a mesoporous phosphate-based glass (MPG) in the P2O5-CaO-Na2O system was synthesised, for the first time, using a combination of sol-gel chemistry and supramolecular templating. A comparison between the structural properties, bioactivity and biocompatibility of the MPG with a non-porous phosphate-based glass (PG) of analogous composition prepared via the same sol-gel synthesis method, but in the absence of a templating surfactant is also presented. Results indicate that the MPG has enhanced bioactivity and biocompatibility compared to the PG, despite having similar local structure and dissolution properties. In contrast to the PG, the MPG shows formation of hydroxyl carbonate apatite (HCA) on its surface after 24 hours of immersion in simulated body fluid. Moreover, MPG shows enhanced viability of Saos-2 osteosarcoma cells after 7 days of culturing. This suggests that textural properties (porosity and surface area) play a crucial role in the kinetics of HCA formation and in interaction with cells. Increased efficiency of drug loading and release over non-porous PG systems was proved using the antimicrobial tetracycline hydrochloride as a drug model. This study represents a significant advance in the field of mesoporous materials for drug delivery and bone tissue regeneration as it reports, for the first time, the synthesis, structural characterisation and biocompatibility of mesoporous calcium phosphate glasses.In the present study, a mesoporous phosphate-based glass (MPG) in the P2O5-CaO-Na2O system was synthesised, for the first time, using a combination of sol-gel chemistry and supramolecular templating. A comparison between the structural properties, bioactivity and biocompatibility of the MPG with a non-porous phosphate-based glass (PG) of analogous composition prepared via the same sol-gel synthesis method, but in the absence of a templating surfactant is also presented. Results indicate that the MPG has enhanced bioactivity and biocompatibility compared to the PG, despite having similar local structure and dissolution properties. In contrast to the PG, the MPG shows formation of hydroxyl carbonate apatite (HCA) on its surface after 24 hours of immersion in simulated body fluid. Moreover, MPG shows enhanced viability of Saos-2 osteosarcoma cells after 7 days of culturing. This suggests that textural properties (porosity and surface area) play a crucial role in the kinetics of HCA formation and in interaction with cells. Increased efficiency of drug loading and release over non-porous PG systems was proved using the antimicrobial tetracycline hydrochloride as a drug model. This study represents a significant advance in the field of mesoporous materials for drug delivery and bone tissue regeneration as it reports, for the first time, the synthesis, structural characterisation and biocompatibility of mesoporous calcium phosphate glasses

Bile microbiome signatures associated with pancreatic ductal adenocarcinoma compared to benign disease: a UK pilot study

Pancreatic ductal adenocarcinoma (PDAC) has a very poor survival. The intra-tumoural microbiome can influence pancreatic tumourigenesis and chemoresistance and, therefore, patient survival. The role played by bile microbiota in PDAC is unknown. We aimed to define bile microbiome signatures that can effectively distinguish malignant from benign tumours in patients presenting with obstructive jaundice caused by benign and malignant pancreaticobiliary disease. Prospective bile samples were obtained from 31 patients who underwent either Endoscopic Retrograde Cholangiopancreatography (ERCP) or Percutaneous Transhepatic Cholangiogram (PTC). Variable regions (V3-V4) of the 16S rRNA genes of microorganisms present in the samples were amplified by Polymerase Chain Reaction (PCR) and sequenced. The cohort consisted of 12 PDAC, 10 choledocholithiasis, seven gallstone pancreatitis and two primary sclerosing cholangitis patients. Using the 16S rRNA method, we identified a total of 135 genera from 29 individuals (12 PDAC and 17 benign). The bile microbial beta diversity significantly differed between patients with PDAC vs. benign disease (Permanova p = 0.0173). The separation of PDAC from benign samples is clearly seen through unsupervised clustering of Aitchison distance. We found three genera to be of significantly lower abundance among PDAC samples vs. benign, adjusting for false discovery rate (FDR). These were Escherichia (FDR = 0.002) and two unclassified genera, one from Proteobacteria (FDR = 0.002) and one from Enterobacteriaceae (FDR = 0.011). In the same samples, the genus Streptococcus (FDR = 0.033) was found to be of increased abundance in the PDAC group. We show that patients with obstructive jaundice caused by PDAC have an altered microbiome composition in the bile compared to those with benign disease. These bile-based microbes could be developed into potential diagnostic and prognostic biomarkers for PDAC and warrant further investigation

Modelling the induced thermotolerance due to heat, acid shock and rising temperatures

This book covers the advances in thermobacteriology, including technological and engineering aspects of thermal processes targeting on the production of food safe products