Publications by authors named "Marilena Loizidou"

54 Publications

Clinical relevance assessment of animal preclinical research (RAA) tool: development and explanation.

PeerJ 2021 27;9:e10673. Epub 2021 Jan 27.

Department of Anesthesiology and Pain Medicine, Blueprint Translational Research Group, Clinical Epidemiology and Regenerative Medicine Programs, Ottawa Hospital Research Institute, Ottawa Hospital, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada.

Background: Only a small proportion of preclinical research (research performed in animal models prior to clinical trials in humans) translates into clinical benefit in humans. Possible reasons for the lack of translation of the results observed in preclinical research into human clinical benefit include the design, conduct, and reporting of preclinical studies. There is currently no formal domain-based assessment of the clinical relevance of preclinical research. To address this issue, we have developed a tool for the assessment of the clinical relevance of preclinical studies, with the intention of assessing the likelihood that therapeutic preclinical findings can be translated into improvement in the management of human diseases.

Methods: We searched the EQUATOR network for guidelines that describe the design, conduct, and reporting of preclinical research. We searched the references of these guidelines to identify further relevant publications and developed a set of domains and signalling questions. We then conducted a modified Delphi-consensus to refine and develop the tool. The Delphi panel members included specialists in evidence-based (preclinical) medicine specialists, methodologists, preclinical animal researchers, a veterinarian, and clinical researchers. A total of 20 Delphi-panel members completed the first round and 17 members from five countries completed all three rounds.

Results: This tool has eight domains (construct validity, external validity, risk of bias, experimental design and data analysis plan, reproducibility and replicability of methods and results in the same model, research integrity, and research transparency) and a total of 28 signalling questions and provides a framework for researchers, journal editors, grant funders, and regulatory authorities to assess the potential clinical relevance of preclinical animal research.

Conclusion: We have developed a tool to assess the clinical relevance of preclinical studies. This tool is currently being piloted.
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http://dx.doi.org/10.7717/peerj.10673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847198PMC
January 2021

Increased expression of IGF-1Ec with increasing colonic polyp dysplasia and colorectal cancer.

J Cancer Res Clin Oncol 2020 Nov 8;146(11):2861-2870. Epub 2020 Aug 8.

Research Department of Surgical Biotechnology, Division of Surgery and Interventional Science, Royal Free Campus, UCL, London, UK.

Purpose: IGF-1Ec is an isoform of Insulin-like growth factor 1 (IGF-1) and has recently been identified to be overexpressed in cancers including prostate and neuroendocrine tumours. The aim of this paper is to investigate the expression of IGF-1Ec in colorectal cancer and polyps compared to normal colon tissues and its association with recurrent disease using semi-quantitative immunohistochemistry.

Methods: Immunohistochemistry for IGF-1Ec expression was performed for colorectal cancer, colorectal polyps and normal colonic tissues. The quantification of IGF-1Ec expression was performed with the use of Image J software and the IHC profiler plugin. Following ethics approval from the National Research Ethics Service (Reference 11/LO/1521), clinical information including recurrent disease on follow-up was collected for patients with colorectal cancer.

Results: Immunohistochemistry was performed in 16 patients with colorectal cancer and 11 patients with colonic polyps and compared to normal colon tissues and prostate adenocarcinoma (positive control) tissues. Significantly increased expression of IGF-1Ec was demonstrated in colorectal cancer (p < 0.001) and colorectal polyps (p < 0.05) compared to normal colonic tissues. Colonic adenomas with high-grade dysplasia had significantly higher expression of IGF-1Ec compared to low-grade dysplastic adenomas (p < 0.001). Colorectal cancers without lymph node metastases at the time of presentation had significantly higher IGF-1Ec expression compared to lymph node-positive disease (p < 0.05). No correlation with recurrent disease was identified with IGF-1Ec expression.

Conclusion: IGF-1Ec is significantly overexpressed in colorectal cancer and polyps compared to normal colon tissues offering a potential target to improve colonoscopic identification of colorectal polyps and cancer and intraoperative identification of colorectal tumours.
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http://dx.doi.org/10.1007/s00432-020-03345-0DOI Listing
November 2020

Cancer cells grown in 3D under fluid flow exhibit an aggressive phenotype and reduced responsiveness to the anti-cancer treatment doxorubicin.

Sci Rep 2020 07 21;10(1):12020. Epub 2020 Jul 21.

School of Life Sciences, University of Westminster, 115 New Cavendish St, London, W1W 6UW, UK.

3D laboratory models of cancer are designed to recapitulate the biochemical and biophysical characteristics of the tumour microenvironment and aim to enable studies of cancer, and new therapeutic modalities, in a physiologically-relevant manner. We have developed an in vitro 3D model comprising a central high-density mass of breast cancer cells surrounded by collagen type-1 and we incorporated fluid flow and pressure. We noted significant changes in cancer cell behaviour using this system. MDA-MB231 and SKBR3 breast cancer cells grown in 3D downregulated the proliferative marker Ki67 (P < 0.05) and exhibited decreased response to the chemotherapeutic agent doxorubicin (DOX) (P < 0.01). Mesenchymal markers snail and MMP14 were upregulated in cancer cells maintained in 3D (P < 0.001), cadherin-11 was downregulated (P < 0.001) and HER2 increased (P < 0.05). Cells maintained in 3D under fluid flow exhibited a further reduction in response to DOX (P < 0.05); HER2 and Ki67 levels were also attenuated. Fluid flow and pressure was associated with reduced cell viability and decreased expression levels of vimentin. In summary, aggressive cancer cell behaviour and reduced drug responsiveness was observed when breast cancer cells were maintained in 3D under fluid flow and pressure. These observations are relevant for future developments of 3D in vitro cancer models and organ-on-a-chip initiatives.
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http://dx.doi.org/10.1038/s41598-020-68999-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374750PMC
July 2020

Radiosensitisation of Hepatocellular Carcinoma Cells by Vandetanib.

Cancers (Basel) 2020 Jul 13;12(7). Epub 2020 Jul 13.

University College London Cancer Institute, University College London, London WC1E 6BT, UK.

Hepatocellular Carcinoma (HCC) is increasing in incidence worldwide and requires new approaches to therapy. The combination of anti-angiogenic drug therapy and radiotherapy is one promising new approach. The anti-angiogenic drug vandetanib is a tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and RET proto-oncogene with radio-enhancement potential. To explore the benefit of combined vandetanib and radiotherapy treatment for HCC, we studied outcomes following combined treatment in pre-clinical models.

Methods: Vandetanib and radiation treatment were combined in HCC cell lines grown and . In addition to 2D migration and clonogenic assays, the combination was studied in 3D spheroids and a syngeneic mouse model of HCC.

Results: Vandetanib IC 50 s were measured in 20 cell lines and the drug was found to significantly enhance radiation cell kill and to inhibit both cell migration and invasion . , combination therapy significantly reduced cancer growth and improved overall survival, an effect that persisted for the duration of vandetanib treatment.

Conclusion: In 2D and 3D studies and in a syngeneic model , the combination of vandetanib plus radiotherapy was more efficacious than either treatment alone. This new combination therapy for HCC merits evaluation in clinical trials.
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http://dx.doi.org/10.3390/cancers12071878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408860PMC
July 2020

Human airway-like multilayered tissue on 3D-TIPS printed thermoresponsive elastomer/collagen hybrid scaffolds.

Acta Biomater 2020 09 11;113:177-195. Epub 2020 Jul 11.

Centre for Biomaterials in Surgical Reconstruction and Regeneration, Division of Surgery & Interventional Science, University College London, London NW3 2PF, United Kingdom. Electronic address:

Developing a biologically representative complex tissue of the respiratory airway is challenging, however, beneficial for treatment of respiratory diseases, a common medical condition representing a leading cause of death in the world. This in vitro study reports a successful development of synthetic human tracheobronchial epithelium based on interpenetrated hierarchical networks composed of a reversely 3D printed porous structure of a thermoresponsive stiffness-softening elastomer nanohybrid impregnated with collagen nanofibrous hydrogel. Human bronchial epithelial cells (hBEpiCs) were able to attach and grow into an epithelial monolayer on the hybrid scaffolds co-cultured with either human bronchial fibroblasts (hBFs) or human bone-marrow derived mesenchymal stem cells (hBM-MSCs), with substantial enhancement of mucin expression, ciliation, well-constructed intercellular tight junctions and adherens junctions. The multi-layered co-culture 3D scaffolds consisting of a top monolayer of differentiated epithelium, with either hBFs or hBM-MSCs proliferating within the hyperelastic nanohybrid scaffold underneath, created a tissue analogue of the upper respiratory tract, validating these 3D printed guided scaffolds as a platform to support co-culture and cellular organization. In particular, hBM-MSCs in the co-culture system promoted an overall matured physiological tissue analogue of the respiratory system, a promising synthetic tissue for drug discovery, tracheal repair and reconstruction. STATEMENT OF SIGNIFICANCE: Respiratory diseases are a common medical condition and represent a leading cause of death in the world. However, the epithelium is one of the most challenging tissues to culture in vitro, and suitable tracheobronchial models, physiologically representative of the innate airway, remain largely elusive. This study presents, for the first time, a systematic approach for the development of functional multilayered epithelial synthetic tissue in vitro via co-culture on a 3D-printed thermoresponsive elastomer interpenetrated with a collagen hydrogel network. The viscoelastic nature of the scaffold with stiffness softening at body temperature provide a promising matrix for soft tissue engineering. The results presented here provide new insights about the epithelium at different surfaces and interfaces of co-culture, and pave the way to offer a customizable reproducible technology to generate physiologically relevant 3D biomimetic systems to advance our understanding of airway tissue regeneration.
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http://dx.doi.org/10.1016/j.actbio.2020.07.013DOI Listing
September 2020

Cancer-associated fibroblasts mediate cancer progression and remodel the tumouroid stroma.

Br J Cancer 2020 Sep 9;123(7):1178-1190. Epub 2020 Jul 9.

Institute of Orthopaedics and Musculoskeletal Sciences, Division of Surgery and Interventional Science, University College London, Stanmore Campus, Brockley Hill, HA7 4LP, London, UK.

Background: Cancer-associated fibroblasts (CAFs) are highly differentiated and heterogeneous cancer-stromal cells that promote tumour growth, angiogenesis and matrix remodelling.

Methods: We utilised an adapted version of a previously developed 3D in vitro model of colorectal cancer, composed of a cancer mass and the surrounding stromal compartment. We compared cancer invasion with an acellular stromal surround, a "healthy" or normal cellular stroma and a cancerous stroma. For the cancerous stroma, we incorporated six patient-derived CAF samples to study their differential effects on cancer growth, vascular network formation and remodelling.

Results: CAFs enhanced the distance and surface area of the invasive cancer mass whilst inhibiting vascular-like network formation. These processes correlated with the upregulation of hepatocyte growth factor (HGF), metallopeptidase inhibitor 1 (TIMP1) and fibulin-5 (FBLN5). Vascular remodelling of previously formed endothelial structures occurred through the disruption of complex networks, and was associated with the upregulation of vascular endothelial growth factor (VEGFA) and downregulation in vascular endothelial cadherin (VE-Cadherin).

Conclusions: These results support, within a biomimetic 3D, in vitro framework, the direct role of CAFs in promoting cancer invasion, and their key function in driving vasculogenesis and angiogenesis.
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http://dx.doi.org/10.1038/s41416-020-0973-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524802PMC
September 2020

The anti-angiogenic tyrosine kinase inhibitor Pazopanib kills cancer cells and disrupts endothelial networks in biomimetic three-dimensional renal tumouroids.

J Tissue Eng 2020 Jan-Dec;11:2041731420920597. Epub 2020 May 18.

Research Department of Surgical Biotechnology, Division of Surgery & Interventional Science, University College London, London, UK.

Pazopanib is a tyrosine kinase inhibitor used to treat renal cell carcinoma. Few in vitro studies investigate its effects towards cancer cells or endothelial cells in the presence of cancer. We tested the effect of Pazopanib on renal cell carcinoma cells (CAKI-2,786-O) in two-dimensional and three-dimensional tumouroids made of dense extracellular matrix, treated in normoxia and hypoxia. Finally, we engineered complex tumouroids with a stromal compartment containing fibroblasts and endothelial cells. Simple CAKI-2 tumouroids were more resistant to Pazopanib than 786-O tumouroids. Under hypoxia, while the more 'resistant' CAKI-2 tumouroids showed no decrease in viability, 786-O tumouroids required higher Pazopanib concentrations to induce cell death. In complex tumouroids, Pazopanib exposure led to a reduction in the overall cell viability (p < 0.0001), disruption of endothelial networks and direct killing of renal cell carcinoma cells. We report a biomimetic multicellular tumouroid for drug testing, suitable for agents whose primary target is not confined to cancer cells.
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http://dx.doi.org/10.1177/2041731420920597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238304PMC
May 2020

Enhanced photodynamic therapy and fluorescence imaging using gold nanorods for porphyrin delivery in a novel in vitro squamous cell carcinoma 3D model.

J Mater Chem B 2020 06 18;8(23):5131-5142. Epub 2020 May 18.

Division of Surgery and Interventional Science, Centre for Nanomedicine and Surgical Theranostics, University College London, Royal Free Campus, Rowland Hill St, London, NW3 2PE, UK.

Nanocomposites of gold nanorods (Au NRs) with the cationic porphyrin TMPyP (5,10,15,20-tetrakis(1- methyl 4-pyridinio)porphyrin tetra(p-toluenesulfonate)) were investigated as a nanocarrier system for photodynamic therapy (PDT) and fluorescence imaging. To confer biocompatibility and facilitate the cellular uptake, the NRs were encapsulated with polyacrylic acid (PAA) and efficiently loaded with the cationic porphyrin by electrostatic interaction. The nanocomposites were tested with and without light exposure following incubation in 2D monolayer cultures and a 3D compressed collagen construct of head and neck squamous cell carcinoma (HNSCC). The results showed that Au NRs enhance the absorption and emission intensity of TMPyP and improve its photodynamic efficiency and fluorescence imaging capability in both 2D cultures and 3D cancer constructs. Au NRs are promising theranostic agents for delivery of photosensitisers for HNSCC treatment and imaging.
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http://dx.doi.org/10.1039/d0tb00810aDOI Listing
June 2020

Synergy between Photodynamic Therapy and Dactinomycin Chemotherapy in 2D and 3D Ovarian Cancer Cell Cultures.

Int J Mol Sci 2020 Apr 30;21(9). Epub 2020 Apr 30.

Division of Surgery & Interventional Science, Faculty of Medical Sciences, University College London, London NW3 2QG, UK.

In this study we explored the efficacy of combining low dose photodynamic therapy using a porphyrin photosensitiser and dactinomycin, a commonly used chemotherapeutic agent. The studies were carried out on compressed collagen 3D constructs of two human ovarian cancer cell lines (SKOV3 and HEY) versus their monolayer counterparts. An amphiphilc photosensitiser was employed, disulfonated tetraphenylporphine, which is not a substrate for ABC efflux transporters that can mediate drug resistance. The combination treatment was shown to be effective in both monolayer and 3D constructs of both cell lines, causing a significant and synergistic reduction in cell viability. Compared to dactinomycin alone or PDT alone, higher cell kill was found using 2D monolayer culture vs. 3D culture for the same doses. In 3D culture, the combination therapy resulted in 10 and 22 times higher cell kill in SKOV3 and HEY cells at the highest light dose compared to dactinomycin monotherapy, and 2.2 and 5.5 times higher cell kill than PDT alone. The combination of low dose PDT and dactinomycin appears to be a promising way to repurpose dactinomycin and widen its therapeutic applications.
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http://dx.doi.org/10.3390/ijms21093203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247344PMC
April 2020

Acceptability and feasibility study of patient-specific 'tumouroids' as personalised treatment screening tools: Protocol for prospective tissue and data collection of participants with confirmed or suspected renal cell carcinoma.

Int J Surg Protoc 2019 2;14:24-29. Epub 2019 Apr 2.

Faculty of Medical Science, University College London, UK.

Introduction: 'Personalised medicine' aims to tailor interventions to the individual, and has become one of the fastest growing areas of cancer research. One of these approaches is to harvest cancer cells from patients and grow them in the laboratory, which can then be subjected to treatments and the response assessed. We have developed a 3D tumour model with a complex protein matrix that mimics the tumour stroma, cell to cell and cell-matrix interactions seen , called a tumouroid. In this study, we test the acceptability and feasibility of using this model to establish patient-derived tumouroids.

Methods And Analysis: This is a first in-human study using prospective tissue and data collection of adult participants with confirmed or suspected renal cell carcinoma. The goals of the study are to assess patient acceptability to the use of patient-derived tumour models for future treatment decisions, and to assess the feasibility of generating patient-specific renal cancer tumouroids that can be challenged with drugs. These goals will be realised through the collection of tumour samples (expected n = 10), participant-completed questionnaires (expected n = 10), and in-depth semi-structured interviews with patients (expected n = 5). Collected multiregional tumour samples will be dissociated to isolate primary cells which are then expanded and incorporated into tumouroids. Drug challenge will ensue and the response will be categorised into "responder", "weak responder", and "non-responder". Statistical analysis will be descriptive.

Ethics And Dissemination: The study has ethical approval (REC reference 17/LO/1744). Findings will be made available to patients, clinicians, funders, and the National Health Service (NHS) through presentations at national and international meetings, peer-reviewed publications, social media and patient support groups.

Trial Registration: Registered on ClinicalTrials.gov (NCT03300102).
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http://dx.doi.org/10.1016/j.isjp.2019.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913561PMC
April 2019

Lymphatic vessels in human adipose tissue.

Cell Tissue Res 2020 Mar 27;379(3):511-520. Epub 2019 Nov 27.

Centro de Medicina Regenerativa, Faculdade de Medicina de Petrópolis, Petrópolis, RJ, Brazil.

Despite being considered present in most vascularised tissues, lymphatic vessels have not been properly shown in human adipose tissue (AT). Our goal in this study is to investigate an unanswered question in AT biology, regarding lymphatic network presence in tissue parenchyma. Using human subcutaneous (S-) and visceral (V-) AT samples with whole mount staining for lymphatic specific markers and three-dimensional imaging, we showed lymphatic capillaries and larger lymphatic vessels in the human VAT. Conversely, in the human SAT, microcirculatory lymphatic vascular structures were rarely detected and no initial lymphatics were found.
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http://dx.doi.org/10.1007/s00441-019-03108-5DOI Listing
March 2020

Mechano-growth Factor Expression in Colorectal Cancer Investigated With Fluorescent Gold Nanoparticles.

Anticancer Res 2019 Apr;39(4):1705-1710

University College London Centre for Nanotechnology and Regenerative Medicine, Division of Surgery and Interventional Science, University College London, Royal Free Campus, London, U.K.

Background/aim: Fluorescent gold nanoparticles demonstrate strong photoluminescence, photostability, and low cellular toxicity, making them attractive agents for biomedical applications. Mechano-growth factor (MGF) is an isoform of IGF1 and its expression has been demonstrated in malignancies including prostate cancer.

Materials And Methods: Near-infrared-emitting gold nanoparticles (AuNPs) were synthesized and conjugated to MGF. Following characterization and confirmation of conjugation, these AuNPs were used to investigate the expression of MGF in colon cancer cell lines (HT29 and SW620) and tissues comparing normal and colon cancer. The prostate cancer cell line PC3 and adenocarcinoma tissues were used as positive controls.

Results: Colon cancer cell lines, adenocarcinoma tissues and polyp tissues demonstrated evidence of MGF peptide expression, which was not found in normal colon tissues and human umbilical vein endothelial cells.

Conclusion: MGF appears to be overexpressed in colon cancer tissues, offering a potential unique target for imaging and drug delivery in colon cancer.
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http://dx.doi.org/10.21873/anticanres.13276DOI Listing
April 2019

Thermoresponsive Stiffness Softening of Hierarchically Porous Nanohybrid Membranes Promotes Niches for Mesenchymal Stem Cell Differentiation.

Adv Healthc Mater 2019 05 4;8(10):e1801556. Epub 2019 Apr 4.

Centre for Biomaterials in Surgical Reconstruction and Regeneration, Division of Surgery & Interventional Science, University College London, 2QG, 10 Pond St, London, NW3 2PS, UK.

Despite the attention given to the development of novel responsive implants for regenerative medicine applications, the lack of integration with the surrounding tissues and the mismatch with the dynamic mechanobiological nature of native soft tissues remain in the current products. Hierarchical porous membranes based on a poly (urea-urethane) (PUU) nanohybrid have been fabricated by thermally induced phase separation (TIPS) of the polymer solution at different temperatures. Thermoresponsive stiffness softening of the membranes through phase transition from the semicrystalline phase to rubber phase and reverse self-assembly of the quasi-random nanophase structure is characterized at body temperature near the melting point of the crystalline domains of soft segments. The effects of the porous structure and stiffness softening on proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBM-MSCs) are investigated. The results of immunohistochemistry, histological, ELISA, and qPCR demonstrate that hBM-MSCs maintain their lineage commitment during stiffness relaxation; chondrogenic differentiation is favored on the soft and porous scaffold, while osteogenic differentiation is more prominent on the initial stiff one. Stiffness relaxation stimulates more osteogenic activity than chondrogenesis, the latter being more influenced by the synergetic coupling effect of softness and porosity.
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http://dx.doi.org/10.1002/adhm.201801556DOI Listing
May 2019

Development data associated with effects of stiffness softening of 3D-TIPS elastomer nanohybrid scaffolds on tissue ingrowth, vascularization and inflammation .

Data Brief 2019 Feb 10;22:885-902. Epub 2019 Jan 10.

Centre for Biomaterials in Surgical Reconstruction and Regeneration, Division of Surgery & Interventional Science, University College London, London NW3 2PF, United Kingdom.

This DiB article contains data related to the research article entitled "Cellular responses to thermoresponsive stiffness memory elastomer nanohybrid scaffolds by 3D-TIPS" (Wu et al., 2018). Thermoresponsive poly (urea-urethane) nanohybrid elastomer (PUU-POSS) scaffolds were implanted in rats for up to 3 months. The porous structure and tensile mechanical properties of the scaffolds are listed and compared before and after and tests. The details of the histological analysis of the explants with different initial stiffness and porous structures at various time points are presented. The images and data presented support the conclusion about the coupled effects of stiffness softening and the hierarchical porous structure modulating tissue ingrowth, vascularization and macrophage polarization in the article (Wu et al., 2018).
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http://dx.doi.org/10.1016/j.dib.2019.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6352546PMC
February 2019

Cellular responses to thermoresponsive stiffness memory elastomer nanohybrid scaffolds by 3D-TIPS.

Acta Biomater 2019 02 14;85:157-171. Epub 2018 Dec 14.

Centre for Biomaterials in Surgical Reconstruction and Regeneration, Division of Surgery & Interventional Science, University College London, London NW3 2PF, United Kingdom. Electronic address:

Increasing evidence suggests the contribution of the dynamic mechanical properties of the extracellular matrix (ECM) to regulate tissue remodeling and regeneration. Following our recent study on a family of thermoresponsive 'stiffness memory' elastomeric nanohybrid scaffolds manufactured via an indirect 3D printing guided thermally-induced phase separation process (3D-TIPS), this work reports in vitro and in vivo cellular responses towards these scaffolds with different initial stiffness and hierarchically interconnected porous structure. The viability of mouse embryonic dermal fibroblasts in vitro and the tissue responses during the stiffness softening of the scaffolds subcutaneously implanted in rats for three months were evaluated by immunohistochemistry and histology. Scaffolds with a higher initial stiffness and a hierarchical porous structure outperformed softer ones, providing initial mechanical support to cells and surrounding tissues before promoting cell and tissue growth during stiffness softening. Vascularization was guided throughout the digitally printed interconnected networks. All scaffolds exhibited polarization of the macrophage response from a macrophage phenotype type I (M1) towards a macrophage phenotype type II (M2) and down-regulation of the T-cell proliferative response with increasing implantation time; however, scaffolds with a more pronounced thermo-responsive stiffness memory mechanism exerted higher inflammo-informed effects. These results pave the way for personalized and biologically responsive soft tissue implants and implantable device with better mechanical matches, angiogenesis and tissue integration. Statement of Significance This work reports cellular responses to a family of 3D-TIPS thermoresponsive nanohybrid elastomer scaffolds with different stiffness softening both in vitro and in vivo rat models. The results, for the first time, have revealed the effects of initial stiffness and dynamic stiffness softening of the scaffolds on tissue integration, vascularization and inflammo-responses, without coupling chemical crosslinking processes. The 3D printed, hierarchically interconnected porous structures guide the growth of myofibroblasts, collagen fibers and blood vessels in real 3D scales. In vivo study on those unique smart elastomer scaffolds will help pave the way for personalized and biologically responsive soft tissue implants and implantable devices with better mechanical matches, angiogenesis and tissue integration.
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http://dx.doi.org/10.1016/j.actbio.2018.12.019DOI Listing
February 2019

Codelivery of a cytotoxin and photosensitiser via a liposomal nanocarrier: a novel strategy for light-triggered cytosolic release.

Nanoscale 2018 Nov;10(43):20366-20376

Division of Surgery and Interventional Science, University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PE, UK.

Endosomal entrapment is a key issue for the intracellular delivery of many nano-sized biotherapeutics to their cytosolic or nuclear targets. Photochemical internalisation (PCI) is a novel light-based solution that can be used to trigger the endosomal escape of a range of bioactive agents into the cytosol leading to improved efficacy in pre-clinical and clinical studies. PCI typically depends upon the endolysosomal colocalisation of the bioactive agent with a suitable photosensitiser that is administered separately. In this study we demonstrate that both these components may be combined for codelivery via a novel multifunctional liposomal nanocarrier, with a corresponding increase in the biological efficacy of the encapsulated agent. As proof of concept, we show here that the cytotoxicity of the 30 kDa protein toxin, saporin, in MC28 fibrosarcoma cells is significantly enhanced when delivered via a cell penetrating peptide (CPP)-modified liposome, with the CPP additionally functionalised with a photosensitiser that is targeted to endolysosomal membranes. This innovation opens the way for the efficient delivery of a range of biotherapeutics by the PCI approach, incorporating a clinically proven liposome delivery platform and using bioorthogonal ligation chemistries to append photosensitisers and peptides of choice.
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http://dx.doi.org/10.1039/c8nr04048fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251340PMC
November 2018

Data of a stiffness softening mechanism effect on proliferation and differentiation of a human bone marrow derived mesenchymal stem cell line towards the chondrogenic and osteogenic lineages.

Data Brief 2018 Dec 28;21:133-142. Epub 2018 Sep 28.

Centre for Biomaterials in Surgical Reconstruction and Regeneration, Division of Surgery & Interventional Science, University College London, London, United Kingdom.

This article contains data related to the research article entitled "Stiffness memory of indirectly 3D-printed elastomer nanohybrid regulates chondrogenesis and osteogenesis of human mesenchymal stem cells" [1] (Wu et al., 2018). Cells respond to the local microenvironment in a context dependent fashion and a continuous challenge is to provide a living construct that can adapt to the viscoelasticity changes of surrounding tissues. Several materials are attractive candidates to be used in tissue engineering, but conventional manufactured scaffolds are primarily static models with well-defined and stable stiffness that lack the dynamic biological nature required to undergo changes in substrate elasticity decisive in several cellular processes key during tissue development and wound healing. A family of poly (urea-urethane) (PUU) elastomeric nanohybrid scaffolds (PUU-POSS) with thermoresponsive mechanical properties that soften by reverse self-assembling at body temperature had been developed through a 3D thermal induced phase transition process (3D-TIPS) at various thermal conditions: cryo-coagulation (CC), cryo-coagulation and heating (CC + H) and room temperature coagulation and heating (RTC + H). The stiffness relaxation and stiffness softening of these scaffolds suggest regulatory effects in proliferation and differentiation of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) towards the chondrogenic and osteogenic lineages.
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http://dx.doi.org/10.1016/j.dib.2018.09.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186969PMC
December 2018

Stiffness memory of indirectly 3D-printed elastomer nanohybrid regulates chondrogenesis and osteogenesis of human mesenchymal stem cells.

Biomaterials 2018 12 10;186:64-79. Epub 2018 Sep 10.

Centre for Biomaterials in Surgical Reconstruction and Regeneration, Division of Surgery & Interventional Science, University College London, London, United Kingdom. Electronic address:

The cellular microenvironment is dynamic, remodeling tissues lifelong. The biomechanical properties of the extracellular matrix (ECM) influence the function and differentiation of stem cells. While conventional artificial matrices or scaffolds for tissue engineering are primarily static models presenting well-defined stiffness, they lack the responsive changes required in dynamic physiological settings. Engineering scaffolds with varying elastic moduli is possible, but often lead to stiffening and chemical crosslinking of the molecular structure with limited control over the scaffold architecture. A family of indirectly 3D printed elastomeric nanohybrid scaffolds with thermoresponsive mechanical properties that soften by reverse self-assembling at body temperature have been developed recently. The initial stiffness and subsequent stiffness relaxation of the scaffolds regulated proliferation and differentiation of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) towards the chondrogenic and osteogenic lineages over 4 weeks, as measured by immunohistochemistry, histology, ELISA and qPCR. hBM-MSCs showed enhanced chondrogenic differentiation on softer scaffolds and osteogenic differentiation on stiffer ones, with similar relative expression to that of human femoral head tissue. Overall, stiffness relaxation favored osteogenic activity over chondrogenesis in vitro.
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http://dx.doi.org/10.1016/j.biomaterials.2018.09.013DOI Listing
December 2018

Therapeutic enhancement of a cytotoxic agent using photochemical internalisation in 3D compressed collagen constructs of ovarian cancer.

Acta Biomater 2018 11 27;81:80-92. Epub 2018 Sep 27.

Division of Surgery & Interventional Science, University College London, London, UK.

Photochemical internalisation (PCI) is a method for enhancing delivery of drugs to their intracellular target sites of action. In this study we investigated the efficacy of PCI using a porphyrin photosensitiser and a cytotoxic agent on spheroid and non-spheroid compressed collagen 3D constructs of ovarian cancer versus conventional 2D culture. The therapeutic responses of two human carcinoma cell lines (SKOV3 and HEY) were compared using a range of assays including optical imaging. The treatment was shown to be effective in non-spheroid constructs of both cell lines causing a significant and synergistic reduction in cell viability measured at 48 or 96 h post-illumination. In the larger spheroid constructs, PCI was still effective but required higher saporin and photosensitiser doses. Moreover, in contrast to the 2D and non-spheroid experiments, where comparable efficacy was found for the two cell lines, HEY spheroid constructs were found to be more susceptible to PCI and a lower dose of saporin could be used. PCI treatment was observed to induce death principally by apoptosis in the 3D constructs compared to the mostly necrotic cell death caused by PDT. At low oxygen levels (1%) both PDT and PCI were significantly less effective in the constructs. STATEMENT OF SIGNIFICANCE: Assessment of new drugs or delivery systems for cancer therapy prior to conducting in vivo studies often relies on the use of conventional 2D cell culture, however 3D cancer constructs can provide more physiologically relevant information owing to their 3D architecture and the presence of an extracellular matrix. This study investigates the efficacy of Photochemical Internalisation mediated drug delivery in 3D constructs. In 3D cultures, both oxygen and drug delivery to the cells are limited by diffusion through the extracellular matrix unlike 2D models, and in our model we have used compressed collagen constructs where the density of collagen mimics physiological values. These 3D constructs are therefore well suited to studying drug delivery using PCI. Our study highlights the potential of these constructs for identifying differences in therapeutic response to PCI of two ovarian carcinoma lines.
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http://dx.doi.org/10.1016/j.actbio.2018.09.041DOI Listing
November 2018

Stiffness memory nanohybrid scaffolds generated by indirect 3D printing for biologically responsive soft implants.

Acta Biomater 2018 10 15;80:188-202. Epub 2018 Sep 15.

UCL Centre for Nanotechnology and Regenerative Medicine, Division of Surgery & Interventional Science, University College London, London NW3 2PF, UK. Electronic address:

Cell and tissue stiffness is an important biomechanical signalling parameter for dynamic biological processes; responsive polymeric materials conferring responsive functionality are therefore appealing for in vivo implants. We have developed thermoresponsive poly(urea-urethane) nanohybrid scaffolds with 'stiffness memory' through a versatile 3D printing-guided thermally induced phase separation (3D-TIPS) technique. 3D-TIPS, a combination of 3D printing with phase separation, allows uniform phase-separation and phase transition of the polymer solution at a large interface of network within the printed sacrificial preform, leading to the creation of full-scale scaffolds with bespoke anatomical complex geometry. A wide range of hyperelastic mechanical properties of the soft elastomer scaffolds with interconnected pores at multi-scale, controlled porosity and crystallinity have been manufactured, not previously achievable via direct printing techniques or phase-separation alone. Semi-crystalline polymeric reverse self-assembly to a ground-stated quasi-random nanophase structure, throughout a hierarchical structure of internal pores, contributes to gradual stiffness relaxation during in vitro cell culture with minimal changes to shape. This 'stiffness memory' provides initial mechanical support to surrounding tissues before gradually softening to a better mechanical match, raising hopes for personalized and biologically responsive soft tissue implants which promote human fibroblast cells growth as model and potential scaffold tissue integration. STATEMENT OF SIGNIFICANCE: Biological processes are dynamic in nature, however current medical implants are often stronger and stiffer than the surrounding tissue, with little adaptability in response to biological and physical stimuli. This work has contributed to the development of a range of thermoresponsive nanohybrid elastomer scaffolds, with tuneable stiffness and hierarchically interconnected porous structure, manufactured by a versatile indirect 3D printing technique. For the first time, stiffness memory of the scaffold was observed to be driven by phase transition and a reverse self-assembly from a semicrystalline phase to a quasi-random nanostructured rubber phase. Early insight into cell response during the stiffness relaxation of the scaffolds in vitro holds promise for personalized biologically responsive soft implants.
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http://dx.doi.org/10.1016/j.actbio.2018.09.016DOI Listing
October 2018

Recommendations for clinical translation of nanoparticle-enhanced radiotherapy.

Br J Radiol 2018 Dec 17;91(1092):20180325. Epub 2018 Sep 17.

17 Department of Medical Physics and Bioengineering, University College London , London , UK.

A multi-disciplinary cooperative for nanoparticle-enhanced radiotherapy (NERT) has been formed to review the current status of the field and identify key stages towards translation. Supported by the Colorectal Cancer Healthcare Technologies Cooperative, the cooperative comprises a diverse cohort of key contributors along the translation pathway including academics of physics, cancer and radio-biology, chemistry, nanotechnology and clinical trials, clinicians, manufacturers, industry, standards laboratories, policy makers and patients. Our aim was to leverage our combined expertise to devise solutions towards a roadmap for translation and commercialisation of NERT, in order to focus research in the direction of clinical implementation, and streamline the critical pathway from basic science to the clinic. A recent meeting of the group identified barriers to and strategies for accelerated clinical translation. This commentary reports the cooperative's recommendations. Particular emphasis was given to more standardised and cohesive research methods, models and outputs, and reprioritised research drivers including patient quality of life following treatment. Nanoparticle design criteria were outlined to incorporate scalability of manufacture, understanding and optimisation of biological mechanisms of enhancement and in vivo fate of nanoparticles, as well as existing design criteria for physical and chemical enhancement. In addition, the group aims to establish a long-term and widespread international community to disseminate key findings and create a much-needed cohesive body of evidence necessary for commercial and clinical translation.
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http://dx.doi.org/10.1259/bjr.20180325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319829PMC
December 2018

Photodynamic therapy in 3D cancer models and the utilisation of nanodelivery systems.

Nanoscale 2018 Jan;10(4):1570-1581

Division of Surgery and Interventional Science, Department of Nanotechnology, University College London, Royal Free Campus, Rowland Hill St, London NW3 2PE, UK.

Photodynamic therapy (PDT) is the subject of considerable research in experimental cancer models mainly for the treatment of solid cancerous tumours. Recent studies on the use of nanoparticles as photosensitiser carriers have demonstrated improved PDT efficacy in experimental cancer therapy. Experiments typically employ conventional monolayer cell culture but there is increasing interest in testing PDT using three dimensional (3D) cancer models. 3D cancer models can better mimic in vivo models than 2D cultures by for example enabling cancer cell interactions with a surrounding extracellular matrix which should enable the treatment to be optimised prior to in vivo studies. The aim of this review is to discuss recent research using PDT in different types of 3D cancer models, from spheroids to nano-fibrous scaffolds, using a range of photosensitisers on their own or incorporated in nanoparticles and nanodelivery systems.
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http://dx.doi.org/10.1039/c7nr07739dDOI Listing
January 2018

Elements of the niche for adult stem cell expansion.

J Tissue Eng 2017 Jan-Dec;8:2041731417725464. Epub 2017 Aug 24.

Institute of Orthopaedics & Musculoskeletal Science, University College London, London, UK.

Adult stem cells are crucial for tissue homeostasis. These cells reside within exclusive locations in tissues, termed niches, which protect adult stem cell fidelity and regulate their many functions through biophysical-, biochemical- and cellular-mediated mechanisms. There is a growing understanding of how these mechanisms and their components contribute towards maintaining stem cell quiescence, self-renewal, expansion and differentiation patterns. In vitro expansion of adult stem cells is a powerful tool for understanding stem cell biology, and for tissue engineering and regenerative medicine applications. However, it is technically challenging, since adult stem cell removal from their native microenvironment has negative repercussions on their sustainability. In this review, we overview specific elements of the biomimetic niche and how recreating such elements can help in vitro propagation of adult stem cells.
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http://dx.doi.org/10.1177/2041731417725464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574483PMC
August 2017

Endolysosomal targeting of a clinical chlorin photosensitiser for light-triggered delivery of nano-sized medicines.

Sci Rep 2017 07 20;7(1):6059. Epub 2017 Jul 20.

Division of Surgery and Interventional Science, University College London, Royal Free Campus, Rowland Hill Street, London, NW3 2PE, UK.

A major problem with many promising nano-sized biotherapeutics including macromolecules is that owing to their size they are subject to cellular uptake via endocytosis, and become entrapped and then degraded within endolysosomes, which can significantly impair their therapeutic efficacy. Photochemical internalisation (PCI) is a technique for inducing cytosolic release of the entrapped agents that harnesses sub-lethal photodynamic therapy (PDT) using a photosensitiser that localises in endolysosomal membranes. Using light to trigger reactive oxygen species-mediated rupture of the photosensitised endolysosomal membranes, the spatio-temporal selectivity of PCI then enables cytosolic release of the agents at the selected time after administration so that they can reach their intracellular targets. However, conventional photosensitisers used clinically for PDT are ineffective for photochemical internalisation owing to their sub-optimal intracellular localisation. In this work we demonstrate that such a photosensitiser, chlorin e, can be repurposed for PCI by conjugating the chlorin to a cell penetrating peptide, using bioorthogonal ligation chemistry. The peptide conjugation enables targeting of endosomal membranes so that light-triggered cytosolic release of an entrapped nano-sized cytotoxin can be achieved with consequent improvement in cytotoxicity. The photoproperties of the chlorin moiety are also conserved, with comparable singlet oxygen quantum yields found to the free chlorin.
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http://dx.doi.org/10.1038/s41598-017-06109-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519633PMC
July 2017

Engineering a vascularised 3D in vitro model of cancer progression.

Sci Rep 2017 03 9;7:44045. Epub 2017 Mar 9.

UCL Institute of Orthopaedics and Musculoskeletal Sciences, UCL Division of Surgery and Interventional Science, Stanmore Campus, HA7 4LP, UK.

The hallmark of tumours is the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment. These microenvironmental cues, which include hypoxia, also regulate the angiogenic processes within a tumour, facilitating the spread of cancer cells. We engineered compartmentalized biomimetic colorectal tumouroids with stromal surrounds that comprised a range of ECM densities, composition and stromal cell populations. Recapitulating tissue ECM composition and stromal cell composition enhanced cancer cell invasion. Manipulation of ECM density was associated with an altered migration pattern from glandular buds (cellular aggregates) to epithelial cell sheets. Laminin appeared to be a critical component in regulating endothelial cell morphology and vascular network formation. Interestingly, the disruption of vascular networks by cancer cells was driven by changes in expression of several anti-angiogenic genes. Cancer cells cultured in our biomimetic tumouroids exhibited intratumoural heterogeneity that was associated with increased tumour invasion into the stroma. These findings demonstrate that our 3D in vitro tumour model exhibits biomimetic attributes that may permit their use in studying microenvironment clues of tumour progression and angiogenesis.
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http://dx.doi.org/10.1038/srep44045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343474PMC
March 2017

Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors.

Sci Rep 2017 02 22;7:43006. Epub 2017 Feb 22.

Department of Biomedical Sciences, Faculty of Science and Technology, University of Westminster, 115 New Cavendish St, W1W 6UW, UK.

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells B: 6.83 Hz; glycosylated cells B: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.
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http://dx.doi.org/10.1038/srep43006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320443PMC
February 2017

A Validated Multiscale In-Silico Model for Mechano-sensitive Tumour Angiogenesis and Growth.

PLoS Comput Biol 2017 01 26;13(1):e1005259. Epub 2017 Jan 26.

University College London, Centre for Medical Image Computing, Department of Medical Physics & Biomedical Engineering, London, United Kingdom.

Vascularisation is a key feature of cancer growth, invasion and metastasis. To better understand the governing biophysical processes and their relative importance, it is instructive to develop physiologically representative mathematical models with which to compare to experimental data. Previous studies have successfully applied this approach to test the effect of various biochemical factors on tumour growth and angiogenesis. However, these models do not account for the experimentally observed dependency of angiogenic network evolution on growth-induced solid stresses. This work introduces two novel features: the effects of hapto- and mechanotaxis on vessel sprouting, and mechano-sensitive dynamic vascular remodelling. The proposed three-dimensional, multiscale, in-silico model of dynamically coupled angiogenic tumour growth is specified to in-vivo and in-vitro data, chosen, where possible, to provide a physiologically consistent description. The model is then validated against in-vivo data from murine mammary carcinomas, with particular focus placed on identifying the influence of mechanical factors. Crucially, we find that it is necessary to include hapto- and mechanotaxis to recapitulate observed time-varying spatial distributions of angiogenic vasculature.
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http://dx.doi.org/10.1371/journal.pcbi.1005259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5268362PMC
January 2017

Detection of cell surface calreticulin as a potential cancer biomarker using near-infrared emitting gold nanoclusters.

Nanotechnology 2016 Jul 3;27(28):285101. Epub 2016 Jun 3.

Research Department of Nanotechnology, Division of Surgery and Interventional Science, University College London, Pond Street, London NW3 2QG, UK.

Calreticulin (CRT) is a cytoplasmic calcium-binding protein. The aim of this study was to investigate CRT presence in cancer with the use of fluorescent gold nanoclusters (AuNCs) and to explore AuNC synthesis using mercaptosuccinic acid (MSA) as a coating agent. MSA-coated AuNCs conferred well-dispersed, bio-stable, water-soluble nanoparticles with bioconjugation capacity and 800-850 nm fluorescence after broad-band excitation. Cell-viability assay revealed good AuNC tolerability. A native CRT amino-terminus corresponding peptide sequence was synthesised and used to generate rabbit site-specific antibodies. Target specificity was demonstrated with antibody blocking in colorectal and breast cancer cell models; human umbilical vein endothelial cells served as controls. We demonstrated a novel route of AuNC/MSA manufacture and CRT presence on colonic and breast cancerous cell surface. AuNCs served as fluorescent bio-probes specifically recognising surface-bound CRT. These results are promising in terms of AuNC application in cancer theranostics and CRT use as surface biomarker in human cancer.
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http://dx.doi.org/10.1088/0957-4484/27/28/285101DOI Listing
July 2016

The next level of 3D tumour models: immunocompetence.

Drug Discov Today 2016 09 22;21(9):1421-1428. Epub 2016 Apr 22.

Research Department of Materials and Tissues, Institute of Orthopaedics, Division of Surgery and Interventional Science, UCL, London, UK. Electronic address:

The complexity of the tumour microenvironment encompasses interactions between cancer and stromal cells. Moving from 2D cell culture methods into 3D models enables more-accurate investigation of those interactions. Current 3D cancer models focus on cancer spheroid interaction with stromal cells, such as fibroblasts. However, over recent years, the cancer immune environment has been shown to have a major role in tumour progression. This review summarises the state-of-art on immunocompetent 3D cancer models that, in addition to cancer cells, also incorporate immune cells, including monocytes, cancer-associated macrophages, dendritic cells, neutrophils and lymphocytes.
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http://dx.doi.org/10.1016/j.drudis.2016.04.010DOI Listing
September 2016

Demonstration of Calreticulin Expression in Hamster Pancreatic Adenocarcinoma with the Use of Fluorescent Gold Quantum Dots.

Anticancer Res 2016 Mar;36(3):861-7

Division of Surgery and Interventional Science, Royal Free Campus, University College London, London, UK.

Background: There is dire need for discovery of novel pancreatic cancer biomarkers and of agents with the potential for simultaneous diagnostic and therapeutic capacity. This study demonstrates calreticulin expression on hamster pancreatic adenocarcinoma via bio-conjugated gold quantum dots (AuQDs).

Materials And Methods: Hamster pancreatic adenocarcinoma cells were cultured, fixed and incubated with fluorescent AuQDs, bio-conjugated to anti-calreticulin antibodies. Anti-calreticulin and AuQDs were produced in-house. AuQDs were manufactured to emit in the near-infrared. Cells were further characterized under confocal fluorescence.

Results: AuQDs were confirmed to emit in the near-infrared. AuQD bio-conjugation to calreticulin was confirmed via dot-blotting. Upon laser excitation and post-incubation with bio-conjugated AuQDs, pancreatic cancer cell lines emitted fluorescence in near-infrared.

Conclusion: Hamster pancreatic cancer cells express calreticulin, which may be labelled with AuQDs. This study demonstrates the application of nanoparticle-based theranostics in pancreatic cancer. Such biomarker-targeting nanosystems are anticipated to play a significant role in the management of pancreatic cancer.
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March 2016