Helen E. Colley

Polymersome-Mediated Delivery of Combination Anticancer Therapy to Head and Neck Cancer Cells: 2D and 3D in Vitro Evaluation

Polymersomes have the potential to encapsulate and deliver chemotherapeutic drugs into tumor cells, reducing off-target toxicity that often compromises anticancer treatment. Here, we assess the ability of the pH-sensitive poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)- poly 2-(diisopropylamino)ethyl methacrylate (PDPA) polymersomes to encapsulate chemotherapeutic agents for effective combinational anticancer therapy. Polymersome uptake and ability to deliver encapsulated drugs into healthy normal oral cells and oral head and neck squamous cell carcinoma (HNSCC) cells was measured in two and three-dimensional culture systems. PMPC-PDPA polymersomes were more rapidly internalized by HNSCC cells compared to normal oral cells. Polymersome cellular uptake was found to be mediated by class B scavenger receptors. We also observed that these receptors are more highly expressed by cancer cells compared to normal oral cells, enabling polymersome-mediated targeting. Doxorubicin and paclitaxel were encapsulated into pH-sensitive PMPC-PDPA polymersomes with high efficiencies either in isolation or as a dual-load for both singular and combinational delivery. In monolayer culture, only a short exposure to drug-loaded polymersomes was required to elicit a strong cytotoxic effect. When delivered to three-dimensional tumor models, PMPC-PDPA polymersomes were able to penetrate deep into the center of the spheroid resulting in extensive cell damage when loaded with both singular and dual-loaded chemotherapeutics. PMPC-PDPA polymersomes offer a novel system for the effective delivery of chemotherapeutics for the treatment of HNSCC. Moreover, the preferential internalization of PMPC polymersomes by exploiting elevated scavenger receptor expression on cancer cells opens up the opportunity to target polymersomes to tumors.

Tissue-engineered Oral Mucosa

Advances in tissue engineering have permitted the three-dimensional (3D) reconstruction of human oral mucosa for various in vivo and in vitro applications. Tissue-engineered oral mucosa have been further optimized in recent years for clinical applications as a suitable graft material for intra-oral and extra-oral repair and treatment of soft-tissue defects. Novel 3D in vitro models of oral diseases such as cancer, Candida, and bacterial invasion have been developed as alternatives to animal models for investigation of disease phenomena, their progression, and treatment, including evaluation of drug delivery systems. The introduction of 3D oral mucosal reconstructs has had a significant impact on the approaches to biocompatibility evaluation of dental materials and oral healthcare products as well as the study of implant-soft tissue interfaces. This review article discusses the recent advances in tissue engineering and applications of tissue-engineered human oral mucosa.

Development of tissue-engineered models of oral dysplasia and early invasive oral squamous cell carcinoma

Background:Current organotypic models of dysplasia and oral squamous cell carcinoma (OSCC) lack the complexity that mimics in vivo tissue. Here we describe a three-dimensional in vitro model of the oral epithelium that replicates tumour progression from dysplasia to an invasive phenotype.Methods:The OSCC cell lines were seeded as a cell suspension (D20, Cal27) or as multicellular tumour spheroids (FaDu) with oral fibroblasts on to a de-epidermised acellular dermis to generate tissue-engineered models and compared with patient biopsies.Results:The D20 and Cal27 cells generated a model of epithelial dysplasia. Overtime Cal27 cells traversed the basement membrane and invaded the connective tissue to reproduce features of early invasive OSCC. When seeded onto a model of the normal oral mucosa, FaDu spheroids produced a histological picture mimicking carcinoma in situ with severe cellular atypia juxtaposed to normal epithelium.Conclusion:It is possible to culture in vitro models with the morphological appearance and histological characteristics of dysplasia and tumour cell invasion seen in vivo using native dermis. Such models could facilitate study of the molecular processes involved in malignant transformation, invasion and tumour growth as well as in vitro testing of new treatments, diagnostic tests and drug delivery systems for OSCC.

Internalization and biodistribution of polymersomes into oral squamous cell carcinoma cells in vitro and in vivo.

The prognosis for oral squamous cell carcinoma (OSCC) is not improving despite advances in surgical treatment. As with many cancers, there is a need to deliver therapeutic agents with greater efficiency into OSCC to improve treatment and patient outcome. The development of polymersomes offers a novel way to deliver therapy directly into tumor cells. Here we examined the internalization and biodistribution of two different fluorescently labeled polymersome formulations; polyethylene oxide (PEO)-poly 2-(diisopropylamino)ethyl methacrylate (PDPA) and poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)-PDPA, into SCC4 OSCC cells in vitro and in vivo. In vitro SCC4 monolayers internalized PMPC-PDPA and PEO-PDPA at similar rates. However, in vivo PMPC-PDPA polymersomes penetrated deeper and were more widely dispersed in SCC4 tumors than PEO-PDPA polymersomes. In the liver and spleen PMPC-PDPA mainly accumulated in tissue macrophages. However, in tumors PMPC-PDPA was found extensively in the nucleus and cytoplasm of tumor cells as well as in tumor-associated macrophages. Use of PMPC-PDPA polymersomes may enhance polymersome-mediated antitumor therapy.

An Orally Bioavailable, Indole-3-glyoxylamide Based Series of Tubulin Polymerization Inhibitors Showing Tumor Growth Inhibition in a Mouse Xenograft Model of Head and Neck Cancer

A number of indole-3-glyoxylamides have previously been reported as tubulin polymerization inhibitors, although none has yet been successfully developed clinically. We report here a new series of related compounds, modified according to a strategy of reducing aromatic ring count and introducing a greater degree of saturation, which retain potent tubulin polymerization activity but with a distinct SAR from previously documented libraries. A subset of active compounds from the reported series is shown to interact with tubulin at the colchicine binding site, disrupt the cellular microtubule network, and exert a cytotoxic effect against multiple cancer cell lines. Two compounds demonstrated significant tumor growth inhibition in a mouse xenograft model of head and neck cancer, a type of the disease which often proves resistant to chemotherapy, supporting further development of the current series as potential new therapeutics.

Effect of Age and Diabetes on the Response of Mesenchymal Progenitor Cells to Fibrin Matrices

Mesenchymal stem cells are showing increasing promise in applications such as tissue engineering and cell therapy. MSC are low in number in bone marrow, and therefore in vitro expansion is often necessary. In vivo, stem cells often reside within a niche acting to protect the cells. These niches are composed of niche cells, stem cells, and extracellular matrix. When blood vessels are damaged, a fibrin clot forms as part of the wound healing response. The clot constitutes a form of stem cell niche as it appears to maintain the stem cell phenotype while supporting MSC proliferation and differentiation during healing. This is particularly appropriate as fibrin is increasingly being suggested as a scaffold meaning that fibrin-based tissue engineering may to some extent recapitulate wound healing. Here, we describe how fibrin modulates the clonogenic capacity of MSC derived from young/old human donors and normal/diabetic rats. Fibrin was prepared using different concentrations to modulate the stiffness of the substrate. MSC were expanded on these scaffolds and analysed. MSC showed an increased self-renewal on soft surfaces. Old and diabetic cells lost the ability to react to these signals and can no longer adapt to the changed environment.

Tissue-engineered oral mucosa to study radiotherapy-induced oral mucositis.

Abstract Purpose: Oral mucositis is a severe and often dose-limiting side-effect of cancer therapy that occurs in patients receiving radiotherapy for head and neck cancers. Although radiation-induced effects on keratinocytes have been studied, little is known about its effect on fibroblasts or endothelial cells or, more importantly, when all these cells are combined in an engineered oral mucosal model. Materials and methods: Monolayer cultures of normal oral keratinocytes, normal oral fibroblasts, human dermal microvascular endothelial cells or tissue-engineered oral mucosa (TEOM) were exposed to 20 Gy irradiation. Cell damage and cytokine release was measured for 72 h for monolayer cultures and for up to 21 d for TEOM. Results: Compared to non-irradiated cells, the viability of all monolayer and co-cultures was significantly reduced 72 h post-irradiation while levels of secreted interleukin IL-6 and CXCL8 were increased. The viability of irradiated TEOM models was significantly reduced compared to controls at all time-points. Histologically, irradiated TEOM displayed thinner epithelium, increased apoptosis and more extensive damage than non-irradiated models. IL-6, CXCL8 and granulocyte macrophage colony-stimulating factor release was reduced whereas IL-1α levels were increased in irradiated TEOM models compared to controls. Conclusions: TEOM models comprising of mixed cell populations may prove useful in examining the pathobiology of radiation-induced mucositis.

Cigarette smoke condensate promotes pro-tumourigenic stromal–epithelial interactions by suppressing miR-145

BACKGROUND Exposure to factors released from tobacco during chewing or smoking is recognized as a major risk factor for oral carcinogenesis and influences the phenotype of oral epithelial cells and fibroblasts within the underlying stroma. Micro(mi)RNA can regulate the expression of genes within cells, and previous studies show that tobacco products can alter the miRNA profiles in lung epithelial cells. However, the molecular alterations occurring in oral fibroblasts exposed to tobacco constituents remain to be elucidated. METHODS Oral fibroblasts were exposed to cigarette smoke condensate (CSC) and miRNA expression compared to untreated controls using tiling low-density arrays (TLDA). Expression of miRNA-145 was confirmed by quantitative (q)RT-PCR. The effect of CSC on fibroblast cell viability, motility and matrix metalloproteinase (MMP)-2 expression was measured using MTS, a wound scratch assay and qRT-PCR, respectively. Oral cancer cell migration in response to culture supernatants from mock, control or pre-miR-145-transfected CSC-treated fibroblasts was analysed by chemotaxis assay. RESULTS TLDA analysis identified widespread changes in the miRNA expression profile of fibroblasts exposed to CSC. Pri-, pre- and mature miRNA-145 were significantly down-regulated in response to CSC, and this was accompanied by up-regulated expression of MMP-2 and increased migration of fibroblasts compared to untreated controls. Re-expression of miR-145 abrogated the ability of fibroblasts to promote oral cancer cell chemotaxis in response to CSC. CONCLUSION These findings suggest that tobacco constituents influence the expression of miRNA within oral fibroblasts promoting a phenotype that increases oral cancer migration and sheds new light on the mechanisms underlying oral cancer pathogenesis.

Culture on fibrin matrices maintains the colony-forming capacity and osteoblastic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into a number of mesenchymal tissues including bone, cartilage, and tendon. Low numbers in vivo means exponential growth is needed in culture to enable therapeutic applications. MSC can expand rapidly in culture but usually lose their extensive capacity for differentiation that makes them therapeutically attractive. To try and maintain their capacity for differentiation and expansion in vitro, we cultured MSC on fibrin gels of different concentrations to create more physiological growth conditions for the cells. The cells were then re-plated onto tissue culture plastic and analysed. The cells that had been pre-cultured for seven days on fibrin, proliferated and maintained their differential potential to the osteogenic lineage better than tissue culture plastic expanded MSC. A concentration relationship between colony number and fibrin concentration was seen with decreasing numbers as fibrin concentration increased. These data support the concept that substrate signals significantly influence MSC growth and differentiation and that growth on a fibrin matrix could be used to maintain a stem cell phenotype during MSC expansion.

Combined mathematical modelling and experimentation to predict polymersome uptake by oral cancer cells

UNLABELLED This study is motivated by understanding and controlling the key physical properties underlying internalisation of nano drug delivery. We consider the internalisation of specific nanometre size delivery vehicles, comprised of self-assembling amphiphilic block copolymers, called polymersomes that have the potential to specifically deliver anticancer therapeutics to tumour cells. The possible benefits of targeted polymersome drug delivery include reduced off-target toxic effects in healthy tissue and increased drug uptake by diseased tissue. Through a combination of in vitro experimentation and mathematical modelling, we develop a validated model of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway, incorporating receptor binding, clustering and recycling. The model predicts how the characteristics of receptor targeting, and the size and concentration of polymersomes alter uptake by tumour cells. The number of receptors per cell was identified as being the dominant mechanism accounting for the difference between cell types in polymersome uptake rate. FROM THE CLINICAL EDITOR This article reports on a validated model developed through a combination of in vitro experimentation and mathematical modeling of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway. The model incorporates receptor binding, clustering, and recycling and predicts how the characteristics of receptor targeting, the size and concentration alter polymersome uptake by cancer cells.