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For multimodality therapies such as the combination of hyperthermia and radiation, quantification of biological effects is key for dose prescription and response prediction. Tumour spheroids have a microenvironment that more closely resembles that of tumours in vivo and may thus be a superior in vitro cancer model than monolayer cultures. Here, the response of tumour spheroids formed from two established human cancer cell lines (HCT116 and CAL27) to single and combination treatments of radiation (0-20 Gy), and hyperthermia at 47 °C (0-780 CEM 43 ) has been evaluated. Response was analysed in terms of spheroid growth, cell viability and the distribution of live/dead cells. Time-lapse imaging was used to evaluate mechanisms of cell death and cell detachment. It was found that sensitivity to heat in spheroids was significantly less than that seen in monolayer cultures. Spheroids showed different patterns of shrinkage and regrowth when exposed to heat or radiation: heated spheroids shed dead cells within four days of heating and displayed faster growth post-exposure than samples that received radiation or no treatment. Irradiated spheroids maintained a dense structure and exhibited a longer growth delay than spheroids receiving hyperthermia or combination treatment at (thermal) doses that yielded equivalent levels of clonogenic cell survival. We suggest that, unlike radiation, which kills dividing cells, hyperthermia-induced cell death affects cells independent of their proliferation status. This induces microenvironmental changes that promote spheroid growth. In conclusion, 3D tumour spheroid growth studies reveal differences in response to heat and/or radiation that were not apparent in 2D clonogenic assays but that may significantly influence treatment efficacy.

Despite a continuing debate about the existence of cancer stem cells (CSCs), recent discoveries have provided further support for their existence and their roles in drug resistance, cancer recurrence and metastasis. CSC characteristics, such as self-renewal and tumour initiation, and supporting cellular processes, particularly the epithelial-to-mesenchymal transition, are attracting a great deal of attention from cancer researchers as they offer opportunities for discovering novel therapeutic targets for future drug development. However, the identification of potential CSC targets presents clear obstacles due to a lack of truly specific CSC markers and the reality of CSC plasticity, making this task a significant challenge. Agents that target developmental signalling pathways, such as Notch, Wnt and Hedgehog, are now in clinical trials whilst alternative approaches including immune-based therapies and microRNA-mediated pathway inhibitors are producing promising pre-clinical results. Here, we discuss the contribution of CSCs to cancer metastasis and the scope of opportunities for therapeutic intervention. In particular, we consider CSC-targeting agents for which there is experimental evidence of anti-metastatic properties and which may have potential to eventually limit relapse and impede metastasis in patients., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cell migration is a key hallmark of malignant cells that contributes to the progression of cancers from a primary, localized mass to an invasive and/or metastatic phenotype. Traditional methods for the evaluation of tumor cell migration in vitro generally employ two-dimensional (2D), homogeneous cultures that do not take into account tumor heterogeneity, three-dimensional (3D) cell-cell contacts between tumor and/or host cells or interactions with extracellular matrix proteins. Here we describe a 3D tumor spheroid-based migration assay which more accurately reflects the solid tumor microenvironment and can accommodate both extracellular matrix and host cell interactions. It is a rapid and highly reproducible 96-well plate-based technique and we demonstrate its utility for the evaluation of therapeutic agents/drugs with anti-migratory properties.

Motility and invasion are key hallmarks that distinguish benign from malignant tumors, enabling cells to cross tissue boundaries, disseminate in blood and lymph and establish metastases at distant sites. Similar properties are also utilized by activated endothelial cells during tumor-induced angiogenesis. It is now appreciated that these processes might provide a rich source of novel molecular targets with the potential for inhibitors to restrain both metastasis and neoangiogenesis. Such therapeutic strategies require assays that can rapidly and quantitatively measure cell movement and the ability to traverse physiological barriers. The need for high-throughput, however, must be balanced by assay designs that accommodate, as far as possible, the complexity of the in vivo tumor microenvironment. This chapter aims to give an overview of some commonly used migration and invasion assays to aid in the selection of a balanced portfolio of techniques for the rapid and accurate evaluation of novel therapeutic agents.

Primary human tumours can often be eradicated by surgery if detected early; however metastatic disease renders complete cure less likely and the development of resistance to therapy results in tumour escape and increased risk of death. Interactions of tumour cells with each other, surrounding normal cells and extracellular matrix or basement membrane components are crucial to all stages of cancer progression. Changes in both cell-cell and cell-substrate proteins are linked to tumour cell migratory and invasive ability, induction of angiogenesis (on which sustained tumour growth and dissemination depends) and apoptosis resistance in response to drugs or radiotherapy. Hypoxia within solid tumours is a key driver of many aspects of progression, and may also nurture cancer stem-like cells which are increasingly linked to relapse and treatment failure. This review will briefly outline the cellular and molecular mechanisms underlying tumour progression, focussing on the acquisition of metastatic capacity and resistance to therapy., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Invasive capacity is the single most important trait that distinguishes benign from malignant lesions. Tumour cells, during intravasation and extravasation of blood and lymphatic channels and when establishing colonies at secondary sites, must move through tissue boundaries that normal adult cells (other than, for example activated leukocytes) do not cross. Similar mechanisms are also utilised by activated endothelial cells during the generation of new blood vessels that enable the sustained growth and dissemination of tumours. It is now increasingly recognised that these processes--cell motility and invasion--might provide a rich source of novel targets for cancer therapy and that appropriate inhibitors may restrain both metastasis and neoangiogenesis. This new paradigm demands screening assays that can rapidly and quantitatively measure cell movement and the ability to traverse physiological barriers. We also need to consider whether simple reductionist in vitro approaches can reliably model the complexity of in vivo tumour invasion/neoangiogenesis. There are both opportunities and challenges ahead in developing a balanced portfolio of assays that will be able to evaluate accurately and finally deliver novel anti-invasive agents with therapeutic potential for clinical use.