three-dimensional-3d-tumor-spheroid-invasion-assay

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

The Institute of Cancer Research

Squamous cell carcinoma of the head and neck (SCCHN) is associated with high morbidity and mortality. Despite significant surgical advances and refinement in the delivery of chemotherapy and radiotherapy, prognosis has improved little in recent decades. Better local control has led to the late presentation of distant metastases and novel therapeutic agents are urgently required to prevent relapse, control disseminated disease and thus improve survival. PIK3CA encodes the p110alpha isoform of phosphoinositide 3-kinase (PI3-K) and is important in SCCHN, aberrations in its activity occurring early in the oncogenic process. PI3-K signalling promotes cell survival, proliferation, invasion and angiogenesis, all contributing to tumour progression. Activation of the PI3-K pathway may also mediate resistance to chemotherapy, radiotherapy and novel therapeutic agents such as epidermal growth factor receptor inhibitors. Elements of this signalling matrix, therefore, offer attractive therapeutic targets in SCCHN as inhibition of many malignant characteristics, as well as sensitisation to multiple treatment modalities, could be anticipated.

The phosphoinositide 3-kinase signalling pathway as a therapeutic target in squamous cell carcinoma of the head and neck.

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.

Cell migration/invasion assays and their application in cancer drug discovery.

Syk, a non-receptor tyrosine kinase, is an important component of immunoreceptor signaling in hematopoietic cells. It has been implicated in key regulatory pathways including phosphoinositide 3-kinase and phospholipase Cgamma (PLCgamma) activation in B cells and integrin signaling in platelets and bronchial epithelial cells. Recently, potential roles in cancer have been reported. In breast cancers, reduced Syk expression was associated with invasion, and its overexpression in cell lines was shown to inhibit cell motility. In contrast, Syk has been shown to mediate chemomigration in nasopharyngeal carcinoma cells. Its role in squamous cell carcinomas of the head and neck (SCCHN) has not yet been investigated. Syk mRNA and protein expression was detected in 6 of 10 SCCHN cell lines. When Syk was transfected into Syk-negative cells (SIHN-011A), chemomigration was enhanced in vitro and this was associated with activation of PLCgamma1. Conversely, abrogation of Syk activity by pharmacologic inhibition or small interfering RNA in HN6 cells with high levels of endogenous expression inhibited migration, haptotaxis, and engagement with matrix proteins; this was accompanied by decreased levels of phosphorylated AKT. Similar effects were seen in Syk-positive CAL 27 cells but not in Syk-negative SIHN-011A cells. Immunoprecipitation suggested co-association of Syk with epidermal growth factor receptor and GRB-2. Syk expression in SCCHN patient tissues was examined by semiquantitative real-time PCR (n = 45) and immunohistochemistry (n = 38) in two independent cohorts. Higher levels of Syk expression were observed in tumors and lymph node metastases relative to normal tissues. High Syk expression significantly correlated with worse survival and may be of prognostic value in SCCHN due to its potential role in cell migration and invasion.

Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck.

Dramatic responses to epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitors may be seen in non-small cell lung cancers (NSCLCs) with a sensitizing mutation of the EGFR TK domain. It is not known how to predict response in patients with squamous cell carcinoma of the head and neck (SCCHN), where EGFR TK mutations are less frequent and where response rates in unselected patients are disappointing. We have characterized the intrinsic sensitivity of a panel of 18 SCCHN cell lines to gefitinib, an EGFR TK inhibitor, and have investigated correlations between putative markers of response and intrinsic sensitivity. Induction of G1 arrest was only seen in cell lines with GI(50) < 1 microM. Expression of EGFR, by three techniques, correlated with sensitivity to gefitinib. ERB-B2 expression appeared to influence sensitivity to gefitinib but ERB-B3 expression did not. While EGFR tyrosine kinase mutations were not detected, EGFR gene amplification was confirmed by fluorescence in situ hybridization in the most sensitive cell line. The number of cytosine adenine dinucleotide repeats in intron 1 of the EGFR gene did not correlate with sensitivity. E-cadherin expression was detected in cell lines with a range of sensitivities, whereas amphiregulin was secreted predominantly by sensitive cell lines. MET expression was an independent predictor of sensitivity to gefitinib, although neither expression nor phosphorylation of insulin-like growth factor 1 receptor correlated with intrinsic resistance. Breast receptor kinase (BRK) was more highly expressed in the sensitive cell lines, but siRNA knockdown of neither BRK nor MET affected sensitivity. Our data suggest that overexpression of EGFR and multiple related cell surface receptors may be associated with sensitivity to gefitinib and that differences between our data and the literature highlight that biomarkers of response are tumour type- and cell line-dependent.

Determinants of response to epidermal growth factor receptor tyrosine kinase inhibition in squamous cell carcinoma of the head and neck.

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.

Tumour-microenvironmental interactions: paths to progression and targets for treatment.

Vascular endothelial growth factors (VEGF-C and VEGF-A) play important roles in tumour-induced lymphangiogenesis and angiogenesis, respectively, key processes implicated in promoting tumour growth and metastatic spread. Previous work from our laboratory has shown that EGFR overexpression in squamous carcinomas of the head and neck (SCCHN) is linked to high levels of VEGF-A and VEGF-C (but low levels of VEGF-D) and is associated with poor prognosis. The present study explored the signalling pathways regulating the induction of VEGF-C and VEGF-A in the SCCHN cell lines CAL 27 and Detroit 562. The addition of exogenous EGF induced the expression of VEGF-C and VEGF-A in a concentration-dependent manner and this was blocked by a selective EGFR inhibitor, gefitinib. In both cell lines stimulated with endogenous or exogenous ligand, inhibition of MEK1/2 (with U0126 or PD98059) or PI3K (with PI-103 or LY294002) resulted in a marked reduction of EGFR-induced VEGF-A expression, whereas exogenous EGF-induced VEGF-C upregulation was blocked by inhibitors of MEK but not PI3K. Inhibition of p38 MAPK suppressed EGF-induced VEGF-C upregulation in CAL 27 cells, but inhibited EGF-induced VEGF-A upregulation in Detroit 562. Taken together, our evidence suggests that both endogenous and exogenous EGFR activation induces VEGF-A expression requiring both PI3K and MAPK signalling whereas VEGF-C expression is dependent on MAPK, but not the PI3K or mTOR pathways in SCCHN cell lines. p38 MAPK appears to be differentially linked to either VEGF-A or VEGF-C regulation in different cellular contexts.

MAPK and PI3K signalling differentially regulate angiogenic and lymphangiogenic cytokine secretion in squamous cell carcinoma of the head and neck.

Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and β-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCβ1, and VGSCβ3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca(2+)](i). We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.

Angiogenic functions of voltage-gated Na+ Channels in human endothelial cells: modulation of vascular endothelial growth factor (VEGF) signaling.

Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z⁺ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28⁺ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.

Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells.

Chimeric antigen receptor (CAR) engineered T-cells occupy an increasing niche in cancer immunotherapy. In this context, CAR-mediated CD3ζ signaling is sufficient to elicit cytotoxicity and interferon-γ production while the additional provision of CD28-mediated signal 2 promotes T-cell proliferation and interleukin (IL)-2 production. This compartmentalisation of signaling opens the possibility that complementary CARs could be used to focus T-cell activation within the tumor microenvironment.

Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signaling.

Receptor tyrosine kinases (RTK) are key targets for novel cancer therapeutics since they activate multiple oncogenic signalling pathways. Also, they are inherently 'druggable' due to their small ATP-dependent kinase domains (inhibitable by small molecules) and cell surface location which renders them accessible to monoclonal antibody-based therapies. The epidermal growth factor receptor (EGFR) is overexpressed in the majority of SCCHN cases and this review focuses primarily on the progress made in targeting the EGFR for the therapy of SCCHN by both small molecules and antibody-based therapies. We then discuss the overlapping and distinct molecular markers of response, innate or acquired resistance to each modality, and how these may be overcome. We also consider other RTKs overexpressed in this disease that may impact on responses and/or provide additional targets for combination therapy.

Molecular markers of response and resistance to EGFR inhibitors in head and neck cancers.

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.

Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays.

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.

Tumor spheroid-based migration assays for evaluation of therapeutic agents.

Acquired resistance to tyrosine kinase inhibitors (TKIs) is becoming a major challenge in the treatment of many cancers. Epidermal growth factor receptor (EGFR) is overexpressed in squamous carcinomas, notably those of the head and neck (HNSCC), and can be targeted with several TKIs. We aimed to identify soluble proteins suitable for development as markers of EGFR TKI resistance in cancer patients to aid in early and minimally invasive assessment of therapeutic responses.

A novel serum protein signature associated with resistance to epidermal growth factor receptor tyrosine kinase inhibitors in head and neck squamous cell carcinoma.

Article	Year
The phosphoinositide 3-kinase signalling pathway as a therapeutic target in squamous cell carcinoma of the head and neck. Expert opinion on therapeutic targets\| PubMed ID: 16083342	2005
Cell migration/invasion assays and their application in cancer drug discovery. Biotechnology annual review\| PubMed ID: 16216785	2005
Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck. Cancer research\| PubMed ID: 17699797	2007
Determinants of response to epidermal growth factor receptor tyrosine kinase inhibition in squamous cell carcinoma of the head and neck. The Journal of pathology\| PubMed ID: 19197951	2009
Tumour-microenvironmental interactions: paths to progression and targets for treatment. Seminars in cancer biology\| PubMed ID: 20599506	2010
MAPK and PI3K signalling differentially regulate angiogenic and lymphangiogenic cytokine secretion in squamous cell carcinoma of the head and neck. European journal of cancer (Oxford, England : 1990)\| PubMed ID: 21074412	2011
Angiogenic functions of voltage-gated Na+ Channels in human endothelial cells: modulation of vascular endothelial growth factor (VEGF) signaling. The Journal of biological chemistry\| PubMed ID: 21385874	2011
Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells. Molecular medicine (Cambridge, Mass.)\| PubMed ID: 22354215	2012
Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signaling. Journal of clinical immunology\| PubMed ID: 22526592	2012
Molecular markers of response and resistance to EGFR inhibitors in head and neck cancers. Frontiers in bioscience (Landmark edition)\| PubMed ID: 23276940	2013
Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23436416	2013
Tumor spheroid-based migration assays for evaluation of therapeutic agents. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23436417	2013
A novel serum protein signature associated with resistance to epidermal growth factor receptor tyrosine kinase inhibitors in head and neck squamous cell carcinoma. European journal of cancer (Oxford, England : 1990)\| PubMed ID: 23582742	2013

Carol Box

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