Peptide gels of fully-defined composition and mechanics for probing cell-cell and cell-matrix interactions in vitro.

Current materials used for in vitro 3D cell culture are often limited by their poor similarity to human tissue, batch-to-batch variability and complexity of composition and manufacture. Here, we present a "blank slate" culture environment based on a self-assembling peptide gel free from matrix motifs. The gel can be customised by incorporating matrix components selected to match the target tissue, with independent control of mechanical properties. Therefore the matrix components are restricted to those specifically added, or those synthesised by encapsulated cells. The flexible 3D culture platform provides full control over biochemical and physical properties, allowing the impact of biochemical composition and tissue mechanics to be separately evaluated in vitro. Here, we demonstrate that the peptide gels support the growth of a range of cells including human induced pluripotent stem cells and human cancer cell lines. Furthermore, we present proof-of-concept that the peptide gels can be used to build disease-relevant models. Controlling the peptide gelator concentration allows peptide gel stiffness to be matched to normal breast (<1 kPa) or breast tumour tissue (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, the peptide gels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions affect the size, shape and organisation of breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and other diseases.

PO-11 - Thrombin and cancer stem-like cells: in vitro support for breast cancer anticoagulation.

INTRODUCTION: Cancer stem-like cells (CSCs) are tumour initiating, resistant to chemotherapy and play a role in metastasis. We have previously shown thrombin pathway signalling regulates CSC activity: thrombin pathway activation with FVIIa or through TF overexpression increases CSC activity whilst TF siRNA knockdown decreases it. We hypothesise that NOACs, by targeting specific factors in the thrombin pathway, may inhibit CSC activity. AIM: In breast cancer cell lines, to determine the effect of i) thrombin and ii) dabigatran (Boehringer Ingelheim), a direct thrombin inhibitor on cancer stem cell activity. MATERIALS AND METHODS: MDA-MB-231, MCF-7, SKBR3 and MDA-MB-157 cell lines representing the spectrum of breast cancer subtypes were cultured with: i) 0.1 NIH Units/ml human thrombin (Sigma) ii) 0.5µM Dabigatran (clinically relevant plasma concentration). Mammosphere culture is an established in vitro assay to measure CSC activity. Mammosphere forming efficiency (MFE) was calculated from the proportion of plated cells forming mammospheres in non-adherent culture, (each experiment, n=≥4, two-tailed independent t-test). RESULTS: Thrombin increased MFE in the high PAR-1 expressing MDA-MB-231 and MDA-MB-157 cell lines, but not the low PAR-1 expressing MCF-7/SKBR3 cell lines as compared to untreated controls (MDA-MB-231 mean (range): thrombin treated: 0.91 (0.70-1.11) vs control: 0.73 (0.51-0.93)%, p=<0.04; MDA-MB-157: thrombin treated: 0.58 (0.45-0.69) vs control: 0.37 (0.31-0.47)%, p=<0.001). Dabigatran abrogated the stimulatory effect of thrombin on MFE in thrombin-treated MDA-MB-231 and MDA-MB-157 cells (MDA-MB-231 mean (range) thrombin+Dabigatran: 1.10 (0.83-1.41) vs thrombin only: 1.45 (1.32-1.66)%, p=<0.01); MDA-MB-157 thrombin+Dabigatran: 0.48 (0.39-0.56) vs thrombin only: 0.58 (0.45-0.69)%, p=<0.05). CONCLUSIONS: The stimulation of mammosphere formation by thrombin and reduction by thrombin inhibitor treatment indicates a functional relationship between cancer stem-like cells and coagulation. This suggests possible clinical utility of novel oral anticoagulants in targeting this critical cancer cell subpopulation.

Cyclooxygenase-2 inhibition: effects on tumour growth, cell cycling and lymphangiogenesis in a xenograft model of breast cancer.

Cyclooxygenase-2 (COX-2) is associated with poor-prognosis breast cancer. We used a nude mouse xenograft model to determine the effects of COX-2 inhibition in breast cancer. Oestrogen receptor (ER)-positive MCF7/HER2-18 and ER-negative MDAMB231 breast cancer cell lines were injected into nude mice and allowed to form tumours. Mice then received either chow containing Celecoxib (a COX-2 inhibitor) or control and tumour growth measured. Tumour proliferation, apoptosis, COX-2, lymphangiogenesis and angiogenesis were assessed by immunohistochemistry (IHC), Western blotting or Q-PCR. Celecoxib inhibited median tumour growth in MCF7/HER2-18 (58.7%, P=0.029) and MDAMB231 (46.3%, P=0.0002) cell lines compared to control. Cyclooxygenase-2 expression decreased following Celecoxib treatment (MCF7/HER2-18 median control 65.3% vs treated 22.5%, P=0.0001). Celecoxib increased apoptosis in MCF7/HER2-18 tumours (TUNEL 0.52% control vs 0.73% treated, P=0.0004) via inactivation of AKT (median pAKT(ser473) 57.3% control vs 35.5% treated, P=0.0001--confirmed at Western blotting). Q-PCR demonstrated decreased podoplanin RNA (lymphangiogenesis marker) in the MCF7/HER2-18 - median 2.9 copies treated vs 66.6 control (P=0.05) and MDAMB231-treated groups--median 160.7 copies vs 0.05 control copies (P=0.015), confirmed at IHC. Cyclooxygenase-2 is associated with high levels of activated AKT(ser473) and lymphangiogenesis in breast cancer. Cyclooxygenase-2 inhibition decreases tumour growth, and may potentially decrease recurrence, by inactivating AKT and decreasing lymphangiogenesis.

Breast stem cells and cancer.

Recent results have increased our understanding of normal stem cells and the signalling pathways which regulate them during the development of the mammary gland. Tumours in many tissues are now thought to develop from dysregulated stem cells and depend on activated stem cell self-renewal pathways such as Notch for their tumourigenic capacity. These cancer stem cells are recognised by specific cell surface proteins that they express and their capacity to grow tumours in vivo or spheres in vitro. We have described human breast DCIS mammospheres grown from cancer stem cells and demonstrated their dependence on the EGF and Notch receptor pathways. Stem cell self-renewal pathways such as these may represent novel therapeutic targets to prevent recurrence of pre-invasive and invasive breast cancer.