Emma Davies

Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.

Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point.

There are a number of small-molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway that have either been approved or are in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest, as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in preclinical models. This article reports on our recent work to identify novel, potent, and selective reversible ERK1/2 inhibitors from a low-molecular-weight, modestly active, and highly promiscuous chemical start point, compound 4. To guide and inform the evolution of this series, inhibitor binding mode information from X-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo antitumor efficacy experiments.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

Selumetinib-based therapy in uveal melanoma patient-derived xenografts

The prognosis of metastatic uveal melanoma (UM) is among the worst of all human cancers. The identification of near-ubiquitous GNAQ/GNA11 mutations and the activation of MAPK signaling in UM have raised hopes of more effective, targeted therapies, based on MEK inhibition, for example. We evaluated the potential of drug combinations to increase the efficacy of the MEK inhibitor selumetinib (AZD6244, ARRY-142886), in UM cell lines and Patient-Derived Xenografts. We first evaluated the combination of selumetinib and DTIC. We found that DTIC did not improve the in vitro or in vivo antitumor efficacy of selumetinib, consistent with the outcome of the SUMIT clinical trial assessing the efficacy of this combination in UM. We then tested additional selumetinib combinations with the chemotherapy agent docetaxel, the ERK inhibitor AZ6197, and the mTORC1/2 inhibitor, vistusertib (AZD2014). Combinations of selumetinib with ERK and mTORC1/2 inhibitors appeared to be the most effective in UM PDX models.

Abstract 2087: The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) appears as an efficient targeted therapy when used in an adjuvant setting in patient-derived xenografts of uveal melanoma

Uveal melanomas (UM) constitute the most common primary intraocular tumors in adults and are characterized by a constitutive activation of the MAPK pathway due to mutations of the GTPase genes GNAQ or GNA11 in almost 80% of cases. The most commonly used treatments for UM are alkylating agents such as dacarbazine (DTIC) and temozolomide (TMZ). The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) has shown clinical activity compared to DTIC/TMZ in a recent Phase II clinical trial and has recently completed a Phase III clinical trial in combination with DTIC (NCT01974752). In parallel with this trial we sought to evaluate the efficacy of DTIC + selumetinib in UM patient-derived xenografts (PDXs). Three models were included in the study (MP34, MP55, and MM26), all bearing a GNAQ or GNA11 mutation. Selumetinib was administered orally at 25 mg/kg/day, 5 days a week, and DTIC at a dose of 40 mg/kg/day on Days 1 to 5 every 4 weeks. A significant tumor growth inhibition (TGI) of 54% was observed in the MP34 model but not in the two remaining PDXs. In one model, MM26, DTIC induced a strong TGI of about 99% with 6/9 complete remissions (CRs). The combination of selumetinib + DTIC did not significantly increase efficacy compared to monotherapy in any of the models; in the MM26 PDX, the combination induced a similar TGI (99%) and CR rate (5/9) as DTIC alone. In this experiment, after two courses of DTIC + selumetinib, selumetinib was continued alone, showing a significant increased growth delay (p In conclusion, we have observed that response of UM PDX models to DTIC was not increased when combined with selumetinib; these results are similar to those seen in the Phase III study of this drug combination. The observation that MEK inhibition was effective in delaying progression in the DTIC-sensitive PDX and published clinical studies demonstrating MEK inhibitor monotherapy activity indicate that MEK inhibition may have value as a treatment for UM; perhaps in the adjuvant setting in two specific clinical situations, i.e. patients with irradiated or enucleated high-risk primary intraocular or surgically resected metastatic UM. Citation Format: Bere Diallo, Gerald Massonnet, Rania El-Botty, Chloe Raymondie, Guillaume Carita, Sergio Roman-Roman, Paul Smith, Emma Davies, Didier Decaudin, Fariba Nemati. The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) appears as an efficient targeted therapy when used in an adjuvant setting in patient-derived xenografts of uveal melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2087.

Abstract 1698: Systems pathology for characterization of cancer model systems in a multicenter IMI-PREDECT project

Despite of our increased understanding of cancer biology, the majority of anti-cancer therapies fail at late-stage clinical trials. Thus, there is an urgent need to develop and validate novel preclinical models that could predict drug efficacy in humans. For this purpose, as a part of IMI-PREDECT public-private research consortium, this study describes methodology and infrastructure for characterization and comparison of preclinical models for drug target validation applying a systems pathology approach. Formalin-fixed paraffin-embedded (FFPE) samples from 1050 different in vitro and in vivo models of breast, prostate and lung cancers, as well as 364 clinical tumors from the same origin, were collected from 26 PREDECT collaborators across the EU. We established standard operating procedures for central processing of FFPE samples, including tissue microarray (TMA) construction and immunohistochemistry (IHC) with 15 different antibodies (CK8, Ki67, p-histone H3, ER, AR, p-AKT, p-ERK, p-p38, γH2AX, cleaved caspase 3, p-MET, HIF1α, p63, vimentin, E-cadherin). We constructed 50 TMA blocks, from which sections were cut and stained as well as digitized using whole slide imaging. Images were hosted on a WebMicroscope digital pathology platform and sample metadata on a PREDECT Metadata database (MBase). We developed image analysis methods for the detection and quantification of IHC biomarkers in the 48,800 stained TMA spots. As a proof-of-concept, we compared MCF-7 on several preclinical platforms including cell cultures, xenografts and xenograft tissue slices. Our results of the integrated biomarker phenotype suggest that of the various MCF-7 in vivo and ex vivo complex cell culture models, the xenograft tissue slice model was the most similar model platform to human clinical samples. In summary, we established a systems pathology approach to analyse and compare novel preclinical cancer models with IHC and digital imaging. The intention is that this large database will be made publicly available on the web as images and summary data that could be broadly useful to the community of cancer researchers and drug developers in comparing cancer model systems. The established infrastructure and workflow integrating molecular and digital pathology in a large-scale consortium setting could be applied to quantitative characterisation of consortium data in collaborations similar to PREDECT. Citation Format: Sami Blom, Yinhai Wang, Tauno Metsalu, Tiina Vesterinen, Teijo Pellinen, Anne Grote, Nina Linder, Jenni Saila, Katja Valimaki, Ruusu-Maria Kovanen, Outi Monni, Panu Kovanen, Emma Davies, Kristin Stock, Marta Estrada, Georgios Sflomos, Sylvia Grunewald, Catarina Brito, Julia Schuler, Ronald de Hoogt, Cathrin Brisken, Heiko van der Kuip, Wytske van Weerden, Simon Barry, Wolgang Sommergruber, Elizabeth Anderson, Matthias Nees, Juha Klefstrom, Jaak Vilo, Emmy Verschuren, Ralph Graeser, John Hickman, Johan Lundin, Olli Kallioniemi. Systems pathology for characterization of cancer model systems in a multicenter IMI-PREDECT project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1698. doi:10.1158/1538-7445.AM2015-1698