Amos Folarin

Hedgehog signalling is required for correct anteroposterior patterning of the zebrafish otic vesicle

Currently, few factors have been identified that provide the inductive signals necessary to transform the simple otic placode into the complex asymmetric structure of the adult vertebrate inner ear. We provide evidence that Hedgehog signalling from ventral midline structures acts directly on the zebrafish otic vesicle to induce posterior otic identity. We demonstrate that two strong Hedgehog pathway mutants, chameleon (contf18b) and slow muscle omitted (smub641) exhibit a striking partial mirror image duplication of anterior otic structures, concomitant with a loss of posterior otic domains. These effects can be phenocopied by overexpression of patched1 mRNA to reduce Hedgehog signalling. Ectopic activation of the Hedgehog pathway, by injection of sonic hedgehog or dominant-negative protein kinase A RNA, has the reverse effect: ears lose anterior otic structures and show a mirror image duplication of posterior regions. By using double mutants and antisense morpholino analysis, we also show that both Sonic hedgehog and Tiggy-winkle hedgehog are involved in anteroposterior patterning of the zebrafish otic vesicle.

Three-dimensional analysis of tumour vascular corrosion casts using stereoimaging and micro-computed tomography

OBJECTIVE In order to perform effective translational research for cancer therapy, we need to employ pre-clinical models which reflect the clinical situation. The purpose of this study was to quantitatively compare the vascular architecture of human colorectal cancer and experimental tumour models to determine the suitability of animal models for vascular studies and antivascular therapy. METHODS In this study we investigated the three-dimensional properties of colonic tumour vasculature in both human clinical tissues (normal mucosa control [n=20], carcinoma [n=20] and adenoma [n=6]) and murine colorectal xenografts (LS147T [n=6] and SW1222 [n=6]). Scanning Electron Microscope Stereoimaging (SEM) and X-ray Micro-Computed Tomography (Micro-CT) methods were employed for 3D analyses of the vascular corrosion casts from these tissues. RESULTS Morphological measurements showed that there were significant differences in the underlying morphology in the different tissues. Of the studied xenografts, LS147T is more consistently similar to the vascular architecture of the human carcinoma than SW1222. The only reversal of this is for the inter-vessel distance. CONCLUSION While SEM stereoimaging provided better surface detailed resolution of the corrosion casts, it was complimented by the fully 3D micro-CT method. Comparison made between the xenografts and clinical tumours showed that the LS147T xenografts shared many similarities with the clinical tumour vasculature. This study provides insight into how to select the most suitable pre-clinical models for translational studies of clinical cancer therapy.

Predicting Response to Radioimmunotherapy from the Tumor Microenvironment of Colorectal Carcinomas

Solid tumors have a heterogeneous pathophysiology, which directly affects antibody-targeted therapies. Here, we consider the influence of selected tumor parameters on radioimmunotherapy, by comparing the gross biodistribution, microdistribution, and therapeutic efficacy of either radiolabeled or fluorescently labeled antibodies (A5B7 anti-carcinoembryonic antigen antibody and a nonspecific control) after i.v. injection in two contrasting human colorectal xenografts in MF1 nude mice. The LS174T is moderately/poorly differentiated, whereas SW1222 has a well-differentiated glandular structure. Biodistribution studies (1.8 MBq (131)I-labeled A5B7, four mice per group) showed similar gross tumor uptake at 48 h in the two models (25.1% and 24.0% injected dose per gram, respectively). However, in therapy studies (six mice per group), LS174T required a 3-fold increase in dose (18 versus 6 MBq) to equal SW1222 growth inhibition ( approximately 55 versus approximately 60 days, respectively). To investigate the basis of this discrepancy, high-resolution multifluorescence microscopy was used to study antibody localization in relation to tumor parameters (5 min, 1 and 24 h, four mice per time point). Three-dimensional microvascular corrosion casting and transmission electron microscopy showed further structural differences between xenografts. Vascular supply, overall antigen distribution, and tumor structure varied greatly between models, and were principally responsible for major differences in antibody localization and subsequent therapeutic efficacy. The study shows that multiparameter, high-resolution imaging of both therapeutic and tumor microenvironment is required to comprehend complex antibody-tumor interactions, and to determine which tumor regions are being successfully treated. This will inform the design of optimized clinical trials of single and combined agents, and aid individual patient selection for antibody-targeted therapies.

Clinically driven design of multi-scale cancer models: the ContraCancrum project paradigm

The challenge of modelling cancer presents a major opportunity to improve our ability to reduce mortality from malignant neoplasms, improve treatments and meet the demands associated with the individualization of care needs. This is the central motivation behind the ContraCancrum project. By developing integrated multi-scale cancer models, ContraCancrum is expected to contribute to the advancement of in silico oncology through the optimization of cancer treatment in the patient-individualized context by simulating the response to various therapeutic regimens. The aim of the present paper is to describe a novel paradigm for designing clinically driven multi-scale cancer modelling by bringing together basic science and information technology modules. In addition, the integration of the multi-scale tumour modelling components has led to novel concepts of personalized clinical decision support in the context of predictive oncology, as is also discussed in the paper. Since clinical adaptation is an inelastic prerequisite, a long-term clinical adaptation procedure of the models has been initiated for two tumour types, namely non-small cell lung cancer and glioblastoma multiforme; its current status is briefly summarized.

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF−/−, or p53−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.

Amplicons on chromosome 12q13-21 in glioblastoma recurrences

There is limited knowledge on the in vivo behavior of amplified regions in human tumors. First evidence indicates that amplicon structures are largely maintained in recurrent tumors. Here, we investigated the fate of amplified regions in several independent cases of recurrent glioblastoma and the possible association of 12q13–21 amplifications and survival. We analyzed 12q13–21 amplicon numbers and sizes in glioblastoma and their recurrences by array‐CGH. The majority of the 12q13–21 amplicons found in the original tumor are lost in the subsequent recurrence. Likewise, the majority of the amplicons found in the first recurrence are lost in the second recurrence. The remaining amplicons of recurrences often expanded or were maintained in size. Because of re‐emergences and de novo appearances of amplicons, however, the overall number of amplicons did not decrease in the recurrences. Understanding genetic changes including gene amplifications in the development of tumor recurrences will contribute to rational therapeutic strategies for an improved patient survival. We recognized a significant longer survival time in glioblastoma patients that lack amplifications of either CDK4, CYP27B1, XRCC6BP1 (KUB3), or MDM2.

A high-content platform to characterise human induced pluripotent stem cell lines

Graphical abstract

Clinically Oriented Translational Cancer Multilevel Modeling: The ContraCancrum Project

The ContraCancrum project aims at developing a composite multilevel platform for simulating malignant tumor development and tumor and normal tissue response to therapeutic modalities and treatment schedules. The project aims at having an impact primarily in (a) the better under-standing of the natural phenomenon of cancer at different levels of biocomplexity and most importantly (b) the disease treatment optimization procedure in the patient’s individualized context by simulating the response to various therapeutic regimens. Fundamental biological mechanisms involved in tumor development and tumor and normal tissue treatment response such as metabolism, cell cycle, tissue mechanics, cell survival following treatment etc. are modeled also addressing stem cells in the context of both tumor and normal tissue behavior. The simulators exploit several discrete and continuous mathematics methods such as cellular automata, the generic Monte Carlo technique, finite elements, differential equations, novel dedicated algorithms etc. The predictions of the simulators rely on the imaging, histopathological, molecular and clinical data of the patient. ContraCancrum deploys two important clinical studies for validating the models, one on lung cancer and one on gliomas. The crucial validation work is based on comparing the multi-level therapy simulation predictions with multi-level patient data, acquired before and after therapy. ContraCancrum aims to pave the way for translating clinically validated multilevel cancer models into clinical practice.

Quality control, imputation and analysis of genome-wide genotyping data from the Illumina HumanCoreExome microarray

The decreasing cost of performing genome-wide association studies has made genomics widely accessible. However, there is a paucity of guidance for best practice in conducting such analyses. For the results of a study to be valid and replicable, multiple biases must be addressed in the course of data preparation and analysis. In addition, standardizing methods across small, independent studies would increase comparability and the potential for effective meta-analysis. This article provides a discussion of important aspects of quality control, imputation and analysis of genome-wide data from a low-coverage microarray, as well as a straight-forward guide to performing a genome-wide association study. A detailed protocol is provided online, with example scripts available at https://github.com/JoniColeman/gwas_scripts.

A Novel Automated High-Content Analysis Workflow Capturing Cell Population Dynamics from Induced Pluripotent Stem Cell Live Imaging Data

Most image analysis pipelines rely on multiple channels per image with subcellular reference points for cell segmentation. Single-channel phase-contrast images are often problematic, especially for cells with unfavorable morphology, such as induced pluripotent stem cells (iPSCs). Live imaging poses a further challenge, because of the introduction of the dimension of time. Evaluations cannot be easily integrated with other biological data sets including analysis of endpoint images. Here, we present a workflow that incorporates a novel CellProfiler-based image analysis pipeline enabling segmentation of single-channel images with a robust R-based software solution to reduce the dimension of time to a single data point. These two packages combined allow robust segmentation of iPSCs solely on phase-contrast single-channel images and enable live imaging data to be easily integrated to endpoint data sets while retaining the dynamics of cellular responses. The described workflow facilitates characterization of the response of live-imaged iPSCs to external stimuli and definition of cell line–specific, phenotypic signatures. We present an efficient tool set for automated high-content analysis suitable for cells with challenging morphology. This approach has potentially widespread applications for human pluripotent stem cells and other cell types.