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Transcriptomic profiling of human breast and melanoma cells selected by migration through narrow constraints.

ABSTRACT: The metastatic spread of cancer cells is a step-wise process that starts with dissociation from primary tumours and local invasion of adjacent tissues. The ability to invade local tissues is the product of several processes, including degradation of extracellular matrices (ECM) and movement of tumour cells through physically-restricting gaps. To identify properties contributing to tumour cells squeezing through narrow gaps, invasive MDA-MB-231 human breast cancer and MDA-MB-435 human melanoma cells were subjected to three successive rounds of selection using cell culture inserts with highly constraining 3??m pores. For comparison purposes, flow cytometry was also employed to enrich for small diameter MDA-MB-231 cells. RNA-Sequencing (RNA-seq) using the Illumina NextSeq 500 platform was undertaken to characterize how gene expression differed between parental, invasive pore selected or small diameter cells. Gene expression results obtained by RNA-seq were validated by comparing with RT-qPCR. Transcriptomic data generated could be used to determine how alterations that enable cell passage through narrow spaces contribute to local invasion and metastasis.

SUBMITTER: Rudzka DA

PROVIDER: S-EPMC5685158 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Transcriptomic profiling of human breast and melanoma cells selected by migration through narrow constraints.

Rudzka Dominika A DA Clark William W Hedley Ann A Kalna Gabriela G Olson Michael F MF

Scientific data 20171114

The metastatic spread of cancer cells is a step-wise process that starts with dissociation from primary tumours and local invasion of adjacent tissues. The ability to invade local tissues is the product of several processes, including degradation of extracellular matrices (ECM) and movement of tumour cells through physically-restricting gaps. To identify properties contributing to tumour cells squeezing through narrow gaps, invasive MDA-MB-231 human breast cancer and MDA-MB-435 human melanoma ce ...[more]

PMID: 29135975

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Project description:BackgroundNeurons are dynamically coupled with each other through neurite-mediated adhesion during development. Understanding the collective behavior of neurons in circuits is important for understanding neural development. While a number of genetic and activity-dependent factors regulating neuronal migration have been discovered on single cell level, systematic study of collective neuronal migration has been lacking. Various biological systems are shown to be self-organized, and it is not known if neural circuit assembly is self-organized. Besides, many of the molecular factors take effect through spatial patterns, and coupled biological systems exhibit emergent property in response to geometric constraints. How geometric constraints of the patterns regulate neuronal migration and circuit assembly of neurons within the patterns remains unexplored.Methodology/principal findingsWe established a two-dimensional model for studying collective neuronal migration of a circuit, with hippocampal neurons from embryonic rats on Matrigel-coated self-assembled monolayers (SAMs). When the neural circuit is subject to geometric constraints of a critical scale, we found that the collective behavior of neuronal migration is spatiotemporally coordinated. Neuronal somata that are evenly distributed upon adhesion tend to aggregate at the geometric center of the circuit, forming mono-clusters. Clustering formation is geometry-dependent, within a critical scale from 200 µm to approximately 500 µm. Finally, somata clustering is neuron-type specific, and glutamatergic and GABAergic neurons tend to aggregate homo-philically.Conclusions/significanceWe demonstrate self-organization of neural circuits in response to geometric constraints through spatiotemporally coordinated neuronal migration, possibly via mechanical coupling. We found that such collective neuronal migration leads to somata clustering, and mono-cluster appears when the geometric constraints fall within a critical scale. The discovery of geometry-dependent collective neuronal migration and the formation of somata clustering in vitro shed light on neural development in vivo.

Project description:The different clinical behavior of low and high grade gliomas and the chance to develop novel selective agents that specifically target tumor-associated proteins in gliomas stimulate the research of molecules playing a role in glioma progression. Gene expression profiling using microarrays allows the study at the same time of the expression patterns of thousands of genes in tumor cells. In the present study microarrays with about 20,000 genes have been employed to discover the gene expression profile in 39 glial neoplasias (28 glioblastomas (GBM) and 11 low grade gliomas, namely 4 oligodendrogliomas, 5 pilocytic astrocytomas (PA), 2 fibrillary astrocytomas (FA)). Unsupervised classification through hierarchical cluster analysis identified 2 groups of tumours: one group mainly composed of low grade malignant tumours (10 low grade gliomas and 3 GBM), the other one constituted by GBM, with the exception of one low grade case. The nearest shrunken centroid classification method was used to identify genes useful to classify and best characterize high and low grade gliomas. [Tibshirani et al. 2002]; This procedure selected 9 genes as most informative for the classification task: ; Among them 7 genes were overexpressed in low grade gliomas, but underexpressed in GBM; on the contrary 2 genes were overexpressed in GBM, but underexpressed in low grade tumours; Forty five tumors were immunostained for IGFBP-2 . 81,5% GBM resulted immunopositive. On the contrary only one low grade glioma was positive. Gene expression profiling and immunohistochemistry suggest that IGFBP-2 may play a role in glioma progression. IGFBP-2 appears to be a novel immunohistochemical marker of malignancy in glial tumours and probably is the basis for targeted chemotherapy. Experiment Overall Design: We retrieved for this study 185 randomly selected cases of gliomas. All the cases had been received unfixed. A sample of neoplastic tissue had been flash frozen over liquid nitrogen and stored at -80°C into the frozen tissue bank of the Section of Pathology of Bellaria Hospital between 1990 and 2002. The remaining (specular) tissues were formalin fixed and paraffin embedded for routine histologic diagnosis. Experiment Overall Design: In the present investigation, only samples with rRNA 28S/18S ratio > 1.0 measured by Bioanalyzer 2100 (Agilent)and no evidence of ribosomal degradation were included. Some cases were excluded as the tissue samples were composed only by necrosis; other cases did not show neoplastic proliferation. Therefore only 39 glial neoplasias were suitable for the study: i.e. 28 GBMs and 11 low grade CNS tumors, namely 4 oligodendrogliomas (OL), 5 pilocytic astrocytomas (PA), 2 fibrillary astrocytoma (FA) showed a good quality of RNA. Experiment Overall Design: All the tumors were re-staged and graded according to 2000 WHO criteria at time of gene expression analysis by two pathologists (GM and VE). Experiment Overall Design: 39 glial neoplasias were labeled by Cy5. Common reference pool consisted of a mixture of total RNA from the low grade CNS tumor group (4 OL, 5 PA, 2 FA) labeled by Cy3. A series of the most informative genes were confirmed by RT-qPCR. Experiment Overall Design:

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Transcriptomic profiling of human breast and melanoma cells selected by migration through narrow constraints.

Publications

Transcriptomic profiling of human breast and melanoma cells selected by migration through narrow constraints.

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