Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Kathryn E. Ware; Shivee Gilja; Shenghan Xu; Samantha Shetler; Mohit K. Jolly; Xueyang Wang; Suzanne Bartholf Dewitt; Alexander J. Hish; Sarah Jordan; William Eward; Herbert Levine; Andrew J. Armstrong; Jason A. Somarelli

doi:10.3791/55520

A indução de transições mesenquimais-epiteliais em células de sarcoma

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Developmental Biology

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JoVE Journal Developmental Biology

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

A indução de transições mesenquimais-epiteliais em células de sarcoma

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11:42 min

April 7, 2017

DOI: 10.3791/55520-v

Kathryn E. Ware¹, Shivee Gilja¹, Shenghan Xu¹, Samantha Shetler¹, Mohit K. Jolly², Xueyang Wang³, Suzanne Bartholf Dewitt⁴, Alexander J. Hish¹, Sarah Jordan¹, William Eward⁵, Herbert Levine², Andrew J. Armstrong⁴, Jason A. Somarelli¹

¹Department of Medicine,Duke University, ²Department of Bioengineering,Rice University, ³Department of Molecular Genetics and Microbiology,Duke University, ⁴Solid Tumor Program and the Duke Prostate Center,Duke University Medical Center, ⁵Duke University Medical Center

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Overview

This article presents a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells. The technique focuses on the combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2) to better understand the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

Key Study Components

Area of Science

Cell culture techniques
Cancer biology
Phenotypic plasticity

Background

Mesenchymal-epithelial transitions (MET) are crucial in understanding cancer behavior.
Epithelial-like sarcomas often show superior outcomes compared to mesenchymal-like sarcomas.
Studying MET can reveal insights into therapeutic strategies.
This method allows for the exploration of gene regulatory networks associated with MET.

Purpose of Study

To investigate the phenotypic consequences of MET in sarcomas.
To identify why epithelial-like sarcomas have better clinical outcomes.
To develop therapeutic interventions that promote a more indolent epithelial-like state in sarcomas.

Methods Used

Transduction of HEK293T cells with microRNA-200 family members and GRHL2.
Plating of cells in a six-well plate with supplemented DMEM.
Monitoring of downstream gene regulatory networks.
Utilization of undergraduate researchers for demonstration of the procedure.

Main Results

The method provides a reproducible means to study MET in sarcomas.
Insights gained may lead to better understanding of cancer treatment outcomes.
Potential identification of new therapeutic targets for sarcoma treatment.
Demonstration of the procedure by undergraduate students enhances educational value.

Conclusions

Inducing MET in sarcoma cells can elucidate mechanisms of cancer aggressiveness.
This approach may facilitate the development of novel therapeutic strategies.
Understanding phenotypic plasticity is key to improving patient outcomes in sarcoma.

Frequently Asked Questions

What is mesenchymal-epithelial transition (MET)?

MET is a biological process where mesenchymal cells acquire epithelial characteristics, which can influence cancer behavior.

Why is studying MET important in sarcomas?

Studying MET helps to understand the differences in outcomes between various sarcoma types and may lead to better treatment strategies.

What are the main components used in this study?

The study utilizes microRNA-200 family members and grainyhead-like 2 (GRHL2) for inducing MET in sarcoma cells.

How does this method contribute to cancer research?

This method allows researchers to explore gene regulatory networks and phenotypes associated with MET, potentially leading to new therapeutic interventions.

Who conducted the demonstration of the procedure?

The procedure was demonstrated by undergraduate students Jackson Xu and Shivee Gilja in the laboratory.

What are the expected outcomes of this research?

The research aims to identify therapeutic interventions that can shift sarcomas toward a more indolent epithelial-like state.

Apresentamos aqui um método de cultura de células para a indução de transições mesenquimais-epiteliais (MET) em células de sarcoma com base na expressão ectópica combinado de microARN-200 e os membros da família grainyhead-like 2 (GRHL2). Este método é adequado para a melhor compreensão do impacto biológico de plasticidade fenotípica sobre a agressividade e tratamentos do cancro.

O objetivo geral deste procedimento é estudar as consequências fenotípicas das transições mesenquimal-epiteliais em sarcomas. Este método pode ajudar a responder a perguntas-chave sobre por que os sarcomas epiteliais têm resultados superiores em relação aos sarcomas mais mesenquimais. A principal vantagem dessa técnica é que ela fornece um meio fácil e reprodutível para estudar redes reguladoras de genes a jusante e fenótipos associados a transições mesenquimais-epiteliais em sarcomas.

Ao entender melhor a plasticidade fenotípica nos sarcomas, esperamos identificar intervenções terapêuticas que nos permitam mudar os sarcomas para um estado epitelial mais indolente. Demonstrando o procedimento estarão dois alunos de graduação em nosso laboratório, Jackson Xu e Shivee Gilja. No primeiro dia da transdução, coloque três vezes 10 a quinta HEK293T células em cada um dos dois poços de uma placa de seis poços em dois mililitros de DMEM suplementado.

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