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Abstract

Introduction

Protocol

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Materials

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Biology

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Published: August 28th, 2018

DOI:

10.3791/58144

1Indiana University School of Medicine, 2Urology, Indiana University Health Southern Indiana Physicians, 3Indiana University Melvin and Bren Simon Cancer Center, 4Department of Medical and Molecular Genetics, Indiana University School of Medicine

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 µm-thick polycarbonate membrane with 0.4 µm pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

The role of productive reciprocal interactions between cancer cells and the tumor microenvironment in tumor progression has been well established and has become a major focus of research in cancer biology1. Similar instances of bidirectional signaling are crucial during wound healing, immune responses, angiogenesis, stem cell niches, and during development2,3,4,5,6,7,8. A common theme in all these biological proces....

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The protocol follows the guidelines of the Institutional Regulatory Board of Indiana University.

1. Cell Preparation

  1. Isolation and culture of human primary mesothelial cells
    1. Isolate human primary mesothelial cells (HPMCs) from human omentum as described previously17,18 and grow them in complete growth medium [Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum.......

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Metastasizing ovarian cancer cells encounter mesothelial cells at the site of metastasis within the peritoneal cavity19. Productive paracrine and juxtacrine interactions with the mesothelial cells help in inducing adaptive responses in the ovarian cancer cells, which enable successful metastasis17,18,20,21. To test the effectiveness of th.......

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Understanding the mechanism of paracrine and juxtacrine signaling between cells is essential for developing a better knowledge of normal tissue homeostasis and disease conditions7,8. Most paracrine signaling studies are conducted by collecting conditioned medium from one cell type and using it to treat the other cell type. This method has an advantage in its inherent simplicity. However, it does not accurately recapitulate the localized concentrations of the secr.......

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We are indebted to the patients for their participation in the tissue collection for these experiments. A DoD OCRP Ovarian Cancer Academy Award (W81XWH-15-0253) and a pilot award from Colleen's Dream Foundation to Anirban K. Mitra supported this research.

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Name Company Catalog Number Comments
24 mm Transwell permeable support with 0.4 µm Pore Polycarbonate Membrane Insert Corning (Costar) 3412 • 10 µm thick translucent polycarbonate membrane
• Treated for optimal cell attachment
• Packaged 6 inserts in a 6 well plate, 4 plates per case
• Membrane must be stained for cell visibility
• Sterilized by gamma radiation
6 well plate Corning (Falcon) 353046 Flat Bottom, TC-treated, sterile, with Lid
15 cm culture dish Corning (Falcon) 353025 Sterile, TC-treated Cell Culture Dish
DMEM Corning (Cellgro) 10-013-CV
Penicillin Streptomycin Corning 30-002-CI
MEM Nonessential amino acids Corning (Cellgro) 25-025-CI
MEM Vitamins Corning (Cellgro) 25-020-CI
0.25% Trypsin, 2.21 mM EDTA Corning 25-053-CI
Fetal bovine serum Atlanta Biologicals S11150
Pipets Any make is fine
CO2 Incubator Any make is fine
Biosafety level II cabinet Any make is fine
FN1 TaqMan Gene Expression Assay ThermoFisher Scientific Hs01549976_m1
TGFB1 TaqMan Gene Expression Assay ThermoFisher Scientific Hs00998133_m1
CDH1 TaqMan Gene Expression Assay ThermoFisher Scientific Hs01023895_m1
GAPDH TaqMan Gene Expression Assay ThermoFisher Scientific Hs99999905_m1
miRNeasy mini RNA isolation Kit Qiagen 217004
High-Capacity cDNA Reverse Transcription Kit ThermoFisher Scientific 43-688-13
HeyA8 ovarian cancer cells Obtained from Ernst Lengyel Lab, University of Chicago
TGFβ Neutralizing Antibody R&D Systems MAB1835-100

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