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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Published: September 20th, 2016

DOI:

10.3791/54429

1Department of Radiology, New Jersey Medical School, Rutgers University

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert's membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

The tumor microenvironment (TME) is a highly complex system comprised of carcinoma cells that co-exist and evolve alongside host stroma. This stromal component typically consists of fibroblasts, myofibroblasts, endothelial cells, various immune components, as well as an extracellular matrix1. A significant constituent, often the majority of this stroma, are activated fibroblasts, frequently referred to as cancer-associated fibroblasts or carcinoma-associated fibroblasts (CAF)2,3. Unlike normal, non-activated fibroblasts, CAFs contribute to tumor initiation, progression, angiogenesis, invasion, metastasis, and recurrence4-11 in a wide v....

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1. Preparation of Culture Media and Cells

  1. Prepare 500 ml of Eagle's minimum essential medium supplemented with 12.5% (vol/vol) heat-inactivated fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine, and 100 units of penicillin and 100 µg of streptomycin per ml.
    NOTE: The growth medium and supplement(s) can be easily exchanged for the growth requirements of other cell strains or cell lines.
  2. Prepare 70 µl of cell culture medium for each insert (6-well format insert): Eagle's min.......

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Here we adapted a permeable microporous membrane insert to develop an in vitro heterotypic cell co-culture system that mimics the in vivo tumor microenvironment (Figure 1). This system allows for two different cell populations to be grown on either side of the insert's porous-membrane for extended periods of time (up to 120 hr, in our use). Importantly the system is capable of maintaining the purity of the cell populations, as determined by plating G.......

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The protocol described here is a simple, adaptable in vitro procedure (Figure 1) that utilizes a permeable microporous membrane insert to generate highly enriched individual cell populations from a co-culture of heterotypic cells. Significantly, the model is suitable for investigating various modes of intercellular communication. The critical steps include selecting the appropriate pore-size insert for specific experimental interest(s), seeding the first cell population on the bottom side of the.......

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This research was supported by grants from the New Jersey Commission on Cancer Research (Pre-Doctoral Fellowship DFHS13PPCO17), the National Institutes of Health (CA049062), and the National Aeronautics and Space Administration (NNX15AD62G).

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Name Company Catalog Number Comments
For Cell Culture
AG01522 (i.e., AG1522) human diploid fibroblast Coriell 107661 Passage 8-13
MDA-MB-231-luc-D3H1 breast adenocarcinoma cell line PerkinElmer 119261 Parental line: ATCC (#HTB-26)
MDA-MB-231/GFP breast adenocarcinoma cell line Cell Biolabs AKR-201
Eagle's minimal essential medium (MEM) Corning Cellgro 15-010-CV
Fetal Bovine Serum (FBS), Qualified Sigma F6178-500mL
Corning Glutagro Supplement (200mM L-alanyl-L-glutamine) Corning Cellgro 25-015-Cl
Penicillin Streptomycin Solution, 100X Corning Cellgro 30-002-Cl
Transwell Insert (i.e., permeable microporous membrane insert) (0.4 μm pore) Costar 3450
Transwell Insert (i.e., permeable microporous membrane insert) (1 μm pore) Greiner bio-one 657610
Transwell Insert (i.e., permeable microporous membrane insert) (3 μm pore) Costar 3452
6-well Culture Plate Greiner Bio-One Cellstar 657160-01
75 cm2 cell culture flask CellStar 658 170
Phosphate-Buffered Saline (PBS), 1X Corning Cellgro 21-040-CV without calcium & magnesium
0.25% (vol/vol) Trypsin, 2.21 mM EDTA, 1X Corning Cellgro 25-053-Cl
15 mL Centrifuge Tube CellTreat 229411
35 x 10 mm Cell Culture Dish Greiner bio-one 627 160
Name Company Catalog Number Comments
For Immunofluorescent Microscopy
Mouse anti-Caveolin 1 BD Transduction Laboratories 610406 In situ Immunofluorescence - 1:5000
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate ThermoFisher Scientific A-11029 In situ Immunofluorescence - 1:2000
Bovine Serum Albumin - Fraction V Rockland BSA-50 Immunoglobulin and protease free
16% (wt/vol) Formaldehyde Solution ThermoFisher Scientific 28908 Dilute to 4% with 1X PBS
Premium Cover Glass (22x22 mm No.1) Fisher 12548B
Triton X-100 Sigma T8787-50ML
SlowFade Gold antifade reagent with DAPI Invitrogen S36938
Name Company Catalog Number Comments
For Flow Cytometric Analysis
Calcein, AM Molecular Probes C3100MP
Hanks' Balanced Salt Solution (HBSS) Gibco 14025-076
Name Company Catalog Number Comments
For Western Blot Analysis
Mouse anti-Caveolin 1 BD Transduction Laboratories 610406 Western Blot - 1:10000
Tween-20 BioRad 170-6531
Nitrocellulose Membrane (0.2 μm) BioRad 162-0112
Western Lightning Plus-ECL PerkinElmer NEL104001EA
BioRad DC Protein Assay BioRad 500-0116
Sodium dodecyl sulfate (SDS) BioRad 161-0302
Sodium deoxycholate monohydrate (DOC) Sigma D5670
IGEPAL CA-630 (NP40) Sigma I8896
30% Acrylamide/Bis Solution, 37.5:1 BioRad 161-0158

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