Name: a mimic of the tumor microenvironment: a simple method for generating enriched cell populations and investigating intercellular communication
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication at JoVE.com

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).

1. Preparation of Culture Media and Cells
<ol>
	<li>Prepare 500 ml of Eagle&#39;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 &#181;g 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.</li>
	<li>Prepare 70 &#181;l of cell culture medium for each insert (6-well format insert): Eagle&#39;s min...

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...

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 variety of carcinomas, including breast, prostate, lung, pancreas, skin, colon, esophagus, and ovary5,6,12-17. Yet, the exact nature of the contribution of CAFs throughout cancer pathogenesis remains poorly defined. Furthermore, clinical evidence has demonstrated a prognostic value of CAFs, correlating their presence to high-grade malignancies, therapy failure, and overall poor prognosis10,18,19.
Clearly, enhancing our understanding of the initiating events in CAF development, as well as the intercellular communications mediating their role within the TME, may provide exciting new therapeutic targets and enhanced strategies that could improve patient outcomes. Towards this goal, several in vivo and in vitro models have been developed. While in vivo approaches are more reflective of patients&#39; TME, they possess limitations, including the immense complexity and heterogeneity both within and between tumors. Furthermore, tumor samples from human subjects often represent highly developed TME and do not permit an understanding of the TME initiating events. Experimental animal studies offer some advantages, however generalization of animal data to humans should be done with caution due to differences in physiology between humans and animals such as rodents (e.g., thiol chemistry20, metabolic rate21, tolerance to stress22, etc.). Further, unlike the human population, which is genetically heterogeneous in nature, laboratory animals are typically bred to homogeneity. Also, it is often difficult to examine transient physiological variations and cell phenotype changes, as well as to control for specific experimental parameters using animals such as rodents. Thus, in vitro 2- and 3-dimensional (2D and 3D) tissue culture models are frequently utilized to advance the basic understanding of TME development. In spite of their lack of an accurate portrayal of the complexity of in vivo systems, these models offer advantages that greatly facilitate mechanistic investigations. In vitro models allow for a more simplified, focused, and cost-effective analysis of the TME, whereby statistically significant data can be generated in cells free of systemic variations that arise in animals.
There are several varieties of in vitro systems. The two most commonly used TME in vitro models consist of mixed monolayer or spheroid cell cultures. Both culture methods are advantageous for basic studies of intercellular interactions (e.g., normal cells with tumor cells) and for the analysis of various TME specific cell phenotype changes (e.g., emergence of cancer-associated fibroblasts from normal fibroblasts). Additionally, the spheroids are able to create a more reflective tissue-like structure of the TME, and can be representative of tumor heterogeneity23. However spheroids often produce widely varying oxygen tension gradients across layers, which may complicate experimental conclusions24. Unfortunately, both models are extremely limited in their ability to isolate pure cell populations for further characterization and study following co-culture. To do so would require at least one cell type to be fluorescently-tagged or labeled with an identifying maker, and then subjecting the mixed co-culture to extensive processing and cell sorting to separate the cell populations. While a cell sorter is capable of isolating a rather pure cell population, one must be cognizant of cellular stress and potential microbial contamination risks25.
To facilitate the understanding of intercellular communication, great efforts have been devoted towards developing and optimizing in vitro systems that closely mimic the in vivo environment, while permitting a simplified approach. One such tool is the permeable microporous insert, a membrane substrate that was first developed in 195326 and subsequently adapted for diverse applications and studies (e.g., cell polarity27, endocytosis28, drug transport29, tissue modeling30, fertilization31, bystander effect32,33, etc.). This system permits the growth of cells with in vivo-like anatomical and functional differentiation, as well as expression of many in vivo markers34,35 that are not observed when cultured on impermeable plasticware. Furthermore, the extremely thin porous membrane (10 &#181;m thick) permits rapid diffusion of molecules and equilibration times, which simulates the in vivo environment and permits independent cellular functioning at both the apical and basolateral cell domains. An additional advantage of the insert&#39;s utility as a TME system is its physical separation of two heterotypic cell populations grown on either side of the membrane in the same environmental conditions, while maintaining various modes of intercellular communication through the membrane pores. Though physically separated, the two cell populations are metabolically coupled via secreted elements and, as described here, also through gap-junctional channels. Additionally, by maintaining the inserts at in vivo partial oxygen tension (PO2), the model reduces the complications of oxygen and chemical gradients observed in other systems. Rather, it increases the understanding of natural mechanisms controlling the TME. Notably, the two cell populations can be easily isolated with high purity, without fluorescent tagging and/or cell sorting following extended periods of co-culture.
Here we describe an in vitro TME protocol consisting of human breast carcinoma cells and human fibroblasts grown, respectively, on either side of a permeable microporous membrane insert, but yet in continuous bi-directional communication through the membrane pores. We show that by using membranes with different pore sizes, the contribution of a specific type of intercellular communication (e.g., secreted factors versus gap junctions) to the development of the TME can be investigated.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Cell Culture</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">AG01522 (i.e., AG1522) human diploid fibroblast</td>
			<td style="width:180px;">Coriell</td>
			<td style="width:139px;">107661</td>
			<td style="width:279px;">Passage 8-13</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MDA-MB-231-luc-D3H1 breast adenocarcinoma cell line</td>
			<td style="width:180px;">PerkinElmer</td>
			<td style="width:139px;">119261</td>
			<td style="width:279px;">Parental line: ATCC (#HTB-26)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MDA-MB-231/GFP breast adenocarcinoma cell line</td>
			<td style="width:180px;">Cell Biolabs</td>
			<td style="width:139px;">AKR-201</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Eagle&#39;s minimal essential medium (MEM)</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">15-010-CV</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Fetal Bovine Serum (FBS), Qualified</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">F6178-500mL</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Corning Glutagro Supplement (200mM L-alanyl-L-glutamine)</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">25-015-Cl</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Penicillin Streptomycin Solution, 100X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">30-002-Cl</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (0.4 &#956;m pore)</td>
			<td style="width:180px;">Costar</td>
			<td style="width:139px;">3450</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (1 &#956;m pore)</td>
			<td style="width:180px;">Greiner bio-one</td>
			<td style="width:139px;">657610</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:289px;">Transwell Insert (i.e., permeable microporous membrane insert) (3 &#956;m pore)</td>
			<td style="width:180px;">Costar</td>
			<td style="width:139px;">3452</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">6-well Culture Plate</td>
			<td style="width:180px;">Greiner Bio-One Cellstar</td>
			<td style="width:139px;">657160-01</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">75 cm2 cell culture flask</td>
			<td style="width:180px;">CellStar</td>
			<td style="width:139px;">658 170</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Phosphate-Buffered Saline (PBS), 1X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">21-040-CV</td>
			<td style="width:279px;">without calcium &#38; magnesium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">0.25% (vol/vol) Trypsin, 2.21 mM EDTA, 1X</td>
			<td style="width:180px;">Corning Cellgro</td>
			<td style="width:139px;">25-053-Cl</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">15 mL Centrifuge Tube</td>
			<td style="width:180px;">CellTreat</td>
			<td style="width:139px;">229411</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">35 x 10 mm Cell Culture Dish</td>
			<td style="width:180px;">Greiner bio-one</td>
			<td style="width:139px;">627 160</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
			<td style="width:180px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Immunofluorescent Microscopy</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Mouse anti-Caveolin 1</td>
			<td style="width:180px;">BD Transduction Laboratories</td>
			<td style="width:139px;">610406</td>
			<td style="width:279px;">In situ Immunofluorescence - 1:5000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td style="width:180px;">ThermoFisher Scientific</td>
			<td style="width:139px;">A-11029</td>
			<td>In situ Immunofluorescence - 1:2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Bovine Serum Albumin - Fraction V</td>
			<td style="width:180px;">Rockland</td>
			<td style="width:139px;">BSA-50</td>
			<td>Immunoglobulin and protease free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">16% (wt/vol) Formaldehyde Solution</td>
			<td style="width:180px;">ThermoFisher Scientific</td>
			<td style="width:139px;">28908</td>
			<td>Dilute to 4% with 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Premium Cover Glass (22x22 mm No.1)</td>
			<td style="width:180px;">Fisher</td>
			<td style="width:139px;">12548B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Triton X-100</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">T8787-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">SlowFade Gold antifade reagent with DAPI</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:139px;">S36938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
			<td style="width:180px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Flow Cytometric Analysis</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Calcein, AM</td>
			<td style="width:180px;">Molecular Probes</td>
			<td style="width:139px;">C3100MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:139px;">14025-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Name</td>
			<td style="width:180px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">For Western Blot Analysis</td>
			<td style="width:180px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Mouse anti-Caveolin 1</td>
			<td style="width:180px;">BD Transduction Laboratories</td>
			<td style="width:139px;">610406</td>
			<td style="width:279px;">Western Blot - 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Tween-20</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">170-6531</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Nitrocellulose Membrane (0.2 &#956;m)</td>
			<td style="width:180px;">BioRad</td>
			<td>162-0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Western Lightning Plus-ECL</td>
			<td style="width:180px;">PerkinElmer</td>
			<td style="width:139px;">NEL104001EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">BioRad DC Protein Assay</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">500-0116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">161-0302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Sodium deoxycholate monohydrate (DOC)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">D5670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">IGEPAL CA-630 (NP40)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:139px;">I8896</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">30% Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:139px;">161-0158</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mimic of the tumor microenvironment: a simple method for generating enriched cell populations and investigating intercellular communication

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&#39;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.

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&#39;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...

Watch this Scientific Journal Video about A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication at JoVE.com

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

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 ...

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