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 ...

The overall goal of this simple in vitro coculture model is to mimic the characteristics of an in vivo tumor microenvironment, enrich individual cell populations, and investigate various modes of intercellular communication. This method can help answer key questions in the cancer and radiobiology fields, specifically regarding the factors underlying the generation of cancer-associated fibroblasts and the responses to therapeutic agents. The main advantage of this technique is that it allows the generation of individual cell populations from a mixed cell culture without stress-inducing fluorescence tagging or cell sorting.Begin by washing the cell culture of interest two times with five milliliters of PBS. After the second wash, detach the cells with one milliliter of room temperature trypsin-EDTA for two minutes at room temperature. Then quench the reaction with nine milliliters of complete growth medium, gently pipetting the medium over the surface of the flask 10 times to dissociate the cells.After counting, dilute the cells to a 2.5 times 10 to the fifth cells per milliliter concentration in fresh medium, and transfer the cells to a sterile 15 milliliter centrifuge tube. Spin down the cells by centrifugation and resuspend the pellet in fresh growth medium supplemented with 50%fetal bovine serum at 2.5 times 10 to the fifth cells per 70 microliters of medium concentration. Next, transfer inserts of the appropriate experimental pore size from their packaging into individual wells of a multi-well dish and cover the dish.Then holding the dish with both hands, gently invert the dish until the insert bottoms are facing upwards. Remove the bottom of the dish. Using sterile forceps in one hand, hold one insert in place and use the other hand to slowly aspirate 70 microliters of cells into a micropipette tip.Then slowly dispense the cells across the surface of what is now the topside of the insert. After seeding each of the inserts, carefully replace the dish bottom and incubate the inverted dish at 37 degrees Celsius and 5%carbon dioxide with humidity for 30 to 45 minutes. At the end of the incubation, in a laminar flow biological safety cabinet carefully reinvert the dish such that the bottoms of the inserts now face down.Then slowly and carefully immerse the bottom of each insert in two milliliters of pre-warmed complete medium, and place the dish back into the humidified incubator. After 48 hours, replace the medium in the bottom of each well with two milliliters of fresh growth medium. When all of the medium has been refreshed, seed 2.5 times 10 to the fifth cells of the second population of interest onto the top of the inserts in one milliliter of fresh medium, and return the dish to the incubator.24, 48, and 96 hours later, replace the medium in the top of each insert with one milliliter of fresh complete medium. And the medium at the bottom of each well with two milliliters of fresh complete medium. After 120 hours of coculture, transfer one insert at a time into individual 35-millimeter cell culture dishes containing one milliliter of PBS.And wash the bottoms and topsides of the inserts with one milliliter of PBS. To collect the cells grown on the bottom of the insert, place the inserts bottom side down into 200 microliters of room temperature trypsin-EDTA. After two minutes at room temperature, stop the reaction with 800 microliters of complete growth medium.Then holding the insert at a slight angle, gently pipette the supernatant over the surface of the cells 10 times, collecting the cells in the dish. When all of the inserts have been stripped, resuspend the cells at a concentration of two times 10 to the fifth cells per milliliter of growth medium. And add 250 microliters of cells onto sterile individual glass coverslips.Place the coverslips into the humidified incubator for one hour. At the end of the incubation, in a laminar flow biological safety cabinet, carefully add two milliliters of complete growth medium to the dish and incubate at 37 degrees Celsius and 5%percent carbon dioxide with humidity for 48 hours. Then wash the cells three times with PBS.After the third wash, fix the cells in 4%formaldehyde in PBS for ten minutes at room temperature, followed by five washes with Tris-buffered saline. After the last wash, permeabilize the cells with 0.25%Triton X-100 supplemented with 0.1 saponin for five minutes at room temperature, followed by incubation in blocking solution for one hour at room temperature. Next, label the samples with the primary antibody of interest in blocking solution at four degrees Celsius overnight.The next morning, remove the unbound antibody with three minute washes in wash solution, followed by an one hour room temperature incubation in the appropriate secondary antibody in blocking solution. At the end of the incubation, wash the unbound secondary antibody as just demonstrated and mount the coverslips onto individual slides with antifade mounting medium containing DAPI. Seal the coverslip edges with clear nail polish.Then image the cells under 63x oil magnification on an inverted microscope equipped with an external light source for fluorescence excitation. This system allows two different cell populations to be grown on either side of the porous membranes of cell culture inserts for at least 120 hours, maintaining a greater than 99%purity in the cell populations on either side of the membrane, when inserts with 0.4 or one micron pores are used. Inserts with three micron pores however, are large enough to allow the cells to migrate across the membrane as observed in this experiment using a GFP-positive human breast cancer cell line.0.4 micron pore inserts also limit the formation of functional gap junctions between the cell cultures on both sides of the insert, constraining the communication to secreted factors. Inserts with one and three micron pores however, allow the functional coupling of cells through the gap junctions as indicated by the transfer of the fluorescent label across the membrane in these cocultures. Importantly, this system can be used to effectively generate cancer-associated fibroblasts from normal human diploid fibroblasts following their coculture with breast cancer cells, as evidenced by the reduced expression of caveolin-1 on the fibroblasts cocultured with the human breast cancer cells.While attempting this procedure, it's important to select inserts with a pore size adequate for the question being explored. And to see the first cell population on the bottom side of the insert in medium that facilitates their attachment. Following this procedure, other methods like immunoblotting, in-situ immunofluorescence, or most other cell-based assays can be performed to answer additional questions about protein expression alterations, changes in invasion and migration or differences in responses to therapeutic agents.After its development, this technique paved the way for researchers in the field of cancer biology and radiation biology to explore the factors that contribute to the development, the evolution of the tumor microenvironment, and in our case to the spread of the harmful effects of ionizing radiation from targeted cells with radiation to the bystander cells in the vicinity. After watching this video, you should have a good understanding of how to prepare a mixed cell coculture, to maintain the coculture, and to harvest high purity enriched cell populations from the coculture for further analysis. Don't forget that working with human cell strains or cell lines can be hazardous and that the appropriate proper personal protection equipment should be worn and the proper biosafety regulations should be adhered to while performing this procedure.Thanks for watching and good luck with your experiments.

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siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...