Name: phospholipid mediator induced transformation in three-dimensional cultures
Uploaded: 2022-07-27T14:20:00
Description: Watch this Scientific Journal Video about Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures at JoVE.com

We thank the IISER Pune Microscopy Facility for access to equipment and infrastructure and support for the experiments. This study was supported by a grant from the Department of Biotechnology (DBT), Govt. of India (BT/PR8699/MED/30/1018/2013), Science and Engineering Research Board (SERB), Govt. of India (EMR/2016/001974) and partly by IISER, Pune Core funding. A. K. was funded by CSIR-SRF fellowship, L.A. was funded through DST-INSPIRE fellowship, V.C was funded by DBT (BT/PR8699/MED/30/1018/2013).

1. Seeding MCF10A cells in lrECM
<ol>
	<li>Maintain MCF10A cells (adherent breast epithelial cells) in the growth medium. Passage the cells every 4 days. 
	NOTE: Composition of growth medium: high glucose DMEM without sodium pyruvate containing horse serum (5%), insulin (10 &#181;g/mL), hydrocortisone (0.5 &#181;g/mL), epidermal growth factor, EGF (20 ng/mL), of cholera toxin (100 ng/mL), and penicillin-streptomycin (100 units/mL) (see Table of Materials).</li>
	<li>Thaw lrECM (se...

Established cell line-based models are widely used to study the process of carcinogenesis. Monolayer cultures of cells continue to provide insights into the various molecular signaling pathways that mediate characteristic changes in cancer cells32. Studies on the role of well-known oncogenes such as Ras, Myc, and mutated p53 were first reported using monolayer cultures as the model system33,34,35,</s...

A myriad of models are available to study the progression of cancer, each of them being unique and representing a subtype of this complex disease. Each model provides unique and valuable insights into cancer biology and has improved the means to mimic the actual disease condition. Established cell lines grown as a monolayer have provided valuable insights into vital processes in vitro, such as proliferation, invasiveness, migration, and apoptosis1. Though two-dimensional (2D) cell culture has been the traditional tool to investigate the response of mammalian cells to several environmental perturbations, extrapolation of these findings to predict tissue-level responses does not seem sufficiently convincing. The major limitation of the 2D cultures is that the microenvironment created differs largely from that of the breast tissue itself2. 2D culture lacks the interaction of the cells with the extracellular matrix, which is vital for the growth of any tissue. Also, tensile forces experienced by the cell in monolayer cultures hinder the polarity of these cells, thus altering cell signaling and behavior3,4,5. Three-dimensional (3D) culture systems have opened up a new avenue in the field of cancer research with their ability to mimic the in vivo conditions in vitro. Many crucial microenvironmental cues that are lost in 2D cell culture could be re-established using 3D cultures of laminin-rich extracellular matrix (lrECM)6.
Various studies have identified the importance of the tumor microenvironment in carcinogenesis7,8. Inflammation-associated factors are a major part of the microenvironment. Platelet Activating Factor (PAF) is a phospholipid mediator secreted by various immune cells that mediates multiple immune responses9,10. High levels of PAF are secreted by different breast cancer cell lines and are associated with enhanced proliferation11. Studies from our lab have shown that the prolonged presence of PAF in acinar cultures leads to the transformation of breast epithelial cells12. PAF activates the PAF receptor (PAFR), activating the PI3K/Akt signaling axis13. PAFR is also reported to be associated with EMT, invasion, and metastasis14.
The present protocol demonstrates a model system to study PAF-induced transformation, using 3D cultures of breast epithelial cells, as has been previously described by Chakravarty et al.12. The breast epithelial cells grown on the extracellular matrix (3D cultures) tend to form polarized growth-arrested spheroids. These are called acini and closely resemble the acini of breast tissue, the smallest functional unit of the mammary gland, in vivo15. These spheroids (Figure 1A,B) consist of a monolayer of closely packed polarized epithelial cells surrounding a hollow lumen and attached to the basement membrane (Figure 1C). This process of morphogenesis has been well described in literature16. When seeded on lrECM, the cells undergo division and differentiation to form a cluster of cells, which then polarize from Day 4 onwards. By Day 8, the acini consist of a group of polarized cells that are in direct contact with the extracellular matrix and a cluster of unpolarized cells enclosed within the outer polarized cells, with no contact to the matrix. These unpolarized cells are known to undergo apoptosis by Day 12 of culture, forming a hollow lumen. By Day 16, growth-arrested structures are formed16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64146/64146fig01.jpg" /> 
Figure 1: Nuclei of cells in acini stained with a nuclear stain. (A) 3D construction of the acini. (B)&#160;Phase Contrast image of MCF10A acini grown on Matrigel for 20 days. (C) The centermost section shows the presence of a hollow lumen. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64146/64146fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Unlike 2D cultures, acinar cultures aid in distinguishing normal and transformed cells through apparent morphology changes. Non-transformed breast epithelial cells form acini with a hollow lumen, mimicking the normal human breast acini. These spheroids, upon transformation, show a disrupted morphology characterized by a major loss of polarity (one of the hallmarks of cancer), absence of a lumen, or disruption of the hollow lumen (due to evasion of apoptosis) that may be induced due to deregulation of various genes17,18,19,20. These transformations can be studied using commonly used techniques such as immunofluorescence. Thus, the 3D cell culture model can function as a simple method to investigate the process of breast acinar morphogenesis and breast carcinogenesis. Establishing a 3D culture system to understand the effect of a phospholipid mediator, PAF, will assist in high throughput preclinical drug screening.
This work has adapted the 3D &#39;on top&#39; culture protocol16,21 for studying transformation induced by PAF22. The phenotypic changes induced by exposure of the acini to the phospholipid mediator were studied using immunofluorescence. Various polarity and epithelial to mesenchymal transition (EMT) markers12,16 were used in the study. Table 1 mentions their normal localization and their expected phenotype upon transformation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td>Marks</td>
			<td>Normal localization</td>
			<td>Transformed phenotype</td>
		</tr>
		<tr>
			<td>&#945;6-Integrin</td>
			<td>Basolateral</td>
			<td>Basal with weak lateral stain</td>
			<td>Strong lateral / Apical stain</td>
		</tr>
		<tr>
			<td>&#946;-Catenin</td>
			<td>Cell-cell junction</td>
			<td>Basolateral</td>
			<td>Abnormal / nuclear or cytoplasmic localization</td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>EMT</td>
			<td>Absent / weak presence</td>
			<td>Up-regulation</td>
		</tr>
	</tbody>
</table>
Table 1: Markers used in the study. Different markers used with their localization in the presence and absence of PAF treatment.
This method can be best utilized to study/screen plausible drugs and target genes for various breast cancer subtypes. This can provide a drug response data closer to the in vivo scenario, aiding in faster and more reliable drug development. Also, this system can be used to study the molecular signaling associated with drug response and drug resistance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin EDTA</td>
			<td>Invitrogen</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>16% paraformaldehyde</td>
			<td>Alfa Aesar</td>
			<td>AA433689M</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Mouse Alexa Flour 488</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Rabbit Alexa Flour 488</td>
			<td>Invitrogen</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber Coverglass</td>
			<td>Nunc</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>C8052-1MG</td>
			<td>1 mg/mL in dH2O</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica</td>
			<td>Leica SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965126</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644-0.2MG</td>
			<td>100 mg/mL in dH2O</td>
		</tr>
		<tr>
			<td>F(ab&#8217;)2 fragment of antibody raised in goat against mouse antigen</td>
			<td>Jackson Immunoresearch</td>
			<td>115-006-006</td>
			<td></td>
		</tr>
		<tr>
			<td>GM130 antibody</td>
			<td>Abcam</td>
			<td>ab52649</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Abcam</td>
			<td>ab7481</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Invitrogen</td>
			<td>33258</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>16050122</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0888</td>
			<td>1 mg/mL in ethanol</td>
		</tr>
		<tr>
			<td>Image Processing Software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I1882</td>
			<td>10 mg/mL stock dH2O</td>
		</tr>
		<tr>
			<td>lrECM (Matrigel)</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting reagent (Slow fade Gold Anti-fade)</td>
			<td>Invitrogen</td>
			<td>S36937</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Stain&#160; (Hoechst)</td>
			<td>Invitrogen</td>
			<td>33258</td>
			<td></td>
		</tr>
		<tr>
			<td>PAF</td>
			<td>Cayman Chemicals</td>
			<td>91575-58-5</td>
			<td>Methylcarbamyl PAF C-16, procured as a 10 mg/mL in ethanol</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Lonza</td>
			<td>17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Sigma</td>
			<td>B9754</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin antibody</td>
			<td>Abcam</td>
			<td>ab92547</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;6-integrin antibody</td>
			<td>Millipore</td>
			<td>MAB1378</td>
			<td></td>
		</tr>
	</tbody>
</table>

phospholipid mediator induced transformation in three-dimensional cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

MCF10A cells, upon exposure to PAF treatment, form acinar structures with very distinct phenotypes. &#945;6-integrin was found to be mislocalized with more apical staining. A few acini also showed discontinuous staining (Figure 2A). Both these phenotypes indicate the loss of basal polarity, as evidenced from literature24,25. Earlier reports indicate the controversial role of &#945;6-integrin in cancer metastasis. &#945;6-integrin is ...

Watch this Scientific Journal Video about Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures at JoVE.com

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Our modified 3D assay cultures can be used to investigate the role of various components in a cancer microenvironment, leading to the identification of novel biomarkers and drug targets. This method enables one to study changes in the cellular phenotype and molecular signaling that closely resemble the immune system. It can also be modified to meet specific requirements of a study.This method can help identify novel biomarkers that can aid in early detection of breast cancer. Also, molecular insights can be utilized for identifying novel drug targets. This method can be used to study both tumorigenesis and morphogenesis after perturbation of gene or genes that can provide insight into the role played by the gene or genes.To begin, trypsinize the cells by aspirating the medium and washing with 2 milliliters of PBS. Add 900 microliters of 0.05%Trypsin-EDTA and incubate at 37 degrees Celsius for around 15 minutes or till the cells are completely trypsinized. Prepare a bed of lrECM in eight wells of a chambered cover glass slide by coating each well with 60 microliters of lrECM.Place it in a 37 degree Celsius carbon dioxide incubator for a maximum of 15 minutes. To prepare the cell suspension, add 5 milliliters of re-suspension medium to quench the trypsin activity and spin the cells at 112 times g for 10 minutes at 25 degrees Celsius. Aspirate the spent medium and re-suspend the cells in 2 milliliters of assay medium.Mix the suspension well to ensure the formation of a single cell suspension. Count the cells using a hemocytometer and calculate the volume of cell suspension needed to seed 6, 000 cells in each well. According to the number of wells required, dilute the cell suspension in the overlay medium.Add 400 microliters of the diluted cell suspension to the prepared lrECM beds, carefully ensuring not to disturb the lrECM bed. Incubate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator. After 3 hours, add 200 nanomoles of PAF to each well.Add the same concentration of PAF during every media change, and replenish the cells with fresh medium every 4 days. After 20 days of culturing, carefully pipette out the medium from each well and wash the wells with 400 microliters of pre-warmed PBS. Fix the acinar structures by adding 400 microliters of freshly prepared 4%paraformaldehyde and incubating for 20 min at room temperature.Rinse the wells once with ice-cold PBS and permeabilize with PBS containing 0.5%Triton X-100 for 10 minutes at 4 degrees Celsius. After 10 minutes, pipette out the Triton X-100 solution carefully and rinse with 400 microliters of freshly prepared PBS glycine. Add 400 microliters of the primary blocking solution comprising 10%goat serum in immunofluorescence buffer and incubate at room temperature for 60 minutes.Remove the primary blocking solution, add 200 microliters of 2%secondary blocking antibody prepared in the primary blocking solution, and leave it at room temperature for 45 to 60 minutes. Prepare the primary antibody in 2%secondary blocking antibody solution in a 1:100 dilution. Remove the secondary blocking solution, add the freshly prepared antibody, and incubate overnight at 4 degrees Celsius.Carefully pipette the primary antibody solution and wash it thrice with 400 microliters of immunofluorescence buffer. Next, prepare a 1:200 dilution of the fluorophore-conjugated secondary antibody in the primary blocking solution and incubate the slides in the secondary antibody solution for 40 to 60 minutes at room temperature. Rinse the slides with 400 microliters of immunofluorescence buffer for 20 minutes, followed by two washes with PBS for 10 minutes each.Counter-stain the nuclei with PBS containing 0.5 nanograms per milliliter of nuclear stain for 5 to 6 minutes at room temperature. Wash the slides thrice with 400 microliters of PBS to remove the excess stain. Carefully pipette out the entire PBS and ensure removal of the residual solution.Then add one drop of mounting reagent into each well and allow it to stand at room temperature overnight. Finally, image the slides under 40x or 63x magnification on a confocal microscope, taking optical Z-sections of 0.6 millimeter step size. The images represent the nuclei of cells in acini stained with a nuclear stain.The 3D construction of the acini and the phase contrast image of MCF10A acini grown on lrECM for 20 days are shown here. The centermost section shows the presence of a hollow lumen. PAF disrupts the polarity and induces EMT-like changes in MCF10A breast acini.MCF10A cells were grown as 3D on top'cultures in lrECM. The cultures were treated with PAF on day 0, 4, 8, 12, and 16. The cultures were maintained for 20 days and then immunostained for alpha6-integrin and nuclear stain, GM130, a marker for apical polarity, and Vimentin, an EMT marker.The centermost stack of the respective acini has been shown here. The representative data is for 40-50 acini from three biologically independent experiments. The lrECM bed needs to be made carefully such that it is evenly spread without any air bubbles.Also, the cell suspension should be mixed evenly and added slowly to avoid clumping. One can obtain protein and RNA from the 3D cultures that can then be used to investigate the different molecular pathways.

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