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JoVE Journal Cancer Research

Cancer Research

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Published: July 27, 2022 doi: 10.3791/64146
Automatic Translation
1, 1,2, 1,3, 1
1Biology Department, Indian Institute of Science Education and Research, 2Department of Biology, Center for Genomics and Systems Biology, New York University, 3Enveda Therapeutics India Pvt Ltd.

Summary

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.

Abstract

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.

Introduction

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.

Figure 1
Figure 1: Nuclei of cells in acini stained with a nuclear stain. (A) 3D construction of the acini. (B) 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 µm. Please click here to view a larger version of this figure.

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 'on top' 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.

Antibodies Marks Normal localization Transformed phenotype
α6-Integrin Basolateral Basal with weak lateral stain Strong lateral / Apical stain
β-Catenin Cell-cell junction Basolateral Abnormal / nuclear or cytoplasmic localization
Vimentin EMT Absent / weak presence Up-regulation

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.

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Protocol

1. Seeding MCF10A cells in lrECM

  1. 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 µg/mL), hydrocortisone (0.5 µg/mL), epidermal growth factor, EGF (20 ng/mL), of cholera toxin (100 ng/mL), and penicillin-streptomycin (100 units/mL) (see Table of Materials).
  2. Thaw lrECM (see Table of Materials) on ice 20 min before the start of the experiment. The day of seeding the cells is generally considered Day 0.
  3. For trypsinizing the cells, aspirate the medium, and wash with 2 mL of PBS. Add 900 µL of 0.05% Trypsin-EDTA and incubate at 37 °C for ~15 min or till the cells are completely trypsinized.
  4. Prepare a bed of lrECM in eight wells of a chambered cover glass slide (see Table of Materials).
    1. When the cells are trypsinizing, coat each well with 60 µL of lrECM and place it in a 37 °C CO2 incubator for a maximum of 15 min.
  5. Prepare the cell suspension following the steps below.
    1. Following the complete dislodging of cells, add 5 mL of re-suspension medium to quench trypsin activity and spin the cells at 112 x g for 10 min at 25 °C.
      NOTE: Composition of re-suspension medium: high glucose DMEM without sodium pyruvate, supplemented with horse serum (20%) and penicillin-streptomycin (100 units/mL).
    2. Aspirate the spent medium and re-suspend the cells in 2 mL of assay medium. Mix the suspension well to ensure the formation of a single cell suspension.
      NOTE: Composition of assay medium: high glucose DMEM without sodium pyruvate, supplemented with horse serum (2%), hydrocortisone (0.5 µg/mL), cholera toxin (100 ng/mL), insulin (10 µg/mL), and penicillin-streptomycin (100 units/mL).
    3. Count the cells using a hemocytometer and calculate the volume of cell suspension needed to seed 6 x 103 cells in each well.
      NOTE: It is generally preferred to include one extra well in the calculation to account for any pipetting errors.
    4. According to the number of wells required, dilute the cell suspension in overlay medium.
      NOTE: Overlay medium composition for a single well is as follows: 400 µL of assay medium, 8 µL of lrECM (2% final), and 0.02 µL of 100 µg/mL of EGF (5 ng/mL final).
  6. Perform seeding of the cells.
    1. Add 400 µL of the diluted cell suspension to the prepared lrECM beds (step 1.4), carefully ensuring not to disturb the lrECM bed. Incubate at 37 °C in a humidified 5% CO2 incubator.

2. PAF treatment

  1. Add PAF 3 h following the seeding of cells. Prepare a 100 µM stock of PAF in PBS and add the required volume of 0.2 µL in each well (which corresponds to 200 nM).
  2. Add the same concentration of PAF during every media change.

3. Re-feeding with fresh media

  1. Replenish the cells with fresh medium every 4 days (i.e., Day 4, Day 8, Day 12, and Day 16).

4. Immunofluorescence study to detect phenotypic changes induced by prolonged PAF exposure

  1. After 20 days of culturing, carefully pipette out the medium from each well and wash the wells with 400 µL of pre-warmed PBS.
  2. Fix the acinar structures by adding 400 µL of 4% paraformaldehyde (freshly prepared by diluting 16% paraformaldehyde in 1x PBS) and incubating for 20 min at room temperature.
  3. Rinse the wells once with ice-cold PBS and permeabilize with PBS containing 0.5% Triton X-100 for 10 min at 4 °C.
  4. After 10 min, immediately but carefully pipette out the Triton-X 100 solution and rinse with 400 µL of PBS-glycine (freshly prepared by adding a pinch of glycine in 1x PBS). This is repeated thrice for 15 min each.
  5. Add 400 µL of the primary blocking solution comprising 10% goat serum (see Table of Materials) in immunofluorescence (IF) buffer and incubate at room temperature for 60 min.
    NOTE: Composition of IF buffer: 0.05% sodium azide, 0.1% BSA, 0.2% Triton-X 100, and 0.05% Tween (20 in 1 PBS).
  6. Remove the primary blocking solution, add 200 µL of 2% secondary blocking antibody (F(ab')2 fragment of antibody raised in goat against mouse antigen, see Table of Materials) prepared in primary blocking solution, and leave it for 45-60 min at room temperature.
  7. Prepare the primary antibody (see Table of Materials) in 2% secondary blocking antibody solution in a 1:100 dilution. After removing the secondary blocking solution, add the freshly prepared antibody and incubate overnight at 4 °C.
  8. The previous step may elicit liquefaction of the basement membrane. Before proceeding with the experiment, wait until the slide reaches room temperature. Carefully pipette the primary antibody solution and wash it thrice with 400 µL of IF buffer.
  9. During the last wash with IF buffer, prepare a 1:200 dilution of the fluorophore-conjugated secondary antibody (see Table of Materials) in the primary blocking solution. Incubate the slides in the secondary antibody solution for 40-60 min at room temperature.
  10. Rinse the slides with 400 µL of IF buffer for 20 min, followed by two washes with PBS for 10 min each.
  11. Counter-stain the nuclei with PBS containing 0.5 ng/mL of nuclear stain (see Table of Materials) for 5-6 min at room temperature. Wash the slides thrice with 400 µL of PBS to remove excess stain.
  12. Carefully pipette out the entire PBS and ensure removal of the residual solution. Add one drop of mounting reagent (see Table of Materials) into each well and allow it to stand at room temperature overnight.
  13. Store the slides at room temperature and image the slides as soon as possible.
  14. Image under 40x or 63x magnification on a confocal microscope (see Table of Materials), taking optical Z-sections of 0.6 mm step size (NA = 1.4).
  15. Open the acquired images with an image processing software (see Table of Materials). Demonstrate the morphological differences using 3D projections.
    NOTE: The centermost optical Z-section can be best used to show differences in the localization of polarity markers. These can be quantified manually to represent the percentage of spheroids showing that specific staining pattern.
  16. To illustrate differences in the expression of proteins, perform a semi-quantitative analysis measuring the mean grey value or calculating corrected total cell fluorescence (CTCF)23. Represent the data as violin or dot plots.

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Representative Results

MCF10A cells, upon exposure to PAF treatment, form acinar structures with very distinct phenotypes. α6-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 α6-integrin in cancer metastasis. α6-integrin is present as a dimer with either β1- or β4-integrin. The α6β4 subunit has been found to play a significant role in forming hemidesmosomes in epithelial cells26. The downregulation of this integrin has been found in the prostate and some instances of breast cancers, wherein loss of α6β4-integrin was found in ductal carcinoma of the breast (grade III). The loss phenotype is seen in the cells that metastasize to the parenchyma and pleural cavity27,28,29. GM130 is a cis-Golgi localized protein; Golgi bodies are apically localized in MCF10A acinar cultures16,30. PAF treatment led to the mislocalization of GM130, suggesting apical polarity disruption (Figure 2B). Vimentin is an intermediate filament that is involved in cell migration; it is upregulated when epithelial cells undergo epithelial to mesenchymal transition (EMT)31. PAF treatment of MCF10A acinar cultures led to elevated vimentin levels as observed by the staining intensity (Figure 2C), suggesting EMT.

Figure 2
Figure 2: PAF disrupts 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 α6-integrin (green) and nuclear stain (blue) (A), (B) GM130 (green), a marker for apical polarity, and (C) Vimentin (red), an EMT marker. The centermost stack of the respective acini has been shown. The representative data is for 40-50 acini from three biologically independent experiments. Scale bar = 20 µm. Please click here to view a larger version of this figure.

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Discussion

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 system33,34,35,36. However, the 2D culture model lacks a lot of important structural and functional parameters present in vivo. Drug screening studies in 2D cultures often show a significant response (cell death or target inhibition), even at lower concentrations, while failing to show any significant effect when admistered in mice37. A major reason for this is the varying drug availability and uptake in a 2D monolayer culture vs. 3D culture38.

The concept of 3D cultures has emerged as a powerful tool for studying the progression of cancer in the last decade. 3D cultures involve growing cells on an extracellular matrix, resulting in the formation of structures that resemble functional units present in the tissue in vivo16. 3D cultures mimic the in vivo microenvironment to a large extent and thus overcome the limitations of 2D cultures39. Moreover, since the cells grown in 3D are of human origin and the system is more amenable to studying early events, it is simpler and more elegant than the rodent models.

Using this platform, a model to study the transformation process following exposure to a phospholipid mediator such as PAF is demonstrated. Studies from our lab have identified that PAF treatment can transform the non-tumorigenic breast epithelial cell line, MCF10A12. This method is optimized to continuously expose the cells to PAF, similar to the in vivo scenario, where PAF is present in the microenvironment.

MCF10A cells, when grown in 2D, have to be passaged every 4 days. If they are not maintained as per the protocol, they behave very differently, which is visible only when grown on ECM16. The passage number of the cells must be kept as low as possible. This method also has limitations due to the variation in protein concentration in different batches of lrECM. This can be overcome by testing the different batches by simply plating cells on a lrECM bed and observing the size distribution of the acini. If there is no large variability in size, the lrECM batch can be considered appropriate for experiments.

Assessment of the transformation is generally done by immunofluorescence, which is demonstrated in the video. While performing immunofluorescence, the steps involving incubation at 4 °C are critical; pipetting out the solution when the lrECM layer is in a liquid state makes the layer uneven, leading to a loss of acinar structures. One of the ways to tackle the unevenness problem is to store the slides at 4 °C on an even platform overnight or change the magnification at which imaging is done. To avoid pipetting out the acinar structures, carefully remove the chamber from the 4 °C storage and remove three-quarters of the volume added initially, then take out the rest during the subsequent washes.

Another major problem faced is the high background due to lrECM. To overcome this problem one can use a 2% secondary blocking antibody instead of 1%. However, increasing secondary blocking does not serve the purpose of staining apical markers and E-cadherin. In such cases, it is advisable to subject the chamber cover glass after Day 20 to PBS-EDTA for 15 min at 4 °C, and then fix the acini following the same protocol as shown in the video. PBS-EDTA partially dissolves the lrECM, thus reducing the background. However, if the incubation time is exceeded, this treatment can result in a loss of structures while pipetting out the solution. Hence, utmost care has to be taken while carrying out this procedure. It has also been observed that storing the slides for longer periods can lead to an increase in background signal. Therefore, imaging must be done as soon as possible. It is advisable to image a minimum of 15 acini per experiment to determine a change in phenotype.

3D acinar cultures have certain limitations, such as difficulty in tracking single cells during live-cell imaging. Certain studies, such as effects on cell cycle, need to be carried out using live-cell imaging to maintain spatial and temporal information. However, due to constant changes in structure, it is difficult to track such parameters in 3D acinar cultures using regular confocal microscopes. This warrants using advanced microscopic techniques such as super-resolution microscopy or Airyscan microscopy. It would also require dislodging of the acinar structures for certain transformation assays such as wound healing, anchorage-independent growth, and in vivo tumorigenicity assays. This dislodging would result in the loss of important spatial and temporal information. Protein lysates collected from the cultures are often diluted due to the presence of IrECM, posing issues with the loading of sufficient amount of lysates. However, this can be overcome to a greater extent by dislodging the acini structures and collecting them before lysis.

Here, a model system has been demonstrated to study phospholipid mediator/immune-associated factor-induced transformation. This model can elucidate genetic and/or epigenetic pathway(s) that may get de-regulated, leading to drastic changes in morphology. This method can also be used to screen potential therapeutics. Such drug screening studies will provide better success than screenings carried out in 2D cultures38. A major highlight of this method is the adaptiveness as per the requirement of the study. The treatment regimen and analysis methods can be modified as needed. Moreover, this method can provide insights into the molecular signaling affected upon treatment40. The technique will also help to predict the possible occurrence of drug resistance. Furthermore, it will also help in elucidating the mechanism involved in drug resistance and identify plausible targets for overcoming the same. Studies have established possibilities of co-culturing multiple cell types in 3D, which can further be modified to accommodate the treatment regimen41. Such opportunities will provide greater insights into the characteristics and molecular changes that happen in vivo during the initiation and progression of breast cancer.

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Disclosures

The authors declare no potential conflicts of interest.

Acknowledgments

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).

Materials

Name Company Catalog Number Comments
0.05% Trypsin EDTA Invitrogen 25300062
16% paraformaldehyde Alfa Aesar AA433689M
Anti Mouse Alexa Flour 488 Invitrogen A11029
Anti Rabbit Alexa Flour 488 Invitrogen A-11008
BSA Sigma A7030
Chamber Coverglass Nunc 155409
Cholera Toxin Sigma C8052-1MG 1 mg/mL in dH2O
Confocal Microscope Leica Leica SP8
DMEM Gibco 11965126
EDTA Sigma E6758
EGF Sigma E9644-0.2MG 100 mg/mL in dH2O
F(ab’)2 fragment of antibody raised in goat against mouse antigen Jackson Immunoresearch 115-006-006
GM130 antibody Abcam ab52649
Goat Serum Abcam ab7481
Hoechst Invitrogen 33258
Horse Serum Gibco 16050122
Hydrocortisone Sigma H0888 1 mg/mL in ethanol
Image Processing Software ImageJ
Insulin Sigma I1882 10 mg/mL stock dH2O
lrECM (Matrigel) Corning 356231
Mounting reagent (Slow fade Gold Anti-fade) Invitrogen S36937
Nuclear Stain  (Hoechst) Invitrogen 33258
PAF Cayman Chemicals 91575-58-5 Methylcarbamyl PAF C-16, procured as a 10 mg/mL in ethanol
Penicillin-Streptomycin Lonza 17-602E
Sodium Azide Sigma S2002
Tris Base Sigma B9754
Triton X-100 Sigma T8787
Tween 20 Sigma P9416
Vimentin antibody Abcam ab92547
α6-integrin antibody Millipore MAB1378

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Phospholipid Mediator Transformation Three-dimensional Cultures Cancer Microenvironment Biomarkers Drug Targets Cellular Phenotype Molecular Signaling Immune System Early Detection Breast Cancer Tumorigenesis Morphogenesis Gene Perturbation Trypsinize Cells PBS Trypsin-EDTA LrECM Bed Chambered Cover Glass Slide Carbon Dioxide Incubator Re-suspension Medium
Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures
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Kuttanamkuzhi, A., Anandi, L.,… More

Kuttanamkuzhi, A., Anandi, L., Chakravarty, V., Lahiri, M. Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures. J. Vis. Exp. (185), e64146, doi:10.3791/64146 (2022).

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