In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Summary

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Abstract

Tumorigenesis is a multi-step process in which cells acquire capabilities that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.

A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.

Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

Introduction

Multiple successive events are required for neoplasm development. In 2011, Hanahan and Weinberg described 10 capabilities that enable transformed cells’ growth, survival, and dissemination: the so-called “Hallmarks of Cancer”¹. The methodology described here compiles three different tools to evaluate in vitro cellular transformation by focusing on some of the tumoral cells’ distinctive features. These techniques assess the cell proliferation rate, the behavior of cells when cultured in 3D and their capacity to form colonies with anchorage independence.

Cell models are crucial to test hypothesis in vitro. Different approaches have been developed to generate experimental models of cellular transformation for the study of cancer^2,3,4. Since breast cancer is the most common cancer among women worldwide and is responsible for approximately 15% of cancer deaths among women⁵, providing suitable cellular models of mammary epithelial cells is of utmost importance for further investigation. In this article, we have illustrated the potential of three techniques to evaluate cellular transformation using an experimental model of Breast Primary Epithelial Cells (BPECs) transformation initially described by Ince and colleagues in 2007⁶ and later implemented in our laboratory⁷. This experimental model is based on the sequential alteration of three targeted genes (SV40 Large T and small t antigens herein referred to as Ttag, hTERT, and HRAS) to the genome of non-transformed BPECs. Moreover, the method used for BPECs derivation favors the maintenance of mammary epithelial cells with luminal or myoepithelial markers, resulting in a heterogeneous cell culture that retains some of the mammary gland physiological traits.

In the mammary gland, luminal mammary epithelial cells, which are responsible for milk production, are located near the lumen, whereas myoepithelial cells are disposed around luminal cells and take care of contraction movements leading the milk to the nipple. The loss of proper organization between these cell lineages is a feature of tumoral transformation⁸ that can be assessed in vitro after immunofluorescent detection of distinctive lineage markers in 3D cell cultures. Another major characteristic of tumoral cells is their capacity to grow without attachment to other cells and to the extracellular matrix¹. When healthy cells are forced to grow in suspension, mechanisms such as anoikis ‒ a type of cell death induced in response to detachment from the extracellular matrix ‒ are activated⁹. The evasion of cell death is one of the distinctive hallmarks of cancer and thus, transformed cells are capable to inactivate anoikis and survive in an anchor-independent manner. This capacity can be evaluated in vitro with the anchorage-independent assay using soft agar. Furthermore, an inherent feature of tumoral tissues is their sustained proliferative signaling capacity, which can be easily monitored under in vitro conditions by measuring the increase of cell number along time, not only in suspension assays but also by monitoring the growth rate of monolayer adherent cultures.

Despite the best model to test tumorigenic potential is the inoculation of tumoral cells in murine models and evaluation of tumor development in situ, it is important to minimize the number of animals employed in experimental procedures as much as possible. Therefore, having suitable tests to assess transformation in vitro is a top priority. Here, we provide a set of tools to evaluate the tumorigenic potential of partially and fully transformed breast epithelial cells that can be easily implemented in most of the laboratories that work with cellular transformation models.

Protocol

Human samples used in the following experiments were obtained from reduction mammoplasties carried out at Clínica Pilar Sant Jordi (Barcelona) under standard procedure consent. All procedures are performed in a Class II Biological Safety Cabinet unless otherwise stated.

1. In vitro culture of human mammary epithelial cells and growth curve plot build-up

1. In vitro culture of breast primary epithelial cells (BPECs): cell passaging
   NOTE: For BPEC derivation and cell culture follow instructions described by Ince et al., 2007⁶.
   1. Medium preparation.
      1. Supplement WIT basal defined medium with P or T supplements, provided by the manufacturer, depending on whether primary or transformed BPECs are cultured.
      2. Add cholera toxin to the supplemented WIT medium to a final concentration of 100 ng/mL for primary or 25 ng/mL for transformed BPECs.
         CAUTION: Cholera toxin is fatal if swallowed. Use personal protective equipment. Avoid its release to the environment.
   2. Cell culture maintenance and passaging.
      NOTE: For the following steps keep in mind that cells are growing in a T25 flask. Nonetheless, volumes can be adapted to other cell culture formats maintaining the proportionality in terms of surface area.
      1. Check on the cell confluency every day. When the culture is 90% confluent, perform cell passaging.
      2. Acquire 1x PBS, 3x trypsin, medium and a 15 mL conical tube containing 2 mL of Fetal Bovine Serum (FBS) for each flask.
      3. Remove the medium from the flask and keep it in the 15 mL conical tube containing FBS.
      4. Rinse cells with 1x PBS.
      5. Detach cells from the surface by adding 1 mL of 3x trypsin. Incubate for 5 min at 37 ˚C.
      6. Check if cells have been detached. Apply vigorous shaking if cells are not completely detached.
      7. Inactivate trypsin by adding the reserved medium supplemented with FBS.
      8. Harvest the cellular suspension and place it in the 15 mL conical tube.
      9. Centrifuge at 500 x g for 5 min, eliminate the supernatant, and resuspend the pelleted cells by flicking the bottom of the tube with a finger.
      10. Add 1–2 mL of fresh media to the pellet and measure the cell concentration using an automatic cell counter or a hemocytometer. This data will later be used to calculate the population doublings and draw the growth curve. Seed 12,000 cells/cm² (e.g., 300,000 cells for a T25 flask) in modified cell culture surface flasks (see Table of Materials).
          NOTE: Dilute the cell suspension solution if the concentration is too high to ensure proper quantification.
      11. Add medium to a final volume of 5 mL and incubate the cells at 37 ˚C and 5% CO₂ atmosphere.
      12. Replace the cell culture medium every 48 h.
2. Population doubling calculation and data visualization
   1. Using data of the cell count obtained in the step 1.1.2.10, apply the following formula to obtain the accumulated population doubling (PD) values:
      
      Where, PD_i denotes the number of population doublings achieved by the cells until the previous subculture (it refers to the PD accumulated on the previous subculture), N_h is the number of harvested cells, and N_s is the number of seeded cells.
   2. Represent data for a specific interval of time using an XY graph where the number of days in culture (x-axis) and the accumulated PD (y-axis) are represented.
   3. Get the best-fit line and the fitting equation:
      
      NOTE: An increased slope (b) means an increased proliferation rate.

2. Three-dimensional (3D) culture in basement membrane matrix and immunofluorescent protein detection

1. 3D culture in basement membrane matrix
   NOTE: This protocol has been adapted from Debnath et al., 2003¹⁰ and is optimized for 24 well plates (see Table of Materials).
   1. Prepare the material a day before the experiment: pre-chill basement membrane matrix overnight at 4 ˚C and let pipette tips, microcentrifuge tubes, and well plates cool in the freezer.
      NOTE: The matrix must be kept at -20 ˚C for long-term storage. Make aliquots to avoid multiple freeze-thaw cycles.
   2. On the day of the experiment, place pre-cooled material on ice.
   3. Rinse wells with cold sterile 1x PBS in order to reduce surface tension.
   4. Cover the bottom of each well with 100 µL of basement membrane matrix.
      NOTE: Dispense the matrix slowly and spread it throughout the well; it is crucial to avoid bubble formation in the bottom layer to avert monolayer cell culture growth.
   5. Place the plate in the incubator, at 37 ˚C, to let the matrix layer solidify.
      NOTE: It usually takes about 20 min to solidify.
   6. Meanwhile, trypsinize cells as explained previously in step 1.1. Centrifuge the cells at 500 x g for 5 min and resuspend in medium. Prepare a 400,000 cells/mL suspension and gently disaggregate any cell clump by pipetting.
   7. Prepare the medium with 8% basement membrane matrix and mix 1:1 (v/v) with cellular suspension to obtain a 200,000 cells/mL solution in 4% matrix.
      NOTE: Calculate the amount of medium needed to avoid matrix unnecessary waste.
   8. Place 500 µL of cell suspension in matrix solution on top of the already solidified matrix layer to seed a total amount of 100,000 cells in medium with 4% basement membrane matrix.
   9. Incubate cells at 37 ˚C for a few minutes and then, add 500 µL of the medium with 4% basement membrane matrix. Incubate the cells at 37 ˚C in an incubator with 5% CO₂ for 14 days. Seeded cells will group and proliferate to originate the acini-like structures.
      NOTE: Cell motility and aggregation can be monitored by time-lapse during the 3D formation process. Use image analysis software (e.g., Fiji/ImageJ or Imaris) to evaluate these events. The number and size of the acini depend on the aggregation process and the proliferation rate and might vary between cell types. Adjust basement membrane matrix concentration and seeded cells to obtain desired 3D structures.
   10. Add 500 µL of the medium with 4% basement membrane matrix 2–3 times per week.
       NOTE: Avoid disturbance of the layers carrying the plate gently during manipulation.
   11. If desired, the number and size of acini can be measured during the culture period. To do so, take random pictures at different times after seeding using a phase contrast or DIC inverted microscope. Use image analysis software to measure the diameter of 100–200 3D structures.
2. Immunostaining
   NOTE: Sterile conditions are not required during this part of the protocol.
   1. Remove the culture medium.
   2. Tear the basement membrane matrix using a p200 pipette tip with the end cut off. Place ~50 µL of disaggregated matrix on top of a glass slide and smear it in an area of 1–2 cm².
   3. Let the sample dry completely at room temperature or use a heating plate at 37 ˚C to accelerate the process. Fix samples with methanol:acetone (1:1, v/v) at -20 ˚C for 30 min.
      NOTE: Fluorescent signal of previous markers, such as that of fluorescent proteins expressed by the cells, will be erased.
      CAUTION: Methanol is flammable, toxic if inhaled, swallowed or in case it comes in contact with skin. Wear personal protective equipment and work inside a fume hood.
   4. Discard the fixation solution and remove the excess, if any, by reclining the slide on filter paper.
      NOTE: The protocol can be paused here. Once dry, slides can be stored at -20 ˚C for several months.
   5. Block samples epitopes with 5% normal goat serum and 0.1% triton-X-100 in 1x PBS (blocking solution) for 2 h at room temperature.
   6. Meanwhile, prepare antibodies working solutions by diluting primary or secondary antibodies at the desired concentration in blocking solution.
      NOTE: Antibody concentration must be accurately adjusted depending on the cell type and the antibody reference. As a guide, to identify cells from luminal and myoepithelial lineages in BPEC, primary anti-Cytokeratin 14 and anti-Claudin-IV antibodies (see Table of Materials) can be used. The recommended working solution concentration is 1:100 for these primary antibodies and 1:500 for anti-Mouse and anti-Rabbit secondary antibodies (see Table of Materials).
   7. Add 30 µL of primary antibodies working solution and cover it with a strip of laboratory wrapping film to avoid evaporation. Incubate overnight at 4 ˚C in a humid chamber.
   8. Wash three times with 1x PBS for 1 h each.
   9. Repeat step 2.2.7 for secondary antibodies. Incubation should be performed in darkness.
   10. Wash with 1x PBS for 2 h.
       NOTE: Adjust antibodies concentration, incubation time, and washing hardness to improve the signal/noise ratio for specific samples.
   11. Remove the remaining PBS and, once dry, counterstain with DAPI at 0.25 µg/mL diluted in antifade mounting medium. Cover slides with a coverslip by letting it settle without applying pressure. Seal with nail polish.
       NOTE: Samples can be stored at 4 ˚C for several weeks. For long-term storage, keep them at -20 ˚C.
   12. Analyze fluorescent signal distribution for each acinus using a confocal microscope.
       NOTE: Confocal microscope configuration must be accurately determined depending on the equipment used and the antibodies applied to the sample. As a guide, with the equipment and reagents detailed in the Table of Materials, use a 40x objective and the following laser and detector settings: for DAPI use excitation with a 405 laser (3%–5%), detection with a PMT detector (800V, Offset: -9) and a spectral band from 410 nm to 500 nm; for A488 (Claudin-IV) use excitation with a 488 laser (7%–10%), detection with a PMT detector (800V, Offset: -20) and a spectral band from 490 nm to 550 nm; and for Cy3 (Cytokeratin 14) use excitation with a 555 laser (2%–10%), detection with a PMT detector (800V, Offset: -35) and a spectral band from 560 nm to 600 nm.

3. Anchorage-independent assay, MTT staining and automatic colony quantification

1. Anchorage-independent assay: agar and cellular suspension plating
   NOTE: Protocol has been adapted from Borowicz et al., 2014¹¹ to perform experiments in BPECs.
   1. Prepare a 1.2% agar solution diluted in ultrapure water in a sterile bottle. Autoclave the solution and maintain it at 42 ˚C during the experiment. The agar solution can be stored at 4 ˚C; when required, heat the agar solution until it is liquid again.
      CAUTION: Use heat-resistant gloves to avoid burn after autoclave.
      NOTE: From now on, sterile conditions must be maintained.
   2. Prepare a 0.6% agar solution by mixing 1:1 (v/v) complete pre-warmed medium with 1.2% agar solution. Maintain at 42 ˚C to avoid premature solidification.
      NOTE: Medium can be previously double supplemented to obtain a fully supplemented 0.6% agar + medium solution once mixed.
   3. Cover the bottom of a 35 mm well with 1.5 mL of 0.6% agar in medium solution and let it solidify at room temperature. Make sure that the bottom of the plate is completely covered before agar solidification, otherwise, cells may adhere to the plate and grow in monolayer.
      NOTE: Adherent and non-adherent surface plates can be used.
   4. Meanwhile, trypsinize cells and, once centrifuged and resuspended in medium, prepare a 50,000 cells/ml solution and gently disaggregate any cell clump by pipetting repeatedly.
   5. Prepare a 0.3% agar + cell suspension in the medium at a final concentration of 25,000 cells/mL.
      NOTE: Optimal cell concentration may differ among cell types. Try different concentrations until individualized colonies are formed.
      1. Place a 40 µm strainer filter on top of a 50 mL sterile tube and filter the 50,000 cells/mL solution letting it drop into the bottom of the tube.
      2. Remove the filter from the 50 mL sterile tube, tilt the cell-containing tube to a 45˚ angle and drop the same volume of 0.6% agar + medium solution pouring it through the internal wall of the tube. This will allow the agar solution to cool down just enough to not damage the cells and avoid its premature solidification.
   6. Homogenize the mixture and deposit 1 mL of 0.3% agar + cell suspension in the medium (containing 25,000 cells) on top of the previously solidified bottom agar layer.
   7. Visualize seeded cells using an inverted microscope to make sure that the cells are individualized. Otherwise, the experiment should be repeated.
   8. Wait until the agar layer is completely solidified, then carefully add 1 mL of the fresh medium on top without disturbing the delicate agar layers beneath.
   9. Incubate the cells at 37 ˚C and 5% CO₂ in an incubator for 3 weeks.
      NOTE: The time required for colony formation can vary among different cell types, but usually 3 weeks are sufficient.
   10. Change medium twice per week. To do so, gently tilt the plate toward you, aspirate medium in the lower corner, and add 1 mL of fresh medium.
       NOTE: Avoid touching the agar layers as they easily detach from the plate.
2. MTT staining
   1. Prepare Thiazolyl Blue Tetrazolium Bromide (MTT) stock solution at 6 mg/mL in ultrapure water in a sterile bottle and filter solution using 0.2 µm filters. This MTT solution can be stored for up to 6 months at -20 ˚C.
      CAUTION: MTT may cause irritation and is suspected of causing genetic defects. Use safety glasses, gloves, and a respiratory filter.
      NOTE: Avoid repeated freeze-thaw cycles.
   2. Prepare a working solution of MTT at 1 mg/mL by diluting the stock solution with sterile ultrapure water.
   3. Once the colony formation period has concluded, remove the medium from the plate and add 1 mL of 1 mg/mL MTT to each well.
   4. Incubate for 24 h in the incubator. Remove the MTT solution by aspirating it gently. Plates can be stored at 4 ˚C for several weeks.
      NOTE: Avoid light exposure to prevent non-specific crystal formation.
3. Colony quantification
   1. Obtain images of each plate using an inverted microscope. Adjust the magnification in order to acquire the maximum field of view with fewer images and still being able to detect small colonies (usually 4x or 10x objectives).
      NOTE: Make sure that images present a homogeneous background. Neither phase-contrast nor differential interference contrast is required since non-stained colonies will not be quantified.
   2. Upload images to ImageJ/Fiji software¹² to count the number of colonies and the area of each MTT-positive colony.
      NOTE: A script for automatic quantification is provided as a Supplementary File. To execute the code, paste it to the macro editor (Plugins | New | Macro) and follow the instructions.
      1. Obtain a binary mask through thresholding the original image (Image | Adjust | Threshold) to obtain well-delimited colonies (Figure 1).
         NOTE: 8- or 16-bit images are usually required to perform this step. The “Minimum threshold” method is recommended.
      2. Run the Extended Particle Analyzer from Biovoxxel plugin¹³ (Plugins | BioVoxxel | Extended Particle Analyzer) to identify MTT positive colonies (Figure 2). Initial guiding conditions: Size (µm²) = 250–Infinity; Solidity = 0.75–1.00.
   3. Estimate the average diameter (D) from each colony area value (A) according to the formula:
      
   4. Filter results by excluding low proliferative colonies (e.g., low diameter).
      1. Choose a minimum number of divisions per week (m) to be considered (e.g., 1).
      2. Estimate the radius of a colony (R) with n cells according to the following formula:
         
         Where, r is the average radius of individual cells in suspension, n is the number of cells forming a colony that suffered m divisions each week during w weeks in culture. In exponential growth: n = 2^(m*w). ρ is the Packaging efficiency. Note that, in random movement, packaging efficiency is ~0.64 and the densest possible packing fraction for identical spheres is 0.74¹⁴.
      3. Discard all colonies that present a diameter lower than 2R as their cells have not achieved the minimum number of divisions considered in step 3.3.4.1.

Representative Results

An experimental model of cellular transformation with the introduction of three genetic elements in BPECs was chosen to generate representative results of oncogenic transformation^6,7 (Figure 3). Non-transformed BPECs (N) were derived from disease-free breast tissue as described by Ince and colleagues⁶ and cultured following the protocol indicated here. After overcoming STASIS (stress or aberrant signaling induced-senescence, a phenomenon typically observed in mammary epithelial cells in vitro that is overcome around 4 weeks after cell culture establishment), cells were consecutively transduced with the lentiviral particles pRRL-CMV-Ttag-IRES-eGFP and pRRL-CMV-TERT-IRES-CherryFP to obtain partially transformed cells (double transduced; D). Expression of the viral Ttag inhibits p53 and retinoblastoma function, and ectopic expression of the hTERT gene compensates for proliferation-dependent telomere length attrition. After fluorescent selection by cell sorting, cells were transduced with pLenti-CMV/TORasV12-Puro, which confers a sustained mitogenic signal, and then growth in the presence of antibiotics to select transduced cells (triple transduced; T), which were fully transformed according to Ince and colleagues⁶.

As shown in Figure 4, a rise in the slope of the regression line (parameter b in the equation 1.2.3) is observed with the increasing number of genetic modifications introduced in BPEC (N: 0.47, D: 0.93, T: 1.13). For a specific interval of time, partially (D) and fully transformed (T) cells achieved a higher number of population doublings compared to the non-transformed cells (N), thus the cell division rate was increased with the transformation process. The same result can also be expressed as the time needed to duplicate the population of cells (t_d), which can be obtained after replacing y by 1 (PD) in the equation y = bx, thus t_d = 1/b. Contrary to the slope, t_d decreases with transformation. While non-transformed cells needed more than 2 days to duplicate their population (N: t_d = 2.13 days), partially transformed cells did it in half the time (D: t_d = 1.08 days). The addition of HRAS under the regulation of a constitutive promoter led to an increased proliferation activity and cells needed less than 1 day to duplicate (T: t_d = 0.89 days) in agreement with the mitogenic activity of this oncogene.

While monolayer cell culture is a useful tool to study in vitro cell behavior, it is a strongly limited approach because it cannot reproduce most of the physiological conditions. Instead, the three-dimensional cell culture technique described here allows cells from different lineages to aggregate and move freely in a 3D environment forming acinar structures thanks to the cell-cell and cell-matrix junction creation (Figure 5A. Time-lapse). During the following 2 weeks, cells distribute according to their original tissue function and proliferate increasing the acini size (Figure 5B). The proper polarization of each acinus can be accurately assessed thanks to the combination of immunofluorescent detection of luminal (Claudin-IV) and myoepithelial (Cytokeratin 14) lineage markers with three-dimensional signal location by confocal microscopy (Figure 6A). While all the acini formed by non-transformed BPECs were properly organized (Claudin-IV positive cells surrounded by Cytokeratin 14 positive cells; Figure 6Ai), loss of polarization (Figure 6Aii) was observed in acini formed by partially and fully transformed BPECs (Figure 6B).

One of the main properties of a transformed cell is its ability to grow with the independence of contact with the basal lamina. This property was evaluated by growing the cells embedded on agar for 3 weeks, avoiding its anchorage to plate surface (Figure 7A). During the following 3 weeks, those cells with anchorage-independent growth capacity gave rise to colonies composed of multiple cells. As shown in Figure 8, 2 days after being seeded in agar, some cells already suffered 2–3 divisions. After 1 week, cells with death-like morphology can be observed indicating that the cells that are not capable of surviving under these conditions eventually died, most likely by anoikis. Nonetheless, some cells continue dividing and forming little colonies growing over the second and third weeks of culture.

After adding MTT to the culture, only those cells metabolically active, i.e., alive, are capable to cleave the tetrazolium ring of the MTT resulting in purple MTT formazan crystals 24 hours later (Figure 7B). However, these crystals are not only formed by colonies with live cells but also by single cells still alive after 3 weeks in agar (Figure 9). Since the purpose of this technique is to determine the ability of cells to proliferate in a substrate-independent manner, the size of the MTT-positive colonies needs to be evaluated. Measurement of colony diameter can be assessed by automatic image analysis (Figure 7C) so that small colonies or individualized cells can be filtered. As shown in Figure 10, gray dots correspond to those colonies that have suffered less than three divisions in 3 weeks thus measuring less than 65 µm of diameter. These events were included in the data visualization but excluded from the final quantification.

Overall, these results indicate that anchorage independence assay allows discriminating between different degrees of transformation (Figure 10). The number of colonies formed by non-transformed BPECs was negligible in comparison to those formed by partially and fully transformed BPECs (colony number: N = 3; D = 278; T = 243). Also, when the colony size was considered, differences between partially and fully transformed states became evident (colony median size: N = 70 µm; D = 83 µm; T = 114 µm). Taking the colony size into account provides more accurate information regarding the tumorigenic potential of studied cell lines and thus it is highly recommended to consider it.

Figure 1: Screenshot exemplifying image thresholding after MTT staining using Fiji software. Please click here to view a larger version of this figure.

Figure 2: Screenshot of the Extended Particle Analyzer from Biovoxxel plugin conditions and outcome in Fiji software. Please click here to view a larger version of this figure.

Figure 3: Schematic representation of the experimental model of cellular transformation. Please click here to view a larger version of this figure.

Figure 4: Proliferation rate in non-transformed, partially, and fully transformed BPECs. Best-fit lines and 95% confidence bands (dotted lines) of the linear regression are shown. ANCOVA for a linear regression model was applied for the comparison between conditions; * p-value < 0.0001. This figure has been adapted from Repullés et al. 2019⁷. Please click here to view a larger version of this figure.

Figure 5: Acini formation and growth after seeding BPECs in basement membrane matrix. (A) Representative images of a time-lapse for the initial times (from 0 to 7 hours) after seeding BPECs in basement membrane matrix. Scale bar = 100 µm. (B) Acini size over the 14 days of culture in basement membrane matrix for the non-transformed, partially, and fully transformed BPEC. Error bars indicate SEM. No statistical differences between conditions on day 14 (One-way ANOVA and Tukey correction; p-value > 0.05). This figure has been adapted from Repullés et al. 2019⁷. Please click here to view a larger version of this figure.

Figure 6: Acini polarization in non-transformed, partially (D), and fully transformed (T) BPECs after 3D culture in basement membrane matrix. (A) Representative images of a polarized and a non-polarized acinus after Cytokeratin 14 (K14, red) and Claudin-IV (Cl-IV, green) immunofluorescence. Polarized acini (i) were considered when Cytokeratin 14-positive cells surrounded Claudin-IV positive cells; otherwise, acini were considered non-polarized since Cytokeratin 14 and Claudin-IV positive cells were located both in the middle and peripheral locations (ii). Scale bar = 25 µm. (B) Percentage of acini polarized. A minimum number of 10 acini were analyzed for each condition. Please click here to view a larger version of this figure.

Figure 7: Schematic representation of the different steps used for the anchorage-independent growth assay in BPECs. Cells were cultured for 3 weeks in soft agar (A) and then MTT staining was applied (B). Scale bar = 5 mm. (C) MTT positive colonies were quantified using Fiji software. Different steps on image processing are highlighted. Scale bar = 200 µm. Please click here to view a larger version of this figure.

Figure 8: Evolution of cell growth for 3 weeks after seeding individualized cells in 0.3% agar. Images are acquired from a partially transformed BPEC culture. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 9: Representative images of the MTT staining and quantification. Examples with an MTT-positive colony (row 1), two MTT-negative colonies (row 2), and single cells positive or negative for MTT staining (row 3) are shown. A indicates the area of the MTT positive colony or cell. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 10: Colony quantification after anchorage-independent assay in non-transformed (N), partially (D), and fully transformed (T) BPECs. (A) Number and diameter of the colonies obtained for the different conditions. Each dot corresponds to one colony. Soft gray dots represent MTT positive colonies, which were excluded in the results because their diameter was lower than 65 µm (minimum size considered after applying the equation number 4 and taking into consideration at least one division per week). The red line indicates the median colony diameter for each group. Two independent replicas were performed for each condition. Different letters (a, b, c) indicate statistically significant differences for the number of colonies (Fisher’s exact test; p-value < 0.05) and for their median diameter (Kruskal-Wallis test with multiple comparisons correction; p-value < 0.05). This graph has been adapted from Repullés et al. 2019⁷. (B) Representative images of entire wells after MTT staining. Scale bar = 5 mm; Inset scale bar = 2.5 mm. Please click here to view a larger version of this figure.

Supplemental File. Please click here to download this file.

Discussion

The experimental protocols described in this paper provide useful tools to assess the oncogenic transformation of in vitro cultured cells. Each technique evaluates specific aspects of the transformation process, and thus, special attention must be paid when drawing conclusions from a single analysis. Growth curves build-up is an approach that demands information already available when culturing cells for other purposes. That makes this technique cheaper and easier to apply compared to other cell proliferation assays. However, to obtain valid results, special attention must be paid when counting and seeding cells every time they are subcultured. An increased growth rate is indicative of cellular transformation¹, but it should not be used alone because other exogenous factors, such as the addition/removal of antibiotic and antifungal substances or the variation in culture conditions (e.g., temperature, CO₂) can also affect cellular division. It is also important to consider that transduction or the treatment of cells with drugs may affect their growth rate in the upcoming days or weeks and thus give a distorted view of long-term proliferation capacity. In this regard, appropriate controls must be performed.

The 3D culture in basement membrane matrix allows the assessment of cell distribution within a whole functional entity, the acinus. An altered organization is indicative of an intercellular communication impairment that could lead to a loss of function, a characteristic of tumoral tissues. The fact that some acini present non-polarized organization indicates that some cells have initiated the transformation process. Regarding technical issues, it is important to accurately determine the optimal concentration of seeded cells and the concentration of the matrix. These two parameters can influence the number and size of the resulting acini and this could interfere with their organizational capacity. Also, manipulation of basement membrane matrix requires a certain degree of experience as it must be gently handled. Despite being a laborious technique, the growth of cells in three dimensions resembles the physiological context of these cells and allows the evaluation not only of the distribution of breast lineage markers^7,15 but also of other structures that provide information about tumoral features, such as the disruption of the basement membrane¹⁶. 3D cell cultures represent the future in cell culture research. In fact, 3D growths can give rise to more physiological and interesting findings while taking into account microenvironment elements and providing us with a lot of different studies as well as therapeutical targets' identification and evaluation, cell to cell interactions, or stem cell investigations.

Anchorage-independent growth of adherent cells is an unequivocal treat of the transformation process. While some of the partially and fully transformed BPECs were still able to give rise to a structured acinus, they manifest their ability to form colonies when forced to grow individually in suspension. At the technical level, the anchorage assay is also complex since small variations during agar manipulation (e.g., high temperature) or during disaggregation of cells after trypsinization can notoriously affect cell colony formation. However, the material required is cheaper than basement membrane matrix used for 3D cell cultures making the assessment of the anchorage-independent growth more affordable. Also, prior to MTT addition, single colonies can be picked and allowed to grow out of the agar in plates with an adherent surface. These colonies may result in clonal cell lines that may continue growing in suspension or adhere again. DNA, RNA, and/or protein can be extracted from these cell cultures for further analyses.

There is a general limitation regarding the methods described here: they are very time consuming, and it takes several weeks to obtain the results. However, since each test assesses a specific tumor characteristic, conclusions made considering the whole set of results are very sound. Therefore, all the three tests together are powerful indicators of cellular transformation.

Materials

Name	Company	Catalog Number	Comments
1 ml Serological Pipettes	Labclinics	PLC91001
1.5 ml Eppendorfs	Thermo Fisher Scientific	3451	Dark eppendorfs are preferred for MTT long-term storage
10 μl Pipette tips w/o filter	Biologix	20-0010
100 ml glass bottle			With cap, autoclavable
1000 μl Pipette tips w/ filter	Labclinics	LAB1000ULFNL
1000 μl Pipette tips w/o filter	Biologix	20-1000
15 ml Conical tubes	VWR	525-0400
2 ml Serological Pipettes	Labclinics	PLC91002
200 μl Pipette tips w/ filter	Labclinics	FTR200-96
5 ml Serological Pipettes	Labclinics	PLC91005
50 ml Conical Tubes	VWR	525-0304
Acetone	PanReac AppliChem	211007	Used for 3D structure fixation prior to immunofluorescent labelling
Agar	Sigma-Aldrich	A1296	Used for anchorage assay
Anti-Claudin 4 antibody	Abcam	15104, RRID:AB_301650	Working dilution 1:100, host: rabbit
Anti-Cytokeratin 14 [RCK107] antibody	Abcam	9220, RRID:AB_307087	Working dilution 1:100, host: mouse
Anti-mouse Cyanine Cy3 antibody	Jackson ImmunoResearch Inc.	115-165-146, RRID:AB_2338690	Working dilution 1:500, host: goat
Anti-rabbit Alexa Fluor 488 antibody	Thermo Fisher Scientific	A-11034, RRID:AB_2576217	Working dilution 1:500, host: goat
Autoclave
BioVoxxel Toolbox		RRID:SCR_015825
Cell culture 24-well Plate	Labclinics	PLC30024	Used for 3D cultures in Matrigel. Flat Bottom
Cell culture 6-well Plate	Labclinics	PLC30006	Used for anchorage assay
Cell incubator (37 ºC and 5 % CO2)
Cell Strainers	Fisherbrand	11587522	Mesh size: 40 μm
CellSense software	Olympus		Used to image acquisition
Centrifuge
Cholera Toxin from Vibrio cholerae	Sigma-Aldrich	C8052	Used to supplement cell culture medium
Class II Biological Safety Cabinet	Herasafe	HAEREUS HS12
Confocal inverted Microscope	Leica	TCS SP5
Cover glasses	Witeg Labortechnik GmbH	4600122	22 X 22 mm, thickness 0.13 - 0.17 mm
DAPI			2-(4-amidinophenyl)-1H -indole-6-carboxamidine
Fetal Bovine Serum	Biowest	S1810	Used to inactivate trypsine action
Fiji software (ImageJ)	National Institutes of Health	RRID:SCR_002285	Free download, no license needed
Glass Pasteur Pipettes
Glass slides	Fisherbrand	11844782
Goat Serum	Biowest	S2000	Used for immunofluorescence of 3D structures
Heat-Resistant Gloves			Used for agar manipulation after autoclave
Heater bath (37 ºC)			Used to temper solutions prior to cell subculture
Heater bath (42 ºC)			Used to keep agar warm
Heating plate			Used for Matrigel dehydration
Humid chamber			Used for the incubation of antibodies during immunofluorescence
Ice			Used during Matrigel manipulation
Ice-box
Inverted Optic Microscope	Olympus	IX71
Matrigel Matrix	Becton Dickinson	354234	Store at -20 ºC and keep cold when in use. Referred to as basement membrane matrix
Methanol	PanReac AppliChem	131091	Used for 3D structure fixation prior to immunofluorescent labelling
Micropipette			p1000, p200 and p10
Microsoft Office Excel	Microsoft	RRID:SCR_016137	Used to calculate population doubling and to obtain growth rate equation
MilliQ water			Referred to as ultrapure water
Nail Polish			Used to seal samples after mounting
Parafilm M	Bemis	PM-999	Used to cover antibody solution during incubation
PBS pH 7.4 (w/o calcium & magnesium)	Gibco	10010-056	Sterile. Used for cell subculture
PBS tablets	Sigma-Aldrich	P4417	Dilute in milliQ water. No sterility required. Used for immunofluorescence
Pipette Aid
Primaria T25 flasks	Corning	353808	Used for BPEC culture
Scepter Automated Cell Counter	Millipore	PHCC20060	Alternatively, use an haemocytometer
Scissors			Used to cut pipette tips and parafilm
Sterile filters 0.22 μm	Millipore	SLGP033RS	Used to filter MTT solution
Thiazolyl Blue Tetrazolium Bromide (MTT)	Sigma-Aldrich	M2128	Store at -20 ºC
Triton X-100	Sigma-Aldrich	T8787	Used for immunofluorescence of 3D structures
Trypsin-EDTA 10X	Biowest	X0930	Dilute in PBS to obtain 3X solution
Vectashield Antifade Mounting Medium	Vector Laboratories	H-1000
WIT-P-NC Culture Medium	Stemgent	00-0051	Used for primary BPEC culture
WIT-T Culture Medium	Stemgent	00-0047	Used for transformed BPEC culture