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.
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.
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 cancer2,3,4. 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.
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
2. Three-dimensional (3D) culture in basement membrane matrix and immunofluorescent protein detection
3. Anchorage-independent assay, MTT staining and automatic colony quantification
An experimental model of cellular transformation with the introduction of three genetic elements in BPECs was chosen to generate representative results of oncogenic transformation6,7 (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 (td), which can be obtained after replacing y by 1 (PD) in the equation y = bx, thus td = 1/b. Contrary to the slope, td decreases with transformation. While non-transformed cells needed more than 2 days to duplicate their population (N: td = 2.13 days), partially transformed cells did it in half the time (D: td = 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: td = 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.
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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 markers7,15 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.
The authors have nothing to disclose.
The AG laboratory is funded by the Spanish Nuclear Safety Council. T.A. and A.G. are members of a research group recognized by Generalitat de Catalunya (2017-SGR-503). MT holds a contract funded by the Scientific Foundation Asociación Española Contra el Cáncer [AECC-INVES19022TERR]. G.F. contract is funded by a grant from Cellex Foundation.
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 |