Name: Mammosphere Assay Reveals Api5-Induced Stemness in Non-Tumorigenic Breast Epithelial Cell Lines (Video) | JoVE Journal
Uploaded: 2026-02-24T13:00:00.000+00:00
Duration: 8 min 5 s
Description: Learn the mammosphere assay protocol to evaluate cancer stem cell-like properties in non-tumorigenic breast epithelial cells through Api5 overexpression. This method enables robust assessment of stemness and self-renewal in MCF-10A cells.

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5 minutes

This study was supported by grants from CSIR-ASPIRE (37WS(0078)/2023-24/EMR-II/ASPIRE) and SERB-POWER (SPG/2021/002661-G) as well as partial funding from IISER, Pune core funding. S.B. was funded by a UGC-SRF fellowship. We would like to thank Prof. Raymond C. Stevens (Scripps Research Institute, USA) for generously providing the MCF10A cells and Dr Abhijith K (Ahammune Biosciences Pvt. Ltd) for his valuable inputs on the manuscript.

1. Coating of tissue culture plates with Poly(2 -hydroxyethyl methacrylate), pHEMA
<ol>
	<li>Dissolve 1.2 g of pHEMA (molecular weight ̴ 300,000) in 100 mL of 95% ethanol by stirring constantly at 65 °C to prepare a 1.2% pHEMA solution (see Table of Materials).</li>
	<li>Coat standard tissue culture plates with pHEMA by applying 1 mL per well of a 6-well plate. The volume of the pHEMA solution can be adjusted based on the size of the tissue culture vessel.</li>
	<li>Allow the plates to dry in an oven at 40 °C under sterile conditions.</li>
	<li>Expose the plates to UV light for 15 min, followed by three washes with Dulbecco's phosphate-buffered saline (DPBS), before using them to set up the mammosphere formation assay.</li>
</ol>
2. Seeding primary generation mammospheres
<ol>
	<li>Culture 3D dissociated MCF10A cells (Control and Api5 overexpression) obtained by dissociating day 16 3D cultures in the growth medium14. 
	NOTE: Composition of growth medium: High glucose DMEM without sodium pyruvate supplemented with 5% horse serum, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 20 ng/mL recombinant human epidermal growth factor (EGF), 100 ng/mL cholera toxin, and 200 units/mL penicillin-streptomycin (see Table of Materials).</li>
	<li>Remove the spent medium, wash the cells with 1 mL of DPBS, then add 500 µL of 0.05% trypsin-EDTA and incubate at 37 °C in a humidified 5% CO2 incubator for 12-15 min till all the cells are trypsinized.</li>
	<li>After all the cells are dislodged, add 2 mL of resuspension medium to neutralise trypsin activity and spin the cell suspension at 112 × g for 10 min at room temperature. 
	NOTE: Composition of re-suspension medium: High glucose DMEM without sodium pyruvate supplemented with 20% horse serum, and 200 units/mL penicillin-streptomycin.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 1 mL of mammosphere medium. Pass the cell suspension through a 25 G needle to get a single-cell suspension. 
	NOTE: Composition of mammosphere medium: DMEM/F12 supplemented with 20 ng/mL epidermal growth factor (EGF), 1 × B27 supplement without Vitamin A, and 200 units/mL penicillin-streptomycin (see Table of Materials).</li>
	<li>Using a haemocytometer, count the number of cells and calculate the volume of cell suspension required to seed 0.2 × 104 cells per well. 
	NOTE: Maintain a low seeding density to accurately determine the mammosphere formation efficiency, thereby minimising the impact of cell aggregation. When collecting protein lysates for immunoblotting, the cell density can be increased to as high as 2.5 × 105 cells per well.</li>
	<li>Dilute the required cell suspension volume in 2 mL of mammosphere medium and seed in each well of a 6-well plate. Seed cells in triplicate for each condition.</li>
	<li>Incubate at 37 °C in a humidified 5% CO2 incubator for 7 days.</li>
	<li>After seven days in culture, manually count the number of mammospheres with a diameter greater than 50 µm in each well by either using a microscope fitted with a graticule or by using a quadrant grid under a light microscope.</li>
	<li>Image the mammospheres using a digital camera fitted to a light microscope at 20x magnification.</li>
	<li>Calculate the mammosphere formation efficiency (MFE) using the following formula5 
	 <img alt="Mammosphere formation efficiency formula diagram; MFE calculation, scientific research method." src="/files/ftp_upload/69999/69999eq1v3.jpg" style="margin: auto;" data-altupdatedat="1784001604000" data-alt="Equation 1"></li>
</ol>
3. Seeding secondary generation mammospheres
<ol>
	<li>Collect the mammospheres formed in the first generation in a 15 mL tube and centrifuge at 300 × g for 10 min at room temperature.</li>
	<li>Aspirate the supernatant, leaving the mammosphere pellet behind.</li>
	<li>Wash the pellet with DPBS.</li>
	<li>Add 200 µL 0.05% trypsin-EDTA to the mammosphere pellet and incubate at 37 °C in a humidified 5% CO2 incubator for 2-3 min. Pass the mammospheres in the trypsin solution through a 25 G needle three times to obtain a single-cell suspension.</li>
	<li>Add 0.5 mL of resuspension medium to neutralise trypsin activity and spin the cell suspension at 112 × g for 10 min at room temperature.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 0.5 mL of mammosphere medium.</li>
	<li>Using a haemocytometer, count the number of cells and calculate the volume of cell suspension required to seed 0.2 × 104 cells per well. 
	NOTE: Maintain a low cell density to assess the self-renewal capacity, thereby minimising the effect of cell aggregation.</li>
	<li>Dilute the required cell suspension volume in 2 mL of mammosphere medium and seed in each well of a 6-well plate. Seed cells in triplicate for each condition.</li>
	<li>Incubate at 37 °C in a humidified 5% CO2 incubator for 7 days.</li>
	<li>After 7 days in culture, count the number of mammospheres with a diameter greater than 50 µm in each plate using either a microscope fitted with a graticule or a quadrant grid.</li>
	<li>Image the mammospheres using a digital camera fitted to a light microscope at 20x magnification.</li>
	<li>Calculate the self-renewal capacity using the following formula5 
	<img alt="Self-renewal capacity formula diagram, ratio of secondary to primary mammospheres for stem cell research." src="/files/ftp_upload/69999/69999eq2v3.jpg" style="margin: auto;" data-altupdatedat="1784001604000" data-alt="Equation 2"></li>
</ol>
4. Western blotting to detect the changes in pluripotency markers upon Api5 overexpression
<ol>
	<li>Collect the mammospheres formed in the first generation in a 15 mL tube and centrifuge at 300 × g for 10 min at room temperature.</li>
	<li>Aspirate the supernatant, leaving the mammosphere pellet behind.</li>
	<li>Wash the pellet with DPBS.</li>
	<li>Collect the pellet in ice-cold RIPA buffer to obtain the protein lysate and perform immunoblotting according to the standard protocol24. 
	NOTE: Composition of RIPA buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethysulphonyl fluoride (PMSF), 10 mM sodium fluoride, 1 mM sodium orthovanadate.</li>
	<li>Probe for stemness markers- Oct3/4, Nanog, Sox2, and Bmi1, and loading control GAPDH (see Table of Materials for antibody dilutions).</li>
	<li>Quantify protein levels using densitometric analysis on ImageJ. Select the region of interest on Western blot images and generate a density profile for each lane.</li>
	<li>Normalize the relative densities of the protein of interest to those of GAPDH for each lane. Calculate the fold change for each protein.</li>
	<li>Plot the fold change in protein levels using any plotting software.</li>
</ol>

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The authors declare no potential conflict of interest.

While monolayer cultures of established cancer cell lines or tumor-derived cells provide insights into the various molecular pathways regulating the cancer phenotypes, research focused on cancer stem cells cannot be conducted in two-dimensional cultures. Since cancer stem cells are believed to be a major contributing factor for relapse after current treatment strategies, it is necessary to establish robust assays to study these cells1,20,<sup class='xr...

The rise in chemotherapy-resistant breast cancers has led to an increasing emphasis on identifying and targeting the underlying cause of therapy resistance and relapse, a population of cancer cells known as cancer stem cells. These cells are characterised by their ability to evade differentiation and remain quiescent, enabling them to resist different modalities of cancer treatment1,2. Several methods can be employed to identify breast cancer stem cells, including the mammosphere formation assay3,4,5, cell sorting based on cell surface markers6,7, isolation based on efflux properties, in vivo tumorigenicity assays7, and the assessment of stem cell nuclear modulators such as Oct48, Nanog9, Sox210&#160;and Bmi11. Typically, identifying and isolating cancer stem cells involves a combination of one or more of these approaches.
Apoptosis inhibitor 5 (Api5) is an anti-apoptotic protein that has been found to be upregulated in several cancers, including breast cancer12,13,14, cervical cancer15,16,17, B-cell chronic lymphoid leukaemia18, and non-small cell lung cancer19. High levels of Api5 have been found to be associated with chemotherapy resistance in cervical16&#160;and triple-negative breast cancer12. There is increasing evidence that therapy resistance and relapse are often driven by cancer stem cells20,21. An interesting study by Song et al.22&#160;revealed that the overexpression of Api5 in cervical cancer cells results in elevated Nanog levels, contributing to increased cancer stemness. Previous studies from our lab have shown that overexpression of Api5 leads to the transformation of non-tumorigenic breast epithelial cells grown in Matrigel-supported 3D cultures. These transformed cells exhibit disrupted polarity, increased proliferation, anchorage-independent growth, and a partial epithelial-to-mesenchymal transition (EMT)-like phenotype. The phenotypic changes observed upon overexpression of Api5 in 3D cultures were retained after dissociating the spheroids into monolayer cultures14. Given that Api5 is upregulated in chemotherapy-resistant triple-negative breast cancers, we speculate that Api5 may also regulate cancer stemness properties in breast epithelial cells.
In this context, we describe the mammosphere formation assay for identifying cancer stem cells from 3D dissociated non-tumorigenic breast epithelial cells transformed by the overexpression of Api5, followed by western blotting for stem cell markers to confirm the identity of these cells. The mammosphere assay involves culturing breast epithelial cells under non-adherent, non-differentiating conditions, which enables the enrichment of stem-like/progenitor cells in three-dimensional structures known as mammospheres. Breast epithelial cells that do not possess stem-like properties typically undergo anoikis; however, cells with stem-like characteristics give rise to mammospheres4,5. This method was originally developed by Dontu et al3&#160;and is inspired by the neurosphere assay, which is used to study and identify neural stem cells and progenitors23. The mammosphere assay described here allows for the assessment of stem cell activity and self-renewal capacity in minimal media supplemented with B27 without vitamin A and recombinant human epidermal growth factor (EGF). Api5 overexpressing 3D dissociated cells are seeded sparsely on Poly(2-hydroxyethyl methacrylate) (pHEMA) coated plates. This allows the cells to grow in non-adherent conditions, such that each subsequent mammosphere is of clonal origin. The mammospheres are allowed to grow till day seven, after which mammosphere formation efficiency is calculated. To distinguish true stem cells from non-stem cells that lose their proliferative capacity over time, subsequent generations of mammosphere formation are done. Serial passaging of mammospheres involves dissociating the spheres collected from primary generation and replating the single cells to form secondary (and subsequent) generation mammospheres. This determines whether the cells can initiate sphere formation repeatedly and is quantified as the self-renewal capacity of cells5.
This method can be utilised to identify breast cancer stem cells in tumorigenic or transformed breast epithelial cells. The method can also be adapted for isolating breast cancer stem cells from patient samples. The system can help identify new targets against breast cancer stem cells that could be associated with chemotherapy resistance.

<table><tbody><tr><td>0.05% Trypsin EDTA&amp;nbsp;&amp;nbsp;</td><td>Invitrogen</td><td>25300062</td><td></td></tr><tr><td>37 &amp;deg;C humidified incubator</td><td>New Brunswick</td><td>Galaxy 170 R</td><td></td></tr><tr><td>Anti-Bmi1 Rabbit pAb</td><td>ABclonal</td><td>A13472</td><td>1:1000 dilution, overnight incubation at 4 &amp;deg;C</td></tr><tr><td>Anti-GAPDH antibody</td><td>Sigma</td><td>G9545-200UL</td><td>1:10000 dilution, 1 h incubation at room temperature</td></tr><tr><td>Anti-Nanog Rabbit mAb</td><td>ABclonal</td><td>A22625</td><td>1:5000 dilution, overnight incubation at 4 &amp;deg;C</td></tr><tr><td>Anti-Oct3/4 Antibody (C-10)</td><td>Santa Cruz</td><td>sc-5279</td><td>1:300 dilution, overnight incubation at 4 &amp;deg;C</td></tr><tr><td>Anti-Sox2 Antibody (E-4)</td><td>Santa Cruz</td><td>sc-365823</td><td>1:300 dilution, overnight incubation at 4 &amp;deg;C</td></tr><tr><td>B-27 Supplement (50x), minus vitamin A</td><td>Invitrogen</td><td>12587010</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Allegra X-15R</td><td></td></tr><tr><td>Cholera toxin&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>C8052-1MG</td><td>&amp;nbsp;1 mg/mL stock in dH2O</td></tr><tr><td>DMEM/F12</td><td>Thermo</td><td>11330032</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Epidermal growth factor (EGF)&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>E9644-0.2MG&amp;nbsp;</td><td>100 mg/mL stock in dH2O</td></tr><tr><td>Ethanol absolute 99.9%</td><td>Jebsen &amp;amp; Jessen GmbH &amp;amp; Co. Germany</td><td>58051 L05</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td><td>SRL</td><td>43272</td><td></td></tr><tr><td>High glucose DMEM without sodium pyruvate</td><td>Invitrogen</td><td>11965126</td><td></td></tr><tr><td>Horse Serum&amp;nbsp;&amp;nbsp;</td><td>Invitrogen</td><td>16050122</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Qualigens</td><td>Q29145</td><td>Used to adjust pH of Tris-HCl (pH 8.0)</td></tr><tr><td>Hydrocortisone&amp;nbsp;</td><td>Sigma&amp;nbsp;&amp;nbsp;</td><td>H0888</td><td>1 mg/mL stock in ethanol</td></tr><tr><td>ImageJ&amp;nbsp;</td><td></td><td></td><td>Image processing software</td></tr><tr><td>ImageQuant LAS 4000&amp;nbsp;</td><td>Cytiva</td><td></td><td></td></tr><tr><td>Immobilon Western chemiluminiscent HRP substrate</td><td>Merck</td><td>WBKLS0500</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>I1882&amp;nbsp;</td><td>10 mg/mL stock in 1% glacial acetic acid</td></tr><tr><td>Nikon Eclipse TS-100 microscope</td><td>Nikon</td><td>BM-1919</td><td></td></tr><tr><td>MCF10A cells</td><td></td><td></td><td>Gift from Prof. Raymond C. Stevens (Scripps Research Institute, USA)</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Lonza&amp;nbsp;</td><td>17-602E</td><td></td></tr><tr><td>Phenylmethysulphonyl fluoride (PMSF)</td><td>Sigma</td><td>78830-5G</td><td></td></tr><tr><td>Plotting software&amp;nbsp;</td><td>GraphPad Prism 10</td><td></td><td></td></tr><tr><td>Poly(2-hydroxyethyl methacrylate) (pHEMA)</td><td>Santacruz</td><td>sc-253284</td><td>1.2% pHEMA in 95% ethanol</td></tr><tr><td>Sodium chloride (NaCl)</td><td>SRL</td><td>41721</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Amresco</td><td>0613-100G</td><td></td></tr><tr><td>Sodium dodecyl sulphate (SDS)</td><td>SRL</td><td>54468</td><td></td></tr><tr><td>Sodium fluoride (NaF)</td><td>HiMedia</td><td>RM1081-500G</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>Sigma-Aldrich</td><td>S6508-10G</td><td></td></tr><tr><td>Tris buffer</td><td>SRL</td><td>71033</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-250ML</td><td></td></tr></tbody></table>

mammosphere assay reveals api5-induced stemness in non-tumorigenic breast epithelial cell lines

Mammosphere Assay Reveals Api5-Induced Stemness in Non-Tumorigenic Breast Epithelial Cell Lines

Breast cancer remains the most prevalent cancer among women globally, with the highest age-standardised incidence and mortality rates. Despite the advancements in therapies targeting breast cancer, 20% of the patients exhibit relapse of the disease, which may be attributed to a population of therapy-resistant cancer stem cells (CSCs). These CSCs can survive conventional treatments by remaining in a quiescent stage or persist due to incomplete resection of the tumor mass, resulting in recurrence at a later stage. Therefore, recent research has focused on identifying, isolating and targeting this resistant population of cells. In vitro, the mammosphere formation assay serves as a robust three-dimensional suspension culture system for enriching and propagating stem-like cells within breast epithelial populations. Here, we present an approach utilising the mammosphere assay to evaluate the cancer stem cell-like phenotype that is enabled in non-tumorigenic breast epithelial cells by the overexpression of Apoptosis Inhibitor 5 (Api5). The protocol involves propagating mammospheres through serial passages to assess mammosphere formation efficiency and self-renewal capacity. Additionally, protein lysates collected from mammospheres are probed for established stemness markers via immunoblotting. This assay enables the investigation of novel regulators, such as Api5, of cancer stem cell-like properties in breast epithelial cell populations. The approach described can be adapted for comparative studies of molecular interventions or for screening therapeutic agents targeting CSCs in vitro.

To assess mammosphere formation efficiency under non-adherent conditions, control and Api5-overexpressing 3D dissociated MCF10A cells were seeded on polyHEMA-coated plates according to the protocol. Api5 overexpressing cells demonstrated a 1.58-fold increase in the mean mammosphere formation efficiency than the controls (Figure 1B,C). This increase demonstrates that higher expression of Api5 increases the proportion of stem cell-like progenitors that survive and proliferate ...

This protocol describes the method for setting up the mammosphere formation assay to study Apoptosis Inhibitor 5 (Api5)- induced cancer stemness in non-tumorigenic breast epithelial cells. Lysates collected from primary generation mammospheres are analysed for different stemness markers to assess the induction of stem cell-like properties.

Breast cancer remains the most prevalent cancer among women globally, with the highest age-standardised incidence and mortality rates. Despite the ...

We are investigating how overexpression of a protein in a non-immunogenic cell line leads to the acquisition of cancer stem cell-like properties. This protocol uses a minimal medium supplemented with B-27 and EGF, making it more cost effective. To begin, coat each well of a standard six-well tissue culture plate with one milliliter of 1.2%poly 2-hydroxyethyl methacrylate solution, and place the plate in an oven set at 40 degrees Celsius to dry under sterile conditions.Expose the dried plate to ultraviolet light for 15 minutes. Then, wash each well three times with Dulbecco's PBS before using the plate to set up the mammosphere formation assay. Obtain a previously cultured 3D dissociated MCF-10A cell culture dish and remove the spent medium.Wash the cells with one milliliter of Dulbecco's PBS. Then, add 500 microliters of 0.05%trypsin-EDTA and incubate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for 12 to 15 minutes until the cells are trypsinized. Once the cells are dislodged, add two milliliters of resuspension medium to neutralize the trypsin activity.Spin the cell suspension at 112 G for 10 minutes at room temperature. Aspirate the supernatant and resuspend the resulting cell pellet in one milliliter of mammosphere medium. Pass the cell suspension through a 25-gauge needle to create a single-cell suspension.Next, using a hemocytometer, count the number of cells. Calculate the required volume of the cell suspension to seed 2000 cells per well. Dilute the calculated volume of cell suspension in two milliliters of mammosphere medium per well, and seed the diluted suspension into each well of a six-well plate, preparing three replicate wells for each condition.Incubate the plate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for seven days. After seven days, manually count the number of mammospheres larger than 50 micrometers in each well. Image the mammospheres using a digital camera attached to a light microscope at 20 x magnification and calculate the mammosphere formation efficiency using the given formula.Collect the first generation mammospheres in a 15-milliliter tube and centrifuge at 300 G for 10 minutes at room temperature. Aspirate the supernatant carefully, leaving the mammosphere pellet intact at the bottom of the tube. Wash the pellet with Dulbecco's PBS.Centrifuge the cell suspension to obtain a mammosphere pellet, then pipette out the Dulbecco's PBS without disturbing the pellet. Now, add 200 microliters of 0.05%trypsin-EDTA to the washed pellet and incubate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for two to three minutes. Then, pass the mammospheres in trypsin solution through a 25-gauge needle three times to obtain a single-cell suspension.Then, add 0.5 milliliters of resuspension medium to neutralize the trypsin activity. Centrifuge the cell suspension at 112 G for 10 minutes at room temperature. Aspirate the supernatant and resuspend the cell pellet in 0.5 milliliters of mammosphere medium.Using a hemocytometer, count the number of cells and calculate the required volume of cell suspension to seed 2000 cells per well. Dilute the required cell suspension volume in two milliliters of mammosphere medium per well, and seed into each well of a six-well plate, preparing three replicate wells for each condition. Incubate the plate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for seven days.After seven days, count the number of mammospheres with a diameter greater than 50 micrometers in each well. Image the mammospheres under 20 x magnification and calculate the self-renewal capacity using the given formula. Collect the first generation mammospheres into a 15-milliliter tube and centrifuge at 300 G for 10 minutes at room temperature.Aspirate the supernatant carefully, ensuring the mammosphere pellet remains undisturbed at the bottom of the tube. Wash the pellet gently with Dulbecco's PBS. After removing the PBS, collect the washed pellet in ice-cold RIPA buffer to obtain the protein lysate and perform immunoblotting using the standard protocol.Finally, quantify the protein levels using densitometric analysis in image J.Select the region of interest on the western blot image and generate a density profile for each lane and the pluripotency marker. Normalize the relative densities of the protein of interest to those of GAPDH for each lane, then calculate the fold change for each protein. API5 over expressing MCF-10A cells showed a 1.58-fold increase in mean mammosphere formation efficiency compared to control cells under non-adherent, serum-deprived conditions.These cells formed second generation mammospheres with a self-renewal capacity nearly 2.5 times higher than control cells. Immunoblotting showed increased expression of Oct 3/4, Nanog, Sox 2, and BMI1 in API5 over expressing primary mammospheres compared to controls. Densitometric analysis showed a 1.83-fold increase in Oct 3/4, a 1.65-fold increase in Nanog, a 1.68-fold increase in Sox 2, and a 1.62-fold increase in BMI1 levels in API5 over expressing mammospheres compared to controls.A challenge in the protocol is standardizing the cell count to ensure sufficient mammosphere formation without cell aggregation. Mammospheres generated by this protocol can also be used to visualize protein localization in a three-dimensional context using immunofluorescence.

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Cancer Research

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