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

Summary

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.

Abstract

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.

Introduction

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 treatment^1,2. Several methods can be employed to identify breast cancer stem cells, including the mammosphere formation assay^3,4,5, cell sorting based on cell surface markers^6,7, isolation based on efflux properties, in vivo tumorigenicity assays⁷, and the assessment of stem cell nuclear modulators such as Oct4⁸, Nanog⁹, Sox2¹⁰ 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 cancer^12,13,14, cervical cancer^15,16,17, 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 and triple-negative breast cancer¹². There is increasing evidence that therapy resistance and relapse are often driven by cancer stem cells^20,21. An interesting study by Song et al.²² 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 mammospheres^4,5. This method was originally developed by Dontu et al³ 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.

Protocol

1. Coating of tissue culture plates with Poly(2 -hydroxyethyl methacrylate), pHEMA

1. 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).
2. 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.
3. Allow the plates to dry in an oven at 40 °C under sterile conditions.
4. 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.

2. Seeding primary generation mammospheres

1. 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).
2. 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.
3. 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.
4. 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).
5. 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.
6. 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.
7. Incubate at 37 °C in a humidified 5% CO₂ incubator for 7 days.
8. 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.
9. Image the mammospheres using a digital camera fitted to a light microscope at 20x magnification.
10. Calculate the mammosphere formation efficiency (MFE) using the following formula⁵
     

3. Seeding secondary generation mammospheres

1. Collect the mammospheres formed in the first generation in a 15 mL tube and centrifuge at 300 × g for 10 min at room temperature.
2. Aspirate the supernatant, leaving the mammosphere pellet behind.
3. Wash the pellet with DPBS.
4. 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.
5. 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.
6. Aspirate the supernatant and resuspend the cell pellet in 0.5 mL of mammosphere medium.
7. 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.
8. 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.
9. Incubate at 37 °C in a humidified 5% CO₂ incubator for 7 days.
10. 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.
11. Image the mammospheres using a digital camera fitted to a light microscope at 20x magnification.
12. Calculate the self-renewal capacity using the following formula⁵
    

4. Western blotting to detect the changes in pluripotency markers upon Api5 overexpression

1. Collect the mammospheres formed in the first generation in a 15 mL tube and centrifuge at 300 × g for 10 min at room temperature.
2. Aspirate the supernatant, leaving the mammosphere pellet behind.
3. Wash the pellet with DPBS.
4. 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.
5. Probe for stemness markers- Oct3/4, Nanog, Sox2, and Bmi1, and loading control GAPDH (see Table of Materials for antibody dilutions).
6. 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.
7. Normalize the relative densities of the protein of interest to those of GAPDH for each lane. Calculate the fold change for each protein.
8. Plot the fold change in protein levels using any plotting software.

Results

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 ...

Discussion

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 cells^1,20,

Materials

List of materials used in this article

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