Accepted Manuscript

Title: Resistance of primary breast cancer cells with enhanced

pluripotency and stem cell activity to sex hormonal
stimulation and suppression

Authors: Mostafa Nasr, Mohamed Farghaly, Tarek Elsaba,

Mohamed El-Mokhtar, Radwa Radwan, Mahmoud El-Sabahy,
Ahmed Abdelkareem, Hussein Fakhry, Noha Mousa

PII: S1357-2725(18)30228-0
DOI: https://doi.org/10.1016/j.biocel.2018.10.005
Reference: BC 5437

To appear in: The International Journal of Biochemistry & Cell Biology

Received date: 24-6-2018

Revised date: 9-10-2018
Accepted date: 11-10-2018

Please cite this article as: Nasr M, Farghaly M, Elsaba T, El-Mokhtar M, Radwan R, El-
Sabahy M, Abdelkareem A, Fakhry H, Mousa N, Resistance of primary breast cancer
cells with enhanced pluripotency and stem cell activity to sex hormonal stimulation
and suppression, International Journal of Biochemistry and Cell Biology (2018),
https://doi.org/10.1016/j.biocel.2018.10.005

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 Resistance of Primary Breast Cancer Cells with Enhanced Pluripotency and Stem Cell
Activity to Sex Hormonal Stimulation and Suppression

Mostafa Nasr1#, Mohamed Farghaly2#, Tarek Elsaba3, Mohamed El-Mokhtar2, Radwa Radwan4,
Mahmoud El-Sabahy4,5, Ahmed Abdelkareem2, Hussein Fakhry3, Noha Mousa1*

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1
Zewail City of Science and Technology, Egypt

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2
Faculty of Medicine, Assiut University, Egypt

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3
South Egypt Cancer Institute, Assiut University, Egypt

4
Department of Pharmaceutics, Faculty of Pharmacy, Assiut International Center of

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Nanomedicine, Al-Rajhy Liver Hospital, Assiut University, Assiut, Egypt

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Laboratory for Synthetic-Biologic Interactions, Department of Chemistry, Texas A&M
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University, College Station, TX, USA
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# Authors contributed equally to this work.

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*Corresponding Author:
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Noha Mousa, M.D., Ph.D.

Zewail City of Science and Technology, Cairo, Egypt, 12587

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Women Health Clinic, Assiut, Egypt, 71511

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Email: noha.mousa@utoronto.ca, nmousa@zewailcity.edu.eg

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Abstract

Female sex steroid hormones have a fundamental role in breast cancer. Meanwhile, current
evidence supports the contribution of breast cancer stem cells in carcinogenesis, metastasis, and
resistance to cytotoxic chemotherapy. Nevertheless, the interaction between breast cancer stem
cells with sex hormones or key hormonal antagonists remains elusive. Objective: To investigate

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the effect of diverse sex hormonal stimulation and suppression regimens on the proliferation of a
primary human breast cancer cells with stem cell activity. Methods: Cells were exposed to

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estradiol, progesterone, letrozole, ulipristal acetate, or a combination of ulipristal acetate-letrozole,
continually for 6 months. Additionally, nanoparticle-linked letrozole and ulipristal acetate

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formulations were included in a subsequent short-term exposure study. Phenotypic, pathologic,
and functional characteristics of unexposed cells were investigated. Results: The proliferation of

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breast cancer cells was comparable among all hormonal stimulation and suppression groups (P =
0.8). In addition, the nanoparticle encapsulated hormonal antagonists were not able to overcome
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the observed resistance of cells. Cell characterization showed a mesenchymal-like phenotype
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overexpressing three master pluripotency markers (Oct 4, SOX2, and Nanog), and 92% of cells
high low
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were expressing ALDH1A1. Notably, the CD44 /CD24 cell population presented only
0.97%- 5.4% over repeat analyses. Most cells lacked the expression of mesenchymal markers;
however, they showed differentiation into osteogenic and adipogenic lineages. Upon transfer to
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serum-free culture, the long-term maintained mesenchymal-like cancer cells showed remarkable
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morphologic plasticity as they switched promptly into an epithelial-like phenotype with significant
mammosphere formation capacity (P= 0.008). Conclusion: Breast cancer cells can develop a
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pluripotent program with enhanced stemness activity that may together contribute to universal
resistance to sex hormonal stimulation or deprivation. Isolation and characterization of patient-
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derived breast cancer stem cells in large clinical studies is therefore crucial to identify new targets
for endocrine therapies, potentially directed towards stemness and pluripotency markers. Such
direction may help overcoming endocrine resistance and draw attention to breast cancer stem cells’
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behaviour under endogenous and exogenous sex hormones throughout a woman’s reproductive
life.

Keywords: Breast cancer, stem cells, sex steroids, letrozole, ulipristal actate

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Introduction:

The role of female reproductive hormones in breast cancer (BC) carcinogenesis is supported by a
wealth of clinical and experimental evidence (Brisken, 2013; Cancer, 1997; Chlebowski et al.,
2003; Key et al., 2002). Estrogen receptor antagonists and aromatase inhibitors have been the
standard BC endocrine therapies for decades and were further proposed for BC chemoprevention

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(Goss et al., 2011; Group, 2005; Powles et al., 2007). Similarly, progesterone receptor antagonists
are showing promise as potential BC therapies (Communal et al., 2016; Esber et al., 2015). In the

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meantime, breast cancer stem cells (BCSC) have been characterized in BC studies, suggesting
their association with resistance to cytotoxic chemotherapies (Lawson et al., 2015; Owens and

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Naylor, 2013; Singh and Settleman, 2010; Tanei et al., 2009), however, the interaction between
BCSC and hormonal interventions, including endocrine therapies, remains considerably

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understudied.

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Current evidence provides no or inadequate information on how BCSC may respond to
clinically used sex hormones and their antagonists. For instance, the effect of aromatase inhibitors
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which are increasingly recommended as the new gold standard BC endocrine therapies on BCSC,
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remains unknown (Cuzick et al., 2014; Group, 2005; Group, 2015; Liu, Yan et al., 2014; Simoes
et al., 2015). Similarly, no available studies of the effect of the selective progesterone receptor
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modulator ulipristal acetate (UPA) on BCSC, despite its growing application in reproductive
disorders in women and its potential value for BC applications (Communal et al., 2016; Esber et
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al., 2015; Fernandez et al., 2017). In a few studies, short-term exposure of BCSC to estradiol or
tamoxifen – often limited to a few days– was reported with conflicting outcomes. One report
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showed that estradiol led to significant expansion of BCSC in BC cell lines (Fillmore et al., 2010).
Also, estrogen, in carcinogenic concentrations, could enhance BCSC function by promoting
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epithelial-mesenchymal transition (EMT), increasing the expression of pluripotency markers, and

reducing apoptosis (Vazquez et al., 2016). Likewise, a brief exposure to estradiol was reported to
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promote self-renewal and EMT in ER positive BC cell lines through a pathway involving Glioma-
associated oncogene 1 (Gli1)(Sun et al., 2014). By contrast, Simões et al reported that estradiol
suppressed the expression of the pluripotency markers (Nanog, OCT4 and Sox-2) in BCSC derived
from the MCF-7 cell line, indicating reduced stemness and enhanced differentiation (Simões et al.,
2011).

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In this study, we investigated the long-term effect of key anti-hormonal medications (letrozole,
ulipristal acetate, and their combination) and their corresponding female sex hormones (estradiol
and progesterone) on a primary human BC cell line that demonstrated enhanced stem cell activity.
We initially hypothesized that there may be a differential response in cell proliferation between
the opposing hormonal interventions.

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Methods

Primary breast cancer cell line generation: A primary breast cancer cell line with stem cell

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activity (BC-SCa) was generated at our lab following standard protocols and was maintained over
20 months before cryopreservation. The sample preparation and explant culture protocol are

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detailed in the supplementary data file.

Hormonal groups: Starting at passage 2, cells were divided into 4 groups according to the type of

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hormonal intervention: Estradiol (E2) at a dose of 10 ng/ml (Sigma-Aldrich, USA); Progesterone
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(30 ng/ml; Sigma-Aldrich, USA); Letrozole (285 ng/ml; TOCRIS, UK), and a control group of
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media without hormones. Cells were exposed to the hormonal intervention continually over
subsequent passages. At passage 9, two additional groups were added: Ulipristal acetate at a dose
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of 50 ng/ml (US Biological, USA), and a combined group containing Ulipristal acetate and
Letrozole (same concentration as the corresponding individual groups). The doses of these
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hormonal agents were estimated based on previous literature with optimization based on our
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team’s experience (Desta et al., 2011; Mousa et al., 2012; Mousa et al., 2009; Pohl et al., 2013;
Russo et al., 2002). Alcohol 70% was used to initially dissolve the hormonal agents, but the final
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alcohol concentration in culture media was kept at around 0.1%(Lippman et al., 1976). All groups
were, at least, cultured and passaged in triplicates throughout the study.
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Nano-particle hormonal antagonist preparation: Ulipristal acetate and Letrozole-loaded PLGA

nanoparticles were, independently, prepared by the oil-in-water single emulsion/solvent
evaporation technique following previously reported methods with modifications (Hariharan et al.,
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2006).

Cell proliferation:

Traditional hormonal interventions: The Alamar Blue ® assay (Invitrogen, USA) was used to
evaluate the proliferation and metabolic activity of cells under the effect of the free hormonal

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medications (Zachari et al., 2014). Cells were obtained at passages 13 or 14 and were seeded in
triplicates at a density of 1,000 cells per well (200 μl of media/well), whereas 6 replicates were
used for the control groups (both positive and negative controls). The positive control included L-
ascorbic acid (5 μl of 25ug/ml; Sigma, USA). After 16 hours of cell incubation at standard culture
conditions, 20 μl of the Alamar Blue reagent was added to each well. The plate was incubated in
the dark for an additional 4 hours. A plate reader (FLUstar Omega, Germany) was used to measure

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fluorescence intensity (at a wave length of 544 nm for excitation and (590-B0) for emission).

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Nanoparticle-linked antagonists: The MTT assay was performed using MTT cell proliferation kit
(Cell Titer 96® Non-Radioactive Cell Proliferation Assay, Promega, USA) according to the

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manufacturer's instructions. Briefly, cells obtained at passage 17 were plated in 96-well plates at a
cell density of (5000 cells/well) in triplicates. We exposed cells to: Group 1: Letrozole-loaded

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PLGA nanoparticles (0.285 µg/ml); Group 2: Ulipristal acetate-loaded PLGA nanoparticles (0.05
µg/ml); Group 3: Empty PLGA nanoparticles (9.19 µg/ml); Group 4: Empty PLGA nanoparticles
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(2.35 µg/ml); Group 5: Control cells treated with PBS. Cultured BC-SCa were incubated at 37 oC
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in a 5% CO2 incubator (Thermo Fisher Scientific, MA, USA) for 24 h. After incubation, the MTT
dye (15 μl) was added to each well and incubated at 37°C for 4 hours. The stop solution (100 μl)
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was then added and mixed thoroughly for colour development over 1 hour. The absorbance (OD)
was measured using a plate reader (PG Instruments Ltd, Leicestershire, UK) at a wavelength of
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570 nm and a reference wavelength of 630 nm. Cell viability (mean, %) was calculated by dividing
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the OD drug-treated sample/OD control samples.

Flow cytometry analysis: Control cells not exposed to any hormones were always used for this
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analysis. Cell characterization was performed using FACS Calibur (Becton Dickinson, Germany.
The following monoclonal antibodies were used: conjugated antibodies against surface markers
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included anti-CD73, CD271, CD105, CD45, CD34, CD11B (Becton Dickinson, Germany), anti-
CD44 and CD24 (Molecular Probes, USA), intracellular pluripotency markers included
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conjugated OCT 4, unconjugated SOX2 (R & D systems, USA), unconjugated Nanog (Bioss,
USA). For ALDH1A1, a polyclonal unconjugated antibody was used (Thermo Scientific, USA) to
evaluated protein expression following previous reports (Ma et al., 2017; Ma et al., 2016).

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In brief, cultured cells were detached using trypsin EDTA (170.000 U trypsin/l; Lonza, USA), and
centrifuged at 1800 rpm for 10 minutes. The pellet was collected, washed once with PBS. For
intracellular markers, the following additional steps were performed: fixation was done using
paraformaldehyde (4%) in 1XPBS followed by vortexing, incubation for 30 minutes at RT, and
washing with PBS. Permeabilization was performed using Triton (0.1, X-100) in 1x PBS, followed
by incubation for 30 minutes at room temperature (RT), then washing with PBS. Cells were re-

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suspended in a blocking buffer to reduce non-specific binding (BSA (0.5%) in 1X PBS). It was
then allocated into Eppendorf’s tubes for the number of required samples and vortexed before

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incubation on ice for 30 minutes. For surface markers, the cell pellet was re-suspended in 150 μl
of the blocking buffer with 5 μl of the selected stain and incubated on ice for another 30 minutes.

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For intracellular markers, 1μl of the desired primary antibody was added to 100μl of cell
suspension (1:100 ratio) and incubated at 4 Co overnight. Afterwards, a wash step was performed,

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and the corresponding secondary antibodies were added (1:500 v/v) and incubated for 1 hour in

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the dark at RT. Cells were finally washed with blocking buffer and re-suspended in PBS for
analysis. Cells were then analyzed and representative histograms, scatter plots and forward/side
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scatters were obtained using BD CellQuest™ Pro software (Becton Dickinson, Germany).
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Stem cell differentiation: Unexposed cells were split at 70-80% confluency, washed and
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centrifuged. The cell pellet was allocated into 2 wells in 2 independent 6-well plates with equal
seeding density. At day 3, osteogenic or adipogenic differentiation media were added to each well
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(2 ml/ well). Cultured cells were checked every 3 days for 19 days before staining with the specific
differentiation dyes. Each 50 ml of the osteogenic differentiation medium was composed of
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DMEM high glucose with L-glutamine (43.3 ml), FBS (5 ml of 10%), penicillin streptomycin (0.5
ml), β- glycerol phosphate (1ml of 500 Mm; Serva, Germany), L-ascorbic acid (100 μl of
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25mg/ml), and Dexamethasone (7.8 μl of 0.064 mM; Lonza, USA). Each 50 ml of the adipogenic
differentiation media was composed of DMEM high glucose with L-glutamine (43.3 ml), FBS
(5ml of 10%), penicillin streptomycin (0.5 ml), Dexamethasone (78 μl of 0.064 mM), Bovine
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insulin (250 μl of 1 mg/ml; Sigma, USA), Indomethacin(62.5 μl of 40 mM; Alfa Aesar, USA), and
3-isobutyl-1- methylxanthine (55.6 μl of 540 μM; Sigma, USA).To confirm osteogenic
differentiation, the differentiation media were aspirated, cells were washed three times with PBS,
incubated with ethanol (70%) for 1 hour at RT, and washed twice with MilliQ water (Thermo
Scientific, Sweden). Water was removed and 3ml of Alizarin Red (1.5%, pH 4.13; Alpha Chemika,
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India) was added. To confirm adipogenic differentiation, media was aspirated, washed three times
with PBS, incubated for 1 hour at RT with 10% formaldehyde (ThermoFisher, USA), and washed
twice with water. Water was aspirated and isopropanol (Sigma, Germany) was added for 5
minutes. Isopropanol was then aspirated, and 24 ml of Oil O Red was added (150 mg Oil Red O
in 50 ml 99% isopropanol, Alfa Aesar, USA) to 16 ml of distilled water, mixed well, and filtered.
The mixture was incubated for 15 min at RT, washed with water until it is clear, then all water was

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aspirated and discarded.

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Mammosphere formation assay: Cells (at passage 14) were detached and suspended in PBS then
seeded into two 6-well plates in triplicates (each at a seeding density of 1000 cells/well). One plate

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served as a control (DMEM medium with 10% FBS), and the other included the serum-free group
of wells (HUMEC Ready medium without FBS). Cells were regularly examined by the inverted

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microscope for the development of mammospheres. After 16 days, the cell suspension was
aspirated and centrifuged at 300 x g for 5 min, re-suspended in 50µl HUMEC media after gentle
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pipetting, and spread as a thin layer of suspension in a new well plate for imaging and
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mammosphere counting. Well-formed mammospheres (> 40 µm diameter) were counted in both
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groups. Mammosphere forming efficiency (MFE) was calculated as MFE (%) = (number of
mammospheres per well) / (number of cells seeded per well) x 100.
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Culture conditions: During the explant culture and passages, 1 through 9, a biosafety cabinet
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class II (Lamil plus 16, Karstulan Metalli Ltd, Finland) was used. Cultured cells were incubated at
37 Cᵒ in a 5% CO2 incubator (LabTech, Korea). HuMEC medium was used (HuMEC ready
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medium 1X, GIBCO, Thermo Fisher Scientific, USA), supplemented with an antibiotic-
antimycotic. FBS was supplemented at a concentration of 2% and 10% during the initial explant
culture and the following passages, respectively. Starting at passage 9 through passage 17, culture
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work was perfromed in a biosafety cabinet class II A (Nuaire, USA). Cultured cells were incubated
at 37 Cᵒ in a 5% CO2 incubator (Nuaire, USA). Centrifugation was done using a Megafuge 16r
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(Thermo Scientific, Germany). Media change and washing were performed every 3 days or more
often based on cells condition. DMEM Low glucose (1g/l) (Lonza, Belgium) was used to prepare
complete culture media (CCM). In few experiments, DMEM high glucose (4.5g/l) with L-
glutamine (Lonza, Belgium) was used equally among groups. All CCM was supplemented with
10% FBS (Gibco, South America origin) and 1% Penicillin-Streptomycin (Lonza, USA). PBS

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(Lonza, Belgium) was used in cell washing. Both Humec and DMEM media contained the PH
indicator phenol red. Cells were examined using an inverted microscope (Leica DMi8, Germany).

Statistical analysis: Categorical data were presented as percentages. Quantitative independent data
were analyzed using Mann–Whitney U for paired groups or Kruskal-Wallis test for comparing
more than 2 groups. SPSS (IBM, USA), and validated statistical calculators (Marx et al., 2016)

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were used for performing the analysis. The significance level was set at p = 0.05.

Results

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A long-term culture of a primary human breast cancer cell line with stem cell features was

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established successfully for 17 passages over 20 months prior to cell cryopreservation. The initial
explant tissue required 2 weeks before the first layer of explanting cells was observed, and 34 days

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before full confluence. The explanted cells started as adherent spindle-shaped cells of a

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mesenchymal/fibroblast-like phenotype. The original tissue fragments were re-cultured for a
second run showing faster tissue attachment, cellular explanting, and proliferation within 1 week.
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The mesenchymal-like cells, which were maintained in a serum containing media throughout the
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study period, acquired a stellate like morphology during subsequent passages (Supp. Fig.1-B and
Supp. videos).
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Two different proliferation assays showed that the response of the BC-SCa was equivalent
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regardless of the hormonal stimulus or the formulation used. Following 6 months of parallel
estrogenic/progestogenic hormonal stimulation or inhibition (using Letrozole, Ulipristal acetate
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and their combination), the Alamar Blue® proliferation assay showed no significant difference in
cell proliferation in between the sex hormones and their corresponding anti-hormonal groups
(Estradiol vs. Letrozole, p = 0.7; Progesterone vs. Ulipristal acetate, p= 0.5). There was also no
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significant difference among all tested groups overall (p=0.8). None of the studied agents or their
combination resulted in significantly distinct cell proliferation or inhibition in comparison to the
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control non-hormonal group of cells (Fig.1.a). Interestingly, the nano-particle linked Ulipristal
acetate and Letrozole did not differ in their proliferative effect from the control unexposed cells or
empty PLGA nanoparticles (p= 0.7 and 0.6 for Ulipristal acetate and Letrozole-Nanoparticles
respectively) (Fig. 1.b).

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Phenotypic characterization was performed using flow cytometry analysis to determine the identity
of our studied cells by examining the group of cells never exposed to hormones, to exclude
acquired hormonal resistance. The studied cells have shown considerable over-expression of three
intracellular pluripotency markers (Oct4, SOX2, and Nanog at 96.4%, 96.6% and 84.9% of cells,
respectively; Fig. 2). Similarly, ALDH1A1 positive cells reached up to 92% (Fig.3). The great
majority of those cells lacked the expression of the hematopoietic markers (80-90% of cells were

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negative for CD34 and CD45). As well, they had limited expression of mesenchymal stem cell
markers commonly used to identify normal adult stem cells; approximately; 75-90% of cells were

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repeatedly negative for the surface markers CD105, CD73, and CD271 in repeat analyses 3 months
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apart. The CD44 /CD24 represented only a small population of our cells, starting at

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approximately 0.97% which increased over 3 months to 5.4% of cells (Fig.4-A). Differentiation
towards the osteogenic and adipogenic lineages was detected by the uptake of tissue-specific dyes

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(Fig.4-B)

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A remarkable morphologic plasticity of the BC-SCa were observed. Once serum was
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excluded from the culture media, cells showed spontaneous rapid switch (within 24 hours) from
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the mesenchymal-like phenotype to an epithelial-like phenotype which started to form
mammospheres (Fig.5-A). This led us to run a prospectively controlled mammosphere formation
assay which confirmed cell plasticity and the reproducible formation of non-adherent
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mammospheres; such capacity was maintained well over 14 passages (Fig.5-B). The
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mammosphere forming efficiency (MFE %) was significantly higher in the serum-free group at an
average of 5.76% compared to 0.20% of control cells in serum-containing media (p-value 0.008)
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(Fig.5-C).

Discussion
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The present study shows evidence of the lack of proliferative response of human breast cancer
cells with stem cell activity to long-term exposure to sex hormones (estradiol and progesterone)
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and their corresponding hormonal antagonist medications (letrozole, ulipristal acetate, and a
combination of both), as well as nanoparticle encapsulated letrozole and ulipristal acetate.
Characterization of this primary cell line revealed the dominance of structural and functional
markers denoting cancer stem cell activity, including the over-expression of pluripotency markers,
ALDH1A1, and enhanced EMT/MET phenotype plasticity.

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The mechanism of BC resistance to endocrine therapies has been inconclusive (Clarke et al., 2015).
Lack of the ER, as well as mutations of the ER gene (ESR1), were proposed(Robinson et al., 2013),
however, only a minority of resistant BC tumours can be produced by such factors (Musgrove and
Sutherland, 2009). Also, receptor mutations could not explain resistance to all endocrine therapies;
for instance, selective estrogen receptor degraders (SERDs)(Spoerke et al., 2016). The
activation of downstream ER signalling pathways, transcription factors, up-regulation of

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growth factors such as HER2 were also proposed as resistance mechanisms to estrogen
deprivation therapies (Osborne et al., 2005). Most recently, the role of breast cancer stem cells

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in tamoxifen resistance was proposed (Ojo et al., 2015). The present study highlights the innate
stemness of a population of breast cancer cells as a powerful contributor to hormonal resistance.

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This understanding agrees with considerable evidence obtained on mechanisms employed by
cancer stem cell to resist cytotoxic chemotherapy, including the activation of the EMT phenotype,

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the over-expression of the ALDH1A genes, the activation of the efflux ABC transporter pumps,

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and the interference with tissue-specific genes or post-translational and downstream signalling
pathways (Dean et al., 2005).
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Nanoparticle-based medicines have shown evidence of enhanced effectiveness and safety
over free drugs, and potential for cancer therapeutics(Friberg and Nyström, 2016). Due to the poor
water solubility of Letrozole, its rapid metabolism, and side effects, many attempts were made to
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produce a nanoparticle formulation that can provide controlled release and enhance its tumour
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suppressive effects (Azandaryani et al., 2017; Dey et al., 2009). One report showed that such
development was adequate to restore hormonal sensitivity in a letrozole resistant xenograft tumour
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(Nair et al., 2011). However, our findings showed maintained resistance of those cells, regardless
of the formulation used, possibly due to the multiple and diverse mechanisms those cells employ.
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We, therefore, believe that unless there is an active targeting component of the nanoparticle
formulations to target one or more of the observed mechanisms, passive nano drug delivery alone
may not be an adequate pathway.
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The identity of cells examined here was intriguing. Morphologically, they acquired
mesenchymal/fibroblast-like phenotype. Cells were mostly negative for normal mesenchymal
stem cell markers. For decades, fibroblast-like cells were observed in BC, and were recognized as
active players essential to support cancer behaviour (Kalluri and Zeisberg, 2006). Later, those cells

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were described as cancer-associated fibroblasts (CAFs), and were linked to tumour aggressiveness
such as enhanced angiogenesis and cell proliferation, and most recently to tamoxifen resistance
(Brechbuhl et al., 2017). The origin and identity of CAFs remain greatly debatable, and diverse
origins were suggested including bone marrow mesenchymal cells, re-programmed resident tissue
fibroblasts, and EMT (Mishra et al., 2008; Shiga et al., 2015). Cells with similar or overlapping
features were later presented as BC mesenchymal stem cells (BC-MSCs) (Kalluri, 2016; Karnoub

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et al., 2007; Yan et al., 2012; Zhang et al., 2013). Like CAFs, the origin of those cells was
controversial. In fact, current evidence endorses an EMT based mechanism as a source of

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mesenchymal-like CSC resistance (Kalluri, 2016; Luo et al., 2015; Mani et al., 2008; May et al.,
2011; Polyak and Weinberg, 2009). Findings of the present study support the activation of a latent

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embryonic EMT program within a potent CSC pool of the cancer tissue. This EMT program has
maintained cell plasticity over long-term culture

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The link between EMT, CSC, and resistance to other cancer therapies including
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chemotherapies, molecular-targeted therapies, and immune-therapies has been supported by
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substantial evidence. (Du and Shim, 2016; Mallini et al., 2014; Shibue and Weinberg, 2017; Singh
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and Settleman, 2010; Zheng et al., 2015). They could revert readily, once exposed to serum-free
media, without additional stimulants, to epithelial-like cells and efficiently form non-adherent
mammospheres. The EMT/MET switch is also a recognized mechanism in cancer invasiveness
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and metastasis (Kalluri and Weinberg, 2009; Liu et al., 2013).This study provides further evidence
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that the EMT phenotype adopted by cells may also have reduced BC hormonal responsiveness.
Most relevant to our findings, the CD146, a proposed EMT activator, was linked to clinical
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tamoxifen resistance(Liang et al., 2017). Accordingly, targeting EMT could help reverse
tamoxifen resistance (Ward et al., 2013), and should be studied for aromatase inhibitors.
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The reported BC-SCa were positive for 3 master pluripotency markers (OCT-4, Nanog and
Sox-2). It appears that factors in the patient’s original tumour, together with in vitro culture factors,
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as outlined below, may have enriched for cells at the top of the hierarchical cancer organization.
This may also clarify why previous studies reported variable responses to hormonal stimuli, it is
likely that heterogeneous and dynamic BCSC populations were identified using different markers;
each stem cell population may have a unique hormonal signalling profile (Gasch et al., 2017).
Some breast cancer stem cells seem responsive to hormonal stimuli while others with enhanced

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pluripotency may be strictly hormonal resistant. Of note, single-cell gene expression analysis
supports the presence of an embryonic cancer stem cell pool (Akrap et al., 2016). The increased
expression of pluripotency markers has been linked to CSC properties, and gained recent attention
(Kim et al., 2013; Lagadec et al., 2012; van Schaijik et al., 2018). The co-expression of OCT-4
and Nanog was also associated with aggressive and metastatic cancer behaviour through the
activation of EMT (Chiou et al., 2010; Wang et al., 2014). Sox-2 was additionally linked to the

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mammosphere formation capacity of BC cells (Leis et al., 2012; Wang et al., 2014).

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ALDH1 gene over-expression is a well-recognized mechanism of cancer stem cell-based
resistance to chemotherapy. We detected ALDH1A1 expression in the majority of the BC-SCa

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population. Aldehyde dehydrogenase enzymes can provide potent detoxification function
dependent on efficient oxidation of their substrates, including an initial oxidative reaction that may

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have inactivated estradiol and letrozole in BCSC (He et al., 1999; Murai et al., 2009). In contrast,
a small percentage, though increased several folds over time, of the BC-SCa presented here, have
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expressed the CD44+/C24- profile, traditionally used for breast cancer stem cell isolation. Despite
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their popularity, the role and specificity of those surface markers remain debatable(Fillmore and
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Kuperwasser, 2007; Jaggupilli and Elkord, 2012). Moreover, the dissociation between the
CD44+/CD24- phenotype, and ALDH1A1 expression has been reported denoting they may not be
concurrent BCSC markers, but rather identify distinct stem cell populations at different hierarchy
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(Liu, Y. et al., 2014; Ricardo et al., 2011). In fact, ALDH1A1 but not the CD44+/C24-enriched
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cells were found to cause resistance to chemotherapy, hence could be a more predictive marker of
CSC based resistance (Miyoshi et al., 2016; Tanei et al., 2009). In an important study relevant to
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our findings, the ALDH1 expression had a predictive value for the failure of tamoxifen therapy
(Simões et al., 2015). Therefore, since ALDH inhibitors could sensitize CSC for cytotoxic
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chemotherapy (Croker and Allan, 2012), such strategy worth to be examined for hormonal re-
sensitization of BCSC.
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Culture method and its effect on stem cell activity: Breast cancer stem cell generation was achieved
previously by inducing the EMT phenotype using different elaborate protocols, including
immunogenic methods (Santisteban et al., 2009), activation of oncogenic cell signalling pathways
(Morel et al., 2008), or cytokines such as IL-6 (Sullivan et al., 2009). The explant culture method
applied in this study was successful in generating BC-SCa with minimal interventions. This

12
method is known for its reliability in generating limbal stem cells suitable for clinical corneal
repair (Rama et al., 2010), as well as mesenchymal stem cells from various tissues (Jing et al.,
2011). Moreover, the explant method is preferred, by our group, over primary cell culture methods
which start with dissociated cells. Those protocols include enzymatic and/or mechanical mincing
of the original cancer tissue, followed by multiple steps of washing, centrifugation, pipetting, and
cell sieving; all of which can risk the loss of the supposedly rare CSC population. By contrast,

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explant culture allows cells to grow spontaneously from BC tissue, without considerable disruption
of essential micro-environmental factors of the native CSC niche. Keeping the original tissue

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fragment in subcultures may further enhance the stemness of cells by supplying key biochemical
niche factors (Jing et al., 2011). Serum containing media were used in the initial explant culture to

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support tissue attachment and enhance cell migration (Pei et al., 2004) and was continued in
successive subcultures. Taken together, we believe the culture conditions in this study have

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enhanced the generation of the EMT phenotype of our cells.

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Relevant clinical data: This cell line was originally generated from BC tissue of a premenopausal
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patient with ER/PR+/Her2−invasive ductal carcinoma. Relevant clinical features included patient’s
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young age at diagnosis, and the rapid growth of a relatively large size, invasive stage II tumour
within a short period of 2 months (Supp. Table 1). Pathological examination indicated abundant
desmoplastic stroma in the tumour tissue (Supp. Fig. 1-A). Desmoplastic reactions were linked to
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poor clinical prognosis (Maeshima et al., 2002). Moreover, around 30% of the tumour was
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occupied by DCIS of the Comedo type, a pathological feature associated with high-grade
malignancy and increased recurrence (Maeshima et al., 2002).DCIS Comedo lesions may serve as
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precursors for basal BC cells, which in turn represent an aggressive type of the myoepithelial
lineage, commonly negative for Her2/neu as observed in this patient(Shekhar et al., 2013). These
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features suggest aggressive clinical behaviour that may be associated with the potent stem
cell/pluripotency program observed in this study. Although conclusions of this study will not be
used to interfere with the patient’s conventional clinical management plan, however, we
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hypothesize that long-term, anti-estrogens may not be a successful therapy for this patient. The
study presents an early model for a personalized approach of predicting response to hormonal
therapies, which can be extended to a larger number of patients in future studies. A similar model
in Glioblastoma has shown potential value as a CSC-based chemopredictive assay (Howard et al.,
2017).

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Study limitations: Cellular characterization after exposure to individual hormonal interventions
was not performed, as we were first interested to understand the de novo resistance behaviour of
the unexposed cells. Also, the in vivo tumorigenicity capacity of our cells was not evaluated in
this study.

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Conclusion: This report suggests that universal hormonal resistance to key endocrine therapies

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may indicate an efficient stemness behaviour of a dominant population of breast cancer cells with
stem cell features, comparable to what has been shown for cytotoxic chemotherapies. Based on

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this report and other evidence, we suggest that the increased expression of pluripotency markers
and ALDH1A1 in cancer stem cells may serve as potential markers for resistance to hormonal

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therapies. Further clinical studies are needed to develop this primary model into a potential stem
cell-based Hormonopredictive assay and evaluate its predictive accuracy for clinical response to
endocrine therapies.
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A
M
D
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A

14
Figure Legends

Figure 1: Effect of hormonal and anti-hormonal interventions on cell proliferation:

a) This bar graph shows the relative proliferation of cells cultured under the effect of different
hormonal conditions using estradiol, progesterone, and their antagonists independently and in
combination. The relative proliferation (indicated by % numbers above the bars) was evaluated
based on the percentage reduction of the metabolic reagent Alamar Blue ®. The positive
control represents cells incubated with ascorbic acid for maximal reagent reduction, and the

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negative control represents cells in control culture media without hormonal agents.
b) The bar graph shows the % viability of breast cancer stem cells exposed to Ulipristal acetate
and Letrozole-loaded PLGA nanoparticles in comparison to empty PLGA nanoparticles and

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negative control cells.

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Figure 2: Flow cytometry cell characterization for pluripotency markers
Flow cytometric analysis of the cells at passage 14. Cells were analyzed for their expression of the
pluripotency markers (OCT4, Sox2 and Nanog). Both the dot blot graphs (right) and the histogram

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views (left) are demonstrated.

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Figure 3: Flow cytometry cell characterization for ALDH1A1 expression:
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Cells were analyzed for their expression of ALDH1A1. The original dot blot graphs (right) and
the histogram views (left) are demonstrated.
M

Figure 4:
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A) Flowcytometry cell characterization for mesenchymal stem cell, hematopoietic markers

and the CD44+/CD24 low phenotype
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Dot plot graphs showing the results of flow cytometric analysis of the cultured breast cancer cells
at passages 11 (first vertical lane) and a second run of the same passage after 3 months (second
vertical lane). Four-colour flow cytometry was used for the analysis, showing the extent of the
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expression of key mesenchymal stem cell surface markers (CD73, CD105 and CD271), and the
hematopoietic cell markers (CD34 and CD45). This figure shows also the expression of surface
markers known to correlate with cancer stem cell properties in breast cancer (the CD44+/CD24
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low phenotype). Numbers represent the percentage of cells in different quadrants.

B) Osteogenic and adipogenic differentiation assays:

Control cells without the effect of hormonal agents are shown before (left) and after (right) the
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incubation with osteogenic (top lane) and adipogenic media (bottom lane). Differentiation towards
the osteogenic and adipogenic lineages was observed by detecting cells stained with Alizarin Red
and Oil O Red tissue-specific stains, respectively.

15
Figure 5: Mesenchymal-epithelial transition (MET):
A) An observation of a mesenchymal-epithelial transition of the cells on passage 11 once a serum-
free medium was used. The successive images obtained for the same cells, as shown by the
direction of the arrows, showing the intermediate stages between the mesenchymal and the
epithelial phenotype.

B) A prospective controlled study of the mammosphere formation capacity showing the

mesenchymal-epithelial transition and mammosphere formation of cells at passage 14, induced by

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the change to serum-free media.

C) Mammosphere formation assay: Efficiency of mammosphere formation was measured in cells

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cultured in serum-free and serum containing culture media in triplicates. The efficiency was
presented in the bar graph as MFE (%) = (number of mammospheres/well) / (number of cells
seeded per well) x 100.

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Supplementary Fig 1:

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A) Histopathologic examination of the tumour sample:

This image demonstrates the histopathologic examination of the patient’s tumour sample,
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stained by H&E. It shows evidence for an invasive ductal carcinoma. Tissue sections presented
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at 2 magnifications (x40 & x100) illustrate considerable desmoplastic stroma (thick black
arrows), surrounding epithelial like tumour cells (thin black arrows)
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B) Long-term explant culture model for the generation of BC-SCa This figure shows
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the successive stages of the presented long-term explant culture model. 1-2 mm tissue
fragments were cultured in replicates of 6-well plate representing day 1 of culture immediately
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after surgery (top left). After full confluency of the first passage, the same tissue fragment was
successfully re-cultured (2nd panel, right). Insets are showing higher magnification of the
cultured cells obtained by phase contrast microscopy (x40). (Slight cropping, colouring and
scaling of image size has been done for aesthetic purposes, without affecting the original data)
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.
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16
Conflict of interest: None of the authors have any competing interests in the manuscript.

Authors’ contributions: MN and MF performed the experiments and wrote the first draft of the
manuscript. TE provided histopathological examination, selection, and evaluation of tissue

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samples. ME supervised some experiments and critically reviewed the manuscript. AA and HF
helped in obtaining the surgical sample and follow up of clinical outcomes.ME and RR provided

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the nanoparticle formulations and studied their effect on cells. NM conceived and designed the
study, supervised experiments and data analysis, and wrote the manuscript.

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Acknowledgement: We would like to thank Professor Anthony Howell at U of Manchester for
critically reviewing the manuscript. We acknowledge the Science and Technology Development
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Fund (STDF) for funding the project. We would like to thank Dr. Nagwa EL-Badri and
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acknowledge the Center of Excellence for Stem Cell Research and Regenerative Medicine (CESC)
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at Zewail City of Science and Technology for contribution to funding and hosting cell
characterization studies. We acknowledge the Center for Medical Research Excellence (CMRE)
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at Assiut University for the use of their cell culture facility and imaging equipment. We are grateful
to Mr. Ahmed Badawi for his valuable technical help with flow cytometry analysis.
TE
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17
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