Loss of polarization in hypoxic breast epithelial cells.

<p><b>A.</b> Positive HIF-1α IHC staining of hypoxic cells (broken arrow) adjacent to the necrotic zone (star) in ductal carcinoma <i>in situ</i> of the breast. Small duct-like formations (arrows) in non-hypoxic regions close to the basal membrane in two different patient specimens of ductal carcinoma <i>in situ</i>. H/E; haematoxylin/eosin staining. Size bars 20 µm. <b>B</b>. Size and polarization of human breast epithelial cell acini grown on ECM-derived substrate at 21% and 1% oxygen. Actin (phalloidin, red) and nuclear (DAPI, blue) staining of normoxic (upper panel) and hypoxic (lower panel) primary human breast epithelial cells (left panels) and MCF-10A cells (right panel) at the indicated days post-seeding. The primary breast epithelial cell micrographs are from one representative time-series out of three sets of cultured breast cell samples from three different healthy women. All confocal micrographs were acquired at the Z-plane where the depictured acini-like structure had the widest circumference. Size bars 20 µm. <b>C.</b> Number of MCF-10A cell organoids of a given diameter (left) and the average size (right) of MCF-10A cell organoids cultured at 21% or 1% oxygen for 21 days on ECM-derived substrate. <b>D.</b> Number of polarized MCF-10A cell organoids of the given diameter (left) and the percentage of polarized organoids (right) after 21 days of culture on ECM-derived substrate at 21% or 1% oxygen. Data from one representative experiment out of three is shown. Organoids were considered polarized if 50% or more of the cells in the outer layer formed a palisade.</p

Expression of HIF-1α, HIF-2α, and HIF-target genes in normoxic and hypoxic breast epithelial cells. A.

<p>HIF-1α and HIF-2α immunohistochemical staining of MCF-10A acini-like structures after 21 days of 3D-culture at normoxia (21%) or hypoxia (1%). Sh-RNA-treated T47D breast cancer cells grown as monolayer and exposed to normoxia or hypoxia for 24h were used as controls. All cells were fixed in PFA and paraffin-embedded. Size bars 20 µm. <b>B.</b> Immunoblot analysis (left panel) of HIF-1α and HIF-2α in protein extracts of MCF-10A cells cultured in monolayer at 21% and 1% oxygen for the indicated period of time. Normoxic and hypoxic SK-N-BE cell extracts were used as controls. Quantification of the HIF signal intensity relative to the loading control (SDHA) (right panel). <b>C.</b> Relative mRNA expression of <i>HIF1A</i>, <i>HIF2A</i>, and the HIF-target genes <i>BNIP3</i>, <i>BHLHE40</i>, <i>OCT4</i>, and <i>VEGFA</i> in normoxic and hypoxic MCF-10A cells retrieved from 3D-cultures 21 days post-seeding. Data are from three independent experiments and statistical analysis was performed with Student’s paired t-test (p).</p

Proliferation and cell death in hypoxic and normoxic 3D-cultures in ECM-derived substrate. A

<p>Ki-67 immunofluorescence (green) and actin (red) staining of primary human breast epithelial cells in 3D-culture in ECM-derived substrate at 21% and 1% oxygen for 12, and 21 days. Representative images from one of three independent experiments with breast epithelial cells isolated from different healthy individuals are shown. Size bars 20 µm. <b>B.</b> MCF-10A cells stained for Ki-67 (green) and actin (red) after 3, 6, 12, and 21 days of 3D-culture in ECM-derived substrate under normoxic (21%) or hypoxic (1%) conditions. Representative images from one of three independent experiments are shown. Size bars 20 µm. <b>C.</b> Percentage of cells with Ki-67 positive nuclei in normoxic and hypoxic MCF-10A cell organoids 3, 12, and 21 days post-seeding, in three independent experiments. Statistical analysis was performed with Student’s paired t-test (p). In each experiment at least 200 cells were included in the calculation. <b>D.</b> Cell death in MCF-10A cells grown in 3D-culture under normoxic and hypoxic conditions for 12 days, by <i>in situ</i> cell death detection (red), nuclear staining with DAPI (blue). All confocal micrographs were acquired at the Z-plane where the depictured acini-like structure had the widest circumference. Size bars 20 µm. <b>E.</b> Percentage of cells with nuclei positive for <i>in situ</i> cell death detection in normoxic (21%) and hypoxic (1%) 3D-cultures at 9 and 12 days post-seeding. Data from four experiments are shown. In each experiment at least 200 cells were included in the calculation.</p

Functional and structural polarization of human breast epithelial cell organoids grown at 21% and 1% oxygen illustrated by the marker of breast epithelial differentiation and polarization laminin 5. A.

<p>Laminin 5 (green) immunofluorescence staining of human primary breast epithelial cells after 12 and 21 days of 3D-culture on ECM-derived substrate under normoxic (21%) or hypoxic (1%) conditions. Images from one representative of three independent experiments with cells from different individuals are shown. Size bars 20 µm. <b>B.</b> Immunofluorescence of laminin 5 (green) performed after 3, 6, 12, and 21 days of culture of MCF-10 cells in 3D-culture on ECM-derived substrate under normoxic (21%) or hypoxic (1%) conditions. All confocal micrographs were acquired at the Z-plane where the depictured acini-like structure had the widest circumference. Size bars 20 µm. <b>C.</b> The ratio of intra cellular to basal cell membrane mean fluorophore intensity in normoxic (open) and hypoxic (black) MCF-10A cells (left panel) and primary human breast epithelial cells (right panel) 21 days post-seeding, measured in one representative cell in ten different organoids. Statistical analysis was performed with Student’s t-test (p).In agreement with the non-malignant status of both the primary breast epithelial and the MCF-10A cells we found HMFG/MUC1 to have a polarized localization in normoxic acinar cells 21 days post-seeding (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046543#pone-0046543-g005" target="_blank">Fig. 5</a>A, B). The basal, as opposed to apical, localization of this protein is in agreement with MCF-10A cells showing little apical polarization as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046543#pone.0046543-Inman1" target="_blank">[27]</a>. HMFG/MUC1 displayed a decrease in polarized localization in both primary breast epithelial and MCF-10A cells at hypoxia (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046543#pone-0046543-g005" target="_blank">Fig. 5</a>A, B). In the MCF-10A cells there was a significant difference in the ratio of intra-cellular to basal localization at hypoxia compared to normoxia (p<0.0001, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046543#pone-0046543-g005" target="_blank">Fig. 5</a>C). The ratio of cytosolic to basal membrane localization could not be reliably determined in the primary breast epithelial cells. The <i>MUC1</i> mRNA expression was significantly decreased in the hypoxic MCF-10A cells after 21 days of 3D-culture (p = 0.013, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046543#pone-0046543-g005" target="_blank">Fig. 5</a>D).</p

ID1 expression and histone acetylation in breast epithelial cells in normoxic compared to hypoxic 3D-cultures.

<p><b>A.</b> Relative mRNA expression of the transcription modulating ID factors, <i>ID1</i> and <i>ID2</i> in MCF-10A cells after 21 days of 3D-culture on ECM-derived substrate under normoxic and hypoxic conditions (left). Showing data from three independent experiments. Statistical analysis was performed with Student’s paired t-test (p). Immunohistochemical staining for ID1 on paraffin-embedded MCF-10A acini-like structures after 21 days of 3D-culture at normoxia (21%) or hypoxia (1%) (right). Size bars 20 µm. <b>B.</b> Acetylated histone 4 (AcH4) visualized by immunofluorescence (green) in normoxic (21%) and hypoxic (1%) human primary breast epithelial cell (left) and MCF-10A cell (right) acini-like structures at the indicated days post seeding. Actin was visualized by phalloidin staining (red). Size bars 20 µm. <b>C</b>. Percentage of MCF-10A cells in acini-like structures with global histone acetylation, i.e. positive for AcH4, 21 days post-seeding at normoxia (open boxes) and hypoxia (black boxes), showing data from three independent experiments. At least 200 cells were calculated in each experiment. Statistical analysis was performed by Student’s paired t-test (p). <b>D.</b> Immunoblot of AcH4 in MCF-10A cells in 3D-culture at 21% (left) and 1% (right) on ECM-derived substrate for 10 days. SDHA was used as a loading control. <b>E.</b> Non-malignant breast epithelial cells grown on differentiation-inducing ECM have an organized cell shape and a high degree of deacetylated histones; these cells differentiate and become post-mitotic. Contrary, breast epithelial cells grown as monolayer without ECM do not receive/accept signals to induce differentiation, leading to sustained global histone acetylation and opening of the chromatin for transcription, resulting in impaired differentiation and/or dedifferentiation accompanied with cell proliferation. Hypoxia, including hypoxic induction of ID1, promotes a proliferative and undifferentiated state in breast epithelial cells despite contact with the ECM.</p

Localization of Human Milk Fat Globule (HMFG) in human breast epithelial cells at normoxia and hypoxia.

<p><b>A.</b> Cellular localization of HMFG (green) in primary human breast epithelial cells after 21 days of 3D-culture on ECM-derived substrate at normoxic (21%) or hypoxic (1%) conditions. Images from one representative of three independent experiments with cells from different individuals are shown. Size bars 20 µm. <b>B.</b> Staining of HMFG (green) on 3D-cultures of MCF-10A cells after 21 days of culture on ECM-derived substrate at normoxic (21%) or hypoxic (1%) conditions. All confocal micrographs were acquired at the Z-plane where the depictured acini-like structure had the widest circumference. Size bars 20 µm. <b>C.</b> The ratio of intra cellular to basal cell membrane mean fluorophore intensity in normoxic (open) and hypoxic (black) MCF-10A cell organoids at 21 days post-seeding, measured in one representative cell in ten different acini-like structures. Statistical analysis was performed with Student’s t-test (p). <b>D.</b> Relative mRNA levels of <i>MUC1</i> in normoxic (21%) and hypoxic (1%) 3D-cultures of MCF-10A cells 21 days post-seeding, showing data from three independent experiments. Statistical analysis was performed with Student’s paired t-test (p).</p

Functional and structural polarization of the human breast epithelial acini-like structures cultured at 21% and 1% oxygen on ECM-derived substrate illustrated by the marker of breast epithelial polarization, α6-integrin. A.

<p>Immunofluorescence staining of the polarization marker α6-integrin (green) after 12 and 21 days of culture of primary human breast epithelial cells on ECM-derived substrate under normoxic (21%) or hypoxic (1%) conditions. Images from one representative of three independent experiments with cells from different women are shown. Size bars 20 µm. <b>B.</b> α6-integrin (green) staining of normoxic (21%) and hypoxic (1%) MCF-10A cells in 3D-culture at 3, 6, 12, and 21 days post-seeding on ECM-derived substrate. All confocal micrographs were acquired at the Z-plane where the depictured acini-like structure had the widest circumference. Size bars 20 µm. <b>C.</b> The ratio of intra cellular to basal cell membrane mean fluorophore intensity in normoxic (open) and hypoxic (black) MCF-10A cell- (left panel) and primary human breast epithelial cell organoids (right panel) at 21 days post-seeding, measured in one representative cell in ten different acini-like structures. Statistical analysis was performed with Student’s t-test (p).</p