Breast Tissue Collection

Central Adelaide Local Health Network Human Ethics Research Committee approved this study (TQEH Ethics Approval #2011120). Participants aged 18 to 75, who underwent cosmetic reduction, prophylactic mammoplasty, and mastectomy for breast cancer removal, generously donated tissue. Participants who were pregnant or had undergone chemotherapy, as well as those with a large breast tumour or multiple tumours, were excluded from the study. Breast tissue was collected directly from the surgical theatre and used for histology and in vitro culture studies.

Breast Tissue Histology

For histology studies, a portion of breast tissue samples were dissected into small portions and fixed in 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA; Cat#P6148) for 7 days at 4 °C. Tissue were processed using a Leica TP1020 Tissue Processor (Leica Microsystems, Wetzlar, Germany) and embedded in paraffin wax. Sections of 5 μm thickness were cut using a Leica Rotary Microtome (Leica Microsystems), and slides were incubated overnight at 37 °C to promote tissue adhesion.

Breast Tissue Processing for Mammary Epithelial Organoids

For mammary epithelial organoids, fresh breast tissue had excess fat removed prior to being finely minced and enzymatically digested with 100 U/mL of Hyaluronidase (Sigma Aldrich; Cat# H3506) and 480 U/mL of Collagenase (Sigma Aldrich; Cat# C0130) in Advanced DMEM/F12 medium (Life Technologies, Carlsbad, CA, USA; Cat# 12491015) containing 2.5 mg/mL Fungizone (Life Technologies; Cat# 15290018), 100 U/mL Penicillin/Streptomycin (Life Technologies; Cat# 15240062), 10 mM HEPES buffer (Life Technologies; Cat# 15630080), and 100 µg/mL L-Glutamine (Life Technologies; Cat# 25030081). The digestion flask was placed in a 37 °C shaking water bath for 16 h to separate the stromal and epithelial components of the tissue. The digested tissue was diluted with DMEM media at least 1:3 and centrifuged at 80 x g for 1 min at a moderate brake to separate the digested tissue into the top lipid layer, middle layer of stromal single cells, and bottom pellet containing mammary epithelial organoids and undigested breast tissue pieces. These three layers were harvested for further processing.

To isolate mammary epithelial organoids, the bottom pellet containing mammary epithelial cells and undigested tissue was transferred to fresh 50 mL Falcon tubes. DMEM media was added to wash and mix the pellet before sitting the tubes for 30–60 s for gravity settling of undigested tissue and then harvesting the supernatant containing mammary epithelial organoids to a separate tube. Mammary epithelial organoids were washed again with DMEM/F12 media and isolated from the supernatant through gravity settling for four minutes; this step was repeated to wash the organoids until the supernatant was clear. After organoids were verified via microscopic analysis, organoids were centrifuged at 480 x g for 1 min, supernatant removed, and resuspended in Advanced DMEM/F12 medium supplemented with 50% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Cat#10091-148), 6% Dimethyl sulfoxide (DMSO; Sigma-Aldrich, Cat#34869), Penicillin/Streptomycin, Fungizone, glutamine, and Fungizone, and stored in liquid nitrogen.

Haematoxylin and Eosin Staining

Formalin-fixed-paraffin-embedded (FFPE) breast tissue blocks obtained were subjected to hematoxylin and eosin (H&E) staining. First, breast tissue sections were dewaxed using 3 different containers of xylene (Merck Millipore, Darmstadt, Germany; Cat#108298), each for 5 min, secondly, they were rehydrated in gradual dilutions of ethanol (2 × 100%, 1 × 90%, 1 × 70% and 1 × 50%), each for 3 min, followed by 3 min in distilled water. Tissue sections were stained with hematoxylin (Sigma Aldrich, St Louis, MO, USA; Cat#HHS16) and eosin (Sigma Aldrich, St Louis USA; Cat#318906), respectively for 30 and 10 s. Sections were then dehydrated through a gradual increase in ethanol concentration (1 × 90%, 2 × 100%) and cleared with 2 different containers of xylene for 5 min. Finally, slides were coverslips with mounting media (Proscitech, Kirwan, QLD, Australia; Cat#IM022) and left to dry and further analysis.

Histological Index of Mammographic Density

Fibroglandular density was assessed as a surrogate indicator for mammographic density as described previously [24, 25]. Briefly, a panel of three scientists (H.H., L.H., W.I.) observed haematoxylin and eosin-stained breast tissue sections on a semi-quantitative scale. The assessment was based on the relative abundance of fibroglandular tissue and adipose tissue in the section. For each tissue block, one tissue section was assessed, and the panel reached a consensus on density through discussion. Higher density scores were assigned to sections containing a greater percentage of stroma and epithelium. The classification scale ranged from 1 to 5, where 1 represented 0–10%, 2 represented 10–25%, 3 represented 25–50%, 4 represented 50–75%, and 5 represented > 75% of fibroglandular tissue [26]. This method has demonstrated a reliable correlation with mammographic density determined by X-ray [3]. Tissue scoring between 1 and 2 was classified as low mammographic density, while tissue scoring between 4 and 5 was classified as high mammographic density. Tissue scoring 3 was not classified as low or high and was not utilised in this study [26].

Immunohistochemistry Staining

The expression of TLR4, MYD88, NFKB, LPS and HMGB1 were assessed through immunohistochemistry. As described previously, dewaxing and hydrating tissue were applied. Then, antigen retrieval was performed using the high pH retrieval solution (DAKO, Cat#K8004) on paired high and low mammographic density tissue. Later, the tissue was incubated with endogenous peroxidase block for 10 min at room temperature. The sections were washed three times for three minutes in 1x PBS buffer, and then the mouse monoclonal to TLR4 with the concentration of 1/50 (Abcam, Cat#ab22048), 1/500 rabbit monoclonal to MYD88 (Abcam, Cat#ab133739), 1/1000 rabbit polyclonal to NFKB p65 (Abcam, Cat#ab16502), 1/300 mouse monoclonal to E.coli LPS (Abcam, Cat#AB35654), 1/400 rabbit monoclonal to HMGB1 (Abcam, Cat#AB79823 were applied and incubated in a humid chamber, overnight at 4 degrees Celsius. Later, the sections were treated with an HRP-conjugated secondary antibody (Agilent Technologies, Singapore; Cat#K346811-2), after 45 min, diaminobenzidine (DAB) (Agilent Technologies; Cat#K346811-2) was added to the slides and incubated for 10 min at room temperature. The slides were then properly washed in distilled water, counterstained with hematoxylin, dehydrated in different percentages of ethanol from 90% to 100%, and 2 changes of xylene to be coversliped with a mounting medium.

Quantification of Immunohistochemistry Images

All stained immunohistochemistry slides were digitally scanned at a magnification of 40x and resolution of 0.23 μm using the NanoZoomer Scanner 2.0-HT (The University of Adelaide, Adelaide Microscopy). The intensity of the staining was quantified using ImageJ (Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. 2022. ImageJ, Version 1.53). Colour deconvolution was applied to separate the hematoxylin and DAB staining components. In ImageJ, the pixel intensity values for any colour range from 0 to 255, where 0 corresponds to the darkest shade and 255 displays the lightest shade of the colour. For quantitative assessment, multiple random regions of both epithelium and adjacent stromal tissue were identified on each histological section. Within each region, the intensity of staining was measured using a fixed threshold setting, which was applied uniformly across all pairs. For each section, the measurements from all selected regions were averaged to yield a single representative value. The mean intensity value from each slide was taken for statistical analysis.

Immunofluorescence Staining

The localisation of TLR4 on epithelium and stroma was investigated by immunofluorescence staining using a mouse monoclonal antibody to TLR4 (Abcam, Cat#ab22048) at a dilution of 1/50, followed by secondary goat anti-mouse 647 Alexa fluor conjugated antibody (Invitrogen, Lot#WB316325). Keratin 17 (KRT14) 594 Alexa fluor conjugated antibody (Santa Cruz Biotechnology, Sc-53253#LotDo822) at a dilution of 1:100, and Keratin 19 (KRT19) 488 Alexa fluor conjugated antibody (Invitrogen, eBiosciense #Lot 22199998) at a dilution of 1:500, were used to identify basal and luminal epithelial cells respectively. Co-localisation of CD68-positive macrophages with TLR4 expression and LPS-positive cells was also determined using the antibodies mentioned above. To investigate cellular location of HMGB1, immunofluorescence staining was performed using HMGB1 antibody (Abcam, Cat#AB79823) and followed by secondary goat anti-rabbit 568 Alexa fluor conjugated antibody (Invitrogen, Cat#A-11011), on high and low density tissue. Briefly, breast tissue sections were deparaffinized and hydrated, followed by antigen retrieval. Blocking non-specific binding was applied by incubating tissue with 10% goat serum in BSA for 30 min at room temperature, plus TrueBlack Lipofuscin Autofluorescence Quencher (Biotium, Fremont, CA) for 45 s. Primary and conjugated antibodies were incubated overnight at 4 °C, and the secondary antibody was incubated for 1 h at room temperature. After each step slides were washed 3 times for 3 min with PBS. Finally, slides were stained with DAPI for 5 min and coverslipped using fluorescent mounting media. Immunofluorescent images were captured using a Zeiss LSM 900 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Where comparison between high and low fibroglandular density was required, the fluorescence intensity setting was kept constant throughout the entire imaging process. All secondary antibody was applied at a 1/500 dilution.

3D Culture of Human Epithelial Organoids

After collecting from liquid nitrogen, organoids were suspended in Human Mammary Epithelial Cell Basal Medium (Life Technologies; Cat# M171500), supplemented with 1X Mammary Epithelial Growth Supplement (MEGS) (Life Technologies; Cat# S0155). Next, cells were centrifuged at 400 x g for 5 min, and the pellet was resuspended in 1 mL media. Working quickly on ice, organoids were generated using the dome approach, where cell suspensions were mixed well with Matrigel (BD, Growth Factor Reduced, Phenol Red-Free #356231), at a 1:2 ratio. One drop of 25 µL Matrigel-cell mixture with the density of 5× 10⁴ cell was placed in the centre of a prewarmed 24-well suspension sterile cell culture plate (Greiner Bio-One; Cat# 662102). The plate was incubated at 37 °C for 20 min to allow Matrigel to solidify. Once the Matrigel polymerised, 500 µl of complete Medium, supplemented with 10 µM of Y-27,632 dihydrochloride, Rho kinase inhibitor (Abcam, Cat# ab120129), was carefully added to the well on top of the dome. The culture Medium was changed every 3–4 days and 3 days after initial seeding, Y-27,632 was removed from the Medium. Cells were cultured for around 10 days to allow organoids to stabilise and grow before treatment.

3D organoid cultures were incubated with 10 mL of LPS (Sigma-Aldrich, #L2630) at 37 °C, 5% CO2 for 24 h. After gently aspirating the Medium, cold Cell Recovery Solution (Bio-Strategy, #Cat BDAA354253) was added into wells, droplets were then mechanically disassociated with a cell scraper. Once the organoids were dislodged, they were transferred into sterile tubes and incubated on ice for 2 h to fully dissolve the Matrigel.

Quantification of Gene Expression

Total RNA was isolated using RNeasy mini kit (QIAGEN, Australia; cat#74104) as per the manufacturer‘s instructions. For complete DNA removal, RNase-Free DNase Set (QIAGEN, Australia; cat#79254) was used. The concentration of purified RNA was measured by absorbance at 260 nm relative to absorbance at 280 nm using Spectrophotometer ND 1000 Nanodrop with program ND-1000 V3.7.1. Later the appropriate concentration of RNA was reverenced transcript to cDNA using Moloney murine leukemia virus (MMLV) enzyme from iScript synthesis kit (Bio-Rad technologies; Cat#1708890). The 20 µL reaction contains 4 µL iScript reaction Mix, 1µL iScript reverse transcriptase, 2 µL isolated RNA, and 13 µL nuclease-free water. The complete reaction mix was incubated in the thermal cycler as follows 5 min at 25 °C, 20 min at 46 °C, and finally 1 min at 95 °C.

Real-Time PCR was conducted to quantify gene expression of TLR4, MYD88, NFKB, HMGB1, TNFA, TGFB1, CCL2, COX2 and VIM. The 10 µL reaction contained 5 µL of SYBR green master mix (Bio-Rad), 0.5 µL of each forward and reverse primers, 2 µL of cDNA, and 2 µL of nuclease-free water. Reactions were run on the thermocycler machine (Applied Biosystems ViiA 7) with following thermal cycling protocol; DNA denaturation at 95 °C for 3 min, amplification with 40 cycles of 15 s at 95 °C, 60 s at 60 °C, and 72 °C for 30 s. Each run contains no template control of each gene. Beta-Actin was used as a reference gene. Real-time PCR data were analysed using the ΔΔCt method, comparing the expression levels of target genes relative to control groups and normalised to the reference gene.

Statistical Analysis

Statistical analysis was conducted using SPSS software (IBM.SPSS statistics, Chicago, IL, USA) version 28.01.1, and GraphPad Prism 8 (GraphPad software Inc, San Diego, USA) version 10.1.0. when p < 0.05, also showed as an asterisk (*), data are considered significantly different from the control, or data between low and high fibroglandular density are significantly different. All data in this study are presented as mean ± SEM (standard error of mean). The quantification of the paired sample from the immunohistochemistry study was performed by a non-parametric Wilcoxon test. The qPCR RT data were assessed based on fold change. mRNA expression level was analysed, using 2-way ANOVA with Šídák’s multiple comparisons test.