Mast Cell Tryptase & Breast Cancer Growth | Nuclear Remodelling

Antibodies

Anti-mast cell tryptase [AA1] (Abcam, ab2378), anti-Mcpt6 immune serum [53]anti-Ki67 [SolA15] (eBioscience, #14-5689-82), Anti-GAPDH [6C5] (Santa Cruz Biotechnology, sc-32233), Anti-histone H2A (Abcam, ab18255), Anti-histone H2B (Abcam, ab1790), Anti-histone H3 (Abcam, ab1791), Anti-histone H4 (Abcam, ab10158), Anti-monomethyl-histone H3 (Lys4) [D1A9] (Cell Signaling, mAb#5326), Anti-dimethyl-histone H3 (Lys9) (Merck, #07-441), Anti-trimethyl-histone H3 (Lys9) (Abcam, ab8898), Anti-trimethyl-histone H3 (Lys27) (Abcam, ab6002), Anti-acetyl-histone H3 (Lys9) (Abcam, ab10812), Anti-acetyl-histone H3 (Lys14) (Cell Signaling, mAb#4318), Anti-histone H3 pan-acetyl (Active Motif, #39139), Anti-acetyl-histone H3 (Lys27) (Active Motif, #39133), Anti-CYR61/CCN1 [D4H5D] (Cell Signaling, mAb#14479), Anti-CTGF/CCN2 [D8Z8U] (Cell Signaling, mAb#86641), Anti-CMA1/chymase (Atlas Antibodies, HPA052634).

Hs578T cells

The Hs578T triple-negative human breast cancer cell line (ATCC) was cultured in Dulbecco’s modified Eagle’s medium (Gibco), supplemented with 10% heat-inactivated foetal bovine serum (Invitrogen), 50 μg/ml streptomycin sulphate (Sigma-Aldrich), 60 μg/ml penicillin G (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich). Cells were kept at 37 °C in 5% CO₂ and medium was replaced when cells achieved 90-100% confluency.

BT-549 cells

The BT-549 triple-negative human breast cancer cell line (ATCC) was cultured in RPMI-1640 (Gibco), supplemented with 10% heat-inactivated foetal bovine serum (Invitrogen), 50 μg/ml streptomycin sulphate (Sigma-Aldrich), 60 μg/ml penicillin G (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich) and 10 μg/mL recombinant human insulin (Sigma-Aldrich). Cells were kept at 37 °C in 5% CO₂ and medium replaced when cells achieved 90–100% confluency.

BT-20 cells

The BT-549 triple-negative human breast cancer cell line (ATCC) was cultured in cultured in Dulbecco’s modified Eagle’s medium (Gibco), supplemented with 10% heat-inactivated foetal bovine serum (Invitrogen), 50 μg/ml streptomycin sulphate (Sigma-Aldrich), 60 μg/ml penicillin G (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich). Cells were kept at 37 °C in 5% CO₂ and medium replaced when cells achieved 90–100% confluency.

MCF10A cells

The MCF10A immortalized non-tumorigenic human breast epithelial cell line (ATCC) was cultured in DMEM/F12 (Thermo Fisher), supplemented with 5% heat-inactivated foetal bovine serum (Invitrogen), 50 μg/ml streptomycin sulphate (Sigma-Aldrich), 60 μg/ml penicillin G (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 20 ng/mL recombinant human EGF (Sigma-Aldrich), 100 ng/mL cholera toxin (Sigma- Aldrich), 0.5 μg/mL hydrocortisone (Sigma-Aldrich), 10 μg/mL recombinant human insulin (Sigma-Aldrich). Cells were kept at 37 °C in 5% CO₂ and medium replaced when cells achieved 90–100% confluency.

Cell viability

Cells were harvested, washed once with PBS, and resuspended in Annexin binding buffer (BD Pharmingen). They were then stained with Annexin V-FITC and Draq7, followed by analysis using a BD Accuri C6 Plus flow cytometer (BD). Viability was also assessed using PrestoBlue (Invitrogen). Cells were harvested, resuspended in culture medium, and seeded in triplicates into a 96-well plate. A 10X concentrated PrestoBlue solution was added, and the cells were incubated for 1 h at 37 °C in 5% CO₂. Fluorescence was then measured with a TECAN Infinite M200 plate reader at an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

Transmission electron microscopy (TEM)

Samples were initially fixed in a solution of 2.5% glutaraldehyde (Ted Pella) and 1% paraformaldehyde (Merck) in PIPES buffer at pH 7.4 and stored at 4 °C until processing. They were then rinsed in 0.1 M PBS for 10 minutes and incubated for 1 h in 1% osmium tetroxide (TAAB; Berks, UK) in 0.1 M PBS. After another rinse in 0.1 M PBS, samples underwent dehydration through a graded ethanol series (50%, 70%, 95%, and 99.9%), with each step lasting 10 minutes, followed by a 5-minute treatment in propylene oxide (TAAB). Next, samples were placed in a 1:1 mixture of Epon Resin (Ted Pella) and propylene oxide for 1 h, transferred to 100% resin, and left overnight. Finally, samples were embedded in fresh Epon resin within capsules, left for 1–2 h, and polymerized at 60 °C for 48 h. Ultrathin sections (60–70 nm) were then cut using an EM UC7 Ultramicrotome (Leica), mounted on grids, and contrasted with 5% uranyl acetate and Reynold’s lead citrate. Visualization was performed using a Tecnai™ G2 Spirit BioTwin transmission electron microscope (Thermo Fisher/FEI) operating at 80 kV, with imaging captured by an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.)

EdU labelling

Cells (0.1 × 10⁶ cells/ml) were seeded into wells of a 24-well plate. Cells were left untreated or treated with 50 nM human recombinant beta-tryptase (Promega) and incubated at 37 °C for 48 h. Two hours prior to cell harvesting, 10 µM EdU was added to the cultures. After incubation, cells were washed in PBS, trypsinized and harvested by centrifugation (400 x g, 5 min) and stained using the Click-iT^TM Plus EdU Alexa 647 Flow Cytometry Kit (ThermoFisher). Samples were then analysed using a BD Accuri C6 Plus flow cytometer (BD), with data collected from 10,000 events per sample and analysed using FlowJo software (BD Biosciences).

Tryptase labelling

Recombinant human tryptase was labelled using the Alexa Fluor^TM 488 protein labelling kit (Invitrogen), according to manufacturer’s instructions.

Time-lapse microscopy

Hs578T breast cancer cells (0.1 × 10⁶ cells/ml) were seeded into a 24-well plate and left to attach for approximately 2 h at 37 °C and 5% CO₂ into an incubation chamber at a time-lapse Eclipse Ti microscope equipped with NIS-Elements software (Nikon). Cells were either left untreated or treated with 50 nM Alexa-488-labelled tryptase. Plates were then incubated for 1 h. Next, combined bright-field and fluorescent images were acquired every 2 min for a period of 30 min.

Laser-scanning microscopy

Aliquots of 100 µl from cell suspensions (10⁵ cells/ml) were left untreated or treated with 50 or 100 nM recombinant human tryptase in 8-chamber tissue culture glass slides (Corning). After treatment, supernatants were removed, rinsed in PBS, and fixed with 4% paraformaldehyde in PBS for 15 minutes. To permeabilize the cells, 100 μl of 0.1% Triton-X100 in PBS was added to each slide, followed by incubation for 10 min at room temperature. Slides were then washed twice in PBS/1% BSA and blocked with 10% goat serum in PBS for 30 minutes at room temperature. Next, 100 μl of mouse anti-human mast cell tryptase (AA1; Abcam) diluted 1:100 in TBS/1% BSA or isotype control at the same concentration was added and incubated overnight at 4 °C. Following incubation, the slides were washed three times with TBS. Goat anti-mouse Alexa-488-conjugated antibody (diluted 1:1000 in PBS/1% BSA) was then added, followed by incubation for 1 h at room temperature in dark. Slides were washed three times with PBS/1% BSA and stained with ActinRed-555 (Invitrogen) and Hoechst 33342 (NucBlueTM, Invitrogen) for 10 minutes, followed by three washes with PBS. Slides were mounted using SlowFade® Gold Antifade Mounting Medium (Life Technologies), and analysed using a LSM 710 laser-scanning microscope equipped with ZEN 2009 software (Carl Zeiss). Z-stack sections were acquired and used for 3D image assembly and analysis with Imaris software (Oxford Instruments).

Subcellular fractionation

Hs578T cells (1 × 10⁶) were diluted to a final concentration of 0.2 × 10⁶ cells/ml in DMEM (Gibco), supplemented with 1% heat-inactivated foetal bovine serum (Invitrogen), 50 µg/ml streptomycin sulphate (Sigma-Aldrich), 60 µg/ml penicillin G (Sigma-Aldrich), and 2 mM L-glutamine (Sigma-Aldrich). Cells were seeded into a TC-60 dish (Corning) and incubated overnight at 37 °C in a humidified atmosphere containing 5% CO₂. The following day, cells were either left untreated (control) or treated with 50 nM recombinant human tryptase for 48 hours. After treatment, the culture medium was removed, and cells were washed with PBS, trypsinized, and harvested by centrifugation at 400 × g for 5 min. Cell pellets were washed twice with PBS and processed for subcellular fractionation using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific), following the manufacturer’s instructions. The resulting fractions were collected into pre-chilled tubes and stored at −18 °C until further use.

Tryptase ELISA

Subcellular fractions from untreated and 50 nM tryptase-treated Hs578T cells were analysed using the Human Tryptase beta-2/TPSB2 ELISA Kit (Thermo Scientific), following the manufacturer’s protocol. The assay was performed in two independent experiments, each conducted in duplicate. Data from both experiments were pooled for analysis.

PyMT tumour sections

Randomisation or blinding was not used. Mice were on FVB/n genetic background (females; 14 weeks). Paraffin-embedded PyMT murine breast cancer tumour sections [54] were deparaffinized and hydrated. The sections were then fixed with 4% paraformaldehyde in TBS for 15 minutes, washed twice in PBS, permeabilized with 0.1% Triton-X100 in TBS for 10 minutes at room temperature. The slides were then washed twice in TBS/1% BSA and blocked with 10% goat serum in TBS for 30 minutes at room temperature. Next, rabbit anti-mouse tryptase (Mcpt6) immune serum diluted 1:500 in TBS/1% BSA or pre-immune serum control at the same concentration was added and incubated overnight at 4 °C. Following incubation, the slides were washed three times with TBS/1% BSA. Goat anti-rabbit Alexa-488-conjugated antibody in TBS/1% BSA was then added and incubated for 1 h at room temperature in the dark. Next, Rabbit anti-mouse Alexa-647-Ki67 (Novus biologicals) diluted 1:500 TBS/1% BSA or isotype control at the same concentration was added and incubated overnight at 4 °C. Following incubation, the slides were washed three times with TBS/1% BSA and stained with Hoechst 33342 (NucBlue^TM; Invitrogen) for 10 minutes, followed by three washes with TBS. The slides were mounted using SlowFade® Gold Antifade Mounting Medium (Life Technologies). Samples were analysed using a LSM 710 laser-scanning microscope equipped with ZEN 2009 software (Carl Zeiss). Z-stack sections were acquired and used for 3D image assembly and analysis with Imaris software (Oxford Instruments). Data are representative of 7 analysed tissue sections.

Human breast cancer tumour sections

Tissue sections from triple negative breast cancer and adjacent healthy tissue were obtained (Uppsala Biobank; ethical approval: Etikprövningsmyndigheten; no 2020-00238). Paraffin-embedded tissue sections were deparaffinized and hydrated. Sections were then fixed with 4% paraformaldehyde in PBS for 15 minutes, washed twice in PBS, permeabilized with 0.1% Triton-X100 in TBS for 10 min at room temperature. Slides were then washed twice in TBS/1% BSA and blocked with 10% goat serum in TBS for 30 minutes at room temperature. Next, mouse anti-human mast cell tryptase (AA1; Abcam; diluted 1:100 in TBS/1% BSA) or isotype control at the same concentration was added, followed by incubation overnight at 4 °C. Next, the slides were washed three times with TBS/1% BSA. Goat anti-mouse Alexa-488-conjugated antibody (diluted 1:1000 in TBS/1% BSA) was then added and incubated for 1 h at room temperature in the dark. Next, rabbit anti-human Alexa-647-Ki67 (Novus biologicals) diluted 1:500 TBS/1% BSA or isotype control at the same concentration was added and incubated overnight at 4 °C. The slides were then washed three times with TBS/1% BSA and stained with Hoechst 33342 (NucBlue^TMInvitrogen) for 10 min, followed by three washes with TBS. Slides were mounted using SlowFade® Gold Antifade Mounting Medium (Life Technologies). Samples were analysed using a LSM 710 laser-scanning microscope equipped with ZEN 2009 software (Carl Zeiss). Z-stack sections were acquired and used for 3D image assembly and analysis with Imaris software (Oxford Instruments). Data are representative of 15 analysed tissue sections.

Fluorescence microscopy

Frozen tissue sections from a triple-negative human breast cancer tumour were fixed and permeabilized with ice-cold methanol for 15 minutes. Slides were then washed twice with PBS containing 1% BSA (PBS/1% BSA) and blocked with 5% goat serum in PBS for 30 minutes at room temperature. Subsequently, slides were incubated overnight at 4 °C with rabbit anti-human mast cell chymase 1 antibody (CMA1; Atlas Antibodies) diluted 1:500 in PBS/1% BSA. Negative controls were prepared by incubating sections with PBS/1% BSA only. After five washes in PBS/1% BSA, slides were incubated for 1 h at room temperature with goat anti-rabbit Alexa Fluor 488 antibody diluted 1:1000 in PBS/1% BSA. Following three additional washes, slides were incubated overnight at 4 °C with mouse anti-human mast cell tryptase antibody (AA1; Abcam) diluted 1:100 in PBS/1% BSA. Corresponding negative controls were prepared in parallel. Slides were then washed five times in PBS/1% BSA and incubated for 1 h at room temperature with goat anti-mouse Alexa Fluor 555 antibody diluted 1:1000 in PBS/1% BSA. After three final washes in PBS/1% BSA, nuclei were stained with Hoechst 33342 (NucBlue™, Invitrogen) for 10 min. Slides were washed again, dried at room temperature, and mounted using SlowFade® Gold Antifade Mountant (Life Technologies). Samples were imaged using a Nikon 90i fluorescence microscope equipped with NIS-Elements software (Nikon).

Western blot analysis

Equal numbers of cells were collected and solubilized in Laemmli buffer (Bio-Rad). The samples were then subjected to SDS-PAGE using Bio-Rad 4–20% Mini-Protean TGX stain-free gels, followed by transfer to a 0.2 µm nitrocellulose membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad) using the Bio-Rad Trans-Blot Turbo transfer system. Membranes were blocked with EveryBlot blocking buffer (Bio-Rad), incubated with primary antibody overnight at 4 °C, and then with a StarBright B700 secondary antibody (Bio-Rad; diluted 1:5000 in TBS) for 1 h at room temperature. The membranes were scanned using a ChemiDoc^TM MP imaging system equipped with Image Lab^TM Touch Software (Bio-Rad).

Ampliseq transcriptome analysis

Hs578T (1 × 105 cells) were incubated in quadruplicates in wells of a 12-well plate for 6 or 24 h, with or without 50 nM tryptase. Cells were harvested and total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. The samples were analysed at NGI Sweden (Uppsala). cDNA libraries were generated and amplified using the Ion AmpliSeqTM Transcriptome Human Gene Expression Kit (Life Technologies), following the protocol provided by the manufacturer, and sequencing was performed on an Ion S5TM XL Sequencer (Thermo Fisher Scientific). Normalized expression values generated by Torrent SuiteTM Software (version 5.10.1) were used for downstream differential expression analyses using the edgeR package (Robinson, McCarthy, and Smyth 2010) implemented in R (version 3.6.0) (The R Foundation n.d.). The full data set is available: GEO Submission (GSE307724); NCBI tracking system #25363733.

ATAC-seq analysis

Hs578T cells (0.5 × 10⁵ cells) were seeded into wells of a 24-well plate. Triplicates of cells were left untreated or treated with 50 nM recombinant human skin tryptase (Promega) at 37 °C for 48 h. Cells were then harvested by centrifugation at 500 x g for 10 min at 4 °C and cell pellets were resuspended in 50 μl of cold lysis buffer (10 mM Tris HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂0.1% NP-40, 0.1% Tween-20 and 0.01% digitonin) and incubated on ice for 3 min. Pellets were resuspended with 1 ml wash buffer (10 mM Tris HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂0.1% Tween-20) and cell nuclei were pelleted at 500 x g for 10 min at 4 °C. Supernatants were removed and pellets were resuspended in 50 μl of tagmentation master mix (TD buffer, 0.5% tween 20, 0.5% digitonin and TDE1 enzyme). Samples were incubated for 30 min at 37 °C in a thermomixer 5436 (Eppendorf) with a mixing speed of 1000 rpm, followed by adding 250 μl of ChIP DNA binding buffer to the tagmented DNA. The mixture was transferred to a Zymo-Spin^TM IC-XL column in a collection tube, centrifugated for 30 sec at 13,000 x g and the flow-through was discarded. The column was washed twice with 200 μl DNA washing buffer (1 min 13,000 x g) and samples were eluted with 15 μl DNA elution buffer directly to the column matrix (30 sec; 13,000 x g). The eluted DNA samples were transferred to a TwinTec 96-well plate. Samples were sequenced on NextSeq2000 (NextSeq 1000/2000 Control Software 1.5.0.42699/RTA 3.10.30) with a 51nt(Read1)-8nt(Index1)-8nt(Index2)-51nt(Read2) setup using ‘P2’ flowcell. Bcl to FastQ conversion was performed using bcl2fastq_v2.20.0.422 from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8 + .

The ATAC-seq analysis was performed using the Nextflow pipeline NF-core/atacseq version 2.1.2 ( [55]. Briefly, sequence adapters are trimmed by Trim Galore, and aligned by BWA. Picard marked duplicates and merged alignments from multiple libraries to one sample, mitochondrial DNA, duplicates, unmapped regions and other unwanted sequences are filtered by SAMtools, Pysam and BAMTools. Normalized bigWig files were generated by BEDTools, and bedGraphToBigWig. Genome-wide enrichment was done by deepTools and peaks were called by MACS2. Differential accessibility analysis was performed by DESeq2 and Homer was used to annotate gene features. The complete dataset is available at the Sequence Read Archive (NCBI; accession number: PRJNA1180776).

Real-Time qPCR

RNA was extracted using the NucleoSpin RNA kit (Macherey-Nagel) according to the protocol provided by the manufacturer. RNA quality and concentration were determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific). Subsequently, cDNA was prepared by using the iScript cDNA Synthesis kit (Bio-Rad), following the instructions provided by the manufacturer. cDNA was synthesized using the SimpliAmp thermocycler (Life Technologies). RT-qPCRs analyses were performed with technical quadruplicates, using the iTaq universal SYBR green supermix kit (Bio-Rad). Samples were analysed using 384-well plates, with a CFX384 real-time system (Bio-Rad). Samples were analysed using the CFX Maestro software (Bio-Rad). The expression of each gene was given relative to GAPDH, with the use of the formula 2(-ΔCq). The used primers were: CCN1-Forward: 5′- ACCGCTCTGAAGGGGATCT-3′, CCN1-Reverse: 5′ ACTGATGTTTACAGTTGGGCTG-3′, CCN2-Forward: 5′ -AAAAGTGCATCCGTACTCCCA-3′ and CCN2-Reverse: 5′- CCGTCGGTACATACTCCACAG-3 ́.

Statistical analyses

Statistical analyses were performed by using the GraphPad Prism 10 software.