Human participants
Collection and analyzes of breast cancer tissue samples and corresponding blood samples were approved by the ethics committees of the Israeli Ministry of Health (Helsinki applications KMC-0028-22), followed by Weizmann Institutional Review Board approval (IRB, 2314-3).
The breast cancer samples for mass spectrometry-based analysis of histone PTMs, which include 21 tumors, were obtained from patients undergoing surgery at the European Institute of Oncology and are part of a study approved by the institute’s ethics committee (study UID 2550).
Plasma samples and data from luminal breast cancer patients were obtained from the Mayo Clinic (IRB approval number 23-011311). Collection of plasma samples from breast cancer patients treated at Sheba Medical Center was approved by the Institutional Review Board (IRB approval 7509-09). Plasma samples from healthy individuals were obtained from the Weizmann Institute of Science (IRB approval number 1850-1).
All studies were carried out in accordance with applicable guidelines and regulations, and informed consent was obtained from all participants. No direct identifying information of participants is reported in this study. Participant information is detailed in Supplementary Tables 2 and 6. No statistical methods were used to predetermine sample sizes. No randomization or blinding was performed during the collection, processing, or analysis of human samples.
PDX Models
All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute and were performed in accordance with Israeli law. Mice were housed and handled in a pathogen-free, temperature-controlled facility (22°C ± 1°C) on a 12 h light-dark cycle. Animals were fed a regular diet (Teklad Global 2018 rodent diet 18% protein) and had ad libitum access to food and water.
Models TM00112 (PDX-1) and TM00386 (PDX-2) were purchased from the Jackson laboratory and implanted into 10-week-old female NSG mice (Jackson laboratory). Briefly, after euthanasia, tumors were collected from donor mice and cut into small fragments. A small pouch was made in the lower back of each mouse and a fragment was inserted into the pouch. The wound was closed using surgical forceps. The clips were removed 4 to 5 days after surgery. Tumors were measured twice a week with a caliper and body weight once a week. The maximum tumor size approved by the IACUC was 1000 mm3. Mice were euthanized when tumors reached approximately 500 mm3and the tumors were collected for analyses. No statistical method was used to predetermine sample sizes. Animals were not randomized and investigators were not blinded during the experiments.
Cell culture
MCF7 and MDA-MB-468 cell lines were obtained from the Weizmann Institute of Science Tissue Culture Cell Repository. The HCC70, HCC1937, and SUM149 cell lines were provided by I. Ben-Porath. HCC70 and HCC1937 cells were cultured in RPMI-1640 supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin. SUM149 cells were maintained in Ham’s F-12 medium supplemented with 5% FBS, 1 μg ml–1 hydrocortisone (Sigma-Aldrich, H0888), 0.5 µg ml–1 insulin (Sigma-Aldrich, I5500), L-glutamine and penicillin-streptomycin. MDA-MB-468 and MCF7 cells were cultured in DMEM (Thermo Fisher Scientific, 41965-039) supplemented with 10% FBS (Sigma-Aldrich, F7524), 1% L-glutamine (Sartorius, 03-020-1B) and 1% penicillin-streptomycin (Corning, 30-002-CI). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Single cell-derived MCF7 clones and corresponding parental MCF7 cells were provided by U. Ben-David.89 or generated in-house by sorting single cells into 96-well plates using a Sony SH800S cell sorter (Sony Biotechnology) and extended for approximately 4 months before experiments.
The cell lines have not been independently authenticated, but have been routinely tested for mycoplasma contamination and found negative.
Selection of tumor samples for different applications
Due to the limited number of dissociated cells available from each tumor sample provided for research, we were unable to profile identical samples using all of the different methods described in the article. Epigenetic analysis of human tumors based on single-cell CyTOF was the priority for each tumor (besides tumor 23, which was used for WGBS and not profiled by CyTOF). For tumor 18, WGBS and snCUT&Tag were performed due to the high availability of tumor cells. For the remaining samples, we did not have enough cells to perform both methodologies. Notably, although the additional analyzes were not performed on identical samples, each was performed on three independent tumor samples, which ensured the reproducibility of the findings from each approach. The goal of the analysis was to identify common patterns in tumors, rather than comparing tumors and patients.
Clinicopathological and molecular data from tumor samples
Patient and tumor information was extracted from pathology reports. To quantify ER positivity, formalin-fixed paraffin-embedded (FFPE) tumor blocks prepared in parallel with sample collection for CyTOF analysis were collected, sectioned, and stained for ER by IHC. The slides were scanned and the digital images containing DAB signals were analyzed using QuPath software.90. Tissue regions were first segmented using a pixel classifier to identify areas containing tissue (average channels < 245, sigma = 2, at 8.4 µm resolution). In these regions, epithelial and stromal compartments were segmented using a supervised pixel classifier, with a minimum object size of 1000 µm.2 and a minimum hole size of 5,000 µm2. The classifier was trained on representative regions from multiple slides using a multi-layer perceptron model (ANN_MLP) implemented in QuPath, based on hematoxylin and DAB channels at 2.1 µm resolution and multi-scale neighborhood features (scales 1, 2, 4, 8 and 12). Cell nuclei of epithelial regions were segmented using Cellpose-SAM91. For each nucleus, the median DAB intensity was quantified. The distribution of nuclear DAB intensities was calculated for each tumor sample and a threshold per sample was used to account for variability in staining between slides. For each tumor, a kernel density estimate of the median nuclear DAB intensity was calculated, limited to the observed intensity range. The local maxima and minima of the density function were identified and the threshold separating ER-negative and ER-positive nuclei was defined as the lowest local minimum between the two largest density peaks. Tumor-specific thresholds derived from this approach were applied to classify nuclei as ER-positive or ER-negative, and the percentage of ER-positive cells was calculated for each sample.
Mutation calling tumor samples
DNA was extracted from FFPE blocks using a RecoverAll Total Nucleic Acid Isolation kit (Thermo Fisher Scientific) and stored at -20°C. FFPE blocks were examined and annotated for tumor CELLULARITY by a board-certified pathologist, with a minimum accepted threshold of tumor CELLULARITY of 20%. DNA concentrations were quantified using a Qubit 3 fluorometer using a dsDNA HS assay kit (Thermo Fisher Scientific).
Libraries were prepared using a TruSight Oncology 500 (TSO500) assay (Illumina) according to the manufacturer’s instructions. Briefly, 100 ng of DNA from each sample was sheared using a Covaris M220 ultrasound machine (Covaris). Genomic libraries were prepared using a Hamilton Microlab STAR system (Hamilton) and sequenced on an Illumina NextSeq550 instrument with a High Output v.2.5 kit (300 cycles). Initial analysis was performed using DRAGEN TruSight Oncology 500 analysis software (v.2.6.2, Illumina).
Variants were annotated and classified according to AMP guidelines into levels IA, IB, IIC, IID, III and IV.92. Variants classified as level IV, synonymous, non-coding, or with no established biological impact were excluded using Velsera Clinical Genomics Workspace (CGW). The remaining variants were manually examined using in silico prediction tools (REVEL, CADD and SpliceAI) as well as annotations from Franklin (Genoox), UCSC Genome Browser and Velsera CGW. Variants were reported only when sequencing coverage exceeded 100× and variant allele frequency was 1%.
Epigenetics-focused CyTOF analysis of freshly isolated breast tumors, PDX models, and breast cancer cell lines
CyTOF Antibody Panel
Antibody panel combined cell identity markers relevant to breast tissue33.34histone PTMs spanning various chromatin states and the chromatin modifiers EZH2 and BMI-1 (Supplementary Table 1). The chromatin marks analyzed were chosen based on the availability of highly specific and validated antibodies, and quantification of core histones was used for signal normalization.
Cell dissociation and CyTOF staining
After surgery, a tumor tissue sample was preserved in MACS tissue storage solution (Miltenyi Biotec, 130-100-008) and dissociation into single cells was performed using a human tumor dissociation kit (Miltenyi Biotec, 130-095-929) according to the manufacturer’s protocol. Red blood cells were removed using red blood cell lysis solution (10x, Miltenyi Biotec, 130-094-183) and CD45.+ cells were depleted using human CD45 microbeads (Miltenyi Biotec, 130-045-801). The cell suspension was then washed with PBS and either further processed for CyTOF analysis or nuclei were extracted for snCUT & Tag analysis (when cell numbers were appropriate). For cell lines, cells were dissociated using Accutase (Sigma-Aldrich, STC-07922) supplemented with DNase I (Merck, 4716728001).
For CyTOF analysis, cells were washed with Maxpar CyTOF PBS (Standard BioTools) and stained with 1.25 μM cisplatin (Standard BioTools) for 1 min. Cisplatin was neutralized with warm 10% FBS–DMEM. Cells were washed with Maxpar Cell Staining Buffer (Standard BioTools) and blocked using TruStain FcX, Fc Receptor Blocking Solution, Human (422301, BioLegend), followed by 30 min of incubation with an extracellular antibody cocktail (Supplementary Table 1). For tumor samples,…
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