Animal Mammary Neoplasms

A murine tumor dataset of 385 DNA gene expression microarrays from 27 GEMMs of mammary carcinoma was compiled (Table 1A; Table S1 in Additional file 1). Of these, 275 samples were obtained from multiple previous publications (Gene Expression Omnibus accession numbers GSE3165, GSE8516, GSE9343, GSE14457, GSE15263, GSE17916, and GSE27101). The other 110 microarray samples (GSE42640) represent newly obtained tumor samples from multiple participating investigators using methods approved by international animal husbandry guidelines. Total RNA was purified from 20 to 30 mg of mouse mammary tumor using Qiagen’s (Valencia, CA USA) RNeasy Mini Kit following the manufacture’s protocols. RNA quantity and quality were determined using the Nanodrop spectrophotometer and Agilent Bioanalyzer, respectively. Total RNA was reverse transcribed and labeled with cyanine-5 (Cy5) dye for experimental samples and cyanine-3 (Cy3) dye for mouse reference samples [31 (link)] using the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Equal quantities of labeled mouse reference RNA and tumor RNA were co-hybridized overnight to Agilent microarrays, washed, scanned and signal intensities were determined.
All tumor samples were co-hybridized to one of three Agilent Technology gene expression microarray types: 22 K, 4X44K, or 4X180K (Figure 1). Two ‘homogeneous expression’ murine models [31 (link)], namely TgMMTV-Neu and TgC3(I)-Tag, were analyzed on all three array types. Therefore, we used both of these models to normalize expression between microarray types [32 (link)]. Ten microarrays (five TgMMTV-Neu and five TgC3(I)-Tag) from each array type were used for normalization (30 microarrays total). All microarray data were independently extracted from the UNC Microarray Database for each array type as log₂ Cy5/Cy3 ratios, filtering for probes with Lowess normalized intensity values greater than 10 in both channels and for probes with data on greater than 70% of the microarrays [31 (link),34 (link)]. Before normalization, each data set was imputed (via the 10 nearest neighbor gene values) and then reduced to the probes that were present on all three array type datasets (11,690 probes, 11,167 genes). Using the 10 normalization arrays per 3 array platforms, the median expression value was calculated for each probe, on each array type, and a normalization factor was applied independently to each probe so the median was the same for each array type. Probe expression values were ‘median centered’ to obtain the final normalized dataset. A principle component analysis was performed to verify the normalization.

CopywriteR was evaluated against WES, targeted sequencing, and ChIPseq datasets. The WES datasets includes: (1) six PDX-derived human melanoma samples with six matched germline references (T98 to T103; C42 to C47); (2) four mouse SCLC samples with one matched germline reference (T21, T23, T43, and T44; C3); (3) nine mouse mammary tumors with two matched germline references (T2, T3, T7, T19, T20, T50, T56, T60, T62; C1, C39); (4) one melanoma biopsy (FFPE) with matched reference (T97; C41); and (5) 16 kidney renal cell carcinoma (KIRC) samples downloaded from the TCGA together with matching germline references [8 (link)]. The targeted sequencing dataset includes matching pre- and post-vermurafenib treatments melanoma samples, as well as germline references [37 ]. The ChIPseq datasets include: (1) the breast cancer cell line MCF7 which was analyzed with multiple ChIPseq samples with six DNA associated proteins (ER, EGR1, GATA3, CTCF, MAX, and EP300) as well as input material as a control [34 (link)]; and (2) five breast cancer samples enriched for ER-binding sites and matching input control samples [35 (link)]. Detailed information (including origin, sequencing method, sequence depth, and other sequencing statistics) for all samples is documented in Additional file 1.
In addition to sequencing data, PDX-derived melanoma and TCGA data were analyzed using Affymetrix SNP6; mSCLC samples were analyzed on Nimblegen arrays; mouse mammary tumors were analyzed by WG-LCS. The Animal Experimental Committee approved all animal experiments. Samples were collected following approval of the Medical Ethical Committee of the NKI (study code N03LAM) and in compliance with the Helsinki Declaration. Previously unpublished data have been made available through the NCBI Gene Expression Omnibus (GEO) [39 ] (accession number GSE60259), the European Nucleotide Archive (ENA) [40 ] (accession number PRJEB6954), and the European Genome-phenome Archive (EGA) [41 ] (accession number EGAS00001000617).

Proteins were isolated from 66cl4 and 67NR mammary breast tumors from mice by homogenization in lysis buffer and analyzed by LC–MS/MS as described in Additional file 1: Methods. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [29 (link)] partner repository with the dataset identifier PXD037288.

Three 8 to 12-week-old NSG female mice were anesthetized by intraperitoneal injection with 10 mg/kg xylazine and 75 mg/kg ketamine (Graeub). To prevent eyes from drying while anesthetised, ophthalmic ointment (Viscotears) was applied to the eyes of mice. After shaving and disinfecting the area around the nipple, the intraductal injection was performed by injecting 500,000 MCF7 cells (resuspended in 10 µL of PBS) into the cleaved 3rd and 4th teats with a blunt end Hamilton syringe (HAMI80508, specifications: 50 µL 705 N, gauge 30/13 mm/pst3), as previously described^{46 (link)}. Paracetamol was added to the drinking water (200 to 300 mg/kg, i.e. 500 mg/250 mL of drinking water) from 1 day prior to the procedure until for 3 days after the intraductal injection. MCF7 cells were grown intraductally for 12 weeks to form mammary tumours. At the end of the period, mice were euthanized by CO₂ inhalation. Engrafted mammary glands were harvested and fixed in 4% PFA for 2 h, prior to dehydration and paraffin embedding.