Tumor-associated autoantibodies from mouse breast cancer models are found in serum of breast cancer patients

B cell responses to tumor antigens occur early in breast tumors and may identify immunogenic drivers of tumorigenesis. Sixty-two candidate antigens were identified prior to palpable tumor development in TgMMTV-neu and C3(1)Tag transgenic mouse mammary tumor models. Five antigens (VPS35, ARPC2, SERBP1, KRT8, and PDIA6) were selected because their decreased expression decreased survival in human HER2 positive and triple negative cell lines in a siRNA screen. Vaccination with antigen-specific epitopes, conserved between mouse and human, inhibited tumor growth in both transgenic mouse models. Increased IgG autoantibodies to the antigens were elevated in serum from women with ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC). The autoantibodies differentiated women with DCIS from control with AUC 0.93 (95% CI 0.88–0.98, p < 0.0001). The tumor antigens identified early in the development of breast cancer in mouse mammary tumor models were conserved in human disease, and potentially identify early diagnostic markers in human breast tumors.


Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see our Editorial Policies and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

April 2020
Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
For the mouse studies comparing adjuvant immunized mice to antigen vaccinated, eight animals per group was used to provide 95% power to observe a statistically significant difference in tumor size between antigen vaccinated mice as compared to control vaccinated mice (

Replication
The large scale siRNA screen was repeated in its entirety twice. The small scale was performed in triplicate. All the RT PCR assays were performed in quadruplicate. All IFN-g ELISPOT assays were performed with 6 replicates. The mouse studies were not performed twice as the TgMMTV-neu and C3 (1)Tag studies were used as replicates for eachother.
Randomization No randomization was performed, not appropriate for this study

Blinding
No blinding was performed for this study but the validation serum set IS blinded.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Authentication
Cells that were directly obtained directly from ATCC were not authenticated because they were purchased directly from the repository. The MMC cell line was verified to express rat neu by flow cytometry and the M6 cell line was verified to express the SV40 antigen by western blotting, and to be estrogen receptor negative by rtPCR.

nature research | reporting summary
April 2020

Mycoplasma contamination
All cell lines were tested for mycoplasma by the universal mycoplasma detection kit (ATCC) prior to the large scale screens or implant into the mouse.
Commonly misidentified lines (See ICLAC register) none

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
All mice were female as in these models they develop breast cancer. TgMMTV-neu mice (strain name: FVB/N-Tg(MMTVneu)202Mul/ J, strain #002376, Jackson Laboratory, Bar Harbor, ME) were purchased from the Jackson laboratory and maintained under strict inbreeding conditions.(38) Confirmation of the transgenic strain was done using PCR for ERBB2. C3(1) Tag mice (strain name: FVB-Tg-(C3-Tag) cJeg/Jeg male mice provided from Dr. Jeff Green NCI) were mated to FVB/nJ parental females (strain #001800). The C3(1)Tag transgenic mice were confirmed by PCR for SV40 large T antigen.
Wild animals none Field-collected samples none

Ethics oversight
All animal care and use was done in accordance with the University of Washington Institutional Animal Care and Use Committee guidelines.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants

Population characteristics
The serum from control women (n=43) were acquired from volunteers from the Puget Sound Blood Center and serum from women with invasive breast cancer (n=37) were acquired from the Cancer Vaccine Institute specimen repository and were collected using the same methods (table 3). The serum for benign, fibroadenoma, hyperplasia, and DCIS were obtained from a Duke repository with blood drawn at the time of an abnormal mammogram and in patients with no invasive pathology at surgery. The definition of a benign lesion was defined as radial scar, cyst, and no fibroadenoma or atypia. The Duke Repository serum was from women with benign tumors (n=12), fibroadenoma (n=37), hyperplasia (n=12), and DCIS (n=59)

Recruitment
These samples were from repositories therefore the recruitment is not known.

Ethics oversight
These samples had no clinically identifiable data associated with them therefore it did not need IRB approval. Our clinical research coordinator (Jennifer Childs) confirmed that the samples did not need IRB approval with the University of Washington IRB.
Note that full information on the approval of the study protocol must also be provided in the manuscript.