Abstract
Understanding how immune cells respond to early oncogenic events is essential for designing immune-based strategies to intercept breast cancer. Mouse models that induce mammary tumorigenesis through Cre-mediated genetic manipulations can be used to study these early events. However, the immune effects of different induction methods remain unclear. Here, we compare adenovirus-delivered Cre with tamoxifen-inducible CreER systems in models targeting luminal mammary epithelial cells for p53-loss. We find that transient intraductal adenoviral infection produces not only an acute immune response but also long-lasting reshaping of the mammary gland immune microenvironment. Adenovirus exposure induces robust and persistent CD8+ T-cell infiltration dominated by CD103+ tissue-resident T cells displaying heightened activation. This sustained antiviral T-cell signature obscures the p53-loss-driven CD8+ T-cell activation detectable in the CreER/tamoxifen model. Adenoviral infection also transiently skews CD4+ T cells toward IFN-γ-producing antiviral states and affects the myeloid compartment, whereas tamoxifen-induced p53-loss increases macrophage abundance and activates CD8+ T-cells during premalignancy. Despite similar tumor latencies across induction strategies, our findings demonstrate that adenoviral infection exerts long-term immunological effects that can confound interpretation of immune dynamics during early mammary tumorigenesis. These results emphasize the importance of induction-method selection when using genetically engineered mouse models to study cancer-immune interactions.
Data availability
All data generated and analyzed in this study are included in this published article and its Supplementary files.
References
Grivennikov, S. I., Greten, F. R. & Karin, M. Immunity, inflammation, and cancer. Cell 140, 883–899. https://doi.org/10.1016/j.cell.2010.01.025 (2010).
Shalapour, S. & Karin, M. Immunity, inflammation, and cancer: An eternal fight between good and evil. J. Clin. Invest. 125, 3347–3355. https://doi.org/10.1172/JCI80007 (2015).
Goff, S. L. & Danforth, D. N. The role of immune cells in breast tissue and immunotherapy for the treatment of breast cancer. Clin. Breast Cancer 21, e63–e73. https://doi.org/10.1016/j.clbc.2020.06.011 (2021).
Swann, J. B. & Smyth, M. J. Immune surveillance of tumors. J. Clin. Invest. 117, 1137–1146. https://doi.org/10.1172/JCI31405 (2007).
Vesely, M. D., Kershaw, M. H., Schreiber, R. D. & Smyth, M. J. Natural innate and adaptive immunity to cancer. Annu. Rev. Immunol. 29, 235–271. https://doi.org/10.1146/annurev-immunol-031210-101324 (2011).
Zhang, S. et al. Tumor initiation and early tumorigenesis: Molecular mechanisms and interventional targets. Signal Transduct. Target. Ther. 9, 149. https://doi.org/10.1038/s41392-024-01848-7 (2024).
Rodriguez-Bejarano, O. H., Parra-Lopez, C. & Patarroyo, M. A. A review concerning the breast cancer-related tumour microenvironment. Crit. Rev. Oncol. Hematol. 199, 104389. https://doi.org/10.1016/j.critrevonc.2024.104389 (2024).
Veglia, F., Sanseviero, E. & Gabrilovich, D. I. Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity. Nat. Rev. Immunol. 21, 485–498. https://doi.org/10.1038/s41577-020-00490-y (2021).
Anderson, K. G. et al. Leveraging immune resistance archetypes in solid cancer to inform next-generation anticancer therapies. J. Immunother. Cancer https://doi.org/10.1136/jitc-2022-006533 (2023).
Liu, C., Wu, P., Zhang, A. & Mao, X. Advances in rodent models for breast cancer formation, progression, and therapeutic testing. Front. Oncol. 11, 593337. https://doi.org/10.3389/fonc.2021.593337 (2021).
Deng, C. X. Conditional knockout mouse models of cancer. Cold Spring Harb. Protoc. 2014, 1217–1233. https://doi.org/10.1101/pdb.top074393 (2014).
Van Keymeulen, A. et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 479, 189–193. https://doi.org/10.1038/nature10573 (2011).
Wagner, K. U. et al. Cre-mediated gene deletion in the mammary gland. Nucleic Acids Res. 25, 4323–4330 (1997).
Feil, R. et al. Ligand-activated site-specific recombination in mice. Proc. Natl. Acad. Sci. U. S. A. 93, 10887–10890. https://doi.org/10.1073/pnas.93.20.10887 (1996).
Feil, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237, 752–757. https://doi.org/10.1006/bbrc.1997.7124 (1997).
Koren, S. et al. PIK3CA induces multipotency and multi-lineage mammary tumours. Nature 525, 114–118. https://doi.org/10.1038/nature14669 (2015).
Tao, L., Xiang, D., Xie, Y., Bronson, R. T. & Li, Z. Induced p53 loss in mouse luminal cells causes clonal expansion and development of mammary tumours. Nat Commun 8, 14431. https://doi.org/10.1038/ncomms14431 (2017).
Van Keymeulen, A. et al. Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity. Nature 525, 119–123. https://doi.org/10.1038/nature14665 (2015).
Wang, H. et al. Inadequate DNA damage repair promotes mammary transdifferentiation, leading to BRCA1 breast cancer. Cell 178, 135-151 e119. https://doi.org/10.1016/j.cell.2019.06.002 (2019).
Tao, L., van Bragt, M. P. A., Laudadio, E. & Li, Z. Lineage tracing of mammary epithelial cells using cell-type-specific Cre-expressing adenoviruses. Stem Cell Rep 2, 770–779. https://doi.org/10.1016/j.stemcr.2014.04.004 (2014).
Chen, X., Han, S., Zhao, D. & Li, Z. Intraductal injection of adenoviruses to perform lineage tracing in the mammary gland. J Mammary Gland Biol Neoplasia 30, 10. https://doi.org/10.1007/s10911-025-09584-6 (2025).
Xiang, D., Tao, L. & Li, Z. Modeling breast cancer via an intraductal injection of Cre-expressing adenovirus into the mouse mammary gland. J. Vis. Exp. 148, 10.3791/59502. https://doi.org/10.3791/59502 (2019).
Rios, A. C., Fu, N. Y., Lindeman, G. J. & Visvader, J. E. In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322–327. https://doi.org/10.1038/nature12948 (2014).
Shehata, M., van Amerongen, R., Zeeman, A. L., Giraddi, R. R. & Stingl, J. The influence of tamoxifen on normal mouse mammary gland homeostasis. Breast Cancer Res. 16, 411. https://doi.org/10.1186/s13058-014-0411-0 (2014).
Tower, H. et al. Estrogen-induced immune changes within the normal mammary gland. Sci. Rep. 12, 18986. https://doi.org/10.1038/s41598-022-21871-4 (2022).
Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J. & Berns, A. Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. Genes Dev. 14, 994–1004 (2000).
Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001).
Reinert, R. B. et al. Tamoxifen-induced Cre-loxP recombination is prolonged in pancreatic islets of adult mice. PLoS One 7(3), e33529. https://doi.org/10.1371/journal.pone.0033529 (2012).
Fang, F. et al. The cell-surface 5’-nucleotidase CD73 defines a functional T memory cell subset that declines with age. Cell Rep. 37, 109981. https://doi.org/10.1016/j.celrep.2021.109981 (2021).
Szabo, P. A., Miron, M. & Farber, D. L. Location, location, location: Tissue resident memory T cells in mice and humans. Sci. Immunol. https://doi.org/10.1126/sciimmunol.aas9673 (2019).
Afzal, S. et al. Interrelated oncogenic viruses and breast cancer. Front. Mol. Biosci. 9, 781111. https://doi.org/10.3389/fmolb.2022.781111 (2022).
Gupta, I. et al. Incidence of HPVs, EBV, and MMTV-like virus in breast cancer in Qatar. Intervirology 65, 188–194. https://doi.org/10.1159/000525277 (2022).
Harkins, L. E. et al. Detection of human cytomegalovirus in normal and neoplastic breast epithelium. Herpesviridae 1, 8. https://doi.org/10.1186/2042-4280-1-8 (2010).
Lawson, J. S. & Glenn, W. K. Evidence for a causal role by mouse mammary tumour-like virus in human breast cancer. npj Breast Cancer 5, 40. https://doi.org/10.1038/s41523-019-0136-4 (2019).
Hu, H. et al. Epstein-Barr virus infection of mammary epithelial cells promotes malignant transformation. EBioMedicine 9, 148–160. https://doi.org/10.1016/j.ebiom.2016.05.025 (2016).
Zeng, J. et al. Exploring the potential of cytomegalovirus-based vectors: A review. Viruses https://doi.org/10.3390/v15102043 (2023).
DuPage, M., Dooley, A. L. & Jacks, T. Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat. Protoc. 4, 1064–1072. https://doi.org/10.1038/nprot.2009.95 (2009).
Sutherland, K. D. et al. Cell of origin of small cell lung cancer: Inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung. Cancer Cell 19(6), 754–764. https://doi.org/10.1016/j.ccr.2011.04.019 (2011).
Flesken-Nikitin, A., Choi, K. C., Eng, J. P., Shmidt, E. N. & Nikitin, A. Y. Induction of carcinogenesis by concurrent inactivation of p53 and Rb1 in the mouse ovarian surface epithelium. Cancer Res. 63, 3459–3463 (2003).
Park, E. S., Xiang, D., Xie, Y., Bronson, R. T. & Li, Z. Oncogenic events dictate the types and locations of gynecological malignancies originating from Krt8(+) mesothelial and Mullerian-derived epithelial cells. Cancers 14, 841. https://doi.org/10.3390/cancers14030841 (2022).
Buchakjian, M. R. et al. A Trp53fl/flPtenfl/fl mouse model of undifferentiated pleomorphic sarcoma mediated by adeno-Cre injection and in vivo bioluminescence imaging. PLoS One. 12, e0183469. https://doi.org/10.1371/journal.pone.0183469 (2017).
Kirsch, D. G. et al. A spatially and temporally restricted mouse model of soft tissue sarcoma. Nat. Med. 13, 992–997. https://doi.org/10.1038/nm1602 (2007).
Kobayashi, T., Owczarek, T. B., McKiernan, J. M. & Abate-Shen, C. Modelling bladder cancer in mice: Opportunities and challenges. Nat. Rev. Cancer 15, 42–54. https://doi.org/10.1038/nrc3858 (2015).
Puzio-Kuter, A. M. et al. Inactivation of p53 and Pten promotes invasive bladder cancer. Genes Dev. 23, 675–680. https://doi.org/10.1101/gad.1772909 (2009).
Acknowledgements
This research was supported by U.S. National Institutes of Health (NIH) grants R01CA222560, R01CA248306, and R01CA295752, and by the Gray Foundation to ZL.
Funding
This research was supported by U.S. National Institutes of Health (NIH) grants R01CA222560, R01CA248306, and R01CA295752, and by the Gray Foundation to ZL.
Author information
Authors and Affiliations
Contributions
SH and ZL contributed to study conceptualization and project administration. SH and ZL contributed to data curation and formal analysis. ZL conceived and designed the experiments; SH and MZ contributed to validation and visualization; SH and ZL contributed to writing original draft and manuscript revision. SH, DZ, XC, MZ, TL, CW, HC, and ZL contributed to investigation and methodology. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Han, S., Zhao, D., Chen, X. et al. Transient adenovirus-Cre infection causes long-lasting remodeling of the mammary gland immune landscape. Sci Rep (2026). https://doi.org/10.1038/s41598-026-43069-8
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-43069-8
