Abstract 1348: LMW-E induction and crosstalk with immune cells potentiates local immune responses leading to an immunosuppressive microenvironment at the early stages of breast tumorigenesis in mouse models
Authors: Mastoraki Sofia , Lulla Amriti R. , Schneider Sarah , Clise-Dwyer Karen , Green Morgan M. , Fowlkes Natalie W. , Hunt Kelly K. , Watowich Stephanie S. , Keyomarsi Khandan 
Published: June 2022

Introduction: Cyclin E is an independent predictor of poor outcomes and response to treatment in breast cancer (BrCa). Expression of low-molecular-weight cyclin E (LMW-E) is associated with more aggressive disease in all BrCa subtypes. While tumor infiltrating lymphocytes (TILs) are more abundant in LMW-E+ tumors, high-TIL/LMW-E+ tumors have lower probability of pathological complete response (pCR) to neoadjuvant chemotherapy. We hypothesized that LMW-E induces immune changes that create a permissive microenvironment in the mammary gland for promoting tumor initiation and subsequent growth. We aimed to evaluate the role of mammary epithelial expression of LMW-E in the temporal induction of systemic and local immune responses that ultimately prime the mammary gland for tumor development.

Methods: We generated a tri-transgenic mouse model capable of conditionally expressing human LMW-E under the control of the MMTV promoter in a p53 heterozygous background (MPT) upon doxycycline (Dox) administration. Female MPT mice were treated with Dox for 3, 6 and 9 months and age-matched untreated controls were sacrificed at each time point. An independent group of non-transgenic mice in a p53 heterozygous background were maintained +/- Dox as positive and negative controls. Mammary glands and peripheral organs (spleen, lung, bone marrow) were harvested for immune profiling by flow cytometry and multiplex immunofluorescence microscopy (mIF). Serum was also collected for cytokine/chemokine assessment. Immune profiling via flow cytometry was performed using two multi-color panels to assess basic immune and more specialized T-cell subsets. For the mIF experiments, two 5-marker panels were applied to mammary tissue.

Results: Histological examination of MPT mammary glands over time showed a temporal increase in acinar proliferation and mitotic figures, which was further confirmed by the co-localization of panCK+ and Ki-67+ markers. We report that although immune cell frequencies changed with age, these specific changes were dependent on LMW-E induction as compared to the non-transgenic cohort of mice. LMW-E+ mammary glands showed a temporal enrichment in B cells, macrophages, T cells (CD4+, PD1+, CD4+Ki67+, and Tregs), cDCs, and panCK+, panCK+vimentin+ populations over time. By contrast, pDCs increased from 3 to 6 months but decreased in the pre-tumorigenic mammary gland of the 9-month old LMW-E+ mice.

Conclusions: LMW-E induction mediates an increase in epithelial cell proliferation and epithelial-to-mesenchymal transition events that result in local immune alterations, specifically affecting T-cell subsets. Our findings suggest that the immunological changes driven by LMW-E lead to an immunosuppressive microenvironment that may promote tumor formation at the early stages of breast tumorigenesis.

Citation Format: Sofia Mastoraki, Amriti R. Lulla, Sarah Schneider, Karen Clise-Dwyer, Morgan M. Green, Natalie W. Fowlkes, Kelly K. Hunt, Stephanie S. Watowich, Khandan Keyomarsi. LMW-E induction and crosstalk with immune cells potentiates local immune responses leading to an immunosuppressive microenvironment at the early stages of breast tumorigenesis in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1348.