Transient adenovirus-Cre infection causes long-lasting remodeling of the mammary gland immune landscape

Transient adenovirus-Cre infection causes long-lasting remodeling of the mammary gland immune landscape | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transient adenovirus-Cre infection causes long-lasting remodeling of the mammary gland immune landscape Sen Han, Dongyi Zhao, Xueqing Chen, Miao Zhu, Tiantian Li, Chujun Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8398573/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version 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 compresses 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. Biological sciences/Cancer Biological sciences/Immunology Adenovirus-Cre mammary gland immune microenvironment breast cancer mouse model p53-loss CD8+ tissue-resident T cells premalignancy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Development of cancers, such as breast cancer, from their corresponding cellular origins occurs within a dynamic immunological ecosystem in which immune cells can both restrain and promote tumorigenesis. A “fight” between anti-tumorigenic and pro-tumorigenic immune and inflammatory mechanisms can determine the course of tumor development and its phenotype 1 , 2 . In breast cancer, during its early stages of development, innate and adaptive immune surveillance mechanisms, such as cytotoxic T cells, NK cells, dendritic cells, and macrophages, can recognize and eliminate transformed mammary epithelial cells (MECs), limiting malignant progression 3 – 5 . However, as oncogenic signaling, genomic instability, and tissue remodeling intensify, the evolving tumor microenvironment can reshape these immune populations, such as during the transition from the precancer to cancer stages 6 . Myeloid-derived immune cells, particularly tumor-associated macrophages and neutrophils, may adopt pro-tumorigenic phenotypes that support immunosuppression, angiogenesis, and invasion, while regulatory T cells and exhausted T-cell states dampen effective antitumor immunity 7 – 9 . Defining how these immune populations are recruited, activated or suppressed, and reprogrammed during the early phases of mammary tumorigenesis is essential for designing strategies that bolster endogenous immune defenses. By understanding the immunological events that tip the balance from elimination to escape, immune-based preventive approaches may be developed to intercept breast cancer at its roots, before clinically detectable disease emerges. As access to early stages of human cancer development, including that of breast cancer, is very difficult if not impossible, in order to achieve the goal of immune-based cancer interception, animal models that can recapitulate this early phase of tumorigenesis are essential. In breast cancer, several elegant approaches have been developed to model mammary tumor initiation from a defined subpopulation of MECs and to follow their progression in mice 10 . One of the prevalent approaches is the Cre/loxP-based strategy that enables precise temporal and spatial control over oncogenic events within the mammary epithelium 11 . By placing Cre recombinase under the control of mammary-specific promoters, such as MMTV , Wap , or Krt8 12,13 , and activating it through ligand-dependent systems such as CreER (Cre-estrogen receptor fusion, inducible by tamoxifen 14 , 15 ), researchers can trigger defined genetic alterations, including oncogene activation or tumor suppressor loss, at selected MEC subpopulations or developmental stages. This approach faithfully recapitulates the stepwise evolution of breast cancer, permitting the study of early cellular transformation, clonal expansion, and cancer-immune cell interaction within an intact immune microenvironment 16 – 19 . To complement the CreER/tamoxifen-based inducible approach, my group developed a novel approach based on intraductal injection of Cre-expressing adenovirus to mouse mammary glands 20 – 22 . Adenovirus is a DNA virus that does not integrate into the host genome and the adenoviral vector we use only leads to transient expression of the Cre recombinase; thus, it serves a similar purpose as an inducible Cre system. The cell type-specificity (for inducing Cre-mediated recombination) is achieved by using different MEC subpopulation-specific promoters (e.g., Krt8 , Wap ) to drive adenovirus-Cre expression. Breast cancer mouse models based on both CreER/tamoxifen and adenovirus-Cre-based approaches offer excellent in vivo systems to characterize immune cell phenotypes and their dynamic changes at different stages of mammary tumorigenesis. In these induced mice, mammary gland immune cells can be modulated by signals from mutated MECs. However, it is unclear whether the injected tamoxifen or adeno-Cre virus (to induce Cre-mediated recombination and initiation of mammary tumorigenesis) can affect the immune microenvironment. This is possible as administration of tamoxifen in vivo can potentially affect estrogen signaling in the mammary gland 23 , 24 , while estrogen/ER (estrogen receptor) signaling plays a key role in regulating mammary gland immune cell populations 25 . Similarly, injection of adenovirus to mammary glands should trigger virus-induced immune reactions, at least transiently 20 . However, it is unclear whether any of these induction approach-related modulations of mammary gland immune cells is transient or more long-lasting and whether such change would affect immune cell phenotypes and dynamics, and ultimately, mammary tumor development. A thorough understanding of these should have important implications for the proper use of these modeling approaches to study immune cells during mammary tumorigenesis. Results Adenoviral infection increases leukocyte infiltration in the mammary gland In this study, we used a breast cancer mouse model that we previously developed based on induced loss of p53 in luminal MECs 17 . The experimental mice ( PY ) carry Trp53 conditional knockout alleles ( Trp53 fl/fl ) 26 along with a conditional Cre-reporter [ Rosa26-LSL-YFP ( R26Y )] 27 , while R26Y reporter-only mice served as wild-type (WT) controls (Supplementary Fig. 1a). To induce mammary tumor initiation specifically in luminal MECs, we utilized an adenovirus expressing Cre under the control of the luminal keratin 8 ( Krt8 , or K8 ) promoter ( Ad-K8-Cre ) 20 . Intraductal injection of Ad-K8-Cre into the abdominal (#4) mammary glands of PY female mice induced simultaneous YFP reporter expression and Trp53 deletion in luminal MECs, resulting in the development of mammary tumors (predominantly of the Claudin-low subtype) with 100% penetrance 17 . For comparison, we also used a CreER/tamoxifen-based approach to induce p53-loss in the same luminal MEC population. We generated KPY experimental mice and KY controls by crossing the K8-CreER allele, a transgenic construct expressing CreER T2 under the same K8 promoter 12 , with PY and R26Y mice, respectively (Supplementary Fig. 1b). Tamoxifen (TAM) induction in KPY female mice led to development of mammary tumors similar to those observed in Ad-K8-Cre -induced PY mice, also with full penetrance 17 . To investigate how p53-loss in luminal MECs alters the mammary gland immune microenvironment during the premalignant stage, we induced p53 deletion by administering Ad-K8-Cre or TAM to PY (and R26Y ) or KPY (and KY ) female mice at ~ 8 weeks of age, respectively. Based on our previous findings, mammary tumors begin to emerge in induced mice around 5–6 months after induction 17 . Accordingly, in this study we defined 3 and 5 months post-induction as the mid- and late-premalignant stages, respectively (Fig. 1 a). To determine whether intraductally delivered adenovirus itself elicits additional immunogenicity capable of altering the mammary immune microenvironment, we evaluated leukocyte infiltration one week after injection. FACS analysis revealed that adenovirus-injected R26Y mice exhibited a significantly higher proportion of CD45 + leucocytes in the injected abdominal mammary glands (55.5%±7.1%) compared with non-injected thoracic glands from the same mice (35.76%±4.74%) (Fig. 1 b-c). In contrast, the K8-CreER -based model relies on systemic TAM administration via intraperitoneal injection, which could in principle exert global effects on all mammary glands through its modulation of estrogen signaling. To assess this possibility, we compared leukocyte infiltration in mammary glands from TAM-treated KY mice with that of age-matched, untreated KY controls. The data indicate that TAM treatment does not significantly alter leukocyte infiltration (28.36%±3.16% vs. 30.98%±3.32%) (Fig. 1 d-e). Transient adenoviral infection in the mammary gland increases T cell infiltration To determine whether transient adenoviral infection in PY and R26Y mice has any long-term effects on the immune microenvironment of the injected mammary glands, we examined leucocyte infiltration at mid- and late-premalignant stages using FACS analysis. At the mid-stage, both adenovirus-infected PY and R26Y mice displayed higher levels of leukocyte infiltration compared with their counterparts induced via TAM (i.e., KPY and KY ) (Fig. 2 a-b). Induced p53 loss in luminal MECs modestly increased immune activity in the mammary glands of KPY mice relative to KY controls; however, no such difference was detected between adenovirus-induced PY and R26Y mice (Fig. 2 a-b). By the late stage, leukocyte infiltration in induced KPY mice rose to levels comparable to those of adenovirus-induced PY and R26Y mice, whereas KY controls remained substantially lower. In contrast, leukocyte infiltration in adenovirus-induced PY and R26Y mice remained similar to each other throughout both stages (Fig. 2 a-b). These findings indicate that transient adenoviral infection causes a sustained increase in mammary gland leukocyte infiltration. Furthermore, loss of p53 in luminal MECs likely triggers an anti-tumor immune response that enhances mammary gland immune activity. This effect is readily detectable in TAM-induced KPY mice but may be obscured in adenovirus-induced PY mice due to the elevated baseline immune infiltration caused by prior adenoviral exposure. To exclude the possibility that the increased immune activation observed in the KPY model following TAM induction was simply due to a higher recombination efficiency compared with adenoviral delivery, we evaluated the initial induction and subsequent clonal expansion of premalignant MECs in both models. We first quantified the proportion of premalignant cells (YFP + ) within the lineage-negative compartment (Lin − , i.e., CD45 − TER119 − CD31 − ) two weeks after induction. The frequency of YFP + premalignant cells was comparable between experimental and control groups across both induction strategies (Supplementary Fig. 2a-b). Moreover, more than 90% of YFP + cells localized to the luminal MEC gate (Lin − CD24 high CD29 low ), indicating that both induction approaches achieved similar levels of p53 deletion specifically within luminal MECs (Supplementary Fig. 2c). We next assessed the clonal expansion capacity of p53-null MECs during premalignancy by FACS. In the adenovirus-induced model, p53-null MECs (YFP + ) represented 0.29%±0.13% of the Lin − compartment at two weeks post-induction, increasing to 0.85%±0.3% at the mid-stage and 2.89%±0.92% at the late-stage of premalignancy. In contrast, YFP + MECs in induced R26Y mice (which retain WT p53) remained at a steady percentage (~ 0.6% YFP + ) throughout the premalignant period (Supplementary Fig. 2d). A similar pattern was observed in the TAM-induced model (Supplementary Fig. 2e). Together, these results demonstrate that both induction strategies generate comparable initial recombination efficiencies in luminal MECs, and that p53-loss confers a consistent clonal expansion advantage during premalignancy. To evaluate how transient adenoviral infection reshapes the immune landscape of the mammary gland, we quantified major immune cell populations, including T cells (CD45 + CD3 + ), NK cells (CD45 + CD3 − NKp46 + ), macrophages (CD45 + CD11b + F4/80 + ), dendritic cells (CD45 + CD11b + CD11c + ), B cells (CD45 + CD19 + ), and neutrophils (CD45 + CD11b + Ly-6G + ), by FACS analysis (Supplementary Fig. 3). Across all time points, adenovirus-infected mammary glands in both PY and R26Y mice exhibited a marked increase in T cells (Fig. 2 c), indicating that the elevated baseline immune infiltration observed following adenoviral injection is driven primarily by T cell enrichment. Adenoviral infection induces CD8 + tissue-resident T cells in the mammary gland We next examined the CD3 + T cell compartment in greater detail. Regardless of the p53 mutagenesis status, adenoviral infection markedly increased CD3 + T cell infiltration, both in frequency and absolute cell number, in virally infected mammary glands at both mid- and late-premalignant stages, compared with TAM-induced mice (Fig. 3 a). This elevation in T cell abundance was driven primarily by an increase in CD8 + T cells, rather than CD4 + T cells (Fig. 3 b-c). Given that transient adenoviral infection produced a sustained elevation in CD8 + T cells, we investigated whether this reflected an expansion of tissue-resident memory T cells. To do so, we assessed CD103 and CD73 expression, canonical markers of tissue-resident memory T cells 28 , 29 , on both CD8 + and CD4 + T cell populations. In mice analyzed 3 months post-induction, ~ 90% of infiltrating CD8 + T cells in adenovirus-infected mammary glands (both PY and R26Y ) expressed CD103, compared with only ~ 30% in TAM-induced KPY and KY mice (Fig. 3 d). CD73 expression was relatively high on CD8⁺ T cells across all groups (Fig. 3 d). In contrast, neither CD103 nor CD73 showed substantial differences on CD4 + T cells across induction methods (Fig. 3 e). Collectively, these findings indicate that transient adenoviral infection elicits a robust CD8 + T cell response, promoting their infiltration and long-term residency within the mammary gland microenvironment. CD8 + tissue-resident T cell phenotype masks p53 loss-driven CD8 + T cell activation We next characterized CD8 + and CD4 + T cell subsets and functional states during premalignancy by assessing expression of IFN-γ, as well as the activation markers PD-1 and CD69, using FACS analysis. In the TAM-induced model ( KPY ), induced p53-loss in luminal MECs elicited a pronounced increase in IFN-γ, PD-1, and CD69 expression in CD8 + T cells at the late-premalignant stage. At the mid-stage, CD8 + T cells in KPY mice showed increased CD69 expression but no significant elevation in IFN-γ or PD-1 (Fig. 4 a-c). In contrast, in the adenovirus-induced model, induced p53-loss in luminal MECs from PY mice did not significantly alter IFN-γ or PD-1 expression in CD8 + T cells when comparing to induced R26Y controls (Fig. 4 a-c). Instead, CD8 + T cells in both adenovirus-induced PY and R26Y mice displayed consistently elevated activation states at both mid- and late-stages relative to their TAM-induced counterparts (Fig. 4 a-c). This sustained activation profile appears to result from the large pool of tissue-resident CD8 + T cells resulted from the prior adenoviral infection. Supporting this, the majority of PD-1 + CD8 + and CD69 + CD8 + cells in adenovirus-induced PY and R26Y mice co-expressed CD103, whereas most PD-1 + CD8 + and CD69 + CD8 + cells in TAM-induced KPY and KY mice lacked CD103 expression (Supplementary Fig. 4a). In contrast, the CD103 expression in the PD-1 + CD4 + and CD69 + CD4 + cells from both models was low and not significantly different (Supplementary Fig. 4b). In the CD4 + T cell compartment, adenovirus-induced PY and R26Y mice exhibited a transient increase in IFN-γ-producing, activated CD4 + T cells at the mid-premalignant stage, which declined to levels comparable to those of TAM-induced mice by the late stage (Fig. 4 d). In contrast, mammary glands from TAM-induced KPY mice displayed a selective increase in IFN-γ-producing, activated CD4 + T cells during the late-premalignant stage compared with KY controls, a feature not observed in the adenovirus-induced model (Fig. 4 d). A similar late-stage increase in PD1 + CD4 + and CD69 + CD4 + T cells was also uniquely observed in induced KPY mice (Fig. 4 e-f). Taken together, these findings indicate that transient adenoviral infection recruits CD8 + T cells into the injected mammary gland, where they differentiate into tissue-resident T cells. These virus-induced CD8 + T cells display a persistently activated phenotype that likely obscures the CD8 + T cell response elicited by the expanding p53-null mutant MEC population. In parallel, viral infection appears to drive otherwise quiescent CD4 + T cells into a transient, IFN-γ-producing, virus-specific state, which may limit their engagement in immune responses against p53-null MECs. Adenoviral infection-induced CD8 + T cell influx condenses the myeloid compartment Lastly, we examined the myeloid compartment in the mammary gland. In the TAM-induced model, induced p53-loss in luminal MECs resulted in increased macrophage infiltration (both in percentage and absolute number) at mid- and late-premalignant stages (in KPY vs. KY , Fig. 5 a-b). In contrast, adenovirus-induced PY and R26Y mice exhibited a pronounced reduction in the macrophage frequency, whereas absolute macrophage numbers remained more comparable to those observed in TAM-induced mice (Fig. 5 a-b). These macrophage changes appeared to be driven primarily by adenoviral infection rather than p53 status. Nevertheless, related to p53-loss, a modest late-stage increase in macrophage abundance was also detected in adenovirus-induced PY mice (Fig. 5 a-b). Despite these quantitative differences, macrophage polarization marker expression (e.g., CD206, CD86, and I-A/I-E) remained largely similar between adenovirus-induced and TAM-induced models (Fig. 5 c-e). However, we observed a trend toward reduced CD206 + , MHC-II + (i.e., I-A/I-E + ), or CD86 + macrophage subpopulations in mice with induced loss of p53 ( PY or KPY ) compared with their respective controls ( R26Y or KY ) (Fig. 5 c-e). Discussion In this work, by characterizing and comparing mammary gland immune cells during premalignant stages in breast cancer mouse models with the same driver and tumor outcomes but different induction approaches, we observed that transient adenoviral infection not only led to an expected initial immune response, but also reshaped the mammary gland immune microenvironment long-term. The main viral infection-induced long-lasting change was the profound increase in CD8 + tissue-resident T cells, thereby confounding the detection of p53 loss-induced CD8 + T cell responses. Moreover, transient adenoviral exposure appeared to skew CD4 + T cells toward IFN-γ-producing anti-virus-specific states at the mid-stage. Whether such CD4 + T cell state affects their response to p53-deficient MECs during premalignancy is unclear. Although compared to T cells, the myeloid compartment was less affected by the adenoviral exposure, the expansion of T cell population in the adenovirus-induced model may have compressed the myeloid compartment. In the TAM-induced KPY (vs. KY ) mice, induced loss of p53 in luminal MECs led to significant increases in both the percentage and absolute number of macrophages at both mid- and late-stages of premalignancy. However, such changes were less profound in adenovirus-induced PY (vs. R26Y ) mice. Nevertheless, both modeling approaches revealed a similar trend of reductions in CD206 + , MHC-II + (i.e., I-A/I-E + ), or CD86 + macrophage subpopulations, correlating with induced p53-loss. Overall, this study highlights the complexity of immune cell changes when using different cancer-induction approaches and offers practical insights for selecting an appropriate induction system to achieve more accurate interpretations of immune regulation during mammary tumorigenesis and beyond. Despite adenovirus-induced changes in the mammary gland immune landscape, adenovirus-induced PY and TAM-induced KPY mice (under similar genetic background) developed mammary tumors with a similar latency 17 . This does not necessarily suggest that virus-induced changes in mammary gland immune cells have no influence on the course of tumor development. Other factors, such as the time required for p53-deficient MECs to acquire secondary driver mutations, may play a more dominant role in determining the tumor latency. Alternatively, as a tightly regulated host defense system, virus-induced upregulation of CD8 + tissue-resident T cells may be off-set by changes in other immune cell subsets (e.g., macrophages), making the overall immune reactions toward p53-null mutant MECs comparable to those without the virus-induced changes. Viral infections have been found in both normal and neoplastic human breast epithelial cells. Although infections by viruses such as Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), certain human papillomaviruses (HPV), and mouse mammary tumor viruses (MMTV) are more frequently found in human breast cancers 30 – 33 , viral infections of normal human breast epithelial cells have also been reported 32 , 34 . For instance, it was reported that HCMV infection could be detected in glandular epithelium in 63% of normal adult breast cases 32 . In another study, it was shown that EBV infection occurs in breast epithelial cells but not in breast cancer cells 34 . Interestingly, similar to adenovirus, HCMV is a DNA virus that does not typically integrate its DNA into the host genome and persists in the host cell episomally 35 . It is possible that viral infections of human breast epithelial cells, even only transient, could also lead to long-last changes in the immune cell landscape in the affected breast tissues, which may have influences on the risk of breast cancer development. Adenovirus-Cre infection is a commonly used approach to induce cancer initiation in genetically engineered mice, such as those for lung cancer 36 , 37 , ovarian/gynecologic cancers 38 , 39 , sarcoma 40 , 41 , and bladder cancer 42 , 43 . It is anticipated that transient adenoviral infection in their corresponding tissues of origin may also affect their immune landscapes, potentially leading to long-lasting immune cell changes locally. Thus, caution should be taken when interpreting immune-related data from cancer mouse models when this induction approach is used. Methods Animals All animal experiments were conducted in accordance with the approved animal protocol (2020N000122) and overseen by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women’s Hospital (BWH). The reporting in the manuscript follows the recommendations in the ARRIVE guidelines. Trp53 fl/fl mice (B6.129P2- Trp53tm1 Brn /J, strain# 008462), Rosa26-stop-YFP ( R26Y ) mice (B6.129X1- Gt(ROSA)26Sor tm1(EYFP)Cos /J, strain# 006148), and K8-CreER transgenic mice (Tg(Krt8-cre/ERT2)17Blpn/J, strain# 017947) were purchased from The Jackson Laboratory (JAX). Homozygous Trp53 fl/fl ; R26Y homo mice ( PY ) were obtained by breeding Trp53 fl/fl mice with R26Y mice. K8-CreER;Trp53 fl/fl ; R26Y homo mice ( KPY ) were produced by further crossing PY mice with K8-CreER transgenic mice. K8-CreER;R26Y homo control mice ( KY ) were generated by breeding the K8-CreER allele into R26Y homo mice. All mouse lines have been backcrossed into the FVB/NJ background for at least six generations. Mouse modeling and lineage tracing Female mice at 8 weeks of age (~ 15-20g of body weight) were used for lineage tracing. Cre/loxP recombination in luminal mammary epithelial cells (MECs) was induced by two approaches: adenovirus-Cre infection or tamoxifen (TAM) injection. Induced mice were analyzed at 3 or 5 months later (i.e., mid-premalignant stage: ~3 months post-induction, ~ 20-25g of body weight; late-premalignant stage: ~5 months post-induction, ~ 25-35g of body weight). The Keratin 8 ( K8 ) promoter-driven Cre-expressing adenoviral vector was generated in-house and deposited at the University of Iowa Viral Vector Core Facility. The Ad-K8-Cre adenovirus was subsequently obtained from the same facility (WC-Li-535). The concentrated adenovirus was diluted in sterile advanced DMEM/F12 culture medium supplemented with 0.1% Bromophenol blue and 0.01 M CaCl 2 . 5 µL of the diluted adenovirus prep (10 9 pfu/mL) was injected into each abdominal mammary gland, as previously described 22 . For tamoxifen-induced recombination, tamoxifen (T5648, Sigma-Aldrich, Chicago, USA) was dissolved in corn oil by rotating at 37°C overnight to a final concentration of 50 mg/mL. Mice received intraperitoneal (i.p.) injections of 5 mg tamoxifen (100 µL) every other day for two doses. Mammary gland single cell preparation Mice were euthanized by carbon dioxide (CO 2 ) overdose in Euthanex multi-cage chamber units, which were set to introduce 100% CO 2 at a fill rate of 30% displacement of the chamber volume per minute with CO 2 , added to the existing air in the chamber. Both abdominal (#4) mammary glands were harvested from each individual mouse and then pooled as a single sample. Tissues were minced into fine fragments and digested in digestion media (5 mL advanced DMEM/F12 culture medium containing 2% Fetal Bovine Serum (FBS), 1% HEPES, 16.5 µg Collagenase I, and 75 µg DNase I) at 37°C for 1 hour with rotation. The resulting single-cell suspension was treated with Red Blood Cell (RBC) lysis buffer for 5 minutes at room temperature (RT) to deplete RBCs. After washing out the lysis buffer, the remaining cells were subjected to flow cytometry analysis. Flow cytometry (FACS) analysis To assess cytokine expression via intracellular staining, an aliquot of single-cell suspension from the mammary gland digestion was incubated with a cell activation cocktail in RMPI 1640 supplemented with 10% FBS and 1% Penicillin-Streptomycin for 5 hours in the presence of Brefeldin A. The remaining cells were immediately subjected to antibody staining for the surface markers. Briefly, the cells were first incubated with fixable viability dye (1:500 dilution) for 20 minutes at RT in the dark to label the dead cells. After centrifugation and removal of the dye solution, the cells were incubated with anti-mouse CD16/32 antibody (1:200; Cat# 156603, clone S17011E; BioLegend, San Diego, USA) at 4°C for 5 minutes to block non-specific binding. Subsequently, cells were stained with a master mix of fluorophore-conjugated antibodies against surface markers (see Table 1 for details) at 4°C for 30 minutes, with dilutions as specified by the manufacturers' protocols. After washing out the antibody dilution, the cells were resuspended and immediately analyzed by flow cytometry. Table 1 FACS antibodies REAGENT or RESOURCE SOURCE CLONE# IDENTIFIER Rat anti-mouse CD45 (BV605) Biolegend 3—F11 Cat# 103155 Rat anti-mouse TER-119 (BV605) Biolegend TER-119 Cat# 116239 Rat anti-mouse CD31 (BV605) Biolegend 390 Cat# 102427 Rat anti-mouse CD24 (PE) Biolegend M1/69 Cat# 101808 Hamster anti-mouse CD29 (APC) Biolegend HMβ1–1 Cat# 102216 Rat anti-mouse CD8a (BUV395) BD 53 − 6.7 Cat# 565968 Hamster anti-mouse CD3 (BUV737) BD 500A2 Cat# 741716 Rat anti-mouse CD4 (BV711) Biolegend RM4-5 Cat# 100549 Rat anti-mouse NKp46 (BV650) Biolegend 29A1.4 Cat# 137635 Hamster anti-mouse CD69 (PE/Cyanine7) Biolegend H1.2F3 Cat# 104512 Rat anti-mouse PD-1 (PE/Cyanine5) Biolegend 29F.1A12 Cat# 135255 Rat anti-mouse CD45 (Alexa Fluor 700) Biolegend 30-F11 Cat# 103128 Rat anti-mouse IFN-γ (BV605) Biolegend XMG1.2 Cat# 505839 Rat anti-mouse CD73 (BV605) Biolegend TY/11.8 Cat# 127215 Mouse anti-mouse CD103 (BV421) Biolegend QA17A24 Cat# 156915 Rat anti-mouse F4/80 (BUV395) BD T45-2342 Cat# 565614 Rat anti-mouse CD19 (BUV737) BD 1D3 Cat# 612782 Rat anti-mouse CD11b (BV650) Biolegend M1/70 Cat# 101239 Rat anti-mouse I-A/I-E (BV605) Biolegend M5/114.15.2 Cat# 107639 Hamster anti-mouse CD11c (BV510) Biolegend N418 Cat# 117337 Rat anti-mouse Ly-6G (BV421) Biolegend 1A8 Cat# 127627 Rat anti-mouse CD86 (PerCP/Cyanine 5.5) Biolegend GL-1 Cat# 105027 Rat anti-mouse CD206 (PE/Dazzle 594) Biolegend C068C2 Cat# 141732 Zombie NIR Fixable Viability Kit Biolegend N/A Cat# 423106 Zombie UV Fixable Viability Kit Biolegend N/A Cat# 423108 For intracellular staining, pre-activated cells were harvested and processed for surface marker staining first, as described above. Then, the cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (Cat# 424401, BioLegend), and subsequently labeled with the corresponding FACS antibodies. FACS data were acquired on a BD LSR II or BD Symphony A5 analyzer (BD Biosciences, San Jose, USA) and analyzed using Flowjo software (10.10.0). Gating strategies are provided in Supplementary Fig. 3. Statistics The results were presented as mean ± S.E.M. Student’s t tests were used for the comparisons between two groups. Comparisons of multiple groups were performed by two-way ANOVA, and p-values were adjusted using Tukey’s method. No randomization or blinding was used in the in vivo studies. Differences are considered to be significant for * p <0.05, ** p <0.01, and *** p <0.005. Declarations Acknowledgments 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 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. Data availability statement All data generated and analyzed in this study are included in this published article and its Supplementary files. Additional information The authors declare no competing interests. Funding Declaration: This research was supported by U.S. National Institutes of Health (NIH) grants R01CA222560, R01CA248306, and R01CA295752, and by the Gray Foundation to ZL. References Grivennikov, S. I., Greten, F. R. & Karin, M. Immunity, inflammation, and cancer. Cell 140 , 883–899. 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. 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. 10.1016/j.clbc.2020.06.011 (2021). Swann, J. B. & Smyth, M. J. Immune surveillance of tumors. J. Clin. Invest. 117 , 1137–1146. 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. 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 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. 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. 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 . 11 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. 10.3389/fonc.2021.593337 (2021). Deng, C. X. Conditional knockout mouse models of cancer. Cold Spring Harb Protoc 1217–1233, (2014). 10.1101/pdb.top074393 (2014). Van Keymeulen, A. et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 479 , 189–193. 10.1038/nature10573 (2011). doi:nature10573 [pii]. 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. 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. 10.1006/bbrc.1997.7124 (1997). Koren, S. et al. PIK3CA induces multipotency and multi-lineage mammary tumours. Nature 525 , 114–118. 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. 10.1038/ncomms14431 (2017). Van Keymeulen, A. et al. Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity. Nature 525 , 119–123. 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, (2019). 10.1016/j.cell.2019.06.002 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. http://dx.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. 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 (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, doi:nature12948 [pii] (2014). 10.1038/nature12948 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. 10.1186/s13058-014-0411-0 (2014). Tower, H. et al. Estrogen-induced immune changes within the normal mammary gland. Sci. Rep. 12 , 18986. 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). 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. 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. 4 10.1126/sciimmunol.aas9673 (2019). Afzal, S. et al. Interrelated Oncogenic Viruses and Breast Cancer. Front. Mol. Biosci. 9 , 781111. 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, (2022). 10.1159/000525277 Harkins, L. E. et al. Detection of human cytomegalovirus in normal and neoplastic breast epithelium. Herpesviridae 1 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 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, (2016). 10.1016/j.ebiom.2016.05.025 Zeng, J. et al. Exploring the Potential of Cytomegalovirus-Based Vectors: A Review. Viruses 15 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. 10.1038/nprot.2009.95 (2009). doi:nprot.2009.95 [pii]. 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 , 754–764. 10.1016/j.ccr.2011.04.019 (2011). doi:S1535-6108(11)00167-X [pii]. 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. 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. 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. 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. 10.1038/nrc3858 (2015). Puzio-Kuter, A. M. et al. Inactivation of p53 and Pten promotes invasive bladder cancer. Genes Dev 23, 675–680, doi:gad.1772909 [pii] (2009). 10.1101/gad.1772909 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFig.legendsdocx.docx Supplementaryfigures.pdf Cite Share Download PDF Status: Published Journal Publication published 12 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Feb, 2026 Reviews received at journal 02 Feb, 2026 Reviews received at journal 02 Feb, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers invited by journal 07 Jan, 2026 Editor assigned by journal 05 Jan, 2026 Editor invited by journal 29 Dec, 2025 Submission checks completed at journal 26 Dec, 2025 First submitted to journal 26 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8398573","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":570993435,"identity":"cc47d17c-1737-4376-8918-fc546c49b1a4","order_by":0,"name":"Sen Han","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sen","middleName":"","lastName":"Han","suffix":""},{"id":570993436,"identity":"38518991-241c-40cd-b09e-90825ebe971a","order_by":1,"name":"Dongyi Zhao","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dongyi","middleName":"","lastName":"Zhao","suffix":""},{"id":570993437,"identity":"a1ba7fa2-000f-4ee8-a0c5-c86616db45e2","order_by":2,"name":"Xueqing Chen","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xueqing","middleName":"","lastName":"Chen","suffix":""},{"id":570993441,"identity":"1115c210-3c97-4daa-9125-732c92b94aa8","order_by":3,"name":"Miao Zhu","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Miao","middleName":"","lastName":"Zhu","suffix":""},{"id":570993443,"identity":"1cb7e1ff-fec1-416a-a47b-20c868db2f87","order_by":4,"name":"Tiantian Li","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tiantian","middleName":"","lastName":"Li","suffix":""},{"id":570993444,"identity":"f142cc76-cae2-4ac7-a7b6-680538e59357","order_by":5,"name":"Chujun Wang","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chujun","middleName":"","lastName":"Wang","suffix":""},{"id":570993445,"identity":"a8c74ba9-d4c8-42ec-bddc-28c5c3e361f6","order_by":6,"name":"Huabiao Chen","email":"","orcid":"","institution":"Harvard Medical School","correspondingAuthor":false,"prefix":"","firstName":"Huabiao","middleName":"","lastName":"Chen","suffix":""},{"id":570993446,"identity":"a63a1ca2-a8e0-4f80-9f55-dfb071b50458","order_by":7,"name":"Zhe Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDACCeaGAx8bGBIYJCB8xgYGBgMCWhgbDs4kWQszL0la5Gc3Nh623XE4j0G6+dlnnpp7sg3szdsk8GlhnHOw4XDumcPFDDLHjGfzHCs2buA5VoZXC7NEIlBL2+3EBokEY8YZbAlARo4ZXi1sIC2WYC3pnxln/ANqkX+DXwsPSAsjWEuOMcPHNpAtPPi1SAC1HOw98z+xTSKnmOFjX4JxG09asQU+LfIzkg9/+LkjLbFfIn0zQ8K3BNl+9sMbb+DTgvAUBmMUjIJRMApGAfkAAOAGTY7qSuGAAAAAAElFTkSuQmCC","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-12-18 19:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8398573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8398573/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-43069-8","type":"published","date":"2026-03-12T15:58:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":99875932,"identity":"2765ba0f-9817-484c-beb1-985b2701c03d","added_by":"auto","created_at":"2026-01-09 10:15:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116438,"visible":true,"origin":"","legend":"","description":"","filename":"AdTammainSR.v1d.docx","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/c727ad70740671883e37b5c9.docx"},{"id":100358183,"identity":"e3ad44f5-32f7-4e82-b4ac-bc1d04c80428","added_by":"auto","created_at":"2026-01-16 07:20:43","extension":"json","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9224,"visible":true,"origin":"","legend":"","description":"","filename":"40641f6706d04759b1a9c409b3512afd.json","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/220429986f32b007389c7dde.json"},{"id":100358060,"identity":"060e2ec2-47af-4013-b906-0c5e38e7194a","added_by":"auto","created_at":"2026-01-16 07:20:37","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126522,"visible":true,"origin":"","legend":"","description":"","filename":"40641f6706d04759b1a9c409b3512afd1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/aa6dbda136da7a0bfc37ee19.xml"},{"id":100358062,"identity":"99ee10e3-3113-4b50-a49d-fe5bcec3ee59","added_by":"auto","created_at":"2026-01-16 07:20:37","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1992618,"visible":true,"origin":"","legend":"","description":"","filename":"AdTamallfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/7a48e10c220d7f35bb587559.pdf"},{"id":99875937,"identity":"2ddf5681-2de2-437b-ad45-8476aaa50fe6","added_by":"auto","created_at":"2026-01-09 10:15:27","extension":"xml","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124882,"visible":true,"origin":"","legend":"","description":"","filename":"40641f6706d04759b1a9c409b3512afd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/fa9c4ec7c130211fcd0ecfe1.xml"},{"id":99875936,"identity":"7b0dcdfc-5d79-4279-b348-d06f26cf9256","added_by":"auto","created_at":"2026-01-09 10:15:27","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137062,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/d323773269b3fc8d16306d38.html"},{"id":100358281,"identity":"285306ac-2119-4bc4-8d52-7b1549a0b2f7","added_by":"auto","created_at":"2026-01-16 07:20:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":971593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdenoviral infection enhanced mammary gland leucocyte infiltration.\u003c/strong\u003e (a) Schematic diagram of lineage-tracing schemes using Cre-expressing adenovirus (\u003cem\u003eAd-K8-Cre\u003c/em\u003e) or tamoxifen (TAM). (b) One week post intraductal injection with \u003cem\u003eAd-K8-Cre\u003c/em\u003e, FACS analysis of infiltrated leukocytes (CD45\u003csup\u003e+\u003c/sup\u003e) in the mammary glands with and without adenovirus injection in \u003cem\u003eR26Y\u003c/em\u003e mice (n=5, representative FACS plots are shown). (c) Statistical analysis of leukocyte infiltration between the injected (abdominal) and non-injected mammary glands (thoracic) from the same mouse. (d, e) FACS and statistical analysis of leucocyte infiltration in mammary glands of \u003cem\u003eKY\u003c/em\u003e mice with or without TAM injection (at one week after injection, n=5). \u003cem\u003eP\u003c/em\u003e value: ***\u003cem\u003ep\u003c/em\u003e＜0.005, ns = not significant. Data represent mean ± S.E.M.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/acb52320fb2ed2caafcbda58.png"},{"id":99875925,"identity":"93da56b0-4e58-4b4b-8243-a6f9e9f66e9c","added_by":"auto","created_at":"2026-01-09 10:15:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1417313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransient adenoviral infection led to T cell infiltration throughout the premalignant stage.\u003c/strong\u003e (a) Representative FACS plots showing CD45\u003csup\u003e+\u003c/sup\u003e leucocyte infiltration within the abdominal mammary glands at mid- (upper panel) and late-premalignant (lower panel) stages; dead cells were excluded from the parental gate. (b) Statistical analysis for leucocyte infiltration as shown in a (n=5). (c) Representative pie chart for the composition of major immune cell populations, including T cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e), NK cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e-\u003c/sup\u003eNKp46\u003csup\u003e+\u003c/sup\u003e), Macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e), dendritic cells (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e), B cells (CD45\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e), and neutrophils (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy-6G\u003csup\u003e+\u003c/sup\u003e), within the leucocyte compartment of mammary glands. \u003cem\u003eP\u003c/em\u003e value: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ns = not significant. Data represent mean ± S.E.M.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/eb15a7220dbcfacfb19345fc.png"},{"id":100357493,"identity":"841d9d35-bb98-4a95-b12e-e224375b3fc7","added_by":"auto","created_at":"2026-01-16 07:19:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1164018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdenoviral infection resulted in abundant CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e tissue-resident T cells.\u003c/strong\u003e Statistical plots for FACS analysis of mammary glands from female mice with the indicated genotypes 3 months and 5 months after inductions. (a) Quantification of percentages (left panel) and absolute cell number (right panel) of CD3\u003csup\u003e+\u003c/sup\u003e T cells within the CD45\u003csup\u003e+\u003c/sup\u003e compartment of mammary glands from mice with the indicated genotypes (n=5 each). (b and c) Quantification of percentages (left panel) and absolute cell number (right panel) of CD8\u003csup\u003e+\u003c/sup\u003e (b) and CD4\u003csup\u003e+\u003c/sup\u003e (c) T cells within the CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e compartment (n=5). (d and e) Statistical plots for CD103 and CD73 expression in CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells at 3 months after induction (n=5). \u003cem\u003eP\u003c/em\u003e value: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.005, ns = not significant. Data represent mean ± S.E.M.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/38ee45b64f79623cac4a35db.png"},{"id":99875930,"identity":"8469f72b-c7da-4ff1-bd6c-5e5ed89181e3","added_by":"auto","created_at":"2026-01-09 10:15:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":896225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe adenoviral infection-driven CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e tissue-resident T cell phenotype masked p53 loss-driven CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cell responses.\u003c/strong\u003e Statistical plots for FACS analysis of mammary glands from female mice with the indicated genotypes 3 and 5 months after inductions. (a-c) Statistical plots for IFN-γ, PD-1, and CD69 expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells (n=5). (d-f) Statistical plots for IFN-γ, PD-1, and CD69 expression in CD4\u003csup\u003e+\u003c/sup\u003e T cells (n=5). \u003cem\u003eP\u003c/em\u003e value: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.005, ns = not significant. Data represent mean ± S.E.M.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/dce1b11656be6a30640664c3.png"},{"id":100358579,"identity":"cb66de4b-8b7f-4ffa-9d35-5343388d1230","added_by":"auto","created_at":"2026-01-16 07:21:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":828362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncrease in macrophage abundance driven by p53-loss was blunted in virus-induced models.\u003c/strong\u003e (a and b) Statistical plots for FACS analysis in quantifying the percentage (a) and absolute number (b) of macrophages (CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e) within the CD45\u003csup\u003e+\u003c/sup\u003e compartment of mammary glands (n=5). (c-e) Statistical plots for CD206, I-A/I-E, and CD86 expression in mammary gland macrophages (n=5).\u003cem\u003e P\u003c/em\u003e value: *\u003cem\u003ep\u003c/em\u003e＜0.05, **\u003cem\u003ep\u003c/em\u003e＜0.01, ***\u003cem\u003ep\u003c/em\u003e＜0.005, ns = not significant. Data represent mean ± S.E.M.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/83fc9197bbdc89f6d2825388.png"},{"id":104739366,"identity":"32f1a0cc-280c-47f4-a2c3-f895c47f6677","added_by":"auto","created_at":"2026-03-16 16:04:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6898278,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/5d3e6a21-fcc3-4d9b-9885-0259a11e170c.pdf"},{"id":99875924,"identity":"dcfec05d-651f-494c-abdf-d41b55fa3ef7","added_by":"auto","created_at":"2026-01-09 10:15:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14671,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.legendsdocx.docx","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/f6b130c508bb9ab76a73cc62.docx"},{"id":99875928,"identity":"d059ab38-3680-4113-8c85-70e92b5763b6","added_by":"auto","created_at":"2026-01-09 10:15:27","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1025546,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8398573/v1/5b96f6944e34ede193436b2e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transient adenovirus-Cre infection causes long-lasting remodeling of the mammary gland immune landscape","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDevelopment of cancers, such as breast cancer, from their corresponding cellular origins occurs within a dynamic immunological ecosystem in which immune cells can both restrain and promote tumorigenesis. A \u0026ldquo;fight\u0026rdquo; between anti-tumorigenic and pro-tumorigenic immune and inflammatory mechanisms can determine the course of tumor development and its phenotype \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In breast cancer, during its early stages of development, innate and adaptive immune surveillance mechanisms, such as cytotoxic T cells, NK cells, dendritic cells, and macrophages, can recognize and eliminate transformed mammary epithelial cells (MECs), limiting malignant progression \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, as oncogenic signaling, genomic instability, and tissue remodeling intensify, the evolving tumor microenvironment can reshape these immune populations, such as during the transition from the precancer to cancer stages \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Myeloid-derived immune cells, particularly tumor-associated macrophages and neutrophils, may adopt pro-tumorigenic phenotypes that support immunosuppression, angiogenesis, and invasion, while regulatory T cells and exhausted T-cell states dampen effective antitumor immunity \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Defining how these immune populations are recruited, activated or suppressed, and reprogrammed during the early phases of mammary tumorigenesis is essential for designing strategies that bolster endogenous immune defenses. By understanding the immunological events that tip the balance from elimination to escape, immune-based preventive approaches may be developed to intercept breast cancer at its roots, before clinically detectable disease emerges.\u003c/p\u003e \u003cp\u003eAs access to early stages of human cancer development, including that of breast cancer, is very difficult if not impossible, in order to achieve the goal of immune-based cancer interception, animal models that can recapitulate this early phase of tumorigenesis are essential. In breast cancer, several elegant approaches have been developed to model mammary tumor initiation from a defined subpopulation of MECs and to follow their progression in mice \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. One of the prevalent approaches is the Cre/loxP-based strategy that enables precise temporal and spatial control over oncogenic events within the mammary epithelium \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. By placing Cre recombinase under the control of mammary-specific promoters, such as \u003cem\u003eMMTV\u003c/em\u003e, \u003cem\u003eWap\u003c/em\u003e, or \u003cem\u003eKrt8\u003c/em\u003e \u003csup\u003e12,13\u003c/sup\u003e, and activating it through ligand-dependent systems such as CreER (Cre-estrogen receptor fusion, inducible by tamoxifen \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e), researchers can trigger defined genetic alterations, including oncogene activation or tumor suppressor loss, at selected MEC subpopulations or developmental stages. This approach faithfully recapitulates the stepwise evolution of breast cancer, permitting the study of early cellular transformation, clonal expansion, and cancer-immune cell interaction within an intact immune microenvironment \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To complement the CreER/tamoxifen-based inducible approach, my group developed a novel approach based on intraductal injection of Cre-expressing adenovirus to mouse mammary glands \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Adenovirus is a DNA virus that does not integrate into the host genome and the adenoviral vector we use only leads to transient expression of the Cre recombinase; thus, it serves a similar purpose as an inducible Cre system. The cell type-specificity (for inducing Cre-mediated recombination) is achieved by using different MEC subpopulation-specific promoters (e.g., \u003cem\u003eKrt8\u003c/em\u003e, \u003cem\u003eWap\u003c/em\u003e) to drive adenovirus-Cre expression.\u003c/p\u003e \u003cp\u003eBreast cancer mouse models based on both CreER/tamoxifen and adenovirus-Cre-based approaches offer excellent \u003cem\u003ein vivo\u003c/em\u003e systems to characterize immune cell phenotypes and their dynamic changes at different stages of mammary tumorigenesis. In these induced mice, mammary gland immune cells can be modulated by signals from mutated MECs. However, it is unclear whether the injected tamoxifen or adeno-Cre virus (to induce Cre-mediated recombination and initiation of mammary tumorigenesis) can affect the immune microenvironment. This is possible as administration of tamoxifen \u003cem\u003ein vivo\u003c/em\u003e can potentially affect estrogen signaling in the mammary gland \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, while estrogen/ER (estrogen receptor) signaling plays a key role in regulating mammary gland immune cell populations \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Similarly, injection of adenovirus to mammary glands should trigger virus-induced immune reactions, at least transiently \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, it is unclear whether any of these induction approach-related modulations of mammary gland immune cells is transient or more long-lasting and whether such change would affect immune cell phenotypes and dynamics, and ultimately, mammary tumor development. A thorough understanding of these should have important implications for the proper use of these modeling approaches to study immune cells during mammary tumorigenesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAdenoviral infection increases leukocyte infiltration in the mammary gland\u003c/h2\u003e \u003cp\u003eIn this study, we used a breast cancer mouse model that we previously developed based on induced loss of p53 in luminal MECs \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The experimental mice (\u003cem\u003ePY\u003c/em\u003e) carry \u003cem\u003eTrp53\u003c/em\u003e conditional knockout alleles (\u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e)\u003csup\u003e26\u003c/sup\u003e along with a conditional Cre-reporter [\u003cem\u003eRosa26-LSL-YFP\u003c/em\u003e (\u003cem\u003eR26Y\u003c/em\u003e)]\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, while \u003cem\u003eR26Y\u003c/em\u003e reporter-only mice served as wild-type (WT) controls (Supplementary Fig.\u0026nbsp;1a). To induce mammary tumor initiation specifically in luminal MECs, we utilized an adenovirus expressing Cre under the control of the luminal \u003cem\u003ekeratin 8\u003c/em\u003e (\u003cem\u003eKrt8\u003c/em\u003e, or \u003cem\u003eK8\u003c/em\u003e) promoter (\u003cem\u003eAd-K8-Cre\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Intraductal injection of \u003cem\u003eAd-K8-Cre\u003c/em\u003e into the abdominal (#4) mammary glands of \u003cem\u003ePY\u003c/em\u003e female mice induced simultaneous YFP reporter expression and \u003cem\u003eTrp53\u003c/em\u003e deletion in luminal MECs, resulting in the development of mammary tumors (predominantly of the Claudin-low subtype) with 100% penetrance \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. For comparison, we also used a CreER/tamoxifen-based approach to induce p53-loss in the same luminal MEC population. We generated \u003cem\u003eKPY\u003c/em\u003e experimental mice and \u003cem\u003eKY\u003c/em\u003e controls by crossing the \u003cem\u003eK8-CreER\u003c/em\u003e allele, a transgenic construct expressing CreER\u003csup\u003eT2\u003c/sup\u003e under the same \u003cem\u003eK8\u003c/em\u003e promoter \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, with \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice, respectively (Supplementary Fig.\u0026nbsp;1b). Tamoxifen (TAM) induction in \u003cem\u003eKPY\u003c/em\u003e female mice led to development of mammary tumors similar to those observed in \u003cem\u003eAd-K8-Cre\u003c/em\u003e-induced \u003cem\u003ePY\u003c/em\u003e mice, also with full penetrance \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate how p53-loss in luminal MECs alters the mammary gland immune microenvironment during the premalignant stage, we induced p53 deletion by administering \u003cem\u003eAd-K8-Cre\u003c/em\u003e or TAM to \u003cem\u003ePY\u003c/em\u003e (and \u003cem\u003eR26Y\u003c/em\u003e) or \u003cem\u003eKPY\u003c/em\u003e (and \u003cem\u003eKY\u003c/em\u003e) female mice at ~\u0026thinsp;8 weeks of age, respectively. Based on our previous findings, mammary tumors begin to emerge in induced mice around 5\u0026ndash;6 months after induction \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Accordingly, in this study we defined 3 and 5 months post-induction as the mid- and late-premalignant stages, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To determine whether intraductally delivered adenovirus itself elicits additional immunogenicity capable of altering the mammary immune microenvironment, we evaluated leukocyte infiltration one week after injection. FACS analysis revealed that adenovirus-injected \u003cem\u003eR26Y\u003c/em\u003e mice exhibited a significantly higher proportion of CD45\u003csup\u003e+\u003c/sup\u003e leucocytes in the injected abdominal mammary glands (55.5%\u0026plusmn;7.1%) compared with non-injected thoracic glands from the same mice (35.76%\u0026plusmn;4.74%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-c). In contrast, the \u003cem\u003eK8-CreER\u003c/em\u003e-based model relies on systemic TAM administration via intraperitoneal injection, which could in principle exert global effects on all mammary glands through its modulation of estrogen signaling. To assess this possibility, we compared leukocyte infiltration in mammary glands from TAM-treated \u003cem\u003eKY\u003c/em\u003e mice with that of age-matched, untreated \u003cem\u003eKY\u003c/em\u003e controls. The data indicate that TAM treatment does not significantly alter leukocyte infiltration (28.36%\u0026plusmn;3.16% vs. 30.98%\u0026plusmn;3.32%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransient adenoviral infection in the mammary gland increases T cell infiltration\u003c/h3\u003e\n\u003cp\u003eTo determine whether transient adenoviral infection in \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice has any long-term effects on the immune microenvironment of the injected mammary glands, we examined leucocyte infiltration at mid- and late-premalignant stages using FACS analysis. At the mid-stage, both adenovirus-infected \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice displayed higher levels of leukocyte infiltration compared with their counterparts induced via TAM (i.e., \u003cem\u003eKPY\u003c/em\u003e and \u003cem\u003eKY\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). Induced p53 loss in luminal MECs modestly increased immune activity in the mammary glands of \u003cem\u003eKPY\u003c/em\u003e mice relative to \u003cem\u003eKY\u003c/em\u003e controls; however, no such difference was detected between adenovirus-induced \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). By the late stage, leukocyte infiltration in induced \u003cem\u003eKPY\u003c/em\u003e mice rose to levels comparable to those of adenovirus-induced \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice, whereas \u003cem\u003eKY\u003c/em\u003e controls remained substantially lower. In contrast, leukocyte infiltration in adenovirus-induced \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice remained similar to each other throughout both stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). These findings indicate that transient adenoviral infection causes a sustained increase in mammary gland leukocyte infiltration. Furthermore, loss of p53 in luminal MECs likely triggers an anti-tumor immune response that enhances mammary gland immune activity. This effect is readily detectable in TAM-induced \u003cem\u003eKPY\u003c/em\u003e mice but may be obscured in adenovirus-induced \u003cem\u003ePY\u003c/em\u003e mice due to the elevated baseline immune infiltration caused by prior adenoviral exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo exclude the possibility that the increased immune activation observed in the \u003cem\u003eKPY\u003c/em\u003e model following TAM induction was simply due to a higher recombination efficiency compared with adenoviral delivery, we evaluated the initial induction and subsequent clonal expansion of premalignant MECs in both models. We first quantified the proportion of premalignant cells (YFP\u003csup\u003e+\u003c/sup\u003e) within the lineage-negative compartment (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003e, i.e., CD45\u003csup\u003e\u0026minus;\u003c/sup\u003eTER119\u003csup\u003e\u0026minus;\u003c/sup\u003eCD31\u003csup\u003e\u0026minus;\u003c/sup\u003e) two weeks after induction. The frequency of YFP\u003csup\u003e+\u003c/sup\u003e premalignant cells was comparable between experimental and control groups across both induction strategies (Supplementary Fig.\u0026nbsp;2a-b). Moreover, more than 90% of YFP\u003csup\u003e+\u003c/sup\u003e cells localized to the luminal MEC gate (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eCD24\u003csup\u003ehigh\u003c/sup\u003eCD29\u003csup\u003elow\u003c/sup\u003e), indicating that both induction approaches achieved similar levels of p53 deletion specifically within luminal MECs (Supplementary Fig.\u0026nbsp;2c). We next assessed the clonal expansion capacity of p53-null MECs during premalignancy by FACS. In the adenovirus-induced model, p53-null MECs (YFP\u003csup\u003e+\u003c/sup\u003e) represented 0.29%\u0026plusmn;0.13% of the Lin\u003csup\u003e\u0026minus;\u003c/sup\u003e compartment at two weeks post-induction, increasing to 0.85%\u0026plusmn;0.3% at the mid-stage and 2.89%\u0026plusmn;0.92% at the late-stage of premalignancy. In contrast, YFP\u003csup\u003e+\u003c/sup\u003e MECs in induced \u003cem\u003eR26Y\u003c/em\u003e mice (which retain WT p53) remained at a steady percentage (~\u0026thinsp;0.6% YFP\u003csup\u003e+\u003c/sup\u003e) throughout the premalignant period (Supplementary Fig.\u0026nbsp;2d). A similar pattern was observed in the TAM-induced model (Supplementary Fig.\u0026nbsp;2e). Together, these results demonstrate that both induction strategies generate comparable initial recombination efficiencies in luminal MECs, and that p53-loss confers a consistent clonal expansion advantage during premalignancy.\u003c/p\u003e \u003cp\u003eTo evaluate how transient adenoviral infection reshapes the immune landscape of the mammary gland, we quantified major immune cell populations, including T cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e), NK cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003eNKp46\u003csup\u003e+\u003c/sup\u003e), macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e), dendritic cells (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e), B cells (CD45\u003csup\u003e+\u003c/sup\u003eCD19\u003csup\u003e+\u003c/sup\u003e), and neutrophils (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy-6G\u003csup\u003e+\u003c/sup\u003e), by FACS analysis (Supplementary Fig.\u0026nbsp;3). Across all time points, adenovirus-infected mammary glands in both \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice exhibited a marked increase in T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), indicating that the elevated baseline immune infiltration observed following adenoviral injection is driven primarily by T cell enrichment.\u003c/p\u003e\n\u003ch3\u003eAdenoviral infection induces CD8\u003csup\u003e+\u003c/sup\u003e tissue-resident T cells in the mammary gland\u003c/h3\u003e\n\u003cp\u003eWe next examined the CD3\u003csup\u003e+\u003c/sup\u003e T cell compartment in greater detail. Regardless of the p53 mutagenesis status, adenoviral infection markedly increased CD3\u003csup\u003e+\u003c/sup\u003e T cell infiltration, both in frequency and absolute cell number, in virally infected mammary glands at both mid- and late-premalignant stages, compared with TAM-induced mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This elevation in T cell abundance was driven primarily by an increase in CD8\u003csup\u003e+\u003c/sup\u003e T cells, rather than CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c). Given that transient adenoviral infection produced a sustained elevation in CD8\u003csup\u003e+\u003c/sup\u003e T cells, we investigated whether this reflected an expansion of tissue-resident memory T cells. To do so, we assessed CD103 and CD73 expression, canonical markers of tissue-resident memory T cells \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, on both CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cell populations. In mice analyzed 3 months post-induction, ~\u0026thinsp;90% of infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells in adenovirus-infected mammary glands (both \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e) expressed CD103, compared with only\u0026thinsp;~\u0026thinsp;30% in TAM-induced \u003cem\u003eKPY\u003c/em\u003e and \u003cem\u003eKY\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). CD73 expression was relatively high on CD8⁺ T cells across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In contrast, neither CD103 nor CD73 showed substantial differences on CD4\u003csup\u003e+\u003c/sup\u003e T cells across induction methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Collectively, these findings indicate that transient adenoviral infection elicits a robust CD8\u003csup\u003e+\u003c/sup\u003e T cell response, promoting their infiltration and long-term residency within the mammary gland microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCD8\u003csup\u003e+\u003c/sup\u003e tissue-resident T cell phenotype masks p53 loss-driven CD8\u003csup\u003e+\u003c/sup\u003e T cell activation\u003c/h3\u003e\n\u003cp\u003eWe next characterized CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets and functional states during premalignancy by assessing expression of IFN-γ, as well as the activation markers PD-1 and CD69, using FACS analysis. In the TAM-induced model (\u003cem\u003eKPY\u003c/em\u003e), induced p53-loss in luminal MECs elicited a pronounced increase in IFN-γ, PD-1, and CD69 expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells at the late-premalignant stage. At the mid-stage, CD8\u003csup\u003e+\u003c/sup\u003e T cells in \u003cem\u003eKPY\u003c/em\u003e mice showed increased CD69 expression but no significant elevation in IFN-γ or PD-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). In contrast, in the adenovirus-induced model, induced p53-loss in luminal MECs from \u003cem\u003ePY\u003c/em\u003e mice did not significantly alter IFN-γ or PD-1 expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells when comparing to induced \u003cem\u003eR26Y\u003c/em\u003e controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). Instead, CD8\u003csup\u003e+\u003c/sup\u003e T cells in both adenovirus-induced \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice displayed consistently elevated activation states at both mid- and late-stages relative to their TAM-induced counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). This sustained activation profile appears to result from the large pool of tissue-resident CD8\u003csup\u003e+\u003c/sup\u003e T cells resulted from the prior adenoviral infection. Supporting this, the majority of PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e and CD69\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e cells in adenovirus-induced \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice co-expressed CD103, whereas most PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e and CD69\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e cells in TAM-induced \u003cem\u003eKPY\u003c/em\u003e and \u003cem\u003eKY\u003c/em\u003e mice lacked CD103 expression (Supplementary Fig.\u0026nbsp;4a). In contrast, the CD103 expression in the PD-1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD69\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e cells from both models was low and not significantly different (Supplementary Fig.\u0026nbsp;4b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the CD4\u003csup\u003e+\u003c/sup\u003e T cell compartment, adenovirus-induced \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice exhibited a transient increase in IFN-γ-producing, activated CD4\u003csup\u003e+\u003c/sup\u003e T cells at the mid-premalignant stage, which declined to levels comparable to those of TAM-induced mice by the late stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In contrast, mammary glands from TAM-induced \u003cem\u003eKPY\u003c/em\u003e mice displayed a selective increase in IFN-γ-producing, activated CD4\u003csup\u003e+\u003c/sup\u003e T cells during the late-premalignant stage compared with \u003cem\u003eKY\u003c/em\u003e controls, a feature not observed in the adenovirus-induced model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). A similar late-stage increase in PD1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD69\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells was also uniquely observed in induced \u003cem\u003eKPY\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-f). Taken together, these findings indicate that transient adenoviral infection recruits CD8\u003csup\u003e+\u003c/sup\u003e T cells into the injected mammary gland, where they differentiate into tissue-resident T cells. These virus-induced CD8\u003csup\u003e+\u003c/sup\u003e T cells display a persistently activated phenotype that likely obscures the CD8\u003csup\u003e+\u003c/sup\u003e T cell response elicited by the expanding p53-null mutant MEC population. In parallel, viral infection appears to drive otherwise quiescent CD4\u003csup\u003e+\u003c/sup\u003e T cells into a transient, IFN-γ-producing, virus-specific state, which may limit their engagement in immune responses against p53-null MECs.\u003c/p\u003e\n\u003ch3\u003eAdenoviral infection-induced CD8\u003csup\u003e+\u003c/sup\u003e T cell influx condenses the myeloid compartment\u003c/h3\u003e\n\u003cp\u003eLastly, we examined the myeloid compartment in the mammary gland. In the TAM-induced model, induced p53-loss in luminal MECs resulted in increased macrophage infiltration (both in percentage and absolute number) at mid- and late-premalignant stages (in \u003cem\u003eKPY\u003c/em\u003e vs. \u003cem\u003eKY\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). In contrast, adenovirus-induced \u003cem\u003ePY\u003c/em\u003e and \u003cem\u003eR26Y\u003c/em\u003e mice exhibited a pronounced reduction in the macrophage frequency, whereas absolute macrophage numbers remained more comparable to those observed in TAM-induced mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). These macrophage changes appeared to be driven primarily by adenoviral infection rather than p53 status. Nevertheless, related to p53-loss, a modest late-stage increase in macrophage abundance was also detected in adenovirus-induced \u003cem\u003ePY\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). Despite these quantitative differences, macrophage polarization marker expression (e.g., CD206, CD86, and I-A/I-E) remained largely similar between adenovirus-induced and TAM-induced models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). However, we observed a trend toward reduced CD206\u003csup\u003e+\u003c/sup\u003e, MHC-II\u003csup\u003e+\u003c/sup\u003e (i.e., I-A/I-E\u003csup\u003e+\u003c/sup\u003e), or CD86\u003csup\u003e+\u003c/sup\u003e macrophage subpopulations in mice with induced loss of p53 (\u003cem\u003ePY\u003c/em\u003e or \u003cem\u003eKPY\u003c/em\u003e) compared with their respective controls (\u003cem\u003eR26Y\u003c/em\u003e or \u003cem\u003eKY\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, by characterizing and comparing mammary gland immune cells during premalignant stages in breast cancer mouse models with the same driver and tumor outcomes but different induction approaches, we observed that transient adenoviral infection not only led to an expected initial immune response, but also reshaped the mammary gland immune microenvironment long-term. The main viral infection-induced long-lasting change was the profound increase in CD8\u003csup\u003e+\u003c/sup\u003e tissue-resident T cells, thereby confounding the detection of p53 loss-induced CD8\u003csup\u003e+\u003c/sup\u003e T cell responses. Moreover, transient adenoviral exposure appeared to skew CD4\u003csup\u003e+\u003c/sup\u003e T cells toward IFN-γ-producing anti-virus-specific states at the mid-stage. Whether such CD4\u003csup\u003e+\u003c/sup\u003e T cell state affects their response to p53-deficient MECs during premalignancy is unclear. Although compared to T cells, the myeloid compartment was less affected by the adenoviral exposure, the expansion of T cell population in the adenovirus-induced model may have compressed the myeloid compartment. In the TAM-induced \u003cem\u003eKPY\u003c/em\u003e (vs. \u003cem\u003eKY\u003c/em\u003e) mice, induced loss of p53 in luminal MECs led to significant increases in both the percentage and absolute number of macrophages at both mid- and late-stages of premalignancy. However, such changes were less profound in adenovirus-induced \u003cem\u003ePY\u003c/em\u003e (vs. \u003cem\u003eR26Y\u003c/em\u003e) mice. Nevertheless, both modeling approaches revealed a similar trend of reductions in CD206\u003csup\u003e+\u003c/sup\u003e, MHC-II\u003csup\u003e+\u003c/sup\u003e (i.e., I-A/I-E\u003csup\u003e+\u003c/sup\u003e), or CD86\u003csup\u003e+\u003c/sup\u003e macrophage subpopulations, correlating with induced p53-loss. Overall, this study highlights the complexity of immune cell changes when using different cancer-induction approaches and offers practical insights for selecting an appropriate induction system to achieve more accurate interpretations of immune regulation during mammary tumorigenesis and beyond.\u003c/p\u003e \u003cp\u003eDespite adenovirus-induced changes in the mammary gland immune landscape, adenovirus-induced \u003cem\u003ePY\u003c/em\u003e and TAM-induced \u003cem\u003eKPY\u003c/em\u003e mice (under similar genetic background) developed mammary tumors with a similar latency \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This does not necessarily suggest that virus-induced changes in mammary gland immune cells have no influence on the course of tumor development. Other factors, such as the time required for p53-deficient MECs to acquire secondary driver mutations, may play a more dominant role in determining the tumor latency. Alternatively, as a tightly regulated host defense system, virus-induced upregulation of CD8\u003csup\u003e+\u003c/sup\u003e tissue-resident T cells may be off-set by changes in other immune cell subsets (e.g., macrophages), making the overall immune reactions toward p53-null mutant MECs comparable to those without the virus-induced changes.\u003c/p\u003e \u003cp\u003eViral infections have been found in both normal and neoplastic human breast epithelial cells. Although infections by viruses such as Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), certain human papillomaviruses (HPV), and mouse mammary tumor viruses (MMTV) are more frequently found in human breast cancers \u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, viral infections of normal human breast epithelial cells have also been reported \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. For instance, it was reported that HCMV infection could be detected in glandular epithelium in 63% of normal adult breast cases \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In another study, it was shown that EBV infection occurs in breast epithelial cells but not in breast cancer cells \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Interestingly, similar to adenovirus, HCMV is a DNA virus that does not typically integrate its DNA into the host genome and persists in the host cell episomally \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. It is possible that viral infections of human breast epithelial cells, even only transient, could also lead to long-last changes in the immune cell landscape in the affected breast tissues, which may have influences on the risk of breast cancer development.\u003c/p\u003e \u003cp\u003eAdenovirus-Cre infection is a commonly used approach to induce cancer initiation in genetically engineered mice, such as those for lung cancer \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, ovarian/gynecologic cancers \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, sarcoma \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and bladder cancer \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. It is anticipated that transient adenoviral infection in their corresponding tissues of origin may also affect their immune landscapes, potentially leading to long-lasting immune cell changes locally. Thus, caution should be taken when interpreting immune-related data from cancer mouse models when this induction approach is used.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in accordance with the approved animal protocol (2020N000122) and overseen by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women\u0026rsquo;s Hospital (BWH). The reporting in the manuscript follows the recommendations in the ARRIVE guidelines. \u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (B6.129P2- \u003cem\u003eTrp53tm1\u003c/em\u003e\u003csup\u003e\u003cem\u003eBrn\u003c/em\u003e\u003c/sup\u003e/J, strain# 008462), \u003cem\u003eRosa26-stop-YFP\u003c/em\u003e (\u003cem\u003eR26Y\u003c/em\u003e) mice (B6.129X1-\u003cem\u003eGt(ROSA)26Sor\u003c/em\u003e\u003csup\u003etm1(EYFP)Cos\u003c/sup\u003e/J, strain# 006148), and \u003cem\u003eK8-CreER\u003c/em\u003e transgenic mice (Tg(Krt8-cre/ERT2)17Blpn/J, strain# 017947) were purchased from The Jackson Laboratory (JAX). Homozygous \u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eR26Y\u003c/em\u003e\u003csup\u003e\u003cem\u003ehomo\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003ePY\u003c/em\u003e) were obtained by breeding \u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice with \u003cem\u003eR26Y\u003c/em\u003e mice. \u003cem\u003eK8-CreER;Trp53\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eR26Y\u003c/em\u003e\u003csup\u003e\u003cem\u003ehomo\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003eKPY\u003c/em\u003e) were produced by further crossing \u003cem\u003ePY\u003c/em\u003e mice with \u003cem\u003eK8-CreER\u003c/em\u003e transgenic mice. \u003cem\u003eK8-CreER;R26Y\u003c/em\u003e\u003csup\u003e\u003cem\u003ehomo\u003c/em\u003e\u003c/sup\u003e control mice (\u003cem\u003eKY\u003c/em\u003e) were generated by breeding the \u003cem\u003eK8-CreER\u003c/em\u003e allele into \u003cem\u003eR26Y\u003c/em\u003e\u003csup\u003e\u003cem\u003ehomo\u003c/em\u003e\u003c/sup\u003e mice. All mouse lines have been backcrossed into the FVB/NJ background for at least six generations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMouse modeling and lineage tracing\u003c/h2\u003e \u003cp\u003eFemale mice at 8 weeks of age (~\u0026thinsp;15-20g of body weight) were used for lineage tracing. Cre/loxP recombination in luminal mammary epithelial cells (MECs) was induced by two approaches: adenovirus-Cre infection or tamoxifen (TAM) injection. Induced mice were analyzed at 3 or 5 months later (i.e., mid-premalignant stage: ~3 months post-induction, ~\u0026thinsp;20-25g of body weight; late-premalignant stage: ~5 months post-induction, ~\u0026thinsp;25-35g of body weight).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eKeratin 8\u003c/em\u003e (\u003cem\u003eK8\u003c/em\u003e) promoter-driven Cre-expressing adenoviral vector was generated in-house and deposited at the University of Iowa Viral Vector Core Facility. The \u003cem\u003eAd-K8-Cre\u003c/em\u003e adenovirus was subsequently obtained from the same facility (WC-Li-535). The concentrated adenovirus was diluted in sterile advanced DMEM/F12 culture medium supplemented with 0.1% Bromophenol blue and 0.01 M CaCl\u003csub\u003e2\u003c/sub\u003e. 5 \u0026micro;L of the diluted adenovirus prep (10\u003csup\u003e9\u003c/sup\u003e pfu/mL) was injected into each abdominal mammary gland, as previously described \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor tamoxifen-induced recombination, tamoxifen (T5648, Sigma-Aldrich, Chicago, USA) was dissolved in corn oil by rotating at 37\u0026deg;C overnight to a final concentration of 50 mg/mL. Mice received intraperitoneal (i.p.) injections of 5 mg tamoxifen (100 \u0026micro;L) every other day for two doses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMammary gland single cell preparation\u003c/h2\u003e \u003cp\u003eMice were euthanized by carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) overdose in Euthanex multi-cage chamber units, which were set to introduce 100% CO\u003csub\u003e2\u003c/sub\u003e at a fill rate of 30% displacement of the chamber volume per minute with CO\u003csub\u003e2\u003c/sub\u003e, added to the existing air in the chamber. Both abdominal (#4) mammary glands were harvested from each individual mouse and then pooled as a single sample. Tissues were minced into fine fragments and digested in digestion media (5 mL advanced DMEM/F12 culture medium containing 2% Fetal Bovine Serum (FBS), 1% HEPES, 16.5 \u0026micro;g Collagenase I, and 75 \u0026micro;g DNase I) at 37\u0026deg;C for 1 hour with rotation. The resulting single-cell suspension was treated with Red Blood Cell (RBC) lysis buffer for 5 minutes at room temperature (RT) to deplete RBCs. After washing out the lysis buffer, the remaining cells were subjected to flow cytometry analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry (FACS) analysis\u003c/h2\u003e \u003cp\u003eTo assess cytokine expression via intracellular staining, an aliquot of single-cell suspension from the mammary gland digestion was incubated with a cell activation cocktail in RMPI 1640 supplemented with 10% FBS and 1% Penicillin-Streptomycin for 5 hours in the presence of Brefeldin A. The remaining cells were immediately subjected to antibody staining for the surface markers.\u003c/p\u003e \u003cp\u003eBriefly, the cells were first incubated with fixable viability dye (1:500 dilution) for 20 minutes at RT in the dark to label the dead cells. After centrifugation and removal of the dye solution, the cells were incubated with anti-mouse CD16/32 antibody (1:200; Cat# 156603, clone S17011E; BioLegend, San Diego, USA) at 4\u0026deg;C for 5 minutes to block non-specific binding. Subsequently, cells were stained with a master mix of fluorophore-conjugated antibodies against surface markers (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for details) at 4\u0026deg;C for 30 minutes, with dilutions as specified by the manufacturers' protocols. After washing out the antibody dilution, the cells were resuspended and immediately analyzed by flow cytometry.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFACS antibodies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eREAGENT or RESOURCE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSOURCE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCLONE#\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIDENTIFIER\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD45 (BV605)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026mdash;F11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 103155\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse TER-119 (BV605)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTER-119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 116239\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD31 (BV605)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 102427\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD24 (PE)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM1/69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 101808\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHamster anti-mouse CD29 (APC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHMβ1\u0026ndash;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 102216\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD8a (BUV395)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u0026thinsp;\u0026minus;\u0026thinsp;6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 565968\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHamster anti-mouse CD3 (BUV737)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 741716\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD4 (BV711)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRM4-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 100549\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse NKp46 (BV650)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29A1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 137635\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHamster anti-mouse CD69 (PE/Cyanine7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH1.2F3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 104512\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse PD-1 (PE/Cyanine5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29F.1A12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 135255\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD45 (Alexa Fluor 700)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30-F11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 103128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse IFN-γ (BV605)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXMG1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 505839\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD73 (BV605)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTY/11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 127215\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-mouse CD103 (BV421)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQA17A24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 156915\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse F4/80 (BUV395)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT45-2342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 565614\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD19 (BUV737)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1D3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 612782\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD11b (BV650)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM1/70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 101239\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse I-A/I-E (BV605)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM5/114.15.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 107639\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHamster anti-mouse CD11c (BV510)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN418\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 117337\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse Ly-6G (BV421)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1A8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 127627\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD86 (PerCP/Cyanine 5.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 105027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat anti-mouse CD206 (PE/Dazzle 594)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC068C2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 141732\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZombie NIR Fixable Viability Kit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 423106\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZombie UV Fixable Viability Kit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiolegend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCat# 423108\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor intracellular staining, pre-activated cells were harvested and processed for surface marker staining first, as described above. Then, the cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (Cat# 424401, BioLegend), and subsequently labeled with the corresponding FACS antibodies.\u003c/p\u003e \u003cp\u003eFACS data were acquired on a BD LSR II or BD Symphony A5 analyzer (BD Biosciences, San Jose, USA) and analyzed using Flowjo software (10.10.0). Gating strategies are provided in Supplementary Fig.\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eThe results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e tests were used for the comparisons between two groups. Comparisons of multiple groups were performed by two-way ANOVA, and p-values were adjusted using Tukey\u0026rsquo;s method. No randomization or blinding was used in the \u003cem\u003ein vivo\u003c/em\u003e studies. Differences are considered to be significant for *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, and ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.005.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by U.S. National Institutes of Health (NIH) grants R01CA222560, R01CA248306, and R01CA295752, and by the Gray Foundation to ZL.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSH 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.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed in this study are included in this published article and its Supplementary files.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding Declaration:\u0026nbsp;\u003c/strong\u003eThis research was supported by U.S. National Institutes of Health (NIH) grants R01CA222560, R01CA248306, and R01CA295752, and by the Gray Foundation to ZL.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGrivennikov, S. I., Greten, F. R. \u0026amp; Karin, M. Immunity, inflammation, and cancer. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e140\u003c/b\u003e, 883\u0026ndash;899. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2010.01.025\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2010.01.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShalapour, S. \u0026amp; Karin, M. Immunity, inflammation, and cancer: an eternal fight between good and evil. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e125\u003c/b\u003e, 3347\u0026ndash;3355. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/JCI80007\u003c/span\u003e\u003cspan address=\"10.1172/JCI80007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoff, S. L. \u0026amp; Danforth, D. N. The Role of Immune Cells in Breast Tissue and Immunotherapy for the Treatment of Breast Cancer. \u003cem\u003eClin. Breast Cancer\u003c/em\u003e. \u003cb\u003e21\u003c/b\u003e, e63\u0026ndash;e73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.clbc.2020.06.011\u003c/span\u003e\u003cspan address=\"10.1016/j.clbc.2020.06.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSwann, J. B. \u0026amp; Smyth, M. J. Immune surveillance of tumors. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e, 1137\u0026ndash;1146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/JCI31405\u003c/span\u003e\u003cspan address=\"10.1172/JCI31405\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVesely, M. D., Kershaw, M. H., Schreiber, R. D. \u0026amp; Smyth, M. J. Natural innate and adaptive immunity to cancer. \u003cem\u003eAnnu. Rev. Immunol.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 235\u0026ndash;271. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-immunol-031210-101324\u003c/span\u003e\u003cspan address=\"10.1146/annurev-immunol-031210-101324\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, S. et al. Tumor initiation and early tumorigenesis: molecular mechanisms and interventional targets. \u003cem\u003eSignal. Transduct. Target. Ther.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41392-024-01848-7\u003c/span\u003e\u003cspan address=\"10.1038/s41392-024-01848-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-Bejarano, O. H., Parra-Lopez, C. \u0026amp; Patarroyo, M. A. A review concerning the breast cancer-related tumour microenvironment. \u003cem\u003eCrit. Rev. Oncol. Hematol.\u003c/em\u003e \u003cb\u003e199\u003c/b\u003e, 104389. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.critrevonc.2024.104389\u003c/span\u003e\u003cspan address=\"10.1016/j.critrevonc.2024.104389\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeglia, F., Sanseviero, E. \u0026amp; Gabrilovich, D. I. Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 485\u0026ndash;498. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41577-020-00490-y\u003c/span\u003e\u003cspan address=\"10.1038/s41577-020-00490-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson, K. G. et al. Leveraging immune resistance archetypes in solid cancer to inform next-generation anticancer therapies. \u003cem\u003eJ. Immunother Cancer\u003c/em\u003e. \u003cb\u003e11\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/jitc-2022-006533\u003c/span\u003e\u003cspan address=\"10.1136/jitc-2022-006533\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, C., Wu, P., Zhang, A. \u0026amp; Mao, X. Advances in Rodent Models for Breast Cancer Formation, Progression, and Therapeutic Testing. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 593337. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2021.593337\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2021.593337\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng, C. X. Conditional knockout mouse models of cancer. \u003cem\u003eCold Spring Harb Protoc\u003c/em\u003e 1217\u0026ndash;1233, (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/pdb.top074393\u003c/span\u003e\u003cspan address=\"10.1101/pdb.top074393\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Keymeulen, A. et al. Distinct stem cells contribute to mammary gland development and maintenance. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e479\u003c/b\u003e, 189\u0026ndash;193. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature10573\u003c/span\u003e\u003cspan address=\"10.1038/nature10573\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011). doi:nature10573 [pii].\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWagner, K. U. et al. Cre-mediated gene deletion in the mammary gland. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 4323\u0026ndash;4330 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeil, R. et al. Ligand-activated site-specific recombination in mice. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e93\u003c/b\u003e, 10887\u0026ndash;10890. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.93.20.10887\u003c/span\u003e\u003cspan address=\"10.1073/pnas.93.20.10887\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeil, R., Wagner, J., Metzger, D. \u0026amp; Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. \u003cem\u003eBiochem. Biophys. Res. Commun.\u003c/em\u003e \u003cb\u003e237\u003c/b\u003e, 752\u0026ndash;757. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1006/bbrc.1997.7124\u003c/span\u003e\u003cspan address=\"10.1006/bbrc.1997.7124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoren, S. et al. PIK3CA induces multipotency and multi-lineage mammary tumours. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e525\u003c/b\u003e, 114\u0026ndash;118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature14669\u003c/span\u003e\u003cspan address=\"10.1038/nature14669\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao, L., Xiang, D., Xie, Y., Bronson, R. T. \u0026amp; Li, Z. Induced p53 loss in mouse luminal cells causes clonal expansion and development of mammary tumours. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 14431. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ncomms14431\u003c/span\u003e\u003cspan address=\"10.1038/ncomms14431\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Keymeulen, A. et al. Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e525\u003c/b\u003e, 119\u0026ndash;123. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature14665\u003c/span\u003e\u003cspan address=\"10.1038/nature14665\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, H. et al. Inadequate DNA Damage Repair Promotes Mammary Transdifferentiation, Leading to BRCA1 Breast Cancer. \u003cem\u003eCell\u003c/em\u003e 178, 135\u0026ndash;151 e119, (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2019.06.002\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2019.06.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao, L., van Bragt, M. P. A., Laudadio, E. \u0026amp; Li, Z. Lineage Tracing of Mammary Epithelial Cells Using Cell-Type-Specific Cre-Expressing Adenoviruses. \u003cem\u003eStem Cell. Rep.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 770\u0026ndash;779. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.stemcr.2014.04.004\u003c/span\u003e\u003cspan address=\"10.1016/j.stemcr.2014.04.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, X., Han, S., Zhao, D. \u0026amp; Li, Z. Intraductal Injection of Adenoviruses to Perform Lineage Tracing in the Mammary Gland. \u003cem\u003eJ. Mammary Gland Biol. Neoplasia\u003c/em\u003e. \u003cb\u003e30\u003c/b\u003e, 10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10911-025-09584-6\u003c/span\u003e\u003cspan address=\"10.1007/s10911-025-09584-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiang, D., Tao, L. \u0026amp; Li, Z. Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland. \u003cem\u003eJ. Vis. Exp.\u003c/em\u003e \u003cb\u003e148\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3791/59502\u003c/span\u003e\u003cspan address=\"10.3791/59502\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRios, A. C., Fu, N. Y., Lindeman, G. J. \u0026amp; Visvader, J. E. In situ identification of bipotent stem cells in the mammary gland. \u003cem\u003eNature\u003c/em\u003e 506, 322\u0026ndash;327, doi:nature12948 [pii] (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature12948\u003c/span\u003e\u003cspan address=\"10.1038/nature12948\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShehata, M., van Amerongen, R., Zeeman, A. L., Giraddi, R. R. \u0026amp; Stingl, J. The influence of tamoxifen on normal mouse mammary gland homeostasis. \u003cem\u003eBreast Cancer Res.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 411. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13058-014-0411-0\u003c/span\u003e\u003cspan address=\"10.1186/s13058-014-0411-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTower, H. et al. Estrogen-induced immune changes within the normal mammary gland. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 18986. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-022-21871-4\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-21871-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarino, S., Vooijs, M., van Der Gulden, H., Jonkers, J. \u0026amp; Berns, A. Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. \u003cem\u003eGenes Dev.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 994\u0026ndash;1004 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. \u003cem\u003eBMC Dev. Biol.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 4 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang, F. et al. The cell-surface 5'-nucleotidase CD73 defines a functional T memory cell subset that declines with age. \u003cem\u003eCell. Rep.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 109981. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.celrep.2021.109981\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2021.109981\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzabo, P. A., Miron, M. \u0026amp; Farber, D. L. Location, location, location: Tissue resident memory T cells in mice and humans. \u003cem\u003eSci. Immunol.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/sciimmunol.aas9673\u003c/span\u003e\u003cspan address=\"10.1126/sciimmunol.aas9673\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfzal, S. et al. Interrelated Oncogenic Viruses and Breast Cancer. \u003cem\u003eFront. Mol. Biosci.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 781111. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmolb.2022.781111\u003c/span\u003e\u003cspan address=\"10.3389/fmolb.2022.781111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta, I. et al. Incidence of HPVs, EBV, and MMTV-Like Virus in Breast Cancer in Qatar. \u003cem\u003eIntervirology\u003c/em\u003e 65, 188\u0026ndash;194, (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1159/000525277\u003c/span\u003e\u003cspan address=\"10.1159/000525277\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarkins, L. E. et al. Detection of human cytomegalovirus in normal and neoplastic breast epithelium. \u003cem\u003eHerpesviridae\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/2042-4280-1-8\u003c/span\u003e\u003cspan address=\"10.1186/2042-4280-1-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawson, J. S. \u0026amp; Glenn, W. K. Evidence for a causal role by mouse mammary tumour-like virus in human breast cancer. \u003cem\u003eNPJ breast cancer\u003c/em\u003e. \u003cb\u003e5\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41523-019-0136-4\u003c/span\u003e\u003cspan address=\"10.1038/s41523-019-0136-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, H. et al. Epstein-Barr Virus Infection of Mammary Epithelial Cells Promotes Malignant Transformation. \u003cem\u003eEBioMedicine\u003c/em\u003e 9, 148\u0026ndash;160, (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ebiom.2016.05.025\u003c/span\u003e\u003cspan address=\"10.1016/j.ebiom.2016.05.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng, J. et al. Exploring the Potential of Cytomegalovirus-Based Vectors: A Review. \u003cem\u003eViruses\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/v15102043\u003c/span\u003e\u003cspan address=\"10.3390/v15102043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuPage, M., Dooley, A. L. \u0026amp; Jacks, T. Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. \u003cem\u003eNat. Protoc.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 1064\u0026ndash;1072. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nprot.2009.95\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2009.95\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009). doi:nprot.2009.95 [pii].\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSutherland, 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. \u003cem\u003eCancer Cell.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 754\u0026ndash;764. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ccr.2011.04.019\u003c/span\u003e\u003cspan address=\"10.1016/j.ccr.2011.04.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011). doi:S1535-6108(11)00167-X [pii].\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlesken-Nikitin, A., Choi, K. C., Eng, J. P., Shmidt, E. N. \u0026amp; Nikitin, A. Y. Induction of carcinogenesis by concurrent inactivation of p53 and Rb1 in the mouse ovarian surface epithelium. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cb\u003e63\u003c/b\u003e, 3459\u0026ndash;3463 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, E. S., Xiang, D., Xie, Y., Bronson, R. T. \u0026amp; Li, Z. Oncogenic Events Dictate the Types and Locations of Gynecological Malignancies Originating from Krt8(+) Mesothelial and Mullerian-Derived Epithelial Cells. \u003cem\u003eCancers\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 841. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers14030841\u003c/span\u003e\u003cspan address=\"10.3390/cancers14030841\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchakjian, M. R. et al. A Trp53fl/flPtenfl/fl mouse model of undifferentiated pleomorphic sarcoma mediated by adeno-Cre injection and in vivo bioluminescence imaging. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, e0183469. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0183469\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0183469\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKirsch, D. G. et al. A spatially and temporally restricted mouse model of soft tissue sarcoma. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 992\u0026ndash;997. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nm1602\u003c/span\u003e\u003cspan address=\"10.1038/nm1602\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi, T., Owczarek, T. B., McKiernan, J. M. \u0026amp; Abate-Shen, C. Modelling bladder cancer in mice: opportunities and challenges. \u003cem\u003eNat. Rev. Cancer\u003c/em\u003e. \u003cb\u003e15\u003c/b\u003e, 42\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrc3858\u003c/span\u003e\u003cspan address=\"10.1038/nrc3858\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuzio-Kuter, A. M. et al. Inactivation of p53 and Pten promotes invasive bladder cancer. \u003cem\u003eGenes Dev\u003c/em\u003e 23, 675\u0026ndash;680, doi:gad.1772909 [pii] (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gad.1772909\u003c/span\u003e\u003cspan address=\"10.1101/gad.1772909\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adenovirus-Cre, mammary gland immune microenvironment, breast cancer mouse model, p53-loss, CD8+ tissue-resident T cells, premalignancy","lastPublishedDoi":"10.21203/rs.3.rs-8398573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8398573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding 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\u003csup\u003e+\u003c/sup\u003e T-cell infiltration dominated by CD103\u003csup\u003e+\u003c/sup\u003e tissue-resident T cells displaying heightened activation. This sustained antiviral T-cell signature obscures the p53-loss-driven CD8\u003csup\u003e+\u003c/sup\u003e T-cell activation detectable in the CreER/tamoxifen model. Adenoviral infection also transiently skews CD4\u003csup\u003e+\u003c/sup\u003e T cells toward IFN-γ-producing antiviral states and compresses the myeloid compartment, whereas tamoxifen-induced p53-loss increases macrophage abundance and activates CD8\u003csup\u003e+\u003c/sup\u003e 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.\u003c/p\u003e","manuscriptTitle":"Transient adenovirus-Cre infection causes long-lasting remodeling of the mammary gland immune landscape","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-09 10:15:18","doi":"10.21203/rs.3.rs-8398573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-03T22:19:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T23:46:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T17:05:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62861491101207847432451232447508288609","date":"2026-01-12T11:52:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334061212025471362461017027432999139390","date":"2026-01-12T11:18:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T16:35:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T10:25:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-29T16:39:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-26T19:51:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-26T19:43:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"beed2dd4-e10f-4d6f-9977-9fe23f66a4b6","owner":[],"postedDate":"January 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":60762325,"name":"Biological sciences/Cancer"},{"id":60762326,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2026-03-16T16:01:50+00:00","versionOfRecord":{"articleIdentity":"rs-8398573","link":"https://doi.org/10.1038/s41598-026-43069-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-12 15:58:13","publishedOnDateReadable":"March 12th, 2026"},"versionCreatedAt":"2026-01-09 10:15:18","video":"","vorDoi":"10.1038/s41598-026-43069-8","vorDoiUrl":"https://doi.org/10.1038/s41598-026-43069-8","workflowStages":[]},"version":"v1","identity":"rs-8398573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8398573","identity":"rs-8398573","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}