Background: n-3 PUFA derived from marine sources, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit potential for breast cancer prevention. In contrast, higher dietary intakes of n-6 PUFA, such as linoleic acid (LA), have been implicated in promoting mammary tumourigenesis. However, there is a need for further exploration into how n-3 PUFA influence breast cancer development in comparison to different amounts and sources of LA. Objective: The purpose of this study was to compare the effects of n-3 PUFA-enriched diets versus n-6 PUFA diets differing in LA content, including corn oil (50% LA) and safflower oil (70% LA), on mammary tumour development in a HER2+ breast cancer model. Methods: Using the HER2+ breast cancer MMTV-neu(ndl)YD5 transgenic mouse model, this study determined the effects of: (1) 10% w/w corn oil (CO, n-6 PUFA, n = 14), (2) 10% w/w safflower oil (SO, n-6 PUFA, n = 14), (3) 3% w/w menhaden oil + 7% w/w CO (3% FO 7% CO, n-3 PUFA, n = 12), and (4) 3% w/w menhaden oil + 7% w/w SO (3% FO 7% SO, n-3 PUFA, n = 14) on puberty onset, tumour incidence, tumour volume, and tumour number in utero until 20 weeks of age. Results: Mice fed the n-3 PUFA-enriched diets showed a lower trajectory of tumour development compared to the n-6 PUFA diets, although the differences for palpated tumour volume and number over time reached significance only between the 10% CO and 3% FO 7% CO groups. This suggests that high LA content in CO may represent a threshold for promoting tumour growth whereby further LA content marginally influences additional tumour development. Exposure to the CO n-6 PUFA diet further resulted in earlier onset of puberty compared to the n-3 PUFA-enriched diet containing CO. To investigate the underlying mechanisms, a qPCR analysis of mammary glands and tumour tissue revealed that the n-3 PUFA diets downregulated the expression of pro-tumourigenic immune markers, including CD206 and F4/80 in the mammary glands and the cannabinoid receptor CB2 in tumours, compared to the n-6 PUFA diets. Conclusions: These findings indicate that the presence of dietary n-3 PUFA plays a key role in modulating mammary tumour development, which may be further influenced by the underlying n-6 PUFA background. The associated changes in immune markers suggest that n-3 PUFA exert anticancer effects in part by shifting the tumour immune microenvironment toward an anti-tumour phenotype and modulating cannabinoid receptor signalling. Collectively, this work informs future human studies investigating the role of dietary fat composition in breast cancer risk.

Breast cancer remains a leading cause of death among women, with the HER2+ subtype being particularly aggressive due to acquired resistance to HER2-targeted therapies. Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2, represses the expression of genetic programs crucial for differentiation, proliferation, and apoptosis. To investigate the role of EZH2 in HER2+ tumor progression, we crossed a genetically engineered mouse model of HER2-driven breast cancer with a conditional Ezh2 knockout strain and showed that Ezh2 is essential for accelerating tumor initiation and metastatic dissemination. Combined bulk and single cell RNA sequencing analyses revealed a significant downregulation of basal cell populations in the absence of Ezh2, and an upregulation of luminal progenitor cell populations, driven by crucial transcription factors such as Esr1. Further, inhibition of EZH2 in vitro resulted in increased expression of ER in HER2+ human breast cancer cell lines and conferred sensitivity to Tamoxifen. These findings demonstrate that EZH2 dictates cancer plasticity and provides rationale for combining EZH2 inhibitors with endocrine therapies to improve HER2+ breast cancer outcomes.

Immunosuppression and metastasis are critical hallmarks of breast cancer, often linked to poor patient outcomes. The secreted cytokine chitinase-3-like 1 (CHI3L1) is frequently overexpressed in breast cancer samples and promotes an immunosuppressed tumor microenvironment. Notably, CHI3L1 expression is elevated in metastatic patient samples when compared with the matched primary breast tumor. To investigate its role in breast cancer metastasis, we generated an inducible genetically engineered mouse model that overexpresses CHI3L1 in the mammary epithelium. Ectopic expression of CHI3L1 in the polyomavirus middle T (PyMT) mouse model of breast cancer suppressed antitumor immune responses, accelerated mammary tumor onset, and enhanced lung metastasis. Mechanistically, elevated CHI3L1 expression in the mammary epithelium enhanced neutrophil recruitment, which subsequently degraded the extracellular matrix and increased the number of circulating tumor cells. These findings reveal a key mechanism driving metastatic dissemination and argue that therapeutically targeting Chi3l1 could enhance antitumor immunity and suppress metastasis.

Patients with estrogen receptor + (ER + , ESR1 + ) breast cancer are most at risk of relapse, where activating mutations in ESR1 promote metastasis and therapeutic resistance. These patients are also disadvantaged in responding to immunotherapies, the mechanisms of which remain to be elucidated. Here, we engineered a transgenic mouse model carrying either Y541S or D542G mutation in ESR1 , mirroring the 2 most common mutations seen in patients. ESR1 mut tumors do not differ in the total number of immune cells yet display downregulation in immune pathways and decreased immune-modulatory cytokines, including IL-17a and IL-1β. T cells and macrophages have lower IFN-γ and antigen presentation, respectively. Mechanistically, ESR1 mut negatively regulates immune modulator expression and upregulates Stat5 to dampen cytokine expression. In concordance, validation on ESR1 mut patient tumors shows decreased IL-17a and IL-1β. Collectively, our findings reveal that ESR1 mutations contribute to an immunosuppressive tumor microenvironment by dampening cytokine secretion and immune cell activity.

Abstract Co-expression of the estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) contributes to breast cancer heterogeneity and therapeutic resistance. However, the molecular mechanisms promoting ER positivity within HER2+ cancers remains largely unknown. Here we show, across HER2+ transgenic mouse models the oncogenic HER2 splice variant lacking exon 16 (HER2∆16) promotes the development of aggressive luminal tumors by facilitating an ER-mediated transcriptional program which is sensitive to endocrine therapies. HER2∆16 is detected across human HER2+ breast tumors and cell lines with higher levels correlating with increased expression of ER and downstream transcriptional targets. Notably, in human cell lines HER2∆16 expression is elevated upon acquired resistance to HER2-targeted therapy and can sensitize cells to the ER-antagonist tamoxifen. Overall, these findings offer valuable insights into the role of HER2∆16 in promoting luminal cell identity and estrogen receptor positivity in breast cancer, providing a useful platform to model HER2+/ER+ disease.

Pulmonary nodules on radiography in setting of recent neutropenia or immune suppression for graft versus host disease (GVHD) are concerning for bacterial or invasive fungal disease (IFD). Determining etiology often requires invasive tests that may be inconclusive and pose risk. Non-invasive assays that detect pathogens may improve time to optimal therapy and outcomes. This study sought to evaluate the utility of a commercial metagenomic microbial cell-free DNA (mcfDNA) sequencing assay (Karius SpectrumTM) to identify causative pathogens in this setting. Subjects aged 120 days to 22 years with recent neutropenia or GVHD and with newly detected chest imaging findings meeting EORTC/MSGERC guidelines for possible IFD were enrolled at 27 North American hospitals from 2019 to 2024. Plasma was collected ≤144 hours after imaging and shipped frozen for mcfDNA testing by Karius Laboratories. Microbiology, radiology, pathology, and procedure reports were collected for 49 days post imaging. A three-physician Central Review Board (CRB) reviewed collected data to identify pathogen(s) explaining imaging findings; this served as the reference standard (RS). The CRB separately labeled mcfDNA resulted pathogens as explanatory or not of imaging findings. Operating characteristics (OC) of mcfDNA were relative to the RS; true positives concurred with at least one RS organism. Analyses include 146 subjects enrolled through May 2023 with a valid mcfDNA result and complete CRB review. Clinical data review identified ≥1 pathogen that explained qualifying imaging for 67 (46%) subjects (33 fungus, 38 bacteria). OC of mcfDNA were: sensitivity, 44.8% (95% confidence interval, 32.9–56.7%); specificity, 77.2% (68.0–86.5%); positive predictive value, 62.5% (48.8–76.2%); negative predictive value (NPV), 62.2% (52.7–71.8%). 18 (12%) mcfDNA tests yielded an explanatory pathogen missed by RS. 7 (5%) subjects had discordant explanatory pathogens identified by the RS and mcfDNA. OC of mcfDNA assay limit it as a stand-alone test for detecting causative pathogens of pulmonary nodules in children with neutropenia or GVHD. NPV is too low to support using this assay to “rule out” infection. This assay offers adjunctive diagnostic utility alongside traditional testing. Brian T. Fisher, DO, MPH/MSCE, Merck: Grant/Research Support|Pfizer: Grant/Research Support Gabriela Maron, MD, MS, SymBio Pharamaceuticals: Advisor/Consultant|SymBio Pharamaceuticals: Grant/Research Support Lara A. Danziger-Isakov, MD, MPH, Aicuris: Grant/Research Support|Ansun BioPharma: Grant/Research Support|Astellas: Advisor/Consultant|Astellas: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer (Any division): Grant/Research Support|Takeda: Grant/Research Support Surabhi Vora, MD, MPH, Astellas: Advisor/Consultant James B. Wood, MD, MSCI, Karius: Grant/Research Support|MeMed: Grant/Research Support Monica I. Ardura, DO, MSCS, Miravista: Grant/Research Support J. Christopher Day, MD, Pfizer: Grant/Research Support William J. Muller, MD, PhD, Ansun Biopharma: Grant/Research Support|Astellas Pharma: Advisor/Consultant|Astellas Pharma: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|Clarivate Analytics (US) LLC: Grant/Research Support|Eli Lilly and Company: Grant/Research Support|Enanta Pharmaceuticals: Advisor/Consultant|Enanta Pharmaceuticals: Grant/Research Support|F. Hoffmann-La Roche: Grant/Research Support|Finley Law Firm, P.C.: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Melinta Therapeutics, Inc.: Grant/Research Support|Merck: Grant/Research Support|Moderna: Grant/Research Support|Nabriva Therapeutics, plc: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|Pfizer: Grant/Research Support|Sanofi Pasteur LLC: Honoraria|Schueler, Dallavo, & Casieri: Advisor/Consultant|Tetraphase Pharmaceuticals, Inc.: Grant/Research Support|Vindico CME: Honoraria Daniel Dulek, MD, Astellas Pharma: Advisor/Consultant|Eurofins-Viracor: Grant/Research Support Kiran K. Belani, MD ,Ctrop med hyg ABP and FIDSA,, AMGEN: Advisor/Consultant Antonio C. Arrieta, MD, FIDSA, FPIDS, Astellas Inc: Grant/Research Support|Astellas Inc: Honoraria|Pfizer: Grant/Research Support

AXL is activated by its ligand GAS6 and is expressed in triple-negative breast cancer cells. In the current study, we report AXL expression in HER2-positive (HER2+) breast cancers where it correlates with poor patient survival. Using murine models of HER2+ breast cancer, Axl, but not its ligand Gas6, was found to be essential for metastasis. We determined that AXL is required for intravasation, extravasation, and growth at the metastatic site. We found that AXL is expressed in HER2+ cancers displaying epithelial-to-mesenchymal transition (EMT) signatures where it contributes to sustain EMT. Interfering with AXL in a patient-derived xenograft (PDX) impaired transforming growth factor β (TGF-β)-induced cell invasion. Last, pharmacological inhibition of AXL specifically decreased the metastatic burden of mice developing HER2+ breast cancer. Our data identify AXL as a potential anti-metastatic co-therapeutic target for the treatment of HER2+ breast cancers.

It is now recognized that mitochondria play a crucial role in tumorigenesis; however, it has become clear that tumor metabolism varies significantly between cancer types. The failure of recent clinical trials aimed at directly targeting tumor respiration through oxidative phosphorylation inhibitors underscores the critical need for further studies providing an in-depth evaluation of mitochondrial bioenergetics. Accordingly, we comprehensively assessed the bulk tumor and mitochondrial metabolic phenotype in murine HER2-driven mammary cancer tumors and benign mammary tissue. Transcriptomic and proteomic profiling revealed a broad downregulation of mitochondrial genes/proteins in tumors, including OXPHOS subunits comprising Complexes I–IV. Despite reductions in tumor mitochondrial proteins, mitochondrial respiration was several-fold higher compared to benign mammary tissue, which persisted regardless of normalization method (wet weight, total protein content, and when corrected for mitochondrial content). This upregulated respiratory capacity could not be explained by OXPHOS uncoupling, suggesting HER2 signaling regulates intrinsic mitochondrial bioenergetics. In further support, lapatinib, an EGFR/HER2 tyrosine kinase inhibitor, attenuated mitochondrial respiration in NF639 murine mammary tumor epithelial cells. Together, this data highlights that the typical correlation between mitochondrial content and respiratory capacity may not apply to all tumor types and implicates HER2-linked activation of mitochondrial respiration supporting tumorigenesis in this model.

Background/Objectives: Dietary changes or increased physical activity to promote health accompanies breast cancer treatment to improve outcomes and prevent cancer recurrence. However, primary breast cancer prevention studies are limited and while there is a growing benefit to diet and exercise, the combinatory benefits of diet and exercise are limited. Therefore, the objective of this exploratory study was to determine the effects of exposure to diets high in n-3 or n-6 polyunsaturated fatty acids (PUFA) combined with exercise on mammary tumour development in female MMTV-neu(ndl)YD5 mice. Methods: Animals were fed maternal diets containing 10% safflower oil (n-6 PUFA-enriched) or 3% menhaden oil + 7% safflower oil (n-3 PUFA enriched) for 20 weeks (wks). Within diets, mice were further divided into sedentary control or exercise groups (n = 8-12). Exercised mice were run on a treadmill from 8 to 12 wks of age before tumours developed. Once tumours were detected, volume and multiplicity were measured throughout the study. Results: Mice consuming n-3 PUFA had reduced tumour volume by 55% (p < 0.001) and weight by 42% (p = 0.02) compared to mice fed n-6 PUFA. Exercised n-3 PUFA-fed mice experienced 10% longer tumour-free status (TD50) than their sedentary counterparts and 24% longer TD50 than sedentary mice fed the n-6 PUFA enriched diet (p = 0.001). Exercised n-6 PUFA-fed mice established tumours 11% sooner than their sedentary counterparts and developed the largest and greatest number of tumours throughout the study (p < 0.0001). Conclusions: Overall, diet quality plays a significant role in mammary tumour outcomes. The effect of exercise on tumour outcomes was not as clear, but this exploratory study highlights the need to consider the temporal trajectory of tumour development that impacts not only lifespan but healthspan.

Supplementary Excel S6 shows SciScore-STAR-Table for the information of antibodies and reagents used in this manuscript

Supplementary Excel S5 shows sialylopeptides of glycoproteins.

Supplementary Table S1 shows clinical characteristics and multivariable Cox regression model analysis of patients. Supplementary Table S2 shows the clinical characteristics of patients with breast cancer.

High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics, yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput, low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation, allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities, demonstrating efficient delivery of various cargo, with high rates of transfection, gene knockout via non-homologous end joining, and precise knock-in through homology-directed repair. To showcase its utility in functional genomics, we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4⁺ T cells, identifying novel regulators of exhaustion, including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.

Supplementary Figure S1-S17 show the methods of CTC analysis and CTC-associated survival (S1); glycosylation profiles of patient CTCs (S2); phenotypic effects of neuraminidase treatment and CD44 depletion on tumor clustering and sialylation (S3); N-glycomic analysis of α2,6-SA peaks in WT and ST6KO cells (S4); Depletion of ST6GAL1 or ST3GAL1 promotes clustering and chemo-evasion (S5); ST6GAL1 expression and cell sensitivity to therapeutics (paclitaxel, doxorubicine and palbociclib)(S6); he altered gene pathways in ST6GAL1-KO cells (S7); ST6GAL1 is associated with cell growth and patient survival (S8); dynamic glycosylation profile in PDXs and breast tumors (S9); ST6GAL1 suppresses metastatic seeding (S10); Counts and Ki67 expression of CTCs and DTCs in situ (S11); depletion of ST6GAL1 promotes transendothelial migration and seeding (S12);α2,6-sialylation of CD44 by ST6GAL1(S13); ST6GAL1 substrates regulate cluster formation via altered binding affinity (S14); the down-regulation of ST6GAL1 substrates inhibits metastatic seeding (S15); anti-PODXL inhibits tumor cell cluster formation and metastatic seeding(S16); and anti-PODXL effects on CTCs and seeding of PDX-M1 (S17).

Breast cancer is the second most common cancer and a major cause of mortality affecting women worldwide. Immunosuppression is a key hallmark of cancer progression, whereby cancer cells suppress the immune system that is programmed to fight the disease. This suppressed tumor immune microenvironment (TIME) facilitates cancer metastatic spread to distant organs, which is associated with treatment resistance, recurrence, and poor prognosis. The Signal transducer and activator of transcription 3 (Stat3) suppresses the immune system, drives cancer progression and metastasis. Genetic ablation of Stat3 in the murine inducible breast tumor model expressing polyoma virus middle T (PyMT), results in delayed tumor onset and reduced tumor growth and metastasis. We have also previously demonstrated that the secreted protein Chitinase 3-like-1 (Chi3l1) is a direct target of Stat3 which promotes immunosuppression through its actions on neutrophils. Chi3l1 recruits neutrophils and induces the formation of Neutrophil Extracellular Traps (NETs) that block T cells from infiltrating the tumor. Targeting both Chi3l1 or neutrophils resulted in increased T cell infiltration and delayed tumor onset. However, the roles of Chi3l1 during the metastatic cascade remained unclear. To further investigate the role of Chi3l1 in metastasis, we generated a novel inducible mouse model that overexpresses Chi3l1 specifically in the mammary epithelial cells. Overexpression of the Chi3l1 protein in the Stat3 knockout PyMT model showed a rescue of the delay in tumor onset. Consistent with our previous studies, Chi3l1 overexpression caused a dramatic influx of neutrophils and the generation of an immune suppressed TIME. In addition, we observe an increase in circulating tumor cells which correlated with a quicker onset and increased incidence of pulmonary metastasis. These results hint that in breast cancer, Chi3l1 overexpression creates a pro-tumorigenic TIME which favorizes tumor initiation and progression as well as induces intravasation of tumor cells into the blood stream, which leads to metastasis to distant sites. Our future studies will investigate whether this phenotype is driven through neutrophils and whether targeting neutrophils would inhibit Chi3l1-mediated metastasis. Adeline Masse, Tarek Taifour, Yu Gu, Virginie Sanguin-Gendreau, Dongmei Zuo, Bin Xiao, Hailey Proud, Nancy U. Lin, Melissa E. Hughes, Kalie Smith, Rinath Jeselsohn, William J. Muller. The secreted cytokine Chitinase 3-like-1 promotes breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7225.

Breast cancer is one of the most common malignancies in the world, with 2.3 million women diagnosed in 2022. It is largely driven by dysregulation of cellular signaling pathways such as mTOR, which, as a key regulator of cellular growth, is frequently hyperactivated in many cancers. Despite evidence implicating mTOR as a promising target for anti-cancer therapies, many inhibitors in clinical trials yield unfavorable outputs, largely due to its complex regulatory mechanisms that are yet fully understood. DEPTOR, a recently identified direct inhibitor of mTOR, has thus become an intriguing focus of study. Interestingly, while many tumors exhibit high mTOR activity and, as expected, also show low DEPTOR expression, paradoxically, high DEPTOR levels in several cancers corresponds with the worst prognoses for patients. These observations point to dual roles for this protein, and unknown pro-tumor functions that are potentially independent of mTOR. Our work aims to expand our understanding of the functions and interacting partners of DEPTOR, and characterize its role in breast cancer, about which knowledge is currently lacking. In a mouse model of luminal B breast cancer, we observe a severe defect in early mammary cell expansion, delay in tumor onset, and reduction in tumor penetrance upon genetic ablation of DEPTOR, indicating DEPTOR acts in an oncogenic capacity in this system. However, these results are not accompanied by a significant change in activity of major mTOR effectors, and gene expression profiles indicate changes in factors related to transcription and chromatin accessibility but not translation, despite the latter being largely modulated by mTOR to drive cell growth. Additionally, while the innate proliferative ability of early DEPTOR-deficient mammary cancer cells appears unchanged, their immune microenvironment (TIME) is altered to favour an anti-tumor immune response. Thus, we will investigate how DEPTOR-dependent transcriptional regulation may reprogram the TIME to influence tumor initiation and progression. Considering mTOR’s ubiquity in cancer, elucidating the activities of a core regulatory component such as DEPTOR will provide important insights for its therapeutic targeting. Further, by investigating DEPTOR’s mTOR-independent roles, this work will highlight the scope of a poorly understood but widely acting player in cancer and its potential as a novel therapeutic target. Alice Jisoo Nam, Bin Xiao, Virginie Sanguin-Gendreau, Dongmei Zuo, William J. Muller. The role of DEPTOR in promoting breast cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 189.

Human breast cancers that have HER2 amplification/overexpression frequently carry PIK3CA mutations, and are often associated with a worse prognosis. However, the role of PIK3CA mutations in the initiation and maintenance of these breast cancers remains elusive. In the present study, we generated a compound mouse model that genetically mimics HER2-positive breast cancer with coexisting PIK3CA(H1047R). Induction of PIK3CA(H1047R) expression in mouse mammary glands with constitutive expression of activated Her2/Neu resulted in accelerated mammary tumorigenesis with enhanced metastatic potential. Interestingly, inducible expression of mutant PIK3CA resulted in a robust activation of phosphatidylinositol-3-kinase (PI3K)/AKT signaling but attenuation of Her2/Her3 signaling, and this can be reversed by deinduction of PIK3CA(H1047R) expression. Strikingly, although these Her2(+) PIK3CA(H1047R) initiated primary mammary tumors are refractory to HER2-targeted therapy, all tumors responded to inactivation of the oncogenic PIK3CA(H1047R), a situation closely mimicking the use of a highly effective inhibitor specifically targeting the mutant PIK3CA/p110a. Notably, these tumors eventually resumed growth, and a fraction of them escaped PI3K dependence by compensatory ERK activation, which can be blocked by combined inhibition of Her2 and MEK. Together, these results suggest that PIK3CA-specific inhibition as a monotherapy followed by combination therapy targeting MAPK and HER2 in a timely manner may be an effective treatment approach against HER2-positive cancers with coexisting PIK3CA-activating mutations.

Supplementary Video S5 shows the clustering video of ST6WT cells presented in Fig.2B.