Breast cancer brain metastases (BCBM) recur after surgery partly because individual tumor cells undergo epithelial-to-mesenchymal transition (EMT), escape the metastatic mass, and infiltrate the brain parenchyma, where they resist complete resection and adjuvant therapy. Margarido et al. (2022) showed by intravital microscopy in mice and multiplex immunohistochemistry in patients that infiltrating BCBM cells are Zeb1-positive and E-cadherin-negative yet retain the epithelial marker pan-keratin — possibly a hybrid epithelial/mesenchymal (E/M) state — and explicitly left open the role of such states. The objective is to determine which E/M states BCBM tumor cells adopt; what the distinct contributions of specific hybrid states are to parenchymal invasion, relapse following surgery, and therapy resistance in the brain setting; and how EMT or mesenchymal/hybrid cells might be therapeutically blocked or eliminated in BCBM. No study to date has resolved the E/M-state spectrum in BCBM, provided a state-resolved functional test in the brain compartment, or established an EMT-blocking therapeutic strategy for BCBM.

Background

Breast cancer brain metastasis (BCBM) — seeding and growth of breast cancer cells within the brain — is among the most frequent intracranial malignancies and a major cause of death in metastatic breast cancer. Standard local treatment is surgical resection, followed by radiotherapy and/or chemotherapy. A key failure mode of surgery is local recurrence from tumor cells that have infiltrated beyond the resectable mass into the brain parenchyma.

Epithelial-to-mesenchymal transition (EMT) is a reversible cell-state program in which epithelial cancer cells downregulate cell-cell adhesion molecules such as E-cadherin (a component of epithelial adherens junctions) and acquire motility, invasiveness, and resistance to chemotherapy and radiotherapy, under transcription factors including Zeb1. Margarido et al. (2022) combined intravital microscopy (imaging of living tissue through a cranial window in mice) using an E-cadherin fluorescent reporter with patient-sample analysis to show that BCBM cells escaping the metastatic mass undergo EMT, that cells revert toward an epithelial state when re-forming tumor masses in the brain, that these invasive cells impair complete surgical resection, and that the infiltrating cells are likely to survive adjuvant chemo- or radiotherapy because EMT itself confers resistance. In multiplex immunohistochemistry (simultaneous staining of several markers in one tissue section) of human BCBM resections, the infiltrating tumor cells expressed Zeb1 and lost E-cadherin yet still expressed pan-keratin (a broad epithelial cytoskeletal marker) — a profile the authors explicitly flagged as possibly indicating a hybrid state, while stating that their mouse experiments 'have neither investigated the presence nor the role of cells with different E/M states'.

A large body of prior work established that EMT is not a binary epithelial-versus-mesenchymal switch: carcinoma cells can stably occupy hybrid epithelial/mesenchymal (E/M) states, which across cancer types are associated with enhanced metastatic ability and therapy resistance (Jolly et al. 2019) and, in basal-like breast cancer, are required for tumorigenicity (Kröger et al. 2019) and enriched in primary tumors (Sahoo et al. 2024). Conceptual reviews frame EMT as a plasticity spectrum of hybrid states (Bornes et al. 2021; Dong & Blanpain 2026). Quantification frameworks exist for placing cells on this spectrum, from bulk-tumor EMT scoring across cancers (Tan et al. 2014) to single-cell mass-cytometry maps of EMT states and trajectories (Karacosta et al. 2019), and partial-EMT dynamics have been traced during triple-negative breast cancer metastasis (Grasset et al. 2022).

None of this state-resolved knowledge has been established in the brain-metastasis compartment. Single-cell and spatial maps of BCBM published since the source study have focused on the immunosuppressive microenvironment and therapy targets (Zou et al. 2023; Xie et al. 2024; Shi et al. 2026) or on pan-cancer brain-metastasis programs in which EMT appears as one undifferentiated module rather than a resolved state spectrum (Xing et al. 2025). The most direct EMT-state mapping in metastatic breast cancer — per-cell EMT scoring identifying three spatial EMT phenotypes across 67 metastatic breast cancer biopsies (Klughammer et al. 2024) — included only one brain-metastasis biopsy among 67. Margarido et al. (2022) therefore concluded: 'Future studies should shed light on the role of specific hybrid E/M states in BCBM and their distinct contributions to patient relapse following surgery. Additionally, developing therapeutic strategies to block EMT and/or mesenchymal cells is required to target this process and to ultimately improve BCBM patients' outcomes.'

Problem Statement

The problem, as posed by the source study, is to establish the role of epithelial/mesenchymal state diversity in breast cancer brain metastasis. Its components serve one overarching objective — determining whether EMT-state diversity drives BCBM progression and constitutes a usable therapeutic handle: (1) Define the spectrum of E/M states that tumor cells adopt in BCBM, including whether the Zeb1-positive, E-cadherin-negative, pan-keratin-positive cells infiltrating the brain parenchyma constitute a genuine hybrid E/M state, and which further hybrid or endpoint states are present along the invasion-colonization sequence. (2) Determine the distinct functional contributions of specific hybrid E/M states to invasion into the brain parenchyma, to relapse of patients following surgery, and to resistance against chemotherapy and radiotherapy in the brain-metastasis setting. (3) Develop and evaluate therapeutic strategies that block EMT induction or eliminate mesenchymal/hybrid cells in BCBM, so that infiltration-driven incomplete resection and post-surgical relapse can be prevented. This question is specific to E/M states within the BCBM brain compartment, and establishing the role those states play requires resolving the E/M-state spectrum together with the functional contributions of the specific hybrid states. The therapeutic component (3) is an integral part of this objective rather than a separable add-on.

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