Elucidating the role of inflammation in breast cancer metastasis

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Project 1: To investigate the hypothesis that DVT promotes TNBC brain metastasis by shaping a tumor-permissive brain microenvironment, I am leveraging a collaboration with Dr. Yogen Kanthi at the National Heart, Lung, and Blood Institute (NHLBI). Dr. Kanthi has been using a well-established DVT model known as the Electrolytic Inferior Vena Cava Model (EIM), which reliably induces DVT in mice by applying a localized electrolytic injury to the inferior vena cava. Building on this model, our team has developed a novel and clinically relevant animal model termed the "DVT-TNBC Brain Metastasis Mouse Model." In this model, DVT is first induced using the EIM, followed by intracardiac injection of syngeneic, brain-tropic TNBC cells to mimic hematogenous dissemination and colonization in the brain. We utilized two well-characterized brain-tropic TNBC cell lines: 4T1-BR5 and E0771-BR5. Our preliminary data revealed that mice with DVT developed significantly larger brain metastatic lesions compared to control mice without DVT. This effect was consistent across both TNBC models, suggesting that DVT fosters a microenvironment conducive to brain colonization by TNBC cells. To dissect the cellular landscape within brain metastases, we performed immunofluorescence staining and found no significant differences between DVT and non-DVT groups in the activation status of microglia and astrocytes, infiltration of CD3+ T cells, or angiogenesis. However, we observed a significant increase in neutrophil infiltration within brain metastases in the DVT group. This finding suggests that DVT may drive brain metastasis at least in part through neutrophil-mediated mechanisms. To directly test the functional role of neutrophils, we are currently conducting neutrophil depletion experiments using anti-Ly6G antibodies in the DVT-TNBC brain metastasis model. These experiments aim to determine whether depletion of neutrophils reduces brain metastatic burden in the context of DVT, thereby providing mechanistic insight into the pro-metastatic role of neutrophils. In parallel, to explore the impact of DVT on the functional states of glial cells in the brain metastatic niche, we are preparing to conduct single-nucleus RNA sequencing of brain tissue harvested from DVT and non-DVT groups. This transcriptomic profiling will allow us to define cell-type-specific responses and uncover novel pathways activated by DVT-induced neuroinflammation. Ultimately, this project aims to uncover how DVT alters the brain microenvironment to support metastatic seeding and outgrowth. By identifying the molecular and cellular interactions between neutrophils, glial cells, and TNBC cells, this work will provide a foundation for the development of novel therapeutic strategies targeting the metastatic cascade in patients with TNBC and comorbid thrombotic disease. Project 2: Tor recapitulates the patients with circulating TNBC cells complicated with asymptomatic small ischemic strokes, we have developed a novel and clinically relevant experimental system using microsphere-induced microinfarctions. In this model, Fluoresbrite® YG Microspheres (20 µm diameter) are injected into the right carotid artery of immunocompetent female mice. This procedure induces the occlusion of small cerebral vessels, mimicking the silent small ischemic strokes that are frequently observed in aging populations. To investigate the temporal relationship between stroke and metastatic seeding, microspheres are injected at two different timepoints relative to cancer cell dissemination: -Post-stroke group: microspheres are injected 2 days prior to intracardiac administration of brain-tropic syngeneic TNBC cells (either 4T1-BR5 or E0771-BR5). -Pre-stroke group: microspheres are injected 7 days after cancer cell injection. Mice injected with PBS serve as controls for both groups. Ischemia induced by microspheres is confirmed using Hypoxyprobe, a marker of tissue hypoxia. Immunofluorescent analysis of brain sections reveals robust activation of glial cells, including astrocytes (GFAP+) and microglia (Iba1+), surrounding the microsphere occlusion sites as early as 2 days post-injection, indicating a localized neuroinflammatory response. Additionally, there is a marked reduction in tight junction proteins (claudin-5) in the vasculature around microspheres, suggesting compromised BBB integrity in ischemic regions. Most critically, the evaluation of brain metastases 12 days following cancer cell injection demonstrates that the post-stroke group with DVT, where ischemia precedes metastatic seeding, develops significantly larger brain metastatic lesions compared to controls. In contrast, the pre-stroke group, in which ischemia is introduced after metastatic colonization, does not show a significant difference in tumor burden. These findings underscore the importance of the premetastatic and early metastatic niche in facilitating brain metastasis and suggest that stroke-induced neuroinflammation and BBB disruption could be key contributors to this enhanced metastatic potential. Through this approach, we have successfully established a novel small ischemic stroke-TNBC brain metastasis mouse model that recapitulates clinically relevant interactions between cerebrovascular injury and cancer metastasis. This model provides a powerful platform for dissecting the molecular and cellular mechanisms through which asymptomatic small ischemic events reshape the brain microenvironment to promote metastatic colonization. The ultimate goal of this work is to identify therapeutic targets that could be leveraged to prevent or mitigate breast cancer brain metastases in patients at risk due to subclinical ischemic strokes. Project 3: To address the urgent need for predictive biomarkers that can identify breast cancer patients at high risk for developing brain metastases, I am currently developing a clinical study focused on analyzing CSF in patients with advanced breast cancer. The study targets patients with metastatic TNBC or HER2-positive breast cancer who do not yet have radiographically detectable brain metastases at the time of enrollment. cfDNA isolated from CSF will be subjected to whole-exome sequencing (WES) to detect the presence of cancer-related gene mutations. The study will determine the proportion of patients in whom at least one cancer-related gene mutation is detected in cfDNA from CSF and evaluate the association between these genomic findings and subsequent development of brain metastases, as monitored through clinical follow-up. These analyses aim to uncover whether molecular alterations in the CSF precede radiographic detection and may therefore serve as early indicators of occult CNS dissemination. In addition to cfDNA analysis, the study includes the characterization of immune cell populations and circulating tumor cells (CTCs). These exploratory endpoints will provide further insight into the immune landscape in patients at high risk for brain metastasis and may uncover novel cellular or immunogenomic signatures associated with metastatic progression. The clinical study protocol is currently under review by the Scientific Research Committee, and I anticipate initiating patient enrollment in Fiscal Year 2026. This research is expected to provide critical insights into the utility of CSF liquid biopsy for early detection and risk stratification of brain metastases in patients with metastatic TNBC and HER2-positive breast cancer, potentially guiding future preventive interventions.