More than half of all brain metastases show infiltrating rather than displacing growth at the macro‐metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer‐binding factor‐1 (LEF1) is an epithelial‐mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain‐colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma.

Brain metastasis in breast cancer remains difficult to treat and its incidence is increasing. Therefore, the development of new therapies is of utmost clinical relevance. Recently, toll-like receptor (TLR) 4 was correlated with IL6 expression and poor prognosis in 1 215 breast cancer primaries. In contrast, we demonstrated that TLR4 stimulation reduces microglia-assisted breast cancer cell invasion. However, the expression, prognostic value, or therapeutic potential of TLR signaling in breast cancer brain metastasis have not been investigated. We thus tested the prognostic value of various TLRs in two brain-metastasis gene sets. Furthermore, we investigated different TLR agonists, as well as MyD88 and TRIF-deficient microenvironments in organotypic brain-slice ex vivo co-cultures and in vivo colonization experiments. These experiments underline the ambiguous roles of TLR4, its adapter MyD88, and the target nitric oxide (NO) during brain colonization. Moreover, analysis of the gene expression datasets of breast cancer brain metastasis patients revealed associations of TLR1 and IL6 with poor overall survival. Finally, our finding that a single LPS application at the onset of colonization shapes the later microglia/macrophage reaction at the macro-metastasis brain-parenchyma interface (MMPI) and reduces metastatic infiltration into the brain parenchyma may prove useful in immunotherapeutic considerations.

Lipids are a ubiquitous class of structurally complex molecules involved in various biological processes. In the fast-growing field of lipidomics, preanalytical issues are frequently neglected. Here, we investigated the stability of lipid profiles of murine liver, brain, lung, heart, and spleen homogenates by quantitative flow injection analysis using tandem mass spectrometry and high-resolution mass spectrometry. Storage of tissue homogenates at room temperature showed substantial alterations of the lipid profiles reflecting lipolytic action. Therefore, ratios of ceramide to sphingomyelin, lysophosphatidylethanolamine to phosphatidylethanolamine, lysophosphatidylcholine to phosphatidylcholine, and diglyceride to triglyceride were applied to monitor sample stability and the effect of sodium dodecyl sulfate (SDS) as a potential stabilizing agent. The addition of SDS led to a concentration-dependent stabilization of lipid profiles in liver, brain, and heart homogenates, while in lung and spleen homogenates, in particular, the lysophosphatidylethanolamine to phosphatidylethanolamine ratio increased upon addition of SDS. In conclusion, we demonstrated that lipid class ratios reflecting lipolytic activity could be applied to evaluate both the stability of samples and the influence of stabilizers.

While the histological growth pattern (HGP) of liver metastases is frequently evaluated, the same attention is often absent for brain metastases despite evidence suggesting its prognostic significance. This oversight may stem from the lack of a standardized method for assessing the HGP at the macro-metastasis / brain parenchyma interface (MMPI_brain). MetInfilt is the first prospective, imaging-guided trial aimed at standardizing the collection and analysis of the HGP at the MMPI_brain. We recruited fifty patients. The MMPI_brain was identified using preoperative contrast-enhanced T1-weighted MRI. Intraoperative confocal microscopy (CONVIVO) visualized the MMPI_brain, while a YELLOW 560 nm filter in the surgical microscope facilitated precise tissue sampling. Samples from the MMPI_brain and the core of the metastasis were collected for postoperative histological confirmation. The protocol achieved successful tissue acquisition from the MMPI_brain in 93.2 % of patients, meeting the study's primary endpoint. Preoperative MRI patterns strongly correlated with infiltrative HGPs, and CONVIVO accurately visualized the MMPI_brain intraoperatively. Exploratory analyses suggest that infiltrative HGPs might negatively impact patient prognosis and represent a potential risk of meningeal metastasis. Our neurosurgical protocol allows the successful and precise acquisition of tissue from the MMPI_brain through presurgical imaging, intraoperative microscopy, and fluorescence-assisted sampling. The evaluation of the HGP in our limited patient cohort highlights its potential clinical significance and supports the urgent necessity to investigate it further for the benefit of patients with brain metastases. Z-2019-1307-9.

Background: Neurological failure contributes to 15–50% of deaths in patients with brain metastases, yet the underlying mechanisms remain poorly understood. Clinical causes range from local compression to meningeal metastasis. In this context, a link between infiltrative histopathological growth patterns (HGPs) and meningeal metastasis was recently described and prompted this reverse translation study. Methods: We conducted a retrospective postmortem histological assessment and a prospective MRI-based proof-of-concept study to explore neurological decline mechanisms in two experimental brain metastasis models with different HGPs: (i) the non-infiltrative TUBO model, characterized by well-defined tumor borders and a multilayered astrocytic capsule; and (ii) the infiltrative E0771-LG model, exhibiting diffuse infiltration and widespread astrogliosis. Results: In the TUBO model, neurological death resulted from local metastatic expansion compressing vital structures, while the E0771-LG model caused mortality mainly through widespread secondary dissemination. We provide the first direct evidence of contralateral recolonization by secondary metastasis-initiating cells (secMICs), and highlight the high efficiency of secondary spread. Additionally, we show that secMICs exploit distinct anatomical structures to reach distant brain regions, bypassing classical vascular dissemination routes. Notably, the HGP and its associated features are intrinsic to tumor cells and are established early during metastatic colonization. Conclusions: This study identifies the HGP as a potential surrogate for predicting the underlying cause of organ failure in brain metastases. Additionally, it highlights the significant role of secondary dissemination and recolonization in brain metastasis, processes that have been largely overlooked in clinical practice. These findings address a critical knowledge gap and may inform future treatment strategies.