Abstract Introduction: Younger women with breast cancer (BC) have increased risk to develop brain metastases (BM) irrespective of the tumor subtype. We have shown that pre-menopausal levels of 17-β-Estradiol (E2) promotes BM of estrogen-unresponsive BC cells by modulating astrocyte function. Yet, microglia (the brain resident-macrophages) express estrogen-receptors (ERs), mediate the neuroprotective and homeostatic effects of E2 in the brain, and play pro and anti-tumorigenic roles through brain metastases progression. Thus, we hypothesized that E2 acts on ER+ microglia to regulate its neuroinflammatory properties and suppress anti-tumoral responses in the brain. Results: To assess how E2 modulates brain immune cells, we performed multiparametric flow cytometry across different stages of BM progression in spontaneous and experimental models of BMs. From early to late stages of BM colonization, brains of E2-treated mice showed reduced neutrophils, lymphocyte infiltration, B and CD8+ T cells compared to E2-suppressed mice. scRNAseq of immune cells from BM-bearing mice identified 6 functional microglia subclusters. The migration Mg cluster, marked by microglial homeostatic genes known to promote activation and surveillance movements, the TNF-α cluster, marked by genes associated with promotion of microglial activation, and the Interferon Mg cluster, marked by genes involved in the regulation of type I Interferons were less abundant in E2-treated mice than E2-suppressed mice. Overrepresentation analysis showed microglia from E2-treated mice enriched in translation programs, while microglia from E2-suppresed mice had a significant enrichment of immune leukocyte activation and antigen presentation processes, suggesting E2 suppresses key microglia functions as antigen-presenters and activators of T cells. To determine how microglia from E2-treated or E2-depleted mice differentially influence T cell function, we performed in vitro coculture assays to assess T cell activation, effector-associated markers, and expansion. Microglia from E2-stimulated BM bearing mice showed decreased ability to induce interferon cytotoxic function and expansion of activated T cells, supporting a pro-tumorigenic T cell immune response. Since BMs are usually treated with brain radiotherapy (RT), either as whole brain radiotherapy (WBRT) or stereotactic radiosurgery (SRS), we tested whether E2-suppression impacted radiotherapy response in experimental metastases models. E2-suppression (ovarian suppression in combination with an aromatase inhibitor) significantly increased the effectiveness of radiation in decreasing BM progression. These studies provide a novel mechanism whereby E2 promotes rapid progression of BMs and provides a rationale for the clinical -testing of endocrine therapies in the management of BMs of E2-unresponsive tumors. Citation Format: Karen L.F. Alvarez-Eraso, Maria J. Contreras-Zárate, Andrew Goodspeed, James Costello, Jenny A. Jaramillo-Gómez, Stella Koliavas, R. Alejandro Marquez-Ortiz, Morgan S. Fox, D. Ryan Ormond, Peter Kabos, Mercedes Rincon, Diana M. Cittelly. Estrogen induces a pro-tumoral phenotype shift in microglia that contributes to E2-unresponsive breast cancer brain metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr PR007.

The central nervous system (CNS) is a common site of metastatic spread for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases remain limited. Tyrosine kinase inhibitors have shown promising results in increasing the overall response in brain metastases, owing to their brain penetrance and increased effectiveness; however, their use is limited to the small group of tumors carrying specific oncogenic drivers. Among these, inhibitors with activity against neurotrophic tyrosine receptor kinases (NTRKs) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs' targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the brain metastatic niche and can, in a paracrine manner, activate NTRK function in cancer cells. Here we show that NTRKs are expressed in primary tumors, brain metastases, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous BDNF and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro , and effectively prevented brain metastatic colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for brain metastases of WT-NTRK+ lung cancers, and therefore, NTRK inhibitors can be used to target non-fusion NTRK function to prevent or decrease brain metastases. SIGNIFICANCE:These studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

BACKGROUND:Young age is an independent risk factor for the development of breast cancer brain metastases (BM). Prior work showed that 17β-estradiol (E2), the predominant premenopausal hormone, promotes BM of tumors intrinsically unresponsive to E2, in part through modulating estrogen receptor-alpha expressing (ERα⁺) glial cells. However, how E2 reshapes the brain tumor microenvironment (TME), particularly microglia‑mediated immunity, and its impact to BM progression remains unclear. METHODS:scRNA sequencing and multiparametric flow cytometry were used to define the impact of E2 and E2-suppression on brain immune-cell populations across different stages of BM progression using spontaneous and experimental models of BM. Depletion of microglia and T-cell co-cultures were used to study microglia's role in E2-induced BM. The effects of E2-suppression alone or in combination with whole brain radiotherapy were tested in preclinical models mimicking late-stage BM. RESULTS:E2 repressed immune surveillance and immune activation programs in microglia from early to late stages of brain metastatic progression, suppressing recruitment of effector immune cells to BM. Estrogen suppression, in turn reactivated anti-tumoral signaling in microglia and increased recruitment of effector immune cells to the brain. Microglia from E2-treated BM-bearing mice showed a reduced capacity to promote T-cell expansion, effector potential, and CD8⁺T cell-mediated tumor cell killing. Conversely, E2-suppression reactivated an effective anti-tumoral response and synergized with RT to significantly decrease BM progression. CONCLUSION:These findings reveal a previously unrecognized mechanism by which E2 accelerates BC‑BM progression through microglial immunosuppression and support evaluation of endocrine therapies as adjunct treatments for ER⁻ breast cancer brain metastases.

Abstract The purpose of these studies is to define mechanisms that drive estrogen receptor positive (ER+) breast cancer brain metastasis (BCBM) colonization and outgrowth. Most ER+ BCBMs arise in postmenopausal women due to prior endocrine therapies or age, yet most in vivo ER+ breast cancer (BC) models require estrogen (E2) supplementation and use young hosts. Thus, the mechanisms underlying metastatic progression in the aged/E2-depleted brain tumor microenvironment (TME) remain unknown. FGFR1-amplification (amp), an established driver of ER+ BC endocrine therapy resistance, was the only genomic alteration associated with increased late recurrence in post-menopausal women with ER+ BC on aromatase inhibitors, and FGFR aberrations are enriched in BCBM patients compared to non-BM metastatic BC. While FGFR1amp often causes auto-activation, TME-dependent FGFR1 activation has emerged as an important mechanism modulating its activity. Here, we tested the hypothesis that canonical and non-canonical activation of FGFR1 by astrocytes and neurons promotes FGFR1-dependent ER+ BCBM in E2-high young and E2-low aged hosts. Intracardiac injection of ER+ cells showed that FGFR1amp lines had higher BM incidence, and were E2-dependent in young but not aged hosts. FGFR1 knockdown (KD) did not alter proliferation but decreased BCBM in both E2-high/young and E2-low/aged mice. Spatial transcriptomics of the ER+ BCBMs TME showed that aging and E2-depletion reduced FGF2 expression and FGF/FGFR signaling in surrounding glial cells, suggesting that TME-driven FGFR1 activation may be distinct in young and aged hosts. In the normal brain, NCAM1, an adhesion molecule expressed on neurons and astrocytes, can activate FGFR1. We found that NCAM1 activated FGFR1 kinase activity and downstream signaling in ER+ BC cells, and induced differential expression of neuronal gene signatures including neural cell adhesion. FGFR1 KD reduced ER+ BC cell migration along neurites in co-culture with primary neurons and decreased synaptic puncta. NCAM1 KD in neuron-like SH-SY5Y cells also decreased ER+ cell migration, suggesting that FGFR1/NCAM1 facilitates ER+ BC interactions with neurons, contributing to early brain colonization. Activated FGFR1 was higher in young versus aged mice at early stages of colonization but absent in late-stage ER+ BCBMs, suggesting that paracrine FGFR1 activation and interactions with neurons and astrocytes are critical for early colonization. Consistently, an FDA-approved FGFR inhibitor blocked ER+ BCBM when administered at early stages of colonization in young mice but had no effect at late stages or in aged mice. Together, these studies suggest a novel mechanism whereby canonical and non-canonical activation of FGFR1 promote ER+ BCBM colonization in young and aged/E2-depleted hosts, and caution that FGFR1 inhibition may only be effective to prevent but not to treat BMs. Citation Format: Morgan S. Fox, Jenny A. Jaramillo-Gómez, R. Alejandro Marquez-Ortiz, Karen L. Alvarez-Eraso, Maria J. Contreras-Zárate, Trinh C. Pham, Elaina N. Barela, Stella Koliavas, Peter Kabos, Carol A. Sartorius, Elizabeth A. Wellberg, Diana M. Cittelly. FGFR1 drives progression of ER+ breast cancer brain metastases in young and aged hosts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2076.

Abstract Introduction: Younger women with breast cancer (BC) have increased risk to develop brain metastases (BM) irrespective of the tumor subtype. We have shown that pre-menopausal levels of 17-β-Estradiol (E2) promotes BM of estrogen-unresponsive BC cells by modulating astrocyte function. Yet, microglia (the brain resident-macrophages) express estrogen-receptors (ERs), mediate the neuroprotective and homeostatic effects of E2 in the brain, and play pro and anti-tumorigenic roles through brain metastases progression. Thus, we hypothesized that E2 acts on ER+ microglia to regulate its neuroinflammatory properties and suppress anti-tumoral responses in the brain. Results: To assess how E2 modulates brain immune cells, we performed multiparametric flow cytometry across different stages of BM progression in spontaneous and experimental models of BMs. From early to late stages of BM colonization, brains of E2-treated mice showed reduced neutrophils, lymphocyte infiltration, B and CD8+ T cells compared to E2-suppressed mice. scRNAseq of immune cells from BM-bearing mice identified 6 functional microglia subclusters. The migration Mg cluster, marked by microglial homeostatic genes known to promote activation and surveillance movements, the TNF-α cluster, marked by genes associated with promotion of microglial activation, and the Interferon Mg cluster, marked by genes involved in the regulation of type I Interferons were less abundant in E2-treated mice than E2-suppressed mice. Overrepresentation analysis showed microglia from E2-treated mice enriched in translation programs, while microglia from E2-suppresed mice had a significant enrichment of immune leukocyte activation and antigen presentation processes, suggesting E2 suppresses key microglia functions as antigen-presenters and activators of T cells. To determine how microglia from E2-treated or E2-depleted mice differentially influence T cell function, we performed in vitro coculture assays to assess T cell activation, effector-associated markers, and expansion. Microglia from E2-stimulated BM bearing mice showed decreased ability to induce interferon cytotoxic function and expansion of activated T cells, supporting a pro-tumorigenic T cell immune response. Since BMs are usually treated with brain radiotherapy (RT), either as whole brain radiotherapy (WBRT) or stereotactic radiosurgery (SRS), we tested whether E2-suppression impacted radiotherapy response in experimental metastases models. E2-suppression (ovarian suppression in combination with an aromatase inhibitor) significantly increased the effectiveness of radiation in decreasing BM progression. These studies provide a novel mechanism whereby E2 promotes rapid progression of BMs and provides a rationale for the clinical -testing of endocrine therapies in the management of BMs of E2-unresponsive tumors. Citation Format: Karen L.F. Alvarez-Eraso, Maria J. Contreras-Zárate, Andrew Goodspeed, James Costello, Jenny A. Jaramillo-Gómez, Stella Koliavas, R. Alejandro Marquez-Ortiz, Morgan S. Fox, D. Ryan Ormond, Peter Kabos, Mercedes Rincon, Diana M. Cittelly. Estrogen induces a pro-tumoral phenotype shift in microglia that contributes to E2-unresponsive breast cancer brain metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B063.

The central nervous system (CNS) is a common site of metastasis for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases (BM) remain limited. Tyrosine kinase inhibitors have shown promising results in increasing overall response in BM, owing to their brain penetrance and increased effectiveness; however, their use is limited to a small group of tumors carrying specific oncogenic drivers. Among these, inhibitors targeting neurotrophic tyrosine receptor kinases (NTRK) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs' targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the BM niche and can, in a paracrine manner, activate NTRK function in cancer cells. In this study, we show that NTRKs are expressed in primary tumors, BM, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous brain-derived neurotrophic factor (BDNF) and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented BM colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for BM of WT-NTRK+ lung cancers; therefore, NTRK inhibitors can be used to target nonfusion NTRK function to prevent or decrease BM. SIGNIFICANCE:These studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide a rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

Patients with invasive lobular carcinoma of the breast (ILC) are at high risk of long-term recurrence and metastatic progression with poor prognoses due to delayed detection and treatment-refractory disease. Unfortunately, few models are available to investigate metastatic ILC (mILC) and understand the unique metastatic patterns and phenotypes, including abdominal metastases, leptomeningeal disease, and mixed osteosclerotic/lytic bone metastases. Therefore, we expanded upon the previously established mammary intraductal (MIND) cell line xenograft model by supplementing mice with low-dose estradiol to promote disease progression. We observed spontaneous multi-organ spread from the mammary gland to common and mILC-specific tissues, with micro-metastatic disease as early as 12 weeks post-engraftment and macro-metastatic disease in 24-30 weeks, without the need for primary tumor resection. Primary and metastatic tumors remain highly endocrine responsive, allowing for the evaluation of novel therapeutics in the setting of disseminated metastasis. Derivative cell lines were isolated from various metastatic lesions, a total of 13 derivates from 7 sites across three hosts, and were found to have shared gene expression changes related to metabolism and intercellular signaling. Focusing on bone-derived variant cells as bone is the most common site for mILC to present, we found that bone-derived variant lines maintain multi-organ metastatic potential upon rechallenge by MIND or intratibial injection, despite increased aggressiveness and maintained endocrine response. Notably, bone lesions from either challenge route showed mixed osteosclerotic/lytic features characteristic to clinical ILC. Accordingly, we found that conditioned medium from ILC cells and the mILC bone-derived variants induce osteoblast differentiation and suppressed osteoclast differentiation in vitro, consistent with their effect on bone remodeling in vivo and in clinical disease. Together, the models developed herein can be utilized to understand the unique metastatic processes of mILC, and to investigate new therapeutic combinations in the setting of endocrine-responsive primary and metastatic ILC.

Abstract Estrogen receptor-positive breast cancer represents a significant proportion of breast cancer brain metastasis but remains understudied. Here we show that FGFR1-amplification, a well-established driver of estrogen receptor-positive breast cancer endocrine resistance, promotes estrogen receptor-positive breast cancer brain metastatic colonization in young and aged female mice, through both canonical FGF2/FGFR1 signaling and non-canonical NCAM1/FGFR1 interactions. Astrocytic FGF2-mediated paracrine activation of FGFR1 promotes breast cancer brain metastasis in estrogen-treated young mice, but FGF2 levels and signaling decrease in the brain with aging and estrogen-depletion. Neuronal and astrocytic NCAM1, which remain unchanged in young and aged brains, promote adhesion to neurons, migration, and growth of estrogen receptor-positive cells, suggesting that interactions with astrocytes and neurons facilitate early estrogen receptor-positive breast cancer brain metastasis colonization through FGFR1. Importantly, FDA-approved FGFR inhibitors effectively block early colonization but not late-stage brain metastases, suggesting prevention of FGFR1+ brain metastases as a window of opportunity for FGFR1 inhibitors.

Intracranial metastases (ICM), specifically parenchymal brain metastases, remain a major clinical challenge in solid tumor oncology, despite recent advances in cancer therapies which have led to improvements in survival for these patients. Improving outcomes even further in this patient population will require a multi-disciplinary approach, including pre-clinical and translational studies, clinical trials, and studies of patient reported outcomes and quality of life. At the 2023 and 2024 joint Society for Neuro-Oncology (SNO) and American Society of Clinical Oncology (ASCO) CNS Metastases Conferences, two ICM collaborative group think tanks convened, composed of diverse, multi-disciplinary stakeholders, including basic and translational researchers, clinical trialists, and clinicians from academia and the community setting. Here we summarize the key knowledge gaps and consensus recommendations put forth by these two think tanks. Advances in ICM research and improvements in patient outcomes will require close inter-specialty and inter-institutional collaboration between stakeholders, including pre-clinical and translational researchers, clinical investigators, industry, and regulatory bodies.

Supplementary Figure 5. MUC5AC silencing inhibits cMET and CD44v6 expression and Astrocytes induce MUC5AC expression in breast cancer cells. A-B. Western blots showing knockdown of MUC5AC in MDA-231BR HER2 (A) and MUC5AC knockout in HCC1954BR (B) reduces expression of cMET and CD44v6. C. IHC staining for cMET and CD44v6 on brain tissue sections from mice injected with shSCR and shMUC5AC MDA-231BR HER2 cells. D. SKBR3 cells were treated with human astrocyte conditioned media for 24h and cells were lysed and subjected for MUC5AC expression. HGF induced signaling in MDA-231BR HER2 (E) and HCC1954BR (F); cells were treated with HGF (100ng/mL) for indicated times. After treatment cells were lysed and subjected for cMET and p-cMET expression using Western blot.

PURPOSE:Breast cancer brain metastasis remains a significant clinical problem. Mucins have been implicated in metastasis; however, whether they are also involved in breast cancer brain metastasis remains unknown. We queried databases of patients with brain metastasis and found mucin 5AC (MUC5AC) to be upregulated and therefore sought to define the role of MUC5AC in breast cancer brain metastasis. EXPERIMENTAL DESIGN:In silico dataset analysis, RNA-sequence profiling of patient samples and cell lines, analysis of patient serum samples, and in vitro/in vivo knockdown experiments were performed to determine the function of MUC5AC in breast cancer brain metastasis. Coimmunoprecipitation was used to unravel the interactions that can be therapeutically targeted. RESULTS:Global in silico transcriptomic analysis showed that MUC5AC is significantly higher in patients with breast cancer brain metastasis. Analysis of archived breast cancer brain metastasis tissue further revealed significantly higher expression of MUC5AC in all breast cancer subtypes, and high MUC5AC expression predicted poor survival in HER2+ breast cancer brain metastasis. We validated these observations in breast cancer brain metastatic cell lines and tissue samples. Interestingly, elevated levels of MUC5AC were detected in the sera of patients with breast cancer brain metastasis. MUC5AC silencing in breast cancer brain metastatic cells reduced their migration and adhesion in vitro and in brain metastasis in the intracardiac injection mouse model. We found high expression of cMET and CD44v6 in breast cancer brain metastasis, which increased MUC5AC expression via hepatocyte growth factor signaling. In addition, MUC5AC interacts with cMET and CD44v6, suggesting that MUC5AC promotes breast cancer brain metastasis via the cMET/CD44v6 axis. Inhibition of the MUC5AC/cMET/CD44v6 axis with the blood-brain barrier-permeable cMET inhibitor bozitinib (PLB1001) effectively inhibits breast cancer brain metastasis. CONCLUSIONS:Our study establishes that the MUC5AC/cMET/CD44v6 axis is critical for breast cancer brain metastasis, and blocking this axis will be a novel therapeutic approach for breast cancer brain metastasis.

Supplementary Figure 1. Expression of MUC5AC is high in breast cancer brain metastasis. A. Heatmap showing the expression of upregulated genes in BC BrM cells as compared to primary BC cells (MDA-231BR vs MDA-231P). B. Comparison of MUC5AC expression (z-score) between BC and brain metastasis samples, using online available Gene Expression Omnibus (GEO) public datasets, probe (214385_s_at). C. Heatmap showing the expression of goblet cells marker genes in BC and breast cancer brain metastasis cell lines (MDA-231BR vs MDA-231P). Data represents upregulation of marker proteins. D. Pie chart representing H-score for MUC5AC expression in BrM tissues originating from BC (Breast cancer), LC (Lung cancer) and other cancers.

Estrogen receptor positive (ER+) breast cancer (BC) represents a significant proportion of BC brain metastasis (BCBM) but remains understudied. Here, we report that FGFR1-amplification, a well-established driver of ER+ BC endocrine resistance, promotes ER+ BCBM colonization in young and aged mice, through brain-dependent mechanisms. FGFR1-dependent brain colonization in young and aged mice occurs via canonical FGF2/FGFR1 signaling and non-canonical NCAM1/FGFR1 interactions. Astrocytic FGF2-mediated paracrine activation of FGFR1 promoted BCBMs in estrogen-treated young mice, but FGF2 signaling decreased in the brain with aging and estrogen-depletion. Neuronal and glial NCAM1, which remain unchanged in young and aged brains, promoted adhesion to neurons and migration of ER+ BC cells, suggesting that interactions with astrocytes and neurons facilitate early ER+ BCBM colonization through FGFR1. Importantly, FDA-approved FGFR inhibitors effectively blocked early but not late metastatic progression only in young mice, suggesting limited efficacy of FGFR inhibitors to block non-kinase-dependent FGFR1 functions in vivo .

AbstractPurpose: Breast cancer brain metastasis remains a significant clinical problem. Mucins have been implicated in metastasis; however, whether they are also involved in breast cancer brain metastasis remains unknown. We queried databases of patients with brain metastasis and found mucin 5AC (MUC5AC) to be upregulated and therefore sought to define the role of MUC5AC in breast cancer brain metastasis. Experimental Design: In silico dataset analysis, RNA-sequence profiling of patient samples and cell lines, analysis of patient serum samples, and in vitro/in vivo knockdown experiments were performed to determine the function of MUC5AC in breast cancer brain metastasis. Coimmunoprecipitation was used to unravel the interactions that can be therapeutically targeted. Results: Global in silico transcriptomic analysis showed that MUC5AC is significantly higher in patients with breast cancer brain metastasis. Analysis of archived breast cancer brain metastasis tissue further revealed significantly higher expression of MUC5AC in all breast cancer subtypes, and high MUC5AC expression predicted poor survival in HER2+ breast cancer brain metastasis. We validated these observations in breast cancer brain metastatic cell lines and tissue samples. Interestingly, elevated levels of MUC5AC were detected in the sera of patients with breast cancer brain metastasis. MUC5AC silencing in breast cancer brain metastatic cells reduced their migration and adhesion in vitro and in brain metastasis in the intracardiac injection mouse model. We found high expression of cMET and CD44v6 in breast cancer brain metastasis, which increased MUC5AC expression via hepatocyte growth factor signaling. In addition, MUC5AC interacts with cMET and CD44v6, suggesting that MUC5AC promotes breast cancer brain metastasis via the cMET/CD44v6 axis. Inhibition of the MUC5AC/cMET/CD44v6 axis with the blood–brain barrier–permeable cMET inhibitor bozitinib (PLB1001) effectively inhibits breast cancer brain metastasis. Conclusions: Our study establishes that the MUC5AC/cMET/CD44v6 axis is critical for breast cancer brain metastasis, and blocking this axis will be a novel therapeutic approach for breast cancer brain metastasis.

Brain metastases (BrM) arising from solid tumors is an ever-increasing and often devastating clinical challenge impacting hundreds of thousands of patients annually worldwide. As systemic anticancer therapies, and thus survival, improve, the risk for central nervous system (CNS) recurrence has increased. Historically, patients with BrM were excluded from clinical trials; however, there has been a shift toward increasing inclusion over the past decade. To most effectively design the next generation of clinical trials for patients with BrM, a multidisciplinary team spanning local and systemic therapies is imperative. CIMARa (Consortium for Intracranial Metastasis Academic Research), formalized in June 2021, is an inclusive group of multidisciplinary clinical investigators, research scientists, and advocates who share the collective goal of improving outcomes for patients with BrM. CIMARa aims to improve outcomes through the development, coordination, and awareness of multi-institutional clinical trials testing novel therapeutic agents for this unique patient population alongside the translation of preclinical research to the clinical setting.

Supplementary Figure 6. PLB1001 inhibit cMET and CD44v6 expression. A. Representative IHC images showing MUC5AC expression in G5-3 PDXs B. Representative fluorescence microscopy images showing GFP+ in G5-3 PDXs C. Representative IHC images showing expression of MUC5AC, cMET, CD44v6 and cleaved caspase3 in G5-3 PDX treated with vehicle or PLB1001 (50mg/kg).

Supplementary Figure 3. Predictive value of IL13RA2 or EFNB1 mRNA levels in primary tumors known to metastasize to brain. Dataset contains 21 matched breast cancer primary and brain metastasis samples, reported in (https://pubmed.ncbi.nlm.nih.gov/29961873/). A. Kaplanmeier plots for IL13RA2 mRNA expression in primary tumors. B. Kaplan-Meier plots to EFNB1 mRNA in primary tumors. C. Kaplan-meier plots for primary tumors with low-IL13RA2/highEFNB1 compared to samples with high-IL13RA2/low-EFNB1. For all plots, average mRNA expression was determined for each target and expression ranked as low (below mean) or high (above mean). Survival curves comparisons were made using Log-rank (Mantel-Cox) test. p<0.05 was considered significant (italic bold). OS: overall survival; DFS: disease free survival; BMFS: brain metastasis free survival; SPBM: survival post brain metastasis.