Brain Metastatic Cells Research Articles

Carcinoma-Astrocyte Gap Junction Interruption by a Dual-Targeted Biomimetic Liposomal System to Attenuate Chemoresistance and Treat Brain Metastasis.

Brain metastasis contributes substantially to the morbidity and mortality of various malignancies and is characterized by high chemoresistance. Intracellular communication between carcinoma cells and astrocytes through gap junctions, which are assembled mainly by the connexin 43 protein, has been shown to play a vital role in this process. However, effectively blocking the gap junctions between the two cell types remains extremely challenging because of insufficient drug delivery to the target site. Herein, we designed a connexin blocker-carbenoxolone (CBX)-loaded biomimetic liposomal system with artificial liposomes fused with brain metastatic cell and reactive astrocyte membranes (LAsomes) to block gap junctions and attenuate chemoresistance. LAsomes effectively penetrated the blood-brain barrier via semaphorin 4D (SEMA 4D)─Plexin B1 interactions and actively migrated to their source cells via homotypic recognition. Consequently, LAsomes effectively inhibited material transfer and Ca2+ flow from metastatic cells to astrocytes via gap junctions, thereby markedly increasing the sensitivity of metastatic tumor cells to chemotherapy. These results reveal that closing the gap junctions may be a promising therapeutic strategy for intractable brain metastasis.

ITGB4 is a prognostic biomarker and correlated with lung adenocarcinoma brain metastasis.

The aim of this study is to explore the prognostic value and immune signature of ITGB4 expression in lung adenocarcinoma (LUAD) brain metastasis. We comprehensively screened genes associated with LUAD brain metastasis by integrating datasets from the GEO database and TMT-based quantitative proteomics profiles. Univariable survival and Multivariate Cox analysis was used to compare several clinical characteristics with survival, and a risk model was constructed. The biological functions were explored via GO and KEGG analysis. Gene set enrichment analysis (GSEA) was performed using the TCGA dataset. In addition, we use TIMER to explore the collection of ITGB4 Expression and Immune Infiltration Level in LUAD. The ability of ITGB4 to regulate tumor metastasis was further assessed by migration, invasion assay and Western-blot in H1975-BrM4 cells. We found that ITGB4 was the only gene with high clinical diagnostic and prognostic value in LUAD. Enrichment analysis indicated that ITGB4 is associated with brain metastasis, infiltration of immune cells, and the response to immunotherapy. ITGB4 expression can effectively predict the outcomes of patients with LUAD who are receiving anti-PD-1 therapy. ITGB4 knockdown inhibited the invasion, migration of H1975-BrM4 brain metastasis cells, as well as epithelial-mesenchymal transition (EMT) abilities. The heightened expression of ITGB4 protein was shown to promote EMT and enhance the metastatic potential. ITGB4 promotes the progression in H1975-BrM4 cells via MEK/ERK signaling pathway. Our findings indicate that the expression of ITGB4 is linked to the occurrence of brain metastasis and infiltration of immune cells, suggesting that ITGB4 might be a clinical treatment target for LUAD.

The tumor-neutrophil interactions in the microenvironment of brain metastases with different primary sites.

Brain metastases (BrM) originating from lung and breast cancer can recruit and activate neutrophils to acquire a tumor-promoting phenotype. It is currently unclear if this phenomenon also occurs in BrM arising from other primary sites. Here, we investigated the effect of tumor cells isolated from melanoma, lung and gastrointestinal (GI) cancer BrM on neutrophil biology and functions. We found that lung and GI, but not melanoma BrM cells produced CXCL8/IL-8, and promoted neutrophil recruitment. Similarly, lung and GI, but not melanoma BrM cells, prolonged the survival of neutrophils, and stimulated them to release MMP9 and CCL4/MIP1β. In situ, lung and GI BrM tissues contained significantly higher numbers of tumor-infiltrating neutrophils compared to melanoma BrM. The levels of neutrophil infiltration significantly correlated with the proliferation index of these tumors. Our findings identify variabilities in the immune microenvironment of BrM with different primary sites, which may ultimately affect their pathophysiology and progression.

CSIG-12. YAP1-TEAD SIGNALING CONTRIBUTES TO LUNG CANCER COLONIZATION OF THE BRAIN

Abstract BACKGROUND Lung cancer is the most common primary tumor to metastasize to the brain. Poor understanding of the brain metastasis process limits therapeutic options. Despite emerging data on the role of YAP signaling pathway in the development of brain tumors, a gap in knowledge remains regarding its role in brain colonization. We hypothesize that YAP1 and the associated TEAD transcriptional factors are responsible for lung cancer progression and that intervention with YAP/TEAD-targeted therapy will prevent brain dissemination. METHODS Lung cancer cells at different stages of cancer progression (VMRC-LCD and NCI-H1975, lung cancer cell lines; NCI-H1915 and LN001, brain metastatic cancer cells) were genetically engineered to overexpress or silence/knockout YAP1 using lentiviral transduction of constructs expressing shRNA/sgRNA or YAP1. We tested several tumor cell capabilities using well-established in vitro assays, including invasion and migration assays, and an in vivo brain metastasis (BM) model to determine the biological effects of YAP1/TEAD suppression at different stages of lung cancer progression. Dose-response assays were performed to determine IC50 of the TEAD inhibitor, verteporfin. RESULTS YAP overexpression enhances the proliferative phenotype in metastatic and BM cell lines but not in non-metastatic cells. However, YAP overexpression only increases the migration ability of BM cell lines such as LN001 and NCI-H1915 compared to the metastatic NCI-H1975 and non-metastatic lung cancer cells. Moreover, we showed the efficacy of the YAP1/TEAD inhibitor in patient-derived lung BM cells. Finally, our in vivo preliminary data showed YAP1 involvement in lung cancer cell colonization of the brain but not in sustaining brain metastasis growth. CONCLUSIONS Our findings showed YAP1 involvement in lung cancer cell colonization of the brain, suggesting YAP1 inhibition is a clinically actionable target for preventing lung cancer dissemination and providing a strong rationale to further evaluate the benefit of a YAP1/TEAD-targeted therapy in in vivo models of BM.

TMIC-48. THREE DIMENSIONAL CANCER CELL-MONOCYTE CO-CULTURE SPHEROIDS RECAPITULATE IMMUNOSUPPRESSIVE AND PRO-TUMORIGENIC MICROENVIRONMENTS IN BRAIN METASTATIC BREAST CANCER

Abstract Brain metastasis occurs in 15 % of breast cancer patients and is one of the major causes of breast cancer related morbidity. Brain metastatic breast cancer (BMBC) microenvironment is a heterogeneous niche of cellular and acellular components of which the innate immune cells, microglia, and macrophages, comprise the major fraction. However, the role of these innate immune cells in the tumor microenvironment (TME) in mediating cancer progression and response to therapy is not well understood. To address this need, herein, we developed three dimensional (3D) co-culture spheroids using brain metastatic triple negative breast cancer (TNBC) cell line MDA-MB-231Br or patient derived brain metastatic TNBC cell line F2-7 with THP1 monocytes. Under co-culture conditions MDA-MB-231Br co-culture spheroids assumed highly protrusive morphology with enhanced mesenchymal and proliferative characteristics. Additionally, phenotypic determination of cocultured macrophages revealed elevated levels of markers associated with alternatively activated macrophages i.e. CD163, CD206, and c-Myc. Furthermore, we found that under coculture conditions immunosuppressive and evasive markers PD1, PDL1, CD47 and CD24 were significantly upregulated. Similarly, F2-7 and monocyte cocultures also led to an immunosuppressive environment with elevated levels of M2 type markers and associated anti-inflammatory cytokines. We further found that the presence of M2 type macrophages in the microenvironment of BMBC spheroids led to acquired resistance against Paclitaxel, a clinically relevant chemotherapeutic drug against BMBC. In sum, in our coculture conditions, brain metastatic TNBC cells polarized monocytes into immunosuppressive and pro-tumorigenic tumor associated macrophages leading to a pro-tumoral microenvironment. Thus, 3D co-culture spheroids of BMBC cells and monocytes can serve as robust platform for studying cancer cell and innate immune cell interactions occurring in the BMBC environment.

TMIC-01. TARGETABLE GENE EXPRESSION CHANGES MEDIATE EXTRAVASATION OF BREAST CANCER CELLS FROM THE CIRCULATION AND LOCAL PROLIFERATION IN THE BRAIN

Abstract As breast cancer migrates into the brain, it acquires distinct genomic, immunologic and phenotypic changes through interactions with the local microenvironment. We sought to determine how targetable gene expression changes mediate breast cancer extravasation into and proliferation within the brain. We created a brain metastatic cell line (“brain-seeking”) via serial intracardiac injection of mCherry+ 4T1 breast cancer cells in Balb/c mice and flow cytometry isolation of micrometastases. We conducted in vitro and in-vivo studies to uncover immunological and genetic differences between primary breast cancer and brain metastases. In culture, we observed significant upregulation in adhesion (p=0.01) and invasion (p=0.0004) along with upregulation of adhesion-promoting genes such as Sdc3, Serpine2 or ECM1 in brain-seeking cells. We observed significant differences in survival of Balb/c mice injected intracranially with brain-seeking cells vs. breast cancer cells (p=0.04). In the mammary fat pad (MFP), parental 4T1 cells caused increased mortality vs. brain-seeking cells (p=0.003). This rejection of brain-seeking 4T1 cells in the MFP could be due to prior adaptation to the brain microenvironment, while rejection of parental 4T1 cells in the brain could be due to lacking said adaptations. Consistent with this finding, of 20,382 analyzed genes in both cell lines, 108 related to immune regulation were significantly upregulated in brain-seeking cells including Cxcl1, Dusp1, C3. Flow cytometry showed increased cytotoxic and helper T cells in intracranially injected parental vs. brain-seeking cells (p <0.001). In the MFP, however, 4T1 parental cells displayed CD3+ T cell depletion and an increased number of neutrophils (p <0.05) while the “brain-seeking cells” showed increased numbers of CD3+ and CD4+ cells (p <0.01) and macrophages (p<0.05). This suggests that brain metastases evolve from their primary tumor through adhesive and invasive adaptations promoting extravasation, along with unique immunological adaptations that allow their proliferation free of immune surveillance in the brain microenvironment.

ACSS2 regulates ferroptosis in an E2F1-dependent manner in breast cancer brain metastatic cells.

Brain metastasis diagnosis in breast cancer patients is considered an end-stage event. The median survival after diagnosis is measured in months, thus there is an urgent need to develop novel treatment strategies. Breast cancers that metastasize to the brain must adapt to the unique brain environment and are highly dependent on acetate metabolism for growth and survival. However, the signaling pathways that regulate survival in breast cancer brain metastatic (BCBM) tumors are not known. Primary brain tumor cells can convert acetate to acetyl-CoA via phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by the cyclin-dependent kinase-5 (CDK5) regulated by the nutrient sensor O-GlcNAc transferase (OGT). Here, we show that breast cancer cells selected to metastasize to the brain contain increased levels of O-GlcNAc, OGT and ACSS2-Ser267 phosphorylation compared to parental breast cancer cells. Moreover, OGT and CDK5 are required for breast cancer cell growth in the brain parenchyma in vivo. Importantly, ACSS2 and ACSS2-S267D phospho-mimetic mutant are critical for in vivo breast cancer growth in the brain but not in the mammary fat pad. Mechanistically, we show that ACSS2 regulates BCBM cell survival by suppressing ferroptosis via regulation of E2F1-mediated expression of anti-ferroptotic proteins SLC7A11 and GPX4. Lastly, we show treatment with a novel brain-permeable small molecule ACSS2 inhibitor induced ferroptosis and reduced BCBM growth ex vivo and in vivo. These results suggest a crucial role for ACSS2 in protecting from ferroptosis in breast cancer brain metastatic cells and suggests that breast cancer brain metastatic cells may be susceptible to ferroptotic inducers.

ENPP1 induces blood-brain barrier dysfunction and promotes brain metastasis formation in HER2-positive breast cancer.

Brain metastasis (BrM) is a devastating end-stage neurological complication that occurs in up to 50% of HER2+ breast cancer patients. Understanding how disseminating tumor cells manage to cross the blood-brain barrier (BBB) is essential for developing effective preventive strategies. We identified the ecto-nucleotidase ENPP1 as specifically enriched in the secretome of HER2+ brain metastatic cells, prompting us to explore its impact on BBB dysfunction and BrM formation. We used in vitro BBB and in vivo premetastatic mouse models to evaluate the effect of tumor-secreted ENPP1 on brain vascular permeability. BBB integrity was analyzed by real-time fluorescence imaging of 20kDa Cy7.5-dextran extravasation and immunofluorescence staining of adherens and tight junction proteins. Pro-metastatic effects of ENPP1 were evaluated in an experimental brain metastatic model. Systemically secreted ENPP1 from primary breast tumors impaired the integrity of BBB with loss of tight and adherens junction proteins early before the onset of BrM. Mechanistically, ENPP1 induced endothelial cell dysfunction by impairing insulin signaling and its downstream AKT/GSK3β/β-catenin pathway. Genetic ablation of ENPP1 from HER2+ brain metastatic cells prevented endothelial cell dysfunction and reduced metastatic burden while prolonging the overall and metastasis-free survival of mice. Furthermore, plasmatic ENPP1 levels correlate with brain metastatic burden and inversely with overall survival. We demonstrated that metastatic breast cancer cells exploit the ENPP1 signaling for cell transmigration across the BBB and brain colonization. Our data implicate ENPP1 as a potential biomarker for poor prognosis and early detection of BrM in HER2+ breast cancer.

MicroRNA-522-3p promotes brain metastasis in non-small cell lung cancer by targeting Tensin 1 and modulating blood-brain barrier permeability

Brain metastases account for more than 50 % of intracranial central nervous system tumors. The blood-brain barrier (BBB) is mainly composed of endothelial cells, which exhibit low endocytosis and high efflux pumps. Although they are connected by continuous tight junctions and serve as a protective insulation, the BBB does not prevent the development of brain metastases from non-small cell lung cancer (NSCLC). Improving understanding on the mechanisms underlying the development of brain metastasis and the differential molecular characteristics relative to the primary tumor are therefore key in the treatment of brain metastases. This study evaluated the differential expression of miR-522-3p in NSCLC and brain metastases using the Gene Expression Omnibus database. NSCLC brain metastasis model was constructed to screen for cell lines that demonstrated high potential for brain metastasis; We also observed differential expression of miRNA-522-3p in the paraffin-embedded specimens of non-small cell lung cancer and brain metastases from our hospital. The molecular biological functions of miRNA-522-3p were verified using 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay and Transwell invasion assays. RNA-seq was employed to identify downstream target proteins, and the dual-luciferase reporter assay confirmed Tensin 1 (TNS1), a protein that links the actin cytoskeleton to the extracellular matrix, as the downstream regulatory target protein. In vitro blood-brain barrier models and co-culture models were constructed to further identify the role of miRNA-522-3p and TNS1; the expression of BBB-related proteins (ZO-1 and OLCN) was also identified. In vivo experiments were performed to verify the effects of miRNA-522-3p on the time and incidence of NSCLC brain metastasis. The results showed significantly high expression in GSE51666; consistent results were obtained in brain metastasis cells and paraffin samples. RNA-seq combined with miRNA target protein prediction demonstrated TNS1 to be directly downstream of miR-522-3p and to be associated with cell proliferation and invasion. By regulating ZO-1 and OCLN expression, mi-522-3p/TNS1 may increase tumor cell penetration through the BBB while decreasing its permeability. In vivo, miR-522-3p was further demonstrated to significantly promote the formation of brain metastases. miR-522-3p/TNS1 can affect BBB permeability and encourage the growth of brain metastases by modifying the BBB TJ proteins. This axis offers new therapeutic targets for the prevention of brain metastasis.

Camouflaging nanoreactor traverse the blood-brain barrier to catalyze redox cascade for synergistic therapy of glioblastoma

The blood-brain barrier (BBB) is a complex and highly restrictive barrier that prevents most biomolecules and drugs from entering the brain. However, effective strategies for delivering drugs to the brain are urgently needed for the treatment of glioblastoma. Based on the efficient BBB penetration properties of exosomes derived from brain metastatic breast cancer cells (EB), this work prepared a nanoreactor (denoted as MAG@EB), which was constructed by self-assembly of Mn2+, arsenate and glucose oxidase (GOx) into nanoparticles wrapped with EB. MAG@EB can enhance the efficiency of traversing the BBB, target and accumulate at in situ glioblastoma sites. The GOx-driven glycolysis effectively cuts off the glucose supply while also providing an abundance of H2O2 and lowering pH. Meanwhile, the released Mn2+ mediated Fenton-like reaction converts elevated H2O2 into highly toxic ·OH. Besides, AsV was reduced to AsIII by glutathione, and the tumor suppressor gene P53 was activated by AsIII to kill glioblastoma cells. Glioblastoma succumbed to the redox cascade triggered by MAG@EB, as the results demonstrated in vivo and in vitro, yielding a remarkable therapeutic effect. This work provides a promising therapeutic option mediated by cascaded nanoreactors for the future treatment of glioblastoma.

Multifaceted interactions between cancer cells and glial cells in brain metastasis.

Cancer brain metastasis has a poor prognosis, is commonly observed in clinical practice, and the number of cases is increasing as overall cancer survival improves. However, experiments in mouse models have shown that brain metastasis itself is an inefficient process. One reason for this inefficiency is the brain microenvironment, which differs significantly from that of other organs, making it difficult for cancer cells to adapt. The brain microenvironment consists of unique resident cell types such as neurons, oligodendrocytes, astrocytes, and microglia. Accumulating evidence over the past decades suggests that the interactions between cancer cells and glial cells can positively or negatively influence the development of brain metastasis. Nevertheless, elucidating the complex interactions between cancer cells and glial cells remains challenging, in part due to the limitations of existing experimental models for glial cell culture. In this review, we first provide an overview of glial cell culture methods and then examine recent discoveries regarding the interactions between brain metastatic cancer cells and the surrounding glial cells, with a special focus on astrocytes and microglia. Finally, we discuss future perspectives for understanding the multifaceted interactions between cancer cells and glial cells for the treatment of metastatic brain tumors.

SpatialGE: A user-friendly web application to democratize spatial transcriptomics analysis.

Spatial transcriptomics (ST) is a powerful tool for understanding tissue biology and disease mechanisms. However, its potential is often underutilized due to the advanced data analysis and programming skills required. To address this, we present spatialGE, a web application that simplifies the analysis of ST data. The application spatialGE provides a user-friendly interface that guides users without programming expertise through various analysis pipelines, including quality control, normalization, domain detection, phenotyping, and multiple spatial analyses. It also enables comparative analysis among samples and supports various ST technologies. We demonstrate the utility of spatialGE through its application in studying the tumor microenvironment of melanoma brain metastasis and Merkel cell carcinoma. Our results highlight the ability of spatialGE to identify spatial gene expression patterns and enrichments, providing valuable insights into the tumor microenvironment and its utility in democratizing ST data analysis for the wider scientific community.

Targeting fatty acid synthase in preclinical models of TNBC brain metastases synergizes with SN-38 and impairs invasion

Fatty acid synthesis (FAS) has been shown to play a key role in the survival of brain-metastatic (BM) breast cancer. We demonstrate that the fatty acid synthase inhibitor TVB-2640 synergizes with the topoisomerase inhibitor SN-38 in triple-negative breast cancer (TNBC) BM cell lines, upregulates FAS and downregulates cell cycle progression gene expression, and slows the motility of TNBC BM cell lines. The combination of SN-38 and TVB-2640 warrants further consideration as a potential therapeutic option in TNBC BMs.

Clinical, molecular, and immunologic profiling of brain metastases (BM) in head and neck squamous cell carcinoma (HNSCC).

6036 Background: BM is a rare complication of HNSCC that carries a high rate of morbidity and poor prognosis. Clinical risk factors, molecular characteristics, and the immunogenicity of HNSCC BM are not well defined, leaving a critical knowledge gap in this field. We performed one of the largest multi-institutional analyses summarizing the clinical, molecular, and immunologic profile of 61 cases of BM-HNSCC. Methods: We conducted a pooled analysis of the clinical characteristics pertaining to BM-HNSCC from 3 academic institutions (n=24). Next-generation sequencing (NGS) and immune profiling (IP) of primary and BM specimens was conducted on a subset of cases (n=19 and n=16, respectively); there were 3 paired samples for NGS and 0 for IP. Four samples (2 BM and 2 non-BM) were submitted to Phase Genomics, Inc for evaluation of structural variants in BM genomes by proximity ligation sequencing (PLS). These results were complimented by a comparative analysis of genomic alterations in an additional cohort of BM (n=37) and local samples (n=4082) submitted for NGS at Foundation Medicine, Inc (FMI). Statistical comparisons were done using Fisher’s exact testing of 2x2 contingency tables with p-values controlled for FDR by the Benjamini-Hochberg procedure. Results: Clinical features were as follows: median age at diagnosis 59 years, 75% male, 55% current/former smokers, 75% oropharyngeal primary, and 84% HPV+ or p16+. The most frequently altered genes in BM specimens (62% HPV/p16+) were ATM (54%), KMT2A (54%), PTEN (46%), RB1 (46%), and TP53 (46%). BM and non-BM samples demonstrated significant levels of structural rearrangement ranging from 9 to 90 variants by PLS. IP identified lower densities of CD8+, PD1+, PDL1+, and FOXP3+ cells in BMs compared to primary tumors. PDL1 combined positive scores were <1% in 12/13 unpaired samples (92%; 10 BM and 2 primary). The FMI BM-HNSCC cohort (51% HPV+) identified CDKN2A (40.5%), TP53 (37.8%), and PIK3CA (27.0%) as the most frequently altered genes. Enrichment analysis of the FMI cohort showed MAP2K2 alterations significantly enriched in BM (11.8% vs 6.4%, P=0.005) and TSC1 alterations significantly enriched in the local site (67.3% vs 37.8%, P=0.008). HPV+ was also significantly enriched in the BM cohort (51.25% vs 26.11%, P=0.001). Overall survival from BM diagnosis was 6m (range 0-27m). Conclusions: HNSCC patients with BM have higher-than-expected proportions of oropharyngeal primary site and HPV/p16-positivity. The most frequent molecular alterations in BM samples are also commonly found in non-BM HNSCC, including targetable PIK3CA alterations. MAP2K2 alterations were significantly enriched in BM compared to non-BM samples, which warrants further investigation. BM samples also tended to have lower markers of immunogenicity. This latter finding could have important clinical implications when considering immunotherapy or immune-modulating drugs.

HLA class-I antigen presentation machinery (APM) alterations mediate immune evasion in lung cancer brain metastases.

e14014 Background: Although ~30% of patients with lung cancer brain metastasis (LBM) achieve intracranial response with immune checkpoint inhibitors (ICI), most have primary resistance, and those who initially respond develop rapid disease progression. The biological factors contributing to this unfavorable clinical scenario are poorly understood and could include distinct immunologic properties of tumors colonizing the brain. Using annotated tumor/patient cohorts, spatially resolved protein analysis, and in vitro models, we investigated the role of HLA class-I APM defects and interferon (IFN)-γ pathway in LBM. Methods: Using multiplex quantitative immunofluorescence, we measured the levels and spatial distribution of HLA class-I APM markers (β2M, tapasin, calreticulin, ERp57, TAP1, and TAP2), IFN-γ signaling (pSTAT1 and IRF-1), and tumor-infiltrating lymphocytes (TILs) (CD8, CD4, and FOXP3) in 56 LBM, 41 primary lung tumors (PLT), and 10 lung-extracranial metastases (ECM) represented in tissue microarrays. A subset of paired LBM/PLT samples from the same patients were also included (n=8). The spatial analysis of the markers was conducted using fluorescence co-localization and single-cell segmentation strategies. Associations between the markers, clinicopathological characteristics, and survival were studied. Protein expression of β2M and IFN-γ-sensitive markers (total STAT1, pSTAT1, IRF1, and PD-L1) before and after treatment with human recombinant IFN-γ was assessed using Western blot in cultured primary lung H2030 adenocarcinoma cells and their in vivo selected brain metastatic counterpart H2030-BrM3. Results: LBM showed significantly lower β2M, TAP2, and tapasin protein levels than PLT. However, LBM also showed higher expression of pSTAT1 and comparable levels of IRF-1 relative to PLT. Similar results were seen in paired LBM/PLT samples, but not in ECM samples. Tumors with low β2M showed reduced CD8+, CD4+, and FOXP3+ TILs. In LBM no significant association between β2M and IFN-γ pathway markers or major clinicopathologic and molecular patient/tumor features were found. LBM samples from patients progressing after radiotherapy and/or chemotherapy showed significantly lower β2M levels. Reduced β2M expression was associated with shorter overall survival in PLT but not in LBM. The brain metastatic H2030-BrM3 cell line showed lower β2M expression both at baseline and after IFN-γ treatment compared with their parental H2030 counterpart. Conclusions: HLA class-I APM downregulation is a prominent feature of LBM, is independent of local adaptive/inducible IFN-γ stimulation, and is associated with unfavorable clinical features. These findings support that HLA class-I APM deficiency plays a major role in immune evasion and immunotherapy resistance in patients with LBM.

Laminin I mediates resistance to lapatinib in HER2-positive brain metastatic breast cancer cells in vitro

The role of extracellular matrix (ECM) prevalent in the brain metastatic breast cancer (BMBC) niche in mediating cancer cell growth, survival, and response to therapeutic agents is not well understood. Emerging evidence suggests a vital role of ECM of the primary breast tumor microenvironment (TME) in tumor progression and survival. Possibly, the BMBC cells are also similarly influenced by the ECM of the metastatic niche; therefore, understanding the effect of the metastatic ECM on BMBC cells is imperative. Herein, we assessed the impact of various ECM components (i.e., Tenascin C, Laminin I, Collagen I, Collagen IV, and Fibronectin) on brain metastatic human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) cell lines in vitro. The highly aggressive TNBC cell line was minimally affected by ECM components exhibiting no remarkable changes in viability and morphology. On the contrary, amongst various ECM components tested, the HER2-positive cell line was significantly affected by Laminin I with higher viability and demonstrated a distinct spread morphology. In addition, HER2-positive BMBC cells exhibited resistance to Lapatinib in presence of Laminin I. Mechanistically, Laminin I-induced resistance to Lapatinib was mediated in part by phosphorylation of Erk 1/2 and elevated levels of Vimentin. Laminin I also significantly enhanced the migratory potential and replicative viability of HER2-positive BMBC cells. In sum, our findings show that presence of Laminin I in the TME of BMBC cells imparts resistance to targeted therapeutic agent Lapatinib, while increasing the possibility of its dispersal and clonogenic survival.

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling.

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also showthat brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

Selective and brain-penetrant ACSS2 inhibitors target breast cancer brain metastatic cells.

Breast cancer brain metastasis (BCBM) typically results in an end-stage diagnosis and is hindered by a lack of brain-penetrant drugs. Tumors in the brain rely on the conversion of acetate to acetyl-CoA by the enzyme acetyl-CoA synthetase 2 (ACSS2), a key regulator of fatty acid synthesis and protein acetylation. Here, we used a computational pipeline to identify novel brain-penetrant ACSS2 inhibitors combining pharmacophore-based shape screen methodology with absorption, distribution, metabolism, and excretion (ADME) property predictions. We identified compounds AD-5584 and AD-8007 that were validated for specific binding affinity to ACSS2. Treatment of BCBM cells with AD-5584 and AD-8007 leads to a significant reduction in colony formation, lipid storage, acetyl-CoA levels and cell survival in vitro. In an ex vivo brain-tumor slice model, treatment with AD-8007 and AD-5584 reduced pre-formed tumors and synergized with irradiation in blocking BCBM tumor growth. Treatment with AD-8007 reduced tumor burden and extended survival in vivo. This study identifies selective brain-penetrant ACSS2 inhibitors with efficacy towards breast cancer brain metastasis.

Murine Models of Laser Interstitial Thermal Therapy for Immuno-Oncology

INTRODUCTION: Cytoreductive surgery is a cornerstone of management for intracranial tumors. However, such treatment may remove an important source of neoantigen. In the case of laser interstital thermal therapy (LITT), thermally-ablated tumor remans in situ, allowing for a potential anti-tumor immune response. METHODS: Brain tumor cell lines (CT-2A) were stereotactically implanted into C57BL/6 mice. A 1064 nm Nd:YAG laser (Neuroblate, Monteris Medical) was used to ablate normal brain and tumor tissue. RESULTS: To establish anatomical boundaries for our ablation, we characterized our CT-2A model on T2 MRI, selecting 10 days post-implantation for LITT based on lesion size. We simultaneously implanted a 400 μm laser fiber and thermocouple probe into the center and edge of the target volume, respectively, for simultaneous ablation and temperature monitoring. To characterize the laser's effects, we first performed a dose titration of laser power/time in normal mouse brains, finding that doses above 2 W for 30 s produced cavitary lesions with charring that resulted in death at 24 h post-LITT. We therefore selected 1 W for 60 s as a safe laser power dose in our tumor model. Ablations at this setting rapidly heated the lesion edge to a peak temperature of mean 43.79 ± 2.993 °C, sparing the surrounding brain. MRI imaging at 1, 3, and 7 days post-LITT revealed an expansile T2 hyperintense lesion, with hemmorhage and necrosis on H&E. LITT increased survival to 28 from 25.5 days (vs sham, p = 0.0172). CONCLUSIONS: Using a clinical laser system, we were able to model the essential clinicopathological effects of LITT in mice: a cytoreductive survival benefit; lesion expansion on MRI; and focal necrosis with hemorrhagic vasculature on H&E. This model allows for detailed immunological studies of LITT and use with existing brain metastasis cell lines.

Abstract 5512: Cerebral endothelial FAK paradoxically inhibits brain metastasis in vivo

Abstract Introduction: In cancer metastasis in the trunk, the secreted proteins from tumors, such as VEGFs and TNF-α, activate focal adhesion kinase (FAK) in vascular endothelial cells (VECs). The activated FAK promotes VE-cadherin phosphorylation and the expression of cell adhesion molecules, and it facilitates the extravasation of cancer cells. In the cerebral blood vessels, the VECs maintain very strong cell adhesion supported by the surrounding pericytes and astrocytes. The strong barrier of cerebral VECs is called the blood-brain barrier (BBB) and is regulated differently from trunk VECs. Although the role of FAK in trunk metastasis is shown in many studies, its importance in brain metastasis is not verified. In this study, we focused on the role of the cerebral endothelial FAK and compared the role of FAK in brain metastasis with that in trunk metastasis. Methods: We generated tamoxifen-inducible VEC-specific FAK knockout (FAK-cKO) mice (VEcad-CreERT2; Ptk2-flox/flox) and brain metastatic (BrM) cell lines of Ex3LL (murine lung cancer cell line). We intracardially injected BrM cells of Ex3LL into the FAK-cKO mice and analyzed the tumor development using in vivo bioluminescence imaging and a CUBIC tissue clearing system. We also analyzed the intracranially injected mice of the parental and BrM cell lines to evaluate the tumor angiogenesis, which is reported to be promoted by the endothelial FAK. Results: Although FAK-cKO in VECs has been reported to inhibit lung metastasis, brain metastasis was accelerated in FAK-cKO mice compared with control mice. The number of brain metastatic sites increased in FAK-cKO mice, but there was no difference in the individual tumor size. It suggests that the brain metastasis was promoted by FAK-cKO, but that the proliferation of brain metastasis was not promoted by FAK-cKO. Blood vessel density was decreased with irregular shape in the brain tumors of FAK-cKO mice, suggesting that the angiogenesis was inhibited by FAK-cKO. It indicates that the angiogenic function of endothelial FAK in the brain vasculature was not different from that in the trunk. Conclusion: FAK-cKO in VECs exacerbated the brain metastasis of the BrM cells although it has been reported to inhibit the metastasis in the trunk. FAK-cKO in VECs decreased tumor angiogenesis both in the brain and the trunk. It suggests that the regulatory mechanism of cell adhesion and the FAK function in the brain vasculature is partly different from that in the other organs in the body trunk. We need further investigation to elucidate the function of endothelial FAK in brain metastasis. Citation Format: Shoko Noda-Narita, Atsu Aiba. Cerebral endothelial FAK paradoxically inhibits brain metastasis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5512.