Microenvironment In Brain Metastases Research Articles

Tertiary Lymphoid Structures in Brain Metastases of Lung Cancer: Prognostic Significance and Correlation With Clinical Outcomes.

The prognosis of patients with brain metastases (BMs) originating from lung cancer remains poor, despite advancements in treatment strategies. The role of tertiary lymphoid structures (TLSs) within the tumor immune microenvironment of BMs has not been extensively explored. This study utilized patient-derived clinical samples from 17 patients with histologically confirmed BMs of lung cancer, undergoing surgical resection. Immunohistochemistry was employed to analyze the presence and characteristics of TLS and tumor-infiltrating lymphocytes (TILs) within BM tissues, correlating these with clinical outcomes. TLSs, albeit in their immature form, were identified within BM tissues, distinguishing them from their mature counterparts in primary lung cancer tissues. A significant correlation between TLS density (but not TIL density) and improved postoperative survival was observed, underscoring the potential of TLS density as an independent prognostic marker. Furthermore, TLS density did not correlate with the Graded Prognostic Assessment (GPA) index, suggesting its unique prognostic value beyond conventional predictors. Our findings reveal the presence of TLSs in lung cancer-derived BMs and highlight their prognostic significance, independent of the GPA index. The identification of TLS within the unique central nervous system tumor microenvironment offers new insights into the immune landscape of BMs and suggests potential avenues for immunotherapeutic interventions targeting these structures to improve patient outcomes.

Aqueous extract of Chuan Xiong Rhizoma enhances inhibitory effect of temozolomide against brain metastasis of melanoma in mice

To investigate the effect of the aqueous extract of Chuan Xiong Rhizoma (CR) on brain metastasis of melanoma B16F10 cells in mice. C57BL/6J mouse models of brain metastasis of melanoma were established by ultrasound-guided intraventricular injection of Luc-labeled B16F10 cells, and brain tumor growth was monitored by in vivo imaging. The mouse models were then randomized for daily gavage of saline or aqueous extract of CR (equivalent crude drug concentration of 1 mg/g). Behavioral tests were used to evaluate the neuroprotective effects of CR in the tumor-bearing mice, and the changes in proteins associated with blood-brain barrier integrity, neuronal cell proliferation and apoptosis, and microglial cell apoptosis and activation were observed using immunofluorescence assay. The efficacy of CR combined with temozolomide (25 mg/kg) against brain metastases of B16F10 cells was observed by in vivo imaging. CR-treated mouse models did not show obvious progression of brain metastases and had a reduced rate of body weight loss and lowered protein expressions of ZO-1, claudin-5, occludin, P-gp, TNF-α, AQP4 and PDGFRβ. In the behavioral tests, the CR-treated mice showed prolonged stay on the wooden stick with a shortened time of sticky stick removal. Immunofluorescence assay showed increased proliferation and decreased apoptosis of neuronal cells and microglia in CR-treated mice. CR treatment significantly increased the levels of CD86, CD206, IL-4 and IL-10 and decreased the levels of CD163 and IL-1β in the microenvironment of brain metastases. The mice receiving combined treatments with CR and temozolomide showed significantly lower intensity of fluorescent signals in the brain than those treated with temozolomide alone. CR does not promote brain metastasis of melanoma while inducing opening of the blood-brain barrier, and its combined use with TMZ results in enhanced inhibition against brain metastasis of melanoma B16F10 cells in mice.

Abstract PO4-14-07: Genomic and immune profiling of breast cancer brain metastases

Abstract BACKGROUND: Brain metastases (BrM) arising from breast cancer (BC) are an increasing consequence of advanced disease, with up to half of patients (pts) with metastatic HER2+ or triple negative breast cancer experiencing central nervous system (CNS) recurrence. The genomic alterations driving CNS recurrence, along with contribution of the immune microenvironment, particularly by BC subtype remains unclear. METHODS: We characterized BrM from a cohort of n=42 BC pts by sequencing whole-exome DNA and total RNA libraries from frozen (n=31) and FFPE (n=34) BCBrM, FFPE extracranial (ECT, n=12) and blood DNA (n=26) tissues from the Duke Brain Tumor Biorepository. Analyses conducted and planned include inference of PAM50 intrinsic subtypes, somatic mutations, copy number alterations, immune cell type decompositions by CIBERSORTx, differential RNA expression, driver analysis, and associations with clinical outcomes. RESULTS: PAM50 subtypes across 31 frozen BrM were 23% Luminal (Lum) A, 13% LumB, 33% HER2-enriched, 27% Basal-like, 3% Normal-like. For 34 FFPE BrM, 15% were LumA, 24% LumB, 33% HER2-enriched, 24% Basal-like, 3% Normal-like. Across 26 paired FFPE and frozen BrM, subtype discrepancy was seen in 23% (6) cases of which frozen to FFPE classification differed: 3 LumA to LumB, 1 LumB to LumA, 1 Normal to LumB, and 1 Basal to LumB. Among 10 paired FFPE BrM and ECT, subtype concordance was observed in 70%. Discordance was seen in 1 case each for ECT to BrM: LumB to HER2, LumB to LumA, Basal to Normal-like. WES demonstrated frequent copy number alterations (CNA) in clinically-relevant genes including TGFB1, NOTCH1, CDK4, and ERBB3, in both BrM and ECT. For pts with matched blood, ERBB3 CNA (gain and loss) were more commonly found in FFPE BrM (7/19, 37%) compared to ECT (1/8, 13%). Among BrM, the most frequently altered clinically-relevant genes included TP53 (~75%), PTEN, EGFR, RB1, PIK3CA, NF1 and ESR1 (all ≤15%); in ECT, TP53 (67%), BRCA2, FBXW7, and ATM (all ≤33%). PI3K pathway genes (e.g. PTEN, PIK3CA) alterations were exclusive to BrM. One pt’s ECT showed an ATM mutation that was not observed in the paired BrM. Conversely, BRAF and CCND2 mutations were observed in BrM, but not ECT for 1 pt each. Inferring the relative abundance of immune populations in frozen BrM illustrated that 23% were CD4+ resting memory T cells, 25% M2 tumor-permissive macrophages, 13% M0 macrophages; M1 tumor-inhibiting macrophages were only 2%. When comparing immune cell populations between FFPE BrM and ECT, ECT had more M1 macrophages (Chi-sq 4.23, P = 0.04), while other immune cell populations were of similar relative abundance. Immune cell fractions did not vary by subtype in BrM or ECT with one exception: M2 macrophages were lower in Basal compared to LumB tissues (frozen BrM: Chi-sq = 9.28, P = 0.05; FFPE BrM: Chi-sq = 4.61, P = 0.33; FFPE EC: Chi-sq = 9.78, P = 0.04). In n=8 pt-matched FFPE BrM/ECT, hallmark pathways upregulated in BrM included MYC and E2F targets and oxidative phosphorylation, while those with lower expression in BrM included epithelial-mesenchymal transition, interferon gamma response, and JAK-STAT signaling. CONCLUSION: This analysis showed moderate discrepancy in subtype call of BrM by tissue preparation (frozen vs. FFPE), with LumB classification showing the highest discrepancy, and more commonly called in FFPE tissues. Subtype concordance between ECT and BrM was relatively high. Analysis of CNA illustrated deletions and amplifications in targetable genes, notably ERBB3 preferentially in BrM compared to ECT. Mutational analysis identified targetable alterations exclusive to BrM; this knowledge could lead to BrM-targeted treatments. Inferred immune cell populations illustrated a tumor permissive microenvironment in BrM. Therapeutic strategies repolarizing macrophages toward a tumor-inhibiting phenotype in BrM are warranted. Analyses of associations between genomic data and clinical outcomes, as well as driver analyses, are ongoing. Citation Format: Amanda Van Swearingen, Marissa Lee, Layne Rogers, Alexander Sibley, Pixu Shi, Xiaodi Qin, Michael Goodin, Kouros Owzar, Carey Anders. Genomic and immune profiling of breast cancer brain metastases [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-07.

Unveiling the hidden role of extracellular vesicles in brain metastases: a comprehensive review.

Extracellular vesicles (EVs) are small, transparent vesicles that can be found in various biological fluids and are derived from the amplification of cell membranes. Recent studies have increasingly demonstrated that EVs play a crucial regulatory role in tumorigenesis and development, including the progression of metastatic tumors in distant organs. Brain metastases (BMs) are highly prevalent in patients with lung cancer, breast cancer, and melanoma, and patients often experience serious complications and are often associated with a poor prognosis. The immune microenvironment of brain metastases was different from that of the primary tumor. Nevertheless, the existing review on the role and therapeutic potential of EVs in immune microenvironment of BMs is relatively limited. This review provides a comprehensive analysis of the published research literature, summarizing the vital role of EVs in BMs. Studies have demonstrated that EVs participate in the regulation of the BMs immune microenvironment, exemplified by their ability to modify the permeability of the blood-brain barrier, change immune cell infiltration, and activate associated cells for promoting tumor cell survival and proliferation. Furthermore, EVs have the potential to serve as biomarkers for disease surveillance and prediction of BMs. Overall, EVs play a key role in the regulation of the immune microenvironment of brain metastasis and are expected to make advances in immunotherapy and disease diagnosis. Future studies will help reveal the specificmechanisms of EVs in brain metastases and use them as new therapeutic strategies.

Abstract A085: Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment

Abstract Breast cancer is the most common cancer diagnosis worldwide, and an unfortunately high number of women die every year due to a lack of effective therapeutic strategies for metastatic breast cancer. Patients with brain metastases in particular have a significantly poorer prognosis than women with metastasis to other sites (e.g., lung, liver, bone). Previous studies by our group identified a ligand-receptor complex in the tumor microenvironment (TME) that promotes metastasis of breast cancer to the brain. This interaction between platelet-derived growth factor-BB (PDGFB), produced by cancerous breast epithelial cells, and its receptor, platelet-derived growth factor receptor beta (PDGFRβ), expressed by mesenchymal cells, is also prognostic of breast cancer metastasis to the brain in patients. The mechanism of how the PDGFB-PDGFRβ pathway mediates brain metastasis is still unclear and is the focus of our current work. To further analyze the biological effects of PDGFB, our group evaluated gene expression changes between primary human breast tumors expressing high PDGFB versus those expressing low PDGFB and found significantly more immune signaling in the low-expressing tumors. These results led us to hypothesize that the PDGFB-PDGFRβ pathway creates an immunosuppressive microenvironment, allowing for increased metastasis of cancerous cells. To test this, flow cytometry was completed on the brains, spleens, lungs, and livers of control mice and mutant mice expressing stromal-specific hyperactive PDGFRβ following intracardiac injection of PDGFB-expressing mammary tumor cells. No significant changes in immune cell populations were observed in non-tumor-bearing mice. However, in the brains of tumor-bearing mutant mice, there are significant changes to the myeloid lineage. Specifically, analysis at day 10 post-injection (prior to detectable metastatic lesions) revealed diminished CD45+CD11b+ populations. Segregating by CD45hi/CD45lo confirmed a profound decrease in CD45loCD11b+F4/80+ microglia, likely a tumor-suppressive population. Looking at day 16 post-injection (detectable metastatic lesions), there is a shift towards an increase in CD45+ CD11b+GR1+ infiltrating myeloid-derived suppressor cells (MDSCs) in experimental mice, likely a tumor-promoting population. Minimal changes were observed in the lungs and livers at any time point. Confirmatory immunofluorescence staining showed a decrease in tumor-infiltrating microglia (Iba1+) and a significant increase in tumor-infiltrating peripheral macrophages (F4/80+), mirroring the flow data. This experiment was then repeated to test the contribution of the ligand (PDGFB), where a significant increase of infiltrating microglia (Iba1+) was observed upon tumor cell knockdown of PDGFB. Together, these results suggest that the PDGFB-PDGFRβ pathway creates an immunosuppressive environment in the brain TME at least in part through reorchestration of the myeloid populations. Citation Format: Alexis A Mossing, Johnathon G Schiebel, Rebecca L Packard, Amrendra Kumar, Sarah A Steck, Nathaniel S Grabinski, Katie A Thies, Bri Wasik, Tasneem Ariswala, Gary Tozbikian, Johnathan Godbout, Steven T Sizemore, Paul R Lockman, Anna E Vilgelm, Gina M Sizemore. Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A085.

CNSC-01. THE ROLE OF SEROTONERGIC RECEPTOR, HTR2B, ON MYELOID DERIVED SUPPRESSOR CELLS IN THE BRAIN METASTATIC ENVIRONMENT

Abstract In the United States, greater than 12% of cancer patients develop brain metastases (br-mets) and median survival remains an abysmal 4-6 months. Throughout the metastatic cascade, tumor cells attract and reprogram stromal cells, such as myeloid derived suppressor cells (MDSCs), which support cancer growth by promoting tissue remodeling, EMT, invasion, immunosuppression, angiogenesis. Once tumor cells have breached the brain’s CNS defenses, infiltrating immune cells can penetrate the glia limitans and enter the CNS parenchyma to help establish the brain metastatic niche. However, there is scant understanding of MDSC’s contribution to the brain metastatic microenvironment. The brain metastatic environment is particularly unique because CNS-resident cells such as microglia, astrocytes, choroid plexus, and neurons function as local mediators to alter invading immune cell plasticity and differentiation. We have demonstrated that MDSCs in the brain microenvironment respond to the neurotransmitter serotonin, upregulate the serotonergic receptor, HTR2B, resulting in increase pNF-κB and its associated signaling genes (IL-6, IL-1B, COX2, CXCL2, NOS2). Furthermore, HTR2B knockdown in MDSCs alters downregulates expression of pNF-κB signaling genes. Additionally, MDSCs in this environment support tumor cell proliferation while hindering their acquisition of neuronal traits. A better understanding of the role of HTR2B in brain metastasis may allow us to develop new treatment strategies for patients of this devastating disease.

Immune Checkpoint Inhibitors Remodel the Immune Microenvironment in Brain Metastases and Promote T Cell Infiltration: Corrigendum.

Immune Checkpoint Inhibitors Remodel the Immune Microenvironment in Brain Metastases and Promote T Cell Infiltration: Corrigendum.

Single-cell sequencing reveals the landscape of the human brain metastatic microenvironment

Brain metastases is the most common intracranial tumor and account for approximately 20% of all systematic cancer cases. It is a leading cause of death in advanced-stage cancer, resulting in a five-year overall survival rate below 10%. Therefore, there is a critical need to identify effective biomarkers that can support frequent surveillance and promote efficient drug guidance in brain metastasis. Recently, the remarkable breakthroughs in single-cell RNA-sequencing (scRNA-seq) technology have advanced our insights into the tumor microenvironment (TME) at single-cell resolution, which offers the potential to unravel the metastasis-related cellular crosstalk and provides the potential for improving therapeutic effects mediated by multifaceted cellular interactions within TME. In this study, we have applied scRNA-seq and profiled 10,896 cells collected from five brain tumor tissue samples originating from breast and lung cancers. Our analysis reveals the presence of various intratumoral components, including tumor cells, fibroblasts, myeloid cells, stromal cells expressing neural stem cell markers, as well as minor populations of oligodendrocytes and T cells. Interestingly, distinct cellular compositions are observed across different samples, indicating the influence of diverse cellular interactions on the infiltration patterns within the TME. Importantly, we identify tumor-associated fibroblasts in both our in-house dataset and external scRNA-seq datasets. These fibroblasts exhibit high expression of type I collagen genes, dominate cell-cell interactions within the TME via the type I collagen signaling axis, and facilitate the remodeling of the TME to a collagen-I-rich extracellular matrix similar to the original TME at primary sites. Additionally, we observe M1 activation in native microglial cells and infiltrated macrophages, which may contribute to a proinflammatory TME and the upregulation of collagen type I expression in fibroblasts. Furthermore, tumor cell-specific receptors exhibit a significant association with patient survival in both brain metastasis and native glioblastoma cases. Taken together, our comprehensive analyses identify type I collagen-secreting tumor-associated fibroblasts as key mediators in metastatic brain tumors and uncover tumor receptors that are potentially associated with patient survival. These discoveries provide potential biomarkers for effective therapeutic targets and intervention strategies.

Fibroblast activation protein as a potential theranostic target in brain metastases of diverse solid tumours

Brain metastases are a very common and serious complication of oncological diseases. Despite the vast progress in multimodality treatment, brain metastases significantly decrease the quality of life and prognosis of patients. Therefore, identifying new targets in the microenvironment of brain metastases is desirable. Fibroblast activation protein (FAP) is a transmembrane serine protease typically expressed in tumour-associated stromal cells. Due to its characteristic presence in the tumour microenvironment, FAP represents an attractive theranostic target in oncology. However, there is little information on FAP expression in brain metastases. In this study, we quantified FAP expression in samples of brain metastases of various primary origin and characterised FAP-expressing cells. We have shown that FAP expression is significantly higher in brain metastases in comparison to non-tumorous brain tissues, both at the protein and enzymatic activity levels. FAP immunopositivity was localised in regions rich in collagen and containing blood vessels. We have further shown that FAP is predominantly confined to stromal cells expressing markers typical of cancer-associated fibroblasts (CAFs). We have also observed FAP immunopositivity on tumour cells in a portion of brain metastases, mainly originating from melanoma, lung, breast, and renal cancer, and sarcoma. There were no significant differences in the quantity of FAP protein, enzymatic activity, and FAP+ stromal cells among brain metastasis samples of various origins, suggesting that there is no association of FAP expression and/or presence of FAP+ stromal cells with the histological type of brain metastases. In summary, we are the first to establish the expression of FAP and characterise FAP-expressing cells in the microenvironment of brain metastases. The frequent upregulation of FAP and its presence on both stromal and tumour cells support the use of FAP as a promising theranostic target in brain metastases.

Abstract 4619: Spatial analysis of the immunosuppressive tumor microenvironment in brain cancer applying a novel multiplex immunofluorescence panel

Abstract Glioblastoma (GBM) and brain metastases (BM) account for >70% of all brain cancers. The annual incidence averages to 10-17 cases per 100.000 person (~ 3 cases for GBM and 7-14 for BM), resulting in 45.000-77.000 new cases in the European Union alone. The standard of care for GBM includes maximal surgical resection followed by radiation and chemotherapy with temozolomide, whilst whole brain irradiation or surgical removal with or without chemotherapy are used for BM. In spite of therapy, the median overall survival remains < 6 months for BM, and 16 months for GBM, suggesting brain cancers as one of the most therapy-resistant tumors. Despite the emergence of novel therapeutic strategies, such as anti-angiogenic therapy, targeting myeloid cells or checkpoint immunotherapy, no significant improvement in overall survival of brain cancer patients was observed. Several studies have suggested the resistance to therapy in brain cancers is largely attributed by the presence of an immunosuppressive brain tumor microenvironment. To investigate the spatial architecture of the tumor microenvironment in BM derived from lung and breast cancers, we employed the Lunaphore 13-plex immune-oncology (IO) Core Panel, consisting of clinically relevant IO biomarkers (CD3, CD4, CD8, CD45, FoxP3, PD1, PD-L1, CD11c, CD20, CD56, CD68, aSMA and Ki-67) for multiplex immunofluorescence (mIF) assays. These findings were then compared to isocitrate dehydrogenase-wildtype glioblastoma, which is a T-cell-poor cancer that is also characterized by an immunosuppressive tumor microenvironment. Further, the IO Core Panel was expanded with previously established antibodies targeting myeloid, blood-brain barrier and tumor cell markers in a proteomic workflow on COMET™, a fully automated, sequential immunofluorescence (seqIF™) platform. Comprehensive mapping of the GBM microenvironment and BM confirmed previous observations obtained by bulk and single cell RNA sequencing analysis that described profound differences in the tumor microenvironment of BM versus GBM. In contrast to GBM, anti-tumor immunity in BM was increased, whereas immunosuppressive cells remained unchanged between the two. Of note, T- and B-cells were not evenly distributed within the tumor tissue, providing an additional level of spatial information that would have been missed by bulk RNA or proteomic analyses. Our study is the first to demonstrate a successful application of Lunaphore’s IO core panel on GBM and BM samples. This highlights potential future use in the development of tools for personalized medicine for GBM and other tumor types. Citation Format: Yvonne Reiss, Jadranka Macas, François Rivest, Victor de Gautard, Saska Brajkovic, Bastian Nicolai, Tatjana Starzetz, Kathleen Sommer, Pinar Cakmak, Jonathan Schup, Jennifer H. Lun, Karl H. Plate. Spatial analysis of the immunosuppressive tumor microenvironment in brain cancer applying a novel multiplex immunofluorescence panel. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4619.

Abstract 6777: Digital Spatial Profiler highlights a T and B cells inflamed tumor microenvironment in brain metastases derived from melanoma vs. non-melanoma solid tumors

Abstract Introduction: Systemic immunotherapies (IO) have recently shown activity in melanoma and non-small cell lung cancer metastatic to the brain, but minimal activity in the treatment of other brain metastases. Further, only a limited percentage of melanoma or lung cancer (MBM/NSCLC)-brain metastasis (BrMs) patients respond to IO. The aim of this study is to explore the differences in the tumor microenvironment (TME) among BrMs from different tumor types including melanoma, NSCLC, breast, and renal cell carcinoma (RCC). Methods: Formalin-fixed paraffin-embedded (FFPE) tumor from 13 MBM and 43 non-MBM [18 NSCLC-BrMs, 20 breast-BrMs and 5 Renal RCC-BrMs] and primary tissue from 7 melanoma, 4 NSCLC and 3 breast cases were identified. RNA was isolated from tumor regions and subjected to whole gene expression profiling (GEP) by RNA-seq. Microenvironment Cell-Populations counter method estimated the abundance of immune and stromal infiltrated cell subpopulations. Inter- and intra-tumor heterogeneity was evaluated in selected regions of interest using a 59-plex immune related protein panel assessed by GeoMx Digital Spatial Profiling (DSP) technology. Results: Whole GEP revealed 3650 transcripts differentially expressed between MBM and non-MBM (False Discovery Rate, FDR, ≤0.01). MBM showed increased expression of genes involved in B cell function, cytolytic activity associated with CD8 and NK cells and complement signaling (C1QA/B/C). Conversely, overexpression of genes involved in epithelial signaling, cell adhesion and neurovascular coupling was observed in non MBM vs MBM. Spatial protein profiling revealed in the TME of MBM vs non-MBM, an increased expression (p <0.1, FC>1.5) of the tumor antigen MART-1, together with a higher infiltration of CD8+ cytotoxic cells (CD8+, 41BB+), antigen presenting cells (HLA-DR+, B2M+, CD40+) and cells involved in the formation of tertiary lymphoid structures (CD20+, CD11c+). Conversely, increased infiltration of Tregs (CD25+, CD127+), neutrophils (CD66b+) and epithelial cells (EpCAM+, PanCK+) was observed in non-MBM vs MBM. Interestingly, LAG3+and PD-L2+cells were also observed to be more enriched in non-MBM vs MBM. GEP in primary tumors (FDR≤0.01) showed limited immune-related signals but revealed overexpression of genes associated with NK cells functions and immune chemoattraction (CCL18/20, CXCL2/5/17) to be overexpressed in PM vs other primary tumors. Conclusion: TME interrogation at molecular and protein levels has shown that MBM are more immune infiltrated than brain metastases derived from non-melanoma solid tumors. Particularly, a cytotoxic and B cells - enriched TME was observed in MBM vs brain metastases derived from NSCLC, breast and RCC. These findings will be validated in larger cohorts and incorporated in therapeutic investigations. Citation Format: Alberto Mendoza-Valderrey, Daria M. Kessler, Ethan Dettmann, Xinmin Li, Sierra Thompson, Steven E. Kolker, Kim A. Margolin, Maria L. Ascierto. Digital Spatial Profiler highlights a T and B cells inflamed tumor microenvironment in brain metastases derived from melanoma vs. non-melanoma solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6777.

The progress of microenvironment-targeted therapies in brain metastases.

The incidence of brain metastases (BrM) has become a growing concern recently. It is a common and often fatal manifestation in the brain during the end-stage of many extracranial primary tumors. Increasing BrM diagnoses can be attributed to improvements in primary tumor treatments, which have extended patients' lifetime, and allowed for earlier and more efficient detection of brain lesions. Currently, therapies for BrM encompass systemic chemotherapy, targeted therapy, and immunotherapy. Systemic chemotherapy regimens are controversial due to their associated side effects and limited efficacy. Targeted and immunotherapies have garnered significant attention in the medical field: they target specific molecular sites and modulate specific cellular components. However, multiple difficulties such as drug resistance and low permeability of the blood-brain barrier (BBB) remain significant challenges. Thus, there is an urgent need for novel therapies. Brain microenvironments consist of cellular components including immune cells, neurons, endothelial cells as well as molecular components like metal ions, nutrient molecules. Recent research indicates that malignant tumor cells can manipulate the brain microenvironment to change the anti-tumoral to a pro-tumoral microenvironment, both before, during, and after BrM. This review compares the characteristics of the brain microenvironment in BrM with those in other sites or primary tumors. Furthermore, it evaluates the preclinical and clinical studies of microenvironment-targeted therapies for BrM. These therapies, due to their diversity, are expected to overcome drug resistance or low permeability of the BBB with low side effects and high specificity. This will ultimately lead to improved outcomes for patients with secondary brain tumors.

Analysis of PD-L1 and CD3 Expression in Glioblastoma Patients and Correlation with Outcome: A Single Center Report.

With the advent of immunotherapies, the field of cancer therapy has been revived with new hope, especially for cancers with dismal prognoses, such as the glioblastoma multiforme (GBM). Currently, immunotherapies should potentiate the host's own antitumor immune response against cancer cells, but it has been documented that they are effective only in small subsets of patients. Therefore, accurate predictors of response are urgently needed to identify who will benefit from immune-modulatory therapies. Brain tumors are challenging in terms of treatments. The immune response in the brain is highly regulated, and the immune microenvironment in brain metastases is active with a high density of tumor-infiltrating lymphocytes (TILs, CD3+ T cells) in certain patients and, therefore, may serve as a potential treatment target. In our study, we performed immunohistochemistry for CD3 and PD-L1 along the routine assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status and the IDH1 and 2 status in a single center cohort of 69 patients with GBM (58 primary tumors and 11 recurrences) who underwent standard multimodal therapies (surgery/radiotherapy/adjuvant temozolamide). We analyzed the association of PD-L1 tumor expression and TILs with overall survival (OS). The PD-L1 expression was observed in 25 of 58 (43%) newly diagnosed primary glioblastoma specimens. The sparse-to-moderate density of TILs, identified with CD3+ expression, was found in 48 of 58 (83%) specimens. Neither PD-L1 expression nor TILs were associated with overall survival. In conclusion, TILs and/or PD-L1 expression are detectable in the majority of glioblastoma samples, and even if they slightly relate to the outcome, they do not show a statistically significant correlation.

Abstract IA020: Improving treatment of primary and metastatic brain tumors: Emerging insights and novel strategies

Abstract Approximately 200,000 patients develop metastasis each year in US alone. This incidence is approximately 20 times higher than glioblastoma – a uniformly fatal disease where all Phase III immune-checkpoint trials have failed to date. Patients diagnosed with metastatic breast cancer have a median survival of two years and account for approximately 40,000 deaths annually in the US alone. About 1/3rd of women with advanced breast cancer are at risk of developing brain metastases (BMs). A notable example is human epidermal growth factor receptor 2 (HER2)-positive breast cancer, where BMs from this disease are refractory to drugs that otherwise control disease in extracranial sites, for two reasons: i) the blood-brain/blood-tumor barrier (BBB/BTB) hinders the delivery of drugs to BMs, and ii) the brain tumor microenvironment (TME) reduces the efficacy of these therapies even when they accrue in BMs (Arvanitis et al, Nature Reviews Cancer 2020). By employing clinically relevant murine models, we demonstrated that theanti-HER2 antibody trastuzumab can upregulate VEGF in the microenvironment of BM (Izumi et al, Nature 2002), and thus, adding anti-VEGF/R agents can improve the efficacy of anti-HER2 therapies (Kodack et al, PNAS 2012). We further showed that HER3 is overexpressed in BMs and confers de novo resistance to anti-HER2 therapies. Blocking HER3 enhanced the efficacy of FDA-approved targeted therapies including trastuzumab and neratinib - a finding supported by independent clinical trials (Kodack et al, Science Translational Medicine 2017). In our attempt to improve survival further, we discovered that breast cancer cells implanted in the brain exhibit increased de novo lipid synthesis compared to breast cancer cells implanted in the breast or liver. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reducesHER2+ breast tumor growth in the brain – unlike those grown in the breast or liver, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies (Ferraro et al, Nature Cancer 2021). In parallel, we have employed focused ultrasound to improve delivery of various therapeutics to BMs by transiently disrupting BBB/BTB (Arvanitis et al, PNAS 2018). Finally, by recognizing the similarities between the TME of BM and glioblastoma, we have developed 3 new strategies to reprogram the immunosuppressive microenvironment of glioblastoma to improve the efficacy of immune-check point blockers against these uniformly fatal tumors (Amoozgar et al, Nature Communications 2021, Amoozgar et al, bioarxiv 2022, Datta et al. bioarxiv 2022). These strategies are likely to also benefit patients with brain metastases that have now become a leading cause of morbidity and mortality with improved detection and extracranial treatments. Citation Format: Rakesh K. Jain. Improving treatment of primary and metastatic brain tumors: Emerging insights and novel strategies [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA020.

Mutational Analysis and Single Cell Sequencing of Melanoma Brain Metastases

<h3>Purpose/Objective(s)</h3> Melanoma brain metastases are a significant source of morbidity and mortality. While radiosurgery and (CNS) penetrant therapies have improved clinical outcomes, understanding the molecular landscape and microenvironment of brain metastases is critical to devise improved treatments for patients. Here, we perform bulk and single cell genomic analysis of melanoma brain metastases to identify molecular mechanisms underlying therapeutic response. <h3>Materials/Methods</h3> 50 consecutive patients who underwent surgical resection with histo-pathologically confirmed melanoma brain metastases and known BRAF^V600E status treated at a single institution were retrospectively identified. In 25 patients (50%) with sufficient brain metastasis tissue for targeted next generation sequencing, DNA mutations were assessed with a CLIA certified sequencing assay spanning over 500 cancer genes. Single cell RNA sequencing was performed on n=5 samples with sufficient frozen tissue. Overall survival (OS) and CNS progression free survival (CNS PFS) were estimated using the Kaplan-Meier method from time of brain metastasis diagnosis. Single cell analysis was performed using the 10x platform and Seurat R package. <h3>Results</h3> The median patient age was 64 years old (27 – 78 years), and the median clinical follow up was 17 months. 21 patients (42%) had BRAF^V600E melanoma brain metastases. BRAF^V600E mutation was associated with worse CNS PFS (p=0.04, log-rank test) and OS (p=0.03, log-rank test). Multivariate analysis incorporating age, Karnofsky performance score, and presence of extracranial disease supports <i>BRAF</i> status as an independent prognostic factor for OS (p<0.05). In patients undergoing targeted next generation sequencing, the most common pathogenic variant was <i>TERT</i> promoter mutation (n=19; 76%) followed by <i>TP53</i> mutation (n=9; 36%). The four TCGA molecular melanoma subgroups were equally represented: <i>NRAS</i> mutant (n=8; 32%), <i>NF1</i> mutant (n=6; 24%), <i>BRAF</i> mutant (n=6; 24%) or triple wildtype (n=5; 20%) Evaluation of clinical outcomes in the context of next generation sequencing results revealed no differences by <i>TERT</i> status but demonstrated worse overall survival in <i>BRAF</i> mutant tumors (median OS: 11 months; p=0.01, log-rank test). Single cell sequencing of 31,219 nuclei across 5 samples revealed distinct subpopulations between <i>BRAF</i> mutant and <i>BRAF</i> wildtype tumors in both the tumor and the brain microenvironment, with increased immune infiltration in <i>BRAF</i> wildtype tumors and greater neuronal subpopulations in <i>BRAF</i> mutant lesions. <h3>Conclusion</h3> Our data suggest <i>BRAF</i> may be useful as a prognostic biomarker and <i>TERT</i> promoter mutations as a recurrent intracranial molecular alteration in melanoma. Single cell analysis shows brain metastases exhibit distinct neuronal and immune populations. Future work will require integration of molecular status with treatment to help guide therapy and interrogation of larger, prospective, multi-institutional cohorts to validate these observations.

Tumor microenvironment and exosomes in brain metastasis: Molecular mechanisms and clinical application.

Metastasis is one of the important biological features of malignant tumors and one of the main factors responsible for poor prognosis. Although the widespread application of newer clinical technologies and their continuous development have significantly improved survival in patients with brain metastases, there is no uniform standard of care. More effective therapeutic measures are therefore needed to improve prognosis. Understanding the mechanisms of tumor cell colonization, growth, and invasion in the central nervous system is of particular importance for the prevention and treatment of brain metastases. This process can be plausibly explained by the "seed and soil" hypothesis, which essentially states that tumor cells can interact with various components of the central nervous system microenvironment to produce adaptive changes; it is this interaction that determines the development of brain metastases. As a novel form of intercellular communication, exosomes play a key role in the brain metastasis microenvironment and carry various bioactive molecules that regulate receptor cell activity. In this paper, we review the roles and prospects of brain metastatic tumor cells, the brain metastatic tumor microenvironment, and exosomes in the development and clinical management of brain metastases.

OS03.5.A Characterization of the inflammatory tumor microenvironment composition in solid cancer patients with brain metastases after progression to immune checkpoint inhibitor therapy

Abstract Background Immunotherapy (IO) has changed the treatment landscape of metastatic cancer patients, however, treatment resistance is frequent. We aimed to characterize the inflammatory tumor microenvironment in brain metastases (BM) after IO to gain a deeper understanding of immunologic escape mechanisms. Material and Methods Solid cancer patients who had BM resection after IO progression (IO cohort) were retrospectively identified. We analyzed tumor-infiltrating immune cell subsets (CD3, CD8, CD45RO, FOXP3) and expression of immune checkpoint molecules (PD-L1, PD-1, LAG-3) by immunohistochemistry. A control cohort of BM tissue samples without prior IO served for comparison (no immunotherapy cohort, NIO). Results Twenty-eight IO patients (12/28, 42.9% females; 16/28, 57.1% males; median 61 years; 14/28, 50% lung cancer; 5/28, 17.9% melanoma; 4/28, 14.3% renal cell carcinoma; 1/28, 3.6% breast cancer; 4/28, 14.3% other cancer entities) and 57 NIO patients (28/57, 49.1% females; 29/57, 50.9% males; median 58 years; 35/57, 61.4% lung cancer; 9/57, 15.8% breast cancer; 4/57, 7.0% melanoma; 3/57, 5.3% renal cell carcinoma; 6/57, 10.5% other cancer entities) were included. IO patients had a median of one (range 0-4) systemic therapy line prior to IO. Median time from last IO application until BM resection was 5.6 months (range 0.2-49.8 months). Patients received a median number of 7 (range 1-56) IO applications (14/28, 50% PD-1-targeting IO; 8/28, 28.6% PD-L1; 2/28, 7.1% CTLA4; 4/28, 14.3% CTLA4+PD-1; 3/28, 10.7% IO+chemotherapy). No statistically significant differences in the densities of investigated TILs or PD-L1 expression between the IO and the NIO cohort were observed. Patients of the IO cohort showed higher PD-L1 expression compared to the NIO cohort (57.1 vs. 42.1%, Chi-square, p&amp;gt;0.05). Overall survival (OS) was similar in both cohorts, with a median OS of 11.0 months (range 5.0-17.0) in the IO cohort and 11.0 months (range 5.5-16.5) in the NIO cohort. Conclusion Our findings show an upregulation of PD-L1 in BM occurring after prior IO therapy in the absence of other overt changes in the inflammatory microenvironment. Ongoing analyses in this cohort are investigating possible molecular driver of resistance by analyzing DNA methylation profiles of pre-and post-IO tissue samples of the IO cohort to potentially gain insights on inflammatory IO resistance mechanisms in BM patients.

Mechanical and metabolic interplay in the brain metastatic microenvironment.

In this Perspective, we provide our insights and opinions about the contribution-and potential co-regulation-of mechanics and metabolism in incurable breast cancer brain metastasis. Altered metabolic activity can affect cancer metastasis as high glucose supply and demand in the brain microenvironment favors aerobic glycolysis. Similarly, the altered mechanical properties of disseminating cancer cells facilitate migration to and metastatic seeding of the brain, where local metabolites support their progression. Cancer cells in the brain and the brain tumor microenvironment often possess opposing mechanical and metabolic properties compared to extracranial cancer cells and their microenvironment, which inhibit the ease of extravasation and metastasis of these cells outside the central nervous system. We posit that the brain provides a metabolic microenvironment that mechanically reinforces the cellular structure of cancer cells and supports their metastatic growth while restricting their spread from the brain to external organs.

Breast cancer extracellular vesicles-derived miR-1290 activates astrocytes in the brain metastatic microenvironment via the FOXA2→CNTF axis to promote progression of brain metastases

Mechanisms underlying breast cancer brain metastasis (BCBM) are still unclear. In this study, we observed that extracellular vesicles (EVs) secreted from breast cancer cells with increased expression of tGLI1, a BCBM-promoting transcription factor, strongly activated astrocytes. EV-derived microRNA/miRNA microarray revealed tGLI1-positive breast cancer cells highly secreted miR-1290 and miR-1246 encapsulated in EVs. Genetic knockin/knockout studies established a direct link between tGLI1 and both miRNAs. Datamining and analysis of patient samples revealed that BCBM patients had more circulating EV-miRs-1290/1246 than those without metastasis. Ectopic expression of miR-1290 or miR-1246 strongly activated astrocytes whereas their inhibitors abrogated the effect. Conditioned media from miR-1290- or miR-1246-overexpressing astrocytes promoted mammospheres. Furthermore, miRs-1290/1246 suppressed expression of FOXA2 transcription repressor, leading to CNTF cytokine secretion and subsequent activation of astrocytes. Finally, we conducted a mouse study to demonstrate that astrocytes overexpressing miR-1290, but not miR-1246, enhanced intracranial colonization and growth of breast cancer cells. Collectively, our findings demonstrate, for the first time, that breast cancer EV-derived miR-1290 and miR-1246 activate astrocytes in the brain metastatic microenvironment and that EV-derived miR-1290 promotes progression of brain metastases through the novel EV-miR-1290→FOXA2→CNTF signaling axis.

The microenvironment of brain metastases from solid tumors.

Brain metastasis (BrM) is an area of unmet medical need that poses unique therapeutic challenges and heralds a dismal prognosis. The intracranial tumor microenvironment (TME) presents several challenges, including the therapy-resistant blood–brain barrier, a unique immune milieu, distinct intercellular interactions, and specific metabolic conditions, that are responsible for treatment failures and poor clinical outcomes. There is a complex interplay between malignant cells that metastasize to the central nervous system (CNS) and the native TME. Cancer cells take advantage of vascular, neuronal, immune, and anatomical vulnerabilities to proliferate with mechanisms specific to the CNS. In this review, we discuss unique aspects of the TME in the context of brain metastases and pathways through which the TME may hold the key to the discovery of new and effective therapies for patients with BrM.