Lung Cancer Brain Metastases Research Articles

Single-cell RNA sequencing and spatial transcriptome reveal potential molecular mechanisms of lung cancer brain metastasis

BackgroundLung cancer is a highly aggressive and prevalent disease worldwide. By the time it is first diagnosed, distant metastases have usually already occurred. Among them, the prognosis of patients with brain metastasis from lung cancer is very poor. Therefore, it is particularly important to identify the evolutionary status of tumor cells during lung cancer brain metastases and discover the underlying mechanisms of lung cancer brain metastases. MethodsIn this study, we analysed three types of data: single-cell RNA sequencing, bulk RNA sequencing, and spatial transcriptome. Firstly, we identified early metastatic epithelial cell clusters (EMEC) using CNV and trajectory analysis in scRNA-seq data. Secondly, we integrated scRNA-seq and spatial transcriptome data with the help of MIA (Multimodal intersection analysis) to explore the biological characteristics of EMEC. Finally, we used bulk RNA-seq data to validate the molecular characteristics of EMEC. ResultA total of 55,763 single cells were obtained and divided into 9 cell types. In brain metastasis, we found a significantly higher proportion of epithelial cells. In addition, we identified a specific subpopulation of epithelial cells, which was named as “early metastatic epithelial cell clusters (EMEC)”. It is enriched in oxidative phosphorylation, coagulation, complement. Moreover, we also found that EMEC underwent cellular communication with other immune cells through ligand-receptor pairs such as MIF-(CD74 + CXCR4) and MIF-(CD74 + CD44). Next, we validated that EMEC were associated with poor clinical prognosis using three independent external datasets. Finally, spatial transcriptome analysis revealed specificity in the spatial distribution of EMEC, which shifted from the peripheral regions to the central regions of the tumour as the depth of tumor invasion progressed. ConclusionThis study reveals the potential molecular mechanisms of lung cancer brain metastasis from both single-cell and spatial transcriptomic perspectives, providing biological insights and clinical reference value for detecting patients suffering from lung cancer brain metastasis.

Hippocampal avoidance whole-brain radiotherapy with simultaneous integrated boost in lung cancer brain metastases and utility of the Hopkins verbal learning test for testing cognitive impairment in Chinese patients: a prospective phase II study

BackgroundThis study aimed to evaluate the efficiency of hippocampal avoidance whole-brain radiotherapy with a simultaneous integrated boost (HA-WBRT-SIB) treating brain metastases (BM) and utility of the Hopkins Verbal Learning Test-Revised (HVLT-R) (Chinese version) in Chinese lung cancer patients.MethodsLung cancer patients with BM undergone HA-WBRT-SIB at our center were enrolled. Brain magnetic resonance imaging, The HVLT total learning score, and side effects were evaluated before radiotherapy and 1, 3, 6, and 12 months after radiotherapy. This study analyzed the overall survival rate, progression-free survival rate, and changes in HVLT-R immediate recall scores.ResultsForty patients were enrolled between Jan 2016 and Jan 2020. The median follow-up time was 14.2 months. The median survival, progression-free survival, and intracranial progression-free survival of all patients were 14.8 months, 6.7 months and 14.8 months, respectively. Multivariate analysis indicated that male sex and newly diagnosed stage IV disease were associated with poor overall survival and progression-free survival, respectively. HVLT-R scores at baseline and 1, 3, and 6 months after radiotherapy were 21.94 ± 2.99, 20.88 ± 3.12, 20.03 ± 3.14, and 19.78 ± 2.98, respectively. The HVLT-R scores at 6 months after radiotherapy decreased by approximately 9.8% compared with those at baseline. No grade 3 toxicities occurred in the entire cohort.ConclusionsHA-WBRT-SIB is of efficiency and cognitive-conserving in treating Chinese lung cancer BM.Trial registrationThis study was retrospectively registered on ClinicalTrials.gov in 24th Feb, 2024. The ClinicalTrials.gov ID is NCT06289023.

Lung cancer brain metastasis and hemorrhagic cerebral venous thrombosis: experiences and lessons

The incidence of lung cancer brain metastasis combined with hemorrhagic cerebral venous sinus thrombosis (CVST) is very rare, and the understanding and treatment experience of this case is insufficient. We reported a case of lung cancer brain metastasis accompanied by venous sinus thrombosis, and describe the diagnosis and treatment plan for colleagues to learn from experience and lessons.

Predictive risk score for isolated brain metastasis in non-small cell lung cancer.

Brain metastasis is common with non-small cell lung cancer (NSCLC). Patients with some early-stage cancers don't benefit from routine brain imaging. Currently clinical stage alone is used to justify additional brain imaging. Other clinical and demographic characteristics may be associated with isolated brain metastasis (IBM). We aimed to define the most salient clinical features associated with synchronous IBM, hypothesizing that clinical and demographic factors could be used to determine the risk of brain metastasis. The National Cancer Database was used to identify patients with NSCLC from 2016-2020. Primary outcome was the presence of IBM relative to patients without evidence of any metastasis. Cohorts were divided into test and validation. The test cohort was used to identify risk factors for IBM using multivariable logistic regression. Using the regression, a scoring system was created to estimate the rate of synchronous IBM. The accuracy of the scoring system was evaluated with receiver operating characteristic (ROC) analysis using the validation cohort. Study population consisted of 396,113 patients: 25,907 IBM and 370,206 without metastatic disease. IBM was associated with age, clinical T stage, clinical N stage, Charlson/Deyo comorbidity score, histology, and grade. A scoring system using these factors showed excellent accuracy in the test and validation cohort in ROC analysis (0.806 and 0.805, respectively). Clinical and demographic characteristics can be used to stratify the risk of IBM among patients with NSCLC and provide an evidence-based method to identify patients who require dedicated brain imaging in the absence of other metastatic disease.

Prognostic Nomograms for Elderly Patients with Small Cell Lung Cancer Brain Metastasis: A Surveillance, Epidemiology, and End Results Population-Based Study with Temporal External Validation

This study aimed to pinpoint independent predictors influencing overall survival (OS) and cancer-specific survival (CSS) in elderly patients with small cell lung cancer (SCLC) brain metastasis (BM), and to create and validate nomograms for OS and CSS prediction. Data from elderly SCLC BM patients were extracted out of the Surveillance, Epidemiology, and End Results database, including 1200 patients identified from 2010 and 2015 who were randomly allocated into a training set and an internal validation set at a proportion of 7:3, and 666 patients diagnosed between 2018 and 2020 as a temporal external validation set. Independent predictors for OS and CSS were determined through univariate Cox analysis, least absolute shrinkage and selection operator analysis, and multivariate Cox analysis sequentially. Nomograms for OS and CSS were constructed, and validated by the internal and temporal external validation sets. Age, N stage, chemotherapy, and liver metastasis were determined as independent predictors of OS and CSS, while radiotherapy and surgery were not. Nomograms were constructed based on these independent predictors. The results of the receiver operator characteristic curves, the areas under the curve and calibration curve demonstrated that the nomograms exhibited commendable discriminative ability and calibration. Moreover, decision curve analysis, net reclassification improvement, and integrated discrimination improvement also suggested that the nomograms possessed superior clinical usefulness and predictive capability relative to the TNM system. Prognostic nomograms for elderly patients with SCLC BM have been developed, demonstrating good performance in terms of accuracy, reliability, and practicality.

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.

Single-cell transcriptome in the examination of the molecular characteristics of macrophages in lung cancer brain metastasis.

e20546 Background: Brain metastasis is one of the most common distant metastasis types in lung cancer patients. Its mechanism involves multiple factors, including cell-cell interactions, immune evasion, and the influence of the microenvironment. Single-cell RNA sequencing technology has significantly advanced our understanding of tumors at a single-cell resolution. It enables an in-depth understanding of the expression profiles of metastatic cells in brain tissue, providing potential predictive biomarkers for future monitoring, diagnosis, and effective treatment. Methods: We collected 50 samples from the GEO database, including 17 normal lung samples, 17 lung cancer samples, and 16 brain metastasis samples. We used the MNN method for batch correction and performed differential analysis using findAllMarkers and FindMarkers. Additionally, we conducted enrichment analysis using ClusterProfiler. Results: After batch correction and re-clustering of the macrophages, we identified 9 clusters. Based on their biological functions, they were classified into 5 macrophage subtypes: RTM_macro, Angio_macro, SEPP1_macro, Prolif_macro, and Mono_like. The angiogenic macrophages (Angio_macro) were most prominent in lung cancer brain metastasis samples (52%), followed by lung cancer (25%), and least present in normal samples (17%). Enrichment analysis revealed that iron death, antigen processing and presentation, and endoplasmic reticulum protein processing were more enriched in brain metastasis. This correlates with the high proportion of angiogenic macrophages present in the brain. The SEPP1_macro subtype showed high expression of SEPP1 and VCAN, genes associated with angiogenesis. Studies suggest that SEPP1 is associated with poor prognosis as it promotes neovascularization and tumor invasion. This subtype was highly expressed in brain metastasis (40%) and lung cancer (45%), but rare in normal samples (4%). In the SEPP1_macro subtype, brain metastasis samples were enriched in signaling pathways such as tumor necrosis factor, PI3K-AKT, NF-kB, and Mitogen-activated protein kinase (MAPK), focusing mainly on the interaction of cytokines and cytokine receptors. Conclusions: In lung cancer and lung cancer brain metastasis, there is a significant presence of macrophages that promote angiogenesis and subpopulations of tumor-promoting cells associated with poor prognosis. The existence of these two cell subsets may indicate their activation during tumor metastasis and their regulatory role in the formation of new blood vessels, thereby promoting tumor growth.

Current trends in lung cancer brain metastases trials.

e14006 Background: Brain metastases are the predominant central nervous system malignancy, primarily originating from lung cancer. In the era of precision medicine, enhanced control of extracranial disease is increasing the incidence of brain metastases, underscoring their status as a growing unmet need in modern Neurooncology. Methods: On December 12, 2023, we searched ClinicalTrials.gov for interventional and therapeutic clinical trials focusing on lung cancer brain metastases (LCBM). We gathered information on trial characteristics, including phase, study design, primary and secondary endpoints, status, enrollment criteria, size, duration, and results. Subsequently, the collected data was subjected to systematic analysis. Results: Among 198 identified trials, the majority were in early phases: 12% in phase I, 6% in phase I/II, and 60% in phase II. Only 27% of trials in phase II & II/III were randomized, with 92% lacking blinding. Trials were concentrated in North America (36%) and Asia/Africa/Australia (33%), predominantly targeting non-small cell lung cancer (NSCLC) (82%). Patients with leptomeningeal metastases were included in only about 10.5% of the trials. Out of 44 completed trials, only 21 resulted in published original articles. Termination occurred in 38 trials, primarily due to insufficient participant enrollment. Tyrosine kinase inhibitors emerged as promising treatments for brain parenchymal and leptomeningeal NSCLC metastases. Conclusions: This study highlights critical deficiencies in LCBM trials, ranging from inadequate trial numbers and recruitment difficulties to the overwhelming focus on NSCLC trials, insufficient randomization and blinding, and a marked scarcity of trials involving patients with leptomeningeal metastases. Addressing these issues is pivotal for advancing the understanding and treatment of LCBM.

Immunotherapy outcomes and profile in lung cancer brain metastases.

8646 Background: Brain metastasis (BM) in NSCLCs have a unique tumor micro-environment (TME) characterized by distinct immune-constituents and the presence of blood-brain barrier, differentiating them from primary NSCLC tumors (PT). Despite the high incidence of BM, earlier trials indicated that less than 15% of BM patients benefit from immune checkpoint inhibitors (ICIs). We characterized the TME of BM to potentially identify a subset of patients who may respond favorably to ICIs. Methods: 36,726 NSCLC samples were analyzed by NGS of DNA (592-gene) and RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). TMB-high was defined as >=10mt/MB, PD-L1 was tested by IHC (22c3), and TME by RNA expression (quanTIseq). Real-world overall survival was obtained from insurance claims data and calculated from treatment start on six common ICIs to last contact (IO-OS), while time-on-treatment (TOT) was from first to last of treatment. Hazard ratios (HRs) were calculated using the Cox proportional hazards model and p values (log-rank test). Fisher’s exact tests and Mann-Whiney U were used and adjusted for multiple comparisons (q<0.05). Results: Samples were comprised of 63.5%, PT 4.9% BM, and 31.6% extracranial metastases. High TMB and loss of heterozygosity (LOH) rates were higher in BM vs. PT (55% vs. 35.7%) and (28.0% vs. 14.3%), q<0.05, respectively, while PD-L1+ rate was similar (53.3% BM vs. 55.6% PT). Significant mutational differences in BM vs. PT included TP53 (77.7% vs 65.1%), KEAP1 (19.4% vs. 12.1%), and STK11 (17.5% vs. 12.0%), q<0.05. Immune deconvolution demonstrated the abundance of neutrophils, Tregs, monocytes, and B cells was higher in PT vs. BM (fold-change: 1.35-1.73, q<0.05), while dendritic cells were higher in BM vs. PT (fold-change: 13, q<0.05). BM had significantly longer IO-OS (24.4 vs. 19.7 mo: HR = 0.83, 95% CI 0.76-0.90, p<0.05) and TOT (7.7 vs. 6.0 mo; HR = 0.83, 95% CI 0.77-0.89, p<0.05) compared to PT. PD-L1+ and TMB High were associated with longer IO-OS in BM vs. PT (HR = 0.71, 95% CI 0.63-0.81, p<0.05) and (HR = 0.83, 95% CI 0.74 – 0.93, p<0.05), respectively. By histology, squamous cell carcinoma (SC) was significantly more prevalent in PT compared to BM (25.9% vs. 8.5%, q<0.05). In adenocarcinoma (AD), BM was associated with longer IO-OS (HR = 0.78, 95% CI 0.70-0.87, p<0.05) compared to PT, while in SC, the survival association was not significant. The longer survival seen in AD was still significant after multivariate analysis with TP53, STK11 and KEAP1 (HR = 0.76, 95% CI 0.68-0.86, q<0.05). In BM, PD-L1+ and TMB High were associated with longer IO-OS in AD and may be considered first line treatment in this population, if asymptomatic. Conclusions: Historically BM have been associated with poor outcomes. We demonstrate that BM has higher TMB, increased infiltration of dendritic cells, and higher LOH than PT. After stratifying patients based on PD-L1 and TMB, patients with BM adenocarcinoma histology had improved post ICI survival.

Stereotactic radiotherapy vs whole brain radiation therapy in EGFR mutated NSCLC: Results & reflections from the prematurely closed phase III HYBRID trial

BackgroundAll known randomized trials of stereotactic radiotherapy (SRT) versus whole brain radiotherapy (WBRT) for brain metastases (BMs) comprise mixed histologies. The phase III HYBRID trial (NCT02882984) attempted to evaluate the non-inferiority of SRT vs. WBRT specifically for EGFR-mutated non-small cell lung cancer (EGFRm NSCLC) BMs. MethodsInclusion criteria were ≤ 5 BMs (any size) from treatment-naïve EGFRm NSCLC. All patients started a first-generation tyrosine kinase inhibitor on the first day of WBRT (37.5 Gy/15 fractions) or SRT (25–40 Gy/5 fractions per tumor volume). The primary endpoint was 18-month intracranial progression-free survival (iPFS; intention-to-treat). ResultsThe trial commenced in June 2015 and was closed in April 2021 after screening 208 patients but enrolling 85 (n = 41 WBRT, n = 44 SRT; median follow-up 31 and 36 months, respectively). Respectively, 9.5 % vs. 10.2 % of patients experienced intracranial progression at 18 months, and the median iPFS was 21.4 vs. 22.3 months (p > 0.05 for all). The SRT arm experienced higher overall survival and cognitive preservation (p < 0.05 for all). The most notable reason for low enrollment was patients not wishing to risk neurocognitive decline from WBRT. ConclusionsAlthough this phase III trial was underpowered, there was no evidence that SRT yielded outcome detriments compared to WBRT for EGFRm NSCLC BMs. Lessons from prematurely closed trials are valuable, as they often provide important experiential perspectives for investigators designing/executing future trials. In the current era, randomized trials involving WBRT without cognitive sparing measures may be at high risk of underaccrual; trial investigators are encouraged to carefully consider our experience when attempting to design such trials. However, trials of molecular-/biologically-stratified patients are highly recommended as the notion of “individualized medicine/oncology” continues to expand.

HIF2A mediates lineage transition to aggressive phenotype of cancer-associated fibroblasts in lung cancer brain metastasis

ABSTRACT Brain metastasis is the most devasting form of lung cancer. Recent studies highlight significant differences in the tumor microenvironment (TME) between lung cancer brain metastasis (LCBM) and primary lung cancer, which contribute significantly to tumor progression and drug resistance. Cancer-associated fibroblasts (CAFs) are the major component of pro-tumor TME with high plasticity. However, the lineage composition and function of CAFs in LCBM remain elusive. By reanalyzing single-cell RNA sequencing (scRNA-seq) data (GSE131907) from lung cancer patients with different stages of metastasis comprising primary lesions and brain metastasis, we found that CAFs undergo distinctive lineage transition during LCBM under a hypoxic situation, which is directly driven by hypoxia-induced HIF-2α activation. Transited CAFs enhance angiogenesis through VEGF pathways, trigger metabolic reprogramming, and promote the growth of tumor cells. Bulk RNA sequencing data was utilized as validation cohorts. Multiplex immunohistochemistry (mIHC) assay was performed on four paired samples of brain metastasis and their primary lung cancer counterparts to validate the findings. Our study revealed a novel mechanism of lung cancer brain metastasis featuring HIF-2α-induced lineage transition and functional alteration of CAFs, which offers potential therapeutic targets.

EZH2 G553C significantly increases the risk of brain metastasis from lung cancer due to salt bridge instability

BackgroundThe incidence and mortality of lung cancer is the highest in China and the world. Brain is the most common distant metastasis site of lung cancer. Its transfer mechanism and predictive biomarkers are still unclear. EZH2 participates in the catalysis of transcriptional inhibition complex, mediates chromatin compactness, leads to the silencing of its downstream target genes, participates in the silencing of multiple tumor suppressor genes, and is related to cell proliferation, apoptosis and cycle regulation. In physiology, EZH2 has high activity in stem cells or progenitor cells, inhibits genes related to cell cycle arrest and promotes self-renewal. To detect the expression and mutation of EZH2 gene in patients with brain metastasis of lung cancer, and provide further theoretical basis for exploring the pathogenesis of brain metastasis of lung cancer and finding reliable biomarkers to predict brain metastasis of lung cancer.MethodsThis study investigated susceptible genes for brain metastasis of lung cancer. The second-generation sequencing technology was applied to screen the differential genes of paired samples (brain metastasis tissues, lung cancer tissues and adjacent tissues) of lung cancer patients with brain metastasi.ResultsIt revealed that there was a significant difference in the G553C genotype of EZH2 between lung cancer brain metastasis tissues and lung cancer tissues (p = 0.045). The risk of lung cancer brain metastasis in G allele carriers was 2.124 times higher than that in C allele carriers. Immunohistochemistry showed that compared with lung cancer patients and lung cancer patients with brain metastasis, the expression level of EZH2 in lung cancer tissues of lung cancer patients was significantly higher than that in adjacent lung tissues (p < 0.0001), and higher than that in brain metastasis tissues (p = 0.0309). RNA in situ immunohybridization showed that EZH2 mRNA expression was gradually high in lung cancer adjacent tissues, lung cancer tissues and lung cancer brain metastasis tissues.ConclusionsEZH2 G553C polymorphism contributes to the prediction of brain metastasis of lung cancer, in which G allele carriers are more prone to brain metastasis.

1415: Clinical significance of PD-L1 expression and EGFR mutation in brain metastasis of lung cancer.

1415: Clinical significance of PD-L1 expression and EGFR mutation in brain metastasis of lung cancer.

Immunotherapy combined with cranial radiotherapy for driver-negative non-small-cell lung cancer brain metastases: a retrospective study.

Background: This study assesses immune checkpoint inhibitors' efficacy for non-small-cell lung cancer (NSCLC) with brain metastases (BM) and explores the role of cranial radiation therapy (CRT) in the immunotherapy era. Methods: The retrospective analysis screened NSCLC patients with BMs from July 2018 to December 2021. Treatment involved chemotherapy combined with immune checkpoint inhibitors as the first-line, with patients divided into CRT and non-CRT groups. Overall survival (OS), progression-free survival and intracranial progression-free survival were calculated and compared. Results: Among 113 patients, 74 who received CRT had significantly better median OS (not reached vs 15.31months), particularly among those with one to three BMs. Factors correlating with better OS included CRT, PD-L1 expression and diagnosis-specific graded prognostic assessment scores. Conclusion: Integrating CRT with anti-PD-1 therapy notably enhanced long-term survival in NSCLC patients with BMs.

Anti-EGFR ScFv functionalized exosomes delivering LPCAT1 specific siRNAs for inhibition of lung cancer brain metastases.

Brain metastasis (BM) is one of the leading causes of cancer-related deaths in patients with advanced non-small cell lung cancer (NSCLC). However, limited treatments are available due to the presence of the blood-brain barrier (BBB). Upregulation of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in NSCLC has been found to promote BM. Conversely, downregulating LPCAT1 significantly suppresses the proliferation and metastasis of lung cancer cells. In this study, we firstly confirmed significant upregulation of LPCAT1 in BM sites compared to primary lung cancer by analyzing scRNA dataset. We then designed a delivery system based on a single-chain variable fragment (scFv) targeting the epidermal growth factor receptor (EGFR) and exosomes derived from HEK293T cells to enhance cell-targeting capabilities and increase permeability. Next, we loaded LPCAT1 siRNA (siLPCAT1) into these engineered exosomes (exoscFv). This novel scFv-mounted exosome successfully crossed the BBB in an animal model and delivered siLPCAT1 to the BM site. Silencing LPCAT1 efficiently arrested tumor growth and inhibited malignant progression of BM in vivo without detectable toxicity. Overall, we provided a potential platform based on exosomes for RNA interference (RNAi) therapy in lung cancer BM.

Mesothelin promotes brain metastasis of non-small cell lung cancer by activating MET

BackgroundBrain metastasis (BM) is common among cases of advanced non-small cell lung cancer (NSCLC) and is the leading cause of death for these patients. Mesothelin (MSLN), a tumor-associated antigen expressed in many solid tumors, has been reported to be involved in the progression of multiple tumors. However, its potential involvement in BM of NSCLC and the underlying mechanism remain unknown.MethodsThe expression of MSLN was validated in clinical tissue and serum samples using immunohistochemistry and enzyme-linked immunosorbent assay. The ability of NSCLC cells to penetrate the blood-brain barrier (BBB) was examined using an in vitro Transwell model and an ex vivo multi-organ microfluidic bionic chip. Immunofluorescence staining and western blotting were used to detect the disruption of tight junctions. In vivo BBB leakiness assay was performed to assess the barrier integrity. MET expression and activation was detected by western blotting. The therapeutic efficacy of drugs targeting MSLN (anetumab) and MET (crizotinib/capmatinib) on BM was evaluated in animal studies.ResultsMSLN expression was significantly elevated in both serum and tumor tissue samples from NSCLC patients with BM and correlated with a poor clinical prognosis. MSLN significantly enhanced the brain metastatic abilities of NSCLC cells, especially BBB extravasation. Mechanistically, MSLN facilitated the expression and activation of MET through the c-Jun N-terminal kinase (JNK) signaling pathway, which allowed tumor cells to disrupt tight junctions and the integrity of the BBB and thereby penetrate the barrier. Drugs targeting MSLN (anetumab) and MET (crizotinib/capmatinib) effectively blocked the development of BM and prolonged the survival of mice.ConclusionsOur results demonstrate that MSLN plays a critical role in BM of NSCLC by modulating the JNK/MET signaling network and thus, provides a potential novel therapeutic target for preventing BM in NSCLC patients.

Brain Metastasis of Non-small Cell Lung Cancer After Disease-Free Survival of 5 years: Case Series and Comprehensive Literature Review

BackgroundIn the treatment of non-small cell lung cancer (NSCLC), a disease-free survival (DFS) of 5 years is a criterion for cure. This study aimed to evaluate the characteristics and outcomes of patients with brain metastases of NSCLC after a DFS of 5 years (late recurrent brain metastasis, LRBM). MethodsWe reviewed 1,281 consecutive patients with brain metastasis of lung cancer at a single institute between November 2014 and December 2022. Relevant articles were retrieved from PubMed. Only peer-reviewed journals published in English were included. ResultsSix patients (0.47%) showed LRBM. Three were male. The median age at lung cancer diagnosis was 45 years. The histological diagnosis of all patients was adenocarcinoma. Driver gene mutations were observed in five patients. The median latency period from lung cancer treatment to the development of brain metastasis was 13 years. All patients had no metastasis to any other organs and underwent craniotomies. The median follow-up duration after craniotomy was 3.5 years. No local intracranial recurrences were observed. Three patients had distant intracranial recurrences at 7, 2, and 0.6 years after craniotomy. Five patients survived for 8, 4, 3, 2, and 0.3 years after craniotomy. One patient experienced re-recurrence in the lung 4 years after craniotomy and died 3.7 years later. In our systematic review, only six studies described LRBM of NSCLC. ConclusionsLRBM is rare in patients with NSCLC. In our institution, many of these patients harbored driver gene mutations, and achieved long-term survival with aggressive local therapy. Multicenter analysis is mandatory.

ADCY4 promotes brain metastasis in small cell lung cancer and is associated with energy metabolism

Brain metastasis (BMs) in small cell lung cancer (SCLC) has a very poor prognosis. This study combined WGCNA with the mfuzz algorithm to identify potential biomarkers in the peripheral blood of patients with BMs. By comparing the significantly differentially expressed genes present in BMs samples, we identified ADCY4 as a target for further study. Expression of ADCY4 was used to cluster mfuzz expression pattern, and 28 hub genes for functional enrichment. PPI network analysis were obtained by comparing with differentially expressed genes in BMs. GABRE, NFE4 and LMOD2 are highly expressed in patients with BMs and have a good diagnostic effect. Immunoinfiltration analysis showed that SCLC patients with BMs may be associated with memory B cells, Tregs, NK cell activation, macrophage M0 and dendritic cell activation. prophytic was used to investigate the ADCY4-mediated anti-tumor drug response. In conclusion, ADCY4 can be used as a promising candidate biomarker for predicting BMs, molecular and immune features in SCLC. PCR showed that ADCY4 expression was increased in NCI–H209 and NCI–H526 SCLC cell lines. In vitro experiments confirmed that the expression of ADCY4 was significantly decreased after anti-PD1 antibody treatment, while the expression of energy metabolism factors were significantly different. This study reveals a potential mechanism by which ADCY4 mediates poor prognosis through energy metabolism -related pathways in SCLC.

Abstract 5521: Nicotine promotes perineural brain metastasis of lung cancer by activating GABAergic neurons

Abstract Tobacco smoking significantly increases the incidence of brain metastasis. However, the pathological mechanism by which smoking promotes brain metastasis through modulating brain microenvironment is yet poorly understood. We previously showed that the incidence of brain metastasis is associated with nicotine intake. Nicotine enhanced polarization of M2 pro-tumor microglia, which promoted brain metastasis. In addition to the immune surveillance role, microglial cells also control neuronal synapse formation. Microglial cells promote neuron synapse formation in the developing brain, whereas microglia depletion suppresses synapse density. Furthermore, microglia-secreted microRNAs (miRNAs) have been shown to promote synaptic formation by targeting synapse-related genes of the DKK-Wnt family. Neurons are one of the most abundant cell types in the brain, and they are highly specialized for cell-to-cell signal activation. However, little is known about the roles of neurons in brain metastasis of lung cancer. We hypothesize that nicotine stimulates microglia to secrete exosomal miR-32-3p, which promotes brain metastasis by augmenting GABAergic synaptic formation and hence releasing GABA that serves as a metabolic substrate to fuel tumor cell growth. We also hypothesize that inhibiting the GABA transporter of tumor cells suppresses brain metastasis by blocking the GABA shunt. We found that lung cancer patients with a smoking history had significantly higher synaptic formation and the expression of GABA transporter in brain metastasis. To investigate the effect of smoking and nicotine intake on brain metastasis in vivo, mice were intracardially transplanted with lung cancer brain metastasis cells (H2030BrM and PC9BrM) followed by administration of nicotine or cigarette smoke exposure in a smoking chamber. We found that smoking and nicotine increased M2 microglia polarization, synaptic formation, and the expression of GABA in mouse brain metastasis. Furthermore, nicotine increased the secretion of miR-32-3p from nicotine-pretreated microglia, which promoted GABA release from GABAergic neurons. Treating H2030BrM and PC9BrM cells with conditioned medium (CM) obtained from neurons exposed to CM derived from nicotine-treated microglia exosomes resulted in increased tumor growth through the activation of the GABA shunt pathway. Blocking GABA transporter 1 (GAT1) by CRISPR/Cas9 or a small molecule of GAT1 inhibitor suppressed the GABAergic neuron-induced tumor progression. Our results indicate that nicotine-activated microglia enhanced GABA release from neurons followed by the promotion of GABA shunt in tumor cells and that a GAT inhibitor serves as a promising therapeutic tool for the treatment of lung cancer patients with brain metastasis. Citation Format: Shih-Ying Wu, Abhishek Tyagi, Ravindra Pramod Deshpande, Kerui Wu, Eleanor Cecile Smith, Kounosuke Watabe. Nicotine promotes perineural brain metastasis of lung cancer by activating GABAergic neurons [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 5521.

Abstract 1383: Investigation of biological role and cytokine axis of cancer-associated fibroblast (CAF) in lung cancer brain metastasis

Abstract Background: Brain metastases (BM) are among the most lethal forms of cancer globally. Lung cancer BM are frequently encountered and are associated with poor prognosis, decreased survival rates, and therapeutic resistances. Cancer-associated fibroblasts (CAFs) have emerged as important players of the brain metastases tumor microenvironment (TME). Mechanistic roles of brain metastases CAFs (hBM-CAFs) in tumorigenesis and therapeutic resistances remain unclear. Material and Methods: In the present study, the patient's BM surgical sample was subjected to primary culture to isolate CAF. The hBM-CAF induced EMT and stemness phenotype was investigated by western blot. Biological role of hBM-CAF in tumorigenesis was evaluated using indirect co-culture method in vitro. FACS analysis was performed to investigate the changes in cisplatin cytotoxicity and radiation susceptibility of lung cancer cell lines when co-cultured with hBM-CAF. CX3CL1 and IL26 expression was confirmed by ELISA along with transcriptomic analysis of hBM-CAF. Molecular events were validated by using neutralizing CX3CL1 and IL26. The tumorigenicity and response to chemotoxicity of lung cancer cells were examined by co-injecting them with hBM-CAF in a mouse xenograft model. Results: Our experiments demonstrated that hBM-CAFs significantly enhanced EMT and stemness phenotype by upregulating transcription factor SNAIL1, TWIST1, Slug and CD133, CD44 respectively in lung cancer cells. We also observed that hBM-CAFs decreased cisplatin cytotoxicity and enhanced radiation resistance, as measured by FACS analysis of apoptotic cells. In vivo experiments using xenograft mouse models revealed that co-injection of lung cancer cells with hBM-CAFs led to faster tumor growth and increased cisplatin resistance. Transcriptomic analysis of hBM-CAFs revealed a distinct genomic signature compared to normal fibroblast (hNFs), with significant overexpression of IL26 and CX3CL1 genes in the hBM-CAFs secretome, as validated by ELISA. Moreover, we found that lung cancer cells expressed high levels of the IL26 receptor IL10Rβ and ILR20α, and CX3CL1 receptor CX3CR1. Our hBM-CAF significantly upregulated JAK-STAT3 and Akt-mTOR pathway. Eventually, we have confirmed that neutralizing IL26 and CX3CL1 inhibit phosphorylation of JAK-STAT3 and Akt-mTOR respectively and revoked the IL26-CX3CL1 induced EMT and stemness phenotype of lung cancer cells. Conclusion: Patient derived hBM-CAF promote aggressive phenotype of lung cancer cells by upregulating EMT and stemness transcriptome factors. Overexpression of IL26 and CX3CL1 cytokine led to the therapeutic resistance through JAK-STAT3 and Akt-mTOR signaling pathway respectively. Targeting IL26-CX3CL1 might be a novel therapeutic efficacy for lung cancer brain metastasis patients. Citation Format: Nah Ihm Kim, SM Abdus Salam, Eshrat Jahan, Eun-Jeong Ahn, Tae-Young Jung, Jae-Hyuk Lee, Joon Haeng Rhee, Sung Sun Kim, Kyung-Hwa Lee, Kyung-Sub Moon. Investigation of biological role and cytokine axis of cancer-associated fibroblast (CAF) in lung cancer brain metastasis [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 1383.