The incidence of brain metastasis (BM) is rising. Improved therapeutic methods to control the primary tumors have led to longer survivals, but have also increased the incidence of late metastases to brain (1, 2). Despite the development of novel targeted therapies, BM remains a devastating complication of cancer. BM patients exhibit poor prognosis due to no effective long-term therapy (3, 4). BM from lung and breast cancer exhibits distinct patterns in the disease course. In lung cancer, BM typically develops within a few months after diagnosis. On the other hand, in breast cancer, metastasis often occurs in one organ before it spreads to other organs, with brain metastasis typically being a late event in the course of disease (5). The median overall survival of the patients diagnosed with BM is approximately 4-15 months in lung cancer and 6-18 months in breast cancer following treatment (2, 6). Due to the limited effectiveness of current therapies and the poor prognosis associated with BM (7–9), it is crucial to identify related genes and molecular mechanisms involved. Understanding these mechanisms can aid in the identification of new therapeutic targets and potentially improve patient survival. The signaling pathways and gene expression profiles in metastatic cancer cells play a significant role in in the formation of organ-specific metastasis. Specific gene expression patterns may dictate tendency for specific targeting specific sites, such as the brain. The unique physiological barriers in the brain, including the blood-brain-barrier (BBB), is hypothesized to limit the ability of metastatic tumor cells to infiltrate the brain (10). Site-specific metastasis may also reflect tissue-specific migration and proliferation signatures that contribute to the differential ability of tumor cells to metastasize and proliferate in specific organs (11, 12). Compared to other sites of cancer metastasis, the normal brain tissue has relatively low collagen content, lower oxygen tension, and high glucose metabolism, which may affect the survival and proliferation of disseminated tumor cells in the brain (13, 14). So far, the molecular signatures and expression patterns involved in brain metastasis remain largely unknown. Employing matched-paired samples from both primary tumors and brain metastases constitutes a distinctive and precise research approach, enabling a thorough investigation of the association between gene expression signatures and metastatic development. Despite its potential, few studies have utilized this method, underscoring the uniqueness of the presented research. Primary lung cancer and their matched-paired were used in previous studies to generate gene expression signature (15). Similarly, 16 primary breast cancers and their matched-paired brain metastases were used to generate gene expression profiles in order to assess clinic-pathological markers, immune-related gene signatures, and identifying therapeutic targets for brain metastases (16). In addition, 21 breast cancer and their matched-paired samples were used previously to perform TrueSeq RNA-sequencing and determine clinically actionable BM target genes (17).

The aim of the present study was to investigate the mechanisms operative in the primary tumors and their matched-paired brain metastasis. The utilization of matched-paired samples provides a unique opportunity to investigate shared molecular characteristics and distinctions, advancing our understanding and potential treatments for malignancies. We compared gene expression profiles of primary tumors with their matched-paired BM. Subsequently, we thoroughly examined the upregulated genes in BM of both tumor types to identify shared and distinctive predominant molecular features in BM. We validated the identified druggable targets by using IHC in the matched-paired samples. The collective findings from our research have the potential to enhance the treatment strategies for patients with BM.

Studying brain metastasis is important for the development of effective treatment options and ultimately improving patient outcomes (21). In the present study, we compared the primary tumors of breast and LUAD to their matched-paired brain metastases. We identified that cancer-related genes were upregulated in the primary tumors compared to their matched-paired brain metastases. In the brain metastases we found common upregulated genes suggestive of the presence of shared molecular mechanisms. In addition, we identified distinct differences of gene expression in BM-BC and BM-LUAD, signifying the utilization of unique mechanisms by different tumor types in brain metastasis. This finding highlights the heterogeneity that exists among brain metastases, revealing a complex interplay between common and unique molecular pathways in this context. Moreover, we found that the upregulated differentially expressed genes in BM-BC and BM-LUAD were associated with the metabolic stress pathway in brain metastasis, with a particular involvement of the glycolysis pathway. Interestingly, the majority of these genes exhibited higher upregulation in BM-BC compared to BM-LUAD, indicating higher glycolysis and glucose uptake in BM-BC. Limited studies have also provided evidence supporting the role of metabolic pathway in cancer metastasis. Dupuy et al. (22) showed that breast cancer cells with high metastatic potential undergo metabolic alterations leading to increased glycolysis and decreased mitochondrial metabolism. Parida et al. (23) showed that variations in metabolic pathways among brain-tropic cells influence their ability to adapt to the brain microenvironment and support tumor growth, highlighting the importance of understanding metabolic factors in brain metastasis. Tchou et al. (24) demonstrated that triple negative breast cancers (TNBCs) with high glycolytic rates exhibit an elevated proliferation index, indicating the association between glycolysis and tumor cell proliferation in this breast cancer subtype. These findings demonstrate the potential significance of metabolic pathway in promoting the survival and proliferation of metastatic cells within the brain microenvironment. However, it is imperative to validate these observations through functional studies, including experiments on glycolysis-related genes’ role in tumor metastasis, such as transwell or wound healing assays, in future research. Given our discovery of elevated glycolysis in BM-BC and the established literature on the impact of metabolic pathways on cancer progression, we hypnotize that the increased glycolysis in BM-BC may be influenced by dietary factors, particularly carbohydrate intake in daily food consumption. A comprehensive investigation into dietary habits and sugar intake patterns could offer valuable insights into the regulation of glycolysis in the context of BM-BC.

Furthermore, we found that the upregulated genes in BM-BC and BM-LUAD were involved in immune response regulation, tumor growth, and proliferation. Limited studies have utilized matched-paired samples to investigate immune related signatures. A study by Iwamoto et al. (16) involved gene expression analyses on 16 matched-paired samples between primary breast cancers and brain metastases. The results aimed to confirm previously reported genes associated with brain metastases and epithelial-mesenchymal transition (EMT) and identify novel therapeutic targets among FDA-approved agents or those investigated in clinical trials for distinct cancers. Their results revealed that immune-related signatures exhibited significantly lower gene expression in brain metastases than in primary breast cancers. Similar to our approach, Tsakonas et al. (2) employed immune-gene expression profiling of 13 primary lung samples and their matched-paired BM. However, detailed information on the specific subtype of the matched-paired samples is not available. Their results revealed significant downregulated of 12 immune-related genes in BM compared to primary tumors. Importantly, we found an FDA-approved druggable target (HDAC3, Figures 5B, C ) that was differentially expressed in LUAD and was upregulated in BM-LUAD compared to primary LUAD. This finding suggests the potential for targeted therapy in BM-LUAD. In our previous study we discovered higher infiltration of immune cells in BM-LUAD as compared to BM-BC (19). Interestingly, despite the higher immune response regulation in BM-BC, we did not find significant immune infiltration between the primary tumors and their matched-paired brain metastases. This could be attributed to factors such as the poor blood-brain barrier (BBB) permeability, differences in genetic characteristics, or insufficient stimulation to facilitate additional infiltration. However, we discovered four immune checkpoint molecules that exhibited differential expression between BM-BC and BM-LUAD, suggesting their potential for developing lineage-specific therapeutic strategies for brain metastasis. Importantly, in line with our previous study, we found higher expression of VISTA and IDO1 in primary LUAD and BM-LUAD as compared to breast cancer, with a predominant higher expression in primary tumors. VISTA is an immune regulatory receptor expressed mainly by myeloid cells and is associated with poor overall survival in various cancers (25, 26). We previously found that VISTA is expressed on tumor cells, T lymphocytes, and to a lesser extent on microglia (19). The results highlight potential for targeted therapy strategies directed against VISTA in the treatment of brain metastasis originating from LUAD.

IDO1 is an immune response regulator involved in the suppression of the anti-tumor immune response and immune evasion. IDO1 is highly expressed in various malignant tumors including lung cancer, and its overexpression is linked to unfavorable clinical outcomes (27–30). Although we found higher expression of IDO1 in BM-LUAD, its level was relatively low as compared to VISTA. Nevertheless, the predominant expression of IDO1 in BM-LUAD should be taken into account regarding the development of combination therapies targeting IDO1. Similarly, the higher expression of IDO1 in 13 primary lung cancer and their matched-paired samples we discovered previously (2), confirming the results of our study. We also found higher expression of NT5E (CD73) in BM-LUAD than in BM-BC. CD73 is a transmembrane glycoprotein and exerts diverse functions in the tumor microenvironment (31). CD73 catalyzes the hydrolysis of adenosine, which contributes to cancer progression, neovascularization, tumor cell proliferation, and immune evasion (32). Moreover, CD73 has been found to be involved in invasion and metastasis across different cancers, such as ovarian and cervical carcinomas (32). Dual blockade of CD73-TGFβ has led to promote a complex inflammatory tumor microenvironment, characterized by decreased levels of myeloid-derived suppressor cells (MDSCs) and M2-macrophages, as well as increased levels of activated dendritic cells, cytotoxic T cells, and B cells (33). The prominent expression of CD73 in BM-LUAD, not in BM-BC, prompts this molecule as another target for the treatment of BM-LUAD. VTCN1, also known as B7-H4, is an immune regulatory molecule belonging to the B7 family (34). VTCN1 is mainly expressed by antigen-presenting cells (APCs) and induces immune suppression by inhibiting the expansion of neutrophil progenitors and T cells (35–37). Overexpression of VTCN1 in tumors correlates with their clinicopathological features, and promotes tumor proliferation, metastasis and immune evasion (38–43). We found higher expression of VTCN1 in BM-BCthan in their primary tumors, and also in BM-LUAD. The significant expression of VTCN1 in BM-BC should be considered as another potential target for inclusion in the development of targeted immunotherapy, especially in targeting BM-BC. In line with the previous article (44), we hypothesize that the immune system may play a role in the development of brain metastasis in breast and lung cancer. This particular immune system involvement requires additional validation, particularly in the case of lung adenocarcinoma.

There are some limitations in our study. The cohort size we analyzed was relatively small, however, it is important to highlight the exceptional uniqueness of the samples we had the opportunity to analyze. Utilizing matched-paired primary tumor and brain metastasis samples is an important and unique approach in cancer research, allowing for direct comparisons of molecular changes between the primary tumor and its corresponding brain metastasis. This not only unravels the specific adaptations that enable tumor cells to invade the brain but also sheds light on the mechanisms governing the selection and survival of metastatic cells in this unique microenvironment. Moreover, a control group consisting of tumors that did not metastasize to the brain was lacking for direct comparisons, however, our study provides valuable insights into various intriguing aspects of brain metastasis.

In conclusion, the comparison of molecular mechanism in primary breast and LUAD with their matched-paired brain metastases, provide valuable insights into the molecular landscape of brain metastasis. The findings of this study highlight the differential gene expression patterns, the significance of metabolic pathways specially the involvement of glycolysis pathway, and the impact of immune response modulation in brain metastasis. In addition, the identification of the FDA-approved druggable target (HDAC3) and the four immune checkpoint molecules in BM suggest that targeted therapies should be tailored for specific tumor lineages in order to effectively manage brain metastasis.

The data presented in the study are deposited in the https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE248830 repository, accession number GSE248830.

This study was approved by the Medical Ethics Committee of the Erasmus Medical Center, Rotterdam, the Netherlands and was carried out in adherence to the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands (MEC 02·953 & MEC-2020–0732). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

SN: Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft. WK: Formal analysis, Writing – review & editing. JK: Conceptualization, Investigation, Project administration, Resources, Supervision, Writing – review & editing. DM: Conceptualization, Investigation, Project administration, Resources, Supervision, Writing – review & editing.