Infiltration In Breast Cancer Research Articles

Abstract PO1-25-01: LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes

Abstract Background: Breast cancer (BC) is a leading cause of cancer death and despite numerous treatment advancements, metastatic BC is incurable. Extracellular vesicles (EVs) are lipid-delimited particles released from cells which contain bioactive protein and nucleic acid cargo. Numerous studies have shown their importance in facilitating communication between cancer cells and the tumour microenvironment, implicating them as mediators of many aspects of carcinogenesis. EV-mediated communication between BC and the innate immune system plays a crucial role in BC progression, metastasis and the maintenance of an immunosuppressive microenvironment. LMTK3 overexpression in BC promotes therapy resistance, disease progression and invasion and metastasis, leading to a poorer prognosis in patients. Analysis of deconvoluted TCGA data indicated that LMTK3 overexpression also correlates with exclusion of M1 macrophages from the tumour microenvironment. This study aimed to explore the role of LMTK3 in EV-mediated communication between BC and immune cells. Methods EVs were collected from conditioned media of wild-type and LMTK3-overexpressing BC cells through differential ultracentrifugation, then characterised by transmission electron microscopy, nanoparticle tracking analysis, quantitative mass spectrometry and Western blotting. Monocytes were treated with EVs and proteomic and phosphoproteomic changes were analysed. 3D coculture models of BC and immune cells were used to assess monocyte and macrophage infiltration, polarisation and anti-cancer activity. Immunofluorescence and flow cytometry was used to assess macrophage polarisation. Bioinformatic analysis of TCGA datasets validated the findings in a clinical setting and the data was also validated in vivo, using syngeneic immunocompetent mouse models. Results LMTK3 overexpression in BC cells promotes the upregulation of a subpopulation of large (80-150nm) CD81+ EVs which are significantly enriched in oncogenic and immunoregulatory proteins, including LDHB, ARHGEF15, PSAT1 and HIC2. EVs from LMTK3-overexpressing but not control BC cells strongly reduce the ability of monocytes to infiltrate BC tumour spheroids and migrate through Matrigel. Proteomics analysis of the monocytes showed an enrichment in M2 polarisation proteins and a downregulation of proteins essential for migration, including S100A8, KLF13 and TRPC3. LMTK3 overexpression in BC also promotes M2 polarisation and exclusion of M1 macrophages in vivo. Summary LMTK3 overexpression in BC correlates with poor prognosis and reduced overall survival. We hypothesise that this is, in part, due to monocyte M2 polarisation and exclusion of M1 macrophages from the TME, mediated by LMTK3-dependent alterations in EV cargo, contributing to tumour immune evasion. Citation Format: Mark Samuels, William Jones, Charlotte Turner, Georgios Giamas. LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes [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 PO1-25-01.

Abstract PO3-25-01: CTLA4 expression profiles and their association with clinical outcomes of breast cancer

Abstract The cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is involved in the progression of various cancers, but its biological roles in breast cancer (BRCA) remain unclear. Therefore, we performed a systematic multiomic analysis to expound on the prognostic value and underlying mechanism of CTLA4 in BRCA. First, the expression of CTLA4 in pan-cancer was evaluated using the platforms of Oncomine. Afterward, the web-based bioinformatics platforms of GEPIA and UCLAN were used to compare the expression of CTLA4 in breast cancer and normal tissues. Next, we assessed the relationship between CTLA4 mRNA expression and prognosis in breast cancer patients using the PrognoScan Database, Kaplan-Meier Plotter, and R2: Kaplan-Meier Scanner. Additionally, the correlation between CTLA4 expression and immune cell infiltration in breast cancer and functional enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed to elucidate the potential biological functions of CTLA4. Finally, we used the STRINGdb and cytoHubba plugin Cytoscape to create a PPI network and identify known and predicted structural essentials for CTLA4 functions and the top five hub genes. CTLA4 was highly expressed in BRCA tumor tissue compared to normal tissue (p< 0.01). The CTLA4 mRNA levels in BRCA based on breast cancer subtypes of luminal, Human epidermal growth factor receptor 2 (HER-2), and Triple-negative breast cancer (TNBC) were considerably higher than in normal tissues (p< 0.001). However, the overexpression of CTLA4 was associated with a better prognosis in BRCA (P< 0.001), and was correlated with clinicopathological characteristics including age, T-stage, estrogen receptors (ER), progesterone receptors (PR), and PAM50 (P< 0.01). The infiltration of multiple immune cells was associated with increased CTLA4 expression in BRCA (P< 0.001). CTLA4 was highly enriched in antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. This study provides suggestive evidence of the prognostic role of CTLA4 in breast cancer, which may be a therapeutic target for breast cancer. Furthermore, CTLA4 may influence breast cancer prognosis through antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. These findings help us understand how CTLA4 plays a role in breast cancer and set the stage for more research. Citation Format: Woo Chul Noh, Young Bum Yoo, Kyoung Sik Park, Sang Eun Nam, Seung Hwan Lim. CTLA4 expression profiles and their association with clinical outcomes of breast cancer [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 PO3-25-01.

IFI30 as a key regulator of PDL1 immunotherapy prognosis in breast cancer

BackgroundIFI30 is a lysosomal thiol reductase involved in antigen presentation and immune regulation in various cancers, including breast cancer. Despite its known involvement, the precise mechanism, function, and relationship with the PD-L1 axis and immune response remain unclear. MethodsWe conducted an extensive investigation into IFI30 mRNA expression in breast cancer utilizing data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Furthermore, we characterized IFI30 mRNA expression across various cell types using publicly available single-cell RNA sequencing datasets, and assessed protein expression through immunohistochemistry using an in-house breast cancer tissue microarray. Functional experiments were performed to elucidate the effects of IFI30 overexpression on PD-L1 expression and inhibitory efficacy in both macrophages and breast tumor cells. ResultsOur study unveiled a marked upregulation of IFI30 expression in breast cancer tissues compared to their normal counterparts, with notable associations identified with tumor stage and prognosis. Additionally, IFI30 expression demonstrated significant correlations with various immune-related signaling pathways, encompassing peptide antigen binding, cytokine binding, and MHC class II presentation. Notably, breast cancer samples exhibiting high IFI30 expression in tumor cells displayed high PD-L1 expression on corresponding cells, alongside a diminished ratio of CD8 + T cell infiltration within the tumor microenvironment. Furthermore, ectopic knockdown of IFI30 in both tumor cells and macrophages resulted in a reduction of PD-L1 expression, while conversely, overexpression of IFI30 led to an increase in PD-L1 expression. ConclusionsThis study offers new insights into the involvement of IFI30 in breast cancer, elucidating its interplay with the PD-L1 axis and immune response dynamics. Our findings suggest that modulation of the IFI30-PD-L1 axis could serve as a promising strategy for regulating T cells infiltration in breast cancer thus treating breast cancer.

Integrative analysis based on the cell cycle-related genes identifies TPX2 as a novel prognostic biomarker associated with tumor immunity in breast cancer.

This study aims to identify the essential cell cycle-related genes associated with prognosis in breast cancer (BRCA), and to verify the relationship between the central gene and immune infiltration, so as to provide detailed and comprehensive information for the treatment of BRCA. Gene expression profiles (GSE10780, GSE21422, GSE61304) and the Cancer Genome Atlas (TCGA) BRCA data were used to identify differentially expressed genes (DEGs) and further functional enrichment analysis. STRING and Cytoscape were employed for the protein-protein interaction (PPI) network construction. TPX2 was viewed as the crucial prognostic gene by the Survival and Cox analysis. Furthermore, the connection between TPX2 expression and immune infiltrating cells and immune checkpoints in BRCA was also performed by the TIMER online database and R software. A total of 18 cell cycle-related DEGs were identified in this study. Subsequently, an intersection analysis based on TCGA-BRCA prognostic genes and the above DEGs identified three genes (TPX2, UBE2C, CCNE2) as crucial prognostic candidate biomarkers. Moreover, we also demonstrated that TPX2 is closely associated with immune infiltration in BRCA and a positive relation between TPX2 and PD-L1 expression was firstly detected. These results revealed that TPX2 is a potential prognostic biomarker and closely correlated with immune infiltration in BRCA, which could provide powerful and efficient strategies for breast cancer immunotherapy.

Spatial and single-cell explorations uncover prognostic significance and immunological functions of mitochondrial calcium uniporter in breast cancer

The mitochondrial calcium uniporter (MCU) is a transmembrane protein facilitating the entry of calcium ions into mitochondria from the cell cytosol. Maintaining calcium balance is crucial for enhancing cellular energy supply and regulating cell death. The interplay of calcium balance through MCU and the sodium-calcium exchanger is known, but its regulation in the breast cancer tumor microenvironment remains elusive. Further investigations are warranted to explore MCU’s potential in BRCA clinical pathology, tumor immune microenvironment, and precision oncology. Our study, employing a multi-omics approach, identifies MCU as an independent diagnostic biomarker for breast cancer (BRCA), correlated with advanced clinical status and poor overall survival. Utilizing public datasets from GEO and TCGA, we discern differentially expressed genes in BRCA and examine their associations with immune gene expression, overall survival, tumor stage, gene mutation status, and infiltrating immune cells. Spatial transcriptomics is employed to investigate MCU gene expression in various regions of BRCA, while spatial transcriptomics and single-cell RNA-sequencing methods explore the correlation between MCUs and immune cells. Our findings are validated through the analysis of 59 BRCA patient samples, utilizing immunohistochemistry and bioinformatics to examine the relationship between MCU expression, clinicopathological features, and prognosis. The study uncovers the expression of key gene regulators in BRCA associated with genetic variations, deletions, and the tumor microenvironment. Mutations in these regulators positively correlate with different immune cells in six immune datasets, playing a pivotal role in immune cell infiltration in BRCA. Notably, high MCU performance is linked to CD8 + T cells infiltration in BRCA. Furthermore, pharmacogenomic analysis of BRCA cell lines indicates that MCU inactivation is associated with increased sensitivity to specific small molecule drugs. Our findings suggest that MCU alterations may be linked to BRCA progression, unveiling new diagnostic and prognostic implications for MCU in BRCA. The study underscores MCU's role in the tumor immune microenvironment and cell cycle progression, positioning it as a potential tool for BRCA precision medicine and drug screening.

Abstract 2568: High infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to treatments or survival in estrogen receptor-positive breast cancer

Abstract Introduction: Psychological stress aggravates cancer cell biology which leads to cancer progression and metastasis through its influence on the sympathetic nervous system. Recently, infiltration of a local anesthetic lidocaine around a tumor prior to its removal was reported to lead to better survival in early-stage breast cancer (BC). Therefore, we hypothesized that neuron infiltration within the bulk tumor of BC could be associated with cancer aggressiveness and worse survival. Methods: A total of 5755 BC patients from five independent cohorts, including METABRIC (n=1903), SCAN-B (n=3273), GSE25066 (n=508), and ISPY2 (GSE173839; n=105), which have both clinical and transcriptomic data, were included in the study. The fraction of neuron infiltration was calculated using the xCell algorithm. Results: High neuron infiltration in BC was not associated with survival in either the METABRIC or SCAN-B cohorts. BC with high neuron infiltration had high expressions of neurotransmitter receptors: adrenergic receptor α1B and 2A, dopamine receptor D2, muscarinic acetylcholine receptor M3 and M4, and TAC1, which encodes TACR1, the receptor for substance P, but had lower expression of adrenergic receptor β2 and cholinergic acetylcholine receptor 1 and 10. Further, levels of neuron infiltration were highest in estrogen receptor-positive/HER2-negative (ER+/HER2-) among the subtypes and lower in tumors with lymph node metastasis, but was not associated with tumor size. Neuron infiltration was associated with higher AJCC staging in METABRIC, which was not validated in SCAN-B cohort. High neuron infiltration in BC was associated with reduced homologous recombination defects, mutation rate, and SNV neoantigen, and with less cell proliferation, evidenced by lower Nottingham histological grade and Ki67 gene expression, as well as less enrichment of Hallmark cell proliferation-related gene sets: E2F targets, G2M checkpoint, and Mitotic spindle. Despite its association with less cell proliferation, BC with high neuron infiltration was not associated with pathological complete response (pCR) in the GSE25066 cohort. High neuron infiltration in BC was associated with higher infiltration of lymph endovascular cells as well as less immune cell infiltration; CD8, CD4 memory, T-helper 1 and 2, dendritic cells, M1 and M2 macrophages, regulatory T cells, and B cells, lower cytolytic activity, and immune response; interferon-alpha and interferon-gamma and IL2 stat signaling. Despite this association, BC with high neuron infiltration was not associated with pCR after immunotherapy in the ISPY2 clinical trial cohort. Conclusion: BC with high infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to systemic therapies or patient survival in ER+/HER2- subtype. Citation Format: Kohei Chida, Masanori Oshi, Arya Mariam Roy, Jun Arima, Pia Sharma, Itaru Endo, Kenichi Hakamada, Kazuaki Takabe. High infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to treatments or survival in estrogen receptor-positive breast cancer [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 2568.

Exploration of a noninvasive radiomics classifier for breast cancer tumor microenvironment categorization and prognostic outcome prediction

Rationale and Objectives: Breast cancer progression and treatment response are significantly influenced by the tumor microenvironment (TME). Traditional methods for assessing TME are invasive, posing a challenge for patient care. This study introduces a non-invasive approach to TME classification by integrating radiomics and machine learning, aiming to predict the TME status using imaging data, thereby aiding in prognostic outcome prediction. Materials and MethodsUtilizing multi-omics data from The Cancer Genome Atlas (TCGA) and The Cancer Imaging Archive (TCIA), this study employed CIBERSORT and MCP-counter algorithms analyze immune infiltration in breast cancer. A radiomics classifier was developed using a random forest algorithm, leveraging quantitative features extracted from intratumoral and peritumoral regions of Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI) scans. The classifer’s ability to predict diverse TME states were and their prognostic implications were evaluated using Kaplan-Meier survival curves. ResultsThree distinct TME states were identified using RNA-Seq data, each displaying unique prognostic and biological characteristics. Notably, patients with increased immune cell infiltration showed significantly improved prognoses (P < 0.05). The classifier, comprising 24 radiomic features, demonstrated high predictive accuracy (AUC of training set = 0.960, 95 % CI: 0.922, 0.997; AUC of testing set = 0.853, 95 % CI: 0.687, 1.000) in differentiating these TME states. Predictions from the classifier also correlated significantly with overall patient survival (P < 0.05). ConclusionThis study offers a detailed analysis of the complex TME states in breast cancer and presents a reliable, noninvasive radiomics classifier for TME assessment. The classifer’s accurate prediction of TME status and its correlation with prognosis highlight its potential as a tool in personalized breast cancer treatment, paving the way for more individualized and less invasive therapeutic strategies.

Low expression of KLRB1 predicts poor survival outcomes and is associated with immune infiltration in breast cancer.

KLRB1 is downregulated in various cancer types. Nevertheless, the specific involvement of KLRB1 in the context of breast cancer (BRCA) has not been fully elucidated. This research aimed to explore its clinical value in BRCA. A dataset comprising 1,109 BRCA samples and 113 healthy samples was retrieved from The Cancer Genome Atlas (TCGA) database to establish the association between KLRB1 expression and pan-cancer. Subsequently, an analysis was executed to explore the link between KLRB1 and BRCA. T-tests and Chi-squared tests were conducted to assess the expression of KLRB1 and its clinical implications in BRCA. The prognosis-predictive value of KLRB1 in BRCA was assessed using the Kaplan-Meier method and Cox regression analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses screened biological pathways to analyze the association between the immune infiltration level and KLRB1 expression in BRCA. Lastly, the conclusion was validated through quantitative polymerase chain reaction (qPCR), immunohistochemistry (IHC), and Cell Counting Kit-8 (CCK8) assays. KLRB1 exhibited low expression in patients with BRCA. Furthermore, KLRB1 demonstrated strong diagnostic potential, as indicated by an area under curve (AUC) of 0.712. Kaplan-Meier survival and Cox regression analyses indicated that attenuated expression of KLRB1 was independently linked to unfavorable clinical outcomes. GO and KEGG enrichment analyses were performed on the top 10 genes that exhibited positive and negative correlations with KLRB1. Analysis of genes positively correlated with KLRB1 revealed associations with signaling receptor activator activity, lymphocyte proliferation, mononuclear cell proliferation, leukocyte proliferation, receptor-ligand activity, immunoglobulin binding, and hematopoietic cell lineage signaling pathway. KLRB1 expression exhibited significant correlations with all immune cells. Furthermore, qPCR and IHC outcomes demonstrated that KLRB1 was significantly downregulated in BRCA tissues. CCK8 findings showed a decrease in the proliferation of BRCA MCF7 cells upon knockout of KLRB1. This research investigated the mechanism and potential therapeutic target of the KLRB1 gene in BRCA. By analyzing the expression and function of the KLRB1 gene, the study aims to find its significant role in the onset and progression of BRCA. This research endeavors to offer novel strategies and approaches for treating BRCA.

Expression and Clinical Significance of NUDCD1, PI3K/AKT/mTOR Signaling Pathway-Related Molecules and Immune Infiltration in Breast Cancer

BackgroundNUDCD1 (NudC Domain Containing 1) performs an essential function in biological processes such as cell progression, migration, cell cycle, and intracellular material transport. Many solid tumors express it highly, which is a prospective biomarker and therapeutic approach. However, the expression and clinical importance of NUDCD1 across breast cancer is unclear. MethodsThe expressions of NUDCD1 in breast cancers and normal breast tissues were studied utilizing the TIMER database and immunohistochemical analysis. Subsequently, we validate the association between the expression of NUDCD1 and clinicopathologic features and prognosis of breast cancer. The immunohistochemical experiments of pathway-related molecules were done on 214 breast cancer tissue microarrays. The investigation of correlation between NUDCD1 expression and tumor immune infiltration was subsequently conducted. ResultsThrough the utilization of bioinformatics analysis and immunohistochemical experiments, it was determined that NUDCD1 exhibited upregulation within breast cancer. Furthermore, it was discovered that an elevated expression of NUDCD1 may potentially be linked to a worse prognosis in breast cancer. Our study reveals that the PI3K/AKT/mTOR signaling pathway may perform a function in NUDCD1 regulating breast cancer progression via enrichment analysis. Furthermore, the expression of NUDCD1 may be associated with the degree of immunological infiltration. ConclusionThe expression of NUDCD1 was explored to be elevated in breast cancer and was observed to be correlated with a poorer prognosis. p-AKT, PI3K, AKT, mTOR, and p-mTOR expression levels underwent significant elevation in breast cancer. The function of NUDCD1 within breast cancer might be associated with the PI3K/AKT/mTOR signaling pathway.

Exploring CCL11 in breast cancer: unraveling its anticancer potential and immune modulatory effects involving the Akt-S6 signaling

BackgroundCCL11, a chemokine known for recruiting immune cells to the tumor microenvironment (TME), has an unclear role in the context of its expression, patient prognosis, and the presence of tumor-infiltrating immune cells (TILs) in breast cancer.MethodsThe expression of CCL11 in invasive breast cancer (BRCA) was analyzed using TCGA database. Survival curve and Cox regression analysis determined the potential of CCL11 as an independent prognostic indicator. GSEA performed functional analysis on genes related to CCL11. CIBERSORT algorithm quantified the infiltration level of immune cells with varying CCL11 expression. Lastly, the correlation between CCL11 expression and anticancer drug sensitivity was examined. Immunohistochemistry (IHC) and qRT-PCR confirmed CCL11 expression in clinical tissue samples. The anti-tumor efficacy of CCL11 was investigated using CCK-8, plate formation, transwell assay, and Western blot.ResultsCCL11 expression was elevated in BRCA tumor tissues compared to adjacent normal tissues. Recurrence-free survival (RFS) was longer in patients with high expression of CCL11. Enrichment and co-expression analyses revealed CCL11's association with numerous immune-related signaling pathways and genes. Validation studies confirmed high CCL11 expression in breast cancer tissues. In vitro experiments substantiated CCL11's anticancer effects in BRCA.ConclusionCCL11 expression correlates with immune cell infiltration in breast cancer, indicating its potential as a prognostic biomarker for BRCA.

Vasculogenic mimicry regulates immune infiltration and mutational status of the tumor microenvironment in breast cancer to influence tumor prognosis.

Vasculogenic mimicry (VM) refers to the direct formation of microcirculatory ducts by invasive malignant tumors via cellular phenotypic transformation. However, there is a lack of VM-based biomarkers for breast cancer. We obtained transcriptomic expression data, single cell sequencing data, and clinical data of patients from The Cancer Genome Atlas Program (TCGA) database and GEO database, performed single cell analysis to obtain specific type annotations of breast cancer cells and analyzed their spatial expression analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analyses as well as Gene Set Enrichment Analysis (GSEA) analyses were performed to clarify the biological pathways and tumor functional enrichment relationships of the major expressed genes of VM in the breast cancer bulk data specimens. VM biomarkers were constructed. Meanwhile, the relationship between VM scores and tumor immune infiltration in breast cancer was analyzed using MCPcounter and ssGSEA methods. In addition, we assessed the specific relationship between NDRG1, a key VM gene in breast cancer, and tumor colonization, adhesion and invasion by biological experiments in breast cancer cell lines. The main cell types of breast cancer (BRCA) samples were annotated by single cell transcriptome analysis. Most of the VM-high group was present in epithelial cells, whereas the VM-low group was present in immune and stromal cells. Multiple tumor pathways such as TGFβ p53 and MAPK were closely associated with VM-mediated breast cancer infiltration and invasion. A prognostic model of breast cancer based on VM key genes was constituted. Prognostic stratification of breast cancer was successfully achieved for the TCGA-BRCA and GSE58812 datasets. Through immune infiltration analysis, we found that differential expression of VM markers was associated with multiple immune cell regulation. In MDA-MB-231 and MDA-MB-453 cell lines, we found that the NDRG1 gene significantly promoted colony formation of breast cancer cells. Our constructed VM-related gene-based model of breast cancer biology holds promise for prognostic prediction and patient stratification of breast cancer. This may provide a potentially clinically valuable aid in promoting a deeper understanding of the biological regulation of VM in breast cancer and exploring the specific mechanisms of tumor angiogenesis and breast cancer development.

KNSTRN Is a Prognostic Biomarker That Is Correlated with Immune Infiltration in Breast Cancer and Promotes Cell Cycle and Proliferation.

Kinetochore-localized astrin/SPAG5-binding protein (KNSTRN) promotes the progression of bladder cancer and lung adenocarcinoma. However, its expression and biological function in breast cancer remain largely unknown. Therefore, this study aimed to analyze KNSTRN expression, prognoses, correlation with immune infiltration, expression-associated genes, and regulated signaling pathways to characterize its role in regulating the cell cycle using both bioinformatics and in vitro functional experiments. Analyses of The Cancer Genome Atlas, Gene Expression Omnibus, TIMER, and The Human Protein Atlas databases revealed a significant upregulation of KNSTRN transcript and protein levels in breast cancer. Kaplan-Meier survival analyses demonstrated a significant association between high expression of KNSTRN and poor overall survival, relapse-free survival, post-progression survival, and distant metastases-free survival in patients with breast cancer. Furthermore, multivariate Cox regression analyses confirmed that KNSTRN is an independent prognostic factor for breast cancer. Immune infiltration analysis indicated a positive correlation between KNSTRN expression and T regulatory cell infiltration while showing a negative correlation with Tgd and natural killer cell infiltration. Gene set enrichment analysis along with single-cell transcriptome data analysis suggested that KNSTRN promoted cell cycle progression by regulating the expression of key cell cycle proteins. The overexpression and silencing of KNSTRN in vitro, respectively, promoted and inhibited the proliferation of breast cancer cells. The overexpression of KNSTRN enhanced the expression of key cell cycle regulators, including CDK4, CDK6, and cyclin D3, thereby accelerating the G1/S phase transition and leading to aberrant proliferation of breast cancer cells. In conclusion, our study demonstrates that KNSTRN functions as an oncogene in breast cancer by regulating immune response, promoting G1/S transition, and facilitating breast cancer cell proliferation. Moreover, KNSTRN has potential as a molecular biomarker for diagnostic and prognostic prediction in breast cancer.

CGAS-STING pathway expression correlates with genomic instability and immune cell infiltration in breast cancer

Genomic instability, as caused by oncogene-induced replication stress, can lead to the activation of inflammatory signaling, involving the cGAS-STING and JAK-STAT pathways. Inflammatory signaling has been associated with pro-tumorigenic features, but also with favorable response to treatment, including to immune checkpoint inhibition. In this study, we aim to explore relations between inflammatory signaling, markers of replication stress, and immune cell infiltration in breast cancer. Expression levels of cGAS-STING signaling components (STING, phospho-TBK1, and phospho-STAT1), replication stress markers (γH2AX and pRPA), replication stress-related proto-oncogenes (Cyclin E1 and c-Myc) and immune cell markers (CD20, CD4, and CD57) are determined immunohistochemically on primary breast cancer samples (n = 380). RNA-sequencing data from TCGA (n = 1082) and METABRIC (n = 1904) are used to calculate cGAS-STING scores. pTBK1, pSTAT1 expression and cGAS-STING pathway scores are all increased in triple-negative breast cancers compared to other subtypes. Expression of γH2AX, pRPA, Cyclin E1, c-Myc, and immune cell infiltration positively correlate with p-STAT1 expression (P < 0.001). Additionally, we observe significant positive associations between expression of pTBK1 and γH2AX, pRPA, c-Myc, and number of CD4+ cells and CD20+ cells. Also, cGAS-STING scores are correlated with genomic instability metrics, such as homologous recombination deficiency (P < 0.001) and tumor mutational burden (P < 0.01). Moreover, data from the I-SPY2 clinical trial (n = 71) confirms that higher cGAS-STING scores are observed in breast cancer patients who responded to immunotherapy combined with chemotherapy. In conclusion, the cGAS-STING pathway is highly expressed in TNBCs and is correlated with genomic instability and immune cell infiltration.

UTP23 Is a Promising Prognostic Biomarker and Is Associated with Immune Infiltration in Breast Cancer.

Breast cancer is one of the malignant tumors with a high incidence and mortality rate among women worldwide, and its prevalence is increasing year by year, posing a serious health risk to women. UTP23 (UTP23 Small Subunit Processome Component) is a nucleolar protein that is essential for ribosome production. As we all know, disruption of ribosome structure and function results in improper protein function, affecting the body's normal physiological processes and promoting cancer growth. However, little research has shown a connection between UTP23 and cancer. We analyzed the mRNA expression of UTP23 in normal tissue and breast cancer using The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database, and the protein expression of UTP23 using The Human Protein Atlas (HPA) database. Next, we examined the relationship between UTP23 high expression and Overall Survival (OS) using Kaplan-Meier Plotters and enriched 980 differentially expressed genes in UTP23 high and low expression samples using GO/KEGG and GSEA to identify potential biological functions of UTP23 and signaling pathways that it might influence. Finally, we also investigated the relationship between UTP23 and immune infiltration and examined the effect of UTP23 on the proliferation of human breast cancer cell lines by knocking down UTP23. We found that UTP23 levels in breast cancer patient samples were noticeably greater than those in healthy individuals and that high UTP23 levels were strongly linked with poor prognoses (P = 0.008). Functional enrichment analysis revealed that UTP23 expression was connected to the humoral immune response. Besides, UTP23 expression was found to be positively correlated with immune cell infiltration. Furthermore, UTP23 knockdown has been shown to inhibit the proliferation of human breast cancer cells MDA-MB-231 and HCC-1806. Taken together, our study demonstrated that UTP23 is a promising target in detecting and treating breast cancer and is intimately linked to immune infiltration.

Integrated Bioinformatics Analysis to Identify a Novel Four-Gene Prognostic Model of Breast Cancer and Reveal Its Association with Immune Infiltration.

Liquid-liquid phase separation (LLPS) impact immune signaling in cancer and related genes have shown prognostic value in breast cancer (BRCA). However, the crosstalk between LLPS and immune infiltration in BRCA remain unclear. Therefore, we aimed to develop a novel prognostic model of BRCA related to LLPS and immune infiltration. BRCA-related, liquid-liquid phase separation (LLPS)-related genes, and differentially expressed genes (DEGs) were identified using public databases. Mutation and drug sensitivity analyses were performed using Gene Set Cancer Analysis database. Univariate cox regression and LASSO Cox regression were used for the construction and verification of prognostic model. Kaplan-Meier analysis was performed to evaluate overall survival (OS). Gene set variation analysis was conducted to analyze key pathways. CIBERSORT was used to assess immune infiltration and its correlation with prognostic genes was determined through Pearson analysis. A total of 6056 BRCA-associated genes, 3775 LLPS-associated genes, and 4049 DEGs, resulting in 314 overlapping genes. Twenty-eight prognostic genes were screened, and some of them were mutational and related to drug sensitivity Subsequently, a prognostic model comprising L1CAM, EVL, FABP7, and CST1 was built. Patients in high-risk group had shorter OS than those in low-risk group. The infiltrating levels of CD8+ T cells, macrophages M0, macrophages M2, dendritic cells activated, and mast cells resting was altered in high-risk group of breast cancer patients compared to low-risk group. L1CAM, EVL, FABP7, and CST1 were related to these infiltrating immune cells. L1CAM, EVL, FABP7, and CST1 were potential diagnostic biomarkers and therapeutic targets for BRCA.

Cancer-associated fibroblast spatial heterogeneity and EMILIN1 expression in the tumor microenvironment modulate TGF-β activity and CD8+ T-cell infiltration in breast cancer.

Rationale: The tumor microenvironment (TME) and its multifaceted interactions with cancer cells are major targets for cancer treatment. Single-cell technologies have brought major insights into the TME, but the resulting complexity often precludes conclusions on function. Methods: We combined single-cell RNA sequencing and spatial transcriptomic data to explore the relationship between different cancer-associated fibroblast (CAF) populations and immune cell exclusion in breast tumors. The significance of the findings was then evaluated in a cohort of tumors (N=75) from breast cancer patients using immunohistochemistry analysis. Results: Our data show for the first time the degree of spatial organization of different CAF populations in breast cancer. We found that IL-iCAFs, Detox-iCAFs, and IFNγ-iCAFs tended to cluster together, while Wound-myCAFs, TGFβ-myCAFs, and ECM-myCAFs formed another group that overlapped with elevated TGF-β signaling. Differential gene expression analysis of areas with CD8+ T-cell infiltration/exclusion within the TGF-β signaling-rich zones identified elastin microfibrillar interface protein 1 (EMILIN1) as a top modulated gene. EMILIN1, a TGF-β inhibitor, was upregulated in IFNγ-iCAFs directly modulating TGFβ immunosuppressive function. Histological analysis of 75 breast cancer samples confirmed that high EMILIN1 expression in the tumor margins was related to high CD8+ T-cell infiltration, consistent with our spatial gene expression analysis. High EMILIN1 expression was also associated with better prognosis of patients with breast cancer, underscoring its functional significance for the recruitment of cytotoxic T cells into the tumor area. Conclusion: Our data show that correlating TGF-β signaling to a CAF subpopulation is not enough because proteins with TGF-β-modulating activity originating from other CAF subpopulations can alter its activity. Therefore, therapeutic targeting should remain focused on biological processes rather than on specific CAF subtypes.

SOX13 is a novel prognostic biomarker and associates with immune infiltration in breast cancer.

The transcription factor, SOX13 is part of the SOX family. SOX proteins are crucial in the progression of many cancers, and some correlate with carcinogenesis. Nonetheless, the biological and clinical implications of SOX13 in human breast cancer (BC) remain rarely known. We evaluated the survival and expression data of SOX13 in BC patients via the UNLCAL, GEPIA, TIMER, and Kaplan-Meier plotter databases. Immunohistochemistry (IHC) was used to verify clinical specimens. The gene alteration rates of SOX13 were acquired on the online web cBioportal. With the aid of the TCGA data, the association between SOX13 mRNA expression and copy number alterations (CNA) and methylation was determined. LinkedOmics was used to identify the genes that co-expressed with SOX13 and the regulators. Immune infiltration and tumor microenvironment evaluations were assessed by ImmuCellAI and TIMER2.0 databases. SOX13 correlated drug resistance analysis was performed using the GDSC2 database. Higher SOX13 expression was discovered in BC tissues in comparison to normal tissues. Moreover, increased gene mutation and amplification of SOX13 were found in BC. Patients with increased SOX13 expression levels showed worse overall survival (OS). Cox analysis showed that SOX13 independently served as a prognostic indicator for poor survival in BC. Further, the expression of SOX13 was also confirmed to be correlated with tumor microenvironment and diverse infiltration of immune cells. In terms of drug sensitivity analysis, we found higher expression level of SOX13 predicts a high IC50 value for most of 198 drugs which predicts drug resistance. The present findings demonstrated that high expression of SOX13 negatively relates to prognosis and SOX13 plays an important role in cancer immunity. Therefore, SOX13 may potentially be adopted as a biomarker for predicting BC prognosis and infiltration of immune cells.

Eukaryotic Translation Initiation Factor 2 Subunit β as a Prognostic Biomarker Associates With Immune Cell Infiltration in Breast Cancer

IntroductionThe present study aims to explore the expression level of eukaryotic translation initiation factor 2 subunit β (EIF2S2) in breast cancer tissue, and its role both in breast cancer prognosis and in the immune microenvironment. MethodsTo assess the association between the expression levels of EIF2S2 and prognosis, the Gene Expression Profiling Interactive Analysis database was initially applied to determine differences in the gene expression of EIF2S2 in various malignant and normal tissues. Furthermore, the expression levels of EIF2S2 were determined in the clinical breast cancer tissues and corresponding para-neoplastic tissues using immunohistochemical analysis. In addition, Kaplan–Meier survival and Cox regression analyses were employed to explore the association between EIF2S2 expression levels and patient prognosis. Finally, the correlation between the expression levels of EIF2S2 and immune cell infiltration in breast cancer was analyzed using the TIMER2.0 database, and subsequently validated by immunohistochemical experiments. ResultsThe Gene Expression Profiling Interactive Analysis database revealed the presence of higher expression levels of EIF2S2 in various different types of cancer compared with normal cells, also correlating its expression with both the age and the tumor stage of patients with breast cancer. The survival analysis results revealed that high expression levels of EIF2S2 could be a risk factor for poor prognosis in patients with breast cancer. Moreover, the EIF2S2 expression level was found to be closely associated with the infiltration levels of various immune cells, including regulatory T cells, CD4+, CD8+, and natural killer cells, in breast cancer. ConclusionsIn conclusion, the present study has demonstrated that an upregulated expression level of EIF2S2 in breast cancer may be associated with poor patient prognosis, affecting immune cell infiltration in breast cancer. Taken together, the findings of the present study have shown that EIF2S2 expression may be a novel therapeutic target for breast cancer.

Exploring the prognostic value of S100A11 and its association with immune infiltration in breast cancer

Breast cancer (BC) is a severe danger to women’s lives and health globally. S100A11 is aberrantly expressed in many carcinomas and serves a crucial function in cancer development. However, the role of S100A11 in BC is unclear. In this study, we utilized multiple databases and online tools, including the TCGA database, cBioPortal, and STRING, to evaluate the significance of S100A11 in BC prognosis and immune infiltration. We found that S100A11 was considerably more abundant in BC tissues. Survival analysis indicated that individuals with S100A11 high expression of BC had shorter overall survival. Multivariate Cox regression analysis revealed that high S100A11 expression independently influenced the poor outcome of patients with BC (HR = 1.738, 95%CI 1.197–2.524). Our nomogram incorporating five factors, including S100A11, age, clinical stage, N, and M, was developed to anticipate the survival probability in BC prognosis. The model demonstrated good consistency and accuracy. Furthermore, the mutation rete of S100A11 was 14%. Survival analysis suggested that breast cancer patients with S100A11 mutation had a worse prognosis. KEGG pathway enrichment analysis revealed that S100A11 may be mainly involved in the IL-17 signaling pathway. Finally, we discovered a correlation between S100A11 expression and immune cell infiltration on BC. S100A11 expression was positively associated with 17 immune checkpoint-related genes. In conclusion, this study indicates that S100A11 may contribute to a worse prognosis for BC and potentially has a significant impact through its influence on immune cell infiltration and the IL-17 signaling pathway.

Identification of an exosome-related signature associated with prognosis and immune infiltration in breast cancer

Exosomes, nanosized vesicles, play a vital role in breast cancer (BC) occurrence, development, and drug resistance. Hence, we proceeded to study the potential prognostic value of exosome-related genes and their relationship to the immune microenvironment in BC. 121 exosome-related genes were provided by the ExoBCD database, and 7 final genes were selected to construct the prognostic signature. Besides, the expression levels of the 7 exosome-related genes were validated by the experiment in BC cell lines. Based on the signature, BC patients from the training and validation cohorts were separated into low- and high-risk groups. Subsequently, the R clusterProfiler package was applied to identify the distinct enrichment pathways between high-risk groups and low-risk groups. The relevance of the tumor immune microenvironment and exosome-related gene risk score were analyzed in BC. Eventually, the different expression levels of immune checkpoint-related genes were compared between the two risk groups. Based on the risk model, the low-risk groups were identified with a higher survival rate both in the training and validation cohorts. A better overall survival was revealed in patients with higher scores evaluated by the estimation of stromal and immune cells in malignant tumor tissues using expression (ESTIMATE) algorithm. Subsequently, BC patients with lower risk scores were indicated by higher expression levels of some immune checkpoint-related genes and immune cell infiltration. Exosomes are closely associated with the prognosis and immune cell infiltration of BC. These findings may contribute to improving immunotherapy and provide a new vision for BC treatment strategies.