3D Collagen Fiber Concentration Regulates Treg Cell Infiltration in Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is characterized by poor prognosis and a lack of effective therapeutic agents owing to the absence of biomarkers. A high abundance of tumor-infiltrating regulatory T cells (Tregs) was associated with worse prognosis in malignant disease. Exploring the association between Treg cell infiltration and TNBC will provide new insights for understanding TNBC immunosuppression and may pave the way for developing novel immune-based treatments. Patients from TCGA were divided into Treg-high (Treg-H) and Treg-low (Treg-L) groups based on the abundance of Tregs according to CIBERSORT analysis. The association between expression level of Tregs and the clinical characteristics as well as prognosis of breast cancer were evaluated. Next, a Treg-related prognostic model was established after survival-dependent univariate Cox and LASSO regression analysis, companied with an external GEO cohort validation. Then, GO, KEGG and GSEA analyses were performed between the Treg-H and Treg-L groups. Masson and Sirius red/Fast Green staining were applied for ECM characterization. Accordingly, Jurkat T cells were encapsulated in 3D collagen to mimic the ECM microenvironment, and the expression levels of CD4, FOXP3 and CD25 were quantified according to immunofluorescence staining. The expression level of Tregs is significantly associated with the clinical characteristics of breast cancer patients, and a high level of Treg cell expression indicates a poor prognosis in TNBC. To further evaluate this, a Treg-related prognostic model was established that accurately predicted outcomes in both TCGA training and GEO validation cohorts of TNBC patients. Subsequently, ECM-associated signaling pathways were identified between the Treg-H and Treg-L groups, indicating the role of ECM in Treg infiltration. Since we found increasing collagen concentrations in TNBC patients with distant migration, we encapsulated Jurkat T cells within a 3D matrix with different collagen concentrations and observed that increasing collagen concentrations promoted the expression of Treg biomarkers, supporting the regulatory role of ECM in Treg infiltration. Our results support the association between Treg expression and breast cancer progression as well as prognosis in the TNBC subtype. Moreover, increasing collagen density may promote Treg infiltration, and thus induce an immunosuppressed TME.

Gao H ，Tian Q ，Zhou Y ，Zhu L ，Lu Y ，Ma Y ，Feng J ，Jiang Y ，Wang B ... -  《Frontiers in Immunology》

Regulatory T cells are associated with the tumor immune microenvironment and immunotherapy response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high risk of distant metastasis, an extremely poor prognosis, and a high risk of death. Regulatory T cells (Tregs) contribute to the formation of a tumor immunosuppressive microenvironment, which plays an important role in the progression and treatment resistance of TNBC. A public single-cell sequencing dataset demonstrated increased infiltration of Tregs in TNBC tissues relative to normal breast tissue. Weighted gene co-expression network analysis was used to identify Treg infiltration-related modules for METABRIC TNBC samples. Subsequently, we obtained two Treg infiltration-associated clusters of TNBC by applying consensus clustering and further constructed a prognostic model based on this Treg infiltration-associated gene module. The ability of the selected gene in the prognostic model, thymidine kinase-1 (TK1), to promote the progression of TNBC was evaluated in vitro. We concluded that two Treg infiltration-associated clusters had different prognoses and sensitivities to drugs commonly used in breast cancer treatment, and multi-omics analysis revealed that the two clusters had different copy number variations of key tumor progression genes. The 7-gene risk score based on TNBC Treg infiltration was a reliable prognostic indicator both in the training and validation cohorts. Moreover, patients with TNBC with high Treg infiltration-related scores lacked the activation of immune activation pathways and exhibited resistance to anti-PD1 immunotherapy. Knocking down TK1 led to impaired proliferation, migration, and invasion of TNBC cells in vitro. In addition, specimens from patients with TNBC with high TK1 expression showed significantly higher Treg infiltration in tumors. Results of spatial transcriptome analysis showed that TK1 positive cells mainly localize in tumor area, and Treg cell infiltration in TNBC tissues was associated with high expression of TK1. Pan-cancer analysis also demonstrated that TK1 is associated with poor prognosis and activation of proliferation pathways in multiple cancers. We established a prognostic model related to Treg infiltration and this model can be used to establish a clinically relevant classification of TNBC progression. Additionally, our work revealed the underestimable potential of TK1 as a tumor biomarker and immunotherapeutic target.

Huang P ，Zhou X ，Zheng M ，Yu Y ，Jin G ，Zhang S ... -  《Frontiers in Immunology》

Immunity and Extracellular Matrix Characteristics of Breast Cancer Subtypes Based on Identification by T Helper Cells Profiling.

The therapeutic effect of immune checkpoint inhibitors on tumors is not only related to CD8+ effector T cells but also sufficiently related to CD4+ helper T (TH) cells. The immune characteristics of breast cancer, including gene characteristics and tumor-infiltrating lymphocytes, have become significant biomarkers for predicting prognosis and immunotherapy response in recent years. Breast cancer samples from The Cancer Genome Atlas (TCGA) database and triple-negative breast cancer (TNBC) samples from GSE31519 in the Gene Expression Omnibus (GEO) database were extracted and clustered based on gene sets representing TH cell signatures. CIBERSORT simulations of immune cell components in the tumor microenvironment and gene set enrichment analyses (GSEAs) were performed in the different clusters to verify the classification of the subtypes. The acquisition of differentially expressed genes (DEGs) in the different clusters was further used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The clinical information from different clusters was used for survival analysis. Finally, the surgical tissues of TNBC samples were stained by immunofluorescence staining and Masson's trichrome staining to explore the correlation of TH cell subtypes with extracellular matrix (ECM). The breast cancer samples from the datasets in TCGA database and GEO database were classified into TH-activated and TH-silenced clusters, which was verified by the immune cell components and enriched immune-related pathways. The DEGs of TH-activated and TH-silenced clusters were obtained. In addition to TH cells and other immune-related pathways, ECM-related pathways were found to be enriched by DEGs. Furthermore, the survival data of TCGA samples and GSE31519 samples showed that the 10-year overall survival (p-value < 0.001) and 10-year event-free survival (p-value = 0.162) of the TH-activated cluster were better, respectively. Fluorescent labeling of TH cell subtypes and staining of the collagen area of surgical specimens further illustrated the relationship between TH cell subtypes and ECM in breast cancer, among which high TH1 infiltration was related to low collagen content (p-value < 0.001), while high TH2 and Treg infiltration contained more abundant collagen (p-value < 0.05) in TNBC. With regard to the relationship of TH cell subtypes, TH2 was positively correlated with Treg (p-value < 0.05), while TH1 was negatively correlated with both of them. The immune and ECM characteristics of breast cancer subtypes based on TH cell characteristics were revealed, and the relationship between different TH cell subsets and ECM and prognosis was explored in this study. The crosstalk between ECM and TH cell subtypes formed a balanced TME influencing the prognosis and treatment response in breast cancer, which suggests that the correlation between TH cells and ECM needs to be further emphasized in future breast cancer studies.

Zhou Y ，Tian Q ，Gao H ，Zhu L ，Zhang Y ，Zhang C ，Yang J ，Wang B ... -  《Frontiers in Immunology》

Regulatory T lymphocyte infiltration in metastatic breast cancer-an independent prognostic factor that changes with tumor progression.

Stenström J ，Hedenfalk I ，Hagerling C 《-》

Regulatory T cells: Their role in triple-negative breast cancer progression and metastasis.

Triple-negative breast cancer (TNBC) is an aggressive and immunogenic subtype of breast cancer. This tumorigenicity is independent of hormonal or HER2 pathways because of a lack of respective receptor expression. TNBC is extremely prone to drug resistance and early recurrence because of T-regulatory cell (Treg) infiltration into the tumor microenvironment (TME) in addition to other mechanisms like genomic instability. Tumor-infiltrating Tregs interact with both tumor and stromal cells as well as extracellular matrix components in the TME and induce an immune-suppressive phenotype. Hence, treatment of TNBC with conventional therapies remains challenging. Understanding the protective mechanism of Tregs in shielding TNBC from antitumor immune responses in the TME will pave the way for developing novel, immune-based therapeutics. The current review focuses on the role of tumor-infiltrating Tregs in tumor progression and metabolic reprogramming of the TME. The authors have extended their focus to oncotargeting Treg-mediated immune suppression in breast cancer. Because of its potential role in the TME, modulating Treg activity may provide a novel strategic intervention to combat TNBC. Both under laboratory conditions and in clinical trials, currently available anticancer drugs and natural therapeutics as potential agents for targeting Tregs are explored.

Malla RR ，Vasudevaraju P ，Vempati RK ，Rakshmitha M ，Merchant N ，Nagaraju GP ... -  《-》