Single-Cell Transcriptome Analysis Reveals the M2 Macrophages and Exhausted T Cells and Intratumoral Heterogeneity in Triple-Negative Breast Cancer

2021 ◽  
Vol 21 ◽  
Author(s):  
Chen Li ◽  
Lingyun Xu
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Single Cell ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Immune Cell ◽  
Therapeutic Potential ◽  
Intratumoral Heterogeneity ◽  
Sequencing Data ◽  
Alternatively Activated Macrophages

Background: Triple-negative breast cancer (TNBC) is a highly heterogeneous and invasive malignancy that is characterized by high recurrence and mortality rates as well as extremely poor prognosis. Objective: This study aimed to analyze T cells and macrophages in the tumor microenvironment with the aim of identifying targets with therapeutic potential. Method: Single-cell sequencing data of TNBC patients from the GSE118389 dataset were analyzed to examine the immune environment and intratumoral heterogeneity of TNBC patients. Result: Polarized alternatively activated macrophages (M2) and exhausted CD8+ T cells were identified in TNBC patients. Immunosuppressive checkpoint analysis revealed that levels of lymphocyte-activation gene 3 (LAG3) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) of exhausted T cells were significantly higher than levels of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). This indicates that these markers are potential immunotherapy targets. Furthermore, analysis of significantly altered immune cell markers showed that several markers are associated with the prognosis of TNBC. Conclusion: Overall, these findings demonstrate inter-tissue heterogeneity of TNBC and provide novel therapeutic targets for the treatment of TNBC.

scholarly journals Epigenetic Quantification of Circulating Immune Cells in Peripheral Blood of Triple-Negative Breast Cancer patients

2021 ◽  
Author(s):  
Mehdi Manoochehri ◽  
Thomas Hielscher ◽  
Nasim Borhani ◽  
Clarissa Gerhäuser ◽  
Olivia Fletcher ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Immune Cells ◽  
Triple Negative ◽  
Immune Cell ◽  
Nk Cell ◽  
Cell Type ◽  
Cell Ratio ◽  
Immune Cell Type

Abstract Background: A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). Methods: Leukocyte subtype-specific un/methylated CpG sites were selected and methylation levels at these sites used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. Results: The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66-4.29), all Padj.<1e-04). A higher neutrophil ratio and lower ratios of NK cells, CD4+ T cells, CD8+ T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28-1.42), all Padj.<1e-04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P=0.019).Conclusion: This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.

scholarly journals Epigenetic quantification of circulating immune cells in peripheral blood of triple-negative breast cancer patients

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Mehdi Manoochehri ◽  
Thomas Hielscher ◽  
Nasim Borhani ◽  
Clarissa Gerhäuser ◽  
Olivia Fletcher ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Immune Cells ◽  
Triple Negative ◽  
Immune Cell ◽  
Nk Cell ◽  
Cell Type ◽  
Cell Ratio ◽  
Immune Cell Type

Abstract Background A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). Methods Leukocyte subtype-specific unmethylated/methylated CpG sites were selected, and methylation levels at these sites were used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. Results The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66–4.29), all Padj. < 1e−04). A higher neutrophil ratio and lower ratios of NK cells, CD4 + T cells, CD8 + T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28–1.42), all Padj. < 1e−04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P = 0.019). Conclusion This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.

scholarly journals Epigenetic Quantification of Circulating Immune Cells in Peripheral Blood of Triple-negative Breast Cancer Patients

2021 ◽  
Author(s):  
Mehdi Manoochehri ◽  
Thomas Hielscher ◽  
Nasim Borhani ◽  
Clarissa Gerhäuser ◽  
Olivia Fletcher ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Immune Cells ◽  
Triple Negative ◽  
Immune Cell ◽  
Nk Cell ◽  
Cell Type ◽  
Cell Ratio ◽  
Immune Cell Type

Abstract Background: A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). Methods: Leukocyte subtype-specific un/methylated CpG sites were selected and methylation levels at these sites used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. Results: The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66-4.29), all Padj.<1e-04). A higher neutrophil ratio and lower ratios of NK cells, CD4+ T cells, CD8+ T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28-1.42), all Padj.<1e-04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P=0.019).Conclusion: This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.

Correlation of the tumor mutational burden with the composition of the immune cell subpopulations in peripheral blood of triple-negative breast cancer patients undergoing neoadjuvant therapy with durvalumab: Results from the prospectively randomized GeparNuevo trial.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 588-588
Author(s):  
Barbara Seliger ◽  
Thomas Karn ◽  
Carsten Denkert ◽  
Andreas Schneeweiss ◽  
Claus Hanusch ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Neoadjuvant Therapy ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Immune Cell ◽  
Effector Memory ◽  
Cell Counts ◽  
Mutational Burden ◽  
Tumor Mutational Burden

588 Background: The GeparNuevo trial is a randomized, double-blind, multi-center phase II trial of neoadjuvant therapy in patients with early-stage triple negative breast cancer (TNBC) investigating the role of durvalumab, an anti-PD-L1 antibody, which blocks PD-L1 binding to PD1 and CD80, in addition to standard chemotherapy with nab-Paclitaxel (nab-Pac) followed by Epirubicin plus Cyclophosphamid (EC; Loibl S et al. ASCO 2018). Since the tumor mutational burden (TMB) has been suggested to be associated with a better outcome of patients undergoing immunotherapy and an increased T cell response, we determined whether there exists a link between TMB and immune cell composition, frequency and function in patients of the GeparNuevo trial. Methods: In order to determine possible predictive and / or prognostic biomarkers, tumor biopsies taken at recruitment from 149 patients out of the 174 enrolled patients underwent deep sequencing in order to determine the TMB. In addition, for 120 patients blood samples were taken at recruitment and during different time points of treatment (after durvalumab pre-treatment, after Nab-Pac and at surgery after EC) and evaluated using multicolor flow cytometry by monitoring the absolute cell counts of T cells, B cells and NK cells as well as the frequency, composition and functionality of different immune cell populations. Results: The TMB of the GeparNuevo cohort was in line with published data with a mean of 1.8 mutations/MB (range 0.02 – 7.65), respectively. Preliminary evaluation demonstrated a significant correlation of TMB with blood parameters, in particular with subsets of CD8+ T cells. Interestingly, the data suggest a negative correlation of TMB with the frequency of effector cells while a positive correlation exists with the effector memory cells at recruitment. In depth analyses of a correlation with treatment arm and clinical responses are currently performed. Conclusions: Using this approach we hope to identify biomarkers, which will allow a better selection of TNBC patients undergoing specific immunotherapies. Clinical trial information: NCT02685059.

scholarly journals Comprehensive genomic and immunophenotypic analysis of CD4 T cell infiltrating human triple-negative breast cancer

2020 ◽  
Author(s):  
He Zhang ◽  
Guohui Qin ◽  
Hui Yu ◽  
Xu Han ◽  
Sha Zhu
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Immune Cell ◽  
Differential Expression Analysis ◽  
Effector Memory ◽  
Activated T Cells ◽  
Cell Functions

AbstractThe aim of this study is to investigate the gene expression module of tumor-infiltrating CD4+T cells and its potential roles in modulating immune cell functions in triple-negative breast cancer. Differentially expressed genes were identified by comparison of the expression profile in CD4+T cells isolated from tumor tissues and peripheral blood of TNBC patients respectively. The differential expression analysis was conducted using R, and then the functional and pathway enrichment of the DEGs were analyzed using GSEA, followed by integrated regulatory network construction and genetic analysis of tumor-infiltrating immune cells based on a scientific deconvolution algorithm. As a result, abundant Treg and exhausted lymphocytes were detected, accompanied by largely decreased of effector/memory and cytotoxic T cells. Immune-related gene correlation analysis showed that the extent of follicular helper T cells gene expression signatures were inversely associated with those of CD4+ naive T cells and CD4+ memory resting T cells, but positively correlated with that of CD4+ memory activated T cells. In addition, we found five core genes including IFNG, CTLA4, FAS, CXCR6, and JUN were significantly over expressed in CD4+ TILs which may contribute to exhaustion of lymphocytes and participate in biological processes associated with regulation of chemotaxis. Study provides a comprehensive understanding of the roles of DEGs associated with the chemotactic and exhausted immunophenotypes of CD4+ TILs that are a valuable resource from which future investigation may be carried out to better understand the mechanisms that promote TNBC progression.

scholarly journals Classification of triple-negative breast cancer based on immune cell infiltration

2020 ◽  
Author(s):  
Wenji Shi ◽  
Dan Wang ◽  
Luz Angela Torres-de la Roche ◽  
Rui Zhuo ◽  
Rudy Leon De Wilde
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
B Cells ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Immune Cell ◽  
Cell Infiltration ◽  
Immune Cell Infiltration ◽  
Immune Phenotype ◽  
Cluster A

Abstract Background Although the classification system of triple-negative breast cancer(TNBC) has become more and more perfect, there is no report on the immune subtype of triple-negative breast cancer based on immune cell infiltration. Results According to immune infiltrating cells, data from 360 patients were divided into cluster A (subtype 1 and subtype 3) and cluster B (subtype 2; with poorly immune phenotype). Expression of memory B cells, naïve B cells, M0 macrophages, M1 macrophages, CD4 memory activated T cells, and CD4 naïve T cells were significantly higher in cluster A (P < 0.05). In contrast, the expression of M2 macrophages and resting mast cells were higher in cluster B (P < 0.05). GSVA results show that B cell receptor pathway and JAK-STAT pathway are activated and more frequently altered in cluster A (P < 0.05). mTOR pathway alterations usually appear in cluster B (P < 0.05). Compared with cluster A, the risk of recurrence in cluster B patients is significantly increased (P < 0.05). Conclusions This analysis of tumor microenvironment revealed the multifaceted nature of TNBC and its impact on patient prognosis, being recurrence more often in those with poorly immune phenotype. These results provide a reference for further exploration of the heterogeneity of TNBC.

scholarly journals Interleukin-17-based cytokine signaling as a determinant for tumor immune microenvironment with prognostic value in triple-negative breast cancer

2021 ◽  
Author(s):  
Victor C. Kok ◽  
Charles C.N. Wang ◽  
Szu-Han Liao ◽  
De-Lun Chen
Keyword(s):  
Breast Cancer ◽  
Overall Survival ◽  
T Cells ◽  
Nk Cells ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Immune Cell ◽  
Receptor Interaction ◽  
Cytokine Signaling ◽  
Interleukin 17

Abstract Background: Only a proportion of triple-negative breast cancer (TNBC) is immunotherapy-responsive. We hypothesized that the tumor microenvironment (TME) might influence the outcomes of TNBC and investigated the relevant signaling pathways. Methods: RNA-seq data of 115 TNBC samples and 112 normal adjacent tissues were retrieved for ESTIMATE, CIBERSORTx, X2K, KEGG, and GSVA analyses. The immune score (IS) and stromal score (SS) were calculated and correlated with the overall survival (OS) in TNBC. Finally, we validated the altered transcription factor (TF) genes in the cBioPortal. Results: The SS is a good predictor of the OS (better survival in SS-low patients; P = 0.0081). In line with these results, when compared with IS-low/SS-high patients, IS-high/SS-low patients showed a better OS (P = 0.045). Moreover, macrophages were polarized toward the M2 phenotypes in IS-low/SS-high patients (P < 0.001). Compared with IS-low/SS-high patients, CIBERSORTx also showed that IS-high/SS-low patients had an increased number of memory B cells, CD8+ T cells (14.8% vs. 3.7%, p = 0.0286), activated CD4+ memory T cells, follicular helper T cells, and activated NK cells in the TME; additionally, fewer resting NK cells were detected in the TME (P = 0.0108). Additionally, there were 284 upregulated and 367 downregulated DEGs (Differentially Expressed Genes) in IS-high/SS-low, and 187 upregulated and 183 downregulated DEGs in IS-low/SS-high patients. KEGG analysis further revealed that the DEGs were enriched in the IL-17 and cytokine-cytokine receptor interaction pathways. Of note, as per the cBioPortal platform, we discovered that 13% of ER-negative, HER2-unamplified BC patients harbored IL17RA deep deletions and 25% harbored TRAF3IP2 amplifications; interestingly, the nine altered TF genes were associated with significantly worse relapse-free survival and OS, in the context of 2,377 and 4,819 BC patients, respectively.Conclusions: The TME with different immune cell components influences the survival of TNBC patients. IS-high/SS-low patients show a better overall survival. Further studies are required to examine whether an immune/stromal state also predicts the response to immunotherapy.

scholarly journals Local, multimodal intralesional therapy renders distant brain metastases susceptible to PD-L1 blockade in a preclinical model of triple-negative breast cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Toshihiro Yokoi ◽  
Takaaki Oba ◽  
Ryutaro Kajihara ◽  
Scott I. Abrams ◽  
Fumito Ito
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Brain Metastases ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Therapeutic Potential ◽  
Mammary Tumors ◽  
Preclinical Model ◽  
Tumor Cell Death ◽  
Intralesional Therapy

AbstractDespite recent progress in therapeutic strategies, prognosis of metastatic triple-negative breast cancer (TNBC) remains dismal. Evidence suggests that the induction and activation of tumor-residing conventional type-1 dendritic cells (cDC1s) is critical for the generation of CD8+ T cells that mediate the regression of mammary tumors and potentiate anti-PD-1/PD-L1 therapeutic efficacy. However, it remains unknown whether this strategy is effective against metastatic TNBC, which is poorly responsive to immunotherapy. Here, using a mouse model of TNBC, we established orthotopic mammary tumors and brain metastases, and treated mammary tumors with in situ immunomodulation (ISIM) consisting of intratumoral injections of Flt3L to mobilize cDC1s, local irradiation to induce immunogenic tumor cell death, and TLR3/CD40 stimulation to activate cDC1s. ISIM treatment of the mammary tumor increased circulating T cells with effector phenotypes, and infiltration of CD8+ T cells into the metastatic brain lesions, resulting in delayed progression of brain metastases and improved survival. Furthermore, although anti-PD-L1 therapy alone was ineffective against brain metastases, ISIM overcame resistance to anti-PD-L1 therapy, which rendered these tumor-bearing mice responsive to anti-PD-L1 therapy and further improved survival. Collectively, these results illustrate the therapeutic potential of multimodal intralesional therapy for patients with unresectable and metastatic TNBC.

scholarly journals CIBERSORT analysis of TCGA and METABRIC identifies subgroups with better outcomes in triple negative breast cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kelly E. Craven ◽  
Yesim Gökmen-Polar ◽  
Sunil S. Badve
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Molecular Mechanisms ◽  
Triple Negative ◽  
Immune Cell ◽  
Molecular Taxonomy ◽  
The Cancer Genome Atlas ◽  
Activated T Cells ◽  
Cancer Immunoediting

AbstractStudies have shown that the presence of tumor infiltrating lymphocytes (TILs) in Triple Negative Breast Cancer (TNBC) is associated with better prognosis. However, the molecular mechanisms underlying these immune cell differences are not well delineated. In this study, analysis of hematoxylin and eosin images from The Cancer Genome Atlas (TCGA) breast cancer cohort failed to show a prognostic benefit of TILs in TNBC, whereas CIBERSORT analysis, which quantifies the proportion of each immune cell type, demonstrated improved overall survival in TCGA TNBC samples with increased CD8 T cells or CD8 plus CD4 memory activated T cells and in Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) TNBC samples with increased gamma delta T cells. Twenty-five genes showed mutational frequency differences between the TCGA high and low T cell groups, and many play important roles in inflammation or immune evasion (ATG2B, HIST1H2BC, PKD1, PIKFYVE, TLR3, NOTCH3, GOLGB1, CREBBP). Identification of these mutations suggests novel mechanisms by which the cancer cells attract immune cells and by which they evade or dampen the immune system during the cancer immunoediting process. This study suggests that integration of mutations with CIBERSORT analysis could provide better prediction of outcomes and novel therapeutic targets in TNBC cases.

scholarly journals Immune cell composition and functional marker dynamics from multiplexed immunohistochemistry to predict response to neoadjuvant chemotherapy in the WSG-ADAPT-TN trial

2021 ◽  
Vol 9 (5) ◽  
pp. e002198
Author(s):  
Monika Graeser ◽  
Friedrich Feuerhake ◽  
Oleg Gluz ◽  
Valery Volk ◽  
Michael Hauptmann ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Neoadjuvant Chemotherapy ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Immune Cell ◽  
Tumor Stroma ◽  
Cd4 Cells ◽  
Cd8 Cells ◽  
Cell Composition

BackgroundThe association of early changes in the immune infiltrate during neoadjuvant chemotherapy (NACT) with pathological complete response (pCR) in triple-negative breast cancer (TNBC) remains unexplored.MethodsMultiplexed immunohistochemistry was performed in matched tumor biopsies obtained at baseline and after 3 weeks of NACT from 66 patients from the West German Study Group Adjuvant Dynamic Marker-Adjusted Personalized Therapy Trial Optimizing Risk Assessment and Therapy Response Prediction in Early Breast Cancer - Triple Negative Breast Cancer (WSG-ADAPT-TN) trial. Association between CD4, CD8, CD73, T cells, PD1-positive CD4 and CD8 cells, and PDL1 levels in stroma and/or tumor at baseline, week 3 and 3-week change with pCR was evaluated with univariable logistic regression.ResultsCompared with no change in immune cell composition and functional markers, transition from ‘cold’ to ‘hot’ (below-median and above-median marker level at baseline, respectively) suggested higher pCR rates for PD1-positive CD4 (tumor: OR=1.55, 95% CI 0.45 to 5.42; stroma: OR=2.65, 95% CI 0.65 to 10.71) and PD1-positive CD8 infiltrates (tumor: OR=1.77, 95% CI 0.60 to 5.20; stroma: OR=1.25, 95% CI 0.41 to 3.84; tumor+stroma: OR=1.62, 95% CI 0.51 to 5.12). No pCR was observed after ‘hot-to-cold’ transition in PD1-positive CD8 cells. pCR rates appeared lower after hot-to-cold transitions in T cells (tumor: OR=0.26, 95% CI 0.03 to 2.34; stroma: OR=0.35, 95% CI 0.04 to 3.25; tumor+stroma: OR=0.00, 95% CI 0.00 to 1.04) and PD1-positive CD4 cells (tumor: OR=0.60, 95% CI 0.11 to 3.35; stroma: OR=0.22, 95% CI 0.03 to 1.92; tumor+stroma: OR=0.32, 95% CI 0.04 to 2.94). Higher pCR rates collated with ‘altered’ distribution (levels below-median and above-median in tumor and stroma, respectively) of T cell (OR=3.50, 95% CI 0.84 to 14.56) and PD1-positive CD4 cells (OR=4.50, 95% CI 1.01 to 20.14).ConclusionOur exploratory findings indicate that comprehensive analysis of early immune infiltrate dynamics complements currently investigated predictive markers for pCR and may have a potential to improve guidance for individualized de-escalation/escalation strategies in TNBC.

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