Nearly 20 million people are diagnosed with cancer each year with breast cancer being the most common among women. Triple negative breast cancer (TNBC), defined by its no/low expression of ER and PR and lack of amplification of HER2, makes up 15-20% of all breast cancer cases. While patients overall have a higher response to chemotherapy, this subgroup is associated with the lowest survival rate indicating significant clinical and molecular heterogeneity demanding alternate treatment options. Therefore, new therapies have been explored, with a large focus on utilizing the immune system. A whole host of immunotherapies have been studied including immune checkpoint inhibitors, now standard of care for eligible patients, and possibly the most exciting and promising is that of a TNBC vaccine. While currently there are no approved TNBC vaccines, this review highlights many promising studies and points to an antigen, p53, which we believe is highly relevant for TNBC.

Neoadjuvant chemotherapy (NAC) significantly enhances clinical outcomes in patients with triple-negative breast cancer (TNBC); however, chemoresistance frequently results in treatment failure. Consequently, understanding the mechanisms underlying resistance and accurately predicting this phenomenon are crucial for improving treatment efficacy.

Ultrasound images from 62 patients, taken before and after neoadjuvant therapy, were collected. Mitochondrial-related genes were extracted from a public database. Ultrasound features associated with NAC resistance were identified and correlated with significant mitochondrial-related genes. Subsequently, a prognostic model was developed and evaluated using the GSE58812 dataset. We also assessed this model alongside clinical factors and its ability to predict immunotherapy response.

A total of 32 significant differentially expressed genes in TNBC across three groups indicated a strong correlation with ultrasound features. Univariate and multivariate Cox regression analyses identified six genes as independent risk factors for TNBC prognosis. Based on these six mitochondrial-related genes, we constructed a TNBC prognostic model. The model's risk scores indicated that high-risk patients generally have a poorer prognosis compared to low-risk patients, with the model demonstrating high predictive performance (p = 0.002, AUC = 0.745). This conclusion was further supported in the test set (p = 0.026, AUC = 0.718). Additionally, we found that high-risk patients exhibited more advanced tumor characteristics, while low-risk patients were more sensitive to common chemotherapy drugs and immunotherapy. The signature-related genes also predicted immunotherapy response with a high accuracy of 0.765.

We identified resistance-related features from ultrasound images and integrated them with genomic data, enabling effective risk stratification of patients and prediction of the efficacy of neoadjuvant chemotherapy and immunotherapy in patients with TNBC.

Deciphering mechanisms for cancer immune escape may provide targets for improving immunotherapy efficacy. By in vivo genome-wide CRISPR loss-of-function screening in a mouse model of triple negative breast cancer (TNBC), we uncovered a non-classical function of Cd28 in cancer cells to promote immune escape. Knocking out Cd28 in cancer cells increased infiltration of type I conventional DC (cDC1) and activated tumor-specific CD8+ T cells, and pharmaceutical inducible knockdown of Cd28 inhibited pre-established tumor growth and overcame anti-PD-1 resistance in vivo. Furthermore, high expression of cancer cell CD28 in human TNBC tissues correlated with elevated PD-L1 expression, less CD8+ T cell infiltration, and poor prognosis. Mechanistically, intracellular CD28 directly bound to Cd274 mRNA and recruited spliceosomal factor SNRPB2 to stabilize Cd274 mRNA in nucleus, promoting PD-L1 expression and immune escape. Therefore, disrupting cancer cell CD28-mediated immune escape may provide a potential approach to improve breast cancer immunotherapy.

Triple negative breast cancers often contain higher numbers of tumour-infiltrating lymphocytes compared with other breast cancer subtypes, with their number correlating with prolonged survival. Since little is known about tumour-infiltrating lymphocyte trafficking in triple negative breast cancers, we investigated the relationship between tumour-infiltrating lymphocytes and the vascular compartment to better understand the immune tumour microenvironment in this aggressive cancer type. We aimed to identify mechanisms and signaling pathways responsible for immune cell trafficking in triple negative breast cancers, specifically of basal type, that could potentially be manipulated to change such tumours from immune "cold" to "hot" thereby increasing the likelihood of successful immunotherapy in this challenging patient population. We characterised the spatial immune environment in 10 basal breast cancers showing a range of tumour-infiltrating lymphocytes using multiplex fluorescent immunohistochemistry and quantitative digital analysis of CD3+ T cells. We examined their relationship to blood vessels and their activation status as defined by VCAM-1, ICAM-1 and PD-L1. Confirmation of the relationship between tumour-infiltrating lymphocytes and endothelial activation was performed through in silico analysis on TCGA BRCA RNA-seq data (N = 808). Significantly higher CD3+ T cell densities were observed in the stromal compartment compared with the neoplastic cell compartment (P = 0.003). ICAM-1 activated blood vessels were spatially associated with higher CD3+ T cell densities only within 30 microns of blood vessels compared with more distal activated and non-activated blood vessels (P = 0.041). In silico analysis confirmed higher numbers of tumour-infiltrating lymphocytes in basal breast cancers and that higher numbers were significantly associated with endothelial cell activation molecules, co-clustering with upregulated ICAM-1 and VCAM-1 amongst others. PD-L1 was also identified in a subset of blood vessels, suggesting an additional immune regulatory mechanism in endothelial cells. Regulating the activation status of tumour-associated vascular endothelial cells may improve T cell trafficking into basal breast tumours and enhance immunotherapeutic response.

Triple-negative breast cancer (TNBC) is challenging to treat because of its lack of specific molecular targets. The IMMUNOPEG study aimed to evaluate a novel structured method for interpreting TNBC immunohistochemistry specimens processed with VENTANA PD-L1 (SP142) assay. The study involved 10 pathologists who evaluated 50 different immunohistochemistry specimens of TNBC with programmed death ligand 1 (PD-L1) expression considered challenging and that were previously evaluated by the scientific committee, using the NAVIFY Digital Pathology platform. Initially, the overall percent agreement (OPA) was 74%, with a negative percent agreement (NPA) of 68.2% for samples classified as negative, and a positive percent agreement (PPA) of 94.5% for positive samples. After training on the method, the OPA improved significantly to 81.6%, with the NPA increasing to 80.5% and the PPA decreasing to 85.5%. The mean percentage of the tumor area occupied by PD-L1-stained immune cells decreased from 2.5% to 1.6% post-training, approaching to the scientific committee's consensus of 1.029%. The study found that the pathologists' confidence in their assessments increased significantly when using the structured method, which was found to be easy to use by 9 out of 10 pathologists. All pathologists agreed that the structured method was useful for assessing PD-L1 expression. The study suggests that this method has potential value in interpreting challenging cases of PD-L1 immunohistochemistry (IHC) in TNBC. Further refinement and a training protocol may be necessary to enhance the method's efficiency. The potential for generalizing this structured method to other IHC procedures and pathologies warrants additional research.

Committed to our growing effort addressed toward the development of potent anti-breast cancer candidates, new 4-hydrazinylpyrimidine-5-carbonitriles featuring a morpholinyl or piperidinyl moiety at the position-2 and derivatized with various functionalities at the hydrazinyl group were designed through structure optimization, and their antiproliferative potency against two human breast cancer (BC) cell lines, relative to the reference drug 5-FU, was evaluated. Compounds showing remarkable cytotoxic activity versus the hormone dependent MCF-7 cell line (IC50 = 1.62 ± 0.06 µM- 9.88 ± 0.38 µM) and the non-hormone dependent MDA-MB-231 cell line (IC50 = 3.26 ± 0.14 µM-12.93 ± 0.55 µM) were further tested by multiple assays for clarification of their potential activity. Promising derivatives revealing low damage to healthy cells were subject to enzymatic inhibitory assessment against ARO and EGFR and their activities compared to letrozole and erlotinib respectively. Compounds 3c, 6a as well as compounds 4c, 4d proved to be good inhibitors of the ARO and EGFR enzymes respectively. Active compounds were also evaluated for their underlying mode of action by further investigation for CDK, Hsp90, PI3K inhibition and compared to normal MCF-10A cells and assessed for their enhancement of the caspase 9 levels. Additionally, cell cycle analysis and apoptotic induction were performed. They demonstrated remarkable activities in the previous assays and emanated as leads as anti-breast cancer candidates. Eventually, molecular docking analysis revealed that hit compounds 3c, 4c, 4d, and 6a could bind favorably to the proposed in silico models of various protein-ligand interactions. Therefore, our promising top candidates, by demonstrating appreciable anti-breast cancer activities, present valuable prospects for optimization, potency enhancement and future application.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer known for its poor prognosis and the absence of viable targets for standard receptor-based therapies. Several studies have suggested that targeting programmed death-ligand 1 (PD-L1) in tumors that express this biomarker, either on tumor cells and/or in the tumor inflammatory infiltrate, may be beneficial in some patients. This study aimed to assess the overall prevalence of PD-L1 positivity using the SP142 antibody clone in patients with advanced TNBC in Malaysia.

This was a multicenter, cross-sectional prevalence study on PD-L1 positivity among patients with advanced-stage TNBC in Malaysia. Patients were identified using medical records and were enrolled in the study if they met the inclusion criteria. PD-L1 evaluation was performed using archived formalin-fixed paraffin-embedded tissue specimens. Demographic and clinical data were also obtained and summarized using descriptive statistics. The association of these parameters with PD-L1 positivity was assessed using chi-square and logistic regression analysis.

Three medical centers provided 138 complete cases for analysis. Of these 138 cases, 52 (37.7%; 95% confidence interval, 29.6%-46.3%) showed positive PD-L1 expression, defined as immune cell PD-L1 expression ≥ 1%. In a univariate analysis, stage III of the disease and tumor samples from resected specimens were significantly associated with a positive PD-L1 status. However, further assessment using a multivariate model revealed that only resected tumor samples remained significantly associated with PD-L1 positivity after controlling for disease staging.

The prevalence of PD-L1 positivity among patients with stage III or IV TNBC was 37.7%. A significant association was noted between PD-L1 positivity and the tumor tissue obtained from resected specimens. Although the mechanism and clinical significance of this association remain unclear, this finding indicates a possible disparity in the PD-L1 status of samples obtained using surgical resection or biopsy.

In triple-negative breast cancer (TNBC), the tumor immune microenvironment (TIME) is a highly heterogeneous ecosystem that exerts indispensable roles in tumorigenesis and tumor progression. Cancer-associated fibroblasts (CAFs) and cancer-associated adipocytes (CAAs) are the main matrix components in the TIME of TNBC. CAFs mediate the edesmoplastic response, which is a major driver of the immunosuppressive microenvironment to promote tumor growth. In addition, CAAs, a type of tumor-educated adipocyte, participate in crosstalk with breast cancer and are capable of secreting various cytokines, adipokines and chemokines, especially C-C Motif Chemokine Ligand 2 (CCL2), resulting in changes of cancer cell phenotype and function. Therefore, how to treat tumors by regulating the CAFs and the secretion of CCL2 by CAAs in TIME is investigated here. Our research group previously found that rhein (Rhe) has been identified as effective against CAFs, while hesperidin (Hes) could effectively diminish CCL2 secretion by CAAs. Inspired by the above, we developed unique PLGA-based nanoparticles loaded with Rhe and Hes (RH-NP) using the emulsion solvent diffusion method. The RH-NP particles have an average size of 114.1 ± 0.98 nm. RH-NP effectively reduces CAFs and inhibits CCL2 secretion by CAAs, promoting increased infiltration of cytotoxic T cells and reducing immunosuppressive cell presence within tumors. This innovative, safe, low-toxic, and highly effective anti-tumor strategy could be prospective in TNBC treatment.

Breast cancer (BRCA) is among the most prevalent cancers and is responsible for numerous patient fatalities. Immunotherapy has emerged as a promising approach to cancer treatment. Recent studies have identified Siglec-15 as a novel immune target that plays a crucial role in tumor immune evasion, suggesting its potential significance in BRCA. We utilized databases such as TCGA to investigate the relevance of SIGLEC15 in BRCA. The expression of the Siglec-15 protein in 74 breast cancer patients was detected using immunohistochemistry, and its association with clinicopathological features and overall survival was evaluated. The co-expression of Siglec-15, CD68, CK, and CD8 in BRCA tissues was identified through multiplex immunofluorescence staining. Our study revealed that SIGLEC15 expression in BRCA was significantly elevated compared to adjacent normal tissues. Kaplan-Meier analysis identified SIGLEC15 as a prognostic protective factor. According to the receiver operating characteristic curve analysis, SIGLEC15 could predict the luminal subtype of BRCA. Enrichment analysis demonstrated that SIGLEC15 involves various biological pathways, including immunity, metabolism, tumors, and infectious diseases. Correlation analysis revealed an association between SIGLEC15 expression and immune infiltration in BRCA. We also confirmed that the Siglec-15 protein is expressed in cancer cells, tumor-infiltrating T cells, and macrophages in BRCA tissues, significantly higher levels than in normal breast tissues. Consequently, SIGLEC15 correlates with tumor immune infiltration, molecular subtypes, and BRCA progression and prognosis. However, further research is required to elucidate the role of SIGLEC15 in breast cancer.

Tuning immune-cold tumor hot has largely attracted attention to improve cancer treatment, including immunotherapy and antibody-drug conjugates (ADCs). Utilizing multiomic analyses and experimental validation, this work identifies a pivotal role for the USP10/B7-H4 proteolytic axis in mediating the interplay between tumor immune responses and ADC efficacy, particularly for sacituzumab govitecan (SG) in treating triple negative breast cancers (TNBCs). Mechanistically, the inhibition of autocrine motility factor receptor (AMFR)-mediated ubiquitylation of B7-H4 by the deubiquitinase USP10 leads to the stabilization of B7-H4, which suppresses tumor immune activity and reduces SG treatment effectiveness. Pharmacological inhibition of USP10 promotes the degradation of B7-H4, enhancing tumor immunogenicity and consequently improving the tumor-killing efficacy of SG. In preclinical TNBC models, suppression of USP10/B7-H4 proteolytic axis is effective in increasing SG killing efficacy and reducing tumor growth, especially for the tumors with the USP10high/B7-H7high signature. Collectively, these findings uncover a novel strategy for targeting the immunosuppressive molecule B7-H4 for cancer therapy.

Cancer stem cells (CSCs) significantly interfere with immunotherapy, leading to challenges such as low response rates and acquired resistance. PD-L1 expression is associated with the CSC population's overexpression of CD44. Mounting evidence suggests that the breast cancer stem cell (BCSC) marker CD44 and the immune checkpoint PD-L1 contribute to treatment failure through their networks. Natural compounds can overcome therapy resistance in breast cancer by targeting mechanisms underlying resistance in BCSCs. This review provides an updated insight into the CD44 and PD-L1 networks of BCSCs in mediating metastasis and immune evasion. The review critically examines existing literature, providing a comprehensive understanding of the topic and emphasizing the impact of natural flavones on the signaling pathways of BCSCs. Additionally, the review discusses the potential of natural compounds in targeting CD44 and PD-L1 in breast cancer (BC). Natural compounds consistently show potential in targeting regulatory mechanisms of BCSCs, inducing loss of stemness, and promoting differentiation. They offer a promising approach for developing alternative therapeutic strategies to manage breast cancer.

Evaluation of the programmed cell death ligand-1 (PD-L1) combined positive score (CPS) is vital to predict the efficacy of the immunotherapy in triple-negative breast cancer (TNBC), but pathologists show substantial variability in the consistency and accuracy of the interpretation. It is of great importance to establish an objective and effective method which is highly repeatable.

We proposed a model in a deep learning-based framework, which at the patch level incorporated cell analysis and tissue region analysis, followed by the whole-slide level fusion of patch results. Three rounds of ring studies (RSs) were conducted. Twenty-one pathologists of different levels from four institutions evaluated the PD-L1 CPS in TNBC specimens as continuous scores by visual assessment and our artificial intelligence (AI)-assisted method.

In the visual assessment, the interpretation results of PD-L1 (Dako 22C3) CPS by different levels of pathologists have significant differences and showed weak consistency. Using AI-assisted interpretation, there were no significant differences between all pathologists (P = 0.43), and the intraclass correlation coefficient (ICC) value was increased from 0.618 [95% confidence interval (CI) = 0.524-0.719] to 0.931 (95% CI = 0.902-0.955). The accuracy of interpretation result is further improved to 0.919 (95% CI = 0.886-0.947). Acceptance of AI results by junior pathologists was the highest among all levels, and 80% of the AI results were accepted overall.

With the help of the AI-assisted diagnostic method, different levels of pathologists achieved excellent consistency and repeatability in the interpretation of PD-L1 (Dako 22C3) CPS. Our AI-assisted diagnostic approach was proved to strengthen the consistency and repeatability in clinical practice.

Among breast cancers, triple-negative breast cancer (TNBC) has been recognized as the most aggressive type with a poor prognosis and low survival rate. Targeted therapy for TNBC is challenging because it lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Chemotherapy, radiation therapy, and surgery are the common therapies for TNBC. Although TNBC is prone to chemotherapy, drug resistance and recurrence are commonly associated with treatment failure. Combination therapy approaches using chemotherapy, mAbs, ADC, and antibody-siRNA conjugates may be effective in TNBC. Recent advances with siRNA-based therapy approaches are promising for TNBC therapy with better prognosis and reduced mortality. This review discusses advances in nanomaterial- and nanobiomaterial-based siRNA delivery platforms for TNBC therapy exploring targeted therapy approaches for major genes, proteins, and TFs upregulated in TNBC tumors, which engage in molecular pathways associated with low TNBC prognosis. Bioengineered siRNA drugs targeting one or several genes simultaneously can downregulate desired genes, significantly reducing disease progression.

Breast cancer is a heterogeneous disease with various morphologies and molecular features, and it is the second leading cause of cancer death in women in developed countries. According to the literature, we currently lack both prognostic biomarkers and therapeutic targets. The most important prognostic factors are disease stage and Nottingham grade. We conducted a retrospective analysis involving 273 patients with BC who underwent neoadjuvant therapy before proceeding to curative surgical treatment between 1 January 2014 and 31 December 2023. Pathological procedures were conducted at the Department of Pathology, Emergency County Hospital of Targu Mureș, Romania. A statistical analysis was performed. Regarding the relationship between Nottingham grade and Ki67, grade I was associated with a Ki67 of less than 14. The relationship between tumor grade and luminal was similar (p = 0.0001): Grade I was associated with luminal A. Regarding TNM stage, it was statistically significantly correlated with TILs (p = 0.01) and RCB (p = 0.0001). Stages III and IV were associated with a high RCB and poor prognosis. Regarding the prognostic value, Nottingham grade 3 and TNM stages III and IV were correlated with low overall survival and disease-free survival, with poor prognosis, and, among the molecular variables, RCB played the most important prognostic role.

Accounting for 10-20% of breast cancer cases, triple-negative breast cancer (TNBC) is associated with a disproportionate number of breast cancer deaths. One challenge in studying TNBC is its genomic profile: with the exception of TP53 loss, most breast cancer tumors are characterized by a high number of copy number alterations (CNAs), making modeling the disease in whole animals challenging. We computationally analyzed 186 CNA regions previously identified in breast cancer tumors to rank genes within each region by likelihood of acting as a tumor driver. We then used a Drosophila p53-Myc TNBC model to identify 48 genes as functional drivers. To demonstrate the utility of this functional database, we established six 3-hit models; altering candidate genes led to increased aspects of transformation as well as resistance to the chemotherapeutic drug fluorouracil. Our work provides a functional database of CNA-associated TNBC drivers, and a template for an integrated computational/whole-animal approach to identify functional drivers of transformation and drug resistance within CNAs in other tumor types.

Immune checkpoint inhibitors (ICIs) in combination with chemotherapy have showed its benefits in clinical studies, and here we conducted a further evaluation on the safety and efficacy of this treatment strategy.

A systematic literature review was conducted in PubMed, Embase and Cochrane Library to identify clinical studies on ICIs and chemotherapy for metastatic breast cancer. The primary efficacy endpoints were progression-free survival (PFS) and overall survival (OS), and adverse events (AEs) were analyzed. Random or fixed effects models were used to estimate pooled Hazard ratio (HR), odds ratio (OR) and the data of 95% confidence interval (CI) depend on the Heterogeneity. Cochrane risk assessment tool was used to assess risk of bias. We also drew forest plots and funnel plots, respectively.

Seven studies with intend-to-treat (ITT) population for 3255 patients were analyzed. ICIs pooled therapy showed clinical benefits compared with chemotherapy alone, improving PFS (HR = 0.81, 95% CI: 0.74-0.90) of patients with metastatic triple negative breast cancer (mTNBC), especially in patients with PD-L1-positive tumors. However, it had no effect on OS (HR = 0.92, 95% CI 0.85-1.01). Besides, mTNBC patients received pooled therapy were less frequently to have AEs (OR = 1.30, 95% CI: 1.09-1.54). In patients with metastatic Human Epidermal Growth Factor Receptor 2 (HER2) negative breast cancer, pooled therapy showed no benefit for PFS (HR = 0.80, 95% CI: 0.50-1.28) and OS (HR = 0.87, 95% CI: 0.48-1.58).

Pooled therapy had improved PFS in mTNBC patients, especially in patients with PD-L1-positive tumors, and it was less likely to cause grade ≥ 3 AEs.

Breast cancer characterized as "cold tumors" exhibit low levels of immune cell infiltration, which limits the efficacy of conventional immunotherapy. Recent studies have focused on strategies using nanotechnology combined with tumor microenvironment modulation to transform "cold tumors" into "hot tumors". This approach involves the use of functionalized nanoparticles that target and modify the tumor microenvironment to promote the infiltration and activation of antitumor immune cells. By delivering immune activators or blocking immunosuppressive signals, these nanoparticles activate otherwise dormant immune responses, enhancing tumor immunogenicity and the therapeutic response. These strategies not only promise to increase the response rate of breast cancer patients to existing immunotherapies but also may pave new therapeutic avenues, providing a new direction for the immunotherapy of breast cancer.

Immune-positron emission tomography (PET) imaging with tracers that target CD8 and granzyme B has shown promise in predicting the therapeutic response following immune checkpoint blockade (ICB) in immunologically "hot" tumors. However, immune dynamics in the low T-cell infiltrating "cold" tumor immune microenvironment during ICB remain poorly understood. This study uses molecular imaging to evaluate changes in CD4 + T cells and CD8 + T cells during ICB in breast cancer models and examines biomarkers of response.

[89Zr]Zr-DFO-CD4 and [89Zr]Zr-DFO-CD8 radiotracers were used to quantify changes in intratumoral and splenic CD4 T cells and CD8 T cells in response to ICB treatment in 4T1 and MMTV-HER2 mouse models, which represent immunologically "cold" tumors. A correlation between PET quantification metrics and long-term anti-tumor response was observed. Further biological validation was obtained by autoradiography and immunofluorescence.

Following ICB treatment, an increase in the CD8-specific PET signal was observed within 6 days, and an increase in the CD4-specific PET signal was observed within 2 days in tumors that eventually responded to immunotherapy, while no significant differences in CD4 or CD8 were found at the baseline of treatment that differentiated responders from nonresponders. Furthermore, mice whose tumors responded to ICB had a lower CD8 PET signal in the spleen and a higher CD4 PET signal in the spleen compared to non-responders. Intratumoral spatial heterogeneity of the CD8 and CD4-specific PET signals was lower in responders compared to non-responders. Finally, PET imaging, autoradiography, and immunofluorescence signals were correlated when comparing in vivo imaging to ex vivo validations.

CD4- and CD8-specific immuno-PET imaging can be used to characterize the in vivo distribution of CD4 + and CD8 + T cells in response to immune checkpoint blockade. Imaging metrics that describe the overall levels and distribution of CD8 + T cells and CD4 + T cells can provide insight into immunological alterations, predict biomarkers of response to immunotherapy, and guide clinical decision-making in those tumors where the kinetics of the response differ.

Granzyme K (GZMK) is a crucial mediator released by immune cells to eliminate tumor cells, playing significant roles in inflammation and tumorigenesis. Despite its importance, the specific role of GZMK in breast cancer and its mechanisms are not well understood.

We utilized data from the TCGA and GEO databases and employed a range of analytical methods including GO, KEGG, GSEA, ssGSEA, and PPI to investigate the impact of GZMK on breast cancer. In vitro studies, including RT-qPCR, CCK-8 assay, cell cycle experiments, apoptosis assays, Celigo scratch assays, Transwell assays, and immunohistochemical methods, were conducted to validate the effects of GZMK on breast cancer cells. Additionally, Cox regression analysis integrating TCGA and our clinical data was used to develop an overall survival (OS) prediction model.

Analysis of clinical pathological features revealed significant correlations between GZMK expression and lymph node staging, differentiation grade, and molecular breast cancer subtypes. High GZMK expression was associated with improved OS, progression-free survival (PFS), and recurrence-free survival (RFS), as confirmed by multifactorial Cox regression analysis. Functional and pathway enrichment analyses of genes positively correlated with GZMK highlighted involvement in lymphocyte differentiation, T cell differentiation, and T cell receptor signaling pathways. A robust association between GZMK expression and T cell presence was noted in the breast cancer tumor microenvironment (TME), with strong correlations with ESTIMATEScore (Cor = 0.743, P < 0.001), ImmuneScore (Cor = 0.802, P < 0.001), and StromalScore (Cor = 0.516, P < 0.001). GZMK also showed significant correlations with immune checkpoint molecules, including CTLA4 (Cor = 0.856, P < 0.001), PD-1 (Cor = 0.82, P < 0.001), PD-L1 (Cor = 0.56, P < 0.001), CD48 (Cor = 0.75, P < 0.001), and CCR7 (Cor = 0.856, P < 0.001). Studies indicated that high GZMK expression enhances patient responsiveness to immunotherapy, with higher levels observed in responsive patients compared to non-responsive ones. In vitro experiments confirmed that GZMK promotes cell proliferation, cell division, apoptosis, cell migration, and invasiveness (P < 0.05).

Our study provides insights into the differential expression of GZMK in breast cancer and its potential mechanisms in breast cancer pathogenesis. Elevated GZMK expression is associated with improved OS and RFS, suggesting its potential as a prognostic marker for breast cancer survival and as a predictor of the efficacy of immunotherapy.

Breast cancer is the most commonly diagnosed cancer in women and is a leading cause of cancer death in women worldwide. Despite the implementation of multiple treatment options, including immunotherapy, breast cancer treatment remains a challenge. In this review, we aim to summarize present challenges in breast cancer immunotherapy and recent advancements in overcoming treatment resistance. We elaborate on the inhibition of signaling cascades, such as the Notch, Hedgehog, Hippo, and WNT signaling pathways, which regulate the self-renewal and differentiation of breast cancer stem cells and, consequently, disease progression and survival. Cancer stem cells represent a rare population of cancer cells, likely originating from non-malignant stem or progenitor cells, with the ability to evade immune surveillance and develop resistance to immunotherapeutic treatments. We also discuss the interactions between breast cancer stem cells and the immune system, including potential agents targeting breast cancer stem cell-associated signaling pathways, and provide an overview of the emerging approaches to breast cancer stem cell-targeted immunotherapy. Finally, we consider the development of breast cancer vaccines and adoptive cellular therapies, which train the immune system to recognize tumor-associated antigens, for eliciting T cell-mediated responses to target breast cancer stem cells.

Although ongoing studies are assessing the efficacy of new systemic therapies for patients with triple negative breast cancer (TNBC), the overwhelming majority have excluded patients with brain metastases (BM). Therefore, we aim to characterize systemic therapies and outcomes in a cohort of patients with TNBC and BM managed with stereotactic radiosurgery (SRS) and delineate predictors of increased survival.

We used our prospective patient registry to evaluate data from 2012 to 2023. We included patients who received SRS for TNBC-BM. A competing risk analysis was conducted to assess local and distant control.

Forty-three patients with 262 tumors were included. The median overall survival (OS) was 16 months (95% CI 13-19 months). Predictors of increased OS after initial SRS include Breast GPA score > 1 (p < 0.001) and use of immunotherapy such as pembrolizumab (p = 0.011). The median time on immunotherapy was 8 months (IQR 4.4, 11.2). The median time to new CNS lesions after the first SRS treatment was 17 months (95% CI 12-22). The cumulative rate for development of new CNS metastases after initial SRS at 6 months, 1 year, and 2 years was 23%, 40%, and 70%, respectively. Thirty patients (70%) underwent multiple SRS treatments, with a median time of 5 months (95% CI 0.59-9.4 months) for the appearance of new CNS metastases after second SRS treatment.

TNBC patients with BM can achieve longer survival than might have been previously anticipated with median survival now surpassing one year. The use of immunotherapy is associated with increased median OS of 23 months.

This literature review provides a comprehensive overview of triple-negative breast cancer (TNBC) and explores innovative targeted therapies focused on specific hallmarks of cancer cells, aiming to revolutionize breast cancer treatment. TNBC, characterized by its lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), presents distinct features, categorizing these invasive breast tumors into various phenotypes delineated by key elements in molecular assays. This article delves into the latest advancements in therapeutic strategies targeting components of the tumor microenvironment and pivotal hallmarks of cancer: deregulating cellular metabolism and the Warburg effect, acidosis and hypoxia, the ability to metastasize and evade the immune system, aiming to enhance treatment efficacy while mitigating systemic toxicity. Insights from in vitro and in vivo studies and clinical trials underscore the promising effectiveness and elucidate the mechanisms of action of these novel therapeutic interventions for TNBC, particularly in cases refractory to conventional treatments. The integration of targeted therapies tailored to the molecular characteristics of TNBC holds significant potential for optimizing clinical outcomes and addressing the pressing need for more effective treatment options for this aggressive subtype of breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel has been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetics, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies.

This work describes the development of novel biomimetic and carrier-free nanobinders (NBs) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive and biomimetic prodrugs. A fine tuning of the preparation conditions allowed to identify NB@5 as the most suitable nanoformulation in terms of reproducibility, stability and in vitro effectiveness.

Our data show that NB@5 effectively binds to HSA in cell-free experiments, demonstrating its protective role in the controlled release of drugs and suggesting the potential to exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully proves that the drugs encapsulated within the NBs are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1.

Recent investigations have reported the benefits of using a tyrosine kinase inhibitor, dasatinib (DA), as well as programmed death-ligand 1 (PD-L1) inhibitors in the management of several solid tumors, including breast cancer. Nevertheless, the outcome of the combination of these inhibitors on HER2-positive breast cancer is not explored yet.

Herein, we investigated the impact of DA and PD-L1 inhibitor (BMS-202) combination on HER2-positive breast cancer cell lines, SKBR3 and ZR75.

Our data reveal that the combination significantly inhibits cell viability of both cancer cell lines as compared to monotreatment. Moreover, the combination inhibits epithelial-mesenchymal transition (EMT) progression and reduces cancer cell invasion by restoring E-cadherin and β-catenin expressions and loss of vimentin, major biomarkers of EMT. Additionally, the combination reduces the colony formation of both cell lines in comparison with their matched control. Also, the combination considerably inhibits the angiogenesis of the chorioallantoic membrane model compared with monotreatment. Molecular pathway analysis of treated cells shows that this combination blocks HER2, AKT, β-catenin, and JNK1/2/3 activities.

Our findings implicate that a combination of DA and BMS-202 could have a significant impact on the management of HER2-positive breast cancer.

Triple-negative breast cancer (TNBC) comprises 15-20% of all breast cancers (BC). Lacking targeted therapy options, TNBC becomes the focal point of clinical investigations aiming not only to identify drugs with enhanced response potential but also to uncover new immunological and/or metabolic pathways conducive to more effective treatments. Currently, neoadjuvant treatment for TNBC relies on standard chemotherapy in conjunction with immunotherapy, given the improved response observed with this drug combination. This review delves into the latest therapeutic updates in TNBC treatment and explores potential advancements shaping the future landscape of this disease in the neoadjuvant setting.

Effective targeted therapeutic strategies for triple-negative breast cancer (TNBC), the most malignant subtype of breast cancer, are currently lacking. Ferroptosis has been reported to be associated with the onset and advancement of various cancer types, including TNBC. However, there are limited studies on the correlation between TNBC and ferroptosis-related genes. In addition, the potential biomarkers of ferroptosis in TNBC need further investigation. The present study aimed to assess the prognostic role of a novel ferroptosis-related gene signature in the context of TNBC. The signature was established utilizing The Cancer Genome Atlas dataset. This three-gene model [transferrin receptor 2 (TFR2), regulator of G protein signaling 4 and zinc finger protein 36] was developed utilizing least absolute shrinkage and selection operator regression analysis and demonstrated satisfactory predictive performance in TNBC. The area under the curve values of the receiver operating characteristic curves in this model concerning the 1-, 2- and 3-year survival prediction were 0.721, 0.840 and 0.856, respectively. The predictive performance of the model was verified using the TNBC dataset GSE25307. Gene set enrichment analysis (GSEA) demonstrated the enrichment of genes in the low-risk group in a number of important metabolic pathways. Single-sample GSEA demonstrated a variation in the expression levels of immune checkpoint molecules between the high- and low-risk groups. The inhibitory impact of TFR2 knockdown on the proliferative capacity of TNBC cells was verified through in vitro experiments. The data also demonstrated that TFR2 knockdown facilitated the ferroptosis of TNBC cells. Additional assessments indicated that the effects of TFR2 knockdown were partially reversed upon treatment with the ferroptosis inhibitor ferrostatin-1. In conclusion, in the present study, a novel and accurate ferroptosis-related predictive signature was established for TNBC with potential future clinical applications. To the best of our knowledge, the present study is the first to report that TFR2 regulated ferroptosis in TNBC cells in vitro.

Immune checkpoint blockade (ICB) therapy offers promise in the treatment of triple-negative breast cancer (TNBC); however, its limited efficacy in certain TNBC patients poses a challenge. In this study, we elucidated the metabolic mechanism at 'sub-subtype' resolution underlying the non-response to ICB therapy in TNBC. Here, an analytic pipeline was developed to reveal the metabolic heterogeneity, which is correlated with the ICB outcomes, within each immune cell subtype. First, we identified metabolic 'sub-subtypes' within certain cell subtypes, predominantly T cell subsets, which are enriched in ICB non-responders and named as non-responder-enriched (NR-E) clusters. Notably, most of NR-E T metabolic cells exhibit globally higher metabolic activities compared to other cells within the same individual subtype. Further, we investigated the extra-cellular signals that trigger the metabolic status of NR-E T cells. In detail, the prediction of cell-to-cell communication indicated that NR-E T cells are regulated by plasmatic dendritic cells (pDCs) through TNFSF9, as well as by macrophages expressing SIGLEC9. In addition, we also validate the communication between TNFSF9+ pDCs and NR-E T cells utilizing deconvolution of spatial transcriptomics analysis. In summary, our research identified specific metabolic 'sub-subtypes' associated with ICB non-response and uncovered the mechanisms of their regulation in TNBC. And the proposed analytical pipeline can be used to examine metabolic heterogeneity within cell types that correlate with diverse phenotypes.

To assess the cost-effectiveness of sacituzumab govitecan for treating relapsed or refractory metastatic triple-negative breast cancer (TNBC) in Singapore.

A three-state partitioned survival model was developed to evaluate the cost-effectiveness of sacituzumab govitecan from a healthcare system perspective over 5 years. Clinical inputs were obtained from the ASCENT trial. Health state utilities were retrieved from the literature and direct costs were sourced from public healthcare institutions in Singapore. Sensitivity and scenario analyses were conducted to explore the impact of uncertainties and assumptions on cost-effectiveness results.

Compared with single-agent chemotherapy, sacituzumab govitecan was associated with a base-case incremental cost-effectiveness ratio (ICER) of S$328,000 (US$237,816) per quality-adjusted life year (QALY) gained. One-way sensitivity analyses showed that the ICER was most sensitive to the cost of sacituzumab govitecan and progression-free utility values. Regardless of variation in these parameters, the ICER remained high, and a substantial price reduction was required to reduce the ICER.

At its current price, sacituzumab govitecan does not represent a cost-effective treatment for relapsed or refractory metastatic TNBC in Singapore. Our findings will be useful to inform funding decisions alongside other factors including clinical effectiveness, safety, and budget impact considerations.

FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1) is a member of the methyltransferase superfamily and is involved in the processing and modification of ribosomal RNA. We herein demonstrate that FTSJ1 favors TNBC progression. The knockdown of FTSJ1 inhibits TNBC cell proliferation and development, induces apoptosis of cancer cells, and increases the sensitivity of TNBC cells to T-cell-mediated cytotoxicity. Furthermore, the high expression of FTSJ1 in TNBC attenuates CD8+T cell infiltration in the tumor microenvironment (TME) correlated with poorer prognosis for clinical TNBC patients. In this study, we establish that FTSJ1 acts as a tumor promotor, is involved in cancer immune evasion, and may serve as a potential immunotherapy target in TNBC.

Despite radiotherapy ability to significantly improve treatment outcomes and survival in triple-negative breast cancer (TNBC) patients, acquired resistance to radiotherapy poses a serious clinical challenge. Protein disulfide isomerase exists in endoplasmic reticulum and plays an important role in promoting protein folding and post-translational modification. However, little is known about the role of protein disulfide isomerase family member 4 (PDIA4) in TNBC, especially in the context of radiotherapy resistance.

We detected the presence of PDIA4 in TNBC tissues and paracancerous tissues, then examined the proliferation and apoptosis of TNBC cells with/without radiotherapy. As part of the validation process, xenograft tumor mouse model was used. Mass spectrometry and western blot analysis were used to identify PDIA4-mediated molecular signaling pathway.

Based on paired clinical specimens of TNBC patients, we found that PDIA4 expression was significantly higher in tumor tissues compared to adjacent normal tissues. In vitro, PDIA4 knockdown not only increased apoptosis of tumor cells with/without radiotherapy, but also decreased the ability of proliferation. In contrast, overexpression of PDIA4 induced the opposite effects on apoptosis and proliferation. According to Co-IP/MS results, PDIA4 prevented Tax1 binding protein 1 (TAX1BP1) degradation by binding to TAX1BP1, which inhibited c-Jun N-terminal kinase (JNK) activation. Moreover, PDIA4 knockdown suppressed tumor growth xenograft model in vivo, which was accompanied by an increase in apoptosis and promoted tumor growth inhibition after radiotherapy.

The results of this study indicate that PDIA4 is an oncoprotein that promotes TNBC progression, and targeted therapy may represent a new and effective anti-tumor strategy, especially for patients with radiotherapy resistance.

Annually, a significant number of individuals succumb to cancer, an anomalous cellular condition characterized by uncontrolled cellular proliferation and the emergence of highly perilous tumors. Identifying underlying molecular mechanism(s) driving disease progression has led to various inventive therapeutic approaches, many of which are presently under pre-clinical and/or clinical trials. Over the recent years, numerous alternative strategies for addressing cancer have also been proposed and put into practice. This article delineates the modern therapeutic drugs employed in cancer treatment and their associated toxicity. Due to inherent drug toxicity associated with most modern treatments, demand rises for alternative therapies and phytochemicals with minimal side effects and proven efficacy against cancer. Analogs of taxol, Vinca alkaloids like vincristine and vinblastine, and podophyllotoxin represent a few illustrative examples in this context. The phytochemicals often work by modifying the activity of molecular pathways that are thought to be involved in the onset and progression of cancer. The principal objective of this study is to provide an overview of our current understanding regarding the pharmacologic effects and molecular targets of the active compounds found in natural products for cancer treatment and collate information about the recent advancements in this realm. The authors' interest in advancing the field of phytochemical research stems from both the potential of these compounds for use as drugs as well as their scientific validity. Accordingly, the significance of herbal formulations is underscored, shedding light on anticancer phytochemicals that are sought after at both pre-clinical and clinical levels, with discussion on the opportunities and challenges in pre-clinical and clinical cancer studies.

The advent of immune checkpoint inhibitors marks significant progress in the evolution of cancer treatment. Recent clinical trials have demonstrated the success of immune-oncologic (IO) agents like pembrolizumab (Keytruda™) in combination with chemotherapy against triple-negative breast cancer (TNBC) [Ann Oncol. 2017 Jun 1;28(6):1388-1398]. There is less literature investigating pembrolizumab in monotherapy and in cases of rare tumor mutational burden.

Here, we report the case of a 65-year-old Native American and African American woman with previous incomplete lines of therapy diagnosed with recurrent TNBC and pulmonary metastases. Next-generation sequencing of the metastatic nodules demonstrated a significantly hypermutated tumor with rare polyploidy. The patient had a durable (14 months) response and ongoing remission of the metastatic lesions after administering the programmed cell death 1 inhibitor pembrolizumab. No serious immune checkpoint inhibitor-related toxicities or disease progression was observed during the treatment.

Our report describes recurrent TNBC with a rare amount of hypermutation and the successful use of an IO agent as a treatment.

Triple negative breast cancer (TNBC) cells resist chemotherapy by hijacking apoptosis. Alternative cell death forms like ferroptosis offer new treatment options. A combined therapy using neratinib (NTB; ferroptosis inducer) and silibinin (SLB; apoptosis inducer) via albumin-based nanocarriers (N-S Alb NPs) was explored to target TNBC. N-S Alb NPs had optimal size (134.26 ± 10.23 nm), PDI (0.224 ± 0.01), and % entrapment efficiency (∼80 % for NTB and ∼87 % for SLB). Transmission electron microscopy confirmed their spherical shape. In vitro release studies showed sustained drug release without hemolysis risk. N-S Alb NPs had higher cellular uptake and cytotoxicity than individual drugs or their mixture. IC50 values for N-S Alb NPs were significantly reduced in MDA-MB-231 (∼2.23-fold) and 4T1 (∼1.85-fold) cell lines and apoptosis index were significantly higher in MDA-MB-231 (∼1.31-fold) and 4T1 cell line (∼1.35-fold) than the physical mixture of both drugs (NTB + SLB). N-S Alb NPs generated more reactive oxygen species (ROS) and caused mitochondrial membrane depolarization, indicating increased cell death. They also exhibited better ferroptosis induction by reducing glutathione (GSH), increasing Fe2+ activity and MDA levels in TNBC cells. Thus, N-S Alb NPs had the ability to promote "mixed" type cell death, showed promise in enhancing the payload capabilities and targeting in TNBC.

Immunotherapy of triple-negative breast cancer (TNBC) has an unsatisfactory therapeutic outcome due to an immunologically "cold" microenvironment. Fusobacterium nucleatum (F. nucleatum) was found to be colonized in triple-negative breast tumors and was responsible for the immunosuppressive tumor microenvironment and tumor metastasis. Herein, we constructed a bacteria-derived outer membrane vesicle (OMV)-coated nanoplatform that precisely targeted tumor tissues for dual killing of F. nucleatum and cancer cells, thus transforming intratumor bacteria into immunopotentiators in immunotherapy of TNBC. The as-prepared nanoparticles efficiently induced immunogenic cell death through a Fenton-like reaction, resulting in enhanced immunogenicity. Meanwhile, intratumoral F. nucleatum was killed by metronidazole, resulting in the release of pathogen-associated molecular patterns (PAMPs). PAMPs cooperated with OMVs further facilitated the maturation of dendritic cells and subsequent T-cell infiltration. As a result, the "kill two birds with one stone" strategy warmed up the cold tumor environment, maximized the antitumor immune response, and achieved efficient therapy of TNBC as well as metastasis prevention. Overall, this strategy based on a microecology distinction in tumor and normal tissue as well as microbiome-induced reversal of cold tumors provides new insight into the precise and efficient immune therapy of TNBC.

Approximately, 15% of global breast cancer cases are diagnosed as triple-negative breast cancer (TNBC), identified as the most aggressive subtype due to the simultaneous absence of estrogen receptor, progesterone receptor, and HER2. This characteristic renders TNBC highly aggressive and challenging to treat, as it excludes the use of effective drugs such as hormone therapy and anti-HER2 agents. In this review, we explore standard therapies and recent emerging approaches for TNBC, including PARP inhibitors, immune checkpoint inhibitors, PI3K/AKT pathway inhibitors, and cytotoxin-conjugated antibodies. The mechanism of action of these drugs and their utilization in clinical practice is explained in a pragmatic and prospective manner, contextualized within the current landscape of standard therapies for this pathology. These advancements present a promising frontier for tailored interventions with the potential to significantly improve outcomes for TNBC patients. Interestingly, while TNBC poses a complex challenge, it also serves as a paradigm and an opportunity for translational research and innovative therapies in the field of oncology.

Breast cancer is currently the most prevalent form of cancer worldwide. Nevertheless, there remains limited clarity regarding our understanding of the tumor microenvironment and metabolic characteristics associated with it. ATP-binding cassette (ABC) transporters are the predominant transmembrane transporters found in organisms. Therefore, it is essential to investigate the role of ABC transporters in breast cancer. Transcriptome data from breast cancer patients were downloaded from the TCGA database. ABC transporter-related genes were obtained from the Genecards database. By LASSO regression, ABC-associated prognostic signature was constructed in breast cancer. Subsequently, immune microenvironment analysis was performed. Finally, cell experiments were performed to verify the function of ABCB7 in the breast cancer cell lines MDA-MB-231 and MCF-7. Using the ABC transporter-associated signature, we calculated a risk score for each breast cancer patient. Patients with breast cancer were subsequently categorized into high-risk and low-risk groups, utilizing the median risk score as the threshold. Notably, patients in the high-risk group exhibited significantly worse prognosis (P<0.05). Additionally, differences were observed in terms of immune cell infiltration levels, immune correlations, and gene expression of immune checkpoints between the two groups. Functional experiments conducted on breast cancer cell lines MDA-MB-231 and MCF-7 demonstrated that ABCB7 knockdown significantly diminished cell activity, proliferation, invasion, and migration. These findings emphasize the significance of understanding ABC transporter-mediated metabolic and transport characteristics in breast cancer, offering promising directions for further research and potential therapeutic interventions.

PD-L1 expression is known to predict the benefits of immune checkpoint inhibitor therapy for triple-negative breast cancer (TNBC). We examined whether the PD-L1 expression evaluated in biopsy specimens accurately reflects its expression in the whole tumor. Immunohistochemistry was performed on 81 biopsy and resection specimens from patients with TNBC to determine their PD-L1 status. We found PD-L1-positive tumors in 23 (28%) biopsy specimens and primarily PD-L1-negative tumors in 58 (72%). The PD-L1 status was reevaluated in matching postoperative specimens of primarily PD-L1-negative tumors. Of them, 31% (18/58) were positive, whereas 69% (40/58) were negative. Considering the pre- and postoperative analyses, 41 (51%) patients had PD-L1-positive tumors, while 40 had PD-L1-negative tumors. We found 18 (22%) more PD-L1-positive tumors while examining the resection specimens compared to biopsies, and the difference was statistically significant (p = 0.0038). Diagnostic biopsies do not fully reflect the PD-L1 expression in TNBC. Our results suggest that a significant subset of TNBC patients may be misclassified as PD-L1-negative and disqualified from anti-PD-L1 therapy.

Metastasis is the leading cause of breast cancer-associated death. Lung metastasis commonly occurs in triple-negative breast cancer (TNBC) metastasis, worsening the TNBC prognosis. Considering their role in tumor progression and metastasis, tumor-associated macrophages (TAMs) are essential therapeutic targets in cancer therapy. Previous studies have demonstrated that honokiol inhibits tumor growth and progression. Here we assessed how honokiol inhibits lung metastasis of TNBC by regulating the polarization of macrophages. We found that honokiol decreased the expression of IL-13-triggered M2 markers like CD206, Arg1, and CCL2, preventing the invasion and migration ability of TNBC cells. The activation of signal transducer and activator of transcription STAT6 and STAT3 was significantly suppressed by honokiol in M2 polarized macrophages. Meanwhile, honokiol increased the expression of LPS/IFNγ-induced M1 markers such as CD11c, iNOS, and IL12 by promoting STAT1 phosphorylation. Besides, honokiol decreased both the ratio of M2/M1 macrophages and the expression of the IL-10/IL-12 gene in lung tissues, thereby inhibiting the proliferation and metastasis of murine breast cancer. Moreover, honokiol reduced the infiltration of macrophages to the lung tissue through the CCL2/CCR2 pathways. These results highlight the potential of honokiol in suppressing TNBC tumor progression and lung metastasis by regulating the polarization and recruitment of macrophages.