Triple negative breast cancer (TNBC) stands out among breast cancers subtypes for its high aggressiveness and invasiveness. Compelling new evidence pointed out the role of epigenetic modifications in TNBC, with recent studies demonstrating that approximately 30 % of human breast cancers could potentially benefit from histone deacetylase 6 (HDAC6) inhibitor therapy. We herein disclose a novel class of spiro-fused compounds acting as potent and selective HDAC6 inhibitors. Structure-based optimization led to derivatives 23c and 24c with high potency and selectivity towards HDAC6 in vitro and in cell-based settings. Following our observation that mRNA expression level of HDAC6 was significantly higher in MDA-MB-231, we have evaluated the effect of the compounds on cell viability. Moreover, we have unveiled for compounds 23c and 24c the involvement of the autophagic machinery in cell death induction. Scratch assay revealed for the newly conceived compounds a very potent effect on inhibiting the migration process in MDA-MB-231 cells. Our results underscore the key role of HDAC6 in TNBC progression, providing a solid groundwork to reshape TNBC therapy.

Triple-negative breast cancer (TNBC) represents a significant therapeutic challenge owing to the scarcity of targeted medicines and elevated recurrence rates. We previously reported the development of the nuclease-resistant RNA sTN58 aptamer, which selectively targets TNBC cells. Here, sTN58 aptamer was employed to capture and purify its binding target from the membrane protein fraction of cisplatin-resistant mesenchymal stem-like TNBC cells. Mass spectrometry in conjunction with aptamer binding assays across various cancer cell lines identified CD44 as the cellular target of sTN58. By binding to CD44, sTN58 inhibits the invasive growth and hyaluronic acid-dependent tube formation in chemoresistant TNBC cells, where CD44 serves as a key driver of tumor cell aggressiveness and stem-like plasticity. Moreover, in vivo studies demonstrated the aptamer's high tumor targeting efficacy and its capacity to significantly inhibit tumor growth and lung metastases following intravenous administration in mice with orthotopic TNBC. Overall, our findings reveal the striking potential of sTN58 as a targeting reagent for the recognition and therapy of cancers overexpressing CD44.

Breast cancer affects a significant number of patients worldwide, and early diagnosis is critical for improving cure rates and prognosis. Deep learning-based breast cancer classification algorithms have substantially alleviated the burden on medical personnel. However, existing breast cancer diagnosis models face notable limitations which are challenging to obtain in clinical settings, such as reliance on a large volume of labeled samples, an inability to comprehensively extract features from breast cancer images, and susceptibility to overfitting on account of imbalanced class distribution. Therefore, we propose the class-aware multi-level attention learning model focused on semi-supervised breast cancer diagnosis to effectively reduce the dependency on extensive data annotation. Additionally, we develop the multi-level fusion attention learning module, which integrates multiple mutual attention components across different layers, allowing the model to precisely identify critical regions for lesion categorization. Finally, we design the class-aware adaptive pseudo-labeling module which adaptively predicts category distribution in unlabeled data, and directs the model to focus on underrepresented categories, ensuring a balanced learning process. Experimental results on the BACH dataset demonstrate that our proposed model achieves an accuracy of 86.7% with only 40% labeled microscopic data, showcasing its outstanding contribution to semi-supervised breast cancer diagnosis.

Triple-negative breast cancer (TNBC) presents therapeutic challenges due to its aggressive, drug-resistance, and low immunological reactivity. Cuproptosis, an emerging therapeutic modality, is a promising strategic intervention for treating TNBC. Nonetheless, the effectiveness of cuproptosis is compromised by tumor adaptations, including the Warburg effect, increased intracellular glutathione (GSH), and copper efflux, thus breaking the barrier of cuproptosis is the basis for developing cuproptosis-based clinical therapies. Herein, a self-accelerating strategy utilizing a pH-responsive copper framework encapsulating glucose oxidase (GOx), modified with polyethylene glycol (PEG) and tumor-penetrating peptide (tLyp1) has been developed. Upon reaching the acidic tumor microenvironment, the released GOx increases intracellular acidity and hydrogen peroxide (H2O2). The elevated intracellular GSH and H2O2 serve as "fuel" to amplify the copper-based catalytic within tumor cells. Concurrently, the reduction of copper efflux proteins (ATP7B) and the depletion of GSH lead to copper overload in tumor cells, leading to cuproptosis via copper overload, mitochondrial disruption, and Fe-S protein instability. This constellation of interrelated events constitutes a potent "Copper Bomb," which concurrently triggers the immune system and effectively kills the tumor. It robustly engages innate and adaptive immunity via the release of mitochondrial DNA, facilitating the cGAS-STING pathway and precipitating immunogenic cell death. This process reverses the immunosuppressive tumor microenvironment, eliminates tumor cells, and suppresses metastasis, thus offering a novel therapeutic modality for the comprehensive treatment of triple-negative breast cancer (TNBC).

Breast cancer patients with estrogen receptor-negative (ERneg) status, encompassing triple negative breast cancer (TNBC) and human epidermal growth factor receptor 2 positive breast cancer, are confronted with a heightened risk of drug resistance, often leading to early recurrence; the biomarkers and biological processes associated with recurrence is still unclear. In this study, we analyzed bulk RNA sequencing (RNA-seq) data from 285 cancer and paracancerous samples from 155 TNBC patients, along with transcriptome data from 11 independent public cohorts comprising 7,449 breast cancer patients and 26 single-cell RNA-seq datasets. Our results revealed differential enrichment of nerve-related pathways between TNBC patients with and without 10-year recurrence-free survival. We developed an early recurrence index (ERI) using a machine learning model and constructed a nomogram that accurately predicts the 10-year survival of ERneg patients (area under the curve [AUC]Training = 0.79; AUCTest = 0.796). Further analysis linked ERI to enhanced neural function and immunosuppression. Additionally, we identified EN1, the most significant ERI gene, as a potential biomarker that may regulate the tumor microenvironment and sensitize patients to immunotherapy.

The immunosuppressive tumor microenvironment (TME) poses a critical barrier to the efficacy of immune checkpoint inhibitors in triple-negative breast cancer (TNBC). Here, we report a self-assembled polymeric nanoplatform coloading the photosensitizer verteporfin and the Wnt/β-catenin inhibitor XAV-939. This dual-functional system enhanced cellular uptake and potentiated photodynamic therapy (PDT)-induced immunogenic cell death, while simultaneously downregulating β-catenin signaling to reverse immunosuppression. In vivo, the nanoplatform substantially improved therapeutic outcomes, converting "cold" tumors into immune-responsive phenotypes characterized by augmented dendritic cell maturation, increased cytotoxic T cell infiltration, reduced regulatory T cell abundance, and enhanced proinflammatory cytokine release. Combined with PD-L1 blockade, this strategy synergistically activated systemic antitumor immunity, resulting in primary tumor regression, metastasis reduction, and systemic abscopal effects against distal tumors. These results highlight the promise of targeted TME reprogramming as a strategy to overcome TNBC's recalcitrance to immunotherapy.

Triple-negative breast cancer (TNBC) is commonly characterized by high-grade and aggressive features, resulting in an augmented likelihood of distant metastasis and inferior prognosis for patients. Tumor immune microenvironment (TME) has been recently considered to be tightly correlated with tumor progression and immunotherapy response. However, the actual heterogenous TME within TNBC remains more explorations.

The thorough analyses of different cell types within TME were conducted on the self-tested single-cell RNA sequencing dataset which contained nine TNBC treatment-naïve patients, including subclusters classification, CellChat algorithm, transcription factors (TFs) expression, pseudotime analysis and functional enrichment assay. The malignant epithelial cluster was confirmed by copy number variations analysis, and subsequently LASSO-Cox regression was carried out to establish a Malignant Cell Index (MCI) model on the basis of five crucial genes (BGN, SDC1, IMPDH2, SPINT1, and UQCRFS1), which was validated in several TNBC cohorts through Kaplan-Meier survival and immunotherapy response analyses. The public spatial transcriptome, proteome data and qRT-PCR, western blotting experiments were exploited to corroborate UQCRFS1 expression in RNA and protein levels. Additionally, functional experiments were implemented to unravel the impacts of UQCRFS1 on TNBC cells.

The diverse subclusters of TME cells within TNBC were clarified to display distinct characteristics in cell-cell interactions, TFs expression, differentiation trajectory and functional pathways. Particularly, IL32high Treg imparted an essential effect on tumor evasion and predicted a worsened prognosis of TNBC patients. Furthermore, MCI model enabled to notify the inferior prognosis and immunotherapy resistance in TNBC. Ultimately, UQCRFS1 knockdown dampened the proliferative and migratory competence in vitro as well as tumor growth in vivo of TNBC cells.

Our study offers innovative perspectives on comprehending the heterogeneity within TME of TNBC, thereby facilitating the elucidation of TNBC biology and providing clinical recommendations for TNBC patients' prognosis, such as IL32high Treg infiltration, MCI evaluation, and UQCRFS1 expression.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, posing significant therapeutic challenges due to the lack of effective targets. Elevating intracellular calcium levels is a promising strategy in cancer therapy, and highly expressed calreticulin (CALR) in tumors has emerged as a potential target for inducing calcium overload. However, few studies on CALR ligands have been reported. Herein, we designed, synthesized, and evaluated pyrazolopyridine derivatives as potential CALR ligands. Among them, the leading compound 2a was identified as a high binding affinity ligand (Kd = 2.6 μM) with potent antitumor activity (IC50 = 0.1 μM). Mechanistic studies demonstrated that 2a could interact with CALR, inducing calcium overload and leading to apoptosis in TNBC cells. Further in vivo pharmacodynamic evaluations confirmed the safety and antitumor activity of 2a. In conclusion, our findings developed a novel CALR ligand and provided a new anti-TNBC strategy via inducing calcium dysregulation.

Triple-negative breast cancer (TNBC) is an aggressive malignancy with high metastasis and recurrence rates. Current treatments like chemotherapy and immunotherapy face challenges due to chemotherapy side effects, limited immunotherapy applicability, and TNBC's immunosuppressive microenvironment.

To achieve a more effective treatment for TNBC, a novel therapeutic strategy has been developed, which uses X-ray excited photodynamic therapy (X-PDT) to activate the tumor immune microenvironment following with the immunotherapy of Anti-CTLA4.

Base on the 4T1 tumor mouse model, this study initially investigated the regulatory effects of X-PDT on the tumor immune microenvironment. Subsequently, the therapeutic efficacy of combining X-PDT with Anti-CTLA4 was evaluated for its inhibitory effects on primary, metastatic, and recurrent tumors. The underlying mechanisms were further elucidated through comprehensive techniques including flow cytometry, ELISA, and immunofluorescence assays.

The synergistic strategy can effectively ablate the primary tumor while inhibiting metastasis and preventing recurrence like a vaccine. It enhances intratumoural dendritic cells (DCs) maturation (from 25.7% to 58.3%, P < 0.05) and immune T cell infiltration activating a strong anti-tumor immune response. The anti-tumor efficacy of synergistic therapy is enhanced by 2.5 times comparing with immunotherapy alone, while the tumor metastasis has been inhibited significantly. The maturation level of mature dendritic cells was increased from 26.7% to 86.3% (P < 0.01). The intratumoural CD8+/CD4+ T cells were increased from 0.51% and 1.54% to 15.4% and 23.1% (P < 0.0001), respectively. The synergistic therapy exerts a powerful vaccine-like long-term immune memory function to prevent tumor recurrence with the elevated level of effector memory T (Tem) cells (from 12.8% to 33.3%, P < 0.05).

Based on the 4T1 mouse model, developed an effective vaccine-like therapeutic strategy combining X-PDT with Anti-CTLA4, which can effectively ablate tumors, inhibit metastasis, and prevent tumor recurrence. This work may provide a novel effective therapeutic modality for the clinical treatment of TNBC.

Chemotherapy-induced hematopoietic toxicity is a multifactorial challenge in the treatment of oncology patients. The resultant bone marrow suppression is a major dose-limiting side effect. In this study, we utilized 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, to investigate the mechanisms underlying bone marrow recovery following chemotherapy. A single injection of 5-FU did not alter mouse bone structure but caused acute damage to bone marrow cellularity and vasculature. Single-cell RNA-sequencing of bone marrow mesenchymal lineage cells revealed a substantial reduction in early mesenchymal progenitors and a marked expansion of marrow adipogenic lineage precursors (MALPs) five days post-treatment. Furthermore, 5-FU upregulated the expression of myofibroblast markers in MALPs, indicating a myofibroblast transformation. Using Adipoq-Cre to label MALPs in vivo, we observed that 5-FU transiently increases the number of MALPs in the bone marrow by promoting their proliferation. Immunostaining confirmed the elevated expression of myofibroblast markers in MALPs. By day 14 after 5-FU injection, bone marrow cellularity and vasculature were largely restored; however, the ablation of MALPs significantly impaired this recovery. Taken together, our study uncovers the critical role of MALPs in facilitating bone marrow repair following chemotherapy-induced injury and identifies them as a potential cellular target for treating chemotherapy-induced myelosuppression.

Residual disease after neoadjuvant chemotherapy (NAC) has important role in triple negative breast cancer (TNBC). The CREATE-X study demonstrated a survival benefit from adjuvant capecitabine (adjC) in breast cancer patients, especially for TNBC populations. Because the landscape of early TNBC treatment has been changing rapidly, an optimal adjuvant strategy for real-world practice is needed. We evaluated the effectiveness of adjC in TNBC patients with residual disease after NAC.

We used de-identified, anonymous data from an institutional clinical data warehouse to retrospectively analyze 934 TNBC patients who received NAC between 2017 and 2023. Among them, 405 patients received at least 1 cycle of adjC, and 77 received no adjuvant treatment. The primary outcomes of the study were distant-disease free survival (DDFS) rate and overall survival (OS) rate at 3 years. The secondary outcomes were subgroup analyses and Cox regression analyses of survival outcomes.

The median follow up period was 34.3 months (range 1.8-71.5). The DDFS rate at 3 years was higher in the capecitabine group: 86.3% of the capecitabine group and 74.4% of the no adjuvant group (P = .019). The OS rates at 3 years were 93.3% and 83.8%, respectively (P = .032). Subgroup analyses indicated a greater benefit from adjC in patients aged 50 years or older and those who received platinum-based NAC, both in terms of DDFS and OS.

Our study showed that adjC was more effective than no adjuvant treatment for TNBC patients with residual disease in terms of DDFS and OS.

Withaferin A (WA), a natural product used in traditional medicine, has recently garnered attention because of its diverse pharmacological effects. However, the direct targets responsible for these effects remain elusive. The discovery of targets is usually serendipitous and research has predominantly concentrated on covalent interactions, overlooking non-covalent targets. The unbiased and proteome-wide mapping of WA-interacting proteins in living cells remains largely unexplored. We have developed a chemical proteomics platform that enabled profiling of the covalent/non-covalent interactome and target occupancy in disease-related cells, which was used to reveal the landscape of the targets of WA in triple-negative breast cancer (TNBC) cells. Analysis of the discovered high-occupancy targets suggested that WA was substantially involved in the RNA metabolism pathway, in addition to other biological processes. Moreover, we biochemically validated a selection of previously unknown high-occupancy targets from various important biological pathways, including the non-covalent target MVK and covalent targets HNRNPF and CKAP4, which all play critical roles in TNBC. Collectively, these findings provided a target map for comprehensive understanding of the anti-TNBC activity of WA, and present WA-targetable proteins as new avenues for pharmacological intervention in TNBC. We anticipate that this platform will be applicable for the unbiased profiling of the targets of WA in various other disease-related cell models, as well as for other bioactive electrophilic natural products in different pathophysiological systems.

Samples' collection for translational analyses in phase III trials requires a huge effort and there is no evidence on how it translates into new knowledge on tumour biology or optimization of patients' selection. We systematically reviewed phase III trials in breast cancer (BC) to evaluate how frequently a translational project has been integrated into their design and how this integration translated into new translational evidence.

Interventional phase III trials evaluating anticancer drugs in BC published in 11 major journals between 2014 and 2018 were included.

89 BC phase III trials were identified, 3 had no sample collection. Among the others, in 36 % the information on sample collection for research purposes was not clear while more than half of the samples had definitive evidence of it. After a median follow-up of 87.9 months, 55.8 % studies published translational data with a mean number of 1.31 (SD 1.7) and 1.07 (SD 1.8), congress abstracts and secondary papers, respectively. There was a higher probability of published translational results for studies with positive outcomes (68.6 % vs 47.1 %), clear evidence of sample collection (72.2 % vs 28.1 %), well-established translational endpoint (73 % vs 42.9 %) and higher impact factor journal (IF) for the clinical publications (64.5 % vs 33.3 %). Secondary translational papers were usually published in lower IF journals with a significant delay from the clinical publication.

Although extremely resource-demanding, sample collections for translational analyses in phase III trials are frequently not well defined, and only 50 % produce new translational evidence, which is delayed in time and published in lower IF journals.

Activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway could effectively initiate antitumor immunity in triple-negative breast cancer. However, current nuclear DNA-mediated activation of STING pathway remains constrained by the tight protection of nuclear membrane and histones, highlighting the need for new strategies to enhance its efficacy. Mitochondrial DNA (mtDNA), in contrast, is more vulnerable to damage. Herein, our nanoplatforms exploited the high glutathione (GSH) environment characteristic of tumors to release abundant Mnb+, which induced mitochondrial dysfunction and the release of endogenous mtDNA. The released mtDNA, in conjunction with Mnb+ itself functioning as a strong cGAS agonist, effectively activated cGAS-STING pathway. Consequently, the cGAS-STING-dependent secretion of type I interferon successively enhanced the maturation of dendritic cells and cross-priming of CD8+ T cells. In a poorly immunogenic 4T1 tumor model, TPP-MMONs efficiently primed systemic antitumor immunity and significantly enhanced the therapeutic efficacy of αPD-L1 therapy, suppressing tumor growth in both localized and metastatic tumor models. These findings provided an innovative and straightforward strategy to enhance TNBC immunogenicity by targeting mitochondrial damage to induce mtDNA-mediated cGAS-STING activation, thereby sensitizing tumors to immune checkpoint inhibitor therapy. STATEMENT OF SIGNIFICANCE: The cGAS-STING pathway is a promising target for overcoming immunoresistance in TNBC. However, current nuclear DNA-based activation strategies are limited by the tight protection of nuclear membrane and histones. Herein, we reported novel manganese-rich, mitochondria-targeting nanoplatforms (TPP-MMONs), which can release abundant Mn²⁺ and significantly induce mitochondrial dysfunction, leading to the release of mtDNA. As a result, the nanoplatforms can effectively stimulate the cGAS-STING pathway, thereby enhancing immune responses and improving the therapeutic efficacy of αPD-L1 therapy, offering new insights into TNBC treatments.

Real-world outcomes are poorly understood for patients with human epidermal growth factor receptor 2 (HER2)-low (immunohistochemistry 1+ or 2+ with negative in situ hybridization) metastatic breast cancer (mBC).

Using for the first time a nationwide electronic health record-derived de-identified database, we assessed demographics, treatment patterns, and outcomes of patients with HER2-low mBC who previously received one line of chemotherapy in the metastatic setting. The post-chemotherapy line was termed the index line of therapy (LOT).

3765 patients [hormone receptor (HR)-positive: 78.8%, HR-negative: 21.0%] met the inclusion criteria (1 January 2011-30 April 2023). 61.7% of HR-positive patients received endocrine therapy prior to the index LOT. The largest patient percentage received single-agent chemotherapy at the index and subsequent two LOTs. For the overall cohort, the median real-world time to treatment discontinuation/death was 4.1 months (95% CI: 3.9-4.2) and the median real-world time to next treatment/death was 5.1 months (95% CI: 4.8-5.3) from the index LOT. Median real-world overall survival (all patients) was 15.8 months (95% confidence interval: 15.2-16.5, median follow-up = 54.5 months) from the index LOT.

These data highlight the unmet clinical needs of patients with HER2-low mBC by characterizing the treatment patterns and poor outcomes in this population on the current standard of care.

The potential of photodynamic therapy (PDT) in combination with chemotherapy to improve treatment outcomes for triple-negative breast cancer (TNBC), for which no targeted therapy is available, is the subject of considerable investigation. In PDT, photosensitizers (PSs) are frequently administered directly but do not selectively target cancer cells. To address the delivery of a PS to TNBC and enhance cellular uptake, the Ru-NH2-modified avidin bioconjugate (RuAvi) via Tyr-specific modification using the Mannich reaction is prepared. The RuAvi is further assembled with the cinnamoyl peptide-F(D)LF(D)LFK-NH2 (FK), which binds to formyl peptide receptor 1, overexpressed in TNBC. Notably, the modified Avi still possesses the ability to efficiently bind biotin for the assembly of up to four copies of the FK peptides. The resultant FK4-RuAvi exhibited an IC50 value of 0.36 ± 0.08 µM, which is ≈3.5-fold lower than that of RuAvi (1.25 ± 0.09 µM), upon irradiation in the triple-negative MDA-MB-231 breast cancer cells. FK4-RuAvi also shows efficient uptake in MDA-MB-231 tumor spheroids and exhibited significant toxicity after irradiation compared to the control RuAvi. The presented strategy has the potential to improve the efficacy of targeted PDT to meet the high demand for targeted therapies to treat TNBC, such as targeted adjuvant treatment after breast cancer surgery.

Triple-negative breast cancer (TNBC), characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2) expression, represents a therapeutic challenge due to its aggressive nature and limited treatment options. Here, we identified the cholesterol-lowering drug lovastatin (LV) as a potent apoptosis-inducing agent in TNBC. Mechanistically, LV disrupts the interaction between the deubiquitinating enzyme USP14 and Survivin, a key anti-apoptotic protein, enhancing polyubiquitination and the proteasomal degradation of Survivin. The overexpression of USP14 was found to stabilize Survivin and rescue LV-induced apoptosis and tumor suppression in vitro and in vivo, whereas USP14 silencing or inhibition with IU1 (a USP14-specific inhibitor) enhanced Survivin turnover and synergized with LV to suppress colony formation in TNBC cells. Clinical relevance was demonstrated through bioinformatic analysis and immunohistochemistry, revealing that elevated Survivin expression in TNBC tissues correlated with poor prognosis and is significantly upregulated in TNBC versus non-TNBC tissues. Our findings identify the USP14-Survivin axis as a potential therapeutic target and highlight LV as a promising candidate for TNBC treatment.

Pyroptosis has attracted significant attention for its role in cancer chemotherapy and immunotherapy. However, few drugs have been reported to induce pyroptosis via the Caspase-3/gasdermin E (GSDME) pathway. Herein, three novel PtIV prodrugs, MRP, DRP, and HRP are rationally designed by conjugating DNA methyltransferase (DNMT) inhibitor (RG108) and/or histone deacetylase (HDAC) inhibitor (PhB) to the PtIV center. These prodrugs can be easily reduced to cisplatin (CDDP) due to the high glutathione (GSH) levels in tumors, liberating the coordinated ligands. Released RG108 reactivates the GSDME gene and reduces pyroptosis in low GSDME-expressing tumor cells. Meanwhile, PhB-induced chromatin loosening enhances CDDP-DNA binding, which not only increases Caspase-3 expression, but also upregulates GSDME. HRP demonstrates superior ability to suppress tumor growth and metastasis while reducing systemic toxicity compared with CDDP. By reactivating GSDME and loosening chromatin, HRP effectively boosts tumor cell pyroptosis and exhibits the most pronounced anticancer performance. These findings highlight HRP's potential as a therapeutic agent for triple-negative breast cancer (TNBC) and offer innovative strategies for combining chemotherapy with immunotherapy. To the best of current knowledge, this is the first report of platinum complexes inducing pyroptosis via the Caspase-3/GSDME pathway in low GSDME-expressing tumor cells.

Inducing immunogenic cell death (ICD) has emerged as a promising strategy for targeting immunologically "cold" tumors. However, most current therapies focus on a single mechanism, limiting their efficacy. In this study, we propose a nano-enabled approach that synergistically activates two complementary immunogenic killing mechanisms: pyroptosis, which elicits a potent inflammatory response, and ICD, characterized by the presentation of 'eat-me' signals and tumor antigens to the immune system. Capsaicin, a naturally occurring compound, was employed to induce pyroptosis via ROS-mediated gasdermin E (GSDME) cleavage, resulting in cell membrane blebbing and subsequent cell death. To simultaneously trigger ICD, we incorporated 2D graphene oxide (GO) engineered with optimized physicochemical properties to induce robust ICD under near-infrared irradiation. Our in vitro and in vivo experiments demonstrated that the combined treatment of capsaicin and GO not only enhanced cancer cell killing but also promoted immune cell infiltration and potentiated anti-tumor immunity, leading to significant tumor suppression. Moreover, the dual-trigger mechanism of pyroptosis and ICD yielded superior anti-tumor efficacy compared to single-modality treatments while maintaining a favorable biosafety profile. These findings highlight the potential of a synergistic nano-enabled strategy for improving cancer immunotherapy.

Breast cancer represents a significant contributor to cancer-related mortality among women worldwide, with triple-negative breast cancer (TNBC) often exhibiting more aggressive clinical features and a heightened lethality rate. The emergence of malignant progression, along with issues of drug resistance, poses substantial challenges in the clinical management of this disease.

The analysis of gene expression profiles at the single-cell level was conducted on circulating tumor cells (CTCs) obtained from TNBC patients, with the objective of identifying specific marker genes associated with CTCs. The TCGA database served as the training cohort for the development of a prognostic CTCs signature model, while the METABRIC dataset was utilized as the validation cohort to assess the robustness of the CTCs signature model. Furthermore, we investigated the differences in prognosis, immune scores, tumor mutational burden, and responses to immunotherapy and chemotherapy across various risk groups established based on the CTCs signature model. Colony formation and transwell assays were conducted to assess the influence of CTCs signature genes on cellular proliferation and invasive capabilities.

Seven marker genes associated with CTCs (BLOC1S3, FOXD2, GZMB, KCNJ13, NTRK3, SOAT2, and ZNF589) were identified and incorporated into a CTCs signature model. The risk score derived from this model stratified TNBC patients into high-risk and low-risk groups. Notably, the overall survival (OS) rate for the low-risk group was significantly higher than that of the high-risk group. Furthermore, the low-risk cohort exhibited more favorable prognostic outcomes and demonstrated heightened sensitivity to both immunotherapy and chemotherapy. Finally, knockdown experiments conducted in TNBC cell lines demonstrated that CTCs signature genes play a crucial role in the regulation of cellular proliferation and invasion.

The CTCs signature model offers novel insights into the prognostic significance of CTC marker genes in TNBC. This understanding may serve as a valuable reference for predicting responses to immunotherapy and chemotherapy, as well as for revealing the molecular mechanisms and therapeutic targets of TNBC.

Embedding natural products into chitosan nanoparticles (CNP) is an effective way to produce a novel combination with better antimicrobial and anticancer activities. Therefore, this study aims to incorporate carob honey (CH) into CNP, determine its potential antimicrobial along with antiproliferative activities, by well diffusion and MTT cell viability assays, respectively. Successful loading of CH in CNP was confirmed after due characterization. The nanoparticles, synthesized by ionic gelation method, produced a small (101.3 ± 4.13 nm), stable (+27.27 ± 0.95 mV), and monodispersed (0.2265 ± 0.0027) CH-loaded CNP (CHCNP). The best antibacterial activity occurred in Klebsiella pneumoniae (K. pneumoniae) (23 ± 0 mm to 16 ± 1.7 mm) followed by Escherichia coli (E. coli) (18 ± 2.0 mm to 10 ± 1 mm). Meanwhile, Aspergillus niger (A. niger) and Aspergillus flavus (A. flavus) were evenly inhibited with inhibition zones in the range of 15 ± 3 mm to 7 ± 0.8 mm and 15 ± 5 mm to 9 ± 1.4 mm, respectively. CHCNP showed a remarkable cytotoxic effect on MDA-MB-231 according to concentration and time, with IC50 of 25 ± 5 to 18 ± 2.6 μg/mL within 24-72 h. These findings demonstrated the feasibility of loading CH in CNP to form a nanoformulation that could potentially serve as a target-specific therapeutic agent in the treatments of microbial infections and breast cancer. However, there is a need for further research on the safety, dosage optimization, in vivo studies and mechanisms of action of the nanoparticles.

Numerous studies have demonstrated that extracellular vesicles (EVs) carry a variety of noncoding RNAs (ncRNAs), which can be taken up by neighboring cells or transported to distant sites via bodily fluids, thereby facilitating intercellular communication and regulating multiple cellular functions. Within the tumor microenvironment, EV-ncRNA, on the one hand, regulate the expression of PD-L1, thereby influencing tumor immune evasion, promoting tumor cell proliferation, and enhancing tumor growth, invasion, and metastasis in vivo. On the other hand, these specific EV-ncRNAs can also modulate the functions of immune cells (such as CD8 + T cells, macrophages, and NK cells) through various molecular mechanisms, inducing an immunosuppressive microenvironment and promoting resistance to anti-PD-1 therapy. Therefore, delving into the molecular mechanisms underlying EV-ncRNA regulation of immune checkpoints presents compelling therapeutic prospects for strategies that selectively target EV-ncRNAs. In this review, we elaborate on the cutting-edge research progress related to EV-ncRNAs in the context of cancer and dissect their pivotal roles in the PD-1/PD-L1 immune checkpoint pathway. We also highlight the promising clinical applications of EV-ncRNAs in anti-PD-1/PD-L1 immunotherapy, bridging basic research with practical clinical applications.

Breast cancer remains one of the most prevalent malignancies worldwide, underscoring an urgent need for innovative therapeutic strategies. Immunotherapy has emerged as a transformative frontier in this context. In triple-negative breast cancer (TNBC), the combination of immunotherapy based on PD-1/PD-L1 immune checkpoint inhibitors (ICIs) with chemotherapy has proven efficacious in both early and advanced clinical trials. These encouraging results have led to the approval of ICIs for TNBC, opening up new therapeutic avenues for challenging-to-treat patient populations. Furthermore, a multitude of ongoing trials are actively investigating the efficacy of immunotherapy-based combinations, including ICIs in conjunction with chemotherapy, targeted therapy and radiation therapy, as well as other novel strategies such as bispecific antibodies, CAR-T cells and cancer vaccines across all breast cancer subtypes, including HR-positive/HER2-negative and HER2-positive disease. This review provides a comprehensive overview of current immunotherapeutic approaches in breast cancer, highlighting pivotal findings from recent clinical trials and the potential impact of these advancements on patient outcomes.

The detection of small extracellular vesicles (sEVs) is currently a pivotal liquid biopsy approach for noninvasive cancer diagnosis. However, the lack of adequate specificity and sensitivity, as well as labor-intensive purification and analysis procedures, present challenges in isolating and profiling sEVs. Here, we present a protein-specific enzymatic optical reporter deposition-based liquid biopsy assay for the rapid and efficient capture and ultrasensitive detection of sEVs using a minimal volume of initial biofluids (10 μL). Biotin aptamers were employed to label sEV proteins for peroxidase conjugation, catalyzing the conversion of fluorescein tyramine into highly reactive free radicals. Efficient signal conversion was achieved by depositing nanoheterolayers composed of covalent tyraminated complexes onto sEV surfaces. The present method offers a detection limit of 6.4 × 103 particles mL-1 with a linear range of 104-1010 particles mL-1 for sEVs. Two machine learning algorithms, principal coordinates analysis and principal component analysis, were subsequently applied for dimensionality reduction. In a clinical cohort of 84 patients, including 6 cancer types and noncancer cases, the assay achieved an overall accuracy of 100% (95% confidence interval) in distinguishing between cancer and noncancer controls and 96% in classifying cancer types. As drugs are frequently administered to patients to modulate the activity of tumor cells, we investigated the efficacy of this strategy in treatment monitoring, achieving an overall accuracy of 100%. This strategy demonstrates a cost-effective, rapid, and low sample volume consumption approach that holds significant potential for precise cancer diagnosis and auxiliary assessment of drug response in clinical settings.

High-mobility group box 1 (HMGB1) plays various roles depending on its subcellular localization. Extracellular HMGB1 interacts with receptors, such as toll-like receptor 4 and receptor for advanced glycation end products (RAGE), promoting cell proliferation, survival, and migration in cancer cells. It also increases the expression of programmed death-ligand 1 (PD-L1) in cancer cells by binding to RAGE. However, the effect of intracellular HMGB1 on the regulation of immune checkpoints such as PD-L1 has not been well characterized. In this study, we aimed to investigate the effects of intracellular HMGB1 on PD-L1 expression in breast cancer cells.

Human and mouse triple-negative breast cancer cells, MDA-MB-231 and 4T1, along with HMGB1-deficient mouse embryonic fibroblast cells, were cultured. HMGB1 overexpression was achieved using a Myc-tagged plasmid, while siHMGB1 constructs were used for gene silencing. Quantitative reverse-transcriptase PCR and western blot analysis were performed to assess gene and protein expressions. Confocal imaging, immunoprecipitation, and proximity ligation assays were used to investigate HMGB1 localization and Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) interactions. In vivo experiments were performed using tumor-bearing mice treated with STAT3 and HMGB1 inhibitors. Statistical analyses were performed using Student's t-tests, one-way analysis of variance, Pearson's correlation, and Kaplan-Meier survival analysis, with significance set at p < 0.05.

In breast cancer cells, HMGB1 translocation from the nucleus to the cytoplasm increased the JAK2-STAT3 interaction and induced STAT3 phosphorylation, leading to increased STAT3 target signaling, including the epithelial-mesenchymal transition (EMT) phenotype and PD-L1 expression. Inhibition of nucleo-cytoplasmic translocation of HMGB1 decreased STAT3 phosphorylation and PD-L1 expression. Furthermore, HMGB1 enhanced breast cancer cell migration, invasion, and EMT, contributing to tumor growth in an in vivo mouse model that were mitigated by the HMGB1-targeted approach.

These findings underscore the critical role of intracellular HMGB1 in modulating PD-L1 expression via the JAK2-STAT3 signaling pathways in breast cancer and suggest that targeting HMGB1 translocation is a promising strategy for breast cancer treatment.

Investigating the essential function of CD300LG within the tumor microenvironment in triple-negative breast cancer (TNBC). Transcriptomic and single-cell data from TNBC were systematically collected and integrated. Four machine learning algorithms were employed to identify distinct target genes in TNBC patients. Specifically, CIBERSORT and ssGSEA algorithms were utilized to elucidate immune infiltration patterns, whereas TIDE and TCGA algorithms predicted immune-related outcomes. Moreover, single-cell sequencing data were analyzed to investigate the function of CD300LG-positive cells within the tumor microenvironment. Finally, immunofluorescence staining confirmed the significance of CD300LG in tumor phenotyping. After machine learning screening and independent dataset validation, CD300LG was identified as a unique prognostic biomarker for triple-negative breast cancer. Enrichment analysis revealed that CD300LG expression is strongly linked to immune infiltration and inflammation-related pathways, especially those associated with the cell cycle. The presence of CD8+ T cells and M1-type macrophages was elevated in the CD300LG higher group, whereas the abundance of M2-type macrophage infiltration showed a significant decrease. Immunotherapy prediction models indicated that individuals with low CD300LG expression exhibited better responses to PD-1 therapy. Additionally, single-cell RNA sequencing and immunofluorescence analyses uncovered a robust association between CD300LG and genes involved in tumor invasion. CD300LG plays a pivotal role in the tumor microenvironment of TNBC and represents a promising therapeutic target.

Individuals with triple-negative breast cancer (TNBC) exhibit elevated lactate levels, which offers a valuable lead for investigating the molecular mechanisms underlying the tumor microenvironment (TME) and identifying more efficacious treatments.

TNBC samples were classified based on lactate-associated genes. A single-cell transcriptomic approach was employed to examine functional differences across cells with varying lactate metabolism. Immunohistochemistry was used to explore the relationship between lactate metabolism and the CXCL12/CXCR4 signaling axis. In addition, utilizing machine learning techniques, we constructed a prognostic model based on lactic acid phenotype genes.

Lactate-associated gene-based stratification revealed increased immune cell infiltration and immune checkpoint expression in Lactate Cluster 1. Elevated lactate metabolism scores were observed in both cancer-associated fibroblasts (CAFs) and malignant cells. CAFs with high lactate metabolism exhibited immune suppression through the CXCL12/CXCR4 axis. Immunohistochemistry confirmed elevated LDHA, LDHB, CXCL12, and CXCR4 levels in the high lactate group.

This study elucidates the complex interplay between lactate and immune cells in TNBC and highlights the CXCL12/CXCR4 axis as a key pathway through which lactate mediates immune suppression, offering new insights into metabolic regulation within the TME. Furthermore, we developed a prognostic model based on lactate metabolism phenotype genes to predict the prognosis of TNBC patients and guide immunotherapy.

Recent studies have identified novel cardiac glycosides from natural sources with potential anti-tumor properties. Toxicarioside H (ToxH) is a novel cardiac glycoside isolated by our collaborative research team. However, its ability to induce ferroptosis in triple-negative breast cancer (TNBC) cells has not been investigated. Therefore, this study evaluates whether ToxH has the capability of inducing ferroptosis and elucidates the underlying molecular mechanisms. Treatment with ToxH led to dose- and time-dependent growth inhibition in BT-549 and MDA-MB-468 cells. Flow cytometry analysis and lactate dehydrogenase assay revealed that ToxH induced various forms of cell death in both BT-549 and MDA-MB-468 cells. Examination through transmission electron microscopy, along with flow cytometry analysis of 7-AAD-stained dead cells and ferroptosis markers BODIPY-C11 and Fe2+ ions, identified various forms of cell death induced by ToxH, including apoptosis, autophagy, apoptotic necrosis, and ferroptosis. Co-treatment with the ferroptosis inhibitor Fer-1 significantly reduced ToxH-induced cell death, indicating that ToxH primarily inhibits TNBC cell growth by inducing ferroptosis. Further investigation into the molecular mechanisms revealed upregulation of Nrf2 and HO-1 expression by ToxH. Effective inhibition of ToxH-induced ferroptosis was achieved through shRNA-mediated knockdown of HO-1 expression. Animal experiments demonstrated that ToxH treatment markedly suppressed tumor growth compared to the control group, while co-administration of Fer-1 led to an increase in tumor growth. These findings suggest that ToxH suppresses TNBC cell growth by modulating the Nrf2/HO-1 signaling pathway to induce ferroptosis. ToxH presents itself as a promising cardiac glycoside compound for TNBC treatment, warranting further translational research.

Triple-negative breast cancer (TNBC) is an aggressive and invasive subtype of breast cancer for which chemotherapy, such as paclitaxel (PTX), remains a primary treatment option. However, resistance to chemotherapy poses a significant challenge, necessitating the development of novel therapeutic strategies. This study aimed to address PTX resistance in TNBC by developing a peptide drug, Charis 1000 (C1K), designed to target transforming growth factor beta (TGF-β) signaling. C1K was synthesized using standard solid-phase peptide synthesis and optimized for enhanced stability. Molecular docking predicted the binding interactions between C1K and TGF-β1, and surface plasmon resonance (SPR) confirmed a moderate binding affinity. The therapeutic potential of C1K was evaluated in TNBC cell lines (4T1, MDA-MB-231, and PTX-resistant MDA-MB-231) and in vivo using a syngeneic 4T1 mouse model. Functional assays demonstrated that C1K inhibited TGF-β-mediated signaling, reduced autophagy, a key mechanism underlying PTX resistance, and significantly enhanced PTX-induced apoptosis. In vivo studies further revealed synergistic effects of C1K and PTX, resulting in enhanced apoptosis in both sensitive and PTX-resistant TNBC cells. These findings suggest that C1K effectively targets TGF-β to inhibit autophagy and potentiate the apoptotic effects of PTX, as a promising combinatorial therapeutic agent for improving treatment outcomes in TNBC patients.

Ferrous iron (Fe2+)-based chemodynamic therapy (CDT) shows great potential for improving chemotherapeutic efficacy and reducing side effects. However, spontaneous oxidation and biological matrixes can influence the catalytic reactive oxygen species (ROS) generation of Fe2+, thereby limiting the efficacy of CDT. Herein, we reported a simple and convenient method to construct hyaluronic acid (HA)-stabilized iron/zinc oxide nanoparticles (IZ@H NPs), which showed intrinsic peroxidase (POD)-like activity and excellent light-activated Fe2+ release performance. Moreover, we demonstrate that catalytic ROS generation follows a cascade amplification manner due to the light-activated release of Fe2+ from IZ@H NPs, leading to formation of iron-DNA complexes (IDCs). After loading doxorubicin (DOX), the nanosystem (termed IZD@H NPs) exhibits tumor cell targeting, robust ROS generation and high cytotoxicity, significantly suppressing tumor growth in xenograft mouse models while maintaining good biosafety. This work gives novel insight into amplifying Fe2+-mediated catalytic ROS generation and presents a new strategy for in vivo Fe2+ delivery to enhance chemodynamic/chemotherapy.

Triple-negative breast cancer cells must evade immune surveillance to metastasize to distant sites, yet this process is not well understood. The Eyes absent (EYA) family of proteins, which are crucial for embryonic development, become dysregulated in cancer, where they have been shown to mediate proliferation, migration, and invasion. Our study reveals an unusual mechanism by which EYA3 reduces the presence of cytotoxic natural killer (NK) cells in the premetastatic niche (PMN) to enhance metastasis, independent of its effects on the primary tumor. We find that EYA3 up-regulates nuclear factor κB signaling to enhance CCL2 expression, which, in contrast to previous findings, suppresses cytotoxic NK cell activation in vitro and their infiltration into the PMN in vivo. These findings uncover an unexpected role for CCL2 in inhibiting NK cell responses at the PMN and suggest that targeting EYA3 could be an effective strategy to reactivate antitumor immune responses to inhibit metastasis.

Triple negative breast cancer (TNBC) refers to a molecular subtype of breast cancers (BC) with high rate of distant metastases and poor prognosis. Epirubicin (EPI) is widely used for the therapy of TNBC but it's limited in clinical use due to cardiotoxicity and chemotherapy resistance. We previously identified F-α-DDB-derivative, a novel synthetic of bifendate, as a potential agent to improve the therapeutic efficacy of TNBC in combination with EPI since F-α-DDB-derivative inhibited MDA-MB-468, but not as much as EPI. In this study, we investigated the antitumor activity of F-α-DDB-derivative in combination with EPI against TNBC in vitro and in vivo and whether co-treatment would induce additional cardiotoxicity. In human TNBC MDA-MB-468 cells, application of F-α-DDB-derivative (11.5, 23.0, 46.0 μg/ml) in combination with EPI (1.5, 3.0, 6.0 μg/ml) exhibited great inhibition on cell viability and cell proliferation. Additionally, F-α-DDB-derivative (5.75, 11.5, 23.0, 46.0 μg/ml) interacted with EPI (0.75, 1.5, 3.0, 6.0 μg/ml) synergistically to induce apoptosis in MDA-MB-468 cells. This suggests that F-α-DDB-derivative may be more sensitive to apoptotic pathways. Furthermore, we revealed that co-administration of EPI and F-α-DDB-derivative (ip, once every other day for 14 days) significantly increased the therapeutic efficacy of EPI (2.0 mg/kg) or F-α-DDB-derivative (20.0 mg/kg) in mice harboring MDA-MB-468 cell xenografts without additional cardiotoxicity compared to that in EPI monotherapy group. These results implicate that co-treatment of EPI and F-α-DDB-derivative may be a potential therapeutic approach for the treatment of TNBC.

TNBC remains the most aggressive and therapy-resistant type of breast cancer, for which efficient targeted therapies have not been developed yet. Here, we identified TRPML3 (ML3) as a potential therapeutic target in TNBC. Our data showed that ML3 is significantly upregulated in TNBC cells compared with nontumorigenic control cells. ML3 knockdown (KD) impairs TNBC cell proliferation by inducing cell cycle arrest and caspase-dependent apoptosis. ML3 KD also inhibits TNBC cell migration and invasion. Mechanistically, ML3 KD reduces lysosomal number and enhances lysosomal acidification, which in turn activates mTORC1, thereby inhibiting autophagy initiation and flux. This disruption negatively impacts mitochondrial function, as evidenced by reduced ATP production, increased ROS and NO production, and mitochondrial fragmentation. Importantly, ML3 KD enhances TNBC cell sensitivity to doxorubicin and paclitaxel. The finding suggests that targeting ML3 disrupts lysosomal and mitochondrial homeostasis and enhance chemosensitivity, presenting ML3 as a potential therapeutic vulnerability in TNBC enhancing chemosensitivity.

Cell division cycle 20 homologue (Cdc20) is an essential mitotic regulator whose overexpression is closely associated with tumorigenesis and poor prognosis in triple-negative breast cancer (TNBC). Targeting Cdc20 has therefore emerged as a promising therapeutic avenue for this aggressive malignancy. In the present study, a receptor-based drug design approach was employed to optimize Apcin analogues as Cdc20 inhibitors. Through a two-step strategy-concept validation followed by structural optimization-we identified compound 14c, which demonstrated remarkable Cdc20 binding affinity (KD: 7.65 μM), potent antiproliferative effects against MDA-MB-231 TNBC cells (IC50: 3.28 μM), and a favorable selectivity index (4.22 for MCF-7 non-TNBC cells and 7.27 for MCF 10A normal cells). 14c effectively inhibited Cdc20 activity, induced G2/M phase arrest, promoted DNA damage accumulation, and stabilized key substrates such as Cyclin B1 and Bim, leading to enhanced apoptosis and suppression of tumor cell proliferation and migration. In vivo, 14c significantly inhibited tumor growth in an MDA-MB-231 xenograft model with a 90 % tumor inhibition rate and no observable toxicity. These results highlight the potential of 14c as a potent Cdc20 inhibitor, offering a promising therapeutic approach for TNBC.

The build-up of senescent cells in tissues is a key indicator of aging, associated with negative prognosis and therapy resistance. Despite immune dysfunction related to aging, also known as immunosenescence, is recognized as a factor in this process, the exact mechanisms are still unclear. In this study, we reported that melatonin deficiency accelerated macrophage senescence in triple-negative breast cancer, whereas melatonin could defend macrophages against senescence through the Nfatc1-Trim26-cgas-Sting pathway. Mechanistically, melatonin enhanced the nuclear translocation of Nfatc1 and elevated Trim26 transcription levels. Trim26, functioning as an E3 ligase, ubiquitinates cgas, thereby inhibiting the activation of the cgas-Sing pathway and consequently preventing cell senescence. Conversely, melatonin deficiency induced cgas-Sting pathway activation to promote macrophage aging. Our results show that melatonin inhibited macrophage senescence and improved chemotherapy responsiveness, with further enhancement when combined with the cgas inhibitor (G150). Overall, our findings indicated that melatonin protects macrophages from immunosenescence, suggesting its therapeutic potential for enhancing chemotherapy response.

Recurrence of breast cancer may be due to the presence of breast cancer stem cells (BCSC). Abnormal tumor cell growth is closely associated with increased reactive oxygen species (ROS) and disruption of redox homeostasis, and BCSCs exhibit low levels of ROS. The detailed mechanism between the low levels of ROS in BCSCs and their maintenance of stemness characteristics has not been reported. A growing number of studies have shown that tumor development is often accompanied by metabolic reprogramming, which is an important hallmark of tumor cells. As the first rate-limiting enzyme of pentose phosphate pathway (PPP), the expression of G6PD is precisely regulated in tumor cells, and there is a certain correlation between PPP and BCSCs. MiR-375 has been shown to inhibit stem cell-like properties in breast cancer, but the exact mechanism is not clear. Here, KLF5, as a transcription factor, was identified to bind to the promoter of G6PD to promote its expression, whereas miR-375 inhibited the expression of KLF5 by binding to the 3'UTR region of KLF5 mRNA and thus reduced the expression of G6PD expression, inhibits PPP to reduce NADPH, and increases ROS levels in breast cancer cells, thereby weakening breast cancer cell stemness. Our study reveals the specific mechanism by which miR-375 targets the KLF5/G6PD signaling axis to diminish the stemness of breast cancer cells, providing a therapeutic strategy against BCSCs.