Triple-negative Breast Cancer Mouse Research Articles

Receptor CDCP1 is a potential target for personalized imaging and treatment of poor outcome HER2+, triple negative and metastatic ER+/HER2- breast cancers.

Receptor CUB-domain containing- protein 1 (CDCP1) was evaluated as a target for detection and treatment of breast cancer. CDCP1 expression was assessed immunohistochemically in tumors from 423 patients (119 triple-negative breast cancer (TNBC); 75 HER2+; 229 ER+/HER2- including 228 primary tumors, 229 lymph node and 47 distant metastases). Cell cytotoxicity induced in vitro by a CDCP1-targeting antibody-drug conjugate (ADC), consisting of the human/mouse chimeric antibody ch10D7 and the microtubule disruptor monomethyl auristatin E (MMAE), was quantified, including in combination with HER2-targeting ADC T-DM1. Detection of CDCP1-expressing primary and metastatic xenografts in mice was examined by PET-CT imaging using 89zirconium-labelled ch10D7 (89Zr-ch10D7). The impact of ch10D7-MMAE on tumor burden and survival in vivo, including in combination with T-DM1, was quantified in cell line and patient-derived xenograft mouse models. CDCP1 is expressed predominantly on the surface of malignant cells of 70% of TNBCs, 80% of HER2+ tumors, and increases in ER+/HER2- tumors from 44.9% in primary tumors to 56.4% in lymph node metastases and 74.3% in distant metastases. PET-CT imaging with 89Zr-ch10D7 is effective for the detection of primary and metastatic CDCP1-expressing TNBCs in mice. ADC ch10D7-MMAE kills CDCP1-expressing cells in vitro and controls primary and metastatic TNBC xenografts in mice, conferring significant survival advantages over chemotherapy. It compares favorably to T-DM1 in vivo, and ch10D7-MMAE combined with T-DM1 showed the most potent efficacy, markedly reducing tumor burden of CDCP1+/HER2+ xenografts and prolonging mouse survival, compared with T-DM1 or ch10D7. CDCP1-directed molecular imaging has potential to identify aggressive breast cancers for CDCP1-targeted treatment.

Monensin Inhibits Triple-Negative Breast Cancer in Mice by a Na+-Dependent Cytotoxic Action Unrelated to Cytostatic Effects

Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, defined by its limited therapeutic options and poor outcomes. This study investigated the therapeutic potential of targeting Na+ homeostasis in TNBC cells to induce TNBC inhibition. For this purpose, BALB/c mice were inoculated with 4T1-Luc2 breast cancer cells and treated with the Na+ ionophore monensin (8 mg/kg) or vehicle alone. Tumor development and cellular Na+ content were assessed using vivo live imaging techniques, while intracellular Na+ variations and cytotoxicity were evaluated through live cell analysis. Monensin treatment increased Na+ levels in cancerous tissues and reduced TNBC mass (monensin: 0.146 ± 0.06; vehicle: 0.468 ± 0.2 cm3; p < 0.001). This treatment induced extensive necrosis in TNBC tumors while preserving the structural and functional integrity of healthy organs and maintaining the proliferative activity of both tumor and normal tissues. Monensin did not alter the expression of proliferating nuclear antigen (PCNA) in 4T1-Luc2 cells but triggered cytotoxicity preceded by intracellular Na+ accumulation. Na+-free conditions prevented both Na+ accumulation and 4T1-Luc2 cell death. Thus, monensin exerts its antitumor effects in TNBC through a Na+-dependent and tumor-specific cytotoxic mechanism, without inducing cytostatic effects on normal or transformed tissues. Collectively, these findings underscore the potential of Na+ ionophores as promising therapeutic agents for TNBC.

Peptide-Drug Conjugate for Therapeutic Reprogramming of Tumor-Associated Macrophages in Breast Cancer.

In triple-negative breast cancer (TNBC), pro-tumoral macrophages promote metastasis and suppress the immune response. To target these cells, a previously identified CD206 (mannose receptor)-binding peptide, mUNO was engineered to enhance its affinity and proteolytic stability. The new rationally designed peptide, MACTIDE, includes a trypsin inhibitor loop, from the Sunflower Trypsin Inhibitor-I. Binding studies to recombinant CD206 revealed a 15-fold lower KD for MACTIDE compared to parental mUNO. Mass spectrometry further demonstrated a 5-fold increase in MACTIDE's half-life in tumor lysates compared to mUNO. Homing studies in TNBC-bearing mice shows that fluorescein (FAM)-MACTIDE precisely targeted CD206+ tumor-associated macrophages (TAM) upon intravenous, intraperitoneal, and even oral administration, with minimal liver accumulation. MACTIDE was conjugated to Verteporfin, an FDA-approved photosensitizer and YAP/TAZ pathway inhibitor to create the conjugate MACTIDE-V. In the orthotopic 4T1 TNBC mouse model, non-irradiated MACTIDE-V-treated mice exhibited anti-tumoral effects comparable to those treated with irradiated MACTIDE-V, with fewer signs of toxicity, prompting further investigation into the laser-independent activity of the conjugate. In vitro studies using bone marrow-derived mouse macrophages showed that MACTIDE-V excluded YAP from the nucleus, increased phagocytic activity, and upregulated several genes associated with cytotoxic anti-tumoral macrophages. In mouse models of TNBC, MACTIDE-V slowed primary tumor growth, suppressed lung metastases, and increased markers of phagocytosis and antigen presentation in TAM and monocytes, increasing the tumor infiltration of several lymphocyte subsets. MACTIDE-V is proposed as a promising peptide-drug conjugate for modulating macrophage function in breast cancer immunotherapy.

Creating Single Atomic Coordination for Hypoxia-Resistant Pyroptosis Nano-Inducer to Boost Anti-Tumor Immunotherapy.

General synthesis and mechanical understanding of type I nano-photosensitizers are of great importance for hypoxia-resistant pyroptosis inducers. Herein, a simple solvothermal treatment is developed to convert non-photosensitive small molecules (hemin) into uniform carbon nanodots (HNCDs) with strong type I photodynamic activity and red fluorescence emission. These HNCDs inherit the single atomic Fe-N4 center of hemin while creating sp2-hybridized carbon surroundings, which synergistically modulated the energy level and electron transfer for converting the type II photodynamic process to type I. After encapsulating HNCDs with bovine serum albumin (BSA)to facilitate in vivo applications, the resulting BSA nanoparticles (HB) can image tumors and significantly induce the pyroptosis of tumor cells even under an extremely hypoxic environment (2% O2). This evokes a strong antitumor immune response, effectively restraining tumor growth and lung metastasis in triple-negative breast cancer mice, with good biocompatibility. This work introduces an applicable pyroptosis nano-inducer to combat hypoxic tumors and highlights the regulation of Fe-N4 centers to develop hypoxia-resistant type I nano-photosensitizers for cancer treatment.

Chlorpyrifos induces lung metastases and modulation of cancer stem cell markers in triple negative breast cancer model.

Breast cancer is a major public health problem, and distant metastases are the main cause of morbidity and mortality. Chlorpyrifos is an organophosphate that promotes Epithelial-Mesenchymal Transition-like phenotype in breast cancer cell lines and modulates the Breast Cancer Stem Cells activating two key processes related to the metastatic cascade. Here, we investigated whether Chlorpyrifos may induce distant metastases in an in vivo triple negative tumor model. Also, we studied the expression of Breast Cancer Stem Cell and Epithelial-Mesenchymal Transition activation-markers in Triple Negative Breast Cancer mice tumors and human cells. We demonstrate that Chlorpyrifos modulates stem cell plasticity as a function of growth conditions in monolayer or three-dimensional culture. Furthermore, Chlorpyrifos decreased the doubling period, increased tumor volume, stimulated the infiltration of adjacent muscle fibers and induced lung and lymphatic node metastases in mice. Finally, Chlorpyrifos modulated the expression of Epithelial-Mesenchymal Transition and Breast Cancer Stem Cell markers in mice exposed to the pesticide. All our findings confirm that Chlorpyrifos promotes breast cancer progression, enhances stemness and Epithelial-Mesenchymal Transition marker expression and generates lung metastases in an in vivo model induced in mice.

A Three-agent Regimen for Triple Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) has a poor prognosis with current treatment options. Novel therapeutic strategies are urgently needed to enhance treatment outcomes for TNBC. This study evaluated the efficacy of a three-agent regimen compared to existing treatment regimens in a TNBC mouse model, and elucidated its potential mechanisms of action. The TNBC xenograft tumor mouse model was established using a 4T1 cell line in female BALB/c mice. Mice were treated with the three-agent regimen and other comparative treatments. Tumor volume was monitored to assess the anti-tumor effects. Biochemical and pathological evaluations were conducted to examine the impact of the regimen on anti-tumor immunity, anti- tumor angiogenesis, and tumor cell apoptosis. The three-agent regimen consisting of SIN+BEV+PAB demonstrated significant anti-tumor efficacy compared to controls, PAB alone, SIN+PAB, and BEV+PAB groups from day 9 of drug administration. The superior anti-tumor effect of SIN+BEV+PAB was primarily attributed to enhanced anti-tumor immunity, evidenced by increased percentages of CD4+ and CD8+ T cells, elevated IFN-γ levels, and decreased percentages of Tregs, reduced levels of TGF-β, IL-6, and IL-10. Additionally, the regimen showed potent anti-angiogenic effects by reducing VEGF expression and micro vessel density (MVD). Furthermore, it promoted tumor cell apoptosis through upregulation of BAX and cleaved caspase3, while downregulating Bcl2. These findings suggest that the novel three-agent combination of SIN+BEV+PAB may prove beneficial in improving treatment outcomes for patients with TNBC. The development of this regimen, which may be eligible for patent protection, could facilitate its introduction as a new therapeutic option for advanced TNBC in clinical practice.

Targeting TGFβ and Over-Expressing GM-CSF in the Tumor with Oncolytic Adenovirus Inhibits Tumor Growth/Metastases and Augments Immune Checkpoint Inhibitor Response in a Triple Negative Breast Cancer Mouse Model

Targeting TGFβ and Over-Expressing GM-CSF in the Tumor with Oncolytic Adenovirus Inhibits Tumor Growth/Metastases and Augments Immune Checkpoint Inhibitor Response in a Triple Negative Breast Cancer Mouse Model

Simultaneous targeting and suppression of heat shock protein 60 to overcome heat resistance and induce mitochondrial death of tumor cells in photothermal immunotherapy

As the most aggressive and metastatic subtype of breast cancer, clinical demands of triple negative breast cancer (TNBC) have far not been met. Heat shock protein 60 (HSP60) is over expressed in tumor cells and impair the efficacy of photothermal therapy. In this work, a conjugate composed of self-designed peptide targeting HSP60 and gold nanorods was constructed, referred to as AuNR-P17. Results showed that AuNR-P17 was able to simultaneously down regulate the level of HSP60 and locate in the mitochondria where HSP60 is enriched in the tumor cells of TNBC, which also impeded the interaction between HSP60 and integrin α3, thereby reducing the tumor cells' heat tolerance and metastatic capabilities. At the same time, AuNR-P17 induced remarkable mitochondrial apoptosis when exposed to the laser irradiation of 808 nm. The dual functions of AuNR-P17 led to the decrement of BCL-2 and the activation of p53 and cleaved caspase-3. The danger associated molecular patterns (DAMPs) generated from the mitochondrial apoptosis elicited strong and long-term specific immune responses against TNBC in vivo and ultimately inhibited the tumor metastasis and recurrence with significantly prolonged survival (>100 days) on TNBC mice. In conclusion, this study demonstrated HSP60 a promising potential therapeutic target for triple negative breast cancer and exhibited powerful capacity of AuNR-P17 in photothermal immune therapy.

Abstract C001: Cytomegalovirus infection modulates distinct immune cell dynamics across tumor-immune microenvironments and systemic macroenvironment during metastasis

Abstract Cytomegalovirus (CMV) is ubiquitously present worldwide and has been implicated in the modulation of cancer development and spread. Notably, CMV proteins have been detected in approximately 90% of lymph node and brain metastases in breast cancer, suggesting its potential influencing tumor invasiveness and metastases. This study investigates the effects of CMV infection on the tumor microenvironment and macroenvironment during metastatic progression in triple-negative breast cancer (TNBC). We employed a syngeneic TNBC mouse model designed to develop spontaneous metastases following primary tumor resection. Using multi-omics sequencing and comprehensive analysis tools, we characterized the tumor-immune landscape across multiple organs relevant to CMV infection and tumor metastasis, including blood, bone marrow, spleen, lungs, brain, and lymph nodes. Our results demonstrate that CMV infection systematically activated the immune response, suppressing primary tumor growth and subsequent metastasis. At the primary tumor site, prior to metastasis, CMV-infected mice exhibited significantly lower proportions of macrophages and plasma cells compared to uninfected counterparts, while CD8+ T cells were significantly enriched. In contrast, the proportions of neutrophils and dendritic cells did not significantly differ between the groups. Distinct patterns of immune cell dynamics were observed at metastatic sites. In the spleen of CMV-infected mice, a notable reduction in macrophages and elevated neutrophil levels were detected. Lung tissues displayed a marked increase in macrophages, CD8+ T cells, and dendritic cells in CMV-infected mice. Interestingly, in the blood, we noted a decreased proportion of natural killer cells in CMV-infected mice. However, no significant difference in plasma cell percentages was observed between CMV-infected and uninfected mice in metastatic lung tissues. The cell-cell interaction analysis further showed a complex crosstalk network between immune cells as well as tumor and immune cells. These findings highlight the profound impact of CMV infection on remodeling the immune landscape at both primary tumors and metastatic sites. The distinct immune cell dynamics orchestrated by CMV infection underscore the virus's role in modifying tumor-immune microenvironments and macroenvironments. Our study offers novel perspectives for developing targeted cancer therapies that consider the complex interactions between viral infections and the tumor immune axis. Further research is warranted to elucidate the mechanisms underlying CMV-mediated immune modulation at molecular level and its potential therapeutic implications in TNBC and other cancer types. Citation Format: Wenjuan Dong, Jianting Sheng, Shan Xu, Matthew Vasquez, Hong Zhao, Stephen T.C Wong. Cytomegalovirus infection modulates distinct immune cell dynamics across tumor-immune microenvironments and systemic macroenvironment during metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C001.

Abstract A020: Elevated extracellular glucose level in tumor microenvironment by hyperglycemia increases glycolysis rate, stiffness, contractility, and motility of breast cancer cells through the cAMP-RhoA-Rock and AMPK-YAP-RhoA axes to promote metastasis

Abstract Glucose is a preferred nutrient for most cells, including cancer cells. The tumor microenvironment is both hypoxic and hypoglycemic. Glucose levels in tumors are ten times lower than in normal tissues due to poor vascularization and high glucose consumption by cancer cells, which adapt to hypoglycemia. Obesity, often accompanied by hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, is linked to increased breast cancer metastasis. High extracellular glucose levels have been shown to promote cancer cell motility, though the mechanisms remain unclear. Given cancer cells' reliance on aerobic glycolysis (the Warburg effect), we hypothesized that hyperglycemia in obesity promotes cancer metastasis by modulating glucose metabolism and related cell mechanics pathways. Using Triple-Negative Breast Cancer (TNBC) cells, we found that elevated extracellular glucose levels increase glycolysis, activating the RhoA GTPase signaling pathway. This results in greater cell stiffness and contractility, correlating with increased motility. Our unpublished data, which I will present, validate these findings in obese-TNBC mouse models and further elucidate the glycolysis-mediated activation of the RhoA-ROCK axis. We also identified a novel long-term effect of hyperglycemia in promoting metastasis via transcriptional regulation of the AMPK-YAP-RhoA axis. To confirm hyperglycemia's pro-metastatic effects, we developed two obese-TNBC mouse models using diet-induced obesity (DIO) in NSG and C57BL/6J mice with orthotopic implantation of human and mouse TNBC cells, respectively. RhoA GTPase activity was measured with a GTP-RhoA FRET sensor or pull-down assay. Cell stiffness was assessed using our innovative parallel microfiltration (PMF) assay or Atomic Force Microscopy (AFM). We disrupted glucose uptake by knocking out glucose transporter 3 (GLUT3) via CRISPR/Cas9 or using a GLUT3-selective inhibitor, G3iA. Cell motility was measured by scratch wound-healing and invasion assays. Bulk RNA-seq identified differentially expressed genes under high glucose conditions in human breast cancer cells. Our results show increased lung metastasis of breast cancer cells under hyperglycemic conditions in vivo. Besides the previously reported cAMP-RhoA-ROCK axis, we discovered the glycolysis-AMPK-YAP axis also regulates RhoA-ROCK activity transcriptionally. GLUT3 inhibition activated AMPK, reduced F-actin levels via the AMPK-VASP axis, and decreased non-muscle myosin activity through the AMPK-YAP-RhoA axis. These changes in cell mechanics correlated with reduced motility. Our study suggests that targeting glucose metabolism and cell mechanics could be a novel approach to inhibit cancer metastasis, particularly in obese patients. Citation Format: Tae-Hyung Kim, Aadil Q Bhat, Jun-Yong Choe, Donghoon Yoon. Elevated extracellular glucose level in tumor microenvironment by hyperglycemia increases glycolysis rate, stiffness, contractility, and motility of breast cancer cells through the cAMP-RhoA-Rock and AMPK-YAP-RhoA axes to promote metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A020.

Soy\'s secret weapon: Genistein\'s fight against triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited treatment options. Genistein a natural isoflavone found in soybeans and legumes is a plant-based compound with anticancer properties that has been shown to be promising for TNBC treatment in preclinical studies. This review examines genistein’s potential as a therapeutic agent against TNBC. In vitro studies reveal its capacity to inhibit cell growth, induce apoptosis, and suppress TNBC metastasis. In vivo, genistein suppresses tumor growth and extends survival in TNBC mice. It targets key oncogenic pathways, including NF-κB, Akt, and PI3K. It has also been shown to regulate apoptosis-related gene expression, promote apoptosis, and activate the DNA damage response. Furthermore, Genistein demonstrated the ability to reactivate estrogen receptor expression in ERα-negative MDA-MB-231 xenograft mice, particularly when combined with the histone deacetylase inhibitor trichostatin A. This reactivation restored ER-dependent cellular sensitivity to tamoxifen and 17-estradiol. Combination therapy with genistein and other drugs or phytochemicals has shown to be particularly effective in preclinical studies. These findings suggest that Genistein holds promise as a potential therapeutic agent for TNBC by targeting key signaling pathways involved in cell proliferation, survival, and apoptosis, as well as modulating ER expression and enhancing sensitivity to existing therapies.

Abstract B012: HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy

Abstract The high rate of locoregional/distant recurrence in triple-negative breast cancer (TNBC) is a major driver of the decreased survival observed in breast cancer patients. Radiation therapy is a reliable treatment modality to limit recurrence for many cancers, but TNBC patients have a higher chance for relapse despite receiving increased doses of radiation compared to patients with other breast cancer subtypes. This study investigated the differential pathways in radioresistant TNBC tumors that impact recurrence. We developed two radioresistant cell line models (468-RR and 4T1-RR) by administering a radiation dose of 50Gy over the course of 8 weeks to the human TNBC cell line, MDA-MB-468, and the mouse TNBC cell line, 4T1, respectively. Migration and invasion were assessed using the scratch-wound assay and transwell assay, respectively. We assessed in vivo metastasis by injecting the cells in the mammary fat pad, tail vein, or left ventricle. RNA-sequencing of the cell lines was done to identify global transcriptomic changes and regulatory pathways induced by radiation therapy. The mRNA stability of integrin b3 was assessed by inhibiting transcription using actinomycin D. Moreover, we implemented the newly developed InduroRT-mediated circRNA-sequencing (IMRT-seq) to identify the circular RNAs processed by HNRNPL that are present in radioresistant cells. We observed enhanced lung and bone metastasis, based on bioluminescent imaging, in both the spontaneous and vascular metastasis models for the radioresistant cells compared to the parental cells. Genset enrichment analysis identified mRNA stability and metabolism as upregulated pathways in radioresistant cells with HNRNPL, a ribonucleoprotein involved in mRNA stability, as one of the top genes. By knocking down HNRNPL, we observed decreased migration capacity of the radioresistant cells. Given the role of HNRNPL in integrin and extracellular matrix genes, which are key components associated with metastasis in cancer cells, we screened for these genes in our RNA-seq dataset and identified integrin b3 as a possible target. More specifically, knocking down HNRNPL decreased the mRNA stability of integrin b3 as assessed by actinomycin d treatment. By expressing integrin b3 in the HNRNPL-depleted cells, we were able to rescue the migration capacity of these cells. We believe a subset of HNRNPL-mediated circular RNAs are responsible for integrin b3 mRNA stability by sequestering miRNAs that target integrin b3. With IMRT-seq we are exploring the potential circRNA-miRNA interactions present in radioresistant TNBC responsible for its metastatic phenotype. The radioresistant cells derived from TNBC cell lines have an enhanced metastatic potential than the treatment-naive cell lines and our data suggest that RNA metabolism is pivotal to this phenotype. The upregulation of HNRNPL upon radiation by Nrf2 increases integrin b3 expression by stabilizing its transcripts and preventing its degradation. This novel mechanism of radioresistant metastasis provides a therapeutic approach to minimize TNBC recurrence. Citation Format: Ayush Kumar, Kensei Kishimoto, Christi Wisniewski, Shivam Goel, Arthur Mercurio. HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B012.

CD74 promotes the formation of an immunosuppressive tumor microenvironment in triple-negative breast cancer in mice by inducing the expansion of tolerogenic dendritic cells and regulatory B cells.

CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor (MIF). MIF binding to CD74 induces a signaling cascade resulting in the release of its cytosolic intracellular domain (CD74-ICD), which regulates transcription in naïve B and chronic lymphocytic leukemia (CLL) cells. In the current study, we investigated the role of CD74 in the regulation of the immunosuppressive tumor microenvironment (TME) in triple-negative breast cancer (TNBC). TNBC is the most aggressive breast cancer subtype and is characterized by massive infiltration of immune cells to the tumor microenvironment, making this tumor a good candidate for immunotherapy. The tumor and immune cells in TNBC express high levels of CD74; however, the function of this receptor in the tumor environment has not been extensively characterized. Regulatory B cells (Bregs) and tolerogenic dendritic cells (tol-DCs) were previously shown to attenuate the antitumor immune response in TNBC. Here, we demonstrate that CD74 enhances tumor growth by inducing the expansion of tumor-infiltrating tol-DCs and Bregs. Utilizing CD74-KO mice, Cre-flox mice lacking CD74 in CD23+ mature B cells, mice lacking CD74 in the CD11c+ population, and a CD74 inhibitor (DRQ), we elucidate the mechanism by which CD74 inhibits antitumor immunity. MIF secreted from the tumor cells activates CD74 expressed on DCs. This activation induces the binding of CD74-ICD to the SP1 promotor, resulting in the up-regulation of SP1 expression. SP1 binds the IL-1β promotor, leading to the down-regulation of its transcription. The reduced levels of IL-1β lead to decreased antitumor activity by allowing expansion of the tol-DC, which induces the expansion of the Breg population, supporting the cross-talk between these 2 populations. Taken together, these results suggest that CD74+ CD11c+ DCs are the dominant cell type involved in the regulation of TNBC progression. These findings indicate that CD74 might serve as a novel therapeutic target in TNBC.

Modulating versatile pathways using a cleavable PEG shell and EGFR-targeted nanoparticles to deliver CRISPR-Cas9 and docetaxel for triple-negative breast cancer inhibition.

Human antigen R (HuR), an RNA-binding protein, is implicated in regulating mRNA stability and translation in cancer, especially in triple-negative breast cancer (TNBC), a highly aggressive form. CRISPR/Cas9-mediated HuR knockout (HuR CRISPR) presents a promising genetic therapeutic approach, but it encounters transfection limitations. Docetaxel (DTX), an effective cytotoxic agent against metastatic breast cancer (BC), faces challenges related to vehicle-associated adverse events in DTX formulations. Therefore, we designed multifunctional nanoparticles with pH-sensitive PEG derivatives and targeting peptides to enable efficient HuR CRISPR and DTX delivery to human TNBC MDA-MB-231 cells and tumor-bearing mice. Our findings indicated that these nanoparticles displayed pH-responsive cytotoxicity, precise EGFR targeting, efficient tumor penetration, successful endosomal escape, and accurate nuclear and cytoplasmic localization. They also demonstrated the ability to spare normal cells and prevent hemolysis. Our study concurrently modulated multiple pathways, including EGFR, Wnt/β-catenin, MDR, and EMT, through the regulation of EGFR/PI3K/AKT, HuR/galectin-3/GSK-3β/β-catenin, and P-gp/MRPs/BCRP, as well as YAP1/TGF-β/ZEB1/Slug/MMPs. The combined treatment arrested the cell cycle at the G2 phase and inhibited EMT, effectively impeding tumor progression. Tissue distribution, biochemical assays, and histological staining revealed the enhanced safety profile of pH-responsive PEG- and peptide-modified nanoformulations in TNBC mice. The DTX-embedded and peptide-modified nanoparticles mitigated the side effects of DTX, enhanced cytotoxicity in TNBC MDA-MB-231 cells, and exhibited remarkable antitumor efficacy and safety in TNBC-bearing mice with HuR CRISPR deletion. Collectively, the combination therapy of DTX and CRISPR/Cas9 offers an effective platform for delivering antineoplastic agents and gene-editing systems to combat tumor resistance and progression in TNBC.

Synergistic Anti-Tumor Effects of Newcastle Disease Virus and Doxorubicin: Evidence from A Murine Breast Cancer Model

BackgroundDespite the recent advances in the diagnosis and treatment of breast cancer, triple-negative breast cancer (TNBC) remains a clinical challenge due to its aggressive nature and resistance to conventional therapies. Virotherapy has emerged as a promising cancer treatment strategy, leveraging the ability of viruses to specifically target and replicate in cancerous cells. This study evaluated the oncolytic potential of a combined therapeutic strategy, utilizing Newcastle disease virus (NDV) and Doxorubicin hydrochloride (Dox) both in vitro and in vivo. MethodsThe in vitro experiments involved exposing human and mouse TNBC cell lines (MDA-MB-231 and 4T1, respectively) to NDV and Dox, individually or in combination. Cell viability assays and flow cytometry analyses were conducted to assess the synergistic effects of NDV and Dox on regulating breast cancer cell behavior in vitro. Furthermore, the immune-stimulating potential of NDV was investigated by examining its effects on dendritic cell (DC) maturation using flow cytometry and T cell proliferation. The in vitro anti-tumor effects of NDV were examined in both parental and tamoxifen-resistant cancer cells to assess its efficacy against chemoresistance. Animal models of breast cancer were treated with NDV in combination with Dox. The body weight changes, tumor volume, and survival rates of the mice were monitored throughout the study. Histopathological analyses were conducted to evaluate the potential toxic effects of the treatments. ResultsBased on the MTT results, NDV at optimal concentrations synergized the effect of Dox to reduce the viability of both MDA-MB231 and 4T1 cell lines (Isobologram combination index of less than 1). Additionally, individual treatment with NDV was able to significantly reduce the viability of patient-derived breast cancer cells, compared to the untreated control (P < 0.05) without affecting the cells of normal adjacent tissue. Furthermore, a combination of NDV and Dox significantly enhanced the percentage of early and late apoptotic cells in MDA-MB-231 (P < 0.0001) and late apoptotic cells in 4T1 (P < 0.0001), in comparison with individual treatment with these agents. Flow cytometry results showed that, compared to wild type MDA-MB-231 cells, NDV-infected MDA-MB-231 cells were better inducers of T cell proliferation and DC maturation as evidenced by increased proliferation index (P < 0.05) and elevated expression of CD1a, CD83, and CD86 (P < 0.0001), respectively. Moreover, co-treatment of both wild-type and (tamoxifen) TAM-resistant MCF-7/TAMR-1 cells with TAM and NDV significantly reduce the viability of the cancer cells (P < 0.0001). In tumor-bearing mice locally engrafted with 4T1 cells, combined treatment of NDV and Dox exhibited a marked reduction in median tumor volume compared to the control group, validating our in vitro findings on their synergistic anti-tumor effects. These findings suggest that combining NDV with Dox can effectively inhibit tumor progression and has the potential to reduce the dose, and consequently the toxic side-effects, of Dox in breast cancer therapy.

Enhanced Antitumor Efficacy of Oncolytic Vaccinia Virus Therapy Through Keratin-Mediated Delivery in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents an aggressive subtype characterized by high rates of recurrence and metastasis, necessitating the exploration of alternative treatment strategies. Oncolytic vaccinia virus (OVV) therapy has emerged as a promising approach, selectively infecting and destroying tumor cells. However, its efficacy is often hampered by inadequate viral distribution within the tumor microenvironment. Here, we investigate the potential of keratin (KTN) as a carrier for OVV delivery to enhance viral distribution and antitumor efficacy. In vitro assays revealed that KTN significantly improves OVV stability, leading to increased tumor cell apoptosis and necrosis. Furthermore, KTN effectively inhibits cancer cell migration by suppressing the epithelial-mesenchymal transition (EMT) process and downregulating metastasis-related proteins. These findings are corroborated in a syngeneic TNBC mouse model, where KTN-mediated OVV delivery enhances cytotoxic T cell-mediated antitumor immune responses without compromising the anti-angiogenic effects of the virus. Notably, KTN alone exhibits antitumor effects by suppressing tumor growth and metastasis, underscoring its potential as a standalone therapeutic agent. In conclusion, our study underscores the promise of KTN-mediated OVV delivery as a promising therapeutic strategy for TNBC. By improving viral distribution, suppressing EMT, and enhancing antitumor immunity, this approach holds significant potential for enhancing patient outcomes in TNBC treatment. Further investigation is warranted to explore the broader utility of KTN in various cancer therapy approaches.

Delivery of IL2RG mRNA via nanoparticles to enhance CD8+ T cell promotes anti-tumor effects against late-stage triple-negative breast cancer

BackgroundThe manipulation of CD8+ T cell functions through genetic modifications presents a novel approach to cancer immunotherapy. This study aimed to explore the effects of IL2RG-overexpressing CD8+ T cells and assess the efficacy of delivering IL2RG mRNA via nanoparticles (NPs) to the tumor microenvironment in triple-negative breast cancer (TNBC).MethodsSingle-cell RNA sequencing (scRNA-seq) was conducted on tissue from early and late-stage TNBC, followed by a meta-analysis of six breast cancer arrays from the GEO database to identify candidate genes. CRISPR/Cas9 was employed for gene knockout and overexpression in CD8+ T cells, which were then analyzed using flow cytometry, ELISA, immunofluorescence, and metabolic assays. A 3D cancer cell spheroid model was used for co-culture experiments. Lipid-coated calcium-phosphate (LCP)@IL2RG NPs were synthesized and injected into TNBC mouse models.ResultsIL2RG was identified as significantly associated with late-stage TNBC. Overexpression of IL2RG in CD8+ T cells led to increased cell activation, cytotoxicity, and glycolysis, while knockout reduced these effects. Overexpressing IL2RG in CD8+ T cells co-cultured with 3D cancer spheroids resulted in enhanced apoptosis and reduced cancer cell invasion. Injection of LCP@IL2RG NPs into TNBC mouse models significantly mitigated tumor growth.ConclusionsOverexpressing IL2RG in CD8+ T cells enhances tumor immunotherapy. Delivering IL2RG mRNA via NPs further amplifies this effect, offering a promising strategy for the treatment of late-stage TNBC.

BAFF overexpression in triple-negative breast cancer promotes tumor growth by inducing IL-10-secreting regulatory B cells that suppress anti-tumor T cell responses.

Despite BAFF's (B cell activating factor, BAFF) known influence on B cell survival and proliferation, its specific effects within the tumor microenvironment remain unclear. We aimed to elucidate how BAFF overexpression in breast cancer cells impacts tumor growth and the functions of T and B cells in the tumor microenvironment. BAFF was overexpressed in the 4T1 mouse triple-negative breast cancer cell line, and tumor growth, immune cell infiltration, and activity were assessed in vitro and in vivo using flow cytometry, co-culture assays, and mouse tumor models with B cell depletion. BAFF overexpression in 4T1 cells promoted tumor growth in vivo, suppressed CD8+ T cell activity, and increased IL-10-secreting CD5+ regulatory B cells in tumors. 4T1/BAFF cells directly enhanced IL-10 production in CD5+ B cells via BAFF/BAFF-receptor interactions, and IL-10 from CD5+ B cells inhibited IFN-γ secretion by T cells. B cell depletion partially reversed the tumor-promoting effects of BAFF overexpression. Our study reveals a novel mechanism by which BAFF can foster tumor progression, with the induction of IL-10-secreting regulatory B cells that suppress anti-tumor T cell responses appearing to be a key component of BAFF's tumor-promoting activity. These findings underscore the complex immunomodulatory effects that BAFF exerts in the tumor microenvironment and point to BAFF-induced regulatory B cells as a potential new therapeutic target in breast cancer that warrants further investigation.

Mechanotransduction-Piloted Whole-Cell Vaccines for Spatiotemporal Modulation of Postoperative Antitumor Immunity.

Whole tumor cell vaccines hold promise by presenting a broader spectrum of autologous-origin tumor antigens to combat postoperative recurrence and metastasis. However, challenges such as intractable adjuvant modification and obscure interactions with antigen-presenting cells in the postoperative microenvironment impede their translation into effective personalized immunotherapies. In this study, we propose cancer vaccines derived from manganese oxide-immobilized resected tumor cells, featuring whole tumor antigens and adjustable stiffness to modulate interactions with antigen-presenting cells in the postoperative microenvironment. These vaccines effectively stimulate dendritic cell phagocytosis and function through sequential stiffness-mediated mechanotransduction and interferon signaling. We evaluated their efficacy using an orthotopic triple-negative breast cancer mouse model and found that combining the vaccines with radiotherapy effectively inhibits postoperative tumor recurrence and metastasis. Our study underscores the potential of utilizing mechanotransduced adjuvants alongside directly inactivated whole-cell vaccines as a universal solution for preventing postoperative tumor recurrence.

Targeting Cholesterol Biosynthesis with Statins Synergizes with AKT Inhibitors in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer patient deaths due to extensive molecular heterogeneity, high recurrence rates, and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. Here, we performed a genome-wide CRISPR/Cas9 screen with PI3Kα and AKT inhibitors to find targetable synthetic lethalities in TNBC. Cholesterol homeostasis was identified as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro in mouse TNBC xenografts and in patient-derived estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single-agent or combination treatment with AKT inhibitor and pitavastatin, which was rescued by inhibition of the cholesterol-trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels, and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. These findings support combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. Significance: Two FDA-approved compounds, AKT inhibitors and pitavastatin, synergize to induce cell death in triple-negative breast cancer, motivating evaluation of the efficacy of this combination in clinical trials.