Triple-negative Breast Cancer Models Research Articles

Erythropoietin induces tumour progression and CD39 expression on immune cells in a preclinical model of triple-negative breast cancer.

The adverse effects observed in some cancer patients treated with erythropoiesis-stimulating agents such as erythropoietin (EPO) might be due to the latter's well-known immunosuppressive functions. Here, we used a mouse model of syngeneic triple-negative breast cancer to explore EPO's immunomodulatory role in a tumour setting. Our results showed that EPO treatment promotes tumour growth, exacerbates the 'immune desert', and results in a 'cold tumour'. EPO treatment changed the immune cell distribution in peripheral blood, secondary lymphoid organs, and the tumour microenvironment (TME). Our in-depth analysis showed that EPO mainly impacts CD4 T cells by accelerating their activation in the spleen and thus their subsequent exhaustion in the TME. This process is accompanied by a general elevation of CD39 expression by several immune cells (notably CD4 T cells in the tumour and spleen), which promotes an immunosuppressive TME. Lastly, we identified a highly immunosuppressive CD39+ regulatory T cell population (ICOS+, CTLA4+, Ki67+) as a potential biomarker of the risk of EPO-induced tumour progression. EPO displays pleiotropic immunosuppressive functions and enhances mammary tumour progression in mice.

Functional exploration of copy number alterations in a Drosophila model of triple-negative breast cancer.

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

Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC.

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

177Lu-SN201 nanoparticle shows superior anti-tumor efficacy over conventional cancer drugs in 4T1 orthotopic model.

In the current in-vivo study we demonstrate the potential of the radiolabeled nanoparticle 177Lu-SN201 as an effective anticancer treatment, as evidenced by significantly prolonged survival and reduced tumor burden in the aggressive, triple negative 4T1 murine breast cancer model. We show with high statistical significance that 177Lu-SN201 is superior at suppressing the tumor growth not only compared to vehicle but also to the commonly used cancer drugs paclitaxel, niraparib, carboplatin, and the combination of the immune checkpoint inhibitors anti PD-1 and anti-CTLA-4. The dosing of the standard drugs were based on examples in the literature where good effects have been seen in various mouse models. The treatment is reasonably well-tolerated, as indicated by clinical chemistry of liver and renal function through the measurement of glutamate pyruvate alanine aminotransferase, alanine amino transferase, blood urea nitrogen, and creatinine levels in plasma samples, despite some weight loss. Overall, 177Lu-SN201 presents as a promising therapeutic candidate for cancer treatment.

Nanomedicine Targeting Cuproplasia in Cancer: Labile Copper Sequestration Using Polydopamine Particles Blocks Tumor Growth In Vivo through Altering Metabolism and Redox Homeostasis.

Copper plays critical roles as a metal active site cofactor and metalloallosteric signal for enzymes involved in cell proliferation and metabolism, making it an attractive target for cancer therapy. In this study, we investigated the efficacy of polydopamine nanoparticles (PDA NPs), classically applied for metal removal from water, as a therapeutic strategy for depleting intracellular labile copper pools in triple-negative breast cancer models through the metal-chelating groups present on the PDA surface. By using the activity-based sensing probe FCP-1, we could track the PDA-induced labile copper depletion while leaving total copper levels unchanged and link it to the selective MDA-MB-231 cell death. Further mechanistic investigations revealed that PDA NPs increased reactive oxygen species (ROS) levels, potentially through the inactivation of superoxide dismutase 1 (SOD1), a copper-dependent antioxidant enzyme. Additionally, PDA NPs were found to interact with the mitochondrial membrane, resulting in an increase in the mitochondrial membrane potential, which may contribute to enhanced ROS production. We employed an in vivo tumor model to validate the therapeutic efficacy of PDA NPs. Remarkably, in the absence of any additional treatment, the presence of PDA NPs alone led to a significant reduction in tumor volume by a factor of 1.66 after 22 days of tumor growth. Our findings highlight the potential of PDA NPs as a promising therapeutic approach for selectively targeting cancer by modulating copper levels and inducing oxidative stress, leading to tumor growth inhibition as shown in these triple-negative breast cancer models.

Celecoxib Augments Paclitaxel-Induced Immunogenic Cell Death in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy that lacks effective targeted therapies. Inducing immunogenic cell death (ICD) in tumor cells represents a promising strategy to enhance therapeutic efficacy by promoting antitumor immunity. Paclitaxel (PTX), a commonly used chemotherapy drug for TNBC, can induce ICD; however, the resulting immunogenicity is limited. Thus, there is an urgent need to explore strategies that improve the effectiveness of ICD in TNBC by incorporating immunoregulatory agents. This study investigated the potential of celecoxib (CXB) to enhance PTX-induced ICD by blocking the biosynthesis of PGE2 in the tumor cells. We observed that the combination of CXB and PTX promoted the maturation of dendritic cells and primed a T cell-dependent immune response, leading to enhanced tumor rejection in a vaccination assay. To further optimize drug delivery in vivo, we developed cRGD-modified liposomes for the targeted codelivery of CXB and PTX. This delivery system significantly improved drug accumulation and triggered robust antitumor immunity in an orthotopic mouse model of TNBC. Moreover, it served as an in situ vaccine to inhibit tumor recurrence and lung metastasis. Overall, our findings provide in-depth insights into the therapeutic mechanism underlying the combination of CXB and PTX, highlighting their potential as effective immune-based therapies for TNBC.

Effect of AURKA targeting on nuclear PD-L1 and the efficacy of immune checkpoint inhibitors in triple-negative breast cancer.

e13153 Background: Aurora-A mitotic kinase (AURKA) plays a role in Triple Negative Breast Cancer (TNBC) progression through activation of epithelial to mesenchymal transition (EMT)-mediated cancer cell plasticity. Cancer cells that undergo EMT acquire a CD44high/CD24low and/or ALDHhigh cancer stem-like phenotype (Cancer Stem Cells) characterized by intrinsic drug resistance. Cancer Stem Cells (CSCs) shows high PD-L1 expression that induces tumor immune escape through CD8+ T-cells exhaustion. Significantly, “undraggable” nuclear PD-L1 may play a key role in induction of immune therapy resistance and cancer cell plasticity. Methods: (1) TNBC Models: MDA-MB 231, unique MDA-MB 231/LM Durvalumab resistant (DUV-R) and TNBC-M40 cells established from metastatic PDXs; (2) In Vitro Studies: TNBC cells were treated with the AURKA inhibitor alisertib (50nM). PD-L1 expression was evaluated by immunoblotting. Apoptosis was measured by immunofluorescence using Cleaved-PARP antibodies. To assess stemness capacity, TNBC cells were cultured under non-adherent conditions to form mammospheres. ALDH activity (functional stemness marker) was measured using the Aldefluor Kit. (3) In Vivo Studies: 1x106 MDA-MB 231/LM cells were injected into the mammary fat pad of humanized NSG-CD34+ female mice. Animals were treated with anti-PD-L1 ICI atezolizumab (20 mg/Kg) and alisertib (25 mg/Kg) as monotherapy or in combination. 1x106 MDA-MB 231/LM cells were also injected into the mammary fat pad of humanized NSG-CD34+ female mice and treated with durvalumab (20 mg/Kg) to develop durvalumab-resistant models. PD-L1, CD44, Vimentin expression and tumor infiltration of CD8+ T-cells were assessed by immunofluorescence. Results: Treatment of MDA-MB 231/LM xenografts with alisertib decreased the levels of CD44 and PD-L1 expression and increased CD8+ T-cells tumor infiltration. Because PD-L1 genetic targeting enhanced in vitro alisertib-induced apoptosis, MDA-MB 231/LM xenografts were treated with alisertib and the anti-PD-L1 ICI atezolizumab. Com- bination of atezolizumab and alisertib resulted in the reduced expression of the EMT marker vimentin that was linked to lack of organ metastasis. Combination of atezolizumab and alisertib also induced a significant reduction of ALDHHigh CSC in ex-vivo 3D-Organoids established from TNBC-M40 cells. MDA-MB 231/LM DUV-R cells showed increased nuclear PD-L1 compared to parental cells. The combination of alisertib with Immune Checkpoint Inhibitors (ICIs) induced apoptosis of MDA-MB 231/LM DUV-R cells through down-regulation of intra-tumoral nuclear PD-L1 expression. Conclusions: This study provides innovative pre-clinical rationale for combining AURKA inhibitors with ICIs to reduce “ undruggable” nuclear PD-L1 expression and impair cancer cell plasticity of metastatic TNBCs that currently have limited effective therapeutic options.

Hemay181: A novel SDC (sugar linked drug conjugate) for solid tumors targeting tumor microenvironment.

e15006 Background: Hemay181 is a novel broad-spectrum conjugated cytotoxic drug for treating advanced solid tumours. The drug exerts its inhibitory effect on tumour growth by releasing cytotoxic drug SN-38 in tumour tissues via β-glucuronidase (βGU) metabolism to inhibit the activity of topoisomerase I (TOP1). The water solubility of Hemay181 is increased > 5000-fold compared to SN-38. Hemay181 also binds to plasma albumin to form a macromolecular drug transporter, significantly improving the drug's pharmacokinetics. In addition, βGU has low activity in normal tissues, localised to the lysosomes, but is highly expressed in the hypoxic tumour microenvironment. Methods: In vitro and In Vivo studies showed Hemay181 inhibited growth in 29 human tumour cell lines and 16 human tumor Xenograft models, including colorectal cancer, gastric cancer, triple-negative breast cancer, head and neck cancer, pancreatic cancer, lung cancer, prostate cancer and melanoma. Results: In the absence of βGU, Hemay181 showed significantly weaker inhibitory activity than Hemay181+βGU or no significant inhibitory activity. In these In vitro studies with the addition of βGU, the activity was similar to SN-38, ranging from 3.9 nM to 159 nM. Hemay181 showed tumor selectivity with low activity against two normal human cell lines. The IC50 values of Hemay181 against Chang liver (normal liver cells) and MRC 5 (embryonic lung cells) were 318 nM and 14566 nM under non-enzymatic digestion conditions. In vivo, Hemay181 had significant inhibitory effects on the growth of human colorectal cancer, pancreatic cancer, head and neck cancer, triple-negative breast cancer, lung cancer, liver cancer, ovarian cancer and gastric cancer tumour models. The lowest effective dose of Hemay181 was 1.875mg/kg in the human colon cancer HCT116 model. Within the dose range of 3.75~60 mg/kg, the highest inhibitory rate of the above different tumour models could reach more than 90%. Hemay181 significantly reduced tumour volume compared to irinotecan, which only slowed the tumour growth rates. There was no significant decrease in the body weight of Hemay181 in these studies. In the colon cancer PDX model (CR0029) (30mg/kg), the tumour inhibition rate (TGI) was 78%. At 60mg/kg, in triple-negative breast cancer (BR1458) and non-small cell lung cancer (LU3075) PDX models, a TGI was 97% and 98%, respectively. At 60mg/kg, there was a significant tumour suppressive effect on larger tumours treated with irinotecan, with a reduction of tumour volume. In vitro , 1 μM Hemay181 is stable in CD1 mouse and SD rat plasma, with a minor degree of metabolism in beagle dog, cynomolgus monkey and human plasma. Conclusions: In summary, the novel broad-spectrum anti-tumour drug Hemay181 has the characteristics of high water solubility, tumour-targeting ability, low toxicity and side effects on normal tissues. It is under evaluation in ongoing clinical studies.

Repurposing hemostatic drug desmopressin in AVPR2-expressing triple-negative breast cancer: Target expression analysis and preclinical antineoplastic effects on tumor growth and metastatic progression.

e15087 Background: Desmopressin (dDAVP) is a hemostatic drug that acts as an agonist of the arginine vasopressin receptor 2 (AVPR2) that is present in vascular and malignant cells. Preclinical data show that dDAVP triggers cytostatic mechanisms, blocking angiogenesis and reducing metastases by limiting the survival of circulating tumor cells. Triple-negative breast cancer (TNBC) is associated with poor prognosis due to limited response to therapy and frequent early metastatic recurrence. Considering the unsatisfied clinical need for novel therapeutic options in TNBC, we evaluated the antitumoral activity of dDAVP on experimental models of TNBC, alone or in addition to chemotherapy. We also assessed the expression and clinical impact of AVPR2 in breast cancer samples. Methods: Human and murine TNBC cell lines were used for in vitro experiments. Target expression was assessed by qPCR and immunohistochemistry (IHC), in experimental or clinical tumors (NCT01606072). Effects of dDAVP were evaluated by proliferation, clonogenic, 3D growth and chemotaxis assays, qPCR arrays, and confocal microscopy. Local and distant tumor progression were assessed in vivo in nude or BALB/c mice. TIMER2.0 and UCSC Xena platforms (BRCA TCGA /n = 1260) were used for bioinformatic studies. Results: dDAVP significantly reduced growth and chemotaxis of AVPR2-expressing TNBC cells. Cytostatic effects of dDAVP were associated with altered actin cytoskeleton dynamics and differential expression of angiogenesis and metastasis-related genes. Cooperative effects were observed in vitro and in vivo after combining dDAVP with taxane or alkylating therapy. Tumor-bearing mice were treated with intravenous injections of dDAVP (0.3 µg/kg, thrice weekly) in combination with weekly cycles of paclitaxel (10 mg/kg i.p.) or carmustine (25 mg/kg i.p.). Combination regimens resulted in greater survival and tumor growth and metastasis inhibition. Bioinformatic analysis of the TCGA database showed that AVPR2 had a positive prognostic impact on overall survival in breast cancer patients, especially in the basal-like molecular subtype. AVPR2 also correlated with different immune cell infiltrates such as NK and dendritic cells, and CD4+ and CD8+ T cells. Moreover, AVPR2 expression was detected by IHC in 6/18 breast cancer clinical samples using a small cohort of surgical specimens. Conclusions: AVPR2 expression is associated with improved outcomes in patients with TNBC. Agonist activation of AVPR2 using dDAVP leads to cancer cell growth inhibition in TNBC cell lines and mouse models. We propose dDAVP as a potential novel therapeutic option for treating TNBC either as monotherapy or in combination with chemotherapy. The therapeutic impact of its addition to immune checkpoint inhibitors remains to be explored.

Novel protein drug conjugate ANXV-chemo and cytotoxic and anti-proliferative effects against human triple-negative breast cancer in vitro.

e15024 Background: Targeted delivery of cytotoxic drugs to tumor cells and tumor microenvironment remains a fundamental challenge in the fight against cancer including triple-negative breast cancer (TNBC). The nonapoptotic externalization of the phospholipid phosphatidylserine (PS) on cancer cells is an attractive biomarker. Cancer cells have up to 90-fold more PS molecules externalized than healthy cells, and TNBC cells have high PS externalization. The Annexin A5 human protein has a high affinity to PS and has shown anti-cancer effects both in vitro and in vivo. Here we utilize the recombinant protein Annexin A5 (ANXV), currently in Phase 2a as a treatment of retinal vein occlusion, as a delivery vehicle to deliver a covalently linked chemotherapy (chemo) to TNBC cells via externalized PS. Methods: Chemo was conjugated to ANXV through a one-step SMCC crosslinking reaction. ANXV-chemo was analyzed via LC-MS and the weighted average of the drug-to-ANXV ratio (DAR) was determined. MDA-MB-231 TNBC cells were utilized for internalization, cytotoxic, and proliferation studies. Incucyte was used to monitor live cells for up to 96 hours (samples tested in quadruplicate). For internalization studies, ANXV-pHrodo red was incubated with the cells, and the fluorescent intensity was measured over 48 hours. For the cytotoxic and proliferation studies, ANXV-chemo (0-100 nM), sacituzumab govitecan-hziy (SG) (0-100 nM), or thefree chemo (0-200nM) were incubated with the cells for up to 96 hours. All concentrations refer to the amount of drug linked to the conjugates or free drug. All data analysis was completed in GraphPad Prism (10.1.1). Results: ANXV-chemo was successfully crosslinked and had a DAR of 2.4 ± 0.23 chemo molecules. ANXV was quickly internalized, and ANXV could be detected within the cells for 48 hours after initial incubation, indicating constant cell uptake of the protein. The ANXV-chemo was nearly 30 times more cytotoxic to the TNBC cells than the free chemo as indicated by the EC50 after 72 hours (ANXV-chemo = 1.2 nM; free chemo = 36.7 nM). When comparing equal molar drug concentrations (4 nM), ANXV-chemo had more significant anti-proliferative and cytotoxic effects than the standard of care SG. By 24 hours, ANXV-chemo significantly increased cell death (p < 0.0021). By 48 hours, ANXV-chemo significantly decreased proliferation (p < 0.0332). After 96 hours, ANXV-chemo inhibited proliferation by 5.5 times (p < 0.0001) and induced 6.5 times more cell death (p < 0.0002) when compared to SG. Conclusions: By covalently linking chemo to ANXV, an efficient and PS-targeted treatment for TNBC has been developed. ANXV-chemo demonstrated significantly better anticancer effects in vitro when compared to the free drug and current targeted standard of care treatments. An investigation of ANXV-chemo as a targeted anticancer agent is currently underway in a murine TNBC model.

A combination of a TLR7/8 agonist and an epigenetic inhibitor suppresses triple-negative breast cancer through triggering anti-tumor immune

BackgroundCombination therapy involving immune checkpoint blockade (ICB) and other drugs is a potential strategy for converting immune-cold tumors into immune-hot tumors to benefit from immunotherapy. To achieve drug synergy, we developed a homologous cancer cell membrane vesicle (CM)-coated metal-organic framework (MOF) nanodelivery platform for the codelivery of a TLR7/8 agonist with an epigenetic inhibitor.MethodsA novel biomimetic codelivery system (MCM@UN) was constructed by MOF nanoparticles UiO-66 loading with a bromodomain-containing protein 4 (BRD4) inhibitor and then coated with the membrane vesicles of homologous cancer cells that embedding the 18 C lipid tail of 3M-052 (M). The antitumor immune ability and tumor suppressive effect of MCM@UN were evaluated in a mouse model of triple-negative breast cancer (TNBC) and in vitro. The tumor immune microenvironment was analyzed by multicolor immunofluorescence staining.ResultsIn vitro and in vivo data showed that MCM@UN specifically targeted to TNBC cells and was superior to the free drug in terms of tumor growth inhibition and antitumor immune activity. In terms of mechanism, MCM@UN blocked BRD4 and PD-L1 to prompt dying tumor cells to disintegrate and expose tumor antigens. The disintegrated tumor cells released damage-associated molecular patterns (DAMPs), recruited dendritic cells (DCs) to efficiently activate CD8+ T cells to mediate effective and long-lasting antitumor immunity. In addition, TLR7/8 agonist on MCM@UN enhanced lymphocytes infiltration and immunogenic cell death and decreased regulatory T-cells (Tregs). On clinical specimens, we found that mature DCs infiltrating tumor tissues of TNBC patients were negatively correlated with the expression of BRD4, which was consistent with the result in animal model.ConclusionMCM@UN specifically targeted to TNBC cells and remodeled tumor immune microenvironment to inhibit malignant behaviors of TNBC.

Syringeable Near-Infrared Light-Activated In Situ Immunogenic Hydrogel Boosts the Cancer-Immunity Cycle to Enhance Anticancer Immunity.

Effective anticancer immunity depends on properly activating multiple stepwise events in the cancer-immunity cycle. An immunologically "cold" tumor microenvironment (TME) engenders immune evasion and refractoriness to conventional checkpoint blockade immunotherapy. Here, we combine nanoparticle formulations and an in situ formed hydrogel scaffold to treat accessible tumors locally and to stimulate systemic immunity against metastatic tumor lesions. The nanoparticles encapsulate poly(ε-caprolactone)-derived cytotoxic chemotherapy and adjuvant of Toll-like receptor 7/8 through a reactive oxygen species (ROS)-cleavable linker that can be self-activated by the coassembled neighboring photosensitizer following near-infrared (NIR) laser irradiation. Further development results in syringeable, NIR light-responsive, and immunogenic hydrogel (iGEL) that can be implanted peritumorally and deposited into the tumor surgical bed. Upon NIR laser irradiation, the generated ROS induces iGEL degradation and bond cleavage in the polymer-drug conjugates, triggering the immunogenic cell death cascade in cancer cells and spontaneously releasing encapsulated agents to rewire the cancer-immunity cycle. Notably, upon application in multiple preclinical models of melanoma and triple-negative breast cancer, which are aggressive and refractory to conventional immunotherapy, iGEL induces durable remission of established tumors, extends postsurgical tumor-free survival, and inhibits metastatic burden. The result of this study is a locally administrable immunogenic hydrogel for triggering host systemic immunity to improve immunotherapeutic efficacy with minimal off-target side effects.

Targeting neddylation and sumoylation in chemoresistant triple negative breast cancer

Triple negative breast cancer (TNBC) accounts for 15–20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.

Breaking the Tumor Chronic Inflammation Balance with a Programmable Release and Multi-Stimulation Engineering Scaffold for Potent Immunotherapy.

Tumor-associated chronic inflammation severely restricts the efficacy of immunotherapy in cold tumors. Here, a programmable release hydrogel-based engineering scaffold with multi-stimulation and reactive oxygen species (ROS)-response (PHOENIX) is demonstrated to break the chronic inflammatory balance in cold tumors to induce potent immunity. PHOENIX can undergo programmable release of resiquimod and anti-OX40 under ROS. Resiquimod is first released, leading to antigen-presenting cell maturation and the transformation of myeloid-derived suppressor cells and M2 macrophages into an antitumor immune phenotype. Subsequently, anti-OX40 is transported into the tumor microenvironment, leading to effector T-cell activation and inhibition of Treg function. PHOENIX consequently breaks the chronic inflammation in the tumor microenvironment and leads to a potent immune response. In mice bearing subcutaneous triple-negative breast cancer and metastasis models, PHOENIX effectively inhibited 80% and 60% of tumor growth, respectively. Moreover, PHOENIX protected 100% of the mice against TNBC tumor rechallenge by electing a robust long-term antigen-specific immune response. An excellent inhibition and prolonged survival in PHOENIX-treated mice with colorectal cancer and melanoma is also observed. This work presents a potent therapeutic scaffold to improve immunotherapy efficiency, representing a generalizable and facile regimen for cold tumors.

Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Exploring the prognostic significance of arm-level copy number alterations in triple-negative breast cancer.

Somatic copy number alterations (SCNAs) are prevalent in cancer and play a significant role in both tumorigenesis and therapeutic resistance. While focal SCNAs have been extensively studied, the impact of larger arm-level SCNAs remains poorly understood. Here, we investigated the association between arm-level SCNAs and overall survival in triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer lacking targeted therapies. We identified frequent arm-level SCNAs, including 21q gain and 7p gain, which correlated with poor overall survival in TNBC patients. Further, we identified the expression of specific genes within these SCNAs associated with survival. Notably, we found that the expression of RIPK4, a gene located on 21q, exhibited a strong correlation with poor overall survival. In functional assays, we demonstrated that targeting Ripk4 in a murine lung metastatic TNBC model significantly reduced tumor burden, improved survival, and increased CD4+ and CD8+ T cell infiltration. RIPK4 enhanced the survival of triple-negative breast cancer cells at secondary sites, thereby facilitating the formation of metastatic lesions. Our findings highlight the significance of arm-level SCNAs in breast cancer progression and identify RIPK4 as a putative driver of TNBC metastasis and immunosuppression.

Exogenous Metabolic Modulators Improve Response to Carboplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks targeted therapies, leaving cytotoxic chemotherapy as the current standard treatment. However, chemotherapy resistance remains a major clinical challenge. Increased insulin-like growth factor 1 signaling can potently blunt chemotherapy response, and lysosomal processes including the nutrient scavenging pathway autophagy can enable cancer cells to evade chemotherapy-mediated cell death. Thus, we tested whether inhibition of insulin receptor/insulin-like growth factor 1 receptor with the drug BMS-754807 and/or lysosomal disruption with hydroxychloroquine (HCQ) could sensitize TNBC cells to the chemotherapy drug carboplatin. Using in vitro studies in multiple TNBC cell lines, in concert with in vivo studies employing a murine syngeneic orthotopic transplant model of TNBC, we show that BMS-754807 and HCQ each sensitized TNBC cells and tumors to carboplatin and reveal that exogenous metabolic modulators may work synergistically with carboplatin as indicated by Bliss analysis. Additionally, we demonstrate the lack of overt in vivo toxicity with our combination regimens and, therefore, propose that metabolic targeting of TNBC may be a safe and effective strategy to increase sensitivity to chemotherapy. Thus, we conclude that the use of exogenous metabolic modulators, such as BMS-754807 or HCQ, in combination with chemotherapy warrants additional study as a strategy to improve therapeutic responses in women with TNBC.

Constructing a novel prognostic model for triple-negative breast cancer based on genes associated with vasculogenic mimicry.

Research has shown a connection between vasculogenic mimicry (VM) and cancer progression. However, the functions of genes related to VM in the emergence and progression of TNBC have not been completely elucidated. A survival risk model was constructed by screening biomarkers using DESeq2 and WGCNA based on public TNBC transcriptome data. Furthermore, gene set enrichment analysis was performed, and tumor microenvironment and drug sensitivity were analyzed. The selected biomarkers were validated via quantitative PCR detection, immunohistochemical staining, and protein detection in breast cancer cell lines. Biomarkers related to the proliferation and migration of TNBC cells were validated via in vitro experiments. The findings revealed that 235 target genes were connected to the complement and coagulation cascade pathways. The risk score was constructed using KCND2, NRP1, and VSTM4. The prognosis model using the risk score and pathological T stage yielded good validation results. The clinical risk of TNBC was associated with the angiogenesis signaling pathway, and the low-risk group exhibited better sensitivity to immunotherapy. Quantitative PCR and immunohistochemistry indicated that the expression levels of KCND2 in TNBC tissues were higher than those in adjacent nontumor tissues. In the TNBC cell line, the protein expression of KCND2 was increased. Knockdown of KCND2 and VSTM4 inhibited the proliferation and migration of TNBC cells in vitro. In this study, three VM-related biomarkers were identified, including KCND2, NRP1, and VSTM4. These findings are likely to aid in deepening our understanding of the regulatory mechanism of VM in TNBC.

Peposertib, a DNA-PK Inhibitor, Enhances the Anti-Tumor Efficacy of Topoisomerase II Inhibitors in Triple-Negative Breast Cancer Models.

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.

Antibody and siRNA Nanocarriers to Suppress Wnt Signaling, Tumor Growth, and Lung Metastasis in Triple‐Negative Breast Cancer

AbstractThe paucity of targeted therapies for triple‐negative breast cancer (TNBC) causes patients with this aggressive disease to suffer a poor clinical prognosis. A promising target for therapeutic intervention is the Wnt signaling pathway, which is activated in TNBC cells when extracellular Wnt ligands bind overexpressed Frizzled7 (FZD7) transmembrane receptors. This stabilizes intracellular β‐catenin proteins that in turn promote transcription of oncogenes that drive tumor growth and metastasis. To suppress Wnt signaling in TNBC cells, this work develops therapeutic nanoparticles (NPs) functionalized with FZD7 antibodies and β‐catenin small interfering RNAs (siRNAs). The antibodies enable TNBC cell specific binding and inhibit Wnt signaling by locking FZD7 receptors in a ligand unresponsive state, while the siRNAs suppress β‐catenin through RNA interference. Compared to NPs coated with antibodies or siRNAs individually, NPs coated with both agents more potently reduce the expression of several Wnt related genes in TNBC cells, leading to greater inhibition of cell proliferation, migration, and spheroid formation. In two murine models of metastatic TNBC, the dual antibody/siRNA nanocarriers outperformed controls in terms of inhibiting tumor growth, metastasis, and recurrence. These findings demonstrate suppressing Wnt signaling at both the receptor and mRNA levels via antibody/siRNA nanocarriers is a promising approach to combat TNBC.