4T1 Murine Breast Cancer Research Articles

The highly metastatic 4T1 breast carcinoma model possesses features of a hybrid epithelial/mesenchymal phenotype.

Epithelial-mesenchymal transitions (EMTs) are thought to promote metastasis via downregulation of E-cadherin (also known as Cdh1) and upregulation of mesenchymal markers such as N-cadherin (Cdh2) and vimentin (Vim). Contrary to this, E-cadherin is retained in many invasive carcinomas and promotes collective cell invasion. To investigate how E-cadherin regulates metastasis, we examined the highly metastatic, E-cadherin-positive murine 4T1 breast cancer model, together with the less metastatic, 4T1-related cell lines 4T07, 168FARN and 67NR. We found that 4T1 cells display a hybrid epithelial/mesenchymal phenotype with co-expression of epithelial and mesenchymal markers, whereas 4T07, 168FARN, and 67NR cells display progressively more mesenchymal phenotypes in vitro that relate inversely to their metastatic capacity in vivo. Using RNA interference and constitutive expression, we demonstrate that the expression level of E-cadherin does not determine 4T1 or 4T07 cell metastatic capacity in mice. Mechanistically, 4T1 cells possess highly dynamic, unstable cell-cell junctions and can undergo collective invasion without E-cadherin downregulation. However, 4T1 orthotopic tumors in vivo also contain subregions of EMT-like loss of E-cadherin. Thus, 4T1 cells function as a model for carcinomas with a hybrid epithelial/mesenchymal phenotype that promotes invasion and metastasis.

Triple negative breast cancer migration is modified by mitochondrial metabolism alteration induced by natural extracts of C. spinosa and P. alliacea

Tumor metabolism is a crucial aspect of cancer development, and mitochondria plays a significant role in the aggressiveness and metastasis of tumors. As a result, mitochondria have become a promising therapeutic target in cancer treatment, leading to the development of compounds known as mitocans. In our group, we have consolidated the search of anticancer therapies based on natural products derived from plants, obtaining extracts such as P2Et from Caesalpinia spinosa and Anamu-SC from Petiveria alliacea, which have been shown to have antitumor activities in different cancer models. These extracts, due to their complex molecular composition, can interfere with multiple functions during tumor progression. To better understand how these natural products operate (P2Et and Anamu-SC), we constructed a model using 4T1 murine breast cancer cells with reduced expression of genes associated with glycolysis (Hexokinase-2) and mitochondrial function (Cqbp). The results indicate that the cells were more sensitive to the Anamu-SC extract, showing significant decreases in glucose consumption, ATP production, and oxygen consumption rate. Additionally, we observed changes in mitochondrial function, which reduced the cells’ ability to migrate, particularly when C1qbp was silenced. This triple-negative breast cancer model allows us to identify potential natural products that can modulate tumor cell metabolism.

The Proapoptotic Action of Pyrrolidinedione-Thiazolidinone Hybrids towards Human Breast Carcinoma Cells Does Not Depend on Their Genotype.

The development of new, effective agents for the treatment of breast cancer remains a high-priority task in oncology. A strategy of treatment for this pathology depends significantly on the genotype and phenotype of human breast cancer cells. We aimed to investigate the antitumor activity of new pyrrolidinedione-thiazolidinone hybrid molecules Les-6287, Les-6294, and Les-6328 towards different types of human breast cancer cells of MDA-MB-231, MCF-7, T-47D, and HCC1954 lines and murine breast cancer 4T1 cells by using the MTT, clonogenic and [3H]-Thymidine incorporation assays, flow cytometry, ELISA, and qPCR. The studied hybrids possessed toxicity towards the mentioned tumor cells, with the IC50 ranging from 1.37 to 21.85 µM. Simultaneously, these derivatives showed low toxicity towards the pseudonormal human breast epithelial cells of the MCF-10A line (IC50 > 93.01 µM). Les-6287 at 1 µM fully inhibited the formation of colonies of the MCF-7, MDA-MB-231, and HCC1954 cells, while Les-6294 and Les-6328 did that at 2.5 and 5 µM, respectively. Les-6287 suppressed DNA biosynthesis in the MCF-7, MDA-MB-231, and HCC1954 cells. At the same time, such an effect on the MCF-10A cells was significantly lower. Les-6287 induces apoptosis using extrinsic and intrinsic pathways via a decrease in the mitochondrial membrane potential, increasing the activity of caspases 3/7, 8, 9, and 10 in all immunohistochemically different human breast cancer cells. Les-6287 decreased the concentration of the metastasis- and invasion-related proteins MMP-2, MMP-9, and ICAM-1. It did not induce autophagy in treated cells. In conclusion, the results of our study suggest that the synthesized hybrid pyrrolidinedione-thiazolidinones might be promising agents for treating breast tumors of different types.

Ru(II)-Photoactive Agents for Targeting ER Stress and Immunogenic Cell Death.

Immunotherapy has emerged as a promising avenue for cancer treatment by bolstering the immune system's ability to recognize and attack cancer cells. Photodynamic therapy shows potential in enhancing antitumor immunity, though the mechanisms behind its success are not fully understood. In this manuscript, we investigate two previously reported green light activated PCT/PDT agents where compound 2 - [Ru(tpy)(Me2bpy)( 3 )] 2+ , (tpy = 2,2':6',2''- terpyridine, Me2bpy = 6,6'-dimethyl-2,2'-bipyridine, 3 = pyridyl-BODIPY-I2,) - shows remarkable photoselectivity in assays containing both 2D cancer cells and 3D cocultures containing BALB/c macrophages and 4T1 murine breast cancer cells. Through flow cytometry and protein analysis, we found complex 2 displays superior evidence of induced endoplasmic reticulum (ER) stress markers and indicators of immunogenic cell death (ICD) compared to its ligand 3 , despite its weaker photoselectivity. Most importantly, these results were supported by in vivo studies where 2 produced anti-tumor immunity against the 4T1 tumor model in BALB/c mice. Complete tumor elimination was achieved in 2/8 mice, and these mice were both protected against a subsequent contralateral rechallenge and showed increased ex vivo peripheral tumor antigen-specific recall, suggesting memory T cells are induced by 2 . Signatures of M1 macrophage polarization were also evident in tumor tissue from the remaining 6/8 mice treated with 2 compared to untreated tumors. These findings demonstrate Ru(II) complexation plays a critical role in ER targeting which triggers ICD, highlighting the potential of Ru(II) agents as future in situ tumor vaccines.

Using chitosan-coated magnetite nanoparticles as a drug carrier for opioid delivery against breast cancer

Over the past decades, opium derivatives have been discovered as new anticancer agents. In our study, Fe3O4 superparamagnetic nanoparticles (SPIONs) decorated with chitosan were loaded with papaverine or noscapine to surmount drug delivery-related obstacles. Modifying the magnetic nanoparticles (MNP) surface with polymeric materials such as chitosan prevents oxidation and provides a site for drug linkage, which renders them a great drug carrier. The obtained systems were characterized by DLS (20–40 nm were achieved for MNPs and drug- loaded MNPs), TEM (spherical with average size of 11–20 nm) FTIR, XRD, and VSM (71.3 − 42.8 emu/g). Contrary to noscapine, papaverine-MNPs attenuated 4T1 murine breast cancer cell proliferation (11.50 ± 1.74 µg/mL) effectively compared to the free drug (62.35 ± 2.88 µg/mL) while sparing L-929 fibroblast cells (138.14 ± 4.38 µg/mL). Furthermore, SPION and SPION-chitosan displayed no cytotoxic activity. Colony-formation assay confirmed the long-term cytotoxicity of nanostructures. Both developed formulations promoted ROS production accompanied by late apoptotic cell death. The biocompatible nanoparticle exerted an augmenting effect to deliver papaverine to metastatic breast cancer cells.

RNA editing enzyme ADAR2 regulates P-glycoprotein expression in murine breast cancer cells through the circRNA-miRNA pathway

Among the various RNA modifications, adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, is the most common nucleotide conversion in mammalian cells. The pathological relevance of ADAR expression has been highlighted in recent human genetic studies. Low expression of the ADAR2 gene is correlated with a poor prognosis in breast cancer patients, but the underlying mechanism remains enigmatic. In this study, we constructed Adar2-knockdown (Adar2-KD) murine breast cancer 4T1 cells and observed their reduced susceptibility to chemotherapeutic drug doxorubicin. Downregulation of ADAR2 induced the expression of P-glycoprotein (P-gp), leading to a reduction in the intracellular accumulation of doxorubicin. The upregulation of P-gp occurred at the post-transcriptional level due to the decreased miR-195a-3p function. The search for the underlying cause of the induction of P-gp expression in Adar2-KD 4T1 cells led to the identification of circular RNA (circRNA) circHif1a as a sponge for miR-195a-3p. The enhanced expression of circHif1a inhibited miR-195a-3p function, resulting in the upregulation of P-gp expression. These results suggest that ADAR2 acts as a suppressor of circHif1a biogenesis and then allows miR-195a-3p to interfere with P-gp translation. Our findings may help to improve drug efficacy by clarifying the mechanism of chemoresistance in breast cancer.

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.

A recombinant adeno-associated virus expressing secretory TGF-β type Ⅱ receptor inhibits triple-negative murine breast cancer 4T1 cell proliferation and lung metastasis in mice

To investigate the effects of an adeno-associated virus (AAV2) vector expressing secretory transforming growth factor-β (TGF-β) type Ⅱ receptor (sTβRⅡ) extracellular domain-IgG2a Fc fusion protein (sTβRⅡ-Fc) on proliferation and migration of triple-negative murine breast cancer 4T1 cells in mice. The pAAV-sTβRⅡ-Fc vector expressing sTβRⅡ-Fc fusion protein constructed by molecular cloning, the capsid protein-expressing vector pAAV2 and the helper vector were co-transfected into HEK 293T cells to prepare the recombinant AAV2-sTβRⅡ virus, which was purified by density gradient centrifugation with iodixanol. Western blotting was used to examine the effects of AAV-sTβRⅡ virus on Smad2/3 phosphorylation in 4T1 cells and on expression levels of E-cadherin, vimentin and p-Smad2/3 in 4T1 cell xenografts in mice. BALB/c mice bearing subcutaneous xenografts of luciferase-expressing 4T1 cells received intravenous injections of AAV-sTβRⅡ virus, AAV-GFP virus or PBS (n=6) through the tail vein, and the proliferation and migration of 4T1 cells were analyzed with in vivo imaging. Ki67 expression in the tumor tissues and sTβRⅡ protein expressions in mouse livers were detected with immunohistochemistry and immunofluorescence staining, and tumor metastases in the vital organs were examined with HE staining. The recombinant pAAV-sTβRⅡ-Fc vector successfully expressed sTβRⅡ in HEK 293T cells. Infection with AAV2-sTβRⅡ virus significantly reduced TGF-β1-induced Smad2/3 phosphorylation in 4T1 cells and effectively inhibited proliferation and lung metastasis of 4T1 xenografts in mice (P<0.05). In the tumor-bearing mice, intravenous injection of AAV-sTβRⅡ virus significantly increased E-cadherin expression, reduced vimentin and Ki67 protein expressions and Smad2/3 phosphorylation level in the tumor tissues (P<0.05 or 0.01), and induced liver-specific sTβRⅡ expression without causing body weight loss or heart, liver, spleen or kidney pathologies. The recombinant AVV2 vector encoding sTβRⅡ extracellular domain is capable of blocking the TGF-β signaling pathway to inhibit the proliferation and lung metastasis of 4T1 cells in mice.

Anti-Netrin-1 decorated nanoparticles combined with chemotherapy for the treatment of triple-negative breast cancer

Nanoparticle's success as drug delivery systems for cancer treatment has been achieved through passive targeting mechanisms. However, tumor heterogeneity and rapid drug clearance limit the treatment efficacy. Improved outcomes and selective drug release can be achieved by grafting ligands at the surface of nanocarriers that bind molecules overexpressed in the tumor microenvironment (TME). In this work, we developed a docetaxel-loaded nanoemulsions (NEs) binding an anti-netrin-1 monoclonal antibody (NP137) to selectively target the netrin-1 protein overexpressed in many different tumors. The goal is to refine a combined approach utilizing NP137 and docetaxel as an improved tumor-targeting chemotherapeutic agent for addressing triple-negative breast cancer (TNBC). Several factors have been considered for the optimization of the active targeted drug delivery system via the click-chemistry conjugation, as the impact of PEGylated surfactant that stabilize the NEs shell on conjugation efficiency, cytocompatibility with EMT6 cell line and colloidal stability over time of NEs. Results showed that a 660 Da PEG chain length contributed to NEs colloidal stability and had no impact on cell viability or on the antibody binding ability for its ligand after surface conjugation. Moreover, docetaxel was encapsulated into the oily core of NEs, with an encapsulation efficiency of 70 %. To validate our treatment strategy in vivo, the 4T1 murine breast cancer model was used. As a result, the comparison of active-targeted and non-targeted NEs revealed that only active-targeted NE could decrease the tumor growth rate.

Ruthenium-Cathepsin Inhibitor Conjugates for Green Light-Activated Photodynamic Therapy and Photochemotherapy.

Dysregulated cathepsin activity is linked to various human diseases including metabolic disorders, autoimmune conditions, and cancer. Given the overexpression of cathepsin in the tumor microenvironment, cathepsin inhibitors are promising pharmacological agents and drug delivery vehicles for cancer treatment. In this study, we describe the synthesis and photochemical and biological assessment of a dual-action agent based on ruthenium that is conjugated with a cathepsin inhibitor, designed for both photodynamic therapy (PDT) and photochemotherapy (PCT). The ruthenium-cathepsin inhibitor conjugate was synthesized through an oxime click reaction, combining a pan-cathepsin inhibitor based on E64d with the Ru(II) PCT/PDT fragment [Ru(dqpy)(dppn)], where dqpy = 2,6-di(quinoline-2-yl)pyridine and dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine. Photochemical investigations validated the conjugate's ability to release a triazole-containing cathepsin inhibitor for PCT and to generate singlet oxygen for PDT upon exposure to green light. Inhibition studies demonstrated the conjugate's potent and irreversible inactivation of purified and intracellular cysteine cathepsins. Two Ru(II) PCT/PDT agents based on the [Ru(dqpy)(dppn)] moiety were evaluated for photoinduced cytotoxicity in 4T1 murine triple-negative breast cancer cells, L929 fibroblasts, and M0, M1, and M2 macrophages. The cathepsin inhibitor conjugate displayed notable selectivity for inducing cell death under irradiation compared to dark conditions, mitigating toxicity in the dark observed with the triazole control complex [Ru(dqpy)(dppn)(MeTz)]2+ (MeTz = 1-methyl-1H-1,2,4-triazole). Notably, our lead complex is among a limited number of dual PCT/PDT agents activated with green light.

Targeting CXCR4/CXCL12 axis via [177Lu]Lu-DOTAGA.(SA.FAPi)2 with CXCR4 antagonist in triple-negative breast cancer

PurposeRadiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role in orchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC).MethodsPublic database was first interrogated to reveal the correlation between CAFs’ scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen.ResultsCAFs’ scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12.ConclusionThe combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.

Epiberberine inhibits bone metastatic breast cancer-induced osteolysis

Ethnopharmacological relevanceThe anti-tumor related diseases of Coptidis Rhizoma (Huanglian) were correlated with its traditional use of removing damp-heat, clearing internal fire, and counteracting toxicity. In the recent years, Coptidis Rhizoma and its components have drawn extensive attention toward their anti-tumor related diseases. Besides, Coptidis Rhizoma is traditionally used as an anti-inflammatory herb. Epiberberine (EPI) is a significant alkaloid isolated from Coptidis Rhizoma, and exhibits multiple pharmacological activities including anti-inflammatory. However, the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis has not been demonstrated clearly. Aim of the studyBone metastatic breast cancer can lead to osteolysis via inflammatory factors-induced osteoclast differentiation and function. In this study, we try to analyze the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis. MethodsTo evaluate whether epiberberine could suppress bone metastatic breast cancer-induced osteolytic damage, healthy female Balb/c mice were intratibially injected with murine triple-negative breast cancer 4T1 cells. Then, we examined the inhibitory effect and underlying mechanism of epiberberine on breast cancer-induced osteoclastogenesis in vitro. Xenograft assay was used to study the effect of epiberberine on breast cancer cells in vivo. Moreover, we also studied the inhibitory effects and underlying mechanisms of epiberberine on RANKL-induced osteoclast differentiation and function in vitro. ResultsThe results show that epiberberine displayed potential therapeutic effects on breast cancer-induced osteolytic damage. Besides, our results show that epiberberine inhibited breast cancer cells-induced osteoclast differentiation and function by inhibiting secreted inflammatory cytokines such as IL-8. Importantly, we found that epiberberine directly inhibited RANKL-induced differentiation and function of osteoclast without cytotoxicity. Mechanistically, epiberberine inhibited RANKL-induced osteoclastogensis via Akt/c-Fos signaling pathway. Furthermore, epiberberine combined with docetaxel effectively protected against bone loss induced by metastatic breast cancer cells. ConclusionsOur findings suggested that epiberberine may be a promising natural compound for treating bone metastatic breast cancer-induced osteolytic damage by inhibiting IL-8 and is worthy of further exploration in preclinical and clinical trials.

Mucosal tumor vaccination delivering endogenous tumor antigens protects against pulmonary breast cancer metastases

BackgroundGenerally, early-stage breast cancer has a good prognosis. However, if it spreads systemically, especially with pulmonary involvement, prospects worsen dramatically. Importantly, tumor-infiltrating T cells contribute to tumor control, particularly intratumoral...

Mannosylated STING Agonist Drugamers for Dendritic Cell-Mediated Cancer Immunotherapy.

The Stimulator of Interferon Genes (STING) pathway is a promising target for cancer immunotherapy. Despite recent advances, therapies targeting the STING pathway are often limited by routes of administration, suboptimal STING activation, or off-target toxicity. Here, we report a dendritic cell (DC)-targeted polymeric prodrug platform (polySTING) that is designed to optimize intracellular delivery of a diamidobenzimidazole (diABZI) small-molecule STING agonist while minimizing off-target toxicity after parenteral administration. PolySTING incorporates mannose targeting ligands as a comonomer, which facilitates its uptake in CD206+/mannose receptor+ professional antigen-presenting cells (APCs) in the tumor microenvironment (TME). The STING agonist is conjugated through a cathepsin B-cleavable valine-alanine (VA) linker for selective intracellular drug release after receptor-mediated endocytosis. When administered intravenously in tumor-bearing mice, polySTING selectively targeted CD206+/mannose receptor+ APCs in the TME, resulting in increased cross-presenting CD8+ DCs, infiltrating CD8+ T cells in the TME as well as maturation across multiple DC subtypes in the tumor-draining lymph node (TDLN). Systemic administration of polySTING slowed tumor growth in a B16-F10 murine melanoma model as well as a 4T1 murine breast cancer model with an acceptable safety profile. Thus, we demonstrate that polySTING delivers STING agonists to professional APCs after systemic administration, generating efficacious DC-driven antitumor immunity with minimal side effects. This new polymeric prodrug platform may offer new opportunities for combining efficient targeted STING agonist delivery with other selective tumor therapeutic strategies.

Combined Blockade of Interleukin-6 and β-Catenin Inhibits Breast and Colon Tumor Growth In Vitro

Background: As the primary regulator of tumor activities, the tumor microenvironment contains several factors contributing to tumor progression and metastasis. Interleukin 6 (IL-6) and β-catenin are two key factors in the tumor microenvironment, which are effective in tumor progression in primary studies. This study aimed to evaluate the impact of IL-6 and β-catenin blockade on tumor growth and progression in vitro. Methods: In this study, small interfering RNA (siRNA) technology was used to target IL-6 and β-catenin using two murine cell lines, 4T1 (breast cancer) and CT26 (colon cancer). The target gene expression was analyzed by applying quantitative real-time polymerase chain reaction assays. The MTT assay was used to measure how a treatment could affect the cells’ survival ability. All data were analyzed by GraphPad Prism 9, and P-values less than 0.05 were considered significant. Results: Both cell lines demonstrated downregulation in the expression patterns of genes involved in apoptosis, metastasis, proliferation, and angiogenesis. Both cell lines showed significantly decreased IL-6 secretion following IL-6 targeting. Moreover, simultaneous targeting of IL-6 and β-catenin factors significantly reduced the survival of cancer cells. Conclusion: These results revealed that targeting β-catenin and IL-6 simultaneously may be a useful treatment strategy for colon and breast cancers. Further investigations are required to determine the effectiveness of these interventions and adjust their safety.

Calcitriol promotes M2 polarization of tumor-associated macrophages in 4T1 mouse mammary gland cancer via the induction of proinflammatory cytokines

Our research found that vitamin D3 (VD3) treatment increased lung metastasis in mice with 4T1 murine breast cancer (BC). This study aims to investigate the impact of VD3 on the activation of tumor-associated macrophages (TAMs) in BC. Mice bearing 4T1, E0771, 67NR BC cells, and healthy mice, were fed diets with varying VD3 contents (100—deficient, 1000—normal, and 5000 IU/kg—elevated). Some mice in the 1000 and 100 IU/kg groups received calcitriol. We studied bone metastasis and characterized TAMs and bone marrow-derived macrophages (BMDMs). 4T1 cells had higher bone metastasis potential in the 5000 IU/kg and calcitriol groups. In the same mice, an elevated tumor osteopontin level and M2 polarization of TAMs (MHCIIlow CD44high phenotype) were observed. Gene expression analysis confirmed M2 polarization of 4T1 (but not 67NR) TAMs and BMDMs, particularly in the 100 IU + cal group (increased Mrc1, Il23, and Il6). This polarization was likely due to COX-2/PGE2 induction in 4T1 calcitriol-treated cells, leading to increased proinflammatory cytokines like IL-6 and IL-23. Future studies will explore COX-2/PGE2 as a primary mediator of calcitriol-stimulated inflammation in the BC microenvironment, especially relevant for BC patients with VD3 deficiency and supplementation.

Copper Deposition in Polydopamine Nanostructure to Promote Cuproptosis by Catalytically Inhibiting Copper Exporters of Tumor Cells for Cancer Immunotherapy.

Cuproptosis is an emerging programmed cell death, displaying great potential in cancer treatment. However, intracellular copper content to induce cuproptosis is unmet, which mainly ascribes to the intracellular pumping out equilibrium mechanism by copper exporter ATP7A and ATP7B. Therefore, it is necessary to break such export balance mechanisms for desired cuproptosis. Mediated by diethyldithiocarbamate (DTC) coordination, herein a strategy to efficiently assemble copper ions into polydopamine nanostructure (PDA-DTC/Cu) for reprogramming copper metabolism of tumor is developed. The deposited Cu2+ can effectively trigger the aggregation of lipoylated proteins to induce cuproptosis of tumor cells. Beyond elevating intracellular copper accumulation, PDA-DTC/Cu enables to break the balance of copper metabolism by disrupting mitochondrial function and restricting the adenosine triphosphate (ATP) energy supply, thus catalytically inhibiting the expressions of ATP7A and ATP7B of tumor cells to enhance cuproptosis. Meanwhile, the killed tumor cells can induce immunogenic cell death (ICD) to stimulate the immune response. Besides, PDA-DTC/Cu NPs can promote the repolarization of tumor-associated macrophages (TAMs ) to relieve the tumor immunosuppressive microenvironment (TIME). Collectively, PDA-DTC/Cu presented a promising "one stone two birds" strategy to realize copper accumulation and inhibit copper export simultaneously to enhance cuproptosis for 4T1 murine breast cancer immunotherapy.

Label-Free In Vivo Monitoring of Lipid Droplets for Real-Time Assessment of Adipose Activator-Induced Tumor Suppression.

To enhance our comprehension of the fundamental mechanisms driving tumor metabolism and metastasis, it is essential to dynamically monitor intratumoral lipid droplet (LD) and collagen processes in vivo. Traditional LD analysis in tumors predominantly relies on observations of in vitro cells or tissue slices, which unfortunately hinder real-time insights into the dynamic behavior of LDs during in vivo tumor progression. In this study, we developed a dual-modality imaging technique that combines coherent anti-Stokes Raman scattering (CARS) and second-harmonic generation (SHG) microscopy for in vivo monitoring of tumor LDs and collagen alterations, assisted by a murine breast cancer 4T1 cell-based dorsal skinfold window. Specifically, we accomplished real-time observations and quantitative analysis of the LD size, density, and collagen alignment within living tumors through CARS/SHG imaging. Additionally, our findings demonstrate that real-time LD monitoring provides a valuable means of assessing the efficacy of anticancer drugs in vivo. We evaluated the impact of adipose activators on lipid metabolism, oxidative stress, and tumor suppression by monitoring changes in LD size and density. Overall, this study highlights the potential of dual-modality CARS/SHG microscopy as a sensitive and flexible tool for antitumor therapeutic strategies.

Propranolol inhibits EMT and metastasis in breast cancer through miR-499-5p-mediated Sox6

PurposeThis study will focus on 4T1 cells, a murine mammary adenocarcinoma cell line, as the primary research subject. We aim to investigate the inhibitory effects and mechanisms of propranolol on epithelial–mesenchymal transition (EMT) in breast cancer cells, aiming to elucidate this phenomenon at the miRNA level.MethodsIn this study, the EMT inhibitory effect of propranolol was observed through in vitro and animal experiments. For the screening of potential target miRNAs and downstream target genes, second-generation sequencing (SGS) and bioinformatics analysis were conducted. Following the screening process, the identified target miRNAs and their respective target genes were confirmed using various experimental methods. To confirm the target miRNAs and target genes, Western Blot (WB), reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence experiments were performed.ResultsIn this study, we found that propranolol significantly reduced lung metastasis in 4T1 murine breast cancer cells (p < 0.05). In vitro and in vivo experiments demonstrated that propranolol inhibited the epithelial–mesenchymal transition (EMT) as evidenced by Western Blot analysis (p < 0.05). Through next-generation sequencing (SGS), subsequent bioinformatics analysis, and PCR validation, we identified a marked downregulation of miR-499-5p (p < 0.05), suggesting its potential involvement in mediating the suppressive effects of propranolol on EMT. Overexpression of miR-499-5p promoted EMT, migration, and invasion of 4T1 cells, and these effects were not reversed or attenuated by propranolol (Validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). Sox6 was identified as a functional target of miR-499-5p, with its downregulation correlating with the observed EMT changes (p < 0.05). Silencing Sox6 or overexpressing miR-499-5p inhibited Sox6 expression, further promoting the processes of EMT, invasion, and migration in 4T1 cells. Notably, these effects were not alleviated by propranolol (validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). The direct interaction between miR-499-5p and Sox6 mRNA was confirmed by dual-luciferase reporter gene assay.ConclusionThese results suggest that propranolol may have potential as a therapeutic agent for breast cancer treatment by targeting EMT and its regulatory mechanisms.

Intratumor injected gold molecular clusters for NIR-II imaging and cancer therapy

Surgical resections of solid tumors guided by visual inspection of tumor margins have been performed for over a century to treat cancer. Near-infrared (NIR) fluorescence labeling/imaging of tumor in the NIR-I (800 to 900 nm) range with systemically administrated fluorophore/tumor-targeting antibody conjugates have been introduced to improve tumor margin delineation, tumor removal accuracy, and patient survival. Here, we show Au25 molecular clusters functionalized with phosphorylcholine ligands (AuPC, ~2 nm in size) as a preclinical intratumorally injectable agent for NIR-II/SWIR (1,000 to 3,000 nm) fluorescence imaging-guided tumor resection. The AuPC clusters were found to be uniformly distributed in the 4T1 murine breast cancer tumor upon intratumor (i.t.) injection. The phosphocholine coating afforded highly stealth clusters, allowing a high percentage of AuPC to fill the tumor interstitial fluid space homogeneously. Intra-operative surgical navigation guided by imaging of the NIR-II fluorescence of AuPC allowed for complete and non-excessive tumor resection. The AuPC in tumors were also employed as a photothermal therapy (PTT) agent to uniformly heat up and eradicate tumors. Further, we performed in vivo NIR-IIb (1,500 to 1,700 nm) molecular imaging of the treated tumor using a quantum dot-Annexin V (QD-P3-Anx V) conjugate, revealing cancer cell apoptosis following PTT. The therapeutic functionalities of AuPC clusters combined with rapid renal excretion, high biocompatibility, and safety make them promising for clinical translation.