4T1 Cells Research Articles

Near-infrared AIEgens for sulfatase imaging in breast cancer in vivo.

Aggregation-induced emission luminogens (AIEgens) enable highly sensitive and in situ visualization of sulfatase to benefit the early diagnosis of breast cancer (BC), but current sulfatase AIEgens always emit visible light (<650 nm). Herein, a near-infrared (NIR) AIEgen QMT-SFA is developed for sulfatase imaging in vivo. Hydrophilic QMT-SFA is cleaved by sulfatase to yield hydrophobic QMT-OH, which subsequently aggregates into nanoparticles to turn the AIE fluorescence "on", enabling sensitive sulfatase imaging in 4T1 cells and mouse models.

Glucosyltriazole amphiphile treatment attenuates breast cancer by modulating the AMPK signaling.

Breast cancer is the second most frequent cancer among women. Out of various subtypes, triple-negative breast cancers (TNBCs)account for 15% of breast cancers and exhibit more aggressive characteristics as well as a worse prognosis due to their proclivity for metastatic progression and limited therapeutic strategies. It has been demonstrated that AMP-activated protein kinase (AMPK)has context-specific protumorigenic implications in breast cancer cells. A set of glucosyltriazole amphiphiles, consisting of acetylated (9a-h) and unmodified sugar hydroxyl groups (10a-h), were synthesized and subjected to in vitro biological evaluation. Among them, 9h exhibited significant anticancer activity against MDA-MB-231,MCF-7, and 4T1 cell lines with IC50 values of 12.5, 15, and 12.55 μM, respectively. Further, compound 9h was evaluated for apoptosis and cell cycle analysis in in vitro models (using breast cancer cells) and antitumour activity in an in vivo model (orthotopic mouse model using 4T1 cells). Annexin-V assay results revealed that treatment with 9h caused 34% and 28% cell death at a concentration of 15 or 7.5 μM, respectively, while cell cycle analysis demonstrated that 9h arrested the cells at the G2/M or G1 phase in MCF-7, MDA-MB-231 and 4T1 cells, respectively. Further, in vivo, investigation showed that compound 9h exhibited equipotent as doxorubicin at 7.5 mg/kg, and superior efficacy than doxorubicin at 15 mg/kg. The mechanistic approach revealed that 9h showed potent anticancer activity in an in vivo orthotopic model (4T1 cells) partly by suppressing the AMPK activation. Therefore, modulating the AMPK activation could be a probable approach for targeting breast cancer and mitigating cancer progression.

Analysis of long-lasting tumor-specific antibodies from murine tumors treated with Aliya pulsed electric fields.

e14543 Background: Pulsed electric field (PEF) tissue destruction has demonstrated meaningful systemic immune responses in mice that are superior to thermal ablation, and synergize effectively with systemic chemotherapy and checkpoint blockade (CPB) immunotherapy. Immune cell populations are increased by PEF, including tumor specific activated CD8 T-cells and plasma B-cells. Antitumor immunity can include the presence of circulating anti-cancer antibodies. However, the presence and role of circulating tumor specific antibodies induced by PEF have not been explored. Serological antibodies are often detected with the ELISA assay, where tumor-associated antigens are coated to a microtiter plate to attract the corresponding serum antibodies. Here, we quantify IgG tumor specific antibodies after PEF using an in-house ELISA. Methods: Mice bearing immune-warm (EMT6) and immune-cold (4T1) orthotopic murine breast tumors were treated with a biphasic PEF dose comparable to Aliya titrated for mouse tumors. To detect tumor specific circulating IgG1 immunoglobulin in serum, an ELISA plate was coated with EMT6 and 4T1 protein lysate, as well as with a known 4T1-expressed tumor-specific antigen (gp70). Serum draws from the treated, untreated, and naïve mice were exposed to the coated wells for 1 hour. Bound IgG were detected by anti-mouse IgG (HRP tagged) antibody added to the wells and incubated 1 hour. A TMB substrate was added and color development was allowed for 15 minutes. The optical density was quantified using a microtiter plate reader. Results: ELISA quantification of serum IgG at multiple timepoints show that 4T1 tumor-specific IgG antibodies were increased 14.3- and 2.6-fold by day 20 in the PEF-treated and untreated group survivors relative to Naïve mice, respectively, (OD=0.86 and 0.15 v. 0.06). Furthermore, IgG antibodies specific to the gp70 antigen of the 4T1 cell line were increased by 44.9- and 2.7-fold in the PEF-treated and untreated groups relative to naïve mice (OD=3.14 and 0.19 v. 0.07). In the EMT6 model, at 3 months post-treatment, only the PEF-ablated group mice survived, and showed 15.5-fold higher antibodies against EMT6 tumor antigens compared to naive mice (OD=0.62 v. 0.04). Conclusions: The specific form of PEF in this study generated tumor-specific IgG long-lived antibodies in mice for immune-warm and immune-cold tumor models, corroborating previous findings of increased murine B-cell and plasma cell populations. This effect has not been well-reported for other ablation technologies. Increases were evident by Day 20 post-PEF and persisted at least 3 months. These murine data suggest the PEF used may invoke potent, long-lived immune responses and anti-tumor immune surveillance, offering a potential approach for in situ tumor vaccination to improve metastatic disease outcomes. Future studies should determine response durability and its role in attaining distant tumor clearance.

Targeting the core program of metastasis with a novel drug combination.

We previously reported that metastases are generally characterized by a core program of gene expression that activates tissue remodeling/vascularization, alters ion homeostasis, induces the oxidative metabolism, and silences extracellular matrix interactions. This core program distinguishes metastases from their originating primary tumors as well as from their destination host tissues. Therefore, the gene products involved are potential targets for anti-metastasis drug treatment. Because the silencing of extracellular matrix interactions predisposes to anoiks in the absence of active survival mechanisms, we tested inhibitors against the other three components. Individually, the low-specificity VEGFR blocker pazopanib (in vivo combined with marimastat), the antioxidant dimethyl sulfoxide (or the substitute atovaquone, which is approved for internal administration), and the ionic modulators bumetanide and tetrathiomolybdate inhibited soft agar colony formation by breast and pancreatic cancer cell lines. The individual candidate agents have a record of use in humans (with limited efficacy when administered individually) and are available for repurposing. In combination, the effects of these drugs were additive or synergistic. In two mouse models of cancer (utilizing 4T1 cells or B16-F10 cells), the combination treatment with these medications, applied immediately (to prevent metastasis formation) or after a delay (to suppress established metastases), dramatically reduced the occurrence of disseminated foci. The combination of tissue remodeling inhibitors, suppressors of the oxidative metabolism, and ion homeostasis modulators has very strong promise for the treatment of metastases by multiple cancers.

Fucoidan from Lessonia trabeculata Induces Apoptosis through Caspase Dependent and Caspase-Independent Activation in 4T1 Breast Adenocarcinoma In Vitro.

Experiments conducted on triple-negative breast cancer have shown that fucoidan from Lessonia trabeculata (FLt) exhibits cytotoxic and antitumor properties. However, further research is necessary to gain a complete understanding of its bioactivity and level of cytotoxicity. The cytotoxic effect of FLt was determined by the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was analyzed using annexin V and caspase 3/7 staining kit and DNA fragmentation. In addition, transcriptional expression of antiapoptotic (Bcl-2 and XIAP) and proapoptotic (caspase 8, caspase 9, and AIF) genes were analyzed in TNBC 4T1 cells. After 72 h of culture, the IC50 for FLt was 561 μg/mL, while doxorubicin (Dox) had an IC50 of 0.04 μg/mL. In addition, assays for FLt + Dox were performed. Annexin V and caspase 3/7 revealed that FLt induces early and late-stage apoptosis. DNA fragmentation results support necrotic death of 4T1 cells. Similarly, transcripts that prevent cell death were decreased, while transcripts that promote cell death were increased. This study showed that FLt induces apoptosis by both caspase-dependent and caspase-independent mechanisms. These findings suggest that FLt may have potential applications in breast cancer treatment. Further research will provide more information to elucidate the mechanism of action of FLt.

Metal-Organic Framework PCN-224 Combined Cobalt Oxide Nanoparticles for Hypoxia Relief and Synergistic Photodynamic/Chemodynamic Therapy.

Photodynamic therapy (PDT) and chemodynamic therapy (CDT) are promising tumor treatments mediated by reactive oxygen species (ROS), which have the advantages of being minimally invasive. However, the hypoxia of tumor microenvironment and poor target ability often reduce the therapeutic effect. Here we propose a tumor targeted nanoplatform PCN-224@Co3O4-HA for enhanced PDT and synergistic CDT, constructed by hyaluronate-modified Co3O4 nanoparticles decorated metal-organic framework PCN-224. Co3O4 can catalyze the decomposition of highly expressed H2O2 in tumor cells to produce oxygen and alleviate the problem of hypoxia. It can also produce hydroxyl radicals according to the Fenton-like reaction for chemical dynamic therapy, significantly improving the therapeutic effect. The cell survival experiment showed that after in vitro treatment, 4T1 and MCF-7 cancer cells died in a large area under the anaerobic state, while the survival ability of normal cell L02 was nearly unchanged. This result effectively indicated that PCN-224@Co3O4-HA could effectively relieve tumor hypoxia and improve the effect of PDT and synergistic CDT. Cell uptake experiments showed that PCN-224@Co3O4-HA had good targeting properties and could effectively aggregate in tumor cells. In vivo experiments on mice, PCN-224@Co3O4-HA presented reliable biosafety performance, and can cooperate with PDT and CDT therapy to prevent the growth of tumor.

The anti-cancer properties of miR-340 plasmid-chitosan complexes (miR-340 CC) on murine model of breast cancer*

MiRNA-340 (miR-340) has been found to have tumour-suppressing effects in breast cancer (BC). However, for clinical use, miRNAs need to be delivered safely and effectively to protect them from degradation. In our previous study, we used chitosan complexes as a safe carrier with anti-cancer properties to deliver miR-340 plasmid into 4T1 cells. This study explored further information concerning the anti-cancer impacts of both chitosan and miR-340 plasmid in a murine model of BC. Mice bearing 4T1 cells were intra-tumorally administered miR-340 plasmid-chitosan complexes (miR-340 CC). Afterwards, the potential of miR-340 CC in promoting anti-tumour immune responses was evaluated. MiR-340 CC significantly reduced tumour size, inhibited metastasis, and prolonged the survival of mice. MiR-340 CC up-regulates P-27 gene expression related to cancer cell apoptosis, and down-regulates gene expressions involved in angiogenesis and metastasis (breast regression protein-39 (BRP-39)) and CD163 as an anti-inflammatory macrophages (MQs) marker. Furthermore, CD47 expression as a MQs immune check-point was remarkably decreased after miR-340 CC treatment. The level of IL-12 in splenocytes of miR-340 CC treated mice increased, while the level of IL-10 decreased, indicating anti-cancer immune responses. Our findings display that miR-340 CC can be considered as a promising therapy in BC.

Self-assembled IR dye/mitoxantrone loaded Porphysomes nanosystem for enhanced combinatorial chemo-photothermal cancer therapy

Chemo-photothermal therapy (PTT) is an emerging non-invasive cancer treatment modality. Light-responsive porphysomes (DPP IR Mtx @Lipo NPs) nanosystems ablate breast cancer cells upon oxidative stress and hyperthermia. The unique self-assembled porphysomes were formed spherical shape in the size range of 150 ± 30 nm formed by the co-assembly of porphyrins along with IR 775 and chemotherapeutic drug, Mitoxantrone (Mtx), forming a camouflaged nanosystem (DPP IR Mtx @Lipo NPs, porphysomes). The advent of the prepared porphysomes aids in proper tuning of NIR absorbance improving singlet oxygen species generation among other anticancer drugs. The eminent release of DPP and adjuvant chemo-drug, Mitoxantrone from the self-assembled porphysomes is triggered by IR 775, a NIR photosensitizer upon laser irradiation. These multifunctional DPP IR Mtx @Lipo NPs have an efficient photothermal conversion efficiency of 65.8% as well as bioimaging properties. In-vitro studies in 2D and 3D models showed a significant cell death of 4T1 cells via the apoptotic pathway when irradiated with NIR laser, causing minimal damage to nearby healthy cells. DPP IR Mtx @Lipo NPs exhibited commingled PDT/PTT interdependent via NIR laser exposure, leading to mitochondrial disruption. Interestingly, the transient transfection using p53-GFP in cancer cells followed by DPP IR Mtx @Lipo NPs treatment causes rapid cell death. The activation of p53-dependent apoptosis pathways was vividly expressed, evidenced by the upregulation of Bax and increased pattern of Caspase-3 cleavage. This effect was pronounced upon transfection and induction with DPP IR Mtx @Lipo NPs, particularly in comparison to non-transfected malignant breast cancer 4T1 cells.

Disrupting Na+ ion homeostasis and Na+/K+ ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo

BackgroundGlycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells.MethodsLive whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]d-glucose to [2H]-labelled l-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET).ResultsIntracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected.ConclusionsGlycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the ‘centrality of the [Na+]i-NKA nexus’ in the mechanistic basis of the Warburg effect.

A Carbon‐Caged Rhodamine Generating Nitrosoperoxycarbonate for Photoimmunotherapy

AbstractPhotoimmunotherapy is a promising cancer treatment modality. While potent 1‐e− oxidative species are known to induce immunogenic cell death (ICD), they are also associated with unspecific oxidation and collateral tissue damage. This difficulty may be addressed by post‐generation radical reinforcement. Namely, non‐oxidative radicals are first generated and subsequently activated into powerful oxidative radicals to induce ICD. Here, we developed a photo‐triggered molecular donor (NPCD565) of nitrosoperoxycarbonate (ONOOCO2−), the first of its class to our knowledge, and further evaluated its feasibility for immunotherapy. Upon irradiation of NPCD565 by light within a broad spectral region from ultraviolet to red, ONOOCO2− is released along with a bright rhodamine dye (RD565), whose fluorescence is a reliable and convenient build‐in reporter for the localization, kinetics, and dose of ONOOCO2− generation. Upon photolysis of NPCD565 in 4T1 cells, damage‐associated molecular patterns (DAMPs) indicative of ICD were observed and confirmed to exhibit immunogenicity by induced maturation of dendritic cells. In vivo studies with a bilateral tumor‐bearing mouse model showcased the potent tumor‐killing capability of NPCD565 of the primary tumors and growth suppression of the distant tumors. This work unveils the potent immunogenicity of ONOOCO2−, and its donor (NPCD565) has broad potential for photo‐immunotherapy of cancer.

A Carbon-Caged Rhodamine Generating Nitrosoperoxycarbonate for Photoimmunotherapy.

Photoimmunotherapy is a promising cancer treatment modality. While potent 1-e- oxidative species are known to induce immunogenic cell death (ICD), they are also associated with unspecific oxidation and collateral tissue damage. This difficulty may be addressed by post-generation radical reinforcement. Namely, non-oxidative radicals are first generated and subsequently activated into powerful oxidative radicals to induce ICD. Here, we developed a photo-triggered molecular donor (NPCD565) of nitrosoperoxycarbonate (ONOOCO2 -), the first of its class to our knowledge, and further evaluated its feasibility for immunotherapy. Upon irradiation of NPCD565 by light within a broad spectral region from ultraviolet to red, ONOOCO2 - is released along with a bright rhodamine dye (RD565), whose fluorescence is a reliable and convenient build-in reporter for the localization, kinetics, and dose of ONOOCO2 - generation. Upon photolysis of NPCD565 in 4T1 cells, damage-associated molecular patterns (DAMPs) indicative of ICD were observed and confirmed to exhibit immunogenicity by induced maturation of dendritic cells. In vivo studies with a bilateral tumor-bearing mouse model showcased the potent tumor-killing capability of NPCD565 of the primary tumors and growth suppression of the distant tumors. This work unveils the potent immunogenicity of ONOOCO2 -, and its donor (NPCD565) has broad potential for photo-immunotherapy of cancer.

Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer

Objective: This study aimed to develop a new delivery strategy that utilized metal organic framework (MOF) loaded with small-interfering RNA (siRNA) targeting ITGAV to overcome tumor matrix barrier, and thus enhance drug penetration and immune accessibility in breast cancer. Methods: MOF@siITGAV particles were constructed and characterized. The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay. The toxicity of MOF@siITGAV was detected by cell counting kit 8 (CCK-8). The blank control group, naked siITGAV group and MOF@siITGAV group were set. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of ITGAV. The level of transforming growth factor β1 (TGF-β1) in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids. Mouse models of triple negative breast cancer were established. The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified. Imminohistochemical (IHC) was used to test the expression of collagen and CD8. Results: MOF@siITGAV particles were constructed with sizes of (198.0±3.3) nm and zeta potential of -(20.2±0.4) mV. MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA. Compared to the blank control group, the expression of ITGAV in the MOF@siITGAV group [(46.5±11.3)%] and the naked siITGAV group [(109.9±19.0)%] was lower. TGF-β1 in the cell culture medium of the blank control group, naked siITGAV group, and MOF@siITGAV group was (474.5±34.4) pg/ml, (437.2±16.5) pg/ml, and (388.4±14.4) pg/ml, respectively. MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity. The cell viability was (99.7±3.5)%, (98.2±5.2)%, (97.3±6.6)%, (92.1±8.1)%, and (92.4±4.1)%, respectively, after MOF@siITGAV treatment with the concentration of 0, 10, 20, 40, 80, and 160 μg/ml, respectively, for 24 h. The tumor growth in the MOF@siITGAV group was suppressed significantly. After 15-day treatment, the tumor volume of the MOF@siITGAV group was (135.3±41.9) mm3, smaller than that of the blank control group [(691.1±193.0) mm3] (P=0.025). The expression of collagen and the number of CD8 positive cells of the MOF@siITGAV group were lower than those of the other two groups. No significant abnormalities were observed in the main organs of mice. Conclusions: Targeting the integrinαv on the surface of cancer cells could destroy extracellular matrix, improve drug delivery, and increase immune infiltration.

Cu+/Ca2+ Dual‐Overload Strategy Assisted by Metabolic‐Symbiotic‐Destruction for Tumor Apoptosis and Tumor Immune Activation

Abstract Ion interference including copper (Cu+)/calcium (Ca2+) overload activate cell‐specific death channels, damage mitochondria and disrupt cellular homeostasis, showing great potential in anti‐tumor therapy. However, the complex metabolic environment and the powerful self‐protection of tumors cause clinical failure of ion interference. Thus, metabolic disruption is expected an innovative strategy for the enhancement of ion interference. Herein, CuS‐α‐CHCA&amp;penthiopyrad@CaCO3‐RGD nanoparticles (CCPCR NPs) are prepared to provide a Cu+/Ca2+ dual‐overload anti‐tumor therapy assisted by metabolic‐symbiosis‐destruction strategy, realizing the collapse of tumor self‐protection. Specifically, precise CuS and CaCO3 delivery triggered irreversible Cu+/Ca2+ dual‐overload and reactive oxygen species (ROS) attack toward 4T1 cells. Meanwhile, α‐CHCA and penthiopyrad disturbed the metabolic symbiotic environment by disrupting the TCA cycle and preventing lactate efflux to aggravate intracellular acidosis and promote Fenton‐like reaction of Cu+, enhancing the sensitivity of tumor cells to copper death and Ca2+ overload. Further, aided by the metabolism of symbiosis, the destroyed tumor cells further activate the polarization of M1 macrophages and the maturation and antigen cross‐presentation of dendritic cells (DCs), which further eliminate tumor cells. In summary, an amplified anti‐tumor dual‐ion interference strategy assisted by metabolic symbiotic destruction is established, which is of great significance in improving the clinical effect of ion therapy for tumors.

Manganese oxide-constructed multifunctional biomimetic nanovaccine for robust tumor-specific T cell priming and chemodynamic therapy

The development of manganese oxide-based chemodynamic immunotherapy is emerging as a key strategy against solid tumors. However, the limited efficacy of nanoplatform in inducing efficient tumor therapeutic effects and creating the prominent antitumor immune responses remains a crucial issue. In this study, we construct a novel multifunctional biomimetic nanovaccine comprising manganese oxide-loaded poly(2-diisopropylaminoethyl methacrylate) (MP) nanoparticles and a coating layer of hybrid cell membrane (RHM) derived from manganese oxide-remodeled 4T1 cells and dendritic cells (DCs) (collectively called MP@RHM) for combination chemodynamic immunotherapy. Compared with the nanovaccines coated with the single cell membrane, the MP@RHM nanovaccine highly efficiently activates both DCs and T cells to boost tumor-specific T cell, owing to the synergistic effects of abundant damage-associated molecular patterns, Mn2+, and T cell-stimulating moieties. Upon peritumoral injection, the MP@RHM nanovaccine targets both the tumor site for focused chemodynamic therapy and the lymph nodes for robust tumor-specific T cell priming, thereby achieving highly efficient chemodynamic immunotherapy. Moreover, as a preventive cancer nanovaccine, MP@RHM generates strong immunological memory to inhibit postoperative tumor metastasis and recurrence. Our study findings highlight a promising approach to construct a multifunctional biomimetic nanovaccine for personalized chemodynamic immunotherapy against solid tumors.

Hydrogen Therapy Reverses Cancer-Associated Fibroblasts Phenotypes and Remodels Stromal Microenvironment to Stimulate Systematic Anti-Tumor Immunity.

Tumor microenvironment (TME) plays an important role in the tumor progression. Among TME components, cancer-associated fibroblasts (CAFs) show multiple tumor-promoting effects and can induce tumor immune evasion and drug-resistance. Regulating CAFs can be a potential strategy to augment systemic anti-tumor immunity. Here, the study observes that hydrogen treatment can alleviate intracellular reactive oxygen species of CAFs and reshape CAFs' tumor-promoting and immune-suppressive phenotypes. Accordingly, a controllable and TME-responsive hydrogen therapy based on a CaCO3 nanoparticles-coated magnesium system (Mg-CaCO3) is developed. The hydrogen therapy by Mg-CaCO3 can not only directly kill tumor cells, but also inhibit pro-tumor and immune suppressive factors in CAFs, and thus augment immune activities of CD4+ T cells. As implanted in situ, Mg-CaCO3 can significantly suppress tumor growth, turn the "cold" primary tumor into "hot", and stimulate systematic anti-tumor immunity, which is confirmed by the bilateral tumor transplantation models of "cold tumor" (4T1 cells) and "hot tumor" (MC38 cells). This hydrogen therapy system reverses immune suppressive phenotypes of CAFs, thus providing a systematic anti-tumor immune stimulating strategy by remodeling tumor stromal microenvironment.

Prostate Specific Membrane Antigen Expression in a Syngeneic Breast Cancer Mouse Model.

Prostate specific membrane antigen (PSMA) has been studied in human breast cancer (BCa) biopsies, however, lack of data on PSMA expression in mouse models impedes development of PSMA-targeted therapies, particularly in improving breast conserving surgery (BCS) margins. This study aimed to validate and characterize the expression of PSMA in murine BCa models, demonstrating that PSMA can be utilized to improve therapies and imaging techniques. Murine triple negative breast cancer 4T1 cells, and human cell lines, MDA-MB-231, MDA-MB-468, implanted into the mammary fat pads of BALB/c mice, were imaged by our PSMA targeted theranostic agent, PSMA-1-Pc413, and tumor to background ratios (TBR) were calculated to validate selective uptake. Immunohistochemistry was used to correlate PSMA expression in relation to CD31, an endothelial cell biomarker highlighting neovasculature. PSMA expression was also quantified by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Accumulation of PSMA-1-Pc413 was observed in 4T1 primary tumors and associated metastases. Average TBR of 4T1 tumors were calculated to be greater than 1.5-ratio at which tumor tissues can be distinguished from normal structures-at peak accumulation with the signal intensity in 4T1 tumors comparable to that in high PSMA expressing PC3-pip tumors. Extraction of 4T1 tumors and lung metastases followed by RT-PCR analysis and PSMA-CD31 co-staining shows that PSMA is consistently localized on tumor neovasculature with no expression in tumor cells and surrounding normal tissues. The selective uptake of PSMA-1-Pc413 in these cancer tissues as well as the characterization and validation of PSMA expression on neovasculature in this syngeneic 4T1 model emphasizes their potential for advancements in targeted therapies and imaging techniques for BCa. PSMA holds great promise as an oncogenic target for BCa and its associated metastases.

Design and one-pot synthesis of new substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine as potential antitumor agents: in vitro and in vivo studies

A new efficient and versatile one-pot three-component synthesis of substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives has been developed. It is based on a multistep cascade reaction from 2-aminothiophenes and 2-hydroxy-4-oxobut-2-enoic acids, and derivatives of cyanoacetic acid catalyzed by diisopropylethylamine. As a result, novel pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives (21 compounds) were synthesized in a mild reaction conditions with a high yield. The structures of the developed compounds were confirmed by NMR and elemental analysis. The influence of electron-withdrawing or electron-donor substituents on the antitumor activity of the developed compounds has been identified. In vitro screening analysis of 21 compounds revealed six lead candidates (12aa, 12dc, 12hc, 12ic, 12lb, and 12mb) that demonstrated the most significant antitumor activity against B16-F10, 4T1 and CT26 cells. Necrosis/apoptosis assay showed that apoptosis was the predominant mechanism of cell death. Molecular docking analysis revealed several potential targets for tested compounds, i.e. phosphatidylinositol 5-phosphate 4-kinase (PI5P4K2C), proto-oncogene serine/threonine-protein kinase (Pim-1), nicotinamide phosphoribosyltransferase (NAMPT) and dihydrofolate reductase (DHFR). The lead compound (12aa) can effectively induce cell apoptosis, possesses a high yield (98 %) and requires low-cost starting chemicals for its synthesis. In vivo experiments with melanoma-bearing mice confirmed that 12aa compound resulted in the significant tumor inhibition on 15 d after the therapy. In particular, tumor volume was ∼0.19 cm3 for 50 mg/kg versus ∼2.39 cm3 in case of untreated mice and tumor weight was ∼71.6 mg for 50 mg/kg versus ∼452.4 mg when considered untreated mice. Thus, our results demonstrated the high potential of the 12aa compound in the treatment of melanoma and can be recommended for further preclinical studies.

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.

Biomimetic “nano-spears” for CAFs-targeting: splintered three “shields” with enhanced cisplatin anti-TNBC efficiency

The treatment dilemma of triple-negative breast cancer (TNBC) revolves around drug resistance and metastasis. Cancer-associated fibroblasts (CAFs) contribute to cisplatin (Cis) resistance and further metastasis in TNBC, making TNBC a difficult-to-treat disease. The dense stromal barrier which restricts drug delivery, invasive phenotype of tumor cells, and immunosuppressive tumor microenvironment (TME) induced by CAFs serve as three “shields” for TNBC against Cis therapy. Here, we designed a silybin-loaded biomimetic nanoparticle coated with anisamide-modified red blood cell membrane (ARm@SNP) as a “nanospear” for CAFs-targeting, which could shatter the “shields” and significantly exhibit inhibitory effect on 4T1 cells in combination with Cis both in vitro and in vivo. The ARm@SNP/Cis elicited 4T1 tumor growth arrest and destroyed three “shields” as follows: disintegrating the stromal barrier by inhibiting blood vessels growth and the expression of fibronectin; decreasing 4T1 cell invasion and metastasis by affecting the TGF-β/Twist/EMT pathway which impeded EMT activation; reversing the immunosuppressive microenvironment by increasing the activity and infiltration of immunocompetent cells. Based on CAFs-targeting, ARm@SNP reversed the resistance of Cis, remodeled the TME and inhibited invasion and metastasis while significantly improving the therapeutic effect of Cis on 4T1 tumor-bearing mice, providing a promising approach for treating intractable TNBC.

Copper sulfide-based hollow nanoarchitectonics for augmented photothermal, chemodynamic, and chemotherapeutic modalities

Despite the advancements, cancer has remained one of the appalling diseases, with numerous casualties due to delayed prognosis and poor survival rate. To this end, combinatorial strategies based on various therapeutic modalities have garnered enormous interest from researchers towards improved therapeutic performances. This study aims to investigate the fabrication of transition metal-based hollow nanoarchitectures for synergistic augmented photothermal (PTT), chemodynamic (CDT), and chemotherapeutic (CT) modalities. Initially, copper sulfide nanoparticles with hollow cavities (hCuS) are synthesized using the facile sacrificial templating-based approach and subsequently loaded with the CT agent (Doxorubicin, DOX), referred to shortly as hCuS-DOX. These hCuS-DOX nanoarchitectures are systematically characterized using various techniques to successfully explore the physicochemical properties. The DOX molecules are encapsulated in hCuS containers through the electrostatic interactions due to their contrary charges and coordination interactions between copper in CuS and nitrogen in DOX molecules. These innovative nanoarchitectures showed pH-responsive release of DOX molecules from the hollow cavities, specifically in the tumor microenvironment, which could be through the protonation of DOX and progressive degradation of hCuS. In addition, these architectures exhibited substantial PTT properties, on the one hand, by raising the temperature to over 60 °C. On the other hand, the released DOX molecules supported the CDT effect by generating enormous levels of deadly free radical species intracellularly, significantly reducing the viability of 4T1 cells. Finally, the apoptosis assay based on calcein AM/propidium iodide staining using various combinations validated the synergistic therapeutic modalities. In summary, these nanoarchitectures potentiated the synergistic therapeutic efficacy against breast carcinoma towards their ability in pharmaceutics.