Mouse Mammary Cells Research Articles

Understanding how the structures of surfactants in hybrid nanoparticles affect the compaction of ct-DNA for cellular uptake: presenting a highly efficient surfactant.

Compaction of calf thymus DNA (ct-DNA) by two single-head-double-tailed surfactants with variable tail lengths i.e., Dihexadecyldimethylammonium bromide (DDAB16) and Dioctadecyldimethylammonium bromide (DDAB18), and one triple-head-double-tailed surfactant N1-dodecyl-N2-(2-(dodecyldimethylammonio)ethyl)-N1,N1,N2,N2-tetramethylethane-1,2-diaminium (MQAS12) has been studied. DDAB18 is found to be the most efficient, while MQAS12 is the least efficient for cellular uptake. Hybrid materials of surfactants and silica nanoparticles have better compaction efficiency due to the cooperative binding. Silica nanoparticles (∼100 nm)-DDAB18 hybrid materials can compact ct-DNA at a much lower concentration than a conventional surfactant, addressing the cytotoxicity issues. Hybrid materials formed with smaller silica nanoparticles (∼40 nm) have also been studied. The results obtained have been used to understand whether Coulombic and/or hydrophobic interactions are responsible for DNA compaction. The hydrophobicity per unit surface area (P) of hybrid nanoparticles has a significant role in DNA compaction. The P largely depends on the surfactants' structures and nanoparticles' sizes. Single-head-double-tailed surfactants with a comparatively smaller headgroup exhibit a large amount of adsorption on the nanoparticles' surfaces, producing a large P. DDAB18 appears to be a DNA intercalative binder. Fluorescence anisotropy decay data of 4,6-diamidino-2-phenylindole (DAPI) reveal the dynamics of ct-DNA at different stages of compaction. Cell viability of mouse mammary gland adenocarcinoma cells (4T1) and human embryonic kidney (HEK) 293 cell lines and in vitro cellular uptake of the gene to 4T1 cells have been investigated. This study provides ideas for designing efficient non-viral vectors. Overall, DDAB18-coated silica nanoparticles appear to be safe and effective DNA compaction agents that can carry nucleic acids for biomedical applications.

Biotinylated platinum(IV)-conjugated graphene oxide nanoparticles for targeted chemo-photothermal combination therapy in breast cancer

Graphene oxide (GO) and GO-based nanocomposites are promising in drug delivery and photothermal therapy due to their exceptional near-infrared optical absorption and high specific surface area. In this study, we have effectively conjugated an oxaliplatin (IV) prodrug, PEGylated graphene oxide, and PEGylated biotin (PB) in a single platform for breast cancer treatment. This platform demonstrates promising prospects for targeted drug delivery and the synergistic application of photothermal-chemotherapy when exposed to NIR-laser irradiation. The resulting nanocomposite (GO(OX)PB (1/1/0.2) NPs) displayed an exceptionally large surface area, minimal particle size (195.7 nm), specific targeting capabilities, a high drug load capacity (43.56 %) and entrapment efficiency (89.48 %) and exhibit excellent photothermal conversion efficiency and photostability when exposed to NIR-laser irradiation (808 nm). The therapeutic effectiveness was assessed both in vitro and in vivo conditions employing human breast cancer cells (MCF-7), mouse mammary gland adenocarcinoma cells (4T1), and 4T1-Luc tumor-bearing mouse models. The findings demonstrated that GO(OX)PB (1/1/0.2) NPs (+L) were highly effective in causing significant cytotoxicity, G2/M phase cell cycle arrest, ROS generation, mitochondrial membrane depolarization, apoptosis, and photothermal effect. This resulted in a greater percentage of cell death compared to free OX, GO(OX)PEG (1/1/0.2) NPs (±L), and GO(OX)PB (1/1/0.2) NPs (−L). The in vivo therapeutic studies on 4T1-Luc tumor-bearing mice revealed that a combination of GO(OX)PB (1/1/0.2) NPs (+L) caused complete disappearance of the tumor, no tumor recurrence, prolonged survival, reduced lung metastasis, and mitigated nephrotoxicity. The serum and blood analysis demonstrated minimal systemic toxicity of GO(OX)PB (1/1/0.2) NPs. The developed nanoplatform, in this context, may serve as a potential nanomedicine to address conventional nephrotoxicity in breast cancer and prevent metastasis by combining chemo-photothermal therapy.

Combination of resveratrol and piperine to target doxorubicin resistance in breast cancer: An in vitro and in vivo study

One of the biggest causes of death worldwide is cancer, which poses a serious threat to healthcare systems all over the world. Triple-negative breast cancer (TNBC), in particular, presents substantial clinical challenges because it does not express Human Epidermal Growth Factor Receptor 2 (HER2) or hormone receptors. Despite concerns about early recurrence and drug resistance, chemotherapy, particularly anthracyclines like Doxorubicin (DOX), remains the mainstay of treatment for TNBC patients. This research investigates the possibility of using natural products, especially piperine (PIP) and resveratrol (RES), to overcome DOX resistance. PIP and RES have shown anticancer effects via various pathways, such as inducing apoptosis and controlling the cell cycle. In this research, in vitro experiments showed that RES and PIP inhibit cell growth in a dose-dependent manner using triple-negative parent mouse mammary breast cancer cells and DOX resistance cells (EMT-6/P and EMT-6/DOX). RES in EMT-6/P cells showed an IC50 value of 146.511 ± 5.35 μM, and in EMT-6/DOX cells, it was 88.635 ± 29.507 μM. In the same way, PIP was found to have IC50 values of 148.819 ± 14.317 and 9.375 μM in the triplicate trials in EMT-6/P and EMT-6/DOX, respectively. Furthermore, on both cell lines, the combination demonstrated very strong synergistic effects; noticeably lower doses were needed for the combined treatment to reduce cell viability by 50%. The IC50 values for the combination treatments of RES and PIP were found to be < 2.289 µM and < 2.325 µM in EMT-6/P cells, respectively, with values < 0.348 µM and 0.243 ± 0.142 µM in EMT-6/DOX cells. The in vivo experiment conducted on Balb/C females indicated that mice bearing EMT-6/Dox cells and treated with the combination of RES and PIP had the highest cure percentage; however, this treatment showed mild toxicity. The study clarifies the potential synergistic activity between PIP and RES in combating DOX-resistant TNBC cells. These findings highlight the significance of investigating natural products as supplemental therapies in cancer treatment and provide insights into new treatment approaches. While the combination shows promise as a therapeutic option for treating breast cancer, especially in cases of DOX resistance, further investigation is needed.

In vivo and in vitro anti-inflammation of Rhapontici Radix extract on mastitis via TMEM59 and GPR161

Ethnopharmacological relevanceRhapontici Radix ethanol extract (RRE) is derived from the dried root of Rhaponticum uniflorum (L.) DC belonging to the Asteraceae family. RRE exhibits significant anti-inflammatory and antioxidant properties; however, the potential of RRE in mastitis treatment requires further investigation. Aim of this studyThis research was performed to examine the protective properties of RRE against mastitis and the mechanisms underlying the effects of RRE. Material and methodsRRE components were analyzed by HPLC-MS/MS and DPPH methods. Isochlorogenic acid B (ICAB) was obtained commercially. MTT assay was utilized to assess RRE or ICAB cytotoxicity in bovine mammary alveolar (MAC-T) cells. Immunohistochemistry were used to investigate the pathological alterations in mammary tissue. The protein levels of inflammatory cytokines and mediators were analyzed using ELISA, and the expression of MAPK and NF-κB signaling pathways, as well as p65 nuclear translocation, were analyzed through Western blotting and immunofluorescence techniques, respectively. Target proteins of RRE were screened by RNA-seq and tandem mass tag analyses. Protein interaction was revealed and confirmed using co-immunoprecipitation and CRISPR/Cas9-based knockdown and overexpression of target genes. ResultsICAB was revealed as one of the main components in RRE, and it was responsible for 84.33% of RRE radical scavenging activity. Both RRE and ICAB mitigated the infiltration of T lymphocytes in the mammary glands of mice, leading to decreased levels of inflammatory mediators (COX-2 and iNOS) and cytokines (TNF-α, IL-6, and IL-1β) in lipopolysaccharide (LPS)-induced MAC-T cells. Furthermore, RRE and ICAB suppressed the LPS-induced phosphorylation of NF-κB inhibitor and p65, thereby impeding p65 nuclear translocation in mouse mammary glands and MAC-T cells. In addition, RRE and ICAB attenuated the LPS-triggered activation of c-Jun N-terminal kinase 1/2, p38, and extracellular regulated protein kinase 1/2. Importantly, co-treated with LPS and ICAB in MAC-T cells, an upregulation of G-protein coupled receptor 161 (GPR161) and transmembrane protein 59 (TMEM59) was observed; the interact between TMEM59 and was found, leading to inhibition of NF-κB activity and inflammatory cytokine production. ConclusionICAB is a prominent antioxidant in RRE. RRE and ICAB reduce mammary inflammation via MAPK and NF-κB pathways and the interaction between TMEM59 and GPR161 mediates the control of ICAB in NF-κB signaling.

Polymeric Micellar Nanoparticles Enable Image-guided Drug Delivery in Solid Tumors.

We report the development of a nanotechnology to co-deliver chemocoxib A with a reactive oxygen species (ROS)-activatable and COX-2 targeted pro-fluorescent probe, fluorocoxib Q (FQ) enabling real time visualization of COX-2 and CA drug delivery into solid cancers, using a di-block PPS 135 - b -POEGA 17 copolymer, selected for its intrinsic responsiveness to elevated reactive oxygen species (ROS), a key trait of the tumor microenvironment. FQ and CA were synthesized independently, then co-encapsulated within micellar PPS 135 - b -POEGA 17 co-polymeric nanoparticles (FQ-CA-NPs), and were assessed for cargo concentration, hydrodynamic diameter, zeta potential, and ROS-dependent cargo release. The uptake of FQ-CA-NPs in mouse mammary cancer cells and cargo release was assessed by fluorescence microscopy. Intravenous delivery of FQ-CA-NPs to mice harboring orthotopic mammary tumors, followed by vital optimal imaging, was used to assess delivery to tumors in vivo . The CA-FQ-NPs exhibited a hydrodynamic diameter of 109.2 ± 4.1 nm and a zeta potential (σ) of -1.59 ± 0.3 mV. Fluorescence microscopy showed ROS-dependent cargo release by FQ-CA-NPs in 4T1 cells, decreasing growth of 4T1 breast cancer cells, but not affecting growth of primary human mammary epithelial cells (HMECs). NP-derived fluorescence was detected in mammary tumors, but not in healthy organs. Tumor LC-MS/MS analysis identified both CA (2.38 nmol/g tumor tissue) and FQ (0.115 nmol/g tumor tissue), confirming the FQ-mediated image guidance of CA delivery in solid tumors. Thus, co-encapsulation of FQ and CA into micellar nanoparticles (FQ-CA-NPs) enabled ROS-sensitive drug release and COX-2-targeted visualization of solid tumors.

Abstract 2007: Ischemic And Non-ischemic Cardiovascular Events Differentially Regulate Cross Disease Communication With Cancer

Cancer and cardiovascular disease (CVD) are the top two leading causes of death worldwide, exhibiting cross-disease communication that increases morbidity. Cancer patients are at significantly increased risk for CVD and recent studies by our lab and others indicate that CVDs like myocardial infarction (MI) or heart failure can in turn accelerate tumor growth. We previously showed this occurs through changes in the innate immune system or via tumor-promoting factors secreted from the damaged myocardium, which together have established the nascent field of Reverse CardioOncology. We hypothesized that ischemic and non-ischemic CVD injuries have distinctly different mechanisms underlying mammary tumor growth. To mimic these events, we modeled non-cardiac hind limb ischemic injury by femoral artery ligation (FAL) and non-ischemic cardiac injury by cardiac cryoinjury (CCI). To test our hypothesis, we introduced primary E0771 mouse mammary cancer cells into the mammary fat pad three days prior to FAL or CCI. Measurements of tumor over time showed significant increases in tumor growth after both FAL and CCI when compared to sham surgeries. However, only the ischemic injury, FAL, induced similar changes in immune cells to what we observed after MI (induced by left anterior descending coronary artery [LAD] ligation). This included significant increases in myeloid populations and decreased lymphocytes within the circulation. Additionally, flow cytometry of immune cells within tumors of mice exposed to FAL showed significantly increased myeloid cells and macrophages, as well as increased immunosuppressive T-regulatory cells. In contrast, while CCI showed significantly increased tumor burden and circulating leukocytes, there were no differences in the above-mentioned subsets of immune cells within the circulation or within the tumor. These data suggest that ischemic injury, via FAL or LAD, induces an immunosuppressive environment within the tumor that accelerates cancer growth that is distinct from the non-ischemic cardiac injury induced by CCI. Taken together, our data suggest that the immune reprogramming after ischemic CVDs is a risk factor for tumor growth and these data help to elucidate the mechanisms that underlie cancer risk in CVD patients.

Abstract LB160: The mouse mammary tumor virus mediates the antineoplastic action of decitabine

Abstract The purpose of this study is to clarify the role of the mouse mammary tumor virus (MMTV) in cancer growth and in mediating the antineoplastic effect of decitabine, a DNA demethylating agent. In vitro studies have shown that decitabine inhibits cancer cell proliferation by stimulating endogenous retroviruses which in turn activates the production of cytostatic interferon beta (IFN-β). We investigated the potential involvement of endogenous MMTV and the mouse IFN-β in the pharmacodynamics of decitabine using stable knockdown cell lines and MMTV hyper-infection. For the first time, we show that knockdown of MMTV or IFN-β in a mouse mammary cancer cell line (4T1) both rendered the cancer cells more resistant to decitabine in mice bearing 4T1-derived mammary cancer. Mechanistically, decitabine enhanced MMTV expression on the RNA level in both control tumors and tumors with MMTV knockdown, although the degree of enhancement was limited in knockdown tumors. Conversely, infection of a mouse colon cancer cell line (MC38) with MMTV released from 4T1 cells rendered the colon cancer cells more sensitive to decitabine. These data confirm the viral mimicry hypothesis as the mechanism of DNA demethylating agents. Practically, our research suggests the possibility of combining the use of an exogenous virus and a DNA demethylating agent as a new approach to cancer treatment. Citation Format: Sarah Light, Maziar Montazer, Cade Wycoff, Richard Tuttle, Andrew Brola, William Wycoff, Ryan Johnson, Yingguang Liu. The mouse mammary tumor virus mediates the antineoplastic action of decitabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB160.

Abstract 3981: Immune targeting progesterone receptor in hormone receptor positive breast cancer

Abstract Background: Hormone receptor positive (HR⁺) breast cancer (BC) is a subtype of BC that is characterized by expression of estrogen receptor (ER) and progesterone receptor (PR). A cornerstone treatment for this subtype of BC is endocrine inhibition therapy (EIT), in which estrogen and ER are suppressed using antagonists to suppress tumor growth. However, patients eventually obtain resistance to EIT. Metastatic HR⁺BC is the deadliest form of BC, with no cure. Current therapies currently only utilize the ER-axis, with no PR-targeted therapies. Little is known about the role of PR in these BCs, but we have found evidence for an immunosuppressive role for PR in HR⁺BC. Yet, it is unclear how PR mediates immunosuppression and if this axis can be therapeutically targeted. We hypothesized that PR confers local immunosuppression through the stimulation of immunosuppressive transcriptional activity and that we can immunologically target this axis using a PR vaccine. Experimental methods: To assess the immune impact of PR, we expressed the isoforms of PR (A, B and A+B) in mouse mammary cancer cells and assessed their capacity to elicit PR signaling. We assessed the impact of PR expression on T cell activity in targeting PR⁺ mammary tumors that express GFP, along with using GFP-specific JEDI CD8⁺ T cells. We explored the ability of PR to protect against GFP xenoantigen immune responses by engrafting PR⁺ mammary cells into immune competent and deficient mice. Lastly, we engineered 1st generation adenoviral vectors encoding mouse and human PR and utilized two antiprogestins (Mifepristone and Onapristone) in vivo. Using our vectors, we vaccinated BALB/c in the footpad and assessed PR-specific immunity using IFNγ ELISPOT. Results: Using PR signaling reporters, we found canonical PR signaling activity in the absence of progesterone (P4) by PR-A and in the presence of P4 by PR-B, but the most profound activation of PR signaling was seen through combined expression of PR-A and B. While expression of these genes did not confer striking cytotoxic T cell resistance in vitro, combination of PR-A+PR-B in 67NR cells permitted successful tumor engraftment in immune competent mice, despite xenoantigen expression. GFP expression did not alter tumor growth in immunodeficient mice, suggesting a key role in modulating local anti-tumor immunity. The use of either Mifepristone or Onapristone significantly suppressed tumor engraftment in immune competent mice in vivo. We found that an Ad-PR vaccine could elicit T cell responses against human PR-specific epitopes, a potential step towards immune targeting these BCs. Conclusions: We found that murine PR-A and PR-B can confer robust P4 signaling that is linked to an immunosuppressive phenotype in vivo. We developed a PR vaccine that elicited PR-specific T cell responses in vivo. We are currently examining the mechanisms that underlie PR-mediated immunosuppression and exploring the potential of PR vaccines in HR⁺BC. Citation Format: Andrea E. Wilson, Junping Wei, Gangjun Lei, Xiao-Yi Yang, Cong-Xiao Liu, Melissa Gajda, Tao Wang, Christy Hagan, Zachary C. Hartman. Immune targeting progesterone receptor in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3981.

Study of the antitumor activity of the combination baicalin and epigallocatechin gallate in a murine model of vincristine-resistant breast cancer

Background: Breast cancer is one of the primary causes of death in women worldwide. Yearly, a quarter of cancer cases in women are breast cancer. The problem is aggravated by the emergence of chemotherapeutic resistance. One of the practices to overcome this problem is using natural products along with chemotherapy. Epigallocatechin Gallate (EGCG) is the main efficient catechin in green tea. Baicalin is the main active chemical constituent of Scutellaria baicalensis. Both natural products have anti-cancer and improving resistance activities. Therefore, a combination of EGCG and baicalin may afford a possible way out to overcome chemotherapeutic resistance. Methods: EGCG and baicalin were tested on Vincristine-sensitive (EMT-6/P) and Vincristine-resistant (EMT-6/V) mouse mammary cell lines. Antiproliferative and apoptotic effects were assessed for EGCG, baicalin, their combination, and Vincristine in vitro using MTT and caspase-3 assays. An in vivo study was conducted to assess the effect of EGCG, baicalin, their combination, vincristine, vincristine along with the combination in both EMT-6/P and EMT-6/V mouse mammary cell lines. The safety profile was also considered using liver enzymes and creatinine assays. Results: in vitro, EGCG and baicalin had synergistic effects in both cell lines. The combination of baicalin and EGCG as 140 and 100 µM respectively, caused an apoptotic effect higher than the single treatment of baicalin 175 µM or EGCG 125 µM, in vincristine-resistant cell lines. In vivo, the combination along with vincristine significantly enhanced the reduction of tumour size in mice implanted with EMT-6/P and EMT-6/V cell lines. According to the safety profile, the combinations of EGCG and baicalin are safe. Conclusion: The combination of baicalin and EGCG can be optimistic in improving vincristine-resistant cells to treatments.

Targeting cisplatin resistance in breast cancer using a combination of Thymoquinone and Silymarin: an in vitro and in vivo study

Background: Breast cancer (BC) is considered the most diagnosed cancer among women globally. This is because of its high possibility of metastasis and high resistance to chemotherapy. Cisplatin is a platinum-based antitumor agent that is used to treat various types of cancer. However, the main obstacle to using this drug is drug resistance. Drug resistance is a cause of most relapses of cancer which eventually lead to death. Nowadays, combining natural products is a trend to overcome drug resistance. Thymoquinone (TQ) is a natural phytochemical that exists mainly in blackseed. It has been used in medicine for decades, especially as an anticancer agent. Silymarin is a milk thistle compound that exhibits anticancer, hepatoprotective, and neuroprotective activity. Hence, the combination of TQ and silymarin could be a probable solution to treat cancer and reduce chemoresistance. Methods: This study tested this combination on cisplatin-sensitive (EMT6/P) and cisplatin-resistant (EMT6/CPR) mouse mammary cell lines. Apoptotic and antiproliferative activity was assessed for TQ and silymarin in vitro using caspase-3 and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays, respectively. An in vivo study was performed to evaluate the effect of TQ and silymarin combination in mice inoculated with EMT6/P and EMT6/CPR cells. The safety profile was also examined using creatinine and liver enzyme assays. Results: In vitro, the TQ and silymarin combination synergized in both cell lines. Also, this combination caused apoptosis induction at a higher rate than the single treatment in both cell lines. In vivo, TQ and silymarin combination resulted in a remarkable reduction in tumor size and enhanced the cure rate in mice implanted with EMT6/P and EMT6/CPR cell lines. According to the safety profile results, TQ and silymarin combination was safe. Conclusion: In conclusion, the combination of TQ and silymarin provides a promising solution in treating BC resistant to cisplatin by inducing apoptosis. Further studies are needed to define the exact anticancer mechanisms of this combination.

Anticancer and antioxidant activities of essential oils of Chiliadenus iphionoides from Jordan: in vitro and in vivo study

Medicinal plants have been used since ancient times and may even be considered the origin of modern medicine. Natural products are a potential source for drug discovery and development of cancer treatments. Several pharmacological therapeutic activities were reported for Chiliadenus iphionoides. This research was designed to evaluate the essential oil of Chiliadenus iphionoides antioxidant potential and antiproliferative activity both in vitro and in vivo. The essential oil of Chiliadenus iphionoides was extracted by hydrodistillation. The chemical composition analysis was performed using gas chromatography. The main compounds identified in Chiliadenus iphionoides essential oil were oxygenated monoterpenes, such as eucalyptol (36.08%). A dose-dependent inhibition of cell proliferation was observed after the treatment of various cell lines (A549 (human lung cancer adenocarcinoma), MDA-MB-231 (triple-negative breast cancer cell line), T47 (human breast cancer cell line), EMT6/P (mouse mammary sarcoma cell line)) with increasing concentrations of the essential oil (0.02–2.5 mg/mL). The essential oil of Chiliadenus iphionoides was more cytotoxic against EMT6 and T47 cells with IC₅₀ values of 0.03±0.03 and 0.08± 0.009 mg/mL, respectively. The essential oil exhibited low cytotoxicity against a normal VERO cell line (IC₅₀ value > 4 mg/ml). Balb/C mice were inoculated with EMT6/p breast cancer cells and then treated with (60 mg/kg/day intraperitoneal injection) of essential extract for ten days. Interestingly, a significant (p < 0.05) reduction occurred in the tumor size of the treated group compared to the control group. Treatment toxicity was evaluated by measuring liver and kidney parameters. The tumor-bearing mice treated with the Chiliadenus iphionoides extract showed normal serum levels of AST, creatinine, and slight elevation in ALT level. The results indicated that Chiliadenus iphionoides have anticancer properties both in vitro and in vivo. We suggest that eucalyptol, a major active component, is a promising candidate for use as an anticancer agent. However, further molecular investigation is required to understand the molecular bearings of Chiliadenus iphionoides activity.

Tristetraprolin promotes survival of mammary progenitor cells by restraining TNFα levels.

Tristetraprolin (TTP) is an RNA binding protein that destabilizes mRNAs of factors involved in proliferation, invasiveness, and inflammation. Disruption of the gene that codes for TTP (Zfp36) led to severe arthritis, autoimmunity, cachexia and dermatitis in mice. It has been shown that these phenotypes were mostly due to excessive TNFα levels in the affected tissues. We have previously reported that TTP expression is required for lactation maintenance. Our results indicated that conditional MG TTP-KO female mice displayed early involution due to the untimely induction of pro-inflammatory pathways led mostly by TNFα overexpression. Here we show that reducing TTP levels not only affects the fully differentiated mammary gland, but also harms morphogenesis of this tissue by impairing the progenitor cell population. We found that Zfp36 expression is linked to mammary stemness in human and mice. In addition, diminishing TTP expression and activity induced apoptosis of stem-like mouse mammary cells, reduced its ability to form mammospheres in culture and to develop into complete glands when implanted into cleared mammary fat pads in vivo. Our results show that survival of the stem-like cells is compromised by increased levels of inflammatory cytokines and stimulation of signaling cascades involving NFκB, STAT3 and MAPK-p38 activation. Moreover, TNFα overexpression and the consequent p38 phosphorylation would be the leading cause of progenitor cell death upon TTP expression restriction. Taken together, our results reveal the relevance of TTP for the maintenance of the mammary progenitor cell compartment by maintaining local TNFα levels at bay.

Restriction site associated DNA sequencing for tumour mutation burden estimation and mutation signature analysis.

Genome-wide measures of genetic disruption such as tumour mutation burden (TMB) and mutation signatures are emerging as useful biomarkers to stratify patients for treatment. Clinicians commonly use cancer gene panels for tumour mutation burden estimation, and whole genome sequencing is the gold standard for mutation signature analysis. However, the accuracy and cost associated with these assays limits their utility at scale. WGS data from 560 breast cancer patients was used for in silico library simulations to evaluate the accuracy of an FDA approved cancer gene panel as well as restriction enzyme associated DNA sequencing (RADseq) libraries for TMB estimation and mutation signature analysis. We also transfected a mouse mammary cell line with APOBEC enzymes and sequenced resulting clones to evaluate the efficacy of RADseq in an experimental setting. RADseq had improved accuracy of TMB estimation and derivation of mutation profiles when compared to the FDA approved cancer panel. Using simulated immune checkpoint blockade (ICB) trials, we show that inaccurate TMB estimation leads to a reduction in power for deriving an optimal TMB cutoff to stratify patients for immune checkpoint blockade treatment. Additionally, prioritisation of APOBEC hypermutated tumours in these trials optimises TMB cutoff determination for breast cancer. The utility of RADseq in an experimental setting was also demonstrated, based on characterisation of an APOBEC mutation signature in an APOBEC3A transfected mouse cell line. In conclusion, our work demonstrates that RADseq has the potential to be used as a cost-effective, accurate solution for TMB estimation and mutation signature analysis by both clinicians and basic researchers.

Hexadecanamide alleviates Staphylococcus aureus-induced mastitis in mice by inhibiting inflammatory responses and restoring blood-milk barrier integrity.

Subacute ruminal acidosis (SARA) has been demonstrated to promote the development of mastitis, one of the most serious diseases in dairy farming worldwide, but the underlying mechanism is unclear. Using untargeted metabolomics, we found hexadecanamide (HEX) was significantly reduced in rumen fluid and milk from cows with SARA-associated mastitis. Herein, we aimed to assess the protective role of HEX in Staphylococcus aureus (S. aureus)- and SARA-induced mastitis and the underlying mechanism. We showed that HEX ameliorated S. aureus-induced mastitis in mice, which was related to the suppression of mammary inflammatory responses and repair of the blood-milk barrier. In vitro, HEX depressed S. aureus-induced activation of the NF-κB pathway and improved barrier integrity in mouse mammary epithelial cells (MMECs). In detail, HEX activated PPARα, which upregulated SIRT1 and subsequently inhibited NF-κB activation and inflammatory responses. In addition, ruminal microbiota transplantation from SARA cows (S-RMT) caused mastitis and aggravated S. aureus-induced mastitis, while these changes were reversed by HEX. Our findings indicate that HEX effectively attenuates S. aureus- and SARA-induced mastitis by limiting inflammation and repairing barrier integrity, ultimately highlighting the important role of host or microbiota metabolism in the pathogenesis of mastitis and providing a potential strategy for mastitis prevention.

Molecular Resonance Imaging of the CAIX Expression in Mouse Mammary Adenocarcinoma Cells.

The carbonic anhydrase isoform IX (hCAIX) is one of the main players in extracellular tumor pH regulation, and it is known to be overexpressed in breast cancer and other common tumors. hCA IX supports the growth and survival of tumor cells, and its expression is correlated with metastasis and resistance to therapies, making it an interesting biomarker for diagnosis and therapy. The aim of this work deals with the development of an MRI imaging probe able to target the extracellular non-catalytic proteoglycan-like (PG) domain of CAIX. For this purpose, a specific nanoprobe, LIP_PepC, was designed by conjugating a peptidic interactor of the PG domain on the surface of a liposome loaded with Gd-bearing contrast agents. A Mouse Mammary Adenocarcinoma Cell Line (TS/A) was chosen as an in vitro breast cancer model to test the developed probe. MRI results showed a high selectivity and sensitivity of the imaging probe toward hCAI-expressing TS/A cells. This approach appears highly promising for the in vivo translation of a diagnostic procedure based on the targeting of hCA IX enzyme expression.

Inhibition of Dipeptidyl Peptidase-4 Activates Autophagy to Promote Survival of Breast Cancer Cells via the mTOR/HIF-1α Pathway.

Autophagy plays a complex role in breast cancer cell survival, metastasis, and chemotherapeutic resistance. Dipeptidyl peptidase (DPP)-4, a therapeutic target for type 2 diabetes mellitus, is also involved in autophagic flux. The potential influence of DPP-4 suppression on cancer biology remains unknown. Here, we report that DPP-4 deficiency promotes breast cancer cell survival via the induction of autophagy by the C-X-C motif chemokine 12 (CXCL12)/C-X-C receptor 4 (CXCR4)/mammalian target of rapamycin (mTOR)/hypoxia inducible factor (HIF)-1α axis. DPP-4 knockdown and DPP-4 inhibitor KR62436 (KR) treatment both increased the levels of LC3II and HIF-1α in cultured human breast and mouse mammary cancer cells. The KR-induced autophagic phenotype in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 and rapamycin. HIF-1α knockdown also suppressed breast cancer autophagy induced by KR. The autophagy inhibitor 3-methyladenine significantly blocked the KR-mediated suppression of cleaved caspase-3 levels and apoptosis in breast cancer cell lines. Finally, we found that the metformin-induced apoptosis of DPP-4-deficient 4T1 mammary cancer cells was associated with the suppression of autophagy. Our findings identify a novel role for DPP-4 inhibition in the promotion of breast cancer survival by inducing CXCL12/CXCR4/mTOR/HIF-1α axis-dependent autophagy. Metformin is a potential drug that counteracts the breast cancer cell survival system.

Hg(II)/Fe(III) Turn‐On Fluorescence Response of Rhodamine‐Anthracene Derivatives with Variable Diamine Chain Length

AbstractTwo rhodamine‐anthracene derivative probes (RA1 and RA2) were designed and synthesized to incorporate different lengths of diamine chains between the rhodamine and anthracene moieties. Among the 16 different metal ions tested, RA1 exhibited the highest fluorescence enhancement in response to Hg2+ with a limit of detection (LOD) of 3.76×10−7 M, while RA2 showed the greatest fluorescence enhancement in the presence of Fe3+ with the LOD of 2.41×10−7 M. Notably, RA1 effectively eliminated the interference caused by Fe3+ during Hg2+ detection, whereas RA2 successfully eliminated the interference of Hg2+ during Fe3+ determination. This response difference between the two ions holds particular importance in scenarios where both heavy metal ions coexist. Additionally, fluorescence microscopy imaging of mouse mammary cancer cells (4T1) under the influence of Fe3+ demonstrated the potential applications of the probe. This study highlights the significance of a simple molecular design approach in tailoring probe selectivity by modifying the intramolecular flexible chain length.

Compaction of Calf Thymus DNA by a Potential One-Head-Two-Tail Surfactant: Properties of Nanomaterials and Biological Testing for Gene Delivery.

A one-head-two-tail cationic surfactant, Dilauryldimethylammonium bromide (DDAB) has shown a great extent of calf thymus DNA (ct-DNA) compaction being adsorbed on the surfaces of negatively charged SiO2 nanoparticles (NPs). DDAB molecules show high adsorption efficiency and induce many positive surface charges per-unit surface area of the SiO2 NPs compared to cationic Gemini (12-6-12) and conventional (DTAB) surfactants in an aqueous medium at pH 7.4, as evident from zeta potential and EDAX data. Transmission electron microscopy and field emission scanning electron microscopy images, along with ethidium bromide exclusion assay and DLS data support the compaction of ct-DNA. Fluorescence microscopic images show that in the presence of SiO2 NPs, DDAB can perform 50% compaction of ct-DNA at a concentration ∼58% and ∼99% lower than that of 12-6-12 and DTAB, respectively. Better ct-DNA compaction by DDAB is evident compared to other Gemini surfactants (12-4-12 and 12-8-12) as well reported before. Time-correlated single photon counting fluorescence intensity decay measurements of a probe DAPI in ct-DNA have revealed the average lifetime value that is decreased by ∼61% at 2.5 μM of DDAB in the presence of SiO2 NPs as compared to a decrease by only ∼29% in its absence, supporting NPs-induced stronger surfactant binding with ct-DNA. Fluorescence lifetime data have also demonstrated the crowding effect of NPs. At 2.5 μM of DDAB, both fast and slow rotational relaxation components of DAPI contribute almost equally to depolarization with the absence of NPs; however, with the presence of NPs, ∼96% weightage of the anisotropy decay is for the fast component. The present DDAB-SiO2 NPs combination has proved to be an excellent gene delivery system based on the cell viability in the mouse mammary gland adenocarcinoma cells (4T1) and human embryonic kidney (HEK) 293 cell lines, and in vitro and in vivo studies.

Lecithin/chitosan nanoparticle drug carrier improves anti-tumor efficacy of Monascus pigment rubropunctatin

Rubropunctatin, a metabolite isolated from the fungi of the genus Monascus, is a natural lead compound applied for the suppression of tumors with good anti-cancer activity. However, its poor aqueous solubility has limited its further clinical development and utilization. Lecithin and chitosan are excellently biocompatible and biodegradable natural materials, which have been approved by the FDA as drug carrier. Here, we report for the first time the construction of a lecithin/chitosan nanoparticle drug carrier of the Monascus pigment rubropunctatin by electrostatic self-assembly between lecithin and chitosan. The nanoparticles are near-spherical with a size 110–120 nm. They are soluble in water and possess excellent homogenization capacity and dispersibility. Our in vitro drug release assay showed a sustained release of rubropunctatin. CCK-8 assays revealed that lecithin/chitosan nanoparticles loaded with rubropunctatin (RCP-NPs) had significantly enhanced cytotoxicity against mouse mammary cancer 4T1 cells. The flow cytometry results revealed that RCP-NPs significantly boosted cellular uptake and apoptosis. The tumor-bearing mice models we developed indicated that RCP-NPs effectively inhibited tumor growth. Our present findings suggest that lecithin/chitosan nanoparticle drug carriers improve the anti-tumor effect of the Monascus pigment rubropunctatin.

Influence of Simulated Microgravity on Mammary Epithelial Cells Grown as 2D and 3D Cultures.

During space travel, astronauts will experience a unique environment that includes continuous exposure to microgravity and stressful living conditions. Physiological adaptation to this is a challenge and the effect of microgravity on organ development, architecture, and function is not well understood. How microgravity may impact the growth and development of an organ is an important issue, especially as space flight becomes more commonplace. In this work, we sought to address fundamental questions regarding microgravity using mouse mammary epithelial cells in 2D and 3D tissue cultures exposed to simulated microgravity. Mouse mammary HC11 cells contain a higher proportion of stem cells and were also used to investigate how simulated microgravity may impact mammary stem cell populations. In these studies, we exposed mouse mammary epithelial cells to simulated microgravity in 2D and then assayed for changes in cellular characteristics and damage levels. The microgravity treated cells were also cultured in 3D to form acini structures to define if simulated microgravity affects the cells' ability to organize correctly, a quality that is of key importance for mammary organ development. These studies identify changes occurring during exposure to microgravity that impact cellular characteristics such as cell size, cell cycle profiles, and levels of DNA damage. In addition, changes in the percentage of cells revealing various stem cell profiles were observed following simulated microgravity exposure. In summary, this work suggests microgravity may cause aberrant changes in mammary epithelial cells that lead to an increase in cancer risk.