4T1 Breast Cancer Cells Research Articles

MIGRATION INHIBITION ACTIVITY BY METHANOL EXTRACT Hibiscus tiliaceus Linn. ON 4T1 BREAST CANCER

The prevalence of breast cancer cases in Indonesia is increasing along with the ability of cancer cells to migrate or move from the primary tumor mass and form new colonies elsewhere. The migration of cancer cells has encouraged the develop anticancer drugs from natural ingredients. Waru leaves have been shown to have cytotoxic activity. This study aims to determine the inhibition activity of migration of breast cancer cells 4T1 from methanol extract of waru leaves. Waru leaves methanol extract was obtained using the maceration method. Cytotoxic test of methanol extract of waru leaves (MEWL) was the migration test used in the scratch wound healing method at concentrations 162.5, 325, and 650 μg/mL at 0, 18, 24, and 42 hours after treatment. Analysis of IC50 using linear regression, while large areas were analyzed using Image-J software. The percentage of data closure was analyzed statistically with the Anova Repeated Measure test. All concentrations of Methanol Extract of Waru Leaves had significant inhibition of cell migration (p<0,05) compared to control at each observation time at 0, 18, 24, and 42 hours after treatment. So, MEWL is able to inhibit migration in 4T1 cells

Targeted codelivery of doxorubicin and oleanolic acid by reduction responsive hyaluronic acid-based prodrug nano-micelles for enhanced antitumor activity and reduced toxicity

Chemotherapy remains one of the most commonly used strategies in cancer treatment but suffers from damages to healthy tissues and organs. How to precisely co-deliver two or more drugs with different mechanisms of action to the tumors for synergistic function is a challenge for chemotherapy. Herein, Oleanolic acid (OA)-conjugated Hyaluronic acid self-assembled nano-micelles loaded with Doxorubicin (DOX) (HSO NPs/DOX) were constructed for CD44 positive cancer targeted codelivery of DOX and OA. HSO NPs/DOX exhibited reduction triggered drug release under high concentration of glutathione, more efficient uptake by 4T1 breast cancer cells than free DOX leading to higher cytotoxicity, pro-apoptotic, and migration inhibitory activities against 4T1 cells. The ex vivo biodistribution experiment demonstrated more HSO NPs/DOX were accumulated in the tumor tissues than free DOX and less in the non-tumor tissues after injections in 4T1 tumor bearing mice. More importantly, synergistic anti-tumor effects of DOX and OA were obtained using HSO NPs/DOX in 4T1 breast tumor-bearing mice and toxicity of DOX to liver and heart were circumvented through regulating the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Silent Information Regulator 1 (Sirt1) expressions. Taken together, HSO NPs/DOX may become a promising codelivery system for chemotherapeutics in cancer therapy.

A Novel Method for the Early Detection of Single Circulating, Metastatic and Self-Seeding Cancer Cells in Orthotopic Breast Cancer Mouse Models.

Metastasis is the main cause of cancer-related deaths, but efficient targeted therapies against metastasis are still missing. Major gaps exist in our understanding of the metastatic cascade, as existing methods cannot combine sensitivity, robustness, and practicality to dissect cancer progression. Addressing this issue requires improved strategies to distinguish early metastatic colonization from metastatic outgrowth. Luciferase-labelled MDA-MB-231, MCF7, and 4T1 breast cancer cells were spiked into samples from tumour-naïve mice to establish the limit of detection for disseminated tumour cells. Luciferase-labelled breast cancer cells (±unlabelled cancer-associated fibroblasts; CAFs) were orthotopically implanted in immunocompromised mice. An ex vivo luciferase assay was used to quantify tumour cell dissemination. In vitro luciferase assay confirmed a linear and positive correlation between cancer cell numbers and the bioluminescence detected at single cell level in blood, brain, lung, liver, and mammary fat pad samples. Remarkably, single luciferase-labelled cancer cells were detectable in all of these sites, as the bioluminescence quantified in the analysed samples was substantially higher than background levels. Ex vivo, circulating tumour cells, metastasis, and tumour self-seeding were detected in all samples from animals implanted with highly metastatic luciferase-labelled MDA-MB-231 cells. In turn, detection of poorly metastatic luciferase-labelled MCF7 cells was scarce but significantly enhanced upon co-implantation with CAFs as early as 20 days after the experiment was initiated. These results demonstrate the feasibility of using an ultrasensitive luciferase-based method to dissect the mechanisms of early metastatic colonization to improving the development of antimetastatic therapies.

Establishment of an animal model of immune-related adverse events induced by immune checkpoint inhibitors.

Immunotherapy, specifically immune checkpoint inhibitors (ICIs), has revolutionized cancer treatment. However, it can also cause immune-related adverse events (irAEs). This study aimed to develop a clinically practical animal model of irAEs using BALB/c mice. Subcutaneous tumors of mouse breast cancer 4T1 cells were generated in inbred BALB/c mice. The mice were treated with programmed death-1 (PD-1) and cytotoxic t-lymphocyte antigen 4 (CTLA-4) inhibitors once every 3 days for five consecutive administration cycles. Changes in tumor volume and body weight were recorded. Lung computed tomography (CT) scans were conducted. The liver, lungs, heart, and colon tissues of the mice were stained with hematoxylin-eosin (H&E) staining to observe inflammatory infiltration and were scored. Serum samples were collected, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ferritin, glutamic-pyruvic transaminase (ALT), tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and interleukin-6 (IL-6). Mouse liver and lung cell suspensions were prepared, and changes in macrophages, T cells, myeloid-derived suppressor cells (MDSCs), and regulatory (Treg) cells were detected by flow cytometry. Mice treated with PD-1 and CTLA-4 inhibitors showed significant reductions in tumor volume and body weight. The tissue inflammatory scores in the experimental group were significantly higher than those in the control group. Lung CT scans of mice in the experimental group showed obvious inflammatory spots. Serum levels of ferritin, IL-6, TNF-α, IFN-γ, and ALT were significantly elevated in the experimental group. Flow cytometry analysis revealed a substantial increase in CD3+T cells, Treg cells, and macrophages in the liver and lung tissues of mice in the experimental group compared with the control group, and the change trend of MDSCs was opposite. The irAE-related animal model was successfully established in BALB/c mice using a combination of PD-1 and CTLA-4 inhibitors through multiple administrations with clinical translational value and practical. This model offers valuable insights into irAE mechanisms for further investigation.

Investigating the Effect of Resveratrol on The Survival Rate of 4T1 Breast Cancer Cells By MTT Method

Cancer is known as an incurable, deadly and deadly disease with pain and disability. During recent decades, changes in people's lifestyles have increased the incidence and prevalence of breast cancer worldwide. With the wide spread of this disease, research has also increased on this issue. Resveratrol is known as a natural polyphenol found in red grapes and some other foods, and with its important anti-cancer properties, it can be effective in reducing the risk of breast cancer and its treatment. Research has shown that resveratrol can have positive effects on breast cancer by reducing DNA methylation, reducing cell proliferation, and reducing cancer growth factors. In this research, resveratrol has been used against 4T1 cancer cells, which were treated with different concentrations of resveratrol after cultivation in 96-well plates and measured by the MTT method. The obtained results showed that resveratrol in all concentrations and at different times significantly reduces the survival of 4T1 cancer cells. By increasing the concentration and treatment time, the decrease in cell viability increases. This indicates strong, dose- and time-dependent effects of resveratrol on breast cancer cells.

Cytotoxic effects of chloridazon-loaded alginate-chitosan nanocapsules on the 4T1 breast cancer cell line.

Cytotoxic effects of chloridazon-loaded alginate-chitosan nanocapsules on the 4T1 breast cancer cell line.

Cytostatic Bacterial Metabolites Interfere with 5-Fluorouracil, Doxorubicin and Paclitaxel Efficiency in 4T1 Breast Cancer Cells.

The microbiome is capable of modulating the bioavailability of chemotherapy drugs, mainly due to metabolizing these agents. Multiple cytostatic bacterial metabolites were recently identified that have cytostatic effects on cancer cells. In this study, we addressed the question of whether a set of cytostatic bacterial metabolites (cadaverine, indolepropionic acid and indoxylsulfate) can interfere with the cytostatic effects of the chemotherapy agents used in the management of breast cancer (doxorubicin, gemcitabine, irinotecan, methotrexate, rucaparib, 5-fluorouracil and paclitaxel). The chemotherapy drugs were applied in a wide concentration range to which a bacterial metabolite was added in a concentration within its serum reference range, and the effects on cell proliferation were assessed. There was no interference between gemcitabine, irinotecan, methotrexate or rucaparib and the bacterial metabolites. Nevertheless, cadaverine and indolepropionic acid modulated the Hill coefficient of the inhibitory curve of doxorubicin and 5-fluorouracil. Changes to the Hill coefficient implicate alterations to the kinetics of the binding of the chemotherapy agents to their targets. These effects have an unpredictable significance from the clinical or pharmacological perspective. Importantly, indolepropionic acid decreased the IC50 value of paclitaxel, which is a potentially advantageous combination.

Improving the Photodynamic Activity of Water-Soluble Porphyrin-Polysaccharide Complexes by Folic Acid Modification.

Studies have shown that folate receptors are highly expressed in various cancer cells. Here, we synthesized folic acid-conjugated pullulan (FAPL) as a solubilizing agent to improve the photodynamic activity of porphyrin derivative-polysaccharide complexes. The porphyrin derivative-FAPL complex exhibited long-term stability in an aqueous solution, attributed to the folic acid modification. Furthermore, in vitro and in vivo experiments highlighted the enhanced photodynamic activity of the porphyrin derivative-FAPL complex toward 4T1 breast-cancer cells, compared with the activities of the porphyrin derivative-pullulan complex and Photofrin. This enhanced activity is attributed to the improvement of intracellular uptake by the folate receptor.

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.

UM171 suppresses breast cancer progression by inducing KLF2

PurposeBreast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs is unmet need. The aim of this study is to show potent anti-neoplastic activity of the UM171 compound on breast cancer cells and its mechanism of action.MethodsThe inhibitory effect of UM171 on several breast cancer (BC) cell lines was examined using MTT and colony-forming assays. Cell cycle and apoptosis assays were utilized to determine the effect of UM171 on BC cell proliferation and survival. Wound healing scratch and transwell migration assays were used to examine the migration of BC cell lines in culture. Xenograft of mouse model with 4T1 cells was used to determine inhibitory effect of UM171 in vivo. Q-RT-PCR and western blotting were used to determine the expression level of genes effected by UM171. Lentivirus-mediated shRNAs were used to knockdown the expression of KLF2 in BC cells.ResultsUM171 was previously identified as a potent agonist of human hematopoietic stem cell renewal and inhibitor of leukemia. In this study, UM171 was shown to inhibit the growth of multiple breast cancer cell lines in culture. UM171-mediated growth inhibition was associated with the induction of apoptosis, G2/M cell cycle arrest, lower colony-forming capacity, and reduced motility. In a xenotransplantation model of mouse triple-negative breast cancer 4T1 cells injected into syngeneic BALB/c mice, UM171 strongly inhibited tumor growth at a level comparable to control paclitaxel. UM171 increased the expression of the three PIM genes (PIM1-3) in breast cancer cells. Moreover, UM171 strongly induced the expression of the tumor suppressor gene KLF2 and cell cycle inhibitor P21CIP1. Accordingly, knockdown of KLF2 using lentivirus-mediated shRNA significantly attenuated the growth suppressor activity of UM171. As PIM1-3 act as oncogenes and are involved in breast cancer progression, induction of these kinases likely impedes the inhibitory effect of KLF2 induction by UM171. Accordingly, combination of UM171 with a PAN-PIM inhibitor LGH447 significantly reduced tumor growth in culture.ConclusionThese results suggested that UM171 inhibited breast cancer progression in part through activation of KLF2 and P21. Combination of UM171 with a PAN-PIM inhibitor offer a novel therapy for aggressive forms of breast cancer.

Photothermal and radiotherapy with alginate-coated gold nanoparticles for breast cancer treatment

Radiation therapy and phototherapy are commonly used cancer treatments that offer advantages such as a low risk of adverse effects and the ability to target cancer cells while sparing healthy tissue. A promising strategy for cancer treatment involves using nanoparticles (NPs) in combination with radiation and photothermal therapy to target cancer cells and improve treatment efficacy. The synthesis of gold NPs (AuNPs) for use in biomedical applications has traditionally involved toxic reducing agents. Here we harnessed dopamine (DA)-conjugated alginate (Alg) for the facile and green synthesis of Au NPs (Au@Alg-DA NPs). Alg-DA conjugate reduced Au ions, simultaneously stabilized the resulting AuNPs, and prevented aggregation, resulting in particles with a narrow size distribution and improved stability. Injectable Au@Alg-DA NPs significantly promoted ROS generation in 4T1 breast cancer cells when exposed to X-rays. In addition, their administration raised the temperature under a light excitation of 808 nm, thus helping to destroy cancer cells more effectively. Importantly, no substantial cytotoxicity was detected in our Au@Alg-DA NPs. Taken together, our work provides a promising route to obtain an injectable combined radio enhancer and photothermally active nanosystem for further potential clinic translation.

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.

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 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.

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

PurposeProstate 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.MethodsMurine 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).ResultsAccumulation 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.ConclusionThe 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.

Mutation of SIVA, a candidate metastasis gene identified from clonally related bilateral breast cancers, promotes breast cancer cell spread in vitro and in vivo.

Metastasis is the most dreaded outcome after a breast cancer diagnosis, and little is known regarding what triggers or promotes breast cancer to spread distally, or how to prevent or eradicate metastasis effectively. Bilateral breast cancers are an uncommon form of breast cancers. In our study, a percentage of bilateral breast cancers were clonally related based on copy number variation profiling. Whole exome sequencing and comparative sequence analysis revealed that a limited number of somatic mutations were acquired in this "breast-to-breast" metastasis that might promote breast cancer distant spread. One somatic mutation acquired was SIVA-D160N that displayed pro-metastatic phenotypes in vivo and in vitro. Over-expression of SIVA-D160N promoted migration and invasion of human MB-MDA-231 breast cancer cells in vitro, consistent with a dominant negative interfering function. When introduced via tail vein injection, 231 cells over-expressing SIVA-D160N displayed enhanced distant spread on IVIS imaging. Over-expression of SIVA-D160N promoted invasion and anchorage independent growth of mouse 4T1 breast cancer cells in vitro. When introduced orthotopically via mammary fat pad injection in syngeneic Balb/c mice, over-expression of SIVA-D160N in 4T1 cells increased orthotopically implanted mammary gland tumor growth as well as liver metastasis. Clonally related bilateral breast cancers represented a novel system to investigate metastasis and revealed a role of SIVA-D160N in breast cancer metastasis. Further characterization and understanding of SIVA function, and that of its interacting proteins, may elucidate mechanisms of breast cancer metastasis, providing clinically useful biomarkers and therapeutic targets.

Development of Liposomes Loaded with Chloroaluminum Phthalocyanine for Application of Photodynamic Therapy in Breast Cancer

Chloraluminium phthalocyanine (ClAlPc) has potential therapeutic effect for the treatment of cancer; however, the molecule is lipophilic and may present self-aggregation which limits its clinical success. Thus, nanocarriers like liposomes can improve ClAlPc solubility, reduce off-site toxicity and increase circulation time. For this purpose, developing suitable liposomes requires the evaluation of different lipid compositions. Herein, we aimed to develop liposomes containing soy phosphatidylcholine (SPC), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPEPEG2000), cholesterol and oleic acid loaded with ClAlPc using the surface response methodology and the Box-Behnken design. Liposomes with particle size from 110.93 to 374.97 nm and PdI from 0.265 to 0.468 were obtained. The optimized formulation resulted in 69.09 % of ClAlPc encapsulated, with particle size and polydispersity index, respectively, at 153.20 nm and 0.309, providing stability and aggregation control. Atomic force microscopy revealed vesicles in a spherical or almost spherical shape, while the analyzes by Differential Scanning Calorimetry (DSC), Powder X-ray Diffraction (PXRD), and Fourier transform infrared spectroscopy (FTIR) suggested that the drug was adequately incorporated into the lipid bilayer of liposomes, in its amorphous state or molecularly dispersed. In vitro studies conducted in breast cancer cells (4T1) showed that liposome improved phototoxicity compared to the ClAlPc solution. ClAlPc-loaded liposomes also enhanced the production of ROS 3-fold compared to the ClAlPc solution. Finally, confocal microscopy and flow cytometry demonstrated the ability of the liposomes to enter cells and deliver the fluorescent ClAlPc photosensitizer with dose and time-dependent effects. Thus, this work showed that Box-Behnken factorial design was an effective strategy for optimizing formulation development. The obtained ClAlPc liposomes can be applied for photodynamic therapy in breast cancer cells.

Abstract PO2-28-03: E-Cigarette Aerosol Exposure: Unraveling the Nexus of Autophagy, Cancer Stem Cells, and EMT in Breast Cancer Metastasis

Abstract Electronic cigarettes (E-cigs) have emerged as a significant public health concern due to their increasing popularity. These products have been associated with various harmful effects on human health. In the United States, breast cancer is the most diagnosed cancer among women, and its recurrence and metastasis are primary contributors to morbidity and mortality. Two critical factors in cancer progression are Epithelial-mesenchymal transition (EMT) and Cancer stem-like cells (CSCs), both of which play pivotal roles in metastasis. As a result, targeting the signaling pathways involved in these processes has become a promising avenue for cancer therapy. Autophagy, a cellular process, has dual roles in cancer development depending on the cancer type and stage. Given these considerations, our hypothesis centers on disrupting the EMT-mediated metastatic cascade in breast cancer by targeting autophagy in Cancer Stem Cells (CSCs). To investigate this hypothesis, we conducted experiments using mouse breast epithelial cells (HC11) and Triple-negative mouse breast cancer cells (4T1) exposed to E-cig aerosol at the air-liquid interphase (ALI) for 1 hr followed by a 24 hr recovery period. Our findings indicate that exposure to E-cig aerosols led to a decrease in epithelial markers (E-Cadherin and β-catenin) and an increase in mesenchymal markers (N-Cadherin and Vimentin) in HC11 cells. This suggests that normal cells may acquire characteristics associated with solitary migration. Furthermore, we observed significant changes in the levels of autophagy-related proteins (LC3B, Beclin-1, ATG12, and ATG16) and upregulation of stem cell markers (OCT4, SOX2, NANOG, KLF4, and c-MYC) in response to E-cig aerosol exposure, both in HC11 and 4T1 cells. Additionally, these alterations extended to changes in the expression of genes associated with the Transforming Growth Factor-beta (TGF-β) signaling pathway, known to play a potential role in promoting EMT. Our results provide a deeper understanding of the interplay between EMT, CSCs, and the autophagy process in the context of ALI exposure in normal and cancer cells. This underscores the need for further research into the shared molecular mechanisms of these three processes to identify common therapeutic targets that can simultaneously affect them. Citation Format: Shilpa Thota, Rizwana Begum, Naveen Chintalaramulu, Biplov Sapkota, Abhishek Pandit. E-Cigarette Aerosol Exposure: Unraveling the Nexus of Autophagy, Cancer Stem Cells, and EMT in Breast Cancer Metastasis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-28-03.

Tumor Targeting with Apatinib-loaded Nanoparticles and Sonodynamic Combined Therapy.

This study implies the enhancement of apatinib killing effect in 4T1 tumor cells through constructing drug-loaded nanoparticles apatinib/Ce6@ZIF- 8@Membranes (aCZM) to enhance tumor therapeutic targeting and reduce toxic side following sonodynamic therapy (SDT). apatinib/Ce6@ZIF-8 (aCZ) were synthesized by in situ encapsulation, and aCZM were constructed by encapsulating the nanoparticles with extracted breast cancer 4T1 cell membranes. aCZM were characterized and tested for the stability by electron microscopy, and the membrane proteins on the nanoparticles' surface were assessed using SDS-PAGE gel electrophoresis. The cell viability of 4T1 cells following treatment with aCZM was tested using cell counting kit-8 (CCK-8). The uptake of nanoparticles was detected by laser confocal microscopy and flow cytometry, and the SDT-mediated production of reactive oxygen species (ROS) was verified by singlet oxygen sensor green (SOSG), electron spin resonance (ESR), and DCFH-DA fluorescent probes. The CCK-8 assay and flow cytometry using Calcein/PI were used to assess the antitumoral effect of aCZM nanoparticles under SDT. The biosafety of aCZM was further verified in vitro and in vivo using the hemolysis assay, routine blood test and H&E staining of vital organs in Balb/c mice. aCZM with an average particle size of about 210.26 nm were successfully synthesized. The results of the SDS-PAGE gel electrophoresis experiment showed that aCZM have a band similar to that of pure cell membrane proteins. The CCK-8 assay demonstrated the absence of effects on cell viability at a low concentration range, and the relative cell survival rate reached more than 95%. Laser confocal microscopy and flow cytometry analysis showed that aCZM treated group has the strongest fluorescence and the highest cellular uptake of nanoparticles. SOSG, ESR, and DCFH-DA fluorescent probes all indicated that the aCZM + SDT treated group has the highest ROS production. The CCK-8 assay also showed that when the ultrasound intensity was fixed at 0.5 W/cm2, the relative cell survival rates in the medium concentration group (10 μg/ml) (5.54 ± 1.26%) and the high concentration group (20 μg/ml) (2.14 ± 1.63%) were significantly lower than those in the low concentration group (5 μg/ml) (53.40 ± 4.25%). Moreover, there was a concentration and intensity dependence associated with the cellkilling effect. The mortality rate of the aCZM in the ultrasound group (44.95 ± 3.03%) was significantly higher than that of the non-ultrasound (17.00 ± 2.26%) group and aCZ + SDT group (24.85 ± 3.08%) (P<0.0001). The live and dead cells' staining (Calcein/PI) also supported this result. Finally, in vitro hemolysis test at 4 and 24 hours showed that the hemolysis rate of the highest concentration group was less than 1%. The blood routine, biochemistry, and H&E staining results of major organs in Balb/c mice undergoing nano-treatments showed no obvious functional abnormalities and tissue damage in 30 days. In this study, a multifunctional bionic drug delivery nanoparticles (aCZM) system with good biosafety and compatibility in response to acoustic dynamics was successfully constructed and characterized. This system enhanced apatinib killing effect on tumor cells and reduced toxic side effects under SDT.