Mammosphere Formation Assay Research Articles

Polyoxometalate inhibition of SOX2-mediated tamoxifen resistance in breast cancer

BackgroundIncreased cancer stem cell (CSC) content and SOX2 overexpression are common features in the development of resistance to therapy in hormone-dependent breast cancer, which remains an important clinical challenge. SOX2 has potential as biomarker of resistance to treatment and as therapeutic target, but targeting transcription factors is also challenging. Here, we examine the potential inhibitory effect of different polyoxometalate (POM) derivatives on SOX2 transcription factor in tamoxifen-resistant breast cancer cells.MethodsVarious POM derivatives were synthesised and characterised by infrared spectra, powder X-ray diffraction pattern and nuclear magnetic resonance spectroscopy. Estrogen receptor (ER) positive breast cancer cells, and their counterparts, which have developed resistance to the hormone therapy tamoxifen, were treated with POMs and their consequences assessed by gel retardation and chromatin immunoprecipitation to determine SOX2 binding to DNA. Effects on proliferation, migration, invasion and tumorigenicity were monitored and quantified using microscopy, clone formation, transwell, wound healing assays, flow cytometry and in vivo chick chorioallantoic membrane (CAM) models. Generation of lentiviral stable gene silencing and gene knock-out using CRISPR-Cas9 genome editing were applied to validate the inhibitory effects of the selected POM. Cancer stem cell subpopulations were quantified by mammosphere formation assays, ALDEFLUOR activity and CD44/CD24 stainings. Flow cytometry and western blotting were used to measure reactive oxygen species (ROS) and apoptosis.ResultsPOMs blocked in vitro binding activity of endogenous SOX2. [P2W18O62]6− (PW) Wells-Dawson-type anion was the most effective at inhibiting proliferation in various cell line models of tamoxifen resistance. 10 µM PW also reduced cancer cell migration and invasion, as well as SNAI2 expression levels. Treatment of tamoxifen-resistant cells with PW impaired tumour formation by reducing CSC content, in a SOX2-dependent manner, which led to stem cell depletion in vivo. Mechanistically, PW induced formation of reactive oxygen species (ROS) and inhibited Bcl-2, leading to the death of tamoxifen-resistant cells. PW-treated tamoxifen-resistant cells showed restored sensitivity to tamoxifen.ConclusionsTogether, these observations highlight the potential use of PW as a SOX2 inhibitor and the therapeutic relevance of targeting SOX2 to treat tamoxifen-resistant breast cancer.

Continuous exposure to doxorubicin induces stem cell-like characteristics and plasticity in MDA-MB-231 breast cancer cells identified with the SORE6 reporter

PurposeCancer stem cells (CSCs) account for recurrence and resistance to breast cancer drugs, rendering them a cause of mortality and therapeutic failure. In this study, we examined the effects of exposure to low concentrations of doxorubicin (Dox) on CSCs and non-CSCs from TNBC.MethodsThe effects of Dox were studied using the SORE6 reporter system. We examined the enrichment of the CSCs population, as well as the proliferation, and death of the reporter-positive fraction (GFP + cells) by flow cytometry. The resistant and stemness phenotypes were analyzed by viability and mammosphere formation assay, respectively. We identified differentially expressed and coregulated genes by RNA-seq analysis, and the correlation between gene expression and clinical outcome was evaluated by Kaplan-Mayer analysis using public databases.ResultsIn MDAMB231 and Hs578t cells, we identified enriched subsets in the CSCs population after continuous exposure to low concentrations of Dox. Cells from these enriched cultures showed resistance to toxic concentrations of Dox and increased efficiency of mammosphere formation. In purified GFP + or GFP- cells, Dox increased the mammosphere-forming efficiency, promoted phenotypic switches in non-CSCs populations to a CSC-like state, reduced proliferation, and induced differential gene expression. We identified several biological processes and molecular functions that partially explain the development of doxorubicin-resistant cells and cellular plasticity. Among the genes that were regulated by Dox exposure, the expression of ITGB1, SNAI1, NOTCH4, STAT5B, RAPGEF3, LAMA2, and GNAI1 was significantly associated with poor survival, the stemness phenotype, and chemoresistance.ConclusionThe generation of chemoresistant cells that have characteristics of CSCs, after exposure to low concentrations of Dox, involves the differential expression of genes that have a clinical impact.

Combination of monensin and erlotinib synergistically inhibited the growth and cancer stem cell properties of triple-negative breast cancer by simultaneously inhibiting EGFR and PI3K signaling pathways.

Cancer stem cells (CSCs) in triple-negative breast cancer (TNBC) are recognized as a highly challenging subset of cells, renowned for their heightened propensity for relapse and unfavorable prognosis. Monensin, an ionophoric antibiotic, has been reported to exhibit significant therapeutic efficacy against various cancers, especially CSCs. Erlotinib is classified as one of the EGFR-TKIs and has been previously identified as a promising therapeutic target for TNBC. Our research aims to assess the effectiveness of combination of monensin and erlotinib as a potential treatment strategy for TNBC. The combination of monensin and erlotinib was assessed for its potential anticancer activity through various in vitro assays, including cytotoxicity assay, colony formation assay, wound healing assay, transwell assay, mammosphere formation assay, and proportion of CSCs assay. Additionally, an in vivo study using tumor-bearing nude mice was conducted to evaluate the inhibitory effect of the monensin and erlotinib combination on tumor growth. The results indicated that combination of monensin with erlotinib synergistically inhibited cell proliferation, the migration rate, the invasion ability and decreased the CSCs proportion, and CSC markers SOX2 and CD133 in vivo and in vitro. Furthermore, the primary proteins involved in the signaling pathways of the EGFR/ERK and PI3K/AKT are simultaneously inhibited by the combination treatment of monensin and erlotinib in vivo and in vitro. The simultaneous inhibition of the EGFR/ERK and PI3K/AKT/mTOR signaling pathways by the combination of monensin and erlotinib exhibited a synergistic effect on suppressing tumor proliferation and cancer cell stemness in TNBC.

Ursolic acid inhibits the proliferation of triple‑negative breast cancer stem‑like cells through NRF2‑mediated ferroptosis.

Ursolic acid (UA), a pentacyclic triterpenoid that has been found in a broad variety of fruits, spices and medicinal plants, has various biological effects such as reducing inflammation, protecting cells from damage, and preserving brain function. However, its impact on ferroptosis in cancer stem‑like cells remains unexplored. The present study investigated the effect of UA on MDA‑MB‑231 and BT‑549 cell‑derived triple‑negative breast CSCs (BCSCs) and its potential ferroptosis pathway. The effects of ferroptosis on BCSCs were demonstrated by the detection of ferroptosis‑related indexes including the intracellular level of glutathione, malondialdehyde, reactive oxygen species and iron. The effects of UA on the biological behaviors of BCSCs were analyzed by Cell Counting Kit‑8, stemness indexes detection and mammosphere formation assay. The mechanism of UA induction on BCSCs was explored by reverse transcription‑quantitative PCR and western blotting. BALB/c‑nude mice were subcutaneously injected with MDA‑MB‑231‑derived BCSCs to establish xenograft models to detect the effects of UA in vivo. The results revealed that BCSCs have abnormal iron metabolism and are less susceptible to ferroptosis. UA effectively reduces the stemness traits and proliferation of BCSCs in spheroids and mice models by promoting ferroptosis. It was observed that UA stabilizes Kelch‑like ECH‑associated protein 1 and suppresses nuclear factor erythroid‑related factor 2 (NRF2) activation. These findings suggested that the ability of UA to trigger ferroptosis through the inhibition of the NRF2 pathway could be a promising approach for treating BCSCs, potentially addressing metastasis and drug resistance in triple‑negative breast cancer (TNBC). This expands the clinical applications of UA and provides a theoretical basis for its use in TNBC treatment.

The transcriptional landscape of cancer stem-like cell functionality in breast cancer

BackgroundCancer stem-like cells (CSCs) have been extensively researched as the primary drivers of therapy resistance and tumor relapse in patients with breast cancer. However, due to lack of specific molecular markers, increased phenotypic plasticity and no clear clinicopathological features, the assessment of CSCs presence and functionality in solid tumors is challenging. While several potential markers, such as CD24/CD44, have been proposed, the extent to which they truly represent the stem cell potential of tumors or merely provide static snapshots is still a subject of controversy. Recent studies have highlighted the crucial role of the tumor microenvironment (TME) in influencing the CSC phenotype in breast cancer. The interplay between the tumor and TME induces significant changes in the cancer cell phenotype, leading to the acquisition of CSC characteristics, therapeutic resistance, and metastatic spread. Simultaneously, CSCs actively shape their microenvironment by evading immune surveillance and attracting stromal cells that support tumor progression.MethodsIn this study, we associated in vitro mammosphere formation assays with bulk tumor microarray profiling and deconvolution algorithms to map CSC functionality and the microenvironmental landscape in a large cohort of 125 breast tumors.ResultsWe found that the TME score was a significant factor associated with CSC functionality. CSC-rich tumors were characterized by an immune-suppressed TME, while tumors devoid of CSC potential exhibited high immune infiltration and activation of pathways involved in the immune response. Gene expression analysis revealed IFNG, CXCR5, CD40LG, TBX21 and IL2RG to be associated with the CSC phenotype and also displayed prognostic value for patients with breast cancer.ConclusionThese results suggest that the characterization of CSCs content and functionality in tumors can be used as an attractive strategy to fine-tune treatments and guide clinical decisions to improve patients therapy response.

Abstract PO5-24-02: Interferon-induced bone marrow stromal antigen 2 (BST2) is a functional tumor-initiating cell marker in triple-negative breast cancer

Abstract Background: A subpopulation of tumor cells known to have intrinsic resistance to therapies and contribute to metastasis function as “cancer stem”, or tumor-initiating, cells (TICs). TICs can be identified by various biomarkers, including fluorescent reporters for signaling pathways that regulate TIC function such as STAT signaling. However, standard TIC reporters have limitations. Their expression is unstable and there is no established method to follow TICs as they undergo cell state changes. Furthermore, we do not completely understand the transcriptional features of TICs. Improved methods of identifying TICs as well as the development of high-resolution transcriptional signatures are essential to understand TIC biology. An improved understanding of TIC biology can inform the development of novel therapeutics to selectively ablate the TIC compartment, thus improving response to standard cancer therapies. Methods: We screened a panel of PDX models to determine whether a STAT reporter identifies TICs in patient-derived xenograft (PDX) models. To augment existing STAT TIC reporters, we developed a two-component STAT signaling-specific lineage-tracing (LT) system. The first component labels active STAT signaling cells by expression of enhanced green fluorescent protein (EGFP+), followed by a self-cleaving P2A peptide and a TAM-inducible Cre-recombinase (4M67-EGFP-P2A-CreERT2). The second component is a constitutively expressed dual-color switching Cre-dependent reporter vector (EFS-loxPdsRedloxP-mNeptune2). We determined which LT cell populations (EGFP+/mNeptune2+, EGFP+/dsRed+, EGFP−/mNeptune2+, and EGFP−/dsRed+) from SUM159 xenografts functioned as TICs. To probe TIC transcriptional features, we performed scRNA seq on the two tumor models with STAT TICs and a PDX model that did not have STAT TICs. We performed lineage trajectory analysis and differential gene expression analysis to characterize candidate TIC transcriptional signatures. To confirm whether these transcriptional features are associated with TICs, we identified a biomarker of this cell state (bone marrow stromal antigen 2 [BST2]) and explored the relationship between this biomarker and TICs. Results: We identified four PDX models with STAT reporter activity, one of which had STAT TICs. We validated our LT system in several models both in vitro and in vivo, then demonstrated EGFP+/mNeptune2+ from SUM159 xenografts are enriched for TICs and that lineage-tagged cells (mNeptune2+) function as proliferative early progenitor cells. scRNA seq of three xenograft models uncovered a distinct antiviral cell state in all three models that represent a more highly enriched TIC subpopulation. Critically, transcriptional features were shared between the antiviral cell states across xenograft models. To determine whether cells in this antiviral state represent TICs, we FACS-enriched BST2 cells (an IFN-stimulated cell surface protein that was a marker of the antiviral cell state) and demonstrated enrichment of the TIC population by mammosphere formation assays and limiting dilution transplantation. We demonstrated that BST2 expression is increased in residual PDX tumors following chemotherapy. Furthermore, we demonstrated similar antiviral cell states are present in human breast cancer scRNA seq datasets. Conclusions: These data demonstrate TICs adopt an antiviral cell state in some triple-negative breast cancers, and BST2 is a function marker of TICs in this cell state. Therefore, targeting genes associated with this cell state or associated antiviral pathways may represent a therapeutic vulnerability to eliminate TICs in some breast cancers. Citation Format: Eric Souto, Ping Gong, John Landua, Ram Srinivasan, Lacey Dobrolecki, Abhinaya Ganesan, Michael Lewis. Interferon-induced bone marrow stromal antigen 2 (BST2) is a functional tumor-initiating cell marker in triple-negative breast cancer [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 PO5-24-02.

Molecular mechanisms of Asparagus racemosus willd. and Withania somnifera (L.) Dunal as chemotherapeutic adjuvants for breast cancer treatment

Ethnopharmacological relevanceDespite various treatment modalities, the progression and metastasis of breast cancer (BC) are grave concerns due to the alarming disease-free survival rate (DFS) and overall survival rate (OS) of affected patients. Over the years, many antibiotics, synthetic compounds, medicinal plant isolates and polyherbal combinations have been used as adjuvants in therapy for the management of primary and secondary tumors. Paclitaxel (PTX)-based chemotherapy for breast cancer causes multiple adverse side effects in patients. Withania somnifera (L.) Dunal (WS) and Asparagus racemosus Willd. (AR) as Ayurveda-inspired plant-based adjuvants were investigated for their anticancer effects on MDA-MB-231 and 4T1 cells in mouse model systems. Aim of the studyThis study focused on evaluating the adjuvant properties of WS and AR plant extracts with PTX and their effectiveness over PTX alone in terms of tumor inhibition. Materials and methodsThe effects of WS and AR on DNA double-strand breaks (DSBs), senescence induction and mitochondrial functions were evaluated in BC cells in vitro. The potential for cancer stem cell (CSC) inhibition was evaluated via mammosphere formation assays and CD44/CD24 immunostaining. In vivo tumor growth studies were conducted in athymic BALB/c mice for MDA-MB-231 cells and in BALB/c mice for 4T1 cells. ResultsInduction of senescence was evident due to DSBs induced by the WS and AR extracts. Mammosphere formation and CD44/CD24 CSC markers were reduced after treatment with WS, AR or the combination of both in MCF-7 cells. WS or AR inhibited epithelial-to-mesenchymal transition (EMT). In vivo studies demonstrated that tumor growth inhibition was more pronounced in the treated group than in the PTX alone group and the untreated control group. ConclusionOur study showed that the use of WS or AR plant hydroalcoholic extracts in combination with paclitaxel (PTX) has better effects on sensitivity and efficacy than PTX alone, as demonstrated in in vitro BC cells and mouse models with BC cell grafts. Hence, scheduling adjuvant therapy with WS or AR alone or combined with PTX can be advantageous for the management of triple-negative BC (TNBC). Further studies are warranted in human clinical conditions to ascertain the efficacy of these treatments.

Ropivacaine as a novel AKT1 specific inhibitor regulates the stemness of breast cancer

BackgroundRopivacaine, a local anesthetic, exhibits anti-tumor effects in various cancer types. However, its specific functions and the molecular mechanisms involved in breast cancer cell stemness remain elusive.MethodsThe effects of ropivacaine on breast cancer stemness were investigated by in vitro and in vivo assays (i.e., FACs, MTT assay, mammosphere formation assay, transwell assays, western blot, and xenograft model). RNA-seq, bioinformatics analysis, Western blot, Luciferase reporter assay, and CHIP assay were used to explore the mechanistic roles of ropivacaine subsequently.ResultsOur study showed that ropivacaine remarkably suppressed stem cells-like properties of breast cancer cells both in vitro and in vivo. RNA-seq analysis identified GGT1 as the downstream target gene responding to ropivacaine. High GGT1 levels are positively associated with a poor prognosis in breast cancer. Ropivacaine inhibited GGT1 expression by interacting with the catalytic domain of AKT1 directly to impair its kinase activity with resultant inactivation of NF-κB. Interestingly, NF-κB can bind to the promoter region of GGT1. KEGG and GSEA analysis indicated silence of GGT1 inhibited activation of NF-κB signaling pathway. Depletion of GGT1 diminished stem phenotypes of breast cancer cells, indicating the formation of NF-κB /AKT1/GGT1/NF-κB positive feedback loop in the regulation of ropivacaine-repressed stemness in breast cancer cells.ConclusionOur finding revealed that local anesthetic ropivacaine attenuated breast cancer stemness through AKT1/GGT1/NF-κB signaling pathway, suggesting the potential clinical value of ropivacaine in breast cancer treatment.

Icariin Regulates EMT and Stem Cell-Like Character in Breast Cancer through Modulating lncRNA NEAT1/TGFβ/SMAD2 Signaling Pathway.

Metastases and drug resistance are the major risk factors associated with breast cancer (BC), which is the most common type of tumor affecting females. Icariin (ICA) is a traditional Chinese medicine compound that possesses significant anticancer properties. Long non-coding RNAs (lncRNAs) are involved in a wide variety of biological and pathological processes and have been shown to modulate the effectiveness of certain drugs in cancer. The purpose of this study was to examine the potential effect of ICA on epithelial mesenchymal transition (EMT) and stemness articulation in BC cells, as well as the possible relationship between its inhibitory action on EMT and stemness with the NEAT1/transforming growth factor β (TGFβ)/SMAD2 pathway. The effect of ICA on the proliferation (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony assays), EMT (Western blotting, immunofluorescence, and wound healing), and stemness (mammosphere formation assays, Western blotting) of BC cells were examined. According to the findings, ICA suppressed the proliferation, EMT, and stem cell-like in MDA-MB-231 cells, and exerted its inhibitory impact by downregulating the TGFβ/SMAD2 signaling pathway. ICA could significantly downregulate the expression of lncRNA NEAT1, and silencing NEAT1 enhanced the effect of ICA in suppressing EMT and expression of different stem cell markers. In addition, silencing NEAT1 was found to attenuate the TGFβ/SMAD2 signaling pathway, thereby improving the inhibitory impact of ICA on stemness and EMT in BC cells. In conclusion, ICA can potentially inhibit the metastasis of BC via affecting the NEAT1/TGFβ/SMAD2 pathway, which provides a theoretical foundation for understanding the mechanisms involved in potential application of ICA for BC therapy.

Establishment and characterization of cisplatin-resistant cell lines from canine mammary gland tumors

The development of cisplatin resistance is one of the major causes of mammary cancer treatment failure, and is associated with changes in Sox4 gene expression. To investigate the characteristic changes that occur in canine mammary gland tumor (CMGT) cells following the development of acquired cisplatin resistance, along with the relationship between these changes and the Sox4 gene. We constructed cisplatin-resistant cell line, CHMpCIS, from the cell line CHMp, which was isolated from the primary lesion of a malignant CMGT. The biological characteristics of these cells were examined by Western blot analysis, Transwell assays, and mammosphere formation assays. Compared to CHMp cells, CHMpCIS cells exhibited elevated cisplatin resistance, apoptotic escape ability, enhanced epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) features, in addition to over-activation of the Wnt/β-catenin signaling pathway and increased Sox4 protein. In CMGT cases, CMGT tissues (CMGTT) expressed higher levels of Sox4 protein and mRNA compared to adjacent tissues (CAMGTT). We found that these changes were inhibited by silencing of Sox4 expression in CHMpCIS cells. Furthermore, activation of the Wnt/β-catenin signaling pathway increased Sox4 expression levels through a positive feedback loop. These results suggested that CHMpCIS cells circumvented the damage caused by cisplatin through altering the expression of the Sox4 gene and activating the Wnt/β-catenin pathway, thereby changing the cellular biological characteristics.

Increased SLC7A3 Expression inhibits Tumor Cell Proliferation and Predicts a Favorable Prognosis in Breast Cancer.

Arginine plays significant and contrasting roles in breast cancer growth and survival. However, the factors governing arginine balance remain poorly characterized. We aimed to identify the molecule that governs arginine metabolism in breast cancer and to elucidate its significance. We analyzed the correlation between the expression of solute carrier family 7 member 3 (SLC7A3), the major arginine transporter, and breast cancer survival in various databases, including GEPIA, UALCAN, Metascape, String, Oncomine, KM-plotter, CBioPortal and Prognosis. Additionally, we validated our findings through bioinformatic analyses and experimental investigations, including colony formation, wound healing, transwell, and mammosphere formation assays. Our analysis revealed a significant reduction in SLC7A3 expression in all breast cancer subtypes compared to adjacent breast tissues. Kaplan-Meier survival analyses demonstrated that high SLC7A3 expression was positively associated with decreased nodal metastasis (HR=0.70, 95% CI [0.55, 0.89]), ER positivity (HR=0.79, 95% CI [0.65, 0.95]), and HER2 negativity (HR=0.69, 95% CI [0.58, 0.82]), and increased recurrence-free survival. Moreover, low SLC7A3 expression predicted poor prognosis in breast cancer patients for overall survival. Additionally, the knockdown of SLC7A3 in MCF-7 and MDA-MB-231 cells resulted in increased cell proliferation and invasion in vitro. Our findings indicate a downregulation of SLC7A3 expression in breast cancer tissues compared to adjacent breast tissues. High SLC7A3 expression could serve as a prognostic indicator for favorable outcomes in breast cancer patients due to its inhibitory effects on breast cancer cell proliferation and invasion.

Interleukin-6 Induces Stem Cell Propagation through Liaison with the Sortilin-Progranulin Axis in Breast Cancer.

Unraveling the complex network between cancer cells and their tumor microenvironment is of clinical importance, as it might allow for the identification of new targets for cancer treatment. Cytokines and growth factors secreted by various cell types present in the tumor microenvironment have the potential to affect the challenging subpopulation of cancer stem cells showing treatment-resistant properties as well as aggressive features. By using various model systems, we investigated how the breast cancer stem cell-initiating growth factor progranulin influenced the secretion of cancer-associated proteins. In monolayer cultures, progranulin induced secretion of several inflammatory-related cytokines, such as interleukin (IL)-6 and -8, in a sortilin-dependent manner. Further, IL-6 increased the cancer stem fraction similarly to progranulin in the breast cancer cell lines MCF7 and MDA-MB-231 monitored by the surrogate mammosphere-forming assay. In a cohort of 63 patient-derived scaffold cultures cultured with breast cancer cells, we observed significant correlations between IL-6 and progranulin secretion, clearly validating the association between IL-6 and progranulin also in human-based microenvironments. In conclusion, the interplay between progranulin and IL-6 highlights a dual breast cancer stem cell-promoting function via sortilin, further supporting sortilin as a highly relevant therapeutic target for aggressive breast cancer.

BRD4/nuclear PD-L1/RelB circuit is involved in the stemness of breast cancer cells

BackgroundBreast cancer (BC) is the most common cancer diagnosed in women worldwide. BC stem cells (BCSCs) have been known to be involved in the carcinogenesis of the breast and contribute to therapeutic resistance. The programmed death-ligand 1 (PD-L1) expression of BC correlated with a poor prognosis. Immunotherapies that target PD-L1 have great potential and have been successful when applied to cancer treatment. However, whether PD-L1 regulates BCSC formation is unknown.MethodsBCSCs were enriched by serum-free suspension culture. The properties of BCSCs were examined by mammosphere formation assay, CD44+/Cd24−, aldehyde dehydrogenase (ALDH) assay, CSC marker analysis, and mammosphere growth assay. To elucidate the functions of bromodomain-containing protein 4 (BRD4), nuclear PD-L1, and RelB proteins in the stemness of BCSCs, mammosphere formation was examined using BRD4 inhibitor and degrader, PD-L1 degrader, and RelB inhibitor. The antitumor function of 3',4',7,8-tetrahydroxyflavone (THF), a specific BRD4 inhibitor, was studied through in vivo tumor model and mouse studies, and the protein levels of c-Myc, PD-L1, and RelB were examined in tumor model under THF treatment.ResultsBRD4 was upregulated in breast CSCs and regulates the stemness of BCs. The downregulation of BRD4 using BRD4 PROTAC, ARV-825, and BRD4 inhibitor, (+)-JQ1, inhibits mammosphere formation and reduces the levels of breast CSC markers (CD44+/CD24− and ALDH1), stem cell marker genes, and mammosphere growth. BRD4 inhibitor (JQ1) and degrader (ARV825) downregulate membrane and nuclear fractions of PD-L1 through the inhibition of PD-L1 transcript levels. The knockdown of PD-L1 inhibits mammosphere formation. Verteporfin, a PD-L1 degrader, inhibits the transcripts and protein levels of PD-L1 and downregulates the transcript and protein levels of RelB. Calcitriol, a RelB inhibitor, and the knockdown of RelB using si-RelB regulate mammosphere formation through interleukin-6 (IL-6) expression. THF is a natural product and a potent selective BRD4 inhibitor, inhibits mammosphere formation, and reduces the levels of CD44+/CD24− and mammosphere growth by downregulating c-Myc, PD-L1, and RelB. 3',4',7,8-THF shows tumoricidal activity and increased levels of CD3+CD4+ and CD3+CD8+ T-cells in the tumor and tumor-draining lymph nodes (TDLNs) in the murine tumor model using 4T1 and MC38 cells.ConclusionsThe results show the first evidence of the essential role of the BRD4/nuclear PD-L1/RelB axis in breast CSC formation. The nuclear PD-L1 regulates RelB, and the RelB/p65 complex induces IL6 and breast CSC formation. Targeting nuclear PD-L1 represents a potential and novel tool for immunotherapies of intractable BC.9Yv5TUoezTtpQRCQc9Q8K4Video

Bovine milk-derived cells express transcriptome markers of pluripotency and secrete bioactive factors with regenerative and antimicrobial activity

The bovine mammary stem/progenitor cell secretome stimulates regeneration in vitro and contains proteins associated with antimicrobial defense. This has led to the exploration of the secretome as a biologic treatment for mastitis, a costly inflammation of the udder commonly caused by bacteria. This study reports on a population of bovine mammary stem/progenitor cells isolated non-invasively from milk (MiDCs). MiDCs were characterized by immunophenotyping, mammosphere formation assays, and single cell RNA sequencing. They displayed epithelial morphology, exhibited markers of mammary stem/progenitor cells, and formed mammospheres, like mammary gland tissue-isolated stem/progenitor cells. Single cell RNA sequencing revealed two sub-populations of MiDCs: epithelial cells and macrophages. Functionally, the MiDC secretome increased fibroblast migration, promoted angiogenesis of endothelial cells, and inhibited the growth of mastitis-associated bacteria, including antibiotic-resistant strains, in vitro. These qualities of MiDCs render them a source of stem cells and stem cell products that may be used to treat diseases affecting the dairy industry, including mastitis.

The Efficacy of CB-103, a First-in-Class Transcriptional Notch Inhibitor, in Preclinical Models of Breast Cancer.

The efficacy of CB-103 was evaluated in preclinical models of both ER+ and TNBC. Furthermore, the therapeutic efficacy of combining CB-103 with fulvestrant in ER+ BC and paclitaxel in TNBC was determined. CB-103 was screened in combination with a panel of anti-neoplastic drugs. We evaluated the anti-tumor activity of CB-103 with fulvestrant in ESR1-mutant (Y537S), endocrine-resistant BC xenografts. In the same model, we examined anti-CSC activity in mammosphere formation assays for CB-103 alone or in combination with fulvestrant or palbociclib. We also evaluated the effect of CB-103 plus paclitaxel on primary tumors and CSC in a GSI-resistant TNBC model HCC1187. Comparisons between groups were performed with a two-sided unpaired Students' t-test. A one-way or two-way ANOVA followed by Tukey's post-analysis was performed to analyze the in vivo efficacy study results. CB-103 showed synergism with fulvestrant in ER+ cells and paclitaxel in TNBC cells. CB-103 combined with fulvestrant or paclitaxel potently inhibited mammosphere formation in both models. Combination of CB-103 and fulvestrant significantly reduced tumor volume in an ESR1-mutant, the endocrine-resistant BC model. In a GSI-resistant TNBC model, CB-103 plus paclitaxel significantly delayed tumor growth compared to paclitaxel alone. our data indicate that CB-103 is an attractive candidate for clinical investigation in endocrine-resistant, recurrent breast cancers with biomarker-confirmed Notch activity in combination with SERDs and/or CDKis and in TNBCs with biomarker-confirmed Notch activity in combination with taxane-containing chemotherapy regimens.

Unravelling the Therapeutic Potential of Antibiotics in Hypoxia in a Breast Cancer MCF-7 Cell Line Model.

Antibiotics inhibit breast cancer stem cells (CSCs) by suppressing mitochondrial biogenesis. However, the effectiveness of antibiotics in clinical settings is inconsistent. This inconsistency raises the question of whether the tumor microenvironment, particularly hypoxia, plays a role in the response to antibiotics. Therefore, the goal of this study was to evaluate the effectiveness of five commonly used antibiotics for inhibiting CSCs under hypoxia using an MCF-7 cell line model. We assessed the number of CSCs through the mammosphere formation assay and aldehyde dehydrogenase (ALDH)-bright cell count. Additionally, we examined the impact of antibiotics on the mitochondrial stress response and membrane potential. Furthermore, we analyzed the levels of proteins associated with therapeutic resistance. There was no significant difference in the number of CSCs between cells cultured under normoxic and hypoxic conditions. However, hypoxia did affect the rate of CSC inhibition by antibiotics. Specifically, azithromycin was unable to inhibit sphere formation in hypoxia. Erythromycin and doxycycline did not reduce the ratio of ALDH-bright cells, despite decreasing the number of mammospheres. Furthermore, treatment with chloramphenicol, doxycycline, and tetracycline led to the overexpression of the breast cancer resistance protein. Our findings suggest that hypoxia may weaken the inhibitory effects of antibiotics on the breast cancer model.

Umbelliprenin induces autophagy and apoptosis while inhibits cancer cell stemness in pancreatic cancer cells.

Umbelliprenin is a sesquiterpene coumarin isolated from Artemisia absinthium L. and shows antitumor effects in various cancers by inducing apoptosis. However, the antitumor effect of umbelliprenin in human pancreatic cancer has not been clarified. The antitumor effects were determined by MTT and AnnexinV/PI double staining assay in vitro and xenograft mice in vivo. Autophagy was determined via immunofluorescence analysis. Apoptotic or autophagic related proteins were measured by immunoblotting. The pancreatic cancer cell stemness were determined by mammosphere formation and ALDEFLUOR assay. It revealed that umbelliprenin inhibited pancreatic cancer cell proliferation in vitro and pancreatic cancer tumor growth in vivo. Moreover, umbelliprenin induced pancreatic cancer cell BxPC3 apoptosis and autophagy as evidenced by upregulated apoptosis and autophagy- related protein expression (p < 0.01). Blocking autophagy by 3-MA or Atg7 knockout enhanced umbelliprenin-induced apoptosis (p < 0.05). Umbelliprenin also reduced pancreatic cancer cell stemness by reducing Oct4, Nanog, and Sox2 mRNA levels (p < 0.01). Mechanistically, umbelliprenin greatly inhibited Akt/mTOR and Notch1 signal pathway. Umbelliprenin may be a novel therapeutic approach for pancreatic cancer treatment.

RON-augmented cholesterol biosynthesis in breast cancer metastatic progression and recurrence.

Recurrence remains a significant clinical barrier to improving breast cancer patient outcomes. The RON receptor is a predictor of metastatic progression and recurrence in breast cancers of all subtypes. RON directed therapies are in development, but preclinical data directly testing the impact of RON inhibition on metastatic progression/recurrence are lacking, and mechanisms to exert this function remain unclear. Herein, we modeled breast cancer recurrence using implantation of RON-overexpressing murine breast cancer cells. Recurrent growth was examined after tumor resection via in vivo imaging and ex vivo culture of circulating tumor cells from whole blood samples from tumor bearing mice. In vitro functional assessment of was performed using mammosphere formation assays. Transcriptomic pathway enrichment identified glycolysis and cholesterol biosynthesis pathways, transcription factor targets, and signaling pathways enriched in RON-overexpressing breast cancer cells. BMS777607, a RON inhibitor, abrogated CTC colony formation tumor cells and tumor recurrence. RON promoted mammosphere formation through upregulated cholesterol production that utilizes glycolysis-derived substrates. In mouse models with RON overexpression, statin-mediated inhibition of cholesterol biosynthesis impeded metastatic progression and recurrence but does not affect the primary tumor. RON upregulates glycolysis and cholesterol biosynthesis gene expression by two pathways: MAPK-dependent c-Myc expression and β-catenin -dependent SREBP2 expression.

Mammosphere formation assay optimization in the characterization of cancer stem cells of the primary breast tumor

Background/Purpose: Breast cancer is the most frequently diagnosed cancer and the leading cause of death from cancer among females worldwide. Breast cancer tumors that feature breast cancer stem cells (BCSCs) are known to cause drug resistance and metastasis. Culturing BCSCs from primary tumors as mammospheres is both difficult and costly. Therefore, the ability to form BCSC mammospheres in-vitro has become essential in assessing their characterization. Flow cytometric analysis of surface markers and measurement of aldehyde dehydrogenase (ALDH) activity are among other methods used to evaluate cancer cells' stem cell activity. &#x0D; Methods:The research material consisted of BCSCs isolated from the tumor tissues collected from two patients with invasive ductal carcinoma breast tumors. Subsequently, several stem cell surface markers, i.e., cluster of differentiation 44 (CD44), CD24, and CD133, were analyzed using flow cytometry during the third passaging of the cells. ALDH assay is performed with negative control verapamil incubated cells. Two mammosphere forming methods, i.e., low attachment and agar-coated wells together with medium seeded in three different cell concentrations, were compared. &#x0D; Results: CD44+, CD24- and CD133+antibody expressions showed that these cells could be tumor-initiating CSCs. ALDH assay results also indicated that these cells possessed stem cell features. In addition, the results of the mammosphere assay revealed that agar-coated wells at a concentration of 7000 cells/cm2 had more prominent floating features and viable spheres.&#x0D; Conclusion: The findings of this study supported the hypothesis that agar-coated culture dishes in mammosphere culture would increase the mammosphere formation efficiency (MFE) value and revealed the importance of the number of cells in elucidating the nature of BCSCs.

Indolocarbazole alkaloid Loonamycin A inhibits triple-negative breast cancer cell stemness and Notch signalling.

Enrichment for therapy-resistant cancer stem cells hampers the treatment of triple-negative breast cancer. Targeting these cells via suppression of Notch signalling can be a potential therapeutic strategy. This study aimed to uncover the mode of action of a new indolocarbazole alkaloid loonamycin A against this incurable disease. The anticancer effects were examined in triple-negative breast cancer cells using in vitro methods, including cell viability and proliferation assays, wound-healing assay, flow cytometry and mammosphere formation assay. RNA-seq technology was used to analyse the gene expression profiles in loonamycin A-treated cells. Real-time RT-PCR and western blot were to evaluate the inhibition of Notch signalling. Loonamycin A has stronger cytotoxicity than its structural analog rebeccamycin. Besides inhibiting cell proliferation and migration, loonamycin A reduced CD44high/CD24low/- sub-population, mammosphere formation, as well as the expression of stemness-associated genes. Co-administration of loonamycin A enhanced antitumour effects of paclitaxel by inducing apoptosis. RNA sequencing results showed that loonamycin A treatment caused the inhibition of Notch signalling, accompanied by the decreased expression of Notch1 and its targeted genes. These results reveal a novel bioactivity of indolocarbazole-type alkaloids and provide a promising Notch-inhibiting small molecular candidate for triple-negative breast cancer therapy.