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A biodegradable polymer packaging system for 'Benitaka' table grapes ( Vitis vinifera L.) was developed to inhibit the development of gray mold during refrigerated storage. The system consisted of packages and sachets containing Na 2 S 2 O 5 to release sulfur dioxide (SO 2 ), both produced with biodegradable films of starch, glycerol, and poly (adipate co-butylene terephthalate) (PBAT) produced via blown extrusion. The films were characterized in terms of thickness, density, mass loss in water, water vapor permeability, sorption isotherms, and mechanical properties. The table grapes were packed with biodegradable plastic bags containing SO 2 -releasing sachets inside. The experimental design was completely randomized, with four repetitions and five treatments: (a) control, without sachet containing Na 2 S 2 O 5 and SiO 2 ; (b) 2 g of Na 2 S 2 O 5 + 2 g of SiO 2 ; (c) 4 g of Na 2 S 2 O 5 + 1 g of SiO 2 ; (d) 4 g of Na 2 S 2 O 5 + 2 g of SiO 2 ; and (e) 4 g of Na 2 S 2 O 5 + 4 g of SiO 2 . The bunches were stored in a refrigerated chamber at 1 ± 1 °C and relative humidity above 90%. The treatments were evaluated 30 and 45 days after the beginning of refrigerated storage and 3 days at room temperature. The grapes were evaluated based on the incidence of gray mold, mass loss, stem browning, shattered berries, and berry bleaching. The data were subjected to the analysis of variance, and the means were compared using Tukey's test at 5%. The biodegradable films had good processability during the production via blown extrusion, with good physical properties to be used in the packaging of grapes and the production of SO 2 -releasing sachets. The biodegradable polymer packaging system (biodegradable plastic bags + SO 2 -releasing sachets) inhibited the development of gray mold on 'Benitaka' table grapes for 45 days at 1 °C, preserving their quality, with low mass loss, few shattered berries, and rachis freshness.

Given the environmental issues caused by the extensive use of conventional petroleum-based packaging, this work proposes functional films based on commercial κ-carrageenan (κc), poly(vinyl alcohol) (PVA), and gallic acid (GA) prepared by the "casting" method. Metallic ions in the κc composition stabilized the films, supporting processability and suitable mechanical properties. However, the incorporated GA amount (6.25 and 10 wt%) in the films created from an aqueous κc solution at 3.0 % wt/v (κc3) prevented crystalline domains in the resulting materials. The κc3/GA6.25 and κc3/GA10 films had less tensile strength (8.50 ± 0.61 and 10.28 ± 0.65 MPa) and high elongation at break (2.36 ± 0.16 and 1.19 ± 0.17 %) compared to the other samples, respectively. Low κc contents (κc2.5/GA6.25 and κc2.5/GA10) promoted stiff films and less permeability to water vapor (5.36 ± 0.51 and 3.76 ± 0.02 [×10 -12  g(Pa × m × s) -1 ], respectively. The κc/GA weight ratio also influenced the film wettability, indicating water contact angles (WCAs) between 55 and 74°. The surface wettability implies a low oil permeability and high water swelling capacity of up to 1600 %. The κc/GA also played an essential role in the film's antimicrobial action against Staphylococcus aureus and Escherichia coli. Thus, the κc3/GA10 film showed suitable physical, chemical, and biological properties, having the potential to be applied as food coatings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Polysaccharides and proteins are compatible macromolecules that can be used to obtain biopolymeric hydrogels through physical interactions. In this study, an environmentally friendly strategy is being proposed to produce gelatin-xanthan gum- cellulose hydrogels, without the addition of chemical synthetic crosslinkers. Xanthan gum was employed as an alternative crosslinking agent, and cellulose was used as a potential reinforcing agent in the polymeric matrix. Firstly, the biopolymers were mixed by the extrusion process, and glycerol was used as a plasticizer. Then, the polymeric mixture was molded by thermopressing to obtain hydrogels as laminated films. All hydrogels formulations resulted in films with smooth surfaces, without pores or cracks, resulting in amorphous polymeric matrices. The obtained hydrogels had a pH-dependent degree of swelling, the highest swelling values were obtained at pH 4 (5.3-7.9 g/g) after 24 h of immersion. Cellulose acted as a reinforcing agent for hydrogels, increasing thermal stability, tensile strength, and Young's modulus of films when employed at the higher level (7%). The strategy employed in this study to obtain nontoxic hydrogels without synthetic crosslinkers was effective, resulting in materials with promising properties to be used as pharmaceutical forms to deliver active compounds in cosmetic or pharmaceutical products.

Background: Breast cancer is a leading cause of death in premenopausal women. Progesterone drives expansion of luminal progenitor cells, leading to the development of poor-prognostic breast cancers. However, it is not known if antagonising progesterone can prevent breast cancers in humans. We suggest that targeting progesterone signalling could be a means of reducing features which are known to promote breast cancer formation., Methods: In healthy premenopausal women with and without a BRCA mutation we studied (i) estrogen and progesterone levels in saliva over an entire menstrual cycle (n = 20); (ii) cancer-free normal breast-tissue from a control population who had no family or personal history of breast cancer and equivalently from BRCA1/2 mutation carriers (n = 28); triple negative breast cancer (TNBC) biopsies and healthy breast tissue taken from sites surrounding the TNBC in the same individuals (n = 14); and biopsies of ER+ve/PR+ve stage T1-T2 cancers and healthy breast tissue taken from sites surrounding the cancer in the same individuals (n = 31); and (iii) DNA methylation and DNA mutations in normal breast tissue (before and after treatment) from clinical trials that assessed the potential preventative effects of vitamins and antiprogestins (mifepristone and ulipristal acetate; n = 44)., Results: Daily levels of progesterone were higher throughout the menstrual cycle of BRCA1/2 mutation carriers, raising the prospect of targeting progesterone signalling as a means of cancer risk reduction in this population. Furthermore, breast field cancerization DNA methylation signatures reflective of (i) the mitotic age of normal breast epithelium and (ii) the proportion of luminal progenitor cells were increased in breast cancers, indicating that luminal progenitor cells with elevated replicative age are more prone to malignant transformation. The progesterone receptor antagonist mifepristone reduced both the mitotic age and the proportion of luminal progenitor cells in normal breast tissue of all control women and in 64% of BRCA1/2 mutation carriers. These findings were validated by an alternate progesterone receptor antagonist, ulipristal acetate, which yielded similar results. Importantly, mifepristone reduced both the TP53 mutation frequency as well as the number of TP53 mutations in mitotic-age-responders., Conclusions: These data support the potential usage of antiprogestins for primary prevention of poor-prognostic breast cancers., Trial Registration: Clinical trial 1 Mifepristone treatment prior to insertion of a levonorgestrel releasing intrauterine system for improved bleeding control - a randomized controlled trial, clinicaltrialsregister.eu, 2009-009014-40 ; registered on 20 July 2009. Clinical trial 2 The effect of a progesterone receptor modulator on breast tissue in women with BRCA1 and 2 mutations, clinicaltrials.gov, NCT01898312 ; registered on 07 May 2013. Clinical trial 3 A pilot prevention study of the effects of the anti- progestin Ulipristal Acetate (UA) on surrogate markers of breast cancer risk, clinicaltrialsregister.eu, 2015-001587-19 ; registered on 15 July 2015., (© 2022. The Author(s).)

An ecofriendly and low-cost film composed by cassava starch, polyvinyl alcohol, and sericin blend (CS-PVA-SS) was synthesized, characterized, and applied as a novel support for Botryosphaeria ribis EC-01 lipase immobilization by enzyme-film-enzyme adsorption. Film revealed thickness between 230 and 309 μm and higher flexibility and malleability in comparison with film without SS. Based on p-nitrophenyl palmitate hydrolysis reaction, the activity retention of immobilized lipase was 987%. For optimal conditions, the yield in ethyl oleate was 95% for immobilized enzyme. Maximum yield was obtained at 49°C, molar ratio oleic acid:ethanol of 1:3, 1.25 g lipase film or 50 U (1.03 ± 0.03 mg protein) and 30 h. Even after seven cycles of use, immobilized lipase showed 52% reduction in ester yield. Biodegradable and biorenewable film is a promising material as a support to immobilize lipases and application in biocatalysis., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.

The objective of the present work was to develop biodegradable polymeric films (starch-PBAT) as support for the immobilization of lipases using sodium montmorillonite (MMT) as a reinforcing agent (2% w/w ) and itaconic acid (IA - 0.5-1.5% w/w ) as a compatibilizing agent. The films were produced through a two steps blow-extrusion. The addition of MMT increased the tensile strength and Tg of the films, while the presence of IA made the films more flexible, reducing their Tg. Lipases from Burkholderia cepacia LTEB11 were immobilized in the films by the adsorption method. The ester yield (% of ethyl oleate synthesis) has shown best results (96%, 6 h) for immobilized enzyme in the MMT film and six cycles of reuse were carried out until a reduction of 50% in the catalytic activity of the enzyme.

Estrogen-receptor-positive breast tumors are treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer stem cells (CSCs) with high aldehyde dehydrogenase activity (ALDH + cells) are enriched following AE treatment. Here, we show that the interleukin-1β (IL-1β) signaling pathway is activated in ALDH + cells, and data from single cells reveals that AE treatment selects for IL-1 receptor (IL1R1)-expressing ALDH + cells. Importantly, CSC activity is reduced by an IL1R1 inhibitor in AE-resistant models. Moreover, IL1R1 expression is increased in the tumors of patients treated with AE therapy and predicts treatment failure. Single-cell gene expression analysis revealed that at least two subpopulations exist within the ALDH + population, one proliferative and one quiescent. Following AE therapy the quiescent population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Targeting of ALDH + IL1R1 + cells merits testing as a strategy to combat AE resistance in patients with residual disease., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX-01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months' endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

Nanotechnology-based approaches hold substantial potential to avoid chemoresistance and minimize side effects. In this work, we have used biocompatible iron oxide magnetic nanoparticles (MNPs) called MF66 and functionalized with the antineoplastic drug doxorubicin (DOX) against MDA-MB-231 cells. Electrostatically functionalized MNPs showed effective uptake and DOX linked to MNPs was more efficiently retained inside the cells than free DOX, leading to cell inactivation by mitotic catastrophe, senescence and apoptosis. Both effects, uptake and cytotoxicity, were demonstrated by different assays and videomicroscopy techniques. Likewise, covalently functionalized MNPs using three different linkers-disulfide (DOX-S-S-Pyr, called MF66-S-S-DOX), imine (DOX-I-Mal, called MF66-I-DOX) or both (DOX-I-S-S-Pyr, called MF66-S-S-I-DOX)-were also analysed. The highest cell death was detected using a linker sensitive to both pH and reducing environment (DOX-I-S-S-Pyr). The greatest success of this study was to detect also their activity against breast cancer stem-like cells (CSC) from MDA-MB-231 and primary breast cancer cells derived from a patient with a similar genetic profile (triple-negative breast cancer). In summary, these nanoformulations are promising tools as therapeutic agent vehicles, due to their ability to produce efficient internalization, drug delivery, and cancer cell inactivation, even in cancer stem-like cells (CSCs) from patients.

Despite significant advancements in the diagnosis and treatment of osteosarcoma, the molecular mechanisms underpinning disease progression remain unclear. This work presents strong clinical and experimental evidence demonstrating that Notch signaling contributes to osteosarcoma progression. First, using a cohort of 12 patients, Notch genes were upregulated in tumors compared with adjacent normal tissue, and high tumor expression of Notch1 intercellular domain (NICD1) and the Notch target gene Hes1 correlated with poor chemotherapy response. Data mining of publicly available datasets confirmed that expression of Notch pathway genes is related to poor prognosis in osteosarcoma. On the basis of in vitro analysis, Notch signaling promoted osteosarcoma proliferation, enhanced chemoresistance, facilitated both migration and invasion, and upregulated stem cell-like characteristics. Xenograft models demonstrated that Notch signaling promotes primary tumor growth and pulmonary metastasis, and Notch inhibition is effective in reducing tumor size and preventing metastasis. Mechanistically, activated Notch signaling induces the expression of ephrinB1 and enhances the tumor-promoting ephrin reverse signaling. Overall, these findings provide functional evidence for Notch pathway genes as candidate biomarkers to predict prognosis in patients with osteosarcoma, and suggest a mechanistic rationale for the use of Notch inhibitors to treat osteosarcoma. IMPLICATIONS: The study provides preclinical evidence for Notch pathway as a molecular marker to predict osteosarcoma prognosis and as a therapeutic target against osteosarcoma. In addition, we identified a novel mechanism that ephrin reverse signaling acts as a key mediator of Notch pathway., (©2019 American Association for Cancer Research.)

Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1β stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1β-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis.

Despite the effectiveness of endocrine therapies to treat estrogen receptor-positive (ER+) breast tumours, two thirds of patients will eventually relapse due to de novo or acquired resistance to these agents. Cancer Stem-like Cells (CSCs), a rare cell population within the tumour, accumulate after anti-estrogen treatments and are likely to contribute to their failure. Here we studied the role of p21-activated kinase 4 (PAK4) as a promising target to overcome endocrine resistance and disease progression in ER + breast cancers. PAK4 predicts for resistance to tamoxifen and poor prognosis in 2 independent cohorts of ER + tumours. We observed that PAK4 strongly correlates with CSC activity in metastatic patient-derived samples irrespective of breast cancer subtype. However, PAK4-driven mammosphere-forming CSC activity increases alongside progression only in ER + metastatic samples. PAK4 activity increases in ER + models of acquired resistance to endocrine therapies. Targeting PAK4 with either CRT PAKi, a small molecule inhibitor of PAK4, or with specific siRNAs abrogates CSC activity/self-renewal in clinical samples and endocrine-resistant cells. Together, our findings establish that PAK4 regulates stemness during disease progression and that its inhibition reverses endocrine resistance in ER + breast cancers., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER + ) breast cancers. Despite its efficacy, ∼40% of women relapse with ET-resistant (ETR) disease. A global transcription analysis in ETR cells reveals a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid metabolism. This altered amino acid metabolism in ETR cells is supported by the activation of autophagy and the enhanced import of acidic amino acids (aspartate and glutamate) mediated by the SLC1A2 transporter. The clinical significance of these findings is validated by multiple orthogonal approaches in a large cohort of ET-treated patients, in patient-derived xenografts, and in in vivo experiments. Targeting these amino acid metabolic dependencies resensitizes ETR cells to therapy and impairs the aggressive features of ETR cells, offering predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in ER + breast cancers., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease., Methods: In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett's multiple comparison test., Results: We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4., Conclusion: This study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.

Purpose: One third of ER-positive breast cancer patients who initially respond to endocrine therapy become resistant to treatment. Such treatment failure is associated with poor prognosis and remains an area of unmet clinical need. Here, we identify a specific posttranslational modification that occurs during endocrine resistance and which results in tumor susceptibility to the apoptosis-inducer TRAIL. This potentially offers a novel stratified approach to targeting endocrine-resistant breast cancer. Experimental Design: Cell line and primary-derived xenograft models of endocrine resistance were investigated for susceptibility to TRAIL. Tumor viability, cancer stem cell (CSC) viability (tumorspheres), tumor growth kinetics, and metastatic burden were assessed. Western blots for the TRAIL-pathway inhibitor, c-FLIP, and upstream regulators were performed. Results were confirmed in primary culture of 26 endocrine-resistant and endocrine-naïve breast tumors. Results: Breast cancer cell lines with acquired resistance to tamoxifen (TAMR) or faslodex were more sensitive to TRAIL than their endocrine-sensitive controls. Moreover, TRAIL eliminated CSC-like activity in TAMR cells, resulting in prolonged remission of xenografts in vivo In primary culture, TRAIL significantly depleted CSCs in 85% endocrine-resistant, compared with 8% endocrine-naïve, tumors, whereas systemic administration of TRAIL in endocrine-resistant patient-derived xenografts reduced tumor growth, CSC-like activity, and metastases. Acquired TRAIL sensitivity correlated with a reduction in intracellular levels of c-FLIP, and an increase in Jnk-mediated phosphorylation of E3-ligase, ITCH, which degrades c-FLIP. Conclusions: These results identify a novel mechanism of acquired vulnerability to an extrinsic cell death stimulus, in endocrine-resistant breast cancers, which has both therapeutic and prognostic potential. Clin Cancer Res; 24(10); 2452-63. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Urogenital atrophy (UA) is a common treatment-limiting side effect of endocrine therapies. Topical estrogen is effective but systemic absorption may counter aromatase inhibitor efficacy. Numerous complementary approaches are marketed for use in UA without rigorous testing of their estrogenicity. We tested multiple essential oils in cancer cell growth and estrogen reporter assays in vitro and assessed clinical outcomes with the essential oil pessaries (EOPs) in breast cancer survivors with UA., Methods: Effects on cell growth were tested in hormone-dependent (MCF-7) and -independent (MDA-MB-231) cell lines using the sulforhodamine-B assay. An estrogen response element (ERE) luciferase reporter assay was used to assess estrogenicity directly. Antifungal activity against two common pathogenic yeasts was assessed using standard microdilution methods. EOPs were offered to breast cancer survivors with symptomatic UA and the service evaluated using serial questionnaires., Results: Two essential oils, Cymbopogon martinii and Pelargonium graveolens , demonstrated marked estrogenicity, stimulating ER+ cell growth and ERE-luciferase reporter activity to levels seen with premenopausal estradiol concentrations. Additional oils were screened for estrogenicity and Lavandula angustifolia and Chamaemelum nobile identified as non/minimally estrogenic. The antifungal activity of this combination of oils was confirmed. A second cohort of breast cancer survivors with UA received the second generation EOP with comparable improvement in symptom scores suggesting that estrogenicity may not be required for optimal therapy of UA., Conclusion: Certain essential oils demonstrate profound estrogenicity and caution should be exercised before their use in breast cancer survivors. Our minimally estrogenic pessary will be formally tested in clinical trials., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest.

Purpose of Review: This review will discuss how the steroid hormones, estrogen and progesterone, as well as treatments that target steroid receptors, can regulate cancer stem cell (CSC) activity. The CSC theory proposes a hierarchical organization in tumors where at its apex lies a subpopulation of cancer cells endowed with self-renewal and differentiation capacity., Recent Findings: In breast cancer (BC), CSCs have been suggested to play a key role in tumor maintenance, disease progression, and the formation of metastases. In preclinical models of BC, only a few CSCs are required sustain tumor re-growth, especially after conventional anti-endocrine treatments. CSCs include therapy-resistant clones that survive standard of care treatments like chemotherapy, irradiation, and hormonal therapy., Summary: The relevance of hormones for both normal mammary gland and BC development is well described, but it was only recently that the activities of hormones on CSCs have been investigated, opening new directions for future BC treatments and CSCs., Competing Interests: Compliance with Ethical StandardsDr. Denis G. Alferez, Dr. Bruno M. Simões, Dr. Sacha J. Howell, and Dr. Robert B. Clarke declare that they have no conflict of interest.This article does not contain any studies with human or animal subjects performed by any of the authors.

Breast cancer specific mortality results from tumour cell dissemination and metastatic colonisation. Identification of the cells and processes responsible for metastasis will enable better prevention and control of metastatic disease, thus reducing relapse and mortality. To better understand these processes, we prospectively collected 307 patient-derived breast cancer samples (n = 195 early breast cancers (EBC) and n = 112 metastatic samples (MBC)). We assessed colony-forming activity in vitro by growing isolated cells in both primary (formation) and secondary (self-renewal) mammosphere culture, and tumour initiating activity in vivo through subcutaneous transplantation of fragments or cells into mice. Metastatic samples formed primary mammosphere colonies significantly more frequently than early breast cancers and had significantly higher primary mammosphere colony formation efficiency (0.9 % vs. 0.6 %; p < 0.0001). Tumour initiation in vivo was significantly higher in metastatic than early breast cancer samples (63 % vs. 38 %, p = 0.04). Of 144 breast cancer samples implanted in vivo, we established 20 stable patient-derived xenograft (PDX) models at passage 2 or greater. Lung metastases were detected in mice from 14 PDX models. Mammosphere colony formation in vitro significantly correlated with the ability of a tumour to metastasise to the lungs in vivo (p = 0.05), but not with subcutaneous tumour initiation. In summary, the breast cancer stem cell activities of colony formation and tumour initiation are increased in metastatic compared to early samples, and predict metastasis in vivo. These results suggest that breast stem cell activity will predict for poor outcome tumours, and therapy targeting this activity will improve outcomes for patients with metastatic disease., Competing Interests: Compliance with Ethical StandardsFundingThis study was funded by Cancer Research UK and Breast Cancer Now. MK was funded by S cience and Technology Development Fund fellowship No. 6123.Research involving Human ParticipantsAll patients underwent fully informed consent in accordance with local research ethics committee guidelines. Ethical approval for metastatic samples was granted by the Central Office for Research Ethics Committee study number 05/Q1402/25. Early breast cancer samples were collected via the Manchester Cancer Research Centre Biobank which is licensed by the Human Tissue Authority (licence number: 30,004) and has been ethically approved as a research tissue bank by the South Manchester Research Ethics Committee (Ref: 07/H1003/161 + 5).Conflict of InterestThe authors declare that they have no conflict of interest.

Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo, and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro, and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro, including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance., Competing Interests: No potential conflicts of interest were disclosed.

Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully  apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

Breast cancer stem cells (BCSCs) play crucial roles in tumor initiation, metastasis and therapeutic resistance. A strict dependency between BCSCs and stromal cell components of tumor microenvironment exists. Thus, novel therapeutic strategies aimed to target the crosstalk between activated microenvironment and BCSCs have the potential to improve clinical outcome. Here, we investigated how leptin, as a mediator of tumor-stromal interactions, may affect BCSC activity using patient-derived samples (n = 16) and breast cancer cell lines, and determined the potential benefit of targeting leptin signaling in these model systems. Conditioned media (CM) from cancer-associated fibroblasts and breast adipocytes significantly increased mammosphere formation in breast cancer cells and depletion of leptin from CM completely abrogated this effect. Mammosphere cultures exhibited increased leptin receptor (OBR) expression and leptin exposure enhanced mammosphere formation. Microarray analyses revealed a similar expression profile of genes involved in stem cell biology among mammospheres treated with CM and leptin. Interestingly, leptin increased mammosphere formation in metastatic breast cancers and expression of OBR as well as HSP90, a target of leptin signaling, were directly correlated with mammosphere formation in metastatic samples (r = 0.68/p = 0.05; r = 0.71/p = 0.036, respectively). Kaplan-Meier survival curves indicated that OBR and HSP90 expression were associated with reduced overall survival in breast cancer patients (HR = 1.9/p = 0.022; HR = 2.2/p = 0.00017, respectively). Furthermore, blocking leptin signaling by using a full leptin receptor antagonist significantly reduced mammosphere formation in breast cancer cell lines and patient-derived samples. Our results suggest that leptin/leptin receptor signaling may represent a potential therapeutic target that can block the stromal-tumor interactions driving BCSC-mediated disease progression.

Over the past decade, there has been growing interest in the Notch signalling pathway within the breast cancer field. This interest stemmed initially from the observation that Notch signalling is aberrantly activated in breast cancer and its effects on various cellular processes including proliferation, apoptosis, and cancer stem cell activity. However more recently, elevated Notch signalling has been correlated with therapy resistance in oestrogen receptor-positive breast cancer. As a result, inhibiting Notch signalling with therapeutic agents is being explored as a promising treatment option for breast cancer patients.

Breast cancer stem cells (BCSCs) are potent tumor-initiating cells in breast cancer, the most common cancer among women. BCSCs have been suggested to play a key role in tumor initiation which can lead to disease progression and formation of metastases. Moreover, BCSCs are thought to be the unit of selection for therapy-resistant clones since they survive conventional treatments, such as chemotherapy, irradiation, and hormonal therapy. The importance of the role of hormones for both normal mammary gland and breast cancer development is well established, but it was not until recently that the effects of hormones on BCSCs have been investigated. This review will discuss recent studies highlighting how ovarian steroid hormones estrogen and progesterone, as well as therapies against them, can regulate BCSC activity., (© 2015 Society for Endocrinology.)

Breast cancers (BCs) typically express estrogen receptors (ERs) but frequently exhibit de novo or acquired resistance to hormonal therapies. Here, we show that short-term treatment with the anti-estrogens tamoxifen or fulvestrant decrease cell proliferation but increase BC stem cell (BCSC) activity through JAG1-NOTCH4 receptor activation both in patient-derived samples and xenograft (PDX) tumors. In support of this mechanism, we demonstrate that high ALDH1 predicts resistance in women treated with tamoxifen and that a NOTCH4/HES/HEY gene signature predicts for a poor response/prognosis in 2 ER+ patient cohorts. Targeting of NOTCH4 reverses the increase in Notch and BCSC activity induced by anti-estrogens. Importantly, in PDX tumors with acquired tamoxifen resistance, NOTCH4 inhibition reduced BCSC activity. Thus, we establish that BCSC and NOTCH4 activities predict both de novo and acquired tamoxifen resistance and that combining endocrine therapy with targeting JAG1-NOTCH4 overcomes resistance in human breast cancers., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo‐ and radiotherapy. Here, we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen‐resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely, ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2‐expressing cells, and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure, and also in the primary tumours of these patients, compared to those of responders. Together, these results suggest that development of tamoxifen resistance is driven by Sox2‐dependent activation of Wnt signalling in cancer stem/progenitor cells.

Telomerase is crucial for the maintenance of stem/progenitor cells in adult tissues and is detected in most malignant cancers, including osteosarcoma. However, the relationship between telomerase expression and cancer stem cells remains unknown. We observed that sphere-derived osteosarcoma cells had higher telomerase activity, indicating that telomerase activity might be enriched in osteosarcoma stem cells. We sorted subpopulations with high or low telomerase activity (TEL) using hTERT transcriptional promoter-induced green fluorescent protein (GFP). The TELpos cells showed an increased sphere and tumor propagating capacity compared to TELneg cells, and enhanced stem cell-like properties such as invasiveness, metastatic activity and resistance to chemotherapeutic agents both in vitro and in vivo. Furthermore, the telomerase inhibitor MST312 prevented tumorigenic potential both in vitro and in vivo, preferentially targeting the TELpos cells. These data support telomerase inhibition as a potential targeted therapy for osteosarcoma stem-like cells.

Introduction: Although oestrogen is essential for the development of the normal breast, adult mammary stem cells are known to be oestrogen receptor alpha (ER) negative and rely on paracrine signals in the mammary epithelium for mediation of developmental cues. However, little is known about how systemic oestrogen regulates breast cancer stem cell (CSC) activity., Methods: Here, we tested the effects of oestrogen on CSC activity in vitro and in vivo and investigated which paracrine signalling pathways locally mediate oestrogen effects., Results: CSC-enriched populations (ESA+CD44+CD24low) sorted from ER positive patient derived and established cell lines have low or absent ER expression. However, oestrogen stimulated CSC activity demonstrated by increased mammosphere and holoclone formation in vitro and tumour formation in vivo. This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA. These data suggest that the oestrogen response is mediated through paracrine signalling from non-CSCs to CSCs. We have, therefore, investigated both epidermal growth factor (EGF) and Notch receptor signals downstream of oestrogen. We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency., Conclusions: These data establish that EGF and Notch receptor signalling pathways operate downstream of oestrogen in the regulation of ER negative CSCs.

Purpose: Breast cancer stem-like cells (CSC) are an important therapeutic target as they are predicted to be responsible for tumor initiation, maintenance, and metastases. Interleukin (IL)-8 is upregulated in breast cancer and is associated with poor prognosis. Breast cancer cell line studies indicate that IL-8 via its cognate receptors, CXCR1 and CXCR2, is important in regulating breast CSC activity. We investigated the role of IL-8 in the regulation of CSC activity using patient-derived breast cancers and determined the potential benefit of combining CXCR1/2 inhibition with HER2-targeted therapy., Experimental Design: CSC activity of metastatic and invasive human breast cancers (n = 19) was assessed ex vivo using the mammosphere colony-forming assay., Results: Metastatic fluid IL-8 level correlated directly with mammosphere formation (r = 0.652; P < 0.05; n = 10). Recombinant IL-8 directly increased mammosphere formation/self-renewal in metastatic and invasive breast cancers (n = 17). IL-8 induced activation of EGFR/HER2 and downstream signaling pathways and effects were abrogated by inhibition of SRC, EGFR/HER2, phosphoinositide 3-kinase (PI3K), or MEK. Furthermore, lapatinib, which targets EGFR/HER2, inhibited the mammosphere-promoting effect of IL-8 in both HER2-positive and negative patient-derived cancers. CXCR1/2 inhibition also blocked the effect of IL-8 on mammosphere formation and added to the efficacy of lapatinib in HER2-positive cancers., Conclusions: These studies establish a role for IL-8 in the regulation of patient-derived breast CSC activity and show that IL-8/CXCR1/2 signaling is partly mediated via a novel SRC and EGFR/HER2-dependent pathway. Combining CXCR1/2 inhibitors with current HER2-targeted therapies has potential as an effective therapeutic strategy to reduce CSC activity in breast cancer and improve the survival of HER2-positive patients.

Breast cancer stem-like cells (CSCs) are an important therapeutic target as they are purported to be responsible for tumor initiation, maintenance, metastases, and disease recurrence. Interleukin-8 (IL-8) is upregulated in breast cancer compared with normal breast tissue and is associated with poor prognosis. IL-8 is reported to promote breast cancer progression by increasing cell invasion, angiogenesis, and metastases and is upregulated in HER2-positive cancers. Recently, we and others have established that IL-8 via its cognate receptors, CXCR1 and CXCR2, is also involved in regulating breast CSC activity. Our work demonstrates that in metastatic breast CSCs, CXCR1/2 signals via transactivation of HER2. Given the importance of HER2 in breast cancer and in regulating CSC activity, a pathway driving the activation of these receptors would have important biological and clinical consequences, especially in tumors that express high levels of IL-8 and other CXCR1/2-activating ligands. Here, we review the IL-8 signaling pathway and the role of HER2 in maintaining an IL-8 inflammatory loop and discuss the potential of combining CXCR1/2 inhibitors with other treatments such as HER2-targeted therapy as a novel approach to eliminate CSCs and improve patient survival.

Since the discovery that neural tissue contains a population of stem cells that form neurospheres in vitro, sphere-forming assays have been adapted for use with a number of different tissue types for the quantification of stem cell activity and self-renewal. One tissue type widely used for stem cell investigations is mammary tissue, and the mammosphere assay has been used in both normal tissue and cancer. Although it is a relatively simple assay to learn, it can be difficult to master. There are methodological and analytical aspects to the assay which require careful consideration when interpreting the results. We describe here a detailed mammosphere assay protocol for the assessment of stem cell activity and self-renewal, and discuss how data generated by the assay can be analysed and interpreted.

There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known, but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG, OCT4, and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor (ER) expression, and increases the number of stem cells and their capacity for invasion, properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of ER-positive tumors.

The identification and characterization of normal and breast cancer stem cells have provided a new vision of breast tumorigenesis. Cancer stem cells may be responsible for breast tumor initiation, progression and development of resistance to therapy. Most breast cancers express the estrogen receptor, and several studies have linked long-term estrogen exposure to enhanced breast cancer risk; however, estrogen receptor-positive tumors usually present a better prognosis than estrogen receptor-negative ones. The finding that estrogen reduces the pool of human breast stem cells may explain the more differentiated phenotype observed in estrogen receptor-positive tumors. In this article, our current understanding of the complex role of estrogen in human breast stem cells is discussed in the context of breast malignancy.