Evaluation of human adipose-derived stromal cell behaviour following exposure to Tamoxifen.

BACKGROUND AND AIM: Adipose-derived stromal cells (ASCs) are a promising cell source for novel tissue engineering approaches to breast reconstruction following cancer resection. However there is limited knowledge on the effect of adjuvant therapies such as hormonal therapy on ASCs, which may affect their efficacy in regenerative strategies. The present study aims to investigate the effects of Tamoxifen and its metabolites Afimoxifene (4-Hydroxy-Tamoxifen) and Endoxifen (N-desmethyl-4-hydroxytamoxifen) on patient-derived ASC viability, apoptosis, adipogenic differentiation and angiogenic potential. METHODS: ASCs were isolated from fat harvested from female breast cancer patients undergoing breast reconstruction surgery or cosmetic procedures. Oestrogen receptor (ER α, ß) expression was analysed using immunofluorescence. ASCs were then treated with various concentrations of Afimoxifene, Endoxifen and Tamoxifen (combination), and the impact on ASC viability and apoptosis determined. ASCs were cultured in adipogenic-differentiation media with or without tamoxifen and derivatives, and adipogenesis was measured using quantitative Real-time Polymerase chain reaction (qRT-PCR) and histological staining (Oil Red O). The effect on secreted VEGF levels was also quantified in ASC conditioned media RESULTS: ASCs were successfully isolated and characterised from human abdominal lipoaspirates or fat tissues (n = 8). ASCs subjected to varying doses of Tamoxifen and metabolites (up to 1000 nM) showed no decline in cell viability or increase in apoptosis, at physiological doses (upto 100 nM). Functional decline in adipogenic differentiation or gene expression was observed at supraphysiological concentrations of Tamoxifen (1000 nM). VEGF165 protein secretion in ASC-cell conditioned media was not significantly impacted irrespective of dosage. CONCLUSION: At physiologically relevant doses, Tamoxifen treatment did not result in any deleterious effect on ASC survival and functionality and is unlikely to negatively impact ASC based breast reconstruction strategies for breast cancer patients receiving this adjuvant hormonal therapy.

Temporal and spatial changes in bone mineral content and mechanical properties during breast-cancer bone metastases.

Cancer cells favour migration and metastasis to bone tissue for 70-80 % of advanced breast cancer patients and it has been proposed that bone tissue provides attractive physical properties that facilitate tumour invasion, resulting in osteolytic and or osteoblastic metastasis. However, it is not yet known how specific bone tissue composition is associated with tumour invasion. In particular, how compositional and nano-mechanical properties of bone tissue evolve during metastasis, and where in the bone they arise, may affect the overall aggressiveness of tumour invasion, but this is not well understood. The objective of this study is to develop an advanced understanding of temporal and spatial changes in nano-mechanical properties and composition of bone tissue during metastasis. Primary mammary tumours were induced by inoculation of immune-competent BALB/c mice with 4T1 breast cancer cells in the mammary fat pad local to the right femur. Microcomputed tomography and nanoindentation were conducted to quantify cortical and trabecular bone matrix mineralisation and nano-mechanical properties. Analysis was performed in proximal and distal femur regions (spatial analysis) of tumour-adjacent (ipsilateral) and contralateral femurs after 3 weeks and 6 weeks of tumour and metastasis development (temporal analysis). By 3 weeks post-inoculation there was no significant difference in bone volume fraction or nano-mechanical properties of bone tissue between the metastatic femora and healthy controls. However, early osteolysis was indicated by trabecular thinning in the distal and proximal trabecular compartment of tumour-bearing femora. Moreover, cortical thickness was significantly increased in the distal region, and the mean mineral density was significantly higher in cortical and trabecular bone tissue in both proximal and distal regions, of ipsilateral (tumour-bearing) femurs compared to healthy controls. By 6 weeks post-inoculation, overt osteolytic lesions were identified in all ipsilateral metastatic femora, but also in two of four contralateral femora of tumour-bearing mice. Bone volume fraction, cortical area, cortical and trabecular thickness were all significantly decreased in metastatic femora (both ipsilateral and contralateral). Trabecular bone tissue stiffness in the proximal femur decreased in the ipsilateral femurs compared to contralateral and control sites. Temporal and spatial analysis of bone nano-mechanical properties and mineralisation during breast cancer invasion reveals changes in bone tissue composition prior to and following overt metastatic osteolysis, local and distant from the primary tumour site. These changes may alter the mechanical environment of both the bone and tumour cells, and thereby play a role in perpetuating the cancer vicious cycle during breast cancer metastasis to bone tissue.

A comparative analysis of extracellular vesicles (EVs) from human and feline plasma.

Extracellular vesicles (EVs) are nanoparticles found in all biological fluids, capable of transporting biological material around the body. Extensive research into the physiological role of EVs has led to the development of the Minimal Information for Studies of Extracellular Vesicles (MISEV) framework in 2018. This framework guides the standardisation of protocols in the EV field. To date, the focus has been on EVs of human origin. As comparative medicine progresses, there has been a drive to study similarities between diseases in humans and animals. To successfully research EVs in felines, we must validate the application of the MISEV guidelines in this group. EVs were isolated from the plasma of healthy humans and felines. EV characterisation was carried out according to the MISEV guidelines. Human and feline plasma showed a similar concentration of EVs, comparable expression of known EV markers and analogous particle to protein ratios. Mass spectrometry analyses showed that the proteomic signature of EVs from humans and felines were similar. Asymmetrical flow field flow fractionation, showed two distinct subpopulations of EVs isolated from human plasma, whereas only one subpopulation was isolated from feline plasma. Metabolomic profiling showed similar profiles for humans and felines. In conclusion, isolation, and characterisation of EVs from humans and felines show that MISEV2018 guidelines may also be applied to felines. Potential comparative medicine studies of EVs may provide a model for studying naturally occurring diseases in both humans and felines.

Implementing subtype-specific pre-clinical models of breast cancer to study pre-treatment aspirin effects.

BACKGORUND: Prior data suggest pre-diagnostic aspirin use impacts breast tumour biology and patient outcome. Here, we employed faithful surgical resection models of HER2+ and triple-negative breast cancer (TNBC), to study outcome and response mechanisms across breast cancer subtypes. METHOD: NOD/SCID mice were implanted with HER2+ MDA-MB-231/LN/2-4/H2N, trastuzumab-resistant HER2+ HCC1954 or a TNBC patient-derived xenograft (PDX). A daily low-dose aspirin regimen commenced until primary tumours reached ~250 mm3 and subsequently resected. MDA-MB-231/LN/2-4/H2N mice were monitored for metastasis utilising imaging. To interrogate the survival benefit of pre-treatment aspirin, 3 weeks post-resection, HCC1954/TNBC animals received standard-of-care (SOC) chemotherapy for 6 weeks. Primary tumour response to aspirin was interrogated using immunohistochemistry. RESULTS: Aspirin delayed time to metastasis in MDA-MB-231/LN/2-4/H2N xenografts and decreased growth of HER2+ /TNBC primary tumours. Lymphangiogenic factors and lymph vessels number were decreased in HER2+ tumours. However, no survival benefit was seen in aspirin pre-treated animals (HCC1954/TNBC) that further received adjuvant SOC, compared with animals treated with SOC alone. In an effort to study mechanisms responsible for the observed reduction in lymphangiogenesis in HER2+ BC we utilised an in vitro co-culture system of HCC1954 tumour cells and mesenchymal stromal cells (MSC). Aspirin abrogated the secretion of VEGF-C in MSCs and also decreased the lymph/angiogenic potential of the MSCs and HCC1954 by tubule formation assay. Furthermore, aspirin decreased the secretion of uPA in HCC1954 cells potentially diminishing its metastatic capability. CONCLUSION: Our data employing clinically relevant models demonstrate that aspirin alters breast tumour biology. However, aspirin may not represent a robust chemo-preventative agent in the HER2+ or TNBC setting.

Hydrogels: 3D Drug Delivery Systems for Nanoparticles and Extracellular Vesicles.

Synthetic and naturally occurring nano-sized particles present versatile vehicles for the delivery of therapy in a range of clinical settings. Their small size and modifiable physicochemical properties support refinement of targeting capabilities, immune response, and therapeutic cargo, but rapid clearance from the body and limited efficacy remain a major challenge. This highlights the need for a local sustained delivery system for nanoparticles (NPs) and extracellular vesicles (EVs) at the target site that will ensure prolonged exposure, maximum efficacy and dose, and minimal toxicity. Biocompatible hydrogels loaded with therapeutic NPs/EVs hold immense promise as cell-free sustained and targeted delivery systems in a range of disease settings. These bioscaffolds ensure retention of the nano-sized particles at the target site and can also act as controlled release systems for therapeutics over a prolonged period of time. The encapsulation of stimuli sensitive components into hydrogels supports the release of the content on-demand. In this review, we highlight the prospect of the sustained and prolonged delivery of these nano-sized therapeutic entities from hydrogels for broad applications spanning tissue regeneration and cancer treatment. Further understanding of the parameters controlling the release rate of these particles and efficient transfer of cargo to target cells will be fundamental to success.

MicroRNAs in Molecular Classification and Pathogenesis of Breast Tumors.

The current clinical practice of breast tumor classification relies on the routine immunohistochemistry-based expression analysis of hormone receptors, which is inadequate in addressing breast tumor heterogeneity and drug resistance. MicroRNA expression profiling in tumor tissue and in the circulation is an efficient alternative to intrinsic molecular subtyping that enables precise molecular classification of breast tumor variants, the prediction of tumor progression, risk stratification and also identifies critical regulators of the tumor microenvironment. This review integrates data from protein, gene and miRNA expression studies to elaborate on a unique miRNA-based 10-subtype taxonomy, which we propose as the current gold standard to allow appropriate classification and separation of breast cancer into a targetable strategy for therapy.

Emerging Evidence of the Functional Impact of the miR379/miR656 Cluster (C14MC) in Breast Cancer.

Many microRNAs exist in clusters that share comparable sequence homology and may target genes in a common pathway. The miR-379/miR-656 (C14MC) cluster is imprinted in the DLK1-Dio3 region of 14q32.3 and contains 42 miRNAs. It plays a functional role in numerous biological pathways including vascular remodeling and early development. With many C14MC miRNAs highlighted as potential tumor suppressors in a variety of cancers, the role of this cluster in breast cancer (BC) has garnered increased attention in recent years. This review focuses on C14MC in BC, providing an overview of the constituent miRNAs and addressing each in terms of functional impact, potential target genes/pathways, and, where relevant, biomarker capacity. Studies have revealed the regulation of key factors in disease progression and metastasis including tyrosine kinase pathways and factors critical to epithelial-mesenchymal transition (EMT). This has potentially important clinical implications, with EMT playing a critical role in BC metastasis and tyrosine kinase inhibitors (TKIs) in widespread use for the treatment of BC. While the majority of studies have reported tumor-suppressing roles for these miRNAs, some have highlighted their potential as oncomiRs. Understanding the collective contribution of miRNAs within C14MC to BC may support improved understanding of disease etiology and present novel approaches to targeted therapy.

Characterization of nanosensitive multifractality in submicron scale tissue morphology and its alteration in tumor progression (Erratum).

An error in the 2nd author's name is corrected.

Characterization of nanosensitive multifractality in submicron scale tissue morphology and its alteration in tumor progression.

SIGNIFICANCE: Assessment of disease using optical coherence tomography is an actively investigated problem, owing to many unresolved challenges in early disease detection, diagnosis, and treatment response monitoring. The early manifestation of disease or precancer is typically associated with subtle alterations in the tissue dielectric and ultrastructural morphology. In addition, biological tissue is known to have ultrastructural multifractality. AIM: Detection and characterization of nanosensitive structural morphology and multifractality in the tissue submicron structure. Quantification of nanosensitive multifractality and its alteration in progression of tumor. APPROACH: We have developed a label free nanosensitive multifractal detrended fluctuation analysis(nsMFDFA) technique in combination with multifractal analysis and nanosensitive optical coherence tomography (nsOCT). The proposed method deployed for extraction and quantification of nanosensitive multifractal parameters in mammary fat pad (MFP). RESULTS: Initially, the nsOCT approach is numerically validated on synthetic submicron axial structures. The nsOCT technique was applied to pathologically characterized MFP of murine breast tissue to extract depth-resolved nanosensitive submicron structures. Subsequently, two-dimensional MFDFA were deployed on submicron structural en face images to extract nanosensitive tissue multifractality. We found that nanosensitive multifractality increases in transition from healthy to tumor. CONCLUSIONS: This method for extraction of nanosensitive tissue multifractality promises to provide a noninvasive diagnostic tool for early disease detection and monitoring treatment response. The novel ability to delineate the dominant submicron scale nanosensitive multifractal properties may also prove useful for characterizing a wide variety of complex scattering media of non-biological origin.

Targeting stromal cell Syndecan-2 reduces breast tumour growth, metastasis and limits immune evasion.

Tumour stromal cells support tumourigenesis. We report that Syndecan-2 (SDC2) is expressed on a nonepithelial, nonhaematopoietic, nonendothelial stromal cell population within breast cancer tissue. In vitro, syndecan-2 modulated TGFß signalling (SMAD7, PAI-1), migration and immunosuppression of patient-derived tumour-associated stromal cells (TASCs). In an orthotopic immunocompromised breast cancer model, overexpression of syndecan-2 in TASCs significantly enhanced TGFß signalling (SMAD7, PAI-1), tumour growth and metastasis, whereas reducing levels of SDC2 in TASCs attenuated TGFß signalling (SMAD7, PAI-1, CXCR4), tumour growth and metastasis. To explore the potential for therapeutic application, a syndecan-2-peptide was generated that inhibited the migratory and immunosuppressive properties of TASCs in association with reduced expression of TGFß-regulated immunosuppressive genes, such as CXCR4 and PD-L1. Moreover, using an orthotopic syngeneic breast cancer model, overexpression of syndecan-2-peptide in TASCs reduced tumour growth and immunosuppression within the TME. These data provide evidence that targeting stromal syndecan-2 within the TME inhibits tumour growth and metastasis due to decreased TGFß signalling and increased immune control.

Effect of Breast Cancer and Adjuvant Therapy on Adipose-Derived Stromal Cells: Implications for the Role of ADSCs in Regenerative Strategies for Breast Reconstruction.

Tissue engineering using Adipose Derived Stromal Cells (ADSCs) has emerged as a novel regenerative medicine approach to replace and reconstruct soft tissue damaged or lost as a result of disease process or therapeutic surgical resection. ADSCs are an attractive cell source for soft tissue regeneration due to the fact that they are easily accessible, multipotent, non-immunogenic and pro-angiogenic. ADSC based regenerative strategies have been successfully translated to the clinical setting for the treatment of Crohn's fistulae, musculoskeletal pathologies, wound healing, and cosmetic breast augmentation (fat grafting). ADSCs are particularly attractive as a source for adipose tissue engineering as they exhibit preferential differentiation to adipocytes and support maintenance of mature adipose graft volume. The potential for reconstruction with an autologous tissue sources and a natural appearance and texture is particularly appealing in the setting of breast cancer; up to 40% of patients require mastectomy for locoregional control and current approaches to post-mastectomy breast reconstruction (PMBR) are limited by the potential for complications at the donor and reconstruction sites. Despite their potential, the use of ADSCs in breast cancer patients is controversial due to concerns regarding oncological safety. These concerns relate to the regeneration of tissue at a site where a malignancy has been treated and the impact this may have on stimulating local disease recurrence or dissemination. Pre-clinical data suggest that ADSCs exhibit pro-oncogenic characteristics and are involved in stimulating progression, and growth of tumour cells. However, there have been conflicting reports on the oncologic outcome, in terms of locoregional recurrence, for breast cancer patients in whom ADSC enhanced fat grafting was utilised as an alternative to reconstruction for small volume defects. A further consideration which may impact the successful translation of ADSC based regenerative strategies for post cancer reconstruction is the potential effects of cancer therapy. This review aims to address the effect of malignant cells, adjuvant therapies and patient-specific factors that may influence the success of regenerative strategies using ADSCs for post cancer tissue regeneration.

Prospective Assessment of Systemic MicroRNAs as Markers of Response to Neoadjuvant Chemotherapy in Breast Cancer.

Neoadjuvant chemotherapy (NACT) is used in locally advanced breast cancer to reduce tumour burden prior to surgical resection. However, only a subset of NACT treated patients will respond to treatment or achieve a pathologic complete response (pCR). This multicenter, prospective study (CTRIAL-IE (ICORG) 10-11 study) evaluated circulating microRNA as novel non-invasive prognostic biomarkers of NACT response in breast cancer. Selected circulating microRNAs (Let-7a, miR-21, miR-145, miR-155, miR-195) were quantified from patients undergoing standard of care NACT treatment (n = 114) from whole blood at collected at diagnosis, and the association with NACT response and clinicopathological features evaluated. NACT responders had significantly lower levels of miR-21 (p = 0.036) and miR-195 (p = 0.017), compared to non-responders. Evaluating all breast cancer cases miR-21 was found to be an independent predictor of response (OR 0.538, 95% CI 0.308-0.943, p < 0.05). Luminal cancer NACT responders were found to have significantly decreased levels of miR-145 (p = 0.033) and miR-21 (p = 0.048), compared to non-responders. This study demonstrates the prognostic ability of miR-21, miR-195 and miR-145 as circulating biomarkers stratifying breast cancer patients by NACT response, identifying patients that will derive the maximum benefit from chemotherapy.

Nanoparticle-Based Delivery of Tumor Suppressor microRNA for Cancer Therapy.

Improved understanding of microRNA expression and function in cancer has revealed a range of microRNAs that negatively regulate many oncogenic pathways, thus representing potent tumor suppressors. Therapeutic targeting of the expression of these microRNAs to the site of tumors and metastases provides a promising avenue for cancer therapy. To overcome challenges associated with microRNA degradation, transient expression and poor targeting, novel nanoparticles are being developed and employed to shield microRNAs for tumor-targeted delivery. This review focuses on studies describing a variety of both natural and synthetic nanoparticle delivery vehicles that have been engineered for tumor-targeted delivery of tumor suppressor microRNAs in vivo.

Extracellular Vesicles for Cancer Therapy: Impact of Host Immune Response.

In recent times, extracellular vesicles (EVs) have come under the spotlight as potential therapeutics for cancer, due to the relative ease of manipulation of contents and potential for tumor targeting. The use of EVs as delivery vehicles may bypass some of the negative effects associated with cell-based carriers, and there has been a major focus on defining EV subtypes, establishing transparent nomenclature, and isolation and characterization techniques. EVs are believed to be a fingerprint of the secreting cell and so researchers harness the positive aspects of a particular cell of origin, and can then further modify EV contents to improve therapeutic efficacy. In this review, we highlight studies employing EVs as cancer therapeutics that have reported on immune response. As we rapidly advance towards potential application in the clinical setting, the question of immune response to EV administration in the cancer setting has become critically important.

Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer.

Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer.

Relative and Absolute Expression Analysis of MicroRNAs Associated with Luminal A Breast Cancer- A Comparison.

MicroRNAs, as small non-coding regulatory RNAs, play crucial roles in various aspects of breast cancer biology. They have prognostic and diagnostic value, which makes them very interesting molecules to investigate. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is the gold standard method to analyse miRNA expression in breast cancer patients. This study investigated two RT-qPCR methods (absolute and relative) to determine the expression of ten miRNAs in whole blood samples obtained from luminal A breast cancer patients compared to healthy controls. Whole blood samples were collected from 38 luminal A breast cancer patients and 20 healthy controls in Paxgene blood RNA tubes. Total RNA was extracted and analysed by relative and absolute RT-qPCR. For relative RT-qPCR, miR-16 was used as an endogenous control. For absolute RT-qPCR, standard curves were generated using synthetic miRNA oligonucleotides to determine the absolute copy number of each miRNA. Of the ten miRNAs that were analysed, the absolute RT-qPCR method identified six miRNAs (miR-16, miR-145, miR-155, miR-451a, miR-21 and miR-486) that were upregulated and one miRNA (miR-195) that was downregulated. ROC curve and AUC analysis of the data found that the combination of three miRNAs (miR-145, miR-195 and miR-486) had the best diagnostic value for luminal A breast cancer with an AUC of 0.875, with 76% sensitivity and 81% specificity. On the other hand, the relative RT-qPCR method identified two miRNAs (miR-155 and miR-486) that were upregulated and miR-195, which was downregulated. Using this approach, the combination of three miRNAs (miR-155, miR-195 and miR-486) was showed to have an AUC of 0.657 with 65% sensitivity and 69% specificity. We conclude that miR-16 is not a suitable normalizer for the relative expression profiling of miRNAs in luminal A breast cancer patients. Compared to relative quantification, absolute quantification assay is a better method to determine the expression level of circulating miRNAs in Luminal A breast cancer.

Role of Extracellular Vesicles (EVs) in Cell Stress Response and Resistance to Cancer Therapy.

Extracellular vesicles (EVs) are nanosized particles released by all cells that have been heralded as novel regulators of cell-to-cell communication. It is becoming increasingly clear that in response to a variety of stress conditions, cells employ EV-mediated intercellular communication to transmit a pro-survival message in the tumor microenvironment and beyond, supporting evasion of cell death and transmitting resistance to therapy. Understanding changes in EV cargo and secretion pattern during cell stress may uncover novel, targetable mechanisms underlying disease progression, metastasis and resistance to therapy. Further, the profile of EVs released into the circulation may provide a circulating biomarker predictive of response to therapy and indicative of microenvironmental conditions linked to disease progression, such as hypoxia. Continued progress in this exciting and rapidly expanding field of research will be dependent upon widespread adoption of transparent reporting standards and implementation of guidelines to establish a consensus on methods of EV isolation, characterisation and nomenclature employed.

Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy.

Triple-negative breast cancer (TNBC) lacks targeted therapies and has a worse prognosis than other breast cancer subtypes, underscoring an urgent need for new therapeutic targets and strategies. IRE1 is an endoplasmic reticulum (ER) stress sensor, whose activation is predominantly linked to the resolution of ER stress and, in the case of severe stress, to cell death. Here we demonstrate that constitutive IRE1 RNase activity contributes to basal production of pro-tumorigenic factors IL-6, IL-8, CXCL1, GM-CSF, and TGFß2 in TNBC cells. We further show that the chemotherapeutic drug, paclitaxel, enhances IRE1 RNase activity and this contributes to paclitaxel-mediated expansion of tumor-initiating cells. In a xenograft mouse model of TNBC, inhibition of IRE1 RNase activity increases paclitaxel-mediated tumor suppression and delays tumor relapse post therapy. We therefore conclude that inclusion of IRE1 RNase inhibition in therapeutic strategies can enhance the effectiveness of current chemotherapeutics.

Engineering Exosomes for Cancer Therapy.

There remains an urgent need for novel therapeutic strategies to treat metastatic cancer, which results in over 8 million deaths annually worldwide. Following secretion, exosomes are naturally taken up by cells, and capable of the stable transfer of drugs, therapeutic microRNAs and proteins. As knowledge of the biogenesis, release and uptake of exosomes continues to evolve, and thus also has interest in these extracellular vesicles as potential tumor-targeted vehicles for cancer therapy. The ability to engineer exosome content and migratory itinerary holds tremendous promise. Studies to date have employed viral and non-viral methods to engineer the parent cells to secrete modified exosomes, or alternatively, to directly manipulate exosome content following secretion. The majority of studies have demonstrated promising results, with decreased tumor cell invasion, migration and proliferation, along with enhanced immune response, cell death, and sensitivity to chemotherapy observed. The studies outlined in this review highlight the exciting potential for exosomes as therapeutic vehicles for cancer treatment. Successful implementation in the clinical setting will be dependent upon establishment of rigorous standards for exosome manipulation, isolation, and characterisation.

S100ß as a serum marker in endocrine resistant breast cancer.

BACKGROUND: Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer. However, its efficacy is limited by intrinsic and acquired resistance. Here the potential of S100ß as a biomarker and inhibition of its signaling network as a therapeutic strategy in endocrine treated patients was investigated. METHODS: The expression of S100ß in tissue and serum was assessed by immunohistochemistry and an enzyme-linked immunosorbent assay, respectively. The S100ß signaling network was investigated in cell line models of endocrine resistance by western blot, PCR, immunoprecipitation, and chromatin-immunoprecipitation. Endocrine resistant xenografts and tumor explants from patients with resistant tumors were treated with endocrine therapy in the presence and absence of the p-Src kinase inhibitor, dasatinib. RESULTS: Tissue and serum levels of S100ß were found to predict poor disease-free survival in endocrine-treated patients (n = 509, HR 2.32, 95% CI is 1.58-3.40, p < 0.0001 and n = 187, HR 4.009, 95% CI is 1.66-9.68, p = 0.002, respectively). Moreover, elevated levels of serum S100ß detected during routine surveillance over the patient treatment period significantly associated with subsequent clinically confirmed disease recurrence (p = 0.019). In vivo studies demonstrated that endocrine treatment induced transcriptional regulation of S100ß which was successfully disrupted with tyrosine kinase inhibition. In endocrine resistant xenografts and tumor explants from patients with endocrine resistant breast cancer, combined endocrine and dasatinib treatment reduced tumor proliferation and down-regulated S100ß protein expression in comparison to endocrine treatment alone. CONCLUSIONS: S100ß has potential as a new surveillance tool for patients with ER-positive breast cancer to monitor ongoing response to endocrine therapy. Moreover, endocrine resistant breast cancer patients with elevated S100ß may benefit from combined endocrine and tyrosine-kinase inhibitor treatment. TRIAL REGISTRATION: ClinicalTrials.gov,  NCT01840293 ). Registered on 23 April 2013. Retrospectively registered.