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.

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.

Oncological Risk in Autologous Stem Cell Donation for Novel Tissue-Engineering Approaches to Postmastectomy Breast Regeneration.

Adipose tissue engineering using adipose-derived stem cells (ADSCs) has emerged as an opportunity to develop novel approaches to postmastectomy breast reconstruction with the potential for an autologous tissue source with a natural appearance and texture. As of yet, the role of ADSCs in breast cancer development and metastasis is not completely understood; therefore, we must consider the oncological safety of employing an autologous source of ADSCs for use in breast regeneration. This study investigated the regenerative properties of ADSCs isolated from breast cancer patients, including those who had received neoadjuvant chemotherapy, and noncancer controls. The ADSCs were characterised for several parameters central to tissue regeneration, including cell viability, proliferation, differentiation potential, and cytokine secretion. A stem cell population was isolated and confirmed by flow cytometry and multilineage differentiation. There was no difference in cell phenotype or surface antigen expression between ADSCs from different sources. Adipose-derived stem cells isolated from the breast of cancer patients exhibited reduced adipogenic differentiation potential compared with ADSCs from other sources. The greatest degree of adipogenic differentiation was observed in ADSCs isolated from the subcutaneous abdominal fat of noncancer controls. The proliferation rate of ADSCs isolated from the breast of cancer patients was increased compared with other sources; however, it was decreased in ADSCs isolated from breast cancer patients who had recently been treated with neoadjuvant chemotherapy. A number of cytokines were detected in the cell conditioned media of ADSCs from different sources, including matrix metalloproteinase-2 (MMP-2), which was detected at higher levels in the secretome of ADSCs from breast cancer patients compared with noncancer controls. This study provides important information relating to the suitability of ADSCs as an autologous cell source for adipose tissue engineering in postcancer reconstruction. Results indicate that while the surface phenotype does not differ, the differentiation capacity, proliferative rate, and secreted cytokine profile are affected by the presence or treatment of breast cancer. These findings support further investigation into the regenerative potential of these ADSCs, if they are to be considered in clinical reconstructive strategies.

Screening of exosomal microRNAs from colorectal cancer cells.

BACKGROUND: Cells release extracellular membrane vesicles including microvesicles known as exosomes. Exosomes contain microRNAs (miRNAs) however the full range within colorectal cancer cell secreted exosomes is unknown. OBJECTIVE: To identify the full range of exosome encapsulated miRNAs secreted from 2 colorectal cancer cell lines and to investigate engineering of exosomes over-expressing miRNAs. METHODS: Exosomes were isolated from HCT-116 and HT-29 cell lines. RNA was extracted from exosomes and microRNA array performed. Cells were engineered to express miR-379 (HCT-116-379) or a non-targeting control (HCT-116-NTC) and functional effects were determined. Exosomes secreted by engineered cells were transferred to recipient cells and the impact examined. RESULTS: Microvesicles 40-100 nm in size secreted by cell lines were visualised and confirmed to express exosomal protein CD63. HT-29 exosomes contained 409 miRNAs, HCT-116 exosomes contained 393, and 338 were common to exosomes from both cell lines. Selected targets were validated. HCT-116-379 cells showed decreased proliferation (12-15% decrease, p < 0.001) and decreased migration (32-86% decrease, p < 0.001) compared to controls. HCT-116-379 exosomes were enriched for miR-379. Confocal microscopy visualised transfer of HCT-116-379 exosomes to recipient cells. CONCLUSIONS: Colorectal cancer cells secrete a large number of miRNAs within exosomes. miR-379 decreases cell proliferation and migration, and miR-379 enriched exosomes can be engineered.

MicroRNA-10a is reduced in breast cancer and regulated in part through retinoic acid.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNA molecules that play a critical role in mRNA cleavage and translational repression, and are known to be altered in many diseases including breast cancer. MicroRNA-10a (miR-10a) has been shown to be deregulated in various cancer types. The aim of this study was to investigate miR-10a expression in breast cancer and to further delineate the role of retinoids and thyroxine in regulation of miR-10a. METHODS: Following informed patient consent and ethical approval, tissue samples were obtained during surgery. miR-10a was quantified in malignant (n = 103), normal (n = 30) and fibroadenoma (n = 35) tissues by RQ-PCR. Gene expression of Retinoic Acid Receptor beta (RARß) and Thyroid Hormone receptor alpha (THRα) was also quantified in the same patient samples (n = 168). The in vitro effects of all-trans Retinoic acid (ATRA) and L-Thyroxine (T4) both individually and in combination, on miR-10a expression was investigated in breast cancer cell lines, T47D and SK-BR-3. RESULTS: The level of miR-10a expression was significantly decreased in tissues harvested from breast cancer patients (Mean (SEM) 2.1(0.07)) Log10 Relative Quantity (RQ)) compared to both normal (3.0(0.16) Log10 RQ, p < 0.001) and benign tissues (2.6(0.17) Log10 RQ, p < 0.05). The levels of both RARß and THRα gene expression were also found to be decreased in breast cancer patients compared to controls (p < 0.001). A significant positive correlation was determined between miR-10a and RARß (r = 0.31, p < 0.001) and also with THRα (r = 0.32, p < 0.001). In vitro stimulation assays revealed miR-10a expression was increased in both T47D and SK-BR-3 cells following addition of ATRA (2 fold (0.7)). While T4 alone did not stimulate miR-10a expression, the combination of T4 and ATRA was found to have a positive synergistic effect. CONCLUSION: The data presented supports a potential tumour suppressor role for miR-10a in breast cancer, and highlights retinoic acid as a positive regulator of the microRNA.

Enhancing pedicle screw fixation in the lumbar spine using allograft bone plug interference fixation.

STUDY DESIGN: A within-subjects controlled laboratory study. OBJECTIVE: To examine a biological alternative to cement augmentation for pedicle screw fixation comparing bilateral axial pullout tests of augmented and nonaugmented (controls) pedicle screws. SUMMARY OF BACKGROUND DATA: Fixation in the osteoporotic spine remains a difficult challenge with failure by loosening or backout. Pedicle screw augmentation has been attempted using polymethylmethacrylate and bioabsorbable calcium cements; however, the potential for extravasation and embolization of cement are becoming increasingly concerning and merit the search for alternative methods to improve screw-anchoring strength. METHODS: Twenty-four (24) fresh human lumbar vertebrae were tested to compare the pullout strength of augmented and nonaugmented pedicle screws. Two different augmentation strategies were employed using allograft bone plugs (ABPs) and evaluated using 12 specimens per group. Bone mineral density of each specimen was obtained using dual-energy x-ray absorptiometry. The augmented versus nonaugmented pedicle was randomized for each vertebra, and bilateral testing enabled paired statistical analyses. Axial pullout tests were performed using an materials testing system servohydraulic test system, and peak force, failure displacement, and stiffness was obtained for each test and correlated with bone mineral density. RESULTS: Augmentation using 6-mm-diameter ABPs with 6.25-mm-diameter pedicle screws resulted in statistically weaker average pullout strength (775±455 N) than the nonaugmented controls (1233±826 N). When using smaller (5 mm diameter) AGPs with the same diameter screws, there was no statistical difference between average pullout strength for the augmented pedicle screws (1772±652 N) and the nonaugmented screws (1780±575 N). CONCLUSIONS: Preliminary study of pedicle screw augmentation using cannulated ABPs showed no improvement of fixation with pedicles in the spine. This was even true in osteoporotic specimens, where augmentation would seem to be of considerable benefit.

miR-379 regulates cyclin B1 expression and is decreased in breast cancer.

MicroRNAs are small non-coding RNA molecules that control gene expression post-transcriptionally, and are known to be altered in many diseases including breast cancer. The aim of this study was to determine the relevance of miR-379 in breast cancer. miR-379 expression was quantified in clinical samples including tissues from breast cancer patients (n=103), healthy controls (n=30) and patients with benign breast disease (n=35). The level of miR-379 and its putative target Cyclin B1 were investigated on all breast tissue specimens by RQ-PCR. Potential relationships with gene expression and patient clinicopathological details were also determined. The effect of miR-379 on Cyclin B1 protein expression and function was investigated using western blot, immunohistochemistry and proliferation assays respectively. Finally, the levels of circulating miR-379 were determined in whole blood from patients with breast cancer (n=40) and healthy controls (n=34). The level of miR-379 expression was significantly decreased in breast cancer (Mean(SEM) 1.9 (0.09) Log10 Relative Quantity (RQ)) compared to normal breast tissues (2.6 (0.16) Log10 RQ, p<0.01). miR-379 was also found to decrease significantly with increasing tumour stage. A significant negative correlation was determined between miR-379 and Cyclin B1 (r=-0.31, p<0.001). Functional assays revealed reduced proliferation (p<0.05) and decreased Cyclin B1 protein levels following transfection of breast cancer cells with miR-379. Circulating miR-379 was not significantly dysregulated in patients with breast cancer compared to healthy controls (p=0.42). This data presents miR-379 as a novel regulator of Cyclin B1 expression, with significant loss of the miRNA observed in breast tumours.

Isolation of secreted microRNAs (miRNAs) from cell-conditioned media.

MicroRNAs (miRNAs) have been found to be stable in the circulation of cancer patients raising their potential as circulating biomarkers of disease. The specific source and role, however, of miRNAs in the circulation is unknown and requires elucidation to determine their true potential. In this study, along with primary tissue explants and primary stromal cells, three breast cancer cell lines were employed, including T47D, MDA-MB-231 and SK-BR-3. Tissue explants were harvested in theatre, with informed patient consent, and included tumour, tumour associated normal, and diseased lymph node samples. Cell-conditioned media containing all factors secreted by the cells were harvested. MiRNAs were extracted from samples using 5 different extraction techniques including the blood protocol, RNeasy® (Qiagen), miRNeasy®mini kit (Qiagen), mirVana™ isolation kit (Ambion) and RNAqueous® kit (Ambion). MiRNAs were successfully isolated from all media samples collected from cell lines, primary cells and fresh tissue explants. However, there was remarkable variation in yield depending on the extraction method used. Aliquots of the same samples were extracted, revealing the two column extraction protocol of the mirVana® miRNA isolation kit to be the most suitable approach. A range of miRNAs, including miR-16, miR-195, miR-497 and miR-10b, were successfully amplified. While miR-16 and miR-195 were detected in media from both cell lines and tissue explants, miR-497 and miR-10b were only detected in secretions from whole tissue explants. The ability to achieve reliable and reproducible miRNA yields from cell-conditioned media is vital for the successful amplification of miRNAs by RQ-PCR.

Influence of stromal-epithelial interactions on breast cancer in vitro and in vivo.

Stromal cell-secreted chemokines including CCL2 have been implicated in the primary tumor microenvironment, as mediators of tumor cell migration, proliferation, and angiogenesis. Expression of CCL2 and its principal receptor CCR2 was analyzed by RQ-PCR in primary tumor cells and breast cancer cell lines. Breast cancer cell lines (MDA-MB-231, T47D) were co-cultured directly on a monolayer of primary breast tumor and normal stromal cells, retrieved using EpCAM+ magnetic beads, and changes in expression of CCL2, CCR2, MMP11, ELK1, VIL2, and Ki67 detected by RQ-PCR. Epithelial cell migration and proliferation in response to stromal cell-secreted factors was also analyzed. In vivo, tumor xenografts were formed by co-injecting T47D cells with primary tumor stromal cells. Following establishment, tumors were harvested and digested, epithelial cells retrieved and analyzed by RQ-PCR. Whole tumor tissue was also analyzed by immunohistochemistry for CD31 and the VIL2 encoded protein Ezrin. Tumor stromal cells expressed significantly higher levels of CCL2 than normal cells, with no CCR2 expression detected. Primary epithelial cells and breast cancer cell lines expressed elevated CCL2, with relative expression of CCR2 found to be higher than the ligand. Interaction of breast cancer epithelial cells with primary tumor, but not normal stromal cells, stimulated increased expression of CCL2 (8-fold), ELK1 (6-fold), VIL2 (6-fold), and MMP11 (17-fold). Factors secreted by stromal cells, including CCL2, stimulated a significant increase in epithelial cell migration, with no effect on cell proliferation in vitro observed. In vivo, the presence of stromal cells resulted in tumors of increased volume, mediated at least in part through neoangiogenesis demonstrated by immunohistochemistry (CD31). Admixed tumor xenografts exhibited increased expression of Ki67, MMP11, VIL2, and ELK1. Elevated Ezrin protein was also detected, with increased cytoplasmic localization. The results presented highlight mechanisms through which breast cancer epithelial cells can harness stromal cell biology to support tumor progression.

Advances in mesenchymal stem cell-mediated gene therapy for cancer.

Mesenchymal stem cells have a natural tropism for tumours and their metastases, and are also considered immunoprivileged. This remarkable combination of properties has formed the basis for many studies investigating their potential as tumour-specific delivery vehicles for suicide genes, oncolytic viruses and secreted therapeutic proteins. The aim of the present review is to discuss the range of approaches that have been used to exploit the tumour-homing capacity of mesenchymal stem cells for gene delivery, and to highlight advances required to realize the full potential of this promising approach.