Sonja Khan

Potential role of mesenchymal stem cells (MSCs) in the breast tumour microenvironment: stimulation of epithelial to mesenchymal transition (EMT)

Bone marrow-derived mesenchymal stem cells (MSCs) are known to specifically migrate to and engraft at tumour sites. Understanding interactions between cancer cells and MSCs has become fundamental to determining whether MSC-tumour interactions should be harnessed for delivery of therapeutic agents or considered a target for intervention. Breast Cancer Cell lines (MDA-MB-231, T47D & SK-Br3) were cultured alone or on a monolayer of MSCs, and retrieved using epithelial specific magnetic beads. Alterations in expression of 90 genes associated with breast tumourigenicity were analysed using low-density array. Expression of markers of epithelial–mesenchymal transition (EMT) and array results were validated using RQ-PCR. Co-cultured cells were analysed for changes in protein expression, growth pattern and morphology. Gene expression and proliferation assays were also performed on indirect co-cultures. Following direct co-culture with MSCs, breast cancer cells expressed elevated levels of oncogenes (NCOA4, FOS), proto-oncogenes (FYN, JUN), genes associated with invasion (MMP11), angiogenesis (VEGF) and anti-apoptosis (IGF1R, BCL2). However, universal downregulation of genes associated with proliferation was observed (Ki67, MYBL2), and reflected in reduced ATP production in response to MSC-secreted factors. Significant upregulation of EMT specific markers (N-cadherin, Vimentin, Twist and Snail) was also observed following co-culture with MSCs, with a reciprocal downregulation in E-cadherin protein expression. These changes were predominantly cell contact mediated and appeared to be MSC specific. Breast cancer cell morphology and growth pattern also altered in response to MSCs. MSCs may promote breast cancer metastasis through facilitation of EMT.

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.

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.

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

BackgroundMicroRNAs (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.MethodsFollowing 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 (RARβ) 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.ResultsThe 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 RARβ 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 RARβ (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.ConclusionThe 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.

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.

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.

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.

Employing mesenchymal stem cells to support tumor-targeted delivery of extracellular vesicle (EV)-encapsulated microRNA-379

Adult Mesenchymal Stem Cells (MSCs) have a well-established tumor-homing capacity, highlighting potential as tumor-targeted delivery vehicles. MSCs secrete extracellular vesicle (EV)-encapsulated microRNAs, which play a role in intercellular communication. The aim of this study was to characterize a potential tumor suppressor microRNA, miR-379, and engineer MSCs to secrete EVs enriched with miR-379 for in vivo therapy of breast cancer. miR-379 expression was significantly reduced in lymph node metastases compared to primary tumor tissue from the same patients. A significant reduction in the rate of tumor formation and growth in vivo was observed in T47D breast cancer cells stably expressing miR-379. In more aggressive HER2-amplified HCC-1954 cells, HCC-379 and HCC-NTC tumor growth rate in vivo was similar, but increased tumor necrosis was observed in HCC-379 tumors. In response to elevated miR-379, COX-2 mRNA and protein was also significantly reduced in vitro and in vivo. MSCs were successfully engineered to secrete EVs enriched with miR-379, with the majority found to be of the appropriate size and morphology of exosomal EVs. Administration of MSC-379 or MSC-NTC cells, or EVs derived from either cell population, resulted in no adverse effects in vivo. While MSC-379 cells did not impact tumor growth, systemic administration of cell-free EVs enriched with miR-379 was demonstrated to have a therapeutic effect. The data presented support miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Exploiting the tumor-homing capacity of MSCs while engineering the cells to secrete EVs enriched with miR-379 holds exciting potential as an innovative therapy for metastatic breast cancer.

Abstract P6-04-01: Engineering mesenchymal stem cells(MSCs) to secrete tumour-suppressing exosomes for breast cancer therapy

Introduction: Mesenchymal Stem Cells(MSCs) are multipotent stromal cells that are known to engraft into tumours, raising their potential as tumour-targeted delivery vehicles. MSCs secrete tiny vesicles known as exosomes, which contain genetic material including microRNAs, and are effectively taken up by recipient cells. This study aimed to characterise a tumour-suppressing microRNA, miR-379, and engineer MSCs to secrete exosomes enriched with the microRNA. Methods: The mechanism of action of miR-379 In Vivo was determined through lentivirus-mediated upregulation of miR-379 in breast tumours, and analysis of changes in tumour angiogenesis, proliferation and progression. Subsequently, MSCs were engineered with lenti-379 and any impact on MSC migration, proliferation and morphology was assessed. MSC-secreted exosomes were isolated and characterised using Transmission Electron Microscopy(TEM) and Western Blot. The exosomal microRNA content was analysed by RQ-PCR, and transfer between cell populations visualised using confocal microscopy. Results: While elevated miR-379 expression did not impact tumour size In Vivo, an increase in tumour necrosis and decrease in invasion was observed. MSCs were successfully transduced with miR-379, resulting in a distinct change in cell morphology. Despite this, MSC-379 cells maintained inherent tumour-targeted migratory capacity, with no impact on proliferation observed. MSC-379 derived exosomes were 30-120nm in size and expressed the exosome-associated protein CD63. A 5-fold increase in miR-379 was observed in engineered exosomes. Successful transfer of RFP-labelled MSC-derived exosomes to breast cancer cells was visualised using confocal microscopy. Conclusion: Engineering tumour-targeted MSCs to secrete exosomes enriched with miR-379 holds exciting potential as a novel therapy for breast cancer. Citation Format: O9Brien KP, Khan S, Thompson K, Joyce D, Lalor P, Dockery P, Ingoldsby H, Kerin MJ, Dwyer RM. Engineering mesenchymal stem cells(MSCs) to secrete tumour-suppressing exosomes for breast cancer therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-04-01.

Abstract 3590: Investigation of exosome-encapsulated microRNA secretion in breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Intercellular communication between breast tumour cells and stromal cells plays a fundamental role in cancer initiation and progression. Mesenchymal Stem Cells(MSCs) are multipotent cells with the proven ability to home to the site of breast tumours and integrate into the tumour architecture. The mechanisms of MSC interaction with breast cancer cells are poorly defined. Exosomes are microvesicles secreted by cells that have the capacity to transport genetic material including microRNAs(miRNAs), which are important regulators of gene expression. However, the miRNAs selectively packaged into exosomes, and their true role in the tumour microenvironment, is poorly understood. Aim: Investigate the presence of MSCs in primary breast tumours, and identify exosome-encapsulated miRNAs secreted by breast cancer cells and MSCs in vitro. Methods: Stromal cells were isolated from primary breast tumours and characterized based on MSC-associated cell surface antigens. Tumour stromal cells were cultured in appropriate conditions to investigate potential for differentiation into osteoblasts or adipocytes, followed by Von Kossa staining of calcium deposition, and Oil Red O staining of fat droplets respectively. MSCs isolated from healthy volunteers and breast cancer cell lines were cultured in exosome-depleted media for 48 hrs, and secreted exosomes isolated using ultracentrifugation. Transmission Electron Microscopy(TEM) and Western Blot were performed to confirm the presence of exosomes in the purified fraction. Extracted miRNA was subjected to global miRNA expression analysis using the Exiqon miRCURY™ LNA Array. Results: A subpopulation of tumour stromal cells expressed the full panel of surface markers associated with MSCs. This subpopulation also had the capacity to differentiate into osteoblasts and adipocytes, demonstrated by calcium deposition and formation of fat droplets. Exosomes were successfully isolated from conditioned media of breast cancer cell lines and MSCs. TEM revealed microvesicles with the appropriate size of 40-100nm. Western Blot confirmed the presence of the exosome-associated protein CD63. miRNA array analysis identified 413 miRNAs encapsulated in MSC-secreted exosomes from a panel of 2089 analysed, while each of the breast cancer cell lines secreted from 381 to 394 miRNAs in exosomes. Of these, 287 were common to all four breast cancer cell lines. Interestingly, a subset of these miRNAs are known to target genes associated with cell cycle regulation and apoptosis (e.g. miR-15a, miR-204, miR-221, miR-223). A panel of the miRNAs also appeared to have differential expression with reference to epithelial subtype. Conclusion: MSCs are present in primary breast tumours, and elucidating their interaction with breast cancer cells will be fundamental to understanding their role in the tumour microenvironment. Bidirectional transfer of exosome-encapsulated miRNAs may play an important role in this intercellular communication. Citation Format: Claire Glynn, Sonja Khan, Cathy Brougham, Cillian Clancy, Doireann Joyce, Peter Dockery, Michael J. Kerin, Roisin M. Dwyer. Investigation of exosome-encapsulated microRNA secretion in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3590. doi:10.1158/1538-7445.AM2014-3590