Shyam A. Patel

Gap Junction–Mediated Import of MicroRNA from Bone Marrow Stromal Cells Can Elicit Cell Cycle Quiescence in Breast Cancer Cells

Bone marrow (BM) metastasis of breast cancer (BC) can recur even decades after initial diagnosis and treatment, implying the long-term survival of disseminated cancer cells in a dormant state. Here we investigated the role of microRNAs (miRNA) transmitted from BM stroma to BC cells via gap junctions and exosomes in tumor cell quiescence. MDA-MB-231 and T47D BC cells arrest in G(0) phase of the cell cycle when cocultured with BM stroma. Analyses of miRNA expression profiles identified numerous miRNAs implicated in cell proliferation including miR-127, -197, -222, and -223 targeting CXCL12. Subsequently, we showed that these CXCL12-specific miRNAs are transported from BM stroma to BC cells via gap junctions, leading to reduced CXCL12 levels and decreased proliferation. Stroma-derived exosomes containing miRNAs also contributed to BC cell quiescence, although to a lesser degree than miRNAs transmitted via gap junctions. This study shows that the transfer of miRNAs from BM stroma to BC cells might play a role in the dormancy of BM metastases.

Mesenchymal Stem Cells Protect Breast Cancer Cells through Regulatory T Cells: Role of Mesenchymal Stem Cell-Derived TGF-β

Mesenchymal stem cells (MSCs) have been shown to support breast cancer growth. Because MSCs also increase the frequency of regulatory T cells (Tregs), this study tested the hypothesis that human MSCs, via Tregs, protect breast cancer cells (BCCs) from immune clearance MSCs suppressed the proliferation of PBMCs when the latter were exposed to gamma-irradiated BCCs. Similarly, MSCs showed significant inhibition of PBMC migration toward BCCs and a corresponding decrease in CXCL12. MSCs also inhibited NK cell and CTL functions, which correlated with reduced numbers of CD8+ and CD56+ cells compared with parallel cultures without MSCs. The reduced NK and CTL activities correlated with a decrease in intracellular and secreted granzyme B. To explain these immunosuppressive findings, we compared Treg levels after coculture with MSCs and found an ∼2-fold increase in Tregs, with associated decreases in antitumor Th1 cytokines and increases in Th2 cytokines. MSC-derived TGF-β1 was largely responsible for the increase in Tregs based on knockdown studies. In the presence of Treg depletion, PBMC proliferation and effector functions were partially restored. Together, these studies show an MSC-mediated increase in Tregs in cocultures of PBMCs and BCCs. The results could be explained, in part, by the increase in Th2-type cytokines and MSC-generated TGF-β1. These findings demonstrate immune protection by MSCs to BCCs. The reduction in immune cell proliferation and recruitment mediated by MSCs has implications for treatment of breast cancer with chemotherapy.

Immunological properties of mesenchymal stem cells and clinical implications

The rapid evolution of experimental data has acknowledged the critical relevance of immune biology in stem cell research. It appears that efficient transfer of stem cells to patients requires robust analyses of the immune properties as well as the responses of the stem cells to immune mediators. This review discusses the biology of adult human mesenchymal stem cells (MSCs) in the context of immunology. MSCs are pluripotent, self-renewing cells with the potential for tissue regeneration, for example the repair of bone, cartilage, tendon, ligament, skeletal muscle, and cardiac muscle. MSCs have also been shown to transdifferentiate into cells of ectodermal origin, such as neurons. MSCs are located in perfused areas of adult bone marrow, whereas hematopoietic stem cells are located in poorly perfused areas of the same organ. MSCs show bimodal, i.e. anti-inflammatory and immune-enhancing, immune responses. MSCs also regulate immune responses such as the regulation of antibody production by B cells, alterations in T cell subtypes, and immune tolerance of allogeneic transplants. MSCs also have the potential for gene delivery. This review explores the diverse clinical potential for MSCs and discusses the limitations and advantages of their immunomodulatory properties.

Delineation of breast cancer cell hierarchy identifies the subset responsible for dormancy

The bone marrow (BM) is a major organ of breast cancer (BC) dormancy and a common source of BC resurgence. Gap junctional intercellular communication (GJIC) between BC cells (BCCs) and BM stroma facilitates dormancy. This study reports on a hierarchy of BCCs with the most immature subset (Oct4hi/CD44hi/med/CD24−/+) demonstrating chemoresistance, dormancy, and stem cell properties: self-renewal, serial passaging ability, cycling quiescence, long doubling time, asymmetric division, high metastatic and invasive capability. In vitro and in vivo studies indicated that this subset was responsible for GJIC with BM stroma. Similar BCCs were detected in the blood of patients despite aggressive treatment and in a patient with a relatively large tumor but no lymph node involvement. In brief, these findings identified a novel BCC subset with stem cell properties, with preference for dormancy and in the circulation of patients. The findings establish a working cellular hierarchy of BCCs based on phenotype and functions.

Tolerance-like mediated suppression by mesenchymal stem cells in patients with dust mite allergy–induced asthma

BACKGROUND Mesenchymal stem cells (MSCs) can suppress and enhance immune functions. MSCs show promise as off-the-shelf cellular therapy for several disorders, including inflammation. OBJECTIVE We investigated the effects of MSCs on the proliferation of PBMCs to allergic subjects (dust mite [DM]), allergic asthmatic subjects, or both. METHODS Proliferation was studied by using tritiated thymidine uptake with or without MSCs. The refractoriness of PBMCs to DM was examined after preconditioning with MSCs and after repeated challenge with low-dose DM. Flow cytometry was used to study regulatory T cells and dendritic cells (DCs), and ELISA was used to study cytokine production. RESULTS Seven subjects with allergic asthma met the inclusion/exclusion criteria. MSCs significantly (P < .05) reduced the proliferation of 6 subjects with allergic asthma but not those with allergy alone. The effect was specific to the allergen because MSCs did not affect challenges to tetanus toxoid. There was no change in CD4/CD25/forkhead box protein 3-positive cells, although there were decreased IFN-γ and increased IL-10 levels. Numbers of mature DCs were increased 6-fold. Refractoriness to DM was achieved by means of repeated exposure to low-dose DM and MSCs and also MSC- preconditioned MSC. CONCLUSION MSCs suppressed the proliferation of DM-challenged PBMCs from allergic asthmatic subjects but not from allergic subjects without asthma. MSCs blunted the maturation of DCs but not regulatory T cells. Repeated exposure to low-dose DM and MSCs, as well as preconditioning of PBMCs with MSCs, caused refractoriness to DM. These findings have implications for the use of MSCs in attenuation of the inflammatory responses to allergic triggers in asthmatic patients with off-the-shelf MSCs.

An AICD-based functional screen to identify APP metabolism regulators

BackgroundA central event in Alzheimers disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered.ResultsTo validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis.ConclusionTaken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.

Stem cells and regenerative medicine: accomplishments to date and future promise

More than 50 years have passed since the first allogeneic hematopoietic stem cell transplant in patients; however, the promise of other stem cell populations for tissue replacement and repair remains unachieved. When considering cell-based interventions for personalized medicine, the factors influencing therapeutic success and safety are more complicated than for traditional small-molecule pharmacological agents and protein biologics. Failure to progress personalized stem cell therapies to the clinic has resulted from complications that include an incomplete understanding of developmental programs and the diversity of host-donor interactions. In order to more rapidly extend the use of stem cells to the clinic, a better understanding of the different stem cell sources and the implications of their host interactions is required. In this review, we introduce the currently available sources and highlight recent literature that instructs the potential and limitations of their use.

The Microenvironmental Effect in the Progression, Metastasis, and Dormancy of Breast Cancer: A Model System within Bone Marrow

Despite diagnostic advances, breast cancer remains the most prevalent cancer among women in the United States. The armamentarium of treatment options for metastatic disease is limited and mostly ineffective with regards to eradicating cancer. However, there have been novel findings in the recent literature that substantiate the function of the microenvironment in breast cancer progression and the support of metastasis to tertiary sites such as bone marrow. The uncovered significance of the microenvironment in the pathophysiology of breast cancer metastasis has served to challenge previously widespread theories and introduce new perspectives for the future research to eradicate breast cancer. This paper delineates the current understanding of the molecular mechanisms involved in the interactions between breast cancer cells and the microenvironment in progression, metastasis, and dormancy. The information, in addition to other mechanisms described in bone marrow, is discussed in the paper.

microRNAs, Gap Junctional Intercellular Communication and Mesenchymal Stem Cells in Breast Cancer Metastasis

The failed outcome of autologous bone marrow transplantation for breast cancer opens the field for investigations. This is particularly important because the bone marrow could be a major source of cancer cells during tertiary metastasis. This review discusses subsets of breast cancer cells, including those that enter the bone marrow at an early period of disease development, perhaps prior to clinical detection. This population of cells evades chemotherapeutic damage even at high doses. An understanding of this population might be crucial for the success of bone marrow transplants for metastatic breast cancer and for the eradication of cancer cells in bone marrow. In vivo and in vitro studies have demonstrated gap junctional intercellular communication (GJIC) between bone marrow stroma and breast cancer cells. This review discusses GJIC in cancer metastasis, facilitating roles of mesenchymal stem cells (MSCs). In addition, the review addresses potential roles for miRNAs, including those already linked to cancer biology. The literature on MSCs is growing and their links to metastasis are beginning to be significant leads for the development of new drug targets for breast cancer. In summary, this review discusses interactions among GJIC, miRNAs and MSCs as future consideration for the development of cancer therapies.

Are we failing to communicate? Internet-based patient education materials and radiation safety

INTRODUCTION Patients frequently turn to the Internet when seeking answers to healthcare related inquiries including questions about the effects of radiation when undergoing radiologic studies. We investigate the readability of online patient education materials concerning radiation safety from multiple Internet resources. METHODS Patient education material regarding radiation safety was downloaded from 8 different websites encompassing: (1) the Centers for Disease Control and Prevention, (2) the Environmental Protection Agency, (3) the European Society of Radiology, (4) the Food and Drug Administration, (5) the Mayo Clinic, (6) MedlinePlus, (7) the Nuclear Regulatory Commission, and (8) the Society of Pediatric Radiology. From these 8 resources, a total of 45 articles were analyzed for their level of readability using 10 different readability scales. RESULTS The 45 articles had a level of readability ranging from 9.4 to the 17.2 grade level. Only 3/45 (6.7%) were written below the 10th grade level. No statistical difference was seen between the readability level of the 8 different websites. CONCLUSIONS All 45 articles from all 8 websites failed to meet the recommendations set forth by the National Institutes of Health and American Medical Association that patient education resources be written between the 3rd and 7th grade level. Rewriting the patient education resources on radiation safety from each of these 8 websites would help many consumers of healthcare information adequately comprehend such material.