Prem S. 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 in Early Entry of Breast Cancer into Bone Marrow

Background An understanding of BC cell (BCC) entry into bone marrow (BM) at low tumor burden is limited when compared to highly metastatic events during heavy tumor burden. BCCs can achieve quiescence, without interfering with hematopoiesis. This occurs partly through the generation of gap junctions with BM stroma, located close to the endosteum. These events are partly mediated by the evolutionary conserved gene, Tac1. Methodogy/Principal Findings This study focuses on the role of mesenchymal stem cells (MSCs), Tac1, SDF-1 and CXCR4 in BCC entry into BM. The model is established in studies with low numbers of tumor cells, and focuses on cancer cells with low metastatic and invasion potential. This allowed us to recapitulate early event, and to study cancer cells with low invasive potential, even when they are part of larger numbers of highly metastatic cells. A novel migration assay showed a facilitating role of MSCs in BCC migration across BM endothelial cells. siRNA and ectopic expression studies showed a central role for Tac1 and secondary roles for SDF-1α and CXCR4. We also observed differences in the mechanisms between low invasive and highly metastatic cells. The in vitro studies were verified in xenogeneic mouse models that showed a preference for low invasive BCCs to BM, but comparable movement to lung and BM by highly metastatic BCCs. The expressions of Tac1 and production of SDF-1α were verified in primary BCCs from paired samples of BM aspirates and peripheral blood. Conclusions/Significance MSC facilitate BCC entry into BM, partly through Tac1-mediated regulation of SDF-1α and CXCR4. We propose a particular population of BCC with preference for BM could be isolated for characterization. This population might be the subset that enter BM at an early time period, and could be responsible for cancer resurgence and resistance to current therapies.

Bone Marrow Stroma Influences Transforming Growth Factor-β Production in Breast Cancer Cells to Regulate c-myc Activation of the Preprotachykinin-I Gene in Breast Cancer Cells

Breast cancer cells (BCCs) have preference for the bone marrow (BM). This study used an in vitro coculture of BCCs and BM stroma to represent a model of early breast cancer metastasis to the BM. The overarching hypothesis states that once BCCs are in the BM, microenvironmental factors induce changes in the expression of genes for cytokines and preprotachykinin-I (PPT-I) in both BCCs and stromal cells. Consequently, the expression of both PPT-I and cytokines are altered to facilitate BCC integration within BM stroma. Cytokine and transcription factor arrays strongly suggested that transforming growth factor-β (TGF-β) and c-myc regulate the expression of PPT-I so as to facilitate BCC integration among stroma. Northern analyses and TGF-β bioassays showed that stromal cells and BCCs influence the level of PPT-I and TGF-β in each other. In cocultures, PPT-I and TGF-β expressions were significantly (P < 0.05) increased and decreased, respectively. TGF-β and PPT-I were undetectable in separate stromal cultures but were expressed as cocultures. Two consensus sequences for c-myc in the 5′ flanking region of the PPT-I gene were shown to be functional using gel shift and reporter gene assays. Mutagenesis of c-myc sites, neutralization studies with anti-TGF-β, and transient tranfections all showed that c-myc is required for TGF-β-mediated induction of PPT-I in BCCs. TGF-β was less efficient as a mediator of BCC integration within stroma for c-myc-BCCs. Because the model used in this study represents BCC integration within BM stroma, these studies suggest that TGF-β is important to the regulation of PPT-I in the early events of bone invasion by BCCs.

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.

Commercialization of kidney transplants: a systematic review of outcomes in recipients and donors

In this study we systematically reviewed outcomes in recipients and donors of commercial kidney transplants. Inherent in a study of this nature is the possibility of center and country bias, in particular there are no publications from China and South America. Publications also tended to report poor outcomes which may reflect bias on the part of the authors or to highlight the ethical issues in this area. We were unable to perform a meta-analysis due to variability in studies making it impossible to synthesize the data other than descriptive. Furthermore, these studies were not large or well conducted. We found that patient and graft survival was generally inferior to the data obtained from the UNOS (United Network for Organ Sharing). Some studies did achieve good outcomes, however, due to lack of details, it was not possible to infer if the donor hospital, surgical technique or immunosuppressive regimen was a factor. There was a higher incidence of unconventional and life-threatening infections such as malaria, invasive fungal infections, pneumonia, HIV and hepatitis. There was also a markedly increased incidence of postoperative surgical interventions in recipients. We suggest the establishment of a database for both recipients and donors to identify unique surgical, medical, infectious and immunosuppressive protocols for the recipients and donors in these hospitals. This could lead to better liaison between the recipient and donor hospitals so that modern surgical and medical practices can be implemented. There should also be improved emotional and psychological support to both the recipient and the donor. However, these steps could be seen as condoning the reprehensible practice of commercialization of human body parts.

Transplantation of A2 kidneys into B and O recipients leads to reduction in waiting time: USRDS experience.

Introduction. Strategy of transplanting kidneys from A2 donors into patients with blood group B and O recipients has been used to alleviate the long waiting times. Materials and Methods. We used an inception cohort of US Renal Data System data base with patients older than 18 years who underwent renal transplantation between January 1995 and July 2006. The primary outcome variable was allograft loss (including death). Bivariate analysis of factors associated with receiving A2 or A2B kidneys was performed with chi-square testing for categorical variables (Fishers exact test used for violations of Cochrans assumptions) and Students t test for continuous variables (Mann-Whitney U test used for nonnormally distributed variables). Results. There were 150,118 first kidney transplants of whom 113 received kidney transplant from A2 to O, and 125 patients received A2 to B kidney transplant. Compared with other recipients from the same blood group, recipients of A2 kidneys had significantly shorter wait times. O recipients had a median wait time of 1.63 years (range 0.00–17.21 years), whereas O recipients who received A2 kidneys had a median wait time of 0.70 years (range 0.02–1.47 years; P<0.001). B recipients had a median wait time of 1.90 years (range 0.00–17.52 years), whereas B recipients who received A2 kidneys had a median wait time of 0.74 years (range 0.10–5.21 years; P<0.001). There was no significant difference in graft loss or death between A2 to O and B versus all other recipients. Conclusions. The results showed that comparatively few patients received A2 to B or O kidney transplant.

An in vitro method to select malignant cells from surgical biopsies of breast cancer patients.

To date, breast cancer (BC) research is mainly studied with cell lines. These cells were passaged multiple times, acquiring phenotypes, additional mutations and epigenetic changes. These changes make the passaged cell lines different from the original malignancy. Thus cell lines, although useful as models could be improved with additional studies with primary BC. It is difficult to obtain malignant cells from breast tissues without contamination from surrounding healthy cells. Selection and expansion of malignant cells from surgical tissues have proved to be daunting tasks. This study describes a reliable and reproducible method for isolating and expanding malignant cells from surgical breast tissues. The method uses co-cultures with BM stroma to select for the cancer cells while the healthy cells undergo rapid cell death. Studies are described to show the cloning efficiencies and sensitivity of the method using surgical samples of varying sizes, different stages of BC, and samples from needle biopsies.