CD63-GFP (green) transfected cells Donor Cells/ Upper well

Nanoparticle Tracking Analysis using the Nanosight

CD63-GFP (green) transfected cells Donor Cells/ Lower well

Figure legends for the supplemental figures

<p>Supplemental Figures 1-8 to support the following: The validity of p63-GFP transfection normalized band densities for western blots; support for dicer knockdown; bioinformatics to support cycling changes of the exosomes; supporting evidence for the source of cellular miR222; support for sensitivity of detecting human breast cancer cells in the femurs of mice; showing the sensitivity of mesenchymal stem cells to carboplatin.</p>

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Abstract In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-β-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)–released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. Significance: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell–derived exosomes, indicating a potential target for therapeutic intervention.

This chapter is concerned with the fate and localization properties of B cells in normal and specifically immunized animals. The effects of antigen on the localization and recirculation of B cells may be due either to a direct interaction between antigen and B cells or to an indirect effect mediated via other cells or cytokines. In their important studies on radioactively labeled lymphocyte recirculation, Gowans and co-workers showed that at least some of the lymphocytes from thoracic duct, particularly when taken from primed donors, became plasma cells upon challenge with antigen in recipients. Antigen localized within lymphoid tissues selectively recruits antigen-specific lymphocytes from the recirculating pool into the antigen-containing organ, as has been shown by two types of experiments: selective depletion studies and selective enrichment studies. Further investigation of the mechanisms involved in the specific accumulation of memory cells in sites of antigen deposition may help to formulate ways to influence, indeed, to direct the migration of antigen-specific lymphocyte subpopulations.

Aging is associated with increased morbidity and high economic costs due to a burdened healthcare system and decreased workforce. Parabiotic animal models indicated that secretome from young cells can restore aged tissue functions. We used a heterochronic co-culture system with young and aged mobilized peripheral blood (MPB) or umbilical cord blood (UCB) and showed hematopoietic restoration, independent of allogeneic difference. Bidirectional communication between the aged and young cells influenced the miRNA cargo of exosomes, resulting in partial reprograming of the aged cells. The restored cells enhanced hematopoiesis (e.g., increased lymphoid:myeloid ratio) in immunodeficient mice bearing autologous aged hematopoietic system. Four exosomal miRNAs targeting PAX and PPMIF were partly responsible for the hematopoietic rejuvenation. Notably, increased natural killer (NK) cells within the restored cells eliminated dormant breast cancer cells in vivo . The findings could be developed as preventive measure and treatment for sustained immune/hematopoietic competence with potential for immunotherapy.

Breast cancer (BC) cells (BCCs) can retain cellular quiescence for decades, a phenomenon referred to as dormancy. BCCs show preference for the bone marrow (BM) where they can remain dormant for decades. Targeting BCCs within the BM is a challenge since the dormant BCCs reside within BM stroma, also residence for hematopoietic stem cells (HSCs). Dormant BCCs could behave as cancer stem cells (CSCs). The CSCs and HSCs are similar by function and also, by commonly expressed genes. The method by which dormant BCCs transition into clinically metastatic cells remains unclear. This study tested the hypothesis that macrophages (MΦs) within BM stroma, facilitates dormancy or reverse this state into metastatic cells. MΦs exhibiting an M2 phenotype constitute ~10% of cultured BM stroma. The M2 MΦs form gap junctional intercellular communication (GJIC) with CSCs, resulting in cycling quiescence, reduced proliferation and carboplatin resistance. In contrast, MΦs expressing the M1 phenotype reversed BC dormancy. Activation of M2a MΦs via the toll-like receptor 4 (TLR4) switched to M1 phenotype. The switch can occur by direct activation of M2a MΦs, or indirectly through activation of mesenchymal stem cells. M1 MΦ-derived exosomes activated NFкB to reverse quiescent BCCs to cycling cells. Using an in vivo model of BC dormancy, injected Mi MOs sensitized BCCs to carboplatin and increased host survival. In summary, we have shown how BM stromal MΦs, through exosomes, regulate the behavior of BCCs, by either inducing or reversing dormancy.

PROBLEM:Medical educators widely accept that health care providers need strong communication skills. The authors sought to develop a course incorporating improvisation to teach health professions students communication skills and build empathy.APPROACH:Teaching health care professionals to communicate more effectively with patients, the public, and each other is a goal of the Alan Alda Center for Communicating Science at Stony Brook University. The authors designed an interprofessional elective for medical, nursing, and dental students that differed in several respects from traditional communication training. The Communicating Science elective, which was offered by the Alda Center from 2012 to 2016, used verbal and nonverbal exercises, role-playing, and storytelling, including improvisation exercises, to teach students to communicate with empathy and clarity.OUTCOMES:In course evaluations completed by 76 students in 2012 and 2013, 100% said they would recommend the course to fellow students, saw the relevance of the course content to their careers, and desired more of the course content in their school's curriculum. As a result of this positive feedback, from 2014 to 2016, 10 hours of instruction pairing empathy and communication training was embedded in the preclinical curriculum at the Stony Brook University School of Medicine.NEXT STEPS:This course could be an effective model, and one that other institutions could employ, for improving communication skills and empathy in the next generation of health care professionals. Next steps include advocating for communication skills training to be embedded throughout the curriculum of a four-year medical school program.

Doctoral students in science disciplines spend countless hours learning how to conduct cutting-edge research but very little time learning to communicate the nature and significance of their science to people outside their field. To narrow this disparity, we created an unusual course titled Communicating Science for doctoral science trainees at Rutgers University. Our goal was to help students develop an advanced ability to communicate their research clearly and accurately and to emphasize its value and significance to diverse audiences. Course design included classroom instruction supplemented with improvisation, video recordings, and ample opportunity for students to practice and receive immediate, constructive feedback in a supportive environment. A multidisciplinary faculty with expertise in science, education, communication, and theater arts taught this course. PhD students came from diverse scientific disciplines, ranging from biology and chemistry to civil engineering. Students also completed a capstone project in which they worked with a professional in the academic or private sector to explore a possible career aspiration. Assessment was in the form of feedback on students' oral and poster presentations, and written abstracts about their research. Student evaluations and comments about course format and content were mostly positive and also provided input for ways to improve the course. We discovered that the diversity of scientific backgrounds among our students enhanced their ability to learn how to communicate their science to others outside their disciplines. We are leveraging the success of our initial course offering to reach other student and faculty groups at Rutgers.

Orthotopic liver transplant (OLT) remains the standard of care for end stage liver disease. To circumvent allo-rejection, OLT subjects receive gluococorticoids (GC). We investigated the effects of GC on endogenous mesenchymal stem (stromal) cells (MSCs) in OLT. This question is relevant because MSCs have regenerative potential and immune suppressor function. Phenotypic analyses of blood samples from 12 OLT recipients, at pre-anhepatic, anhepatic and post-transplant (2 h, Days 1 and 5) indicated a significant decrease in MSCs after GC injection. The MSCs showed better recovery in the blood from subjects who started with relatively low MSCs as compared to those with high levels at the prehepatic phase. This drop in MSCs appeared to be linked to GC since similar change was not observed in liver resection subjects. In order to understand the effects of GC on decrease MSC migration, in vitro studies were performed in transwell cultures. Untreated MSCs could not migrate towards the GC-exposed liver tissue, despite CXCR4 expression and the production of inflammatory cytokines from the liver cells. GC-treated MSCs were inefficient with respect to migration towards CXCL12, and this correlated with retracted cytoskeleton and motility. These dysfunctions were partly explained by decreases in the CXCL12/receptor axis. GC-associated decrease in MSCs in OLT recipients recovered post-transplant, despite poor migratory ability towards GC-exposed liver. In total, the study indicated that GC usage in transplant needs to be examined to determine if this could be reduced or avoided with adjuvant cell therapy.

Abstract Dormant breast cancers resurge as metastatic disease after a long dormancy period in the bone marrow, where cancer cells interact with mesenchymal stem cells (MSC). However, the nature of early interactions between breast cancer cells and MSCs in the bone marrow microenvironment that facilitate adaptation to a quiescent state remains poorly understood. Here, we report that breast cancer cells prime MSC to release exosomes containing distinct miRNA contents, such as miR-222/223, which in turn promotes quiescence in a subset of cancer cells and confers drug resistance. Building on these results, we developed a novel, nontoxic therapeutic strategy to target dormant breast cancer cells based on systemic administration of MSC loaded with antagomiR-222/223. In an immunodeficient mouse model of dormant breast cancer, this therapy sensitized breast cancer cells to carboplatin-based therapy and increased host survival. Overall, our findings illuminate the nature of the regulatory interactions between breast cancer cells and MSCs in the evolution of tumor dormancy and resurgence in the micrometastatic microenvironment of the bone marrow. Cancer Res; 76(19); 5832–44. ©2016 AACR.

Mesenchymal stem cells (MSCs), multipotent cells found in various adult tissues, are an attractive source of cells for cellular therapy and regenerative medicine. Reasons include their ease to expand, plasticity to generate cells of all germ layers, reduced ethical concerns, and ability to be available as ‘off the shelf’ cells for immediate use in transplantation. MSCs can respond differently to varying microenvironments to perform distinct immune functions. The microenvironment can also affect the developmental state of MSCs. Better understanding of how the microenvironment influences MSC multipotency is crucial for effective translational use of these cells in the clinic. This study tested the hypothesis that the changes in an inflammatory microenvironment will influence MSC function. To study these effects, an in vitro model of GvHD was developed by culturing MSCs in a three-dimensional model – rather than in a traditional monolayer – under physiological conditions. The GvHD model was generated based on a modified two-way mixed lymphocyte reaction. The cells were then assessed for phenotype, proliferation, and viability. Following growth in suspension, the MSCs continued to express an MSC phenotype. Proliferation and viability studies suggested that, although the cells were proliferating, they underwent cell death at a quicker rate than their two-dimensional culture counterparts. When cultured in the GvHD model, MSCs exhibited increased viability as compared to culture in the three-dimensional system alone. This in vitro GvHD model will elucidate factors within the inflammatory milieu that alter MSC multipotency. Identifying these factors will allow for more controlled and effective clinical use of MSCs.

The maternal immune system plays a critical role in the establishment, maintenance, and completion of a healthy pregnancy. However, the specific mechanisms utilized to achieve these goals are not well understood. Various cells and molecules of the immune system are key players in the development and function of the placenta and the fetus. Effector cells of the immune system act to promote and yet limit placental development. The T helper 1 (Th1)/T helper 2 (Th2) immune shift during pregnancy is well established. A fine balance between proinflammatory and anti-inflammatory influences is required. We herein review the evidence regarding maternal tolerance of fetal tissues and the underlying cell-mediated immune and humoral (hormones and cytokines) mechanisms. We also note the many unanswered questions in our understanding of these mechanisms. In addition, we summarize the clinical manifestations of an altered maternal immune system during pregnancy related to susceptibility to common viral, bacterial, and parasitic infections, as well as to autoimmune diseases.

Perinatal exposure to infectious agents with associated maternal immune activation (MIA) leads to neuroanatomical and behavioral dysregulation reminiscent of autism spectrum disorders. Persistent microglial activation as well as increased choline acetyltransferase (ChAT) activity in the basal forebrain (BF) are characteristic of autistic subjects. Previous studies have shown that medium from activated microglia promotes cholinergic differentiation of precursors in the BF. We sought to determine whether MIA in vivo would lead to a similar effect on developing BF neurons. Pregnant mice were treated with the viral mimic polyinosinic–polycytidylic acid (poly(I:C)) or saline. Poly(I:C) treatment resulted in increased production of cytokines and chemokines in fetal microglia and increased ChAT activity and cholinergic cell number in the perinatal BF. Whether microglial activation causes these changes is unclear. Examination of fetal brains from mice lacking interleukin-6 (IL-6 KOs) revealed an elevation in non–microglial-derived cytokines and chemokines over wild-type controls. Moreover, IL-6 KO offspring showed an elevation of ChAT activity even in the absence of poly(I:C) administration. These data suggest that elevations in cytokines and/or chemokines caused either by maternal poly(I:C) administration or by the absence of IL-6 are associated with alterations in cholinergic development in the BF.

Epidemiological studies have associated infection during pregnancy with increased risk of neurodevelopmental disorders in children, which is modeled in rodents by stimulating the immune system of pregnant dams with microorganisms or their mimics, such as poly(I:C) or LPS. In two prenatal mouse models, we show that in utero exposure of the fetus to cytokines/inflammatory mediators elicited by maternal immune stimulation with poly(I:C) yields offspring that exhibit a proinflammatory phenotype due to alterations in developmental programming of their immune system. Changes in the innate and adaptive immune elements of these pro-inflammatory offspring result in more robust responses following exposure to immune stimuli than those observed in control offspring from PBS-injected pregnant dams. In the first model, offspring from poly(I:C)-injected immunologically naïve dams showed heightened cellular and cytokine responses 4 h after injection of zymosan, a TLR2 agonist. In the second model, using dams with immunological memory, poly(I:C) injection during pregnancy produced offspring that showed preferential differentiation toward Th17 cell development, earlier onset of clinical symptoms of EAE, and more severe neurological deficits following immunization with MOG35-55. Such "fetal programming" in offspring from poly(I:C)-injected dams not only persists into neonatal and adult life, but also can have profound consequences on health and disease.

There is consensus among investigators studying Autism Spectrum Disorders (ASD) that the etiological basis involves environmental factors acting on the genetic susceptibility of the individual [1-5]. Over 100 candidate genes that may contribute to ASD susceptibility have been identified, and numerous environmental “triggers” have been suggested. Yet, the cause of ASD eludes clear definition and most likely is, as in most diseases, multi-factorial. However, several common immunological themes emerge from clinical and experimental studies of ASD, including persistent neuroinflammation, immune dysregulation, or autoim‐ mune manifestations in many autistic children. Thus, in addition to genetic and environ‐ mental factors, there is compelling evidence that immune factors also play a role in ASD. Abnormalities consistent with immune dysregulation, including abnormal or skewed T helper (Th) cell subsets and cytokine profiles, decreased lymphocyte numbers, decreased T cell mitogen responses, and an imbalance of serum immunoglobulin levels have been re‐ ported in children with ASD [6-11].

Background Immune cell trafficking into the CNS and other tissues plays important roles in health and disease. Rapid quantitative methods are not available that could be used to study many of the dynamic aspects of immune cell-tissue interactions. Methods We used pharmacokinetics and modeling to quantify and characterize the trafficking of radioactively labeled lymphocytes into brain and peripheral tissues. We used variance from two-way ANOVAs with 2 × 2 experimental designs to model the relative influences of lymphocytes and target tissues in trafficking. Results We found that in male CD-1 mice, about 1 in 5,000 intravenously injected lymphocytes entered each gram of brain. Uptake by brain was 2 to 3 times higher in naïve SJL females, but uptake by spleen and clearance from blood was lower, demonstrating a dichotomy in immune cell distribution. Treatment of CD-1 mice with lipopolysaccharide (LPS) increased immune cell uptake into brain but decreased uptake by spleen and axillary nodes. Conclusions Differences in brain uptake and in uptake by spleen between SJL and CD-1 mice were primarily determined by lymphocytes, whereas differences in uptake with LPS were primarily determined by lymphocytes for the brain but by the tissues for the spleen and the axillary lymph node. These results show that immune cells normally enter the CNS and that tissues and immune cells interact in ways that can be quantified by pharmacokinetic models.