Noninvasive assessment of diffusion hypoxia following administration of nitrous oxide-oxygen.

The phenomenon of diffusion hypoxia is commonly believed to occur unless nitrous oxide-oxygen inhalation sedation is followed by "washout" with 100% oxygen for 5 minutes upon termination of the flow of nitrous oxide. When systematically studied, however, this phenomenon generally appears to be unfounded. The present study evaluated the effect of breathing room air instead of 100% oxygen in healthy (ASA 1) human volunteers following administration of sedative concentrations of nitrous oxide. The occurrence of hypoxia was determined objectively, using pulse oximetry and a standardized psychomotor skills test (Trieger test). Diffusion hypoxia was not observed using these criteria.

Hypoxia-induced transcriptional regulation through chromatin modifications and NC2 function

In response to tumor hypoxia, specific genes that promote angiogenesis, proliferation, and survival are induced. Globally, I find that hypoxia induces a mixed pattern of histone modifications that are typically associated with either transcriptional activation or repression. Furthermore, I find that selective activation of hypoxia-inducible genes occurs simultaneously with widespread repression of transcription. I analyzed histone modifications at the core promoters of hypoxia-repressed and -activated genes and find that distinct patterns of histone modifications correlate with transcriptional activity. Additionally, I discovered that trimethylated H3-K4, a modification generally associated with transcriptional activation, is induced at both hypoxia-activated and repressed genes, suggesting a novel pattern of histone modifications induced during hypoxia. ^ In order to determine the mechanism of hypoxia-induced widespread repression of transcription, I focused my studies on negative cofactor 2 (NC2). Previously, we found that hypoxia-induced repression of the alpha-fetoprotein (AFP) gene occurs during preinitiation complex (PIC) assembly, and I find that NC2, an inhibitor of PIC assembly, is induced during hypoxia. Moreover, I find that the beta subunit of NC2 is essential for hypoxia-mediated repression of AFP, as well as the widespread repression of transcription observed during hypoxia. Previous data in Drosophila and S. cerevisiae indicate that NC2 functions as either an activator or a repressor of transcription. The mechanism of NC2-mediated activation remains unclear; although, Drosophila NC2 function correlates with specific core promoter elements. I tested if NC2 activates transcription in mammalian cells using this core promoter-specific model as a guide. Utilizing site-specific mutagenesis, I find that NC2 function in mammalian cells is not dependent upon specific core promoter elements; however, I do find that mammalian NC2 does function in a gene-specific manner as either an activator or repressor of transcription during hypoxia. Furthermore, I find that binding of the alpha subunit of NC2 specifically correlates with NC2-mediated transcriptional activation. NC2α and NC2β are both required for NC2-mediated transcriptional activation; whereas, NC2β alone is required for hypoxia-induced transcriptional repression. Together, these data indicate that hypoxia mediates changes in gene expression through both chromatin modifications and NC2 function. ^

Computer simulation of glioma growth and morphology.

Despite major advances in the study of glioma, the quantitative links between intra-tumor molecular/cellular properties, clinically observable properties such as morphology, and critical tumor behaviors such as growth and invasiveness remain unclear, hampering more effective coupling of tumor physical characteristics with implications for prognosis and therapy. Although molecular biology, histopathology, and radiological imaging are employed in this endeavor, studies are severely challenged by the multitude of different physical scales involved in tumor growth, i.e., from molecular nanoscale to cell microscale and finally to tissue centimeter scale. Consequently, it is often difficult to determine the underlying dynamics across dimensions. New techniques are needed to tackle these issues. Here, we address this multi-scalar problem by employing a novel predictive three-dimensional mathematical and computational model based on first-principle equations (conservation laws of physics) that describe mathematically the diffusion of cell substrates and other processes determining tumor mass growth and invasion. The model uses conserved variables to represent known determinants of glioma behavior, e.g., cell density and oxygen concentration, as well as biological functional relationships and parameters linking phenomena at different scales whose specific forms and values are hypothesized and calculated based on in vitro and in vivo experiments and from histopathology of tissue specimens from human gliomas. This model enables correlation of glioma morphology to tumor growth by quantifying interdependence of tumor mass on the microenvironment (e.g., hypoxia, tissue disruption) and on the cellular phenotypes (e.g., mitosis and apoptosis rates, cell adhesion strength). Once functional relationships between variables and associated parameter values have been informed, e.g., from histopathology or intra-operative analysis, this model can be used for disease diagnosis/prognosis, hypothesis testing, and to guide surgery and therapy. In particular, this tool identifies and quantifies the effects of vascularization and other cell-scale glioma morphological characteristics as predictors of tumor-scale growth and invasion.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

Computer simulation of glioma growth and morphology.

Despite major advances in the study of glioma, the quantitative links between intra-tumor molecular/cellular properties, clinically observable properties such as morphology, and critical tumor behaviors such as growth and invasiveness remain unclear, hampering more effective coupling of tumor physical characteristics with implications for prognosis and therapy. Although molecular biology, histopathology, and radiological imaging are employed in this endeavor, studies are severely challenged by the multitude of different physical scales involved in tumor growth, i.e., from molecular nanoscale to cell microscale and finally to tissue centimeter scale. Consequently, it is often difficult to determine the underlying dynamics across dimensions. New techniques are needed to tackle these issues. Here, we address this multi-scalar problem by employing a novel predictive three-dimensional mathematical and computational model based on first-principle equations (conservation laws of physics) that describe mathematically the diffusion of cell substrates and other processes determining tumor mass growth and invasion. The model uses conserved variables to represent known determinants of glioma behavior, e.g., cell density and oxygen concentration, as well as biological functional relationships and parameters linking phenomena at different scales whose specific forms and values are hypothesized and calculated based on in vitro and in vivo experiments and from histopathology of tissue specimens from human gliomas. This model enables correlation of glioma morphology to tumor growth by quantifying interdependence of tumor mass on the microenvironment (e.g., hypoxia, tissue disruption) and on the cellular phenotypes (e.g., mitosis and apoptosis rates, cell adhesion strength). Once functional relationships between variables and associated parameter values have been informed, e.g., from histopathology or intra-operative analysis, this model can be used for disease diagnosis/prognosis, hypothesis testing, and to guide surgery and therapy. In particular, this tool identifies and quantifies the effects of vascularization and other cell-scale glioma morphological characteristics as predictors of tumor-scale growth and invasion.

An Innovative Phase I Trial Design Allowing for the Identification of Multiple Potential Maximum Tolerated Doses with Combination Therapy of Targeted Agents

Treatment for cancer often involves combination therapies used both in medical practice and clinical trials. Korn and Simon listed three reasons for the utility of combinations: 1) biochemical synergism, 2) differential susceptibility of tumor cells to different agents, and 3) higher achievable dose intensity by exploiting non-overlapping toxicities to the host. Even if the toxicity profile of each agent of a given combination is known, the toxicity profile of the agents used in combination must be established. Thus, caution is required when designing and evaluating trials with combination therapies. Traditional clinical design is based on the consideration of a single drug. However, a trial of drugs in combination requires a dose-selection procedure that is vastly different than that needed for a single-drug trial. When two drugs are combined in a phase I trial, an important trial objective is to determine the maximum tolerated dose (MTD). The MTD is defined as the dose level below the dose at which two of six patients experience drug-related dose-limiting toxicity (DLT). In phase I trials that combine two agents, more than one MTD generally exists, although all are rarely determined. For example, there may be an MTD that includes high doses of drug A with lower doses of drug B, another one for high doses of drug B with lower doses of drug A, and yet another for intermediate doses of both drugs administered together. With classic phase I trial designs, only one MTD is identified. Our new trial design allows identification of more than one MTD efficiently, within the context of a single protocol. The two drugs combined in our phase I trial are temsirolimus and bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway which is fundamental for tumor growth and metastasis. One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxia inducible factor 1α (HIF-1α) which mediates responses to hypoxic conditions. Temsirolimus has resulted in reduced levels of HIF-1α making this an ideal combination therapy. Dr. Donald Berry developed a trial design schema for evaluating low, intermediate and high dose levels of two drugs given in combination as illustrated in a recently published paper in Biometrics entitled “A Parallel Phase I/II Clinical Trial Design for Combination Therapies.” His trial design utilized cytotoxic chemotherapy. We adapted this design schema by incorporating greater numbers of dose levels for each drug. Additional dose levels are being examined because it has been the experience of phase I trials that targeted agents, when given in combination, are often effective at dosing levels lower than the FDA-approved dose of said drugs. A total of thirteen dose levels including representative high, intermediate and low dose levels of temsirolimus with representative high, intermediate, and low dose levels of bevacizumab will be evaluated. We hypothesize that our new trial design will facilitate identification of more than one MTD, if they exist, efficiently and within the context of a single protocol. Doses gleaned from this approach could potentially allow for a more personalized approach in dose selection from among the MTDs obtained that can be based upon a patient’s specific co-morbid conditions or anticipated toxicities.

Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation.

Medulloblastoma, one of the most malignant brain tumors in children, is thought to arise from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer of the cerebellum. However, the mechanism of tumorigenesis remains unknown for the majority of medulloblastomas. In this study, we found that many human medulloblastomas express significantly elevated levels of both myc oncogenes, regulators of neural progenitor proliferation, and REST/NRSF, a transcriptional repressor of neuronal differentiation genes. Previous studies have shown that neither c-Myc nor REST/NRSF alone could cause tumor formation. To determine whether c-Myc and REST/NRSF act together to cause medulloblastomas, we used a previously established cell line derived from external granule layer stem cells transduced with activated c-myc (NSC-M). These immortalized NSCs were able to differentiate into neurons in vitro. In contrast, when the cells were engineered to express a doxycycline-regulated REST/NRSF transgene (NSC-M-R), they no longer underwent terminal neuronal differentiation in vitro. When injected into intracranial locations in mice, the NSC-M cells did not form tumors either in the cerebellum or in the cerebral cortex. In contrast, the NSC-M-R cells did produce tumors in the cerebellum, the site of human medulloblastoma formation, but not when injected into the cerebral cortex. Furthermore, the NSC-M-R tumors were blocked from terminal neuronal differentiation. In addition, countering REST/NRSF function blocked the tumorigenic potential of NSC-M-R cells. To our knowledge, this is the first study in which abnormal expression of a sequence-specific DNA-binding transcriptional repressor has been shown to contribute directly to brain tumor formation. Our findings indicate that abnormal expression of REST/NRSF and Myc in NSCs causes cerebellum-specific tumors by blocking neuronal differentiation and thus maintaining the "stemness" of these cells. Furthermore, these results suggest that such a mechanism plays a role in the formation of human medulloblastoma.

Effects of hypoxic-ischemic encephalopathy and whole-body hypothermia on neonatal auditory function: a pilot study.

We assessed the effects of hypoxic-ischemic encephalopathy (HIE) and whole-body hypothermia therapy on auditory brain stem evoked responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We performed serial assessments of ABRs and DPOAEs in newborns with moderate or severe HIE, randomized to hypothermia ( N = 4) or usual care ( N = 5). Participants were five boys and four girls with mean gestational age (standard deviation) of 38.9 (1.8) weeks. During the first week of life, peripheral auditory function, as measured by the DPOAEs, was disrupted in all nine subjects. ABRs were delayed but central transmission was intact, suggesting a peripheral rather than a central neural insult. By 3 weeks of age, peripheral auditory function normalized. Hypothermia temporarily prolonged the ABR, more so for waves generated higher in the brain stem but the effects reversed quickly on rewarming. Neonatal audiometric testing is feasible, noninvasive, and capable of enhancing our understanding of the effects of HIE and hypothermia on auditory function.

Changes in the PQRST intervals and heart rate variability associated with rewarming in two newborns undergoing hypothermia therapy.

BACKGROUND: Little is known about the effects of hypothermia therapy and subsequent rewarming on the PQRST intervals and heart rate variability (HRV) in term newborns with hypoxic-ischemic encephalopathy (HIE). OBJECTIVES: This study describes the changes in the PQRST intervals and HRV during rewarming to normal core body temperature of 2 newborns with HIE after hypothermia therapy. METHODS: Within 6 h after birth, 2 newborns with HIE were cooled to a core body temperature of 33.5 degrees C for 72 h using a cooling blanket, followed by gradual rewarming (0.5 degrees C per hour) until the body temperature reached 36.5 degrees C. Custom instrumentation recorded the electrocardiogram from the leads used for clinical monitoring of vital signs. Generalized linear mixed models were calculated to estimate temperature-related changes in PQRST intervals and HRV. Results: For every 1 degrees C increase in body temperature, the heart rate increased by 9.2 bpm (95% CI 6.8-11.6), the QTc interval decreased by 21.6 ms (95% CI 17.3-25.9), and low and high frequency HRV decreased by 0.480 dB (95% CI 0.052-0.907) and 0.938 dB (95% CI 0.460-1.416), respectively. CONCLUSIONS: Hypothermia-induced changes in the electrocardiogram should be monitored carefully in future studies.

Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma.

The mammalian target of rapamycin (MTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.

Outcomes of safety and effectiveness in a multicenter randomized, controlled trial of whole-body hypothermia for neonatal hypoxic-ischemic encephalopathy.

BACKGROUND: Whole-body hypothermia reduced the frequency of death or moderate/severe disabilities in neonates with hypoxic-ischemic encephalopathy in a randomized, controlled multicenter trial. OBJECTIVE: Our goal was to evaluate outcomes of safety and effectiveness of hypothermia in infants up to 18 to 22 months of age. DESIGN/METHODS: A priori outcomes were evaluated between hypothermia (n = 102) and control (n = 106) groups. RESULTS: Encephalopathy attributable to causes other than hypoxia-ischemia at birth was not noted. Inotropic support (hypothermia, 59% of infants; control, 56% of infants) was similar during the 72-hour study intervention period in both groups. Need for blood transfusions (hypothermia, 24%; control, 24%), platelet transfusions (hypothermia, 20%; control, 12%), and volume expanders (hypothermia, 54%; control, 49%) was similar in the 2 groups. Among infants with persistent pulmonary hypertension (hypothermia, 25%; control, 22%), nitric-oxide use (hypothermia, 68%; control, 57%) and placement on extracorporeal membrane oxygenation (hypothermia, 4%; control, 9%) was similar between the 2 groups. Non-central nervous system organ dysfunctions occurred with similar frequency in the hypothermia (74%) and control (73%) groups. Rehospitalization occurred among 27% of the infants in the hypothermia group and 42% of infants in the control group. At 18 months, the hypothermia group had 24 deaths, 19 severe disabilities, and 2 moderate disabilities, whereas the control group had 38 deaths, 25 severe disabilities, and 1 moderate disability. Growth parameters were similar between survivors. No adverse outcomes were noted among infants receiving hypothermia with transient reduction of temperature below a target of 33.5 degrees C at initiation of cooling. There was a trend in reduction of frequency of all outcomes in the hypothermia group compared with the control group in both moderate and severe encephalopathy categories. CONCLUSIONS: Although not powered to test these secondary outcomes, whole-body hypothermia in infants with encephalopathy was safe and was associated with a consistent trend for decreasing frequency of each of the components of disability.

Volumetric and anatomical MRI for hypoxic-ischemic encephalopathy: relationship to hypothermia therapy and neurosensory impairments.

OBJECTIVE: To relate volumetric magnetic resonance imaging (MRI) findings to hypothermia therapy and neurosensory impairments. STUDY DESIGN: Newborns > or =36 weeks' gestation with hypoxic-ischemic encephalopathy who participated in the National Institute of Child Health and Human Development hypothermia randomized trial at our center were eligible. We determined the relationship between hypothermia treatment and usual care (control) to absolute and relative cerebral tissue volumes. Furthermore, we correlated brain volumes with death or neurosensory impairments at 18 to 22 months. RESULT: Both treatment groups were comparable before randomization. Total brain tissue volumes did not differ in relation to treatment assignment. However, relative volumes of subcortical white matter were significantly larger in hypothermia-treated than control infants. Furthermore, relative total brain volumes correlated significantly with death or neurosensory impairments. Relative volumes of the cortical gray and subcortical white matter also correlated significantly with Bayley Scales psychomotor development index. CONCLUSION: Selected volumetric MRI findings correlated with hypothermia therapy and neurosensory impairments. Larger studies using MRI brain volumes as a secondary outcome measure are needed.

UNDERSTANDING NANOG'S ROLE IN CANCER BIOLOGY

Understanding Nanog’s Role in Cancer Biology Mark Daniel Badeaux Supervisory Professor Dean Tang, PhD The cancer stem cell model holds that tumor heterogeneity and population-level immortality are driven by a subset of cells within the tumor, termed cancer stem cells. Like embryonic or somatic stem cells, cancer stem cells are believed to possess self-renewal capacity and the ability to give rise to a multitude of varieties of daughter cell. Because of cancer’s implied connections to authentic stem cells, we screened a variety of prostate cancer cell lines and primary tumors in order to determine if any notable ‘stemness’ genes were expressed in malignant growths. We found a promising lead in Nanog, a central figure in maintaining embryonic stem cell pluripotency, and through a variety of experiments in which we diminished Nanog expression, found that it may play a significant role in prostate cancer development. We then created a transgenic mouse model in which we targeted Nanog expression to keratin 14-expressing in order to assess its potential contribution to tumorigenesis. We found a variety of developmental abnormalities and altered differentiation patterns in our model , but much to our chagrin we observed neither spontaneous tumor formation nor premalignant changes in these mice, but instead surprisingly found that high levels of Nanog expression inhibited tumor formation in a two-stage skin carcinogenesis model. We also noted a depletion of skin stem cell populations, which underlies the wound-healing defect our mice harbor as well. Gene expression analysis shows a reduction in c-Jun and Bmp5, two genes whose loss inhibits skin tumor development and reduces stem cell counts respectively. As we further explored Nanog’s activity in prostate cancer, it became apparent that the protein oftentimes was not expressed. Emboldened by the competing endogenous RNA (ceRNA) hypothesis, we identified the Nanog 3’UTR as a regulator of the tumor suppressive microRNA 128a (miR-128a), which includes known oncogenes such as Bmi1 among its authentic targets. Future work will necessarily involve discerning instances in which Nanog mRNA is the biologically relevant molecule, as well as identifying additional mRNA species which may serve solely as a molecular sink for miR-128a.

PURINERGIC SIGNALING REGULATES FILOPODIA-INDUCED ZIPPERING

The molecular mechanisms that mediate endometrial cancer invasion and metastasis remain poorly understood. This is a significant clinical problem, as there is no definitive cure for metastatic disease. The purinergic pathway’s generation of adenosine and its activation of the adenosine receptor A2B (A2BR) induces cell-cell adhesion to promote barrier function. This barrier function is known to be important in maintaining homeostasis during hypoxia, trauma, and sepsis. Loss of this epithelial barrier function provides a considerable advantage for carcinoma progression, as loss of cell-cell adhesions supports proliferation, aberrant signaling, epithelial-to-mesenchymal transition, invasion, and metastasis. The present work provides strong evidence that CD73-generated adenosine actively promotes cell-cell adhesion in carcinoma cells by filopodia-induced zippering. Adenosine-generating ecto-enzyme, CD73, was down-regulated in moderately- and poorly-differentiated, invasive, and metastatic endometrial carcinomas. CD73 expression and enzyme activity in normal endometrium and endometrial carcinomas was significantly correlated to the epithelial phenotype. Barrier function in normal epithelial cells of the endometrium was dependent on stress-induced generation of adenosine by CD73 and adenosine’s activation of A2BR. This same mechanism inhibited endometrial carcinoma cell migration and invasion. Finally, adenosine’s activation of A2BR induced the formation of filopodia that promoted the re-forming of cell-cell adhesions in carcinoma cells. Overall, these studies identified purinergic pathway-induced filopodia to be a novel mechanism of adenosine’s barrier function and a mechanism that has to be avoided/down-regulated by endometrial carcinoma cells attempting to lose attachment with their neighboring cells. These results provide insight into the molecular mechanisms of endometrial cancer invasion. In addition, because loss of cell-cell adhesions has been closely linked to therapy resistance in cancer, these results provide a rational clinical strategy for the re-establishment of cell-cell adhesions to potentially increase therapeutic sensitivity. In contrast to other molecular mechanisms regulating cell-cell adhesions, the purinergic pathway is clinically druggable, with agonists and antagonists currently being tested in clinical trials of various diseases.