CHCHD2 mediates glioblastoma cell proliferation, mitochondrial metabolism, hypoxia­induced invasion and therapeutic resistance.

Glioblastoma (GBM) is the most common and malignant primary brain tumor affecting adults and remains incurable. The mitochondrial coiled­coil­helix­coiled­coil­helix domain­containing protein 2 (CHCHD2) has been demonstrated to mediate mitochondrial respiration, nuclear gene expression and cell migration; however, evidence of this in GBM is lacking. In the present study, it was hypothesized that CHCHD2 may play a functional role in U87 GBM cells expressing the constitutively active epidermal growth factor receptor variant III (EGFRvIII). The amplification of the CHCHD2 gene was found to be associated with a decreased patient overall and progression­free survival. The CHCHD2 mRNA levels were increased in high­vs. low­grade glioma, IDH­wt GBMs, and in tumor vs. non­tumor tissue. Additionally, CHCHD2 protein expression was greatest in invasive, EGFRvIII­expressing patient­derived samples. The CRISPR­Cas9­mediated knockout of CHCHD2 in EGFRvIII­expressing U87 cells resulted in an altered mitochondrial respiration and glutathione status, in decreased cell growth and invasion under both normoxic and hypoxic conditions, and in an enhanced sensitivity to cytotoxic agents. CHCHD2 was distributed in both the mitochondria and nuclei of U87 and U87vIII cells, and the U87vIII cells exhibited a greater nuclear expression of CHCHD2 compared to isogenic U87 cells. Incubation under hypoxic conditions, serum starvation and the reductive unfolding of CHCHD2 induced the nuclear accumulation of CHCHD2 in both cell lines. Collectively, the findings of the present study indicate that CHCHD2 mediates a variety of GBM characteristics, and highlights mitonuclear retrograde signaling as a pathway of interest in GBM cell biology.

Matrix Hyaluronic Acid and Hypoxia Influence a CD133+ Subset of Patient-Derived Glioblastoma Cells.

Glioblastoma (GBM) displays diffusive invasion throughout the brain microenvironment, which is partially responsible for its short median survival rate (<15 months). Stem-like subpopulations (GBM stem-like cells, GSCs) are believed to play a central role in therapeutic resistance and poor patient prognosis. Given the extensive tissue remodeling and processes such as vessel co-option and regression that occur in the tumor microenvironment, it is essential to understand the role of metabolic constraint such as hypoxia on GBM cell populations. This work describes the use of a multidimensional gelatin hydrogel to culture patient-derived GBM cells, to evaluate the influence of hypoxia and the inclusion brain-mimetic hyaluronic acid on the relative activity of GSCs versus overall GBM cells. Notably, CD133+ GBM cell fraction is crucial for robust formation of tumor spheroids in multidimensional cultures. In addition, while the relative size of the CD133+ GBM subpopulation increased in response to both hypoxia and matrix-bound hyaluronan, we did not observe cell subtype-specific changes in invasion signaling pathway activation. Taken together, this study highlights the potential of biomimetic culture systems for resolving changes in the population dynamics and behavior of subsets of GBM specimens for the future development of precision medicine applications. Impact Statement This study describes a gelatin hydrogel platform to investigate the role of extracellular hyaluronic acid and hypoxia on the behavior of a CD133+ subset of cells within patient-derived glioblastoma (GBM) specimens. We report that the relative expansion of the CD133+ GBM stem cell-like population is strongly responsive to extracellular cues, highlighting the significance of biomimetic hydrogel models of the tumor microenvironment to investigate invasion and therapeutic response.

Crosstalk between microglia and patient-derived glioblastoma cells inhibit invasion in a three-dimensional gelatin hydrogel model.

BACKGROUND: Glioblastoma is the most common and deadly form of primary brain cancer, accounting for more than 13,000 new diagnoses annually in the USA alone. Microglia are the innate immune cells within the central nervous system, acting as a front-line defense against injuries and inflammation via a process that involves transformation from a quiescent to an activated phenotype. Crosstalk between GBM cells and microglia represents an important axis to consider in the development of tissue engineering platforms to examine pathophysiological processes underlying GBM progression and therapy. METHODS: This work used a brain-mimetic hydrogel system to study patient-derived glioblastoma specimens and their interactions with microglia. Here, glioblastoma cells were either cultured alone in 3D hydrogels or in co-culture with microglia in a manner that allowed secretome-based signaling but prevented direct GBM-microglia contact. Patterns of GBM cell invasion were quantified using a three-dimensional spheroid assay. Secretome and transcriptome (via RNAseq) were used to profile the consequences of GBM-microglia interactions. RESULTS: Microglia displayed an activated phenotype as a result of GBM crosstalk. Three-dimensional migration patterns of patient-derived glioblastoma cells showed invasion was significantly decreased in response to microglia paracrine signaling. Potential molecular mechanisms underlying with this phenotype were identified from bioinformatic analysis of secretome and RNAseq data. CONCLUSION: The data demonstrate a tissue engineered hydrogel platform can be used to investigate crosstalk between immune cells of the tumor microenvironment related to GBM progression. Such multi-dimensional models may provide valuable insight to inform therapeutic innovations to improve GBM treatment.

Causes of Cancer Re-Admissions: A Patient-Centered Approach.

PURPOSE: Patients with a cancer diagnosis have a high risk of re-admission during the 30 days after discharge. Clinicians, payers, and patients consider this to be an indicator of care quality. The causes of re-admission remain poorly understood. Retrospective chart reviews, used in most re-admission research, provide limited information regarding the causes of, and methods to reduce, re-admissions. This project sought to elicit the insights of those most affected by re-admission: the patients themselves. METHODS: We interviewed patients with cancer who were re-admitted to 2 urban teaching hospitals when they were hospitalized during their second admission. Trained interviewers used a semistructured interview guide to gather information on events just before the second admission, the patients' understanding of the cause of re-admission, and the patients' views about their readiness for discharge at the previous admission. Interviews were transcribed and analyzed, and themes were identified and explored. RESULTS: Three major themes were identified. First, most patients saw their re-admission as caused by problems that could not be treated in an outpatient setting. Second, the patients felt that they did not have sufficient resources at home to manage their care. Furthermore, the patients did not see the outpatient care team as a resource that they could call on for assistance. As a result, most of the decisions to return to the hospital were made by the patients themselves. CONCLUSION: The decision that leads to re-admission often happens at home, in response to unmanageable needs. Strengthening the bond between the care team and the patient, with the aim of providing care in the most appropriate setting, could decrease re-admissions in patients with cancer. Possible interventions include home visits, enhanced discharge planning, and telehealth services.

Maternal Choline Supplementation Modulates Placental Markers of Inflammation, Angiogenesis, and Apoptosis in a Mouse Model of Placental Insufficiency.

Dlx3 (distal-less homeobox 3) haploinsufficiency in mice has been shown to result in restricted fetal growth and placental defects. We previously showed that maternal choline supplementation (4X versus 1X choline) in the Dlx3+/- mouse increased fetal and placental growth in mid-gestation. The current study sought to test the hypothesis that prenatal choline would modulate indicators of placenta function and development. Pregnant Dlx3+/- mice consuming 1X (control), 2X, or 4X choline from conception were sacrificed at embryonic (E) days E10.5, E12.5, E15.5, and E18.5, and placentas and embryos were harvested. Data were analyzed separately for each gestational day controlling for litter size, fetal genotype (except for models including only +/- pups), and fetal sex (except when data were stratified by this variable). 4X choline tended to increase (p < 0.1) placental labyrinth size at E10.5 and decrease (p < 0.05) placental apoptosis at E12.5. Choline supplementation decreased (p < 0.05) expression of pro-angiogenic genes Eng (E10.5, E12.5, and E15.5), and Vegf (E12.5, E15.5); and pro-inflammatory genes Il1b (at E15.5 and 18.5), Tnfα (at E12.5) and Nfκb (at E15.5) in a fetal sex-dependent manner. These findings provide support for a modulatory effect of maternal choline supplementation on biomarkers of placental function and development in a mouse model of placental insufficiency.

Hypoxia activates enhanced invasive potential and endogenous hyaluronic acid production by glioblastoma cells.

Glioblastoma (GBM) is the most common, aggressive, and deadly form of adult brain cancer, and is associated with a short survival rate (median 12-15 months, 5+ year less than 5%). The complex tumor microenvironment includes matrix transitions at the tumor margin, such as gradations in hyaluronic acid (HA). In addition, metabolic stress induced by decreased oxygen content across the tumor may contribute to tumor progression. However, cross-talk between matrix composition and metabolic stress remains unclear. In this study, we fabricated an in vitro brain memetic HA-decorated gelatin hydrogel platform incorporating variable oxygen concentrations to mimic intra-tumoral hypoxia. We observed that EGFR status (wildtype vs. a constitutively active EGFRvIII mutant) of U87 GBM cells affected proliferation and metabolic activity in response to hypoxia and matrix-bound HA. The use of an invasion assay revealed that invasion was significantly enhanced in both cell types under hypoxia. Moreover, we observed compensatory secretion of soluble HA in cases of enhanced GBM cell invasion, consistent with our previous findings using other GBM cell lines. Interestingly, U87 GBM cells adapted to hypoxia by shifting toward a more anaerobic metabolic state, a mechanism that may contribute to GBM cell invasion. Collectively, these data demonstrate that the use of a three-dimensional hydrogel provides a robust method to study the impact of matrix composition and metabolic challenges on GBM cell invasion, a key factor contributing to the most common, aggressive, and deadly form of adult brain cancer.

Maternal choline supplementation during murine pregnancy modulates placental markers of inflammation, apoptosis and vascularization in a fetal sex-dependent manner.

INTRODUCTION: Normal placental vascular development is influenced by inflammatory, angiogenic and apoptotic processes, which may be modulated by choline through its role in membrane biosynthesis, cellular signaling and gene expression regulation. The current study examined the effect of maternal choline supplementation (MCS) on placental inflammatory, angiogenic and apoptotic processes during murine pregnancy. METHOD: Pregnant dams were randomized to receive 1, 2 or 4 times (X) the normal choline content of rodent diets, and tissues were harvested on embryonic day (E) 10.5, 12.5, 15.5 or 18.5 for gene expression, protein abundance and immunohistochemical analyses. RESULTS: The choline-induced changes in the inflammatory and angiogenic markers were a function of fetal sex. Specifically, 4X (versus 1X) choline reduced the transcript (P ≤ 0.05) and protein (P ≤ 0.06) expression of TNF-a and IL-1ß in the male placentas at E10.5 and E18.5, respectively. In the female placentas, 4X (versus 1X) choline modulated the transcript expression of Il1b in a biphasic pattern with reduced Il1b at E12.5 (P = 0.045) and E18.5 (P = 0.067) but increased Il1b at E15.5 (P = 0.031). MCS also induced an upregulation of Vegfa expression in the female placentas at E15.5 (P = 0.034; 4X versus 2X) and E18.5 (P = 0.026; 4X versus 1X). MCS decreased (P = 0.011; 4X versus 1X) placental apoptosis at E10.5. Additionally, the luminal area of the maternal spiral arteries was larger (P ≤ 0.05; 4X versus 1X) in response to extra choline throughout gestation. DISCUSSION: MCS during murine pregnancy has fetal sex-specific effects on placental inflammation and angiogenesis, with possible consequences on placental vascular development.

The Combined Influence of Hydrogel Stiffness and Matrix-Bound Hyaluronic Acid Content on Glioblastoma Invasion.

Glioblastoma (GBM) is the most common and lethal form of brain cancer. Its high mortality is associated with its aggressive invasion throughout the brain. The heterogeneity of stiffness and hyaluronic acid (HA) content within the brain makes it difficult to study invasion in vivo. A dextran-bead assay is employed to quantify GBM invasion within HA-functionalized gelatin hydrogels. Using a library of stiffness-matched hydrogels with variable levels of matrix-bound HA, it is reported that U251 GBM invasion is enhanced in softer hydrogels but reduced in the presence of matrix-bound HA. Inhibiting HA-CD44 interactions reduces invasion, even in hydrogels lacking matrix-bound HA. Analysis of HA biosynthesis suggests that GBM cells compensate for a lack of matrix-bound HA by producing soluble HA to stimulate invasion. Together, a robust method is showed to quantify GBM invasion over long culture times to reveal the coordinated effect of matrix stiffness, immobilized HA, and compensatory HA production on GBM invasion.

Using cost-effectiveness analysis to prioritize policy and programmatic approaches to physical activity promotion and obesity prevention in childhood.

Participation in recommended levels of physical activity promotes a healthy body weight and reduced chronic disease risk. To inform investment in prevention initiatives, we simulate the national implementation, impact on physical activity and childhood obesity and associated cost-effectiveness (versus the status quo) of six recommended strategies that can be applied throughout childhood to increase physical activity in US school, afterschool and childcare settings. In 2016, the Childhood Obesity Intervention Cost Effectiveness Study (CHOICES) systematic review process identified six interventions for study. A microsimulation model estimated intervention outcomes 2015-2025 including changes in mean MET-hours/day, intervention reach and cost per person, cost per MET-hour change, ten-year net costs to society and cases of childhood obesity prevented. First year reach of the interventions ranged from 90,000 youth attending a Healthy Afterschool Program to 31.3 million youth reached by Active School Day policies. Mean MET-hour/day/person increases ranged from 0.05 MET-hour/day/person for Active PE and Healthy Afterschool to 1.29 MET-hour/day/person for the implementation of New Afterschool Programs. Cost per MET-hour change ranged from cost saving to $3.14. Approximately 2500 to 110,000 cases of children with obesity could be prevented depending on the intervention implemented. All of the six interventions are estimated to increase physical activity levels among children and adolescents in the US population and prevent cases of childhood obesity. Results do not include other impacts of increased physical activity, including cognitive and behavioral effects. Decision-makers can use these methods to inform prioritization of physical activity promotion and obesity prevention on policy agendas.

Prepregnancy Obesity and Risks of Stillbirth.

BACKGROUND: We examined the association of maternal obesity with risk of stillbirth, focusing on whether the pattern of results varied by gestational age or maternal race-ethnicity or parity. METHODS: Analyses included 4,012 stillbirths and 1,121,234 liveborn infants delivered in California from 2007-2010. We excluded stillbirths due to congenital anomalies, women with hypertensive disorders or diabetes, and plural births, to focus on fetuses and women without these known contributing conditions. We used Poisson regression to estimate relative risks (RR) and 95% confidence intervals (CI). Separate models were run for stillbirths delivered at 20-23, 24-27, 28-31, 32-36, 37-41 weeks, relative to liveborn deliveries at 37-41 weeks. RESULTS: For stillbirth at 20-23 weeks, RRs were elevated for all race-ethnicity and parity groups. The RR for a 20-unit change in BMI (which reflects the approximate BMI difference between a normal weight and an Obese III woman) was 3.5 (95% CI 2.2, 5.6) for nulliparous white women and ranged from 1.8 to 5.0 for other sub-groups. At 24-27 weeks, the association was significant (p<0.05) only for multiparous non-Hispanic whites; at 28-31 weeks, for multiparous whites and nulliparous whites and blacks; at 32-36 weeks, for multiparous whites and nulliparous blacks; and at 37-41 weeks, for all groups except nulliparous blacks. The pattern of results was similar when restricted to stillbirths due to unknown causes and somewhat stronger when restricted to stillbirths attributable to obstetric causes. CONCLUSION: Increased risks were observed across all gestational ages, and some evidence of heterogeneity of the associations was observed by race-ethnicity and parity.