Systematic Review of Cerebrospinal Fluid Biomarker Discovery in Neuro-Oncology: A Roadmap to Standardization and Clinical Application.

Effective diagnosis, prognostication, and management of CNS malignancies traditionally involves invasive brain biopsies that pose significant risk to the patient. Sampling and molecular profiling of cerebrospinal fluid (CSF) is a safer, rapid, and noninvasive alternative that offers a snapshot of the intracranial milieu while overcoming the challenge of sampling error that plagues conventional brain biopsy. Although numerous biomarkers have been identified, translational challenges remain, and standardization of protocols is necessary. Here, we systematically reviewed 141 studies (Medline, SCOPUS, and Biosis databases; between January 2000 and September 29, 2022) that molecularly profiled CSF from adults with brain malignancies including glioma, brain metastasis, and primary and secondary CNS lymphomas. We provide an overview of promising CSF biomarkers, propose CSF reporting guidelines, and discuss the various considerations that go into biomarker discovery, including the influence of blood-brain barrier disruption, cell of origin, and site of CSF acquisition (eg, lumbar and ventricular). We also performed a meta-analysis of proteomic data sets, identifying biomarkers in CNS malignancies and establishing a resource for the research community.

Breed and loading history influence in vivo skeletal strain patterns in pre-pubertal female chickens.

The influence of loading history on in vivo strains within a given specie remains poorly understood, and although in vivo strains have been measured at the hindlimb bones of various species, strains engendered during modes of activity other than locomotion are lacking, particularly in non-human species. For commercial egg-laying chickens specifically, there is an interest in understanding their bones' mechanical behaviour, particularly during youth, to develop early interventions to prevent the high incidence of osteoporosis in this population. We measured in vivo mechanical strains at the tibiotarsus midshaft during steady activities (ground, uphill, downhill locomotion) and non-steady activities (perching, jumping, aerial transition landing) in 48 pre-pubescent female (egg-laying) chickens from two breeds that were reared in three different housing systems, allowing varying amounts and types of physical activity. Mechanical strain patterns differed between breeds, and were dependent on the activity performed. Mechanical strains were also affected by rearing environment: chickens that were restricted from performing dynamic load bearing activity due to caged-housing generally exhibited higher mechanical strain levels during steady, but not non-steady activities, compared to chickens with prior dynamic load-bearing activity experience. Among chickens with prior experience of dynamic load bearing activity, those reared in housing systems that allowed more frequent physical activity did not exhibit lower mechanical strains. In all groups, the tibiotarsus was subjected to a loading environment consisting of a combination of axial compression, bending, and torsion, with torsion being the predominant source of strain. Aerial transition landing produced the highest strain levels with unusual strain patterns compared to other activities, suggesting it may produce the strongest anabolic response. These results exemplify how different breeds within a given specie adapt to maintain different patterns of mechanical strains, and how benefits of physical activity in terms of resistance to strain are activity-type dependent and do not necessarily increase with increased physical activity. These findings directly inform controlled loading experiments aimed at studying the bone mechanoresponse in young female chickens and can also be associated to measures of bone morphology and material properties to understand how these features influence bone mechanical properties in vivo.

Cerebrospinal fluid methylome-based liquid biopsies for accurate malignant brain neoplasm classification.

BACKGROUND: Resolving the differential diagnosis between brain metastases (BM), glioblastomas (GBM), and central nervous system lymphomas (CNSL) is an important dilemma for the clinical management of the main three intra-axial brain tumor types. Currently, treatment decisions require invasive diagnostic surgical biopsies that carry risks and morbidity. This study aimed to utilize methylomes from cerebrospinal fluid (CSF), a biofluid proximal to brain tumors, for reliable non-invasive classification that addresses limitations associated with low target abundance in existing approaches. METHODS: Binomial GLMnet classifiers of tumor type were built, in fifty iterations of 80% discovery sets, using CSF methylomes obtained from 57 BM, GBM, CNSL, and non-neoplastic control patients. Publicly-available tissue methylation profiles (N = 197) on these entities and normal brain parenchyma were used for validation and model optimization. RESULTS: Models reliably distinguished between BM (area under receiver operating characteristic curve [AUROC] = 0.93, 95% confidence interval [CI]: 0.71-1.0), GBM (AUROC = 0.83, 95% CI: 0.63-1.0), and CNSL (AUROC = 0.91, 95% CI: 0.66-1.0) in independent 20% validation sets. For validation, CSF-based methylome signatures reliably distinguished between tumor types within external tissue samples and tumors from non-neoplastic controls in CSF and tissue. CSF methylome signals were observed to align closely with tissue signatures for each entity. An additional set of optimized CSF-based models, built using tumor-specific features present in tissue data, showed enhanced classification accuracy. CONCLUSIONS: CSF methylomes are reliable for liquid biopsy-based classification of the major three malignant brain tumor types. We discuss how liquid biopsies may impact brain cancer management in the future by avoiding surgical risks, classifying unbiopsiable tumors, and guiding surgical planning when resection is indicated.

Leveraging the CSF proteome toward minimally-invasive diagnostics surveillance of brain malignancies.

Background: Diagnosis and prognostication of intra-axial brain tumors hinges on invasive brain sampling, which carries risk of morbidity. Minimally-invasive sampling of proximal fluids, also known as liquid biopsy, can mitigate this risk. Our objective was to identify diagnostic and prognostic cerebrospinal fluid (CSF) proteomic signatures in glioblastoma (GBM), brain metastases (BM), and primary central nervous system lymphoma (CNSL). Methods: CSF samples were retrospectively retrieved from the Penn State Neuroscience Biorepository and profiled using shotgun proteomics. Proteomic signatures were identified using machine learning classifiers and survival analyses. Results: Using 30 µL CSF volumes, we recovered 755 unique proteins across 73 samples. Proteomic-based classifiers identified malignancy with area under the receiver operating characteristic (AUROC) of 0.94 and distinguished between tumor entities with AUROC ≥0.95. More clinically relevant triplex classifiers, comprised of just three proteins, distinguished between tumor entities with AUROC of 0.75-0.89. Novel biomarkers were identified, including GAP43, TFF3 and CACNA2D2, and characterized using single cell RNA sequencing. Survival analyses validated previously implicated prognostic signatures, including blood-brain barrier disruption. Conclusions: Reliable classification of intra-axial malignancies using low CSF volumes is feasible, allowing for longitudinal tumor surveillance.

Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma.

MYC-driven medulloblastoma (MB) is an aggressive pediatric brain tumor characterized by therapy resistance and disease recurrence. Here, we integrated data from unbiased genetic screening and metabolomic profiling to identify multiple cancer-selective metabolic vulnerabilities in MYC-driven MB tumor cells, which are amenable to therapeutic targeting. Among these targets, dihydroorotate dehydrogenase (DHODH), an enzyme that catalyzes de novo pyrimidine biosynthesis, emerged as a favorable candidate for therapeutic targeting. Mechanistically, DHODH inhibition acts on target, leading to uridine metabolite scarcity and hyperlipidemia, accompanied by reduced protein O-GlcNAcylation and c-Myc degradation. Pyrimidine starvation evokes a metabolic stress response that leads to cell-cycle arrest and apoptosis. We further show that an orally available small-molecule DHODH inhibitor demonstrates potent mono-therapeutic efficacy against patient-derived MB xenografts in vivo. The reprogramming of pyrimidine metabolism in MYC-driven medulloblastoma represents an unappreciated therapeutic strategy and a potential new class of treatments with stronger cancer selectivity and fewer neurotoxic sequelae.

Multi-level cellular and functional annotation of single-cell transcriptomes using scPipeline.

Single-cell RNA-sequencing (scRNA-seq) offers functional insight into complex biology, allowing for the interrogation of cellular populations and gene expression programs at single-cell resolution. Here, we introduce scPipeline, a single-cell data analysis toolbox that builds on existing methods and offers modular workflows for multi-level cellular annotation and user-friendly analysis reports. Advances to scRNA-seq annotation include: (i) co-dependency index (CDI)-based differential expression, (ii) cluster resolution optimization using a marker-specificity criterion, (iii) marker-based cell-type annotation with Miko scoring, and (iv) gene program discovery using scale-free shared nearest neighbor network (SSN) analysis. Both unsupervised and supervised procedures were validated using a diverse collection of scRNA-seq datasets and illustrative examples of cellular transcriptomic annotation of developmental and immunological scRNA-seq atlases are provided herein. Overall, scPipeline offers a flexible computational framework for in-depth scRNA-seq analysis.

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma.

Recurrence of solid tumors renders patients vulnerable to advanced, treatment-refractory disease state with mutational and oncogenic landscape distinctive from initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profile barcoded tumor stem cell populations through therapy at tumor initiation, MRD, and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors show distinct patterns of recurrence in which clonal populations exhibit either a pre-existing fitness advantage or an equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing reveals a tumor-intrinsic immunomodulatory signature with prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from patients with GBM. Our results provide insight into the innate and therapy-driven dynamics of human GBM and the prognostic value of interrogating the MRD state in solid cancers.

The proteomic landscape of glioblastoma recurrence reveals novel and targetable immunoregulatory drivers.

Glioblastoma (GBM) is characterized by extensive cellular and genetic heterogeneity. Its initial presentation as primary disease (pGBM) has been subject to exhaustive molecular and cellular profiling. By contrast, our understanding of how GBM evolves to evade the selective pressure of therapy is starkly limited. The proteomic landscape of recurrent GBM (rGBM), which is refractory to most treatments used for pGBM, are poorly known. We, therefore, quantified the transcriptome and proteome of 134 patient-derived pGBM and rGBM samples, including 40 matched pGBM-rGBM pairs. GBM subtypes transition from pGBM to rGBM towards a preferentially mesenchymal state at recurrence, consistent with the increasingly invasive nature of rGBM. We identified immune regulatory/suppressive genes as important drivers of rGBM and in particular 2-5-oligoadenylate synthase 2 (OAS2) as an essential gene in recurrent disease. Our data identify a new class of therapeutic targets that emerge from the adaptive response of pGBM to therapy, emerging specifically in recurrent disease and may provide new therapeutic opportunities absent at pGBM diagnosis.

Extracellular ATP and its derivatives provide spatiotemporal guidance for bone adaptation to wide spectrum of physical forces.

ATP is a ubiquitous intracellular molecule critical for cellular bioenergetics. ATP is released in response to mechanical stimulation through vesicular release, small tears in cellular plasma membranes, or when cells are destroyed by traumatic forces. Extracellular ATP is degraded by ecto-ATPases to form ADP and eventually adenosine. ATP, ADP, and adenosine signal through purinergic receptors, including seven P2X ATP-gated cation channels, seven G-protein coupled P2Y receptors responsive to ATP and ADP, and four P1 receptors stimulated by adenosine. The goal of this review is to build a conceptual model of the role of different components of this complex system in coordinating cellular responses that are appropriate to the degree of mechanical stimulation, cell proximity to the location of mechanical injury, and time from the event. We propose that route and amount of ATP release depend on the scale of mechanical forces, ranging from vesicular release of small ATP boluses upon membrane deformation, to leakage of ATP through resealable plasma membrane tears, to spillage of cellular content due to destructive forces. Correspondingly, different P2 receptors responsive to ATP will be activated according to their affinity at the site of mechanical stimulation. ATP is a small molecule that readily diffuses through the environment, bringing the signal to the surrounding cells. ATP is also degraded to ADP which can stimulate a distinct set of P2 receptors. We propose that depending on the magnitude of mechanical forces and distance from the site of their application, ATP/ADP profiles will be different, allowing the relay of information about tissue level injury and proximity. Lastly, ADP is degraded to adenosine acting via its P1 receptors. The presence of large amounts of adenosine without ATP, indicates that an active source of ATP release is no longer present, initiating the transition to the recovery phase. This model consolidates the knowledge regarding the individual components of the purinergic system into a conceptual framework of choreographed responses to physical forces.

Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis.

Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.

3D Image Registration Marginally Improves the Precision of HR-pQCT Measurements Compared to Cross-Sectional-Area Registration in Adults With Osteogenesis Imperfecta.

Repositioning error in longitudinal high-resolution peripheral-quantitative computed tomography (HR-pQCT) imaging can lead to different bone volumes being assessed over time. To identify the same bone volumes at each time point, image registration is used. While cross-sectional area image registration corrects axial misalignment, 3D registration additionally corrects rotations. Other registration methods involving matched angle analysis (MA) or boundary transformations (3D-TB) can be used to limit interpolation error in 3D-registering micro-finite-element data. We investigated the effect of different image registration methods on short-term in vivo precision in adults with osteogenesis imperfecta, a collagen-related genetic disorder resulting in low bone mass, impaired quality, and increased fragility. The radii and tibiae of 29 participants were imaged twice on the same day with full repositioning. We compared the precision error of different image registration methods for density, microstructural, and micro-finite-element outcomes with data stratified based on anatomical site, motion status, and scanner generation. Regardless of the stratification, we found that image registration improved precision for total and trabecular bone mineral densities, trabecular and cortical bone mineral contents, area measurements, trabecular bone volume fraction, separation, and heterogeneity, as well as cortical thickness and perimeter. 3D registration marginally outperformed cross-sectional area registration for some outcomes, such as trabecular bone volume fraction and separation. Similarly, precision of micro-finite-element outcomes was improved after image registration, with 3D-TB and MA methods providing greatest improvements. Our regression model confirmed the beneficial effect of image registration on HR-pQCT precision errors, whereas motion had a detrimental effect on precision even after image registration. Collectively, our results indicate that 3D registration is recommended for longitudinal HR-pQCT imaging in adults with osteogenesis imperfecta. Since our precision errors are similar to those of healthy adults, these results can likely be extended to other populations, although future studies are needed to confirm this. © 2022 American Society for Bone and Mineral Research (ASBMR).

CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment.

PURPOSE: Glioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs. EXPERIMENTAL DESIGN: In the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70's role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples. RESULTS: CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells. CONCLUSION: CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.

Identification of five important genes to predict glioblastoma subtypes.

BACKGROUND: Glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, has been classified into three subtypes: classical, mesenchymal, and proneural. While the original classification relied on an 840 gene-set, further clarification on true GBM subtypes uses a 150-gene signature to accurately classify GBM into the three subtypes. We hypothesized whether a machine learning approach could be used to identify a smaller gene-set to accurately predict GBM subtype. METHODS: Using a supervised machine learning approach, extreme gradient boosting (XGBoost), we developed a classifier to predict the three subtypes of glioblastoma (GBM): classical, mesenchymal, and proneural. We tested the classifier on in-house GBM tissue, cell lines, and xenograft samples to predict their subtype. RESULTS: We identified the five most important genes for characterizing the three subtypes based on genes that often exhibited high Importance Scores in our XGBoost analyses. On average, this approach achieved 80.12% accuracy in predicting these three subtypes of GBM. Furthermore, we applied our five-gene classifier to successfully predict the subtype of GBM samples at our centre. CONCLUSION: Our 5-gene set classifier is the smallest classifier to date that can predict GBM subtypes with high accuracy, which could facilitate the future development of a five-gene subtype diagnostic biomarker for routine assays in GBM samples.

High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts.

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10-4 and 10-2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.

Multisite longitudinal calibration of HR-pQCT scanners and precision in osteogenesis imperfecta.

BACKGROUND: For high-resolution peripheral quantitative computed tomography (HR-pQCT) to be used in longitudinal multi-center studies to assess disease and treatment effects, data must be aggregated across multiple timepoints and scanners. This requires an understanding of the factors contributing to scanner precision, and multi-scanner cross-calibration procedures, especially for clinical populations with severe phenotypes, like osteogenesis imperfecta (OI). METHODS: To address this, we first evaluated single- and multi-center short- and long-term precision errors of standard HR-pQCT parameters. Two imaging phantoms were circulated among 13 sites (7 XtremeCT and 6 XtremeCT2) and scanned in triplicate at 3 timepoints/site. Additionally, duplicate in vivo radial and tibial scans were acquired in 29 individuals with OI. Secondly, we investigated subject- and scanner-related factors that contribute to precision errors using regression analysis. Thirdly, we proposed a reference site selection criterion for multisite cross-calibration and demonstrated the external validity of phantom-based calibrations. RESULTS: Our results show excellent short-term single-site precision in both phantoms (CV % < 0.5%) and in density, microarchitecture and finite element parameters of OI participants (CV % = 0.75 to 1.2%). In vivo reproducibility significantly improved with (i) cross sectional area image registration versus no registration and (ii) scans with no motion artifacts. While reproducibility was similar across OI subtypes and anatomical sites, XtremeCT2 scanners achieved ~2.5% better precision than XtremeCT for trabecular parameters. Finally, we demonstrate that multisite longitudinal precision errors resulting from inconsistencies between scanners can be partially corrected through scanner cross-calibration. CONCLUSIONS: This study is the first to assess long-term reproducibility and cross-calibration in a study using first and second generation HR-pQCT scanners. The results presented in this context provide timely guidelines for future use of this powerful clinical imaging modality in multi-center longitudinal clinical trials.

Active hematopoiesis triggers exosomal release of PRDX2 that promotes osteoclast formation.

Hematopoietic disorders, particularly hemolytic anemias, commonly lead to bone loss. We have previously reported that actively proliferating cancer cells stimulate osteoclastogenesis from late precursors in a RANKL-independent manner. We theorized that cancer cells exploit the physiological role of bone resorption to support expanding hematopoietic bone marrow and examined if hematopoietic cells can trigger osteoclastogenesis. Using phlebotomy-induced acute anemia in mice, we found strong correlation between augmented erythropoiesis and increased osteoclastogenesis. Conditioned medium (CM) from K562 erythroleukemia cells and primary mouse erythroblasts stimulated osteoclastogenesis when added to RANKL-primed precursors from mouse bone marrow or RAW264.7 cells. Using immunoblotting and mass spectrometry, PRDX2 was identified as a factor produced by erythroid cells in vitro and in vivo. PRDX2 was detected in K562-derived exosomes, and inhibiting exosomal release significantly decreased the osteoclastogenic capacity of K562 CM. Recombinant PRDX2 induced osteoclast formation from RANKL-primed primary or RAW 264.7 precursors to levels comparable to achieved with continuous RANKL treatment. Thus, increased bone marrow erythropoiesis secondary to anemia leads to upregulation of PRDX2, which is released in the exosomes and acts to induce osteoclast formation. Increased bone resorption by the osteoclasts expands bone marrow cavity, which likely plays a supporting role to increase blood cell production.

Functional genomic landscape of cancer-intrinsic evasion of killing by T cells.

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.

A systematic review and meta-analysis of bone loss in space travelers.

Bone loss in space travelers is a major challenge for long-duration space exploration. To quantify microgravity-induced bone loss in humans, we performed a meta-analysis of studies systematically identified from searching Medline, Embase, Web of Science, BIOSIS, NASA Technical reports, and HathiTrust, with the last update in November 2019. From 25 articles selected to minimize the overlap between reported populations, we extracted post-flight bone density values for 148 individuals, and in-flight and post-flight biochemical bone marker values for 124 individuals. A percentage difference in bone density relative to pre-flight was positive in the skull, +2.2% [95% confidence interval: +1.1, +3.3]; neutral in the thorax/upper limbs, -0.7% [-1.3, -0.2]; and negative in the lumbar spine/pelvis, -6.2 [-6.7, -5.6], and lower limbs, -5.4% [-6.0, -4.9]. In the lower limb region, the rate of bone loss was -0.8% [-1.1, -0.5] per month. Bone resorption markers increased hyperbolically with a time to half-max of 11 days [9, 13] and plateaued at 113% [108, 117] above pre-flight levels. Bone formation markers remained unchanged during the first 30 days and increased thereafter at 7% [5, 10] per month. Upon landing, resorption markers decreased to pre-flight levels at an exponential rate that was faster after longer flights, while formation markers increased linearly at 84% [39, 129] per month for 3-5 months post-flight. Microgravity-induced bone changes depend on the skeletal-site position relative to the gravitational vector. Post-flight recovery depends on spaceflight duration and is limited to a short post-flight period during which bone formation exceeds resorption.

Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts.

The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.

Effects of Long-Term Sclerostin Deficiency on Trabecular Bone Mass and Adaption to Limb Loading Differ in Male and Female Mice.

A new therapeutic option to treat osteoporosis is focused on Wnt signaling and its inhibitor sclerostin, a product of the Sost gene. In this work, we study the effect of sclerostin deficiency on trabecular bone formation and resorption in male and female mice and whether it affects mechano-responsiveness. Male and female 10- and 26-week-old Sost knockout (KO) and littermate controls (LCs) were subjected to in vivo mechanical loading of the left tibia for 2 weeks. The right tibia served as internal control. The mice were imaged using in vivo micro-computed tomography at days 0, 5, 10, and 15 and tibiae were collected for histomorphometric analyses after euthanasia. Histomorphometry and micro-CT-based 3D time-lapse morphometry revealed an anabolic and anti-catabolic effect of Sost deficiency although increased trabecular bone resorption accompanied by diminished trabecular bone formation occurred with age. Loading led to diminished resorption in adult female but not in male mice. A net gain in bone volume could be achieved with mechanical loading in Sost KO adult female mice, which occurred through a further reduction in resorbed bone volume. Our data show that sclerostin deficiency has a particularly positive effect in adult female mice. Sclerostin antibodies are approved to treat postmenopausal women with high risk of osteoporotic fractures. Further studies are required to clarify whether both sexes benefit equally from sclerostin inhibition.