Supplementary Figure S1, related to Experimental Procedures: Molecular characterization of primary BTICs. Supplementary Figure S2, related to Supplementary Figure 1: BTICs recover their proliferative and invasive capacities in vitro. Supplementary Figure S3, related to Supplementary Figure 3: BTICs express G6PC and its inhibition decreases cell migration, invasion and proliferation. Supplementary Figure S4, related to Supplementary Figure 3: G6PC knockdowns exhibit decreased cell invasion and migration. Supplementary Figure S5, related to Supplementary Figure 4: Pharmacological inhibition of G6PC induces an increase in the expression of the astrocytic marker GFAP in the recovery group. Supplementary Figure S6, related to Supplementary Figure 5: G6PC knockdown activates glycogen synthase (GYS1) and inhibits glycogen phosphorylase (PYGL). Supplementary Figure S7, related to Supplementary Figure 6: Invasion and proliferation capacity of BTICs in vivo.

This chapter examines molecular biology, which is generally concerned with the structures, functions, and interactions of the two major groups of macromolecules, the nucleic acids DNA and RNA, and proteins. Nucleic acids can be purified from organisms using a variety of techniques. Restriction endonucleases (REs) can digest the isolated DNA into smaller-sized fragments suitable for analysis and for use in a number of techniques of clinical interest. Molecular biology techniques are used in biomedical science and they rely on the complementary binding of nucleic acids to provide a convenient way of recognizing and isolating specific base sequences within fragments of DNA or RNA molecules include the sequencing of isolated DNA, Southern and Northern blotting, fluorescence in situ hybridization (FISH), the cloning of DNA by recombination and polymerase chain reaction (PCR) technologies, and DNA microarray analysis. The chapter then looks at CRISPR–Cas9, which is a quick and efficient method for editing genes.

The ill-named "logic of monsters" hypothesis of Pere Alberch - one of the founders of modern evo-devo - emphasized the importance of "internal rules" due to strong developmental constraints, linked teratologies to developmental processes and patterns, and contradicted hypotheses arguing that birth defects are related to a chaotic and random disarray of developmental mechanisms. We test these hypotheses using, for the first time, anatomical network analysis (AnNA) to study and compare the musculoskeletal modularity and integration of both the heads and the fore- and hindlimbs of abnormal cyclopic trisomy 18 and anencephalic human fetuses, and of normal fetal, newborn, and adult humans. Our previous works have shown that superficial gross anatomical analyses of these specimens strongly support the "logic of monsters" hypothesis, in the sense that there is an 'order' or 'logic' within the gross anatomical patterns observed in both the normal and abnormal individuals. Interestingly, the results of the AnNA done in the present work reveal a somewhat different pattern: at least concerning the musculoskeletal modules obtained in our AnNA, we observe a hybrid between the "logic of monsters" and the "lack of homeostasis" hypotheses. For instance, as predicted by the latter hypothesis, we found a high level of left-right asymmetry in the forelimbs and/or hindlimbs of the abnormal cyclopic trisomy 18 and anencephalic human fetuses. That is, a network analysis of the organization of/connection between the musculoskeletal structures of these fetuses reveals a more "chaotic" pattern than that detected by superficial gross anatomical comparisons. We discuss the broader developmental, evolutionary, and medical implications of these results.

Glioblastoma multiforme is a heterogeneous and infiltrative cancer with dismal prognosis. Studying the migratory behavior of tumor-derived cell populations can be informative, but it places a high premium on the precision of in vitro methods and the relevance of in vivo conditions. In particular, the analysis of 2D cell migration may not reflect invasion into 3D extracellular matrices in vivo. Here, we describe a method that allows time-resolved studies of primary cell migration with single-cell resolution on a fibrillar surface that closely mimics in vivo 3D migration. We used this platform to screen 14 patient-derived glioblastoma samples. We observed that the migratory phenotype of a subset of cells in response to platelet-derived growth factor was highly predictive of tumor location and recurrence in the clinic. Therefore, migratory phenotypic classifiers analyzed at the single-cell level in a patient-specific way can provide high diagnostic and prognostic value for invasive cancers.

Anatomical and medical studies of gross anatomical birth defects tend to focus mainly on external features, or in hard tissues (i.e., bones, cartilages, teeth). As a new, long term project we have been collecting and systematically comparing detailed musculoskeletal and neural information on normal human development and phenotypes seen in human fetuses, neonates and adults with conditions as diverse as anencephaly, trisomy 13, trisomy 18, trisomy 21, and/or cyclopia. This comparison is based on careful dissections and an extensive review of the scarce literature on the subject. Our dissections and comparisons revealed that certain similar anatomical patterns are frequently found in each of these conditions, supporting the “logic of monsters” hypothesis of Pere Alberch, according to which even in extreme cases of birth defects (e.g. anencephaly, cyclopia) there is still a discernable, marked “order”, instead of random, chaotic phenotypes, because of strong internal constraints limiting the amount of possible developmental outcomes. For instance, the absence of muscles that are normally polymorphic in humans (e.g., fibularis tertius, palmaris longus), the absence of tendons to the 5th digit of the hands/feet, and/or the presence of a few ‘atavistic’ muscles, are seen over and over in different cases of abnormal developmental studied by us. We will discuss the broader implications of our results and of the observed patterns by discussing crucial developmental and medical topics such as the links between developmental delay/arrest, ‘atavisms’, variations, anomalies, polymorphisms and the “logic of monsters”.Support or Funding InformationGrants: This work is funded by start‐up funds to RD from Howard University College of Medicine, and by PhD funds from the Saudi Arabia government to MA.

Signaling for limb bone development usually precedes that for muscle development, such that cartilage is generally present before muscle formation. It remains obscure, however, if: (i) tetrapods share a general, predictable spatial correlation between bones and muscles; and, if that is the case, if (ii) such a correlation would reflect an obligatory association between the signaling involved in skeletal and muscle morphogenesis. We address these issues here by using the results of a multidisciplinary analysis of the appendicular muscles of all major tetrapod groups integrating dissections, muscle antibody stainings, regenerative and ontogenetic analyses of fluorescently-labeled (GFP) animals, and studies of non-pentadactyl human limbs related to birth defects. Our synthesis suggests that there is a consistent, surprising anatomical pattern in both normal and abnormal phenotypes, in which the identity and attachments of distal limb muscles are mainly related to the topological position, and not to the developmental primordium (anlage) or even the homeotic identity, of the digits to which they are attached. This synthesis is therefore a starting point towards the resolution of a centuries-old question raised by authors such as Owen about the specific associations between limb bones and muscles. This question has crucial implications for evolutionary and developmental biology, and for human medicine because non-pentadactyly is the most common birth defect in human limbs. In particular, this synthesis paves the way for future developmental experimental and mechanistic studies, which are needed to clarify the processes that may be involved in the elaboration of the anatomical patterns described here, and to specifically test the hypothesis that distal limb muscle identity/attachment is mainly related to digit topology.

Recent research advances have established mesenchymal stem cells (MSCs) as a promising vehicle for therapeutic delivery. Their intrinsic tropism for brain injury and brain tumors, their lack of immunogenicity, and their ability to breach the blood-brain barrier make these cells an attractive potential treatment of brain disorders, including brain cancer. Despite these advantages, the efficiency of MSC homing to the brain has been limited in commonly used protocols, hindering the feasibility of such therapies. In the present study, we report a reproducible, comprehensive, cell culture-based approach to enhance human adipose-derived MSC (hAMSC) engraftment to brain tumors. We used micro- and nanotechnological tools to systematically model several steps in the putative homing process. By pre-exposing hAMSCs to glioma-conditioned media and the extracellular matrix proteins fibronectin and laminin, we achieved significant enhancements of the individual homing steps in vitro. This homing was confirmed in an in vivo rodent model of brain cancer. This comprehensive, cell-conditioning approach provides a novel method to enhance stem cell homing to gliomas and, potentially, other neurological disorders.

Epidermal growth factor receptor (EGFR) signalling is a potent driver of glioblastoma, a malignant and lethal form of brain cancer. Disappointingly, inhibitors targeting receptor tyrosine kinase activity are not clinically effective and EGFR persists on the plasma membrane to maintain tumour growth and invasiveness. Here we show that endolysosomal pH is critical for receptor sorting and turnover. By functioning as a leak pathway for protons, the Na(+)/H(+) exchanger NHE9 limits luminal acidification to circumvent EGFR turnover and prolong downstream signalling pathways that drive tumour growth and migration. In glioblastoma, NHE9 expression is associated with stem/progenitor characteristics, radiochemoresistance, poor prognosis and invasive growth in vitro and in vivo. Silencing or inhibition of NHE9 in brain tumour-initiating cells attenuates tumoursphere formation and improves efficacy of EGFR inhibitor. Thus, NHE9 mediates inside-out control of oncogenic signalling and is a highly druggable target for pan-specific receptor clearance in cancer therapy.

How do the various anatomical parts (modules) of the animal body evolve into very different integrated forms (integration) yet still function properly without decreasing the individual's survival? This long-standing question remains unanswered for multiple reasons, including lack of consensus about conceptual definitions and approaches, as well as a reasonable bias toward the study of hard tissues over soft tissues. A major difficulty concerns the non-trivial technical hurdles of addressing this problem, specifically the lack of quantitative tools to quantify and compare variation across multiple disparate anatomical parts and tissue types. In this paper we apply for the first time a powerful new quantitative tool, Anatomical Network Analysis (AnNA), to examine and compare in detail the musculoskeletal modularity and integration of normal and abnormal human upper and lower limbs. In contrast to other morphological methods, the strength of AnNA is that it allows efficient and direct empirical comparisons among body parts with even vastly different architectures (e.g. upper and lower limbs) and diverse or complex tissue composition (e.g. bones, cartilages and muscles), by quantifying the spatial organization of these parts—their topological patterns relative to each other—using tools borrowed from network theory. Our results reveal similarities between the skeletal networks of the normal newborn/adult upper limb vs. lower limb, with exception to the shoulder vs. pelvis. However, when muscles are included, the overall musculoskeletal network organization of the upper limb is strikingly different from that of the lower limb, particularly that of the more proximal structures of each limb. Importantly, the obtained data provide further evidence to be added to the vast amount of paleontological, gross anatomical, developmental, molecular and embryological data recently obtained that contradicts the long-standing dogma that the upper and lower limbs are serial homologues. In addition, the AnNA of the limbs of a trisomy 18 human fetus strongly supports Pere Alberch's ill-named "logic of monsters" hypothesis, and contradicts the commonly accepted idea that birth defects often lead to lower integration (i.e. more parcellation) of anatomical structures.

We report the muscular and skeletal abnormalities observed in a very rare and precious 28-week human Trisomy 18 cyclopic fetus, and compare this individual with other humans with Trisomy 18 (Edwards syndrome) as well as with Trisomy 13 (Patau syndrome) and Trisomy 21 (Down syndrome), in the first detailed systematic musculoskeletal comparison of these three important syndromes. Our observations, comparisons, and literature review allow us to examine and discuss similarities and differences from the individual to the syndrome level; to delineate the muscle anomalies caused by aneuploid syndromes; to identify those muscular traits which are diagnostic for each one of the common aneuploid syndromes; to help chart the morphogenetic pathways of these diagnostic anomalies; and to assess the variability (descriptive and quantitative) shown by the diagnostic muscular defects in the different aneuploid syndromes. Importantly, this work comes at a time when there is a rising need for comparisons between vertebrate model organisms and humans in the relatively new and increasingly important field of evolutionary developmental biology, so the novel data presented and discussed here will contribute to a better and transformative understanding of both "normal" and abnormal development, evolution, birth defects, cyclopia and trisomies, and anatomical variations, and will thus have crucial medical implications.

Mosaic evolution is a key mechanism that promotes robustness and evolvability in living beings. For the human head, to have a modular organization would imply that each phenotypic module could grow and function semi-independently. Delimiting the boundaries of head modules and even assessing their existence, is essential to understand human evolution. Here we provide the first study of the human head using anatomical network analysis (AnNA), offering the most complete overview of the modularity of the head to date. Our analysis integrates the many biological dependences that tie hard and soft tissues together, arising as a consequence of development, growth, stresses and loads and motion. We created an anatomical network model of the human head, where nodes represent anatomical units and links represent their physical articulations. The analysis of the human head network uncovers the presence of 10 musculoskeletal modules, deep-rooted in these biological dependences, of developmental and evolutionary significance. In sum, this study uncovers new anatomical and functional modules of the human head using a novel quantitative method that enables a more comprehensive understanding of the evolutionary anatomy of our lineage, including the evolution of facial expression and facial asymmetry.

The results of recent comparative, Bayesian and parsimony‐based cladistic analyses, developmental and regenerative studies of muscles of humans, other primates, and other chordates show that muscles provide particularly useful data for phylogenetic, evolutionary, and developmental analyses. Importantly, the inclusion of soft tissue‐based information in such studies allows researchers to address evolutionary and developmental questions that are not tractable using molecular or skeletal evidence alone. Here we address long‐standing questions about the evolutionary history of modern humans and other chordates, focusing on: 1) the developmental and evolutionary origins of the head, neck, pectoral and forelimb muscles; 2) notions of purpose and progress in evolution and the parallelism between ontogeny and phylogeny; 3) the relationship between trisomies, ‘atavisms’, evolutionary reversions and developmental constraints; 4) the tempo and mode of primate and human evolution (e.g., modularity and ontogenetic constraints); 5) the relationship between modern human anomalies/variations, digit loss/gain, muscle changes and homeotic transformations; 5) similarities and differences among the hind and forelimb structures of modern humans (e.g., serial homology and homoplasy); and 6) the variations and anomalies in the musculature of modern humans and their medical implications.

Na+(K+)/H+ Exchangers (NHEs) are a large family of transport proteins that conduct electroneutral countertransport of cations with protons across lipid bilayers. A newly discovered Na+/H+ Exchanger, NHE9 (SLC9A9), has recently been shown to localize to a perinuclear region corresponding to recycling endosomes, where it is thought to regulate luminal pH and control cargo trafficking and degradation. Given the mounting evidence implicating NHE9 in neurological diseases including autism and ADHD, we explored the functional consequence of altered NHE9 expression levels in brain cancer.Glioblastoma Multiforme (GBM) is the most aggressive form of malignant glioma that accounts for over 50% of all gliomas and is frequently characterized by the amplification and membrane persistence of epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosomal‐mediated degradation, and we postulated that NHE9 might alter this pathway. Here, we have found that levels of NHE9 are amplified in a large subset of GBMs. Using primary GBM cell cultures with naturally high or low levels of NHE9, we have found that altering those levels regulates tumorigencity and migratory capacity in vitro and in nude mouse xenografts. Furthermore, increased NHE9 levels alkalinizes the luminal pH of endosomes, which inhibits EGFR degradation and promotes oncogenic signaling downstream of MAPK and Akt. Finally, we show that inhibiting NHE9 with amiloride‐derived inhibitor EIPA increases EGFR responsiveness to Erlotinib. Our findings demonstrate that NHE9 is a target gene that should be explored as a treatment for a subset of GBMs.Grant Funding Source: Supported by NIH grant R01 DK054214

In the last decades evolutionary developmental biologists have studied the ontogeny of hard and soft tissues in model organisms such as chicken, axolotl, frogs and mice. However, very few researchers have undertaken detailed analyses about the specific relationships between the soft and hard tissues of the tetrapod limbs and particularly of their autopodia (hand/foot) and zeugopodia (forearm/leg). We have therefore addressed this subject by using a wide range of different techniques in a broad taxonomic sample, including developmental and regenerative studies of GFP salamanders, ontogenetic studies of wildtype frogs, comparative studies of all major groups of tetrapods, and dissections of humans with birth defects involving polydactyly and digit reduction. Interestingly, our results show that in almost all cases of both wildtype and non‐wildtype non‐pentadactyly the identity and configuration of muscles is actually highly predictable and is mainly related to the topological position of the digits (e.g., by being the most ulnar/fibular, or instead the most radial/tibial digits) to which the muscles insert, and not to the ontogenetic anlage from which the digits develop or even the homeotic identity of each digit. We will discuss various case studies illustrating this point, and discuss the broader implications of our results for evolutionary, comparative and developmental biology and for human medicine.Grant Funding Source: Faculty Start‐Up Package, Howard University College of Medicine

Abstract Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear especially in a hypoxic tumor microenvironment. In this study, it was determined that glucose-6–phosphatase (G6PC/G6Pase) expression is elevated in GBM when compared with normal brain. Human-derived brain tumor–initiating cells (BTIC) use this enzyme to counteract glycolytic inhibition induced by 2-deoxy-d-glucose (2DG) and sustain malignant progression. Downregulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation). Collectively, these findings establish G6PC as a key enzyme with promalignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target. Implications: This study is the first to demonstrate a functional relationship between the critical gluconeogenic and glycogenolytic enzyme G6PC with the metabolic adaptations during GBM invasion. Visual Overview: http://mcr.aacrjournals.org/content/12/11/1547/F1.large.jpg. Mol Cancer Res; 12(11); 1547–59. ©2014 AACR.

Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear. In this study, we found that the expression of glucose-6-phosphatase-α (G6PC) was elevated in GBM when compared to normal brain ) (p < 0.001). Human-derived brain tumor initiating cells (BTICs) utilize this enzyme to counteract glycolytic inhibition induced by 2-Deoxy-D-glucose (2DG) and sustain malignant progression. Down-regulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation) (p < 0.001). Interestingly, after performing immunohistochemistry staining for G6PC in brain sections derived from animals implanted with G6PC knockdown cells for a survival study, we found a complete rescue of its expression in the tumor cells of these animals. These results explain the absence of survival differences observed between G6PC knockdown and wild type mice. Furthermore, these findings also suggest a very important role for G6PC in cell survival, since only the cells that carried G6PC where able to survive and invade in our animal model. Collectively, our findings establish G6PC as a key enzyme with pro-malignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target.

Abstract Glioblastomas (GBMs) are characterized by a very aggressive behavior and a heterogeneous presentation that includes proliferative, invasive, and necrotic areas. The rapid expansion of GBMs frequently outgrowths the blood supply, originating hypoxic and necrotic regions, which induces metabolic changes in the tumor cells. In a survival adaptation response, tumor cells adjust their glucose metabolism to an anaerobic glycolysis, even in the presence of oxygen. This is known as the Warburg effect and it is enhanced in Brain Tumor Stem Cells (BTSCs), the cellular sub-population considered to be responsible for the tumor origin and recurrence. Glycolysis inhibition can be achieved using glucose analogues like 2-deoxyglucose (2DG). We have previously demonstrated that 2DG treatment of BTSCs induces a decrease in cell proliferation and an increase in cell death and neuronal differentiation. Nevertheless, some BTSC cells are able to survive glycolysis inhibition and recover their aggressive phenotype which might be related to brain tumor recurrence. Here we studied the tumorigenic behavior of human GBM-derived primary BTSCs after recovery from glycolysis inhibition. We observed that cells which were pretreated with 2DG and subsequently allowed to recover for 72h in glucose-containing media, exhibited a more aggressive phenotype; including an increased migration, invasion, and proliferation capability; as well as MMP2 and nestin overexpression. Some of these features recapitulate what occurs in recurrent brain tumors. In studying the potential molecular mechanisms responsible for this behavior, we observed an increased expression of glucose-6-phosphatase isoform α (G6PC) in our primary GBM cells when compared to normal brain. Moreover, 2DG treatment induced an increase in the expression of this isoform. G6PC is a key glucose homeostasis enzyme not previously reported in brain tissue. In addition, when G6PC was inhibited, either pharmacologically (with chlorogenic acid) or genetically (with shRNA), we observed that the recovery capacity of GBM cells was significantly reduced. This was evident by a decrease in their proliferation, migration, and invasion ability, as well as by a decrease in HIF1α, pSTAT3 protein, MMP2 gene expression. In summary, we report for the first time the expression of G6PC in GBM cells. This specific isoform is not expressed in normal brain parenchyma, which makes it a very attractive target for anticancer therapy. Our results suggest a role of G6PC in promoting a mechanism of GBM cell recovery from glycolysis inhibition. By unraveling these mechanisms we describe an important therapeutic target that could impact the recurrence ability of brain tumors. Citation Format: Sara Abbadi, Hugo Guerrero-Cazares, Ameer Abutaleb, Chris L. Smith, William Ruff, Jennifer Schiller, Andre Levchenko, Alfredo Quinones-Hinojosa. Human GBM-derived brain tumor stem cells resist glycolysis inhibition through Glucose 6 phosphatase: a potential clinical implication in the treatment of recurrent brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5422. doi:10.1158/1538-7445.AM2013-5422

Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms.

The architecture of the extracellular matrix (ECM) directs cell behavior by providing spatial and mechanical cues to which cells respond. In addition to soluble chemical factors, physical interactions between the cell and ECM regulate primary cell processes, including differentiation, migration, and proliferation. Advances in microtechnology and, more recently, nanotechnology provide a powerful means to study the influence of the ECM on cell behavior. By recapitulating local architectures that cells encounter in vivo, we can elucidate and dissect the fundamental signal transduction pathways that control cell behavior in critical developmental, physiological, and pathological processes.