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In order to increase the fatigue performance of mechanically loaded PM-components, case hardening procedures can be used to improve the surface layer states in terms of hardness and residual stress...

Despite advances in the treatment of solid tumors, the prognosis of patients with many cancers remains poor, particularly of those with primary and metastatic brain tumors. In the last years, "Cancer Neuroscience" emerged as novel field of research at the crossroads of oncology and classical neuroscience. In primary brain tumors, including glioblastoma (GB), communicating networks that render tumor cells resistant against cytotoxic therapies were identified. To build these networks, GB cells extend neurite-like protrusions called tumor microtubes (TMs). Synapses on TMs allow tumor cells to retrieve neuronal input that fosters growth. Single cell sequencing further revealed that primary brain tumors recapitulate many steps of neurodevelopment. Interestingly, neuronal characteristics, including the ability to extend neurite-like protrusions, neuronal gene expression signatures and interactions with neurons, have now been found not only in brain and neuroendocrine tumors but also in some cancers of epithelial origin. In this review, we will provide an overview about neurite-like protrusions as well as neurodevelopmental origins, hierarchies and gene expression signatures in cancer. We will also discuss how "Cancer Neuroscience" might provide a framework for the development of novel therapies.

Traditionally, monitoring biomechanics parameters requires a significant amount of sensors to track exercises such as gait. Both research and clinical studies have relied on intricate motion capture studios to yield precise measurements of movement. We propose a method that captures motion independently of optical hardware with the specific goal of identifying the phases of gait using joint angle measurement approaches like IMU (inertial measurement units) sensors. We are proposing a machine learning approach to progressively reduce the feature number (joint angles) required to classify the phases of gait without a significant drop in accuracy. We found that reducing the feature number from six (every joint used) to three reduces the mean classification accuracy by only 4.04%, while reducing the feature number from three to two drops mean classification accuracy by 7.46%. We extended gait phase classification by using the biomechanics simulation package, OpenSim, to generalize a set of required maximum joint moments to transition between phases. We believe this method could be used for applications other than monitoring the phases of gait with direct application to medical and assistive technology fields.

Tumor cell extensions called tumor microtubes (TMs) in glioma resemble neurites during neurodevelopment and connect glioma cells to a network that has considerable relevance for tumor progression and therapy resistance. The determination of interconnectivity in individual tumors has been challenging and the impact of tumor cell connectivity on patient survival remained unresolved so far. Here, a connectivity signature from single-cell RNA-sequenced (scRNA-Seq) xenografted primary glioblastoma (GB) cells was established and clinically validated. Thirty-four of 40 connectivity genes were related to neurogenesis, neural tube development or glioma progression, including the TM-network-relevant GAP43 gene. Astrocytic-like and mesenchymal-like GB cells had the highest connectivity signature scores in scRNA-Seq data of patient-derived xenografts and patient samples. In 230 human GBs, high connectivity correlated with the mesenchymal expression subtype, TP53 wildtype, and with dismal patient survival. CHI3L1 was identified as a robust molecular marker of connectivity. Thus, the connectivity signature allows novel insights into brain tumor biology, provides a proof-of-principle that tumor cell connectivity is relevant for patients’ prognosis, and serves as a robust biomarker that can be used for future clinical trials.Statement of significanceIntegration of GB cells into functional networks drives tumor progression and resistance. Here, we established and validated a novel connectivity gene expression signature of single GB cells and whole tumors that can be easily applied to clinical and preclinical samples. It is shown that connectivity is determining prognosis combining molecular, functional and clinical insights into the disease.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods., Ly et al. report a multi-well imaging system (the Picroscope) that can perform longitudinal bright-field z-stack imaging for 24 wells simultaneously. They demonstrate its capability by imaging zebrafish embryos, planaria worms, development of Xenopus tropicalis over a 28-h period and embryonic stem cell monolayer and organoids inside an incubator.

BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

Glioblastomas are known to be highly therapy resistant tumors. The description of tumor microtubes (TMs) as long cellular protrusions that connect glioblastoma cells to a network resistant against all standard therapies advanced the basic biological understanding of the disease and offers a new therapeutic target. An antisense oligonucleotide (ASO) against the production of a protein involved in formation and function of TMs was developed and tested in a xenograft mouse model of human glioblastoma cells. After the implantation of fluorescent tumor cells under a chronic cranial window and the establishment of the tumor, the ASO treatment was started with a continuous micropump application directly into the central nervous system (CNS) and treatment effects were observed in a two-photon microscope. Furthermore, co-treatment with irradiation was tested and tumor size as well as protein expression evaluated in and ex vivo. This led to the conclusion that the setup of combined continuous pump application and two photon microscopy is technically feasible and tolerable. It offers the possibility of applying compounds directly into the CNS under live and longitudinal readouts. The fluorescently tagged ASO was traced in the brain parenchyma over weeks proving a high stability of the compound. In combination with radiotherapy, animals treated with the active ASO against the protein of formation and function of TMs showed a smaller tumor size than animals treated with radiotherapy and the non-targeting ASO. The targeted protein was expressed less in the treated animals then the animals receiving the control ASO and the extend of protein expression correlated with tumor size. Hence, after successful therapies with ASOs in other neurologic diseases and the need for better therapeutic options for glioblastoma patients, the development of an ASO against a protein involved in the TM formation and function offers a new therapeutic option with high translational potential.

New cell culture techniques have led to complex tissue models in biological experiments. For example, 3-D cerebral organoids provide a more realistic model of the human cortical tissue. However, these cell culture experiments are restricted by high costs and limited labor. A massively scalable and cost efficient platform for tissue experiments would benefit genomics, neuroscience, and translational medicine by enabling advanced high throughput tissue screens. Cloud computing and the Internet of Things (IoT) provide new tools for managing multiple experiments in parallel that are remotely controlled through automation. We introduce a cloud-based IoT architecture that takes advantage of these tools to offer an environment where researchers can run thousands of cell culture experiments at once. This technology allows studies with cell cultures to be performed at scales far beyond a single lab setting, democratizing access to advanced tissue models and enabling new avenues of research. IoT Cloud LaboratoryExperiments are automated through cloud connected devices to allow scalability, reproducibility, and online monitoring. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/453595v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@14421e7org.highwire.dtl.DTLVardef@dce92org.highwire.dtl.DTLVardef@75037borg.highwire.dtl.DTLVardef@37afa9_HPS_FORMAT_FIGEXP M_FIG C_FIG

The establishment of neuronal and glial networks in the brain depends on the activities of neural progenitors, which are influenced by cell-intrinsic mechanisms, interactions with the local microenvironment and long-range signaling. Progress in neuroscience has helped identify key factors in CNS development. In parallel, studies in recent years have increased our understanding of molecular and cellular factors in the development and growth of primary brain tumors. To thrive, glioma cells exploit pathways that are active in normal CNS progenitor cells, as well as in normal neurotransmitter signaling. Furthermore, tumor cells of incurable gliomas integrate into communicating multicellular networks, where they are interconnected through neurite-like cellular protrusions. In this Review, we discuss evidence that CNS development, organization and function share a number of common features with glioma progression and malignancy. These include mechanisms used by cells to proliferate and migrate, interact with their microenvironment and integrate into multicellular networks. The emerging intersections between the fields of neuroscience and neuro-oncology considered in this review point to new research directions and novel therapeutic opportunities.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here we propose the Picroscope, which is the first low cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long time periods.

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours

BackgroundGlioma is sensitive to microtubule-targeting agents (MTAs), but most MTAs do not cross the blood brain barrier (BBB). To address this limitation, we developed the new chemical entity, ST-401, a brain-penetrant MTA.MethodsSynthesis of ST-401. Measures of MT assembly and dynamics. Cell proliferation and viability of patient-derived (PD) glioma in culture. Measure of tumor microtube (TM) parameters using immunofluorescence analysis and machine learning-based workflow. Pharmacokinetics (PK) and experimental toxicity in mice. In vivo antitumor activity in the RCAS/tv-a PDGFB-driven glioma (PDGFB-glioma) mouse model.ResultsWe discovered that ST-401 disrupts microtubule (MT) function through gentle and reverisible reduction in MT assembly that triggers mitotic delay and cell death in interphase. ST-401 inhibits the formation of TMs, MT-rich structures that connect glioma to a network that promotes resistance to DNA damage. PK analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft flank tumor mouse model demonstrates significant antitumor activity and no over toxicity in mice. In the PDGFB-glioma mouse model, ST-401 enhances the therapeutic efficacies of temozolomide (TMZ) and radiation therapy (RT).ConclusionOur study identifies hallmarks of glioma tumorigenesis that are sensitive to MTAs and reports ST-401 as a promising chemical scaffold to develop brain-penetrant MTAs.

A low cost remote imaging platform for biological applications was developed. The "Picroscope" is a device that allows the user to perform longitudinal imaging studies on multi-well cell culture plates. Here we present the network architecture and software used to facilitate communication between modules within the device as well as external cloud services. A web based console was created to control the device and view experiment results. Post processing tools were developed to analyze captured data in the cloud. The result is a platform for controlling biological experiments from outside the lab., The authors Pierre V Baudin and Victoria T Ly contributed equally to this work. 21 pages, 12 figures

Most cancer therapeutics developed to date are excluded from the brain and are therefore ineffective in treating glioma, the most devastating type of brain cancer in adults. Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of solid and hematopoietic tumors, and evidence suggests that glioma is sensitive to MTAs; but most MTAs do not cross the blood-brain-barrier. To address this limitation, we developed a brain-penetrant MTA, ST-401, that inhibited the growth of human glioma in culture at nanomolar concentrations. ST-401 bound to the colchicine site, inhibited tubulin assembly and reversibly reduced microtubule (MT) dynamics. Testing of ST-401 on the NCI 60 cancer cell panel indicated that its anti-tumor activity does not correlate with any of the compounds tested thus far through this platform but showed weak correlations for two MTA that work through distinct mechanisms: taxol (p=0.46) and vinblastine (p=0.34). Thus, ST-401 may kill cancer cells through a novel mechanism related to disruption of MT function. We discovered that ST-401 killed patient-derived (PD) glioma isolates in both mitosis and interphase, and inhibited the formation of tumor microtubes, MT-rich structures that connects glioma cells to a network that is resistant to standard therapies. Pharmacokinetic analysis of ST-401 in mice shows brain penetration reaching antitumor concentrations, and in vivo testing of ST-401 in a xenograft model demonstrated significant antitumor activity. In an immunocompetent mouse model of platelet-derived growth factor B-driven glioma, ST-401 significantly enhanced the therapeutic efficacies of standard care therapies temozolomide and radiation therapy. Our study identified novel aspects of glioma tumorigenesis that exhibit enhanced sensitivity to MTAs and shows that the brain-penetrant MTA, ST-401, represents a promising chemical scaffold to develop therapeutics for the treatment of patients diagnosed with glioma.

Both the perivascular niche (PVN) and the integration into multicellular networks by tumor microtubes (TMs) have been associated with progression and resistance to therapies in glioblastoma, but their specific contribution remained unknown. By long-term tracking of tumor cell fate and dynamics in the live mouse brain, differential therapeutic responses in both niches could be determined. Both the PVN, a preferential location of long-term quiescent glioma cells, and network integration facilitated resistance against cytotoxic effects of radiotherapy and chemotherapy - independently of each other, but with additive effects. Perivascular glioblastoma cells were particularly able to actively repair damage to tumor regions. Population of the PVN and resistance in it depended on proficient NOTCH1 expression. In turn, NOTCH1 downregulation induced resistant multicellular networks by TM extension. Our findings identify NOTCH1 as a central switch between the PVN and network niche in glioma, and demonstrate robust cross-compensation when only one niche is targeted.

The natural product antibiotic fidaxomicin is a marketed drug for the treatment of bacterial infections in the gut. Due to its promising in vitro activities against Mycobacterium tuberculosis, the development of next generation fidaxomicin analogs is of great interest. This article reviews the most recent advances, including the elucidation of a unique mode of action by cryo-EM structures, and the efforts towards the clarification of the biosynthetic pathway. Moreover, known fidaxomicin analogs and their reported antibacterial activities are summarized.

BACKGROUND Tumor microtubes (TMs) are ultralong membrane protrusions of tumor cells in astrocytic gliomas, including glioblastomas. TMs are used as routes for brain invasion and for cells to interconnect over long distances resulting in a functional network that allows multicellular communication. This network mediates resistance against the cytotoxicity of radiation and chemotherapy. One explanation for TM network protection is a better homeostasis of calcium ions that would otherwise increase to toxic intracellular levels in response to these therapies. Our working hypothesis is that interfering with the integrity of the glioblastoma cell network is key to a potential breakthrough in glioma therapy. Many cellular structures are polarized and composed of charged elements and are thus potential subjects to electrical forces; this might also influence the complex intercellular calcium waves (ICWs) that are characteristic for glioma networks. We were therefore interested in the effect of TTF on glioma network maintenance. MATERIAL AND METHODS To examine the effect of TTF on glioma TMs we have established a 2D in vitro glioma model using glioblastoma stem cells (GBSCs) grown in high-glucose medium and a 3D model using glioma tumor organoids. Both models reliably reproduce functionality and complexity of morphological features we observe in our mouse model. We analyzed the disruption of tumor network complexity and disruption of functionality by measuring intercellular calcium waves. Tumor cell death and proliferation was investigated in the 2D in vitro glioma model using the inovitroTM-System. RESULTS A peculiar “cricked-TM” phenotype that rarely (0.2% ±0.14) occurred under standard or control conditions was observed in TTF-treated cells (16.22% ±5.12). Cell number was reduced by 75% in two lines of GBSCs after 5 days of TTF exposure; predominantly TM-rich GBSCs (> 4 TMs) were affected. This reduction in tumor cell number corresponded with an increase in cell death (0.3% ±0.09 in untreated cells; 1.4% ±0.45 at day 5 of TTF exposure). The frequency of intercellular calcium transients, a measurement for calcium wave frequency in the glioma networks, was instantly reduced after TTF exposure to 58% ±20.42 of control levels in the primary GBSC 2D culture, and to 57.78% ±12.34 in tumor organoids derived from 3 glioblastoma patients. CONCLUSION This data suggests a potential effect of TTF application on tumor cell networks, at least in vitro. Interestingly, particularly those glioblastoma cells that have so far been proven to be resistant to radio- and chemotherapy appeared to be affected. We will confirm the observed effects of TTFs on tumor cell calcium signaling in our in vivo chronic cranial window mouse model. We anticipate that the results of our project will provide important insights into the underlying mechanism of TTF therapy.

The establishment of neuronal and glial networks in the brain depends on the activities of neural progenitors, which are influenced by cell-intrinsic mechanisms, interactions with the local microenvironment and long-range signaling. Progress in neuroscience has helped identify key factors in CNS development. In parallel, studies in recent years have increased our understanding of molecular and cellular factors in the development and growth of primary brain tumors. To thrive, glioma cells exploit pathways that are active in normal CNS progenitor cells, as well as in normal neurotransmitter signaling. Furthermore, tumor cells of incurable gliomas integrate into communicating multicellular networks, where they are interconnected through neurite-like cellular protrusions. In this Review, we discuss evidence that CNS development, organization and function share a number of common features with glioma progression and malignancy. These include mechanisms used by cells to proliferate and migrate, interact with their microenvironment and integrate into multicellular networks. The emerging intersections between the fields of neuroscience and neuro-oncology considered in this review point to new research directions and novel therapeutic opportunities.

Recent studies have demonstrated extensive cell-to-cell connectivity between tumor cells of gliomas with considerable relevance for tumor progression and therapy resistance. Tumor microtubes (TMs) are neurite-like tumor cell extensions that build these tumor cell networks. Measuring the extent of connectivity in individual tumors has been challenging and depended on anatomical parameters that are difficult to evaluate in patient samples. We performed bulk and single-cell (sc)RNA sequencing of connected vs. unconnected tumor cells from patient-derived xenograft tumors using a newly developed technology that exploits SR101 dye transfer within tumor cell networks. scRNA sequencing was performed with 17 human glioblastoma tumor samples. Three diffuse glioma cohorts from The Cancer Genome Atlas (n = 648), the Chinese Glioma Genome Atlas (n = 668) and the NCT Neuro Master Match (n = 38, IDH-wildtype only) were used to assess clinical properties. A connectivity signature both from bulk and scRNA sequencing data of xenografted primary glioblastoma tumor cells was established. Comparative analysis showed better performance and higher biological relevance of the single-cell derived signature that involves 71 genes. Most of the genes are related to neurogenesis and neural tube development, including several previously recognized TM-relevant genes. Highest connectivity was observed in astrocytic-like and mesenchymal-like tumor cells. Induction of connectivity in vitro was accompanied with increase of the connectivity signature. The connectivity signature was higher in astrocytic as compared to oligodendrocytic gliomas, and highest in IDH-wildtype gliomas. In accordance, connectivity correlated strongly with dismal survival in all three glioma cohorts. The connectivity signature established here is biologically plausible and associates with prognostically relevant glioma subtypes. It provides the first proof-of-principle that tumor cell connectivity is relevant for the clinical course of patients with gliomas, and at the same time serves as a robust biomarker that can be used for future studies, including prospective clinical trials.

BACKGROUND Gliomas form therapy-resistant multicellular networks, using neurite-like protrusions (called Tumor Microtubes [TMs]), and display intercellular calcium transients, that drive brain tumor malignancy. METHODS A refined in vitro model was established to study calcium communication and its molecular toolkit in patient-derived glioma cells. In vitro results were then validated with longitudinal in vivo two-photon calcium imaging in awake mice. RESULTS Here we first describe the discovery of a small subpopulation of glioma cells that display intrinsically active calcium oscillations and behave like pacemakers. Accordingly, these pacemaker-like glioma cells trigger synchronized calcium activity in tumor cells that are connected with them via TMs. The application of network theory shows that glioma cell networks follow a scale-free and small-world topology, governed by a subpopulation of highly connected hub cells. Interestingly, the pacemaker cells most often act as these hubs, hence communicating with many other network members. Graph theory predicts that such network design displays a high degree of robustness, making it resistant to random damage as seen after radio- and chemotherapy – but extremely vulnerable to selective damage to key hubs. In line with this theory, laser ablation of the pacemaker-like hub cells splintered the malignant tumor network into small isolated cell clusters and led to increased cell death, whereas laser ablation of random tumor cells showed no effect. Among others, KCNN4 channels, which are upregulated in gliomas, were identified as drivers of the pacemaker-like hub cells. Suppressing their pacemaking-abilities by KCNN4 inhibition strongly reduced calcium communication and tumor growth. CONCLUSION In summary, coordinated calcium communication in glioma drives brain tumor malignancy. A specific tumor cell subpopulation was identified that acts as hubs of the multicellular network and initiates global calcium activity. The network’s vulnerability to loosing these pacemaker-like hub cells suggests a novel targeted approach against the resistant networks of gliomas.

Most robotics literature model the human's knee and hip as a revolute joint with limited range of rotation. Although somehow close to reality, this approach neglects a critical aspect of these joints, which is their internal flexibility. This paper presents a prototype tensegrity flexural manipulator whose kinematic behavior is inspired by human leg's gait. This prototype, which considers a hybrid (flexible-rigid) structure of the knee and hip would be able to better approximate real behavior and hopefully lead to a better design of artificial (prosthetic) knees and hips. The behavior of the proposed tensegrity manipulator was firstly predicted using OpenSim simulation environment. The paper reports the comparisons between the simulations, physical prototypes and human leg behavior for a variety of ranges of motions and tension analysis.

Early and progressive colonization of the healthy brain is one hallmark of diffuse gliomas, including glioblastomas. We recently discovered ultralong (>10 to hundreds of microns) membrane protrusions [tumor microtubes (TMs)] extended by glioma cells. TMs have been associated with the capacity of glioma cells to effectively invade the brain and proliferate. Moreover, TMs are also used by some tumor cells to interconnect to one large, resistant multicellular network. Here, we performed a correlative gene-expression microarray and in vivo imaging analysis, and identified novel molecular candidates for TM formation and function. Interestingly, these genes were previously linked to normal CNS development. One of the genes scoring highest in tests related to the outgrowth of TMs was tweety-homolog 1 (TTYH1), which was highly expressed in a fraction of TMs in mice and patients. Ttyh1 was confirmed to be a potent regulator of normal TM morphology and of TM-mediated tumor-cell invasion and proliferation. Glioma cells with one or two TMs were mainly responsible for effective brain colonization, and Ttyh1 downregulation particularly affected this cellular subtype, resulting in reduced tumor progression and prolonged survival of mice. The remaining Ttyh1-deficient tumor cells, however, had more interconnecting TMs, which were associated with increased radioresistance in those small tumors. These findings imply a cellular and molecular heterogeneity in gliomas regarding formation and function of distinct TM subtypes, with multiple parallels to neuronal development, and suggest that Ttyh1 might be a promising target to specifically reduce TM-associated brain colonization by glioma cells in patients. SIGNIFICANCE STATEMENT In this report, we identify tweety-homolog 1 (Ttyh1), a membrane protein linked to neuronal development, as a potent driver of tumor microtube (TM)-mediated brain colonization by glioma cells. Targeting of Ttyh1 effectively inhibited the formation of invasive TMs and glioma growth, but increased network formation by intercellular TMs, suggesting a functional and molecular heterogeneity of the recently discovered TMs with potential implications for future TM-targeting strategies.

Background Primary and adaptive resistance against chemo- and radiotherapy and local recurrence after surgery limit the benefits from these standard treatments in glioma patients. Recently we found that glioma cells can extend ultra-long membrane protrusions, "tumor microtubes" (TMs), for brain invasion, proliferation, and interconnection of single cells to a syncytium that is resistant to radiotherapy. We wondered whether TMs also convey resistance to the other 2 standard treatment modalities. Methods Patient-derived glioblastoma stemlike cell (GBMSC) lines were implanted under a cranial window in mice. Longitudinal in vivo two-photon laser scanning microscopy was used to follow tumor growth, including the fate of single glioma cells over months. Results After a cylindrical surgical lesion, GBMSCs increasingly extended TMs toward the lesion area, which contributed to the repopulation of this area over many weeks. In fact, an excessive "healing response" was observed in which tumor cell densities significantly exceeded those of unlesioned brain regions over time. Inhibition of TM formation and function by genetic targeting of growth associated protein-43 robustly suppressed this surgery-induced tumor growth reaction, in contrast to standard postsurgical anti-inflammatory treatment with dexamethasone. After one cycle of temozolomide chemotherapy, intra- and intertumoral heterogeneity of TM formation and interconnection was strongly associated with therapy response: when tumor cells were integrated in TM networks, they were more likely to resist chemotherapy. Conclusion TMs can contribute to the resistance against standard treatment modalities in gliomas. Specific inhibition of TMs is a promising approach to reduce local recurrence after surgery and lower resistance to chemotherapy.

Author(s): CHEN, LEE-HUANG | Advisor(s): Agogino, Alice M. | Abstract: This dissertation presents the design, analysis and testing of various actuated modular spherical tensegrity robots for co-robotic and space exploration applications. Tensegrity structures are of interest in the field of soft robotics due to their flexible and robust nature. They have the ability to passively distribute forces globally providing shock protection from unexpected impact forces. This feature makes them a robust mobile platform suitable for uneven terrain and unpredictable environments in which traditional robots struggle. Robots built from tensegrity structures, which are composed of pure tensile and compression elements, have many potential benefits including high robustness through redundancy, many degrees of freedom in movement and flexible design. However, to fully take advantage of these properties a significant fraction of the tensile elements should be actuated, leading to a potential increase in complexity, messy cable and power routing systems and increased design difficulty.The first part of this dissertation presents an elegant solution to a fully actuated tensegrity robot: the TT-3 (version three) tensegrity robot was developed at University of California Berkeley, in collaboration with NASA Ames. The TT-3 is a lightweight, low cost, modular, and rapidly prototyped spherical tensegrity robot. This dissertation describes in detail the novel design mechanisms, architecture and simulations of TT-3, the first untethered, fully actuated cable-driven six-bar tensegrity spherical robot ever built and tested for mobility. Furthermore, this dissertation discusses the controls and preliminary testing performed to observe TT-3’s system behavior and performance and was evaluated against previous models of tensegrity robots developed at University of California at Berkeley and elsewhere.The second part of this dissertation will present a new platform for prototyping spherical tensegrity robots that significantly reduces the time required for manufacturing and assembly. This simplified tensegrity system design allows for more scientific experiments to be performed in less time. This work describes the design architecture of the TT-4mini, an example of a robot that uses this prototyping platform. In order to demonstrate the platform’s use for scientific experiments, the TT-4mini was shown to achieve uphill climbing, which has not been performed by any other spherical tensegrity robot in hardware. This dissertation discusses preliminary observations on the system’s performance in uphill climbing from simulations and testing, including evidence of climbing surfaces with an incline up to 13 degrees. Furthermore, this new prototyping platform demonstrated the ability to create three complex 12-bar tensegrity structures with simplified procedures.Lastly, the dissertation presents the improved tensegrity robot using the rod-centered architecture: the TT-4 (version four). It is a larger robot; its rod length is 1 meter compared to the 0.65 meter rod length of TT-3. The goal of the TT-4 robot prototype is twofold: to evaluate both the design improvements and the interaction of the robot with a center payload. Because the size of the robot has increased, the volume at the center of the robot available to hold payload has also increased. This allows researchers to further study the configuration, method of attachment, and the dynamics of a payload.These robots are based on a ball-shaped six-bar tensegrity structure and features a unique modular rod-centered distributed actuation and control architecture. This revolutionary new architecture has been demonstrated in both software and hardware testing to increase the performance of tensegrity robots and has the potential to be extensible to a wide range of tensegrity configurations.

A distinct perivascular niche has been implicated in many aspects of tumor biology, particularly stemness, invasion, survival, and therapy resistance of cancer cells. But its overall importance for these factors remains obscure.

The flexibility and structural compliance of the biological shoulder joint allows humans to perform a wide range of motions with their arms. The current paper is a preliminary study in which we propose a structurally compliant robotic manipulator joint inspired by the human shoulder joint, which elastically deforms when actuated. The tensile actuation is similar to the contraction and extension of biological muscles. We present four separate models for the shoulder: a simple saddle, a complex saddle, a suspended tubercle, and interlocked tetrahedrons. The analysis explores the dynamics in each design to compare the inherent advantages and disadvantages, which gives insight into the design and development of better interfaces for biologically inspired human-oriented robotics.

This paper presents the design, analysis and testing of a fully actuated modular spherical tensegrity robot for co-robotic and space exploration applications. Robots built from tensegrity structures (composed of pure tensile and compression elements) have many potential benefits including high robustness through redundancy, many degrees of freedom in movement and flexible design. However to fully take advantage of these properties a significant fraction of the tensile elements should be active, leading to a potential increase in complexity, messy cable and power routing systems and increased design difficulty. Here we describe an elegant solution to a fully actuated tensegrity robot: The TT-3 (version 3) tensegrity robot, developed at UC Berkeley, in collaboration with NASA Ames, is a lightweight, low cost, modular, and rapidly prototyped spherical tensegrity robot. This robot is based on a ball-shaped six-bar tensegrity structure and features a unique modular rod-centered distributed actuation and control architecture. This paper presents the novel mechanism design, architecture and simulations of TT-3, the first untethered, fully actuated cable-driven six-bar tensegrity spherical robot ever built and tested for mobility. Furthermore, this paper discusses the controls and preliminary testing performed to observe the system’s behavior and performance.

Tumor microtubes (TMs), which are ultra-thin and long cellular protrusions extended by glioma cells, have emerged as a novel mechanism of glioma cell growth, invasion and interconnection with consecutive therapy resistance. So far, few molecular drivers of TM formation and function (Gap43, Cx43, Ttyh1 and actin dynamics) have been identified and their downregulation strongly reduced therapy resistance and glioma cell dissemination in vivo. We implemented a multi-level screening pipeline which allows to test drugs for their anti-TM actions. Initially drugs are tested for their general actions on glioma cell growth in vitro before being evaluated by laser scanning microscopy for their impact on TM formation (number of TMs), morphometry (TM length and diameter) and functionality (calcium communication) in 2D models. These parameters as well as anti-invasive properties are further validated in a 3D matrix model, which has shown to correlate well with the phenotype observed in vivo. As a final step of the in vitro pipeline, we developed a brain organoid model with patient-derived glioma cells, which is used to confirm the observed effects of the most promising compounds in a more complex microenvironment. Effective drugs identified in the in vitro screen are then tested in our mouse model in vivo: tumor growth, TM formation (number, morphology, interconnection) and function (calcium communication, invasion, therapy resistance) are longitudinally followed on a single cell level using a chronic cranial window and intravital two photon microscopy. Finally, classical drug effectivity parameters such as survival and tumor size on 7-T MRI scans are measured. First results of compounds, which have demonstrated tumor microtube-inhibiting vs. -promoting effects in this pipeline will be presented. In summary, this novel pipeline enables the rapid development of anti-TM drugs to translate the growing evidence of the importance of TMs to glioma biology into clinical trials.

A technology platform based on standard PCB technology has been developed for cost-effective fabrication of biological and medical test devices. Aluminum, polyimide and Pyralux have been selected as materials with focus on bio- and process compatibility. A process flow consisting of lamination, Al structuring by wet etching, microwell and via formation by laser drilling and via metallization was developed. Surface treatments have been evaluated for hydrophobic and hydrophilic modification of the materials. The technology was demonstrated with a dielectrophoresis enhanced microwell device for single cell handling and detection of cell-to-cell interaction as needed for the improvement of tumor therapy.

Many pharmaceuticals are proteins or their development is based on proteins. Cell-free protein synthesis (CFPS) is an innovative alternative to conventional cell based systems which enables the production of proteins with complex and even new characteristics. However, the short lifetime, low protein production and expensive reagent costs are still limitations of CFPS. Novel automated microfluidic systems might allow continuous, controllable and resource conserving CFPS. The presented microfluidic TRITT platform (TRITT for Transcription - RNA Immobilization & Transfer - Translation) addresses the individual biochemical requirements of the transcription and the translation step of CFPS in separate compartments, and combines the reaction steps by quasi-continuous transfer of RNA templates to enable automated CFPS. In detail, specific RNA templates with 5' and 3' hairpin structures for stabilization against nucleases were immobilized during in vitro transcription by newly designed and optimized hybridization oligonucleotides coupled to magnetizable particles. Transcription compatibility and reusability for immobilization of these functionalized particles was successfully proven. mRNA transfer was realized on-chip by magnetic actuated particle transfer, RNA elution and fluid flow to the in vitro translation compartment. The applicability of the microfluidic TRITT platform for the production of the cytotoxic protein Pierisin with simultaneous incorporation of a non-canonical amino acid for fluorescence labeling was demonstrated. The new reaction mode (TRITT mode) is a modified linked mode that fulfills the precondition for an automated modular reactor system. By continual transfer of new mRNA, the novel procedure overcomes problems caused by nuclease digestion and hydrolysis of mRNA during TL in standard CFPS reactions.

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.