Protrusion and retraction of lamellipodia are common features of eukaryotic cell motility. As a cell migrates through its extracellular matrix (ECM), lamellipod growth increases cell-ECM contact area and enhances engagement of integrin receptors, locally amplifying ECM input to internal signaling cascades. In contrast, contraction of lamellipodia results in reduced integrin engagement that dampens the level of ECM-induced signaling. These changes in cell shape are both influenced by, and feed back onto ECM signaling. Motivated by experimental observations on melanoma cells lines (1205Lu and SBcl2) migrating on fibronectin (FN) coated topographic substrates (anisotropic post-density arrays), we probe this interplay between intracellular and ECM signaling. Experimentally, cells exhibited one of three lamellipodial dynamics: persistently polarized, random, or oscillatory, with competing lamellipodia oscillating out of phase (Park et al., 2017). Pharmacological treatments, changes in FN density, and substrate topography all affected the fraction of cells exhibiting these behaviours. We use these observations as constraints to test a sequence of hypotheses for how intracellular (GTPase) and ECM signaling jointly regulate lamellipodial dynamics. The models encoding these hypotheses are predicated on mutually antagonistic Rac-Rho signaling, Rac-mediated protrusion (via activation of Arp2/3 actin nucleation) and Rho-mediated contraction (via ROCK phosphorylation of myosin light chain), which are coupled to ECM signaling that is modulated by protrusion/contraction. By testing each model against experimental observations, we identify how the signaling layers interact to generate the diverse range of cell behaviors, and how various molecular perturbations and changes in ECM signaling modulate the fraction of cells exhibiting each. We identify several factors that play distinct but critical roles in generating the observed dynamic: (1) competition between lamellipodia for shared pools of Rac and Rho, (2) activation of RhoA by ECM signaling, and (3) feedback from lamellipodial growth or contraction to cell-ECM contact area and therefore to the ECM signaling level.

Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and the N-bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in Drosophila glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.cerebral cavernous malformations | fluvastatin | zoledronic acid | mevalonate pathway | high-throughput screen C erebral cavernous malformations (CCMs) are common vascular malformations that develop in the brain, spinal cord, and, more rarely, the retina (1, 2). The lesions consist of dilated sinusoidal channels lined with a single layer of endothelium devoid of vessel wall elements. CCM disease is often asymptomatic, but on occasion the lesions rupture leading to hemorrhagic stroke. Other common symptoms are headache, seizures, and focal neurological deficits. Treatment options include observation of asymptomatic lesions, antiepileptic medication, and surgical excision of lesions in patients with symptomatic or repetitive hemorrhages or intractable seizures; however pharmacological therapies that improve outcome are lacking. CCM disease is usually sporadic, although 20% of patients carry lossof-function mutations in one of three genes: CCM1 (also known as "KRIT1"), CCM2, and CCM3 (also known as "PDCD10") (3), which encode structurally unrelated cytoplasmic proteins with critical roles in endothelial cells (4) and, uniquely for CCM3, in neurons and astrocytes as well (5, 6). CCM3 loss in neural progenitors results in hyperproliferation/activation of brain astrocytes and enhanced activity of RhoA in vivo and increased proliferation and survival of primary astrocytes in vitro (5, 6). We took advantage of these well-defined cellular phenotypes of CCM3 loss to explore potential pharmacological treatment options for CCM. High-throughput screening of available drugs identified fluvastatin, a 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitor as a top candidate. In combination with the N-bisphosphonate zoledronic acid monohyd...

Patterns of cellular organization in diverse tissues frequently display a complex geometry and topology tightly related to the tissue function. Progressive disorganization of tissue morphology can lead to pathologic remodeling, necessitating the development of experimental and theoretical methods of analysis of the tolerance of normal tissue function to structural alterations. A systematic way to investigate the relationship of diverse cell organization to tissue function is to engineer two-dimensional cell monolayers replicating key aspects of the in vivo tissue architecture. However, it is still not clear how this can be accomplished on a tissue level scale in a parameterized fashion, allowing for a mathematically precise definition of the model tissue organization and properties down to a cellular scale with a parameter dependent gradual change in model tissue organization. Here, we describe and use a method of designing precisely parameterized, geometrically complex patterns that are then used to control cell alignment and communication of model tissues. We demonstrate direct application of this method to guiding the growth of cardiac cell cultures and developing mathematical models of cell function that correspond to the underlying experimental patterns. Several anisotropic patterned cultures spanning a broad range of multicellular organization, mimicking the cardiac tissue organization of different regions of the heart, were found to be similar to each other and to isotropic cell monolayers in terms of local cell–cell interactions, reflected in similar confluency, morphology and connexin-43 expression. However, in agreement with the model predictions, different anisotropic patterns of cell organization, paralleling in vivo alterations of cardiac tissue morphology, resulted in variable and novel functional responses with important implications for the initiation and maintenance of cardiac arrhythmias. We conclude that variations of tissue geometry and topology can dramatically affect cardiac tissue function even if the constituent cells are themselves similar, and that the proposed method can provide a general strategy to experimentally and computationally investigate when such variation can lead to impaired tissue function.

This paper presents a novel implementation of current-mode variable-frequency control. The system architecture features a dual-loop feedback and a unique structure with symmetric signal paths for voltage and current Si signals. The symmetric structure provides practical advantages d(t) L Rs vout(t) for integrated controller design to achieve accurate Adaptive JL-L Voltage Positioning. The system dynamics is analyzed with the S2 it(t) Rc i (t) small-signal model. Design guide lines are derived for simple Current compensation circuits to achieve constant output impedance. ASensing Amplifie LPF(S) Go With variable switching frequency, the system achieves good dynamic response without requiring high switching frequency y at steady state. The system performance was verified by a Comparator two-channel interleaved controller chip implemented with CMOS technology. Vref C;onstan t VLIroltampifer I.

Of rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, higher metastatic propensity of fusion-positive rhabdomyosarcoma (FPRMS), a subtype with PAX3-FOXO1 fusion gene, than fusion-negative subtype (FNRMS) suggest that the fusion gene may stimulate metastasis. Recent single-cell level research revealed that FPRMS has heterogeneity in the expression levels of the fusion gene, i.e., PAX3-FOXO1 fusion protein (P3F-FP) and corresponding phenotypes related to metastasis, e.g., higher cell motility of cells with low P3F-FP (FPLow). However, it remains unclear how the fusion gene and its heterogeneous expression regulate metastasis. Interestingly, FPRMS shows collective invasion, distinct from individual invasion of FNRMS. The collective invasion is prevalently observed in invasive tumors and has higher metastatic potentials than individual invasion. Despite its substantial commonality with well-characterized individual invasion, its features stemming from multi-cellular organization cause distinct features, potentially related to its higher metastatic potentials. Specifically, heterogeneity of a collective cell group, i.e., the leader/follower coordination, regulates collective invasion. The collective cell mass consists of “leaders,” trailblazing invasion paths, and “followers”, spreading through the paths despite their less invasiveness. At the initiation step of metastasis, the mass may enrich the subpopulation of leaders, driving collective invasion. However, it may revert the enrichment at the colonization step, suppressing migration and promoting colonization driven by followers at the secondary tumor site. This well-tuned process upon each metastasis step suggests the significance of dynamic switching between leaders/followers, regulating collective invasion and, in turn, metastasis. Here, we suggest bistability, a system with two stable states, represents the leader/follower coordination in FPRMS. The bistable system exhibits switch-like transitions between the states. i.e., “flip-flopping.” The flip-flopping enables even a small input to drive an “on/off” transition towards leaders or the other, implying the dynamic switching corresponding to each metastasis step. We discovered two distinct subpopulations with leader-like fast migrating FPLow/follower-like slow FPHigh cells, representing bistability in FPRMS. Specifically, the marginal region of FPRMS cell mass where leaders mainly reside showed higher subpopulation of fast FPLow cells. The bistability requires double-positive/negative feedback in the signaling, regulating flip-flopping. We found that YAP signaling may establish double-negative feedback with P3F-FP, modulating the flip-flopping. Thus, we suggest that the fusion gene functions as a toggle switch of bistability, and its "flip-flopping" modulation of expression by YAP signaling may designate cells as leaders or followers, regulating collective invasion and, thus, metastasis. This project will shed light on the metastasis of FPRMS, potentially leading us to suppress its metastasis. Citation Format: JinSeok Park, Anthonios Chronopolous. Flip-flopping of fusion-positive rhabdomyosarcoma regulating intratumoral heterogeneity for metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B029.