Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Balgrist University Hospital

Tumori delle cellule germinali testicolari (TGCT) sono le neoplasie pi&ugrave; frequenti tra i giovani e la loro incidenza &egrave; cresciuta molto rapidamente negli ultimi decenni in Europa e Nord America. Molti studi sono stati effettuati al fine di chiarire i fattori implicati nella comparsa e progressione di questi tumori. Poco si sa circa il ruolo della diafonia cellula-stroma cancro nei processi invasivi TGCT. Qui, passiamo in rassegna diversi fattori che possono essere implicati nella progressione del tumore delle cellule germinali, come matrix metalloproteinases, insulin-like growth factor, transforming growth factor beta, il complesso caderina/catenina e integrine. Paradossalmente, alcune di queste molecole sono anche coinvolti nella regolazione della normale funzione testicolare. Infine, discutiamo le prospettive per future ricerche sul ruolo di stroma in progressione e differenziazione dei tumori delle cellule germinali maschili.

Invasione del tumore germinale e il ruolo di stroma testicolare.

Testicular germ cell tumors (TGCTs) are the most frequent neoplasia among young people and their incidence has grown very quickly during recent decades in North America and Europe. Many studies have been carried out in order to elucidate the factors involved in the appearance and progression of these tumors. Little is known about the role of cancer cell-stroma crosstalk in TGCT invasive processes. Here, we review several factors which may be implicated in germ cell tumor progression, such as matrix metalloproteinases, insulin-like growth factor, transforming growth factor beta, the cadherin/catenin complex and integrins. Paradoxically, some of these molecules are also involved in the regulation of normal testicular function. Finally, we discuss prospects for future research on the role of the stroma in the progression and differentiation of male germ cell tumors.

Germinal tumor invasion and the role of the testicular stroma.

Tumeurs des cellules germinales testiculaires (TGCTs) sont la n&eacute;oplasie la plus fr&eacute;quente chez les jeunes et leur incidence a augment&eacute; tr&egrave;s rapidement au cours de ces derni&egrave;res d&eacute;cennies en Am&eacute;rique du Nord et en Europe. De nombreuses &eacute;tudes ont &eacute;t&eacute; effectu&eacute;es afin d&#39;&eacute;lucider les facteurs impliqu&eacute;s dans l&#39;apparition et la progression de ces tumeurs. On en sait peu sur le r&ocirc;le de la diaphonie cellule-stroma du cancer dans les processus invasifs TGCT. Nous r&eacute;sumons ici, plusieurs facteurs qui pourraient &ecirc;tre impliqu&eacute;s dans la progression des tumeurs des cellules germinales, tels que les m&eacute;talloprot&eacute;inases matricielles, insulin-like growth factor, transforming growth factor beta, la cadh&eacute;rine/cat&eacute;nine complexe et les int&eacute;grines. Paradoxalement, certaines de ces mol&eacute;cules sont &eacute;galement impliqu&eacute;s dans la r&eacute;gulation de la fonction testiculaire normale. Finalement, nous discutons des perspectives pour de futures recherches sur le r&ocirc;le du stroma dans la progression et la diff&eacute;renciation des tumeurs des cellules germinales m&acirc;les.

Invasion tumorale germinal et le r&ocirc;le du stroma testiculaire.

The role of actin in transcription and RNA processing is now widely accepted but the form of nuclear actin remains enigmatic. Monomeric, oligomeric or polymeric forms of actin seem to be involved in nuclear functions. Moreover, uncommon forms of actin such as the "lower dimer" have been observed in vitro. Antibodies have been pivotal in revealing the presence and distribution of different forms of actin in different cellular locations. Because of its high degree of conservation, actin is a poor immunogen and only few specific actin antibodies are available. To unravel the mystery of less common forms of actin, in particular those in the nucleus, we chose to tailor monoclonal antibodies to recognize distinct forms of actin. To increase the immune response, we used a new approach based on peptide nanoparticles, which are designed to mimic an icosahedral virus capsid and allow the repetitive, ordered display of a specific epitope on their surface. Actin sequences representing the highly conserved "hydrophobic loop," which is buried in the filamentous actin filament, were grafted onto the surface of nanoparticles by genetic engineering. After immunization with "loop nanoparticles," a number of monoclonal antibodies were established that bind to the hydrophobic loop both in vitro and in situ. Immunofluorescence studies on cells revealed that filamentous actin filaments were only labeled once the epitope had been exposed. Our studies indicate that self-assembling peptide nanoparticles represent a versatile platform that can easily be customized to present antigenic determinants in repetitive, ordered arrays and elicit an immune response against poor antigens.

Peptide nanoparticles serve as a powerful platform for the immunogenic display of poorly antigenic actin determinants.

Low oxygen availability (hypoxia) is a hallmark of rapidly proliferating tumors and has been suggested to be a characteristic of the embryonic and adult stem cell niche. The idea of relating cancer to stem cells is increasingly popular due to the identification of specific cancer stem cells sharing the typical plasticity and motility of pluripotent stem cells. Hypoxia plays a critical role in early embryonic development and in tumor progression, participating in processes such as angiogenesis, apoptosis, cell migration, invasion and metastasis. Some of the molecular pathways that have been shown to mediate these hypoxia-induced responses, such as the hypoxia inducible factor (HIF)-1alpha and Notch signaling, appear to be active in both embryonic and neoplastic pluripotent stem cells. Nevertheless, the mechanisms underlying these regulatory processes are not yet fully understood. In this review, we attempt to shed some light on the mechanisms involved in hypoxia-dependent processes related to stem cell features and tumor progression, such as the maintenance of the undifferentiated state, cell proliferation, tumor neovascularization, extra-cellular matrix degradation and motility factor up-regulation. With this purpose in mind, we summarize recent observations in embryonic, adult and cancer stem cells that demonstrate the parallelism existing in their hypoxia responses. Finally, based on the observations of our own laboratory and others, we suggest that the comparative analysis of the response to low oxygen levels of embryonic stem cells and cancer stem cells (such as embryonal carcinoma cells), may throw fresh light on our understanding of the mechanisms underlying hypoxia-induced invasiveness and the resistance to anticancer treatments, thereby stimulating the development of novel therapeutic strategies.

Hypoxia and pluripotency in embryonic and embryonal carcinoma stem cell biology.

Testicular germ cell tumors (TGCTs) are the most frequent malignancies in adolescents and young adults. The incidence of TGCTs has doubled over the last few decades and the mechanisms underlying their pervasive growth are still poorly understood. Among them, seminomatous and non-seminomatous tumors have carcinoma in situ of the testis (CIS) as a common precursor lesion. It is currently accepted that the acquisition of genetic alterations and/or exposure to environmental factors are involved in the transition from CIS to invasive tumors. Nevertheless, although several TGCT-associated genetic aberrations have been identified, the mechanisms mediating their effects on TGCT development are still largely unknown. The aim of this review is to analyze the potential role of testicular microenvironmental factors, such as hypoxia and stroma cell-derived factors, in the acquisition by TGCT cells of an aggressive phenotype and the importance of these factors as potential therapeutic targets.

The role of microenvironment in testicular germ cell tumors.

Over the last 15 years, cell transplantation into seminiferous tubules has become a valuable tool to study germinal cell biology and related matters. This is particularly so, because the blood-testis permeability barrier establishes a sealed compartment which protect against certain influences such as immunological rejection. In the light of the functional and genetic similarities between carcinoma in situ (CIS) of the testis and embryonic stem (ES) cells, our laboratory has developed a tumor assay to study cancer invasion processes in testicular germ cell tumors (TGCT) based on the transplantation of ES cells into the seminiferous tubules. Here, we describe this new tumor assay and provide additional information regarding the transplantation techniques used and their application for the study of TGCTs. Finally, we discuss the practical implications of our experimental approach and its potential application for the understanding of TGCT invasive processes and the development of new antineoplastic strategies.

Embryonic stem cell transplantation into seminiferous tubules: a model for the study of invasive germ cell tumors of the testis.

The dynamic rearrangement of the actin cytoskeleton plays a key role in several cellular processes such as cell motility, endocytosis, RNA processing and chromatin organization. However, the supramolecular actin structures involved in the different processes remain largely unknown. One of the less studied forms of actin is the lower dimer (LD). This unconventional arrangement of two actin molecules in an antiparallel orientation can be detected by chemical crosslinking at the onset of polymerization in vitro. Moreover, evidence for a transient incorporation of LD into growing filaments and its ability to inhibit nucleation of F-actin filament assembly implicate that the LD pathway contributes to supramolecular actin patterning. However, a clear link from this actin species to a specific cellular function has not yet been established. We have developed an antibody that selectively binds to LD configurations in supramolecular actin structures assembled in vitro. This antibody allowed us to unveil the LD in different mammalian cells. In particular, we show an association of the antiparallel actin arrangement with the endocytic compartment at the cellular and ultrastructural level. Taken together, our results strongly support a functional role of LD in the patterning of supramolecular actin assemblies in mammalian cells.

An antiparallel actin dimer is associated with the endocytic pathway in mammalian cells.

Cell transplantation into the seminiferous tubules is a useful technique for the study of physiological and pathological conditions affecting the testis. However, the precise three-dimensional organization and, particularly, the complex connectivity of the seminiferous network have not yet been thoroughly characterized. To date, the technical approaches to address these issues have included manual dissection under the stereomicroscope, reconstruction of histological serial sections, and injection of contrast dyes, but all of them have yielded only partial information. Here, using an approach based on the microinjection of a self-polymerizing resin followed by chemical digestion of the surrounding soft tissues, we reveal fine details of the seminiferous tubule scaffold and its connections. These replicas of the testis seminiferous network were studied by scanning electron microscopy. The present results not only establish a morphological basis for more precise microinjection into the mouse seminiferous tubules but also enable a more profound investigation of physiological and embryological features of the testis.

Anatomical basis for cell transplantation into mouse seminiferous tubules.

Formins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. In the present study we show that human FHOD1 lacks actin nucleation and elongation capacity, but acts as an actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1-FH2 element. The bundling phenotype is maintained in the presence of tropomyosin, confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Taken together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments.

FHOD1 is a combined actin filament capping and bundling factor that selectively associates with actin arcs and stress fibers.

The existence of a cellular machinery that is based on the reversible polymerization of globular nucleotide-bound protomers into polar microfilaments is a persistent feature from prokaryotes to higher vertebrates. However, while in bacteria, actin-like proteins with such properties have evolved into a large family with divergent sequences and polymeric structures, eukaryotes express only a small number of highly conserved actins. Indeed, the sequence of actin is one of the best conserved among eukaryotes and yet actin carries out many different functions at distinct cellular sites. Because of the notorious conservation and lack of suitable tools to examine structural plasticity, the vast majority of studies on cellular actin functions consider mainly two structural states, G-actin and F-actin. However, there is more to the structural plasticity of actin than first meets the eye. On one hand, more than 200 actin-binding proteins shape the conformation of actin and thereby regulate functional diversity. On the other hand, unconventional actin conformations that differ from monomeric G-actin are stepping into the limelight. In addition, supramolecular actin structures that extend beyond classical F-actin are emerging. Herein, we recapitulate the current knowledge on the structure and conformations of monomeric actin and its polymerization into higher order structures, paying special attention to less known forms and their involvement in actin function.

Unconventional actin configurations step into the limelight.

Spermatogonial stem cells (SSCs) are pluripotent elements found in the adult seminiferous epithelium between Sertoli cells and a basal lamina which covers the multilayered external wall of peritubular myoid cells. The microenvironment of this pluripotent stem cell niche creates the complex and dynamic system that is necessary for the initiation of spermatogenesis, but this system also contains factors which can potentially collaborate in the progression of testicular germ cell tumors (TGCTs). In this review, we summarize our current knowledge about some important structural and molecular features related to the SSC niche, including growth factors, adhesion molecules, extracellular matrix, mechanical stress and vascularization. We discuss their possible collaborative effects on the generation and progression of TGCTs, which are a type of cancer representing the most frequent neoplasia among young men and whose incidence has grown very quickly during the past decades in North America and Europe. In this regard, a better understanding of the pluripotent stem cell niche where these malignancies arise will provide further insights into the origin of TGCTs and the mechanisms underlying their growth and invasion of adjacent and distant tissues.

The spermatogonial stem cell niche in testicular germ cell tumors.

Tumor initiation and progression are accompanied by complex changes in the cytoarchitecture that at the cellular level involve remodeling of the cytoskeleton. We report on the impact of a mutant β-actin (G245D-actin) on cell structure and multicellular assembly properties. To appreciate the effects of the Gly245Asp substitution on the organization of the actin cytoskeleton, we examined the polymerization properties of G245D-actin in vitro by pyrene polymerization assays and total internal reflection fluorescence microscopy (TIRF). The mutant actin on its own has a significantly reduced polymerization efficiency compared to native actin but also modifies the polymerization of actin in copolymerization experiments. Comparison of the structure of Rat-2 fibroblasts and a stably transfected derivate called Rat-2-sm9 revealed the effects of G245D-actin in a cellular environment. The overall actin levels in Rat-2-sm9 show a 1.6-fold increase with similar amounts of mutant and wild-type actin. G245D-actin expression renders Rat-2-sm9 cells highly tumorigenic in nude mice. In Rat-2-sm9 monolayers, G245D-actin triggers the formation of extensive membrane ruffles, which is a characteristic feature of many transformed cells. To approximate complex cell-cell and cell-matrix interactions that occur in tumors and might modulate the effects of G245D-actin, we extended our studies to scaffold-free 3D spheroid cultures. Bright field and scanning electron microscopy (SEM) show that Rat-2-sm9 and Rat-2 cells share essential features of spheroid formation and compaction. However, the resulting spheroids exhibit distinct phenotypes that differ mainly in surface structure and size. The systematic comparison of transformed and normal spheroids by SEM provides new insights into scaffold-free fibroblast spheroid formation.

A tumorigenic actin mutant alters fibroblast morphology and multicellular assembly properties.

F-actin treadmilling plays a key part in cell locomotion. Because immunofluorescence showed colocalisation of thymosin beta4 (Tβ4) with cofilin-1 and Arp2/3 complex in lamellipodia, we analyzed combinations of these proteins on F-actin-adenosine triphosphate (ATP)-hydrolysis, which provides a measure of actin treadmilling. Actin depolymerising factor (ADF)/cofilin stimulated treadmilling, while Tβ4 decreased treadmilling, presumably by sequestering monomers. Tβ4 added together with ADF/cofilin also inhibited the treadmilling, relative to cofilin alone, but both the rate and extent of depolymerization were markedly enhanced in the presence of both these proteins. Arp2/3 complex reversed the sequestering activity of Tβ4 when equimolar to actin, but not in the additional presence of cofilin-1 or ADF. Transfection experiments to explore the effects of changing the intracellular concentration of Tβ4 in HeLa cells showed that an increase in Tβ4 resulted in reduced actin filaments bundles and narrower lamellipodia, and a conspicuous decrease of cell migration as seen by two different assays. In contrast, cells transfected with a vector leading to Tβ4 knockdown by small interfering RNA (siRNA) displayed prominent actin filament networks within the lamellipodia and the leading lamella and enhanced migration. The experiments reported here demonstrate the importance of the interplay of these different classes of actin-binding proteins on cell behaviour.

Thymosin beta4 inhibits ADF/cofilin stimulated F-actin cycling and hela cell migration: reversal by active Arp2/3 complex.

Testicular germ cell tumors (TGCTs) comprise the vast majority of all testicular malignancies and are the most common type of cancer among young male adults. The nonseminomatous variant of TGCTs is characterized by the presence of embryonic and extraembryonic tissues together with a population of pluripotent cancer stem cells, the so-called embryonal carcinoma. One of the main causes of the resistance of these tumors to therapy is their ability to invade adjacent tissues and metastasize into distant sites of the body. Both of these tumor processes are highly favored by the neovascularization of the malignant tissue. New vessels can be generated by means of angiogenesis or vasculogenesis, and both have been observed to occur during tumor vascularization. Nevertheless, the precise contribution of each process to the neoplastic vascular bed of TGCTs remains unknown. In addition, another process known as tumor-derived vasculogenesis, in which malignant cells give rise to endothelial cells, has also been reported to occur in a number of tumor types, including experimental TGCTs. The participation and cross talk of these 3 processes in tumor vascularization is of particular interest, given the embryonic origin of teratocarcinomas. Thus, in the present review, we discuss the importance of all 3 vascularization processes in the growth, invasion, and metastasis of testicular teratocarcinomas and summarize the current state of knowledge of the TGCT microenvironment and its relationship with vascularization. Finally, we discuss the importance of vascularization as a therapeutic target for this type of malignancy.

Vasculogenesis and angiogenesis in nonseminomatous testicular germ cell tumors.

Polymerization of actin monomers into filaments requires the initial formation of nuclei composed of a few actin subunits; however, their instability has hindered their detailed study. Therefore we used chemically crosslinked actin oligomers to analyse their effect on actin polymerization. Actin dimer (upper dimer, UD), trimer and tetramer intermolecularly crosslinked by phenylene-bismaleimide along the genetic helix (between Lys199 and Cys374) were isolated by gel filtration and found to increasingly stimulate actin polymerization as shown by the pyrene assay and total internal reflection fluorescence microscopy. In contrast, the so-called lower actin dimer (LD) characterized by a Cys374-Cys374 crosslink stimulated actin polymerization only at low but inhibited it at high concentrations. UD and trimer stimulated the repolymerization of actin from complexes with thymosin β4 (Tβ4) or profilin, whereas the LD stimulated repolymerization only from the profilin : actin but not the actin : Tβ4 complex. In vivo, actin polymerization is stimulated by nucleation factors. Therefore the interaction and effects of purified LD, UD and trimer on the actin-nucleating activity of gelsolin, mouse diaphanous related (mDia) formin and the actin-related protein 2/3 (Arp2/3) complex were analysed. Native gel electrophoresis demonstrated binding of LD, UD and trimer to gelsolin and its fragment G1-3, to the FH2 domains of the formins mDia1 and mDia3, and to Arp2/3 complex. UD and trimer increased the nucleating activity of gelsolin and G1-3, but not of the mDia-FH2 domain nor of the Arp2/3 complex. In contrast, LD at equimolar concentration to Arp2/3 complex stimulated its nucleating activity, but inhibited that of mDia-FH2 domains, gelsolin and G1-3, demonstrating differential regulation of their nucleating activity by dimers containing differently oriented actin subunits.

Unai Silvan

University of Zurich

Balgrist University Hospital

Easy and Accurate Mechano-profiling on Micropost Arrays

Unai Silvan

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Balgrist University Hospital

Easy and Accurate Mechano-profiling on Micropost Arrays

University of Zurich