NANOBODY® Molecule, a Giga Medical Tool in Nanodimensions.

Although antibodies remain the most widely used tool for biomedical research, antibody technology is not flawless. Innovative alternatives, such as Nanobody® molecules, were developed to address the shortcomings of conventional antibodies. Nanobody® molecules are antigen-binding variable-domain fragments derived from the heavy-chain-only antibodies of camelids (VHH) and combine the advantageous properties of small molecules and monoclonal antibodies. Nanobody® molecules present a small size (~15 kDa, 4 nm long and 2.5 nm wide), high solubility, stability, specificity, and affinity, ease of cloning, and thermal and chemical resistance. Recombinant production in microorganisms is cost-effective, and VHH are also building blocks for multidomain constructs. These unique features led to numerous applications in fundamental research, diagnostics, and therapy. Nanobody® molecules are employed as biomarker probes and, when fused to radioisotopes or fluorophores, represent ideal non-invasive in vivo imaging agents. They can be used as neutralizing agents, receptor-ligand antagonists, or in targeted vehicle-based drug therapy. As early as 2018, the first Nanobody®, Cablivi (caplacizumab), a single-domain antibody (sdAb) drug developed by French pharmaceutical giant Sanofi for the treatment of adult patients with acquired thrombocytopenic purpura (aTTP), was launched. Nanobody® compounds are ideal tools for further development in clinics for diagnostic and therapeutic purposes.

Cyclic uniaxial mechanical stretching of cells using a LEGO® parts-based mechanical stretcher system.

Mechanical cues are essential for the regulation of cell and tissue physiology. Hence, it has become an utmost necessity for cell biologists to account for those mechanical parameters when investigating biological processes and they need devices to manipulate cells accordingly. Here, we report a simple mechanical cell-stretching system that can generate uniaxial cyclic mechanical stretch on cells in tissue culture. This system is based upon a low-cost battery-powered uniaxial cyclic mechanical stretcher exclusively built out of LEGO® parts combined with a stretchable poly(dimethylsiloxane) tissue culture plate in order to grow and stretch cells. We characterize the system and show that it can be used in a wide variety of downstream applications, including immunofluorescence, western blotting and biochemical assays. We also illustrate how this system can be useful in a study as we investigated the behavior of integrin adhesion complexes upon cell stretching. We therefore present a cost-effective, multipurpose cell-stretching system that should help to increase understanding of mechanical signaling.This article has an associated First Person interview with the first author of the paper.

CD98hc facilitates B cell proliferation and adaptive humoral immunity.

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates.

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with ß-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.

18F-fluorodihydroxyphenylalanine PET/CT in pheochromocytoma and paraganglioma: relation to genotype and amino acid transport system L.

PURPOSE: F-FDOPA is a highly sensitive and specific radiopharmaceutical for pheochromocytoma and paraganglioma (PPGL) imaging. However, 18F-FDOPA might be falsely negative in these tumors, especially those related to mutations in succinate dehydrogenase genes (SDHx). The aim of the present study was to evaluate the relationship between expression of L-DOPA transporters and 18F-FDOPA PET imaging results in PPGL. METHODS: From 2007 to 2015, 175 patients with non-metastatic PPGL were evaluated by 18F-FDOPA PET/CT for initial diagnosis/staging and follow-up. 18F-FDOPA PET/CT was considered as falsely negative for at least one lesion in 10/126 (8%) patients (two sporadic, six SDHD, two SDHB PPGLs). The mRNA and protein expression levels of CD98hc and LATs were evaluated in samples with different genetic backgrounds and imaging phenotypes. The qRT-PCR and immunohistochemical analyses were performed in 14 and 16 tumor samples, respectively. RESULTS: The SDHx mutated samples exhibited a significant decrease in mRNA expression of LAT3 when compared to sporadic PPGLs (P = 0.042). There was also a statistical trend toward decreased CD98hc (P = 0.147) and LAT4 (P = 0.012) levels in SDHx vs sporadic PPGLs. No difference was observed for LAT1/LAT2 mRNA levels. LAT1 protein was expressed in 15 out of 16 (93.75%) SDHx tumors, regardless of the 18F-FDOPA positivity. LAT1 and CD98hc were co-expressed in 6/8 18F-FDOPA-negative PPGLs. In contrast, in one case with absence of LAT1/CD98hc, 18F-FDOPA uptake was positive and attributed to LAT4 expression. CONCLUSIONS: We conclude that down-regulation of LAT1/CD98hc cannot explain the imaging phenotype of SDHx-related PPGLs. A reduced activity of LAT1 remains the primary hypothesis possibly due to a modification of intracellular amino acid content which may reduce 18F-FDOPA uptake.

Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host.

ECM and epithelial stem cells: the scaffold of destiny.

Adult stem cells play a critical role in maintaining tissue homeostasis and promoting longevity. The intricate organization and presence of common markers among adult epithelial stem cells in the intestine, lung, and skin serve as hallmarks of these cells. The specific location pattern of these cells within their respective organs highlights the significance of the niche in which they reside. The extracellular matrix (ECM) not only provides physical support but also acts as a reservoir for various biochemical and biophysical signals. We will consider differences in proliferation, repair, and regenerative capacities of the three epithelia and review how environmental cues emerging from the niche regulate cell fate. These cues are transduced via mechanosignaling, regulating gene expression, and bring us to the concept of the fate scaffold. Understanding both the analogies and discrepancies in the mechanisms that govern stem cell fate in various organs can offer valuable insights for rejuvenation therapy and tissue engineering.

Off the beaten paths: alternative and crosstalk regulation of Rho GTPases.

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.

CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling.

Integrin-dependent assembly of the fibronectin (Fn) matrix plays a central role in vertebrate development. We identify CD98hc, a membrane protein, as an important component of the matrix assembly machinery both in vitro and in vivo. CD98hc was not required for biosynthesis of cellular Fn or the maintenance of the repertoire or affinity of cellular Fn binding integrins, which are important contributors to Fn assembly. Instead, CD98hc was involved in the cell's ability to exert force on the matrix and did so by dint of its capacity to interact with integrins to support downstream signals that lead to activation of RhoA small GTPase. Thus, we identify CD98hc as a membrane protein that enables matrix assembly and establish that it functions by interacting with integrins to support RhoA-driven contractility. CD98hc expression can vary widely; our data show that these variations in CD98hc expression can control the capacity of cells to assemble an Fn matrix, a process important in development, wound healing, and tumorigenesis.

The LEGO® brick road to open science and biotechnology.

LEGO® is a brand of toys that have entertained generations of children. Beyond amusement, LEGO® bricks also constitute a building ecosystem of their own that creators from the general public, as well as scientists and engineers, can use to design and assemble devices for all purposes, including scientific research and biotechnology. We describe several of these constructions to highlight the construction properties of LEGO® and their advantages, caveats, and impact in biotechnology. We also discuss how this emerging trend in LEGO® building pairs with a growing interest in open-access and frugal science which aims to provide access to technology to all scientists regardless of financial wealth and technological prowess.

Lung Adenocarcinoma Tumor Origin: A Guide for Personalized Medicine.

Lung adenocarcinoma, the major form of lung cancer, is the deadliest cancer worldwide, due to its late diagnosis and its high heterogeneity. Indeed, lung adenocarcinoma exhibits pronounced inter- and intra-tumor heterogeneity cofounding precision medicine. Tumor heterogeneity is a clinical challenge driving tumor progression and drug resistance. Several key pieces of evidence demonstrated that lung adenocarcinoma results from the transformation of progenitor cells that accumulate genetic abnormalities. Thus, a better understanding of the cell of origin of lung adenocarcinoma represents an opportunity to unveil new therapeutic alternatives and stratify patient tumors. While the lung is remarkably quiescent during homeostasis, it presents an extensive ability to respond to injury and regenerate lost or damaged cells. As the lung is constantly exposed to potential insult, its regenerative potential is assured by several stem and progenitor cells. These can be induced to proliferate in response to injury as well as differentiate into multiple cell types. A better understanding of how genetic alterations and perturbed microenvironments impact progenitor-mediated tumorigenesis and treatment response is of the utmost importance to develop new therapeutic opportunities.

Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation.

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies.

Cyclic uniaxial cell stretching in tissue culture using a LEGO®-based mechanical stretcher and a polydimethylsiloxane stretchable vessel.

Mechanical signals are essential for the regulation of many biological processes. Therefore, it has become paramount to account for these mechanical parameters when exploring biological processes. Here, we describe a protocol to apply cyclic uniaxial stretch on cells in culture using a LEGO®-based mechanical stretcher and a flexible custom-made polydimethylsiloxane culture vessel as well as validated downstream applications. While this system offers an out-of-the-box limited type of simulation, it provides a reliable and low-cost opportunity to perform cell stretching. For complete details on the use and execution of this protocol, please refer to Boulter et al. (2020).

Blocking the alpha 4 integrin-paxillin interaction selectively impairs mononuclear leukocyte recruitment to an inflammatory site.

Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice], which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice, mice bearing the alpha4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however, the alpha4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyer's patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis.

Tracing the cellular dynamics of sebaceous gland development in normal and perturbed states.

The sebaceous gland (SG) is an essential component of the skin, and SG dysfunction is debilitating1,2. Yet, the cellular bases for its origin, development and subsequent maintenance remain poorly understood. Here, we apply large-scale quantitative fate mapping to define the patterns of cell fate behaviour during SG development and maintenance. We show that the SG develops from a defined number of lineage-restricted progenitors that undergo a programme of independent and stochastic cell fate decisions. Following an expansion phase, equipotent progenitors transition into a phase of homeostatic turnover, which is correlated with changes in the mechanical properties of the stroma and spatial restrictions on gland size. Expression of the oncogene KrasG12D results in a release from these constraints and unbridled gland expansion. Quantitative clonal fate analysis reveals that, during this phase, the primary effect of the Kras oncogene is to drive a constant fate bias with little effect on cell division rates. These findings provide insight into the developmental programme of the SG, as well as the mechanisms that drive tumour progression and gland dysfunction.

Tumor-Stroma Mechanics Coordinate Amino Acid Availability to Sustain Tumor Growth and Malignancy.

Dysregulation of extracellular matrix (ECM) deposition and cellular metabolism promotes tumor aggressiveness by sustaining the activity of key growth, invasion, and survival pathways. Yet mechanisms by which biophysical properties of ECM relate to metabolic processes and tumor progression remain undefined. In both cancer cells and carcinoma-associated fibroblasts (CAFs), we found that ECM stiffening mechanoactivates glycolysis and glutamine metabolism and thus coordinates non-essential amino acid flux within the tumor niche. Specifically, we demonstrate a metabolic crosstalk between CAF and cancer cells in which CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodeling. Collectively, our findings link mechanical stimuli to dysregulated tumor metabolism and thereby highlight a new metabolic network within tumors in which diverse fuel sources are used to promote growth and aggressiveness. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in cancer.

Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway.

Mechanical and metabolic cues independently contribute to the regulation of cell and tissue homeostasis. However, how they cross-regulate each other during this process remains largely unknown. Here, we show that cellular metabolism can regulate integrin rigidity-sensing via the sphingolipid metabolic pathway controlled by the amino acid transporter and integrin coreceptor CD98hc (SLC3A2). Genetic invalidation of CD98hc in dermal cells and tissue impairs rigidity sensing and mechanical signaling downstream of integrins, including RhoA activation, resulting in aberrant tissue mechanical homeostasis. Unexpectedly, we found that this regulation does not occur directly through regulation of integrins by CD98hc but indirectly, via the regulation of sphingolipid synthesis and the delta-4-desaturase DES2. Loss of CD98hc decreases sphingolipid availability preventing proper membrane recruitment, shuttling and activation of upstream regulators of RhoA including Src kinases and GEF-H1. Altogether, our results unravel a novel cross-talk regulation between integrin mechanosensing and cellular metabolism which may constitute an important new regulatory framework contributing to mechanical homeostasis.

Dermal Fibroblast SLC3A2 Deficiency Leads to Premature Aging and Loss of Epithelial Homeostasis.

Skin homeostasis relies on fine-tuning of epidermis-dermis interactions and is affected by aging. While extracellular matrix (ECM) proteins, such as integrins, are involved in aging, the molecular basis of the skin changes needs to be investigated further. Here, we showed that integrin co-receptor, SLC3A2, required for cell proliferation, is expressed at the surface of resting dermal fibroblasts in young patients and is reduced drastically with aging. In vivo SLC3A2 dermal fibroblast deletion induced major skin phenotypes resembling premature aging. Knockout mice (3 months old) presented strong defects in skin elasticity due to altered ECM assembly, which impairs epidermal homeostasis. SLC3A2 dermal fibroblast loss led to an age-associated secretome profile, with 77% of identified proteins belonging to ECM and ECM-associated proteins. ECM not only contributes to skin mechanical properties, but it is also a reservoir of growth factors and bioactive molecules. We demonstrate that dermal fibroblast SLC3A2 is required for ECM to fully exert its structural and reservoir role allowing proper and efficient TGF-ß localization and activation. We identified SLC3A2 as a protective controller of dermal ECM stiffness and quality required to maintain the epidermis to dermis interface as functional and dynamic.

The body's tailored suit: Skin as a mechanical interface.

Skin, by nature, is very similar to the Rouquayrol-Denayrouze suit mentioned by Jules Verne in Twenty Thousand Leagues Under the Sea: it allows "to risk (…) new physiological conditions without suffering any organic disorder". Mechanical cues, to the same extent as other environmental parameters, are such "new physiological conditions". Indeed, skin's primary function is to form a protective barrier to shield inner tissues from the external environment. This requires unique mechanical properties as well as the ability to sense mechanical cues from the environment in order to prevent or repair mechanical damages as well as to function as the primary mechanosensory interface of the whole body.

Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts.

Carcinoma-associated fibroblasts (CAF) mediate the onset of a proinvasive tumour microenvironment. The proinflammatory cytokine LIF reprograms fibroblasts into a proinvasive phenotype, which promotes extracellular matrix remodelling and collective invasion of cancer cells. Here we unveil that exposure to LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signalling, which results in sustained proinvasive activity of CAF. Mechanistically, p300-histone acetyltransferase acetylates STAT3, which, in turn, upregulates and activates the DNMT3b DNA methyltransferase. DNMT3b methylates CpG sites of the SHP-1 phosphatase promoter, which abrogates SHP-1 expression, and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signalling is maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype.