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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

CD98hc (SLC3A2) loss protects against ras-driven tumorigenesis by modulating integrin-mediated mechanotransduction.

CD98hc (SLC3A2) is the heavy chain component of the dimeric transmembrane glycoprotein CD98, which comprises the large neutral amino acid transporter LAT1 (SLC7A5) in cells. Overexpression of CD98hc occurs widely in cancer cells and is associated with poor prognosis clinically, but its exact contributions to tumorigenesis are uncertain. In this study, we showed that genetic deficiency of CD98hc protects against Ras-driven skin carcinogenesis. Deleting CD98hc after tumor induction was also sufficient to cause regression of existing tumors. Investigations into the basis for these effects defined two new functions of CD98hc that contribute to epithelial cancer beyond an intrinsic effect of CD98hc on tumor cell proliferation. First, CD98hc increased the stiffness of the tumor microenvironment. Second, CD98hc amplified the capacity of cells to respond to matrix rigidity, an essential factor in tumor development. Mechanistically, CD98hc mediated this stiffness sensing by increasing Rho kinase (ROCK) activity, resulting in increased transcription mediated by YAP/TAZ, a nuclear relay for mechanical signals. Our results suggest that CD98hc contributes to carcinogenesis by amplifying a positive feedback loop, which increases both extracellular matrix stiffness and resulting cellular responses. This work supports a rationale to explore the use of CD98hc inhibitors as cancer therapeutics.

LIF mediates proinvasive activation of stromal fibroblasts in cancer.

Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF) as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA) expression. We demonstrate that a pulse of transforming growth factor ß (TGF-ß) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-ß-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor) counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

CD98hc (SLC3A2) regulation of skin homeostasis wanes with age.

Skin aging is linked to reduced epidermal proliferation and general extracellular matrix atrophy. This involves factors such as the cell adhesion receptors integrins and amino acid transporters. CD98hc (SLC3A2), a heterodimeric amino acid transporter, modulates integrin signaling in vitro. We unravel CD98hc functions in vivo in skin. We report that CD98hc invalidation has no appreciable effect on cell adhesion, clearly showing that CD98hc disruption phenocopies neither CD98hc knockdown in cultured keratinocytes nor epidermal ß1 integrin loss in vivo. Instead, we show that CD98hc deletion in murine epidermis results in improper skin homeostasis and epidermal wound healing. These defects resemble aged skin alterations and correlate with reduction of CD98hc expression observed in elderly mice. We also demonstrate that CD98hc absence in vivo induces defects as early as integrin-dependent Src activation. We decipher the molecular mechanisms involved in vivo by revealing a crucial role of the CD98hc/integrins/Rho guanine nucleotide exchange factor (GEF) leukemia-associated RhoGEF (LARG)/RhoA pathway in skin homeostasis. Finally, we demonstrate that the deregulation of RhoA activation in the absence of CD98hc is also a result of impaired CD98hc-dependent amino acid transports.

Adipocytes secrete leukotrienes: contribution to obesity-associated inflammation and insulin resistance in mice.

Leukotrienes (LTs) are potent proinflammatory mediators, and many important aspects of innate and adaptive immune responses are regulated by LTs. Key members of the LT synthesis pathway are overexpressed in adipose tissue (AT) during obesity, resulting in increased LT levels in this tissue. We observed that several mouse adipocyte cell lines and primary adipocytes from mice and humans both can secrete large amounts of LTs. Furthermore, this production increases with a high-fat diet (HFD) and positively correlates with adipocyte size. LTs produced by adipocytes play an important role in attracting macrophages and T cells in in vitro chemotaxis assays. Mice that are deficient for the enzyme 5-lipoxygenase (5-LO), and therefore lack LTs, exhibit a decrease in HFD-induced AT macrophage and T-cell infiltration and are partially protected from HFD-induced insulin resistance. Similarly, treatment of HFD-fed wild-type mice with the 5-LO inhibitor Zileuton also results in a reduction of AT macrophages and T cells, accompanied by a decrease in insulin resistance. Together, these findings suggest that LTs represent a novel target in the prevention or treatment of obesity-associated inflammation and insulin resistance.