In addition, the important contribution of astrocytes to HD-related circuit defects has recently been recognized. In parallel, unbiased systems biology studies are providing insights into the possible molecular underpinnings of these functional defects at the level of synaptic signaling and neurotransmitter metabolism. With these approaches, we can now reach a deeper understanding of circuit-based HD mechanisms, which will be crucial for the development of effective and targeted therapeutic strategies.Huntington’s disease (HD) is a hereditary neurodegenerative disorder that typically manifests in midlife with motor, cognitive, and/or psychiatric symptoms. The disease is caused by a CAG triplet expansion in exon 1 of the huntingtin gene and leads to a severe neurodegeneration in the striatum and cortex. Classical electrophysiological studies in genetic HD mouse models provided important insights into the disbalance of excitatory, inhibitory and neuromodulatory inputs, as well as progressive disconnection between the cortex and striatum. However, the involvement of local cortical and striatal microcircuits still remains largely unexplored. Here we review the progress in understanding HD-related impairments in the cortical and basal ganglia circuits, and outline new opportunities that have opened with the development of modern circuit analysis methods. In particular,  HTT) gene on chromosome 4 (Huntington’s disease (HD) is a devastating movement disorder that affects about 1 in 10,000 people. Among the heterogeneous group of neurodegenerative diseases, it takes a special role based on its strictly genetic cause, i.e., an autosomal dominant mutation of the huntingtin  and generally exhibit a slower disease progression than truncated models. The yeast artificial chromosome (YAC) transgenic strategy was used to generate several mouse lines, named corresponding to their number of CAG repeats: YAC18 (control), YAC46, YAC72, and YAC128 . The YACHtt locus, thereby more closely resembling the genetic context of HD patients. In these mice, brain atrophy and motor defects slowly emerge in a protracted manner. Among the knock-in models are the HD allelic series mice with various CAG tract lengths, including CAG140 and the widely used zQ175 line , ventral pallidum, substantia nigra (SN), and subthalamic nucleus (STN). Within the basal ganglia, striatum is the region receiving most of the long-range input, including extensive glutamatergic innervation from CPNs . Althougin vivo studies demonstrating simultaneous activation of dSPN and iSPN cell clusters during motion initiation, as well as similar correlation of their activity with locomotor behavior, suggesting a more sophisticated functional arrangement of basal ganglia circuits than previously thought  and the substantia nigra pars reticulata (SNr). The D2 dopamine receptor-positive SPNs form the indirect pathway and are referred to as iSPNs. This polysynaptic pathway connects to the output nuclei indirectly via the external segment of the globus pallidus (GPe) and STN  Alexand. GPi and thought .In addition to the direct and indirect pathways, a third, hyperdirect pathway exists that bypasses the striatum and connects the frontal cortex to the output nuclei via glutamatergic neurons of the subthalamic nucleus (STN) . Like thApart from SPNs, striatal microcircuits include multiple groups of local interneurons: cholinergic interneurons (CINs) and several types of GABAergic cells that can be distinguished by the expression of molecular markers such as parvalbimun (PV), neuropeptide Y (NPY), neuropeptide Y/somatostatin/nitric oxide synthase (NPY/SST/NOS), calretinin (CR), and tyrosine hydroxylase (TH). Overall, striatal interneurons receive similar types of afferents as striatal SPNs , and provide feedforward inhibition onto SPNs, modulating their activity on different time scales; however, each interneuron subtype has distinct connectivity and physiology. Striatal PV interneurons are fast-spiking cells  that preferentially target the soma and proximal dendrites of SPNs, providing fast and strong inhibitory inputs. All other GABAergic interneurons fire at lower rates and form synapses on distal SPN dendrites . CINs inAlthough striatal interneurons provide the major source of GABAergic inhibition to SPNs, other inhibitory inputs also exist. Both dSPNs and iSPNs send collateral projections to other SPNs belonging to both pathways . AnotherCortical degeneration and dysfunction significantly contribute to impairments in motor and executive functions and cognitive abilities observed in HD. This is underlined by detailed neuropathological studies in human post-mortem brains which revealed a reduction in overall cortical area and cortical white matter, associated with a marked cell loss. It is well-established that CPNs are particularly vulnerable to HD, however region-specific degeneration of interneurons also occurs . InteresA growing body of evidence also highlights the fact that structural and functional alterations in the cortex precede neuronal loss by several years. The cortex of HD mutation carriers shows progressive regional thinning in a topographically predictable manner already up to 15 years before the onset of motor symptoms . On a fuin vivo structural imaging furthermore demonstrated impaired spine turnover and a progressive loss of persistent spines in the somatosensory cortex , transient extracellular signals generated by large populations of neurons. These recordings uncovered altered synchrony between the cortical and striatal networks in HD  for survival. BDNF is mainly produced in the cortex and delivered to the striatum anterogradely via CPN axons . mHTT dein vivo was also confirmed in a recent in vitro approach using compartmentalized microfluidic chambers to reconstitute corticostriatal connectivity in a dish , a monoamine neuromodulator, which, together with GABA signaling, provides crucial counterbalance and adds flexibility to glutamatergic excitation of SPNs. In addition to the disturbances of glutamate and GABA signaling described above, many HD symptoms are therefore associated with altered dopaminergic modulation.The dopaminergic circuit anatomy as well as dopaminergic signaling alterations in HD are described in detail in a number of excellent articles . Two majDA can modulate the function of both excitatory CPNs and inhibitory SPNs on several levels, such as the probability of neurotransmitter release, the post-synaptic receptor sensitivity to the neurotransmitter , and post-synaptic integration and ensuing excitability .Intracellular DA signaling is mediated by a family of G-protein coupled receptors (D1–D5 DA receptors), which are grouped into two classes referred to as D1-like and D2-like. Upon receptor activation, one of the main targets of the recruited heterotrimeric G proteins is protein kinase A (PKA), which is positively or negatively coupled to D1- and D2-like receptors, respectively. As a consequence, the two classes of DA receptors drive complimentary cellular effects. The two types of SPNs show differential expression of DA receptors, with D1 receptors strongly enriched in dSPNs and D2 receptors in iSPNs . D1- andpost-mortem HD brains, ∼40% dopaminergic neuron loss can be observed in the SNc along with a significant decrease in dopaminergic terminals in the striatum and loss of DA transporter (DAT) (In er (DAT) . Transcrer (DAT) .During early disease progression, SPN loss is largely limited to striosomes . As thesAdditionally, D2-mediated DA signaling onto striatal CINs generally reduces acetylcholine release and thereby dampens the inhibition of dSPNs. In HD models, despite the survival of CINs in the striatum, a dysregulation of acetylcholine release has been reported . By thisIn cortical neurons, which receive both dopaminergic and glutamatergic input, a disturbed signal-to-noise ratio and reduced range over which DA and glutamate can be modulated impairs both cognitive and motor functions . An inteImportantly, HD is often accompanied by a range of psychiatric symptoms, such as mood disorders, aggression, compulsive behavior, psychotic episodes, apathy, and sexual disorders . A lack Endocannabinoids (eCBs), such as arachidonoylethanolamide (AEA) and 2-arachidonylglycerol (2-AG), are small lipophilic neuromodulators that are released from the post-synapse to diffuse locally and act retrogradely on presynaptic CB1 receptors where they inhibit neurotransmitter release. In the context of HD, decreased CB1 expression can be detected early in disease in both humans and animal models  Denovan. FurthereCB signaling plays important roles in synaptic plasticity at corticostriatal synapses. It modulates DA signaling to control flexible goal-oriented and reward-driven behavior , two proActivation of CB1 receptors seems to be neuroprotective, possibly via inducing expression of BDNF . It is fIn addition to the changes in various neuronal cell types described above, astroglia has recently emerged as an important contributor to neuronal dysfunction in HD . Indeed,in vivo imaging study of glutamate dynamics, which showed normal glutamate uptake in the striatum of HD mice -derived or directly converted neural cultures have all highlighted dysregulation of synaptic genes. In particular, there was a downregulation of transcripts involved in the post-synaptic scaffold, neurotransmitter signaling, Caed genes . Importaed genes . Quantited genes , in lineed genes  and withed genes , suggest+ channels, but also by unveiling mHTT-induced metabolic disturbances in astrocytes  and striatum (SPNs) have been thoroughly characterized in the last two decades. Recent investigations have also highlighted the contribution of astrocytes to circuit dysfunction in HD. Despite these significant advances in the knowledge about circuit mechanisms of HD, major questions remain that will be particularly critical to address. The microcircuit mechanisms involving cortical and striatal interneurons are still largely unexplored. Moreover, it is not clear how low-level changes in synaptic connectivity and neuronal activity relate to higher-level changes such as network synchronization and LFP oscillations across brain areas. It is further not known whether and how such changes give rise to behavioral symptoms. Progress in this area will be facilitated by new techniques like multi-channel electrophysiology and deep brain imaging that permit simultaneous recording of large neuronal ensembles over prolonged periods of time. Manipulation of neuronal activity Moreover, viewing the described cell types as homogenous populations is clearly an oversimplification. Single-cell RNA-sequencing (scRNA-seq) studies are revealing an ever-greater diversity of cortical neurons , 2018 anIn vitro co-culture systems have been useful in delineating the contribution of various afferents to SPN dysfunction (in vitro approaches to human neurons, thus providing a convenient and physiologically relevant platform for drug testing. In addition, brain organoids (in vitro modeling of disease-related circuit impairments closer to the in vivo setting.function . The avafunction  now openrganoids  and furtrganoids  offer anAt the molecular level, unbiased systems approaches including transcriptomics and proteomics have delivered important insights into the molecular underpinnings of neuronal dysfunction in HD. However, extensive further work will be required to link these molecular alterations to discrete functional deficits.In conclusion, the development of novel methodologies allowing to monitor and manipulate neuronal activity in large populations of neurons during natural behavior, and to detect gene expression changes at a single-cell level, will enable a more complete understanding of circuit mechanisms of HD, and might open the doors for designing more refined therapies for this devastating disorder.Both authors wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The review provides a comprehensive overview of advantages and future prospects of conjunction of nanotransporters with TKIs as a highly promising approach to anticancer therapy.Recent progress in nanomedicine and targeted therapy brings new breeze into the field of therapeutic applications of tyrosine kinase inhibitors (TKIs). These drugs are known for many side effects due to non-targeted mechanism of action that negatively impact quality of patients' lives or that are responsible for failure of the drugs in clinical trials. Some nanocarrier properties provide improvement of drug efficacy, reduce the incidence of adverse events, enhance drug bioavailability, helps to overcome the blood-brain barrier, increase drug stability or allow for specific delivery of TKIs to the diseased cells. Moreover, nanotechnology can bring new perspectives into combination therapy, which can be highly efficient in connection with TKIs. Lastly, nanotechnology in combination with TKIs can be utilized in the field of theranostics, i.e With the incidence of 18.1 million new cases worldwide and death count reaching 9.6 million people in the year of 2018, cancer is a serious player on the field of human health Tyrosine kinase inhibitor (TKI) therapy, however beneficial, has its limitations and disadvantages. Off-target activity causes multitude of adverse events, negatively affecting the quality of patient's life and continuation of therapy This review is focused on two particular themes, concerning TKIs and nanomedicine. In its first part, we indulge in the matters of TKs and their small molecule inhibitors. Second part is dedicated to nanoformulations of TKIs and to the potential nanomedicine has to help overcome the limitations of TKI therapy and to improve it.et al. (2000) - into 20 and 10 subfamilies, respectively, based on kinase domain sequence TKs are transmembrane (receptor) or unattached (non-receptor) enzymes that transfer γ‑phosphate from adenosine triphosphate (ATP) to tyrosine residue of a substrate molecule. This transfer causes allosteric or functional change of the protein and represents an essential mechanism for activation and direction of cell signaling C-terminal tail. In some cases, TK domain can be divided in two parts by a kinase insertion N-terminal lobe, has a conserved sequence, in which a loop with Gly residue serves to localize phosphate on the ATP molecule RTKs are membrane glycoproteins that transfer signals from extracellular into intracellular environment. Therefore, their structure consists of an extracellular hydrophilic domain, a hydrophobic transmembrane segment and an intracellular domain. Extracellular part of RTKs can be very variable according to their corresponding ligand. Binding of the ligand needs to be specific and reversible. A number of low-energy bonds like hydrogen, ionic, hydrophobic or Van der Waals interactions are involved C-terminal tail. This fully activates the RTK et al. further extend the dynamic theory, showing that even when the signal seems to be stably switched on, there are rapid phosphorylation-dephosphorylation cycles happening RTKs are mostly monomeric, undergoing homo- or heteromerization even before or upon ligand binding etc.) or recruit other enzymes, which are involved in various signaling pathways  that lead to growth stimulation, survival, migration, and actin reorganization After the RTK is activated, Src homology-2 (SH2) and phosphotyrosine-binding (PTB) domains of cytoplasmic proteins bind to the phosphorylated tyrosine to initiate the signaling cascade. These adaptive (SH2 containing) and anchoring (PTB containing) proteins have either intrinsic enzymatic activity themselves , consisting mainly of MAPK, PI3K/AKT, Src, PLCγ and JAK/STAT pathways et al. scanned 67 germline disorders related to kinase function, identifying 50 causative kinases, approximately half of them being TKs In the elaborate network of TK signaling , causing tumor progression and therapy resistance Next generation sequencing of 4,853 tumors revealed involvement of FGFR aberrations in 7.1% of samples, mostly in urothelial, breast, endometrial, squamous lung and ovarian cancers - gene amplifications being the most frequent aberration involved VEGFR has complex signaling affairs with both immune and cancer cells PDGFR impacts tumor development by affecting fibroblasts, vascular and cancer cells MET and HER2 gene amplifications are considered important driver oncogene alterations in lung adenocarcinomas In CML, fusion of chromosome 22 and 9 gives rise to chimeric Bcr-Abl protein, where Abl kinase domain is constitutively activated and drives the disease progression From a drug discovery point of view, inhibition of PK activity can be achieved by a molecule that binds either the ligand or the PK itself to prevent ligand binding, dimerization or catalytic activity Table 1).Classification of TKIs has gone through an evolution Table 2). Most FDA-approved TKIs fall within Types I to II. An online PKI database, managed by SB&C Team from Institute of Organic and Analytical Chemistry of the University of Orleans, France, compiles all PKIs, even the ones investigated in clinical trials. At present, there are 117 TKIs listed. One in phase 0, seven in phase I, twenty-nine in phase II, thirty-six in phase III and lastly forty-three in phase IV, out of which five were not approved by FDA Up to this date, there are 55 small molecule PKIs approved by FDA out of which 39 are TKIs , as well as size, morphology and surface charge can have influence on their biological effect and distribution in the organism. NPs larger than 150 nm accumulate mostly in lungs, liver and spleen, but avoid bioaccumulation and clearance by kidneys. Small NPs (under 5 nm) behave in the opposite manner. NPs in between are less likely to be caught in lungs than in liver and spleen Table 3 summarizes recent reports concerning this topic.In the fashion of poorly soluble small molecule anticancer drugs Figure 2). Major determinant in TKI absorption is the pH-dependent solubility. TKIs exhibit weakly basic properties; therefore, the luminal pH of the gastrointestinal tract and the pKa of the drug determine whether they take the ionized or non‑ionized form. In the highly acidic environment of the stomach, the equilibrium is inclined to the ionized form, which normally dissolves more easily than a non‑ionized form. Bioavailability can be impaired by co‑administration of TKIs with an acid-suppressive medication , which increases the stomach pH. The balance of both forms of the drug would shift to the less soluble, non-ionized one and may result in a significant subtherapeutic exposure and treatment failure i) protecting TKIs from the harsh conditions of gastrointestinal tract, ii) improving the intestinal absorption of TKIs and iii) provide TKIs with a controlled release and sustained ionization rates. Interestingly, despite the fact that most of the TKIs get manufactured for the oral administration, nearly all of the FDA-approved nanomedicines are developed for i.v. applications. This implicates an essential need for more advanced nanovehicles able to cope with the obstacles for orally-administered nanomedicines.TKIs are characterized by poor solubility, and thus highly variable bioavailability manifesting through diverse characteristics regarding absorption from the gastrointestinal tract  to plasma proteins (most often to α(1)-acid glycoprotein and/or albumin), which makes these inhibitors ineffective in vivo studies of TKI nanoformulations. Therefore, we cannot compare actual data from clinical usage of TKIs with pharmacokinetic abilities of nanoformulated drugs tested on animals. However, recent studies showed that nanoformulations can markedly improve TKIs pharmacokinetic properties in in vivo settings.Nowadays, mostly rat and mouse models are used for et al., who constructed a phospholipid complex with erlotinib. Pharmacokinetic assessment showed ∼1.7-fold (p < 0.05) higher bioavailability of the nanoformulated complex compared to free drug, with ∼1.3-fold (p < 0.01) higher maximum plasma concentration and longer half-life. et al. prepared a phospholipid complex with ibrutinib, which exhibited 9.14-fold increase in bioavailability compared to free drug. Maximum plasma concentration was also higher and was achieved in shorter time period, while tumor volume was significantly decreased For example, lipid-based nanoformulations, being amphipathic, are able to enhance solubility and bioavailability of TKIs. This phenomenon was demonstrated by Dora et al. used sunitinib maleate in combination with self-nanoemulsifying delivery system (SNEDDS) that is characterized by a high hydrophilic-lipophilic balance. In an in vivo experiment, oral administration of nanoformulation had higher plasma concentration over time, with 1.45 times higher maximum concentration achieved in less than half the time et al. constructed liposomal afatinib with cetuximab (anti-EGFR antibody) coating. These immuno-NPs showed tumor growth inhibition and higher afatinib concentration in tumor tissue, as well as in liver and lungs. Due to the prolonged blood circulation and inhibited binding rate to endogenous proteins, afatinib nanoformulation exhibited higher area under curve (AUC) and plasma terminal half-life, when it could be detected even after 72 h in the bloodstream Nazari-Vanani et al. utilized human serum albumin (HSA), which, as the main protein for plasma transport in physiological conditions, can escape systemic clearance naturally and has higher uptake in cancer cells, because they are in need of nutrients for their uncontrolled growth Gao et al. produced biomimetic galactose-modified polymeric NPs loaded with imatinib that exhibited enhanced oral bioavailability, higher maximum concentration and AUC with increased intestinal permeability and affinity to intestinal epithelial cells. Fast entrance into the bloodstream resulted in fast clearance in liver, and NPs were not able to extend plasma residence time. Authors suggest NP modification with functional biomaterials to maintain imatinib plasma concentration for longer period. However, benefit of this nanoformulation lies in fast bloodstream penetration, as it might result in adverse events reduction Li e.g. involvement of ligands mediating active targeting of specific subsets of cancer cells or utilization of tumor microenvironment stimuli-responsive domains) could further improve the pharmacokinetics of TKI nanoformulations.Taken together, inherent physico-chemical properties of TKIs are substantially complicating their treatment efficiency. Abovementioned studies clearly demonstrate that distinct types of nanomedicines can markedly improve the pharmacokinetic behavior of TKIs. This in particular is due to improved solubility of TKIs incorporated into delivery vehicles, followed by a subsequent enhancement of TKIs hydrophilicity and improved stability in the blood stream that results in a prolonged blood circulation of nanoformulations and their higher residence time in tissues adjacent to tumor mass. Even passive, non-targeted nanoformulations can be then accumulated in the tumor mass through “enhanced permeability and retention” (EPR) effect Although targeted therapy is, in general, regarded as more moderate than classic chemotherapy, it has more intricate mechanisms of action concerning their structure and targets. Even in targeted therapy, severe adverse events occur and resulting dose adjustments or treatment discontinuation impair its better treatment efficacy et al. summarized toxicity characteristics of eleven TKI drugs available on the market. They pooled the side effects classification scale into two groups, the first including rare and uncommon events, the second involving common and very common events. All eleven inhibitors caused nerve, lung and airways disorders with the higher abundance. Also, in the same abundance category, ten caused skin disorders, nine caused gastrointestinal disorders, carcinogenic, mutagenic and toxic effects on reproductive system, and eight of them caused eye, blood and lymphatic disorders. Other adverse effects were related to liver, bile, vascular system, kidneys or musculoskeletal system. Pazopanib, as the most severe inhibitor from the list, had 12 out of 14 indications with the frequency of common, very common. On the other hand, sorafenib had only 5 common or very common effects out of 14 Eckstein As demonstrated in the previous paragraph, pooling of adverse events grading quite commonly occurs in studies and articles. This bad habit may distort the actual graveness of some events. For example, in Common terminology criteria for adverse events (CTCAE v5.0), grade 1 plus grade 2 and grade 3 plus grade 4 events are regularly pooled, where main focus goes to the grade 3/4 group. However, in the case of long-term drug administration, group 1/2 should not be overlooked, if we want to improve patient's quality of life. It is important to keep in mind that the milder but more common side effects put a significant impairment on patient's everyday psyche in vitro and in vivo models, number of papers provide evidence that drug-loaded NPs show enhanced properties compared to free drugs. For example, Cryer et al. developed gold NPs conjugated with an afatinib analog that mediated cancer cell-specific growth inhibition and attenuated inflammatory cytokine release. The selectivity to cancer cells is plausibly due to highly efficient uptake of gold NPs concomitantly inhibiting the efflux of afatinib to the extracellular space et al. evaluated performance of imatinib mesylate-loaded poly(lactide-co-glycolide) NPs that exhibited increased cytotoxicity in vitro and significantly reduced cardiotoxicity in vivo compared to free drug. et al. worked with lapatinib-loaded HSA NPs on HER2 positive In vivo, the nanoformulation increased delivery to brain tissue and significantly prolonged median survival period of tested mice.In this sense, well-thought-out design in drug development, substantial drug toxicity profiling and proper administration are needed to minimize adverse events. Nanoformulations have good potential to reduce these events even more by specific biodistribution of anti-cancer drugs. In preclinical et al. prepared cyclic arginylglycylaspartic acid (cRGD) and polyethylene glycol (PEG) modified liposomes with apatinib that specifically recognize integrin αvβ3 in vitro, plus it performed better targeting, faster clearance from murine organism, significantly reduced tumor weight and no visible damage to internal organs. Lu et al. intended to overcome adverse effects of combination therapy with afatinib and cetuximab by encapsulating the TKI into liposomes and using cetuximab as targeting and therapeutic agents bound to the surface of liposomes in vivo prolonged half-life, better drug delivery, prevention of afatinib binding to hemoglobin, enhanced tumor selectivity and growth inhibition. This study suggested the drug dosage could be significantly decreased in clinical application, therefore potentially reducing adverse events incidence.The relative selectivity of nanodrugs in cancer is based on the so-called passive targeting by the abovementioned EPR effect. This mechanism is based on substantial extravasation of the nanodrugs into the interstitial fluid at the tumor site. Nanodrugs can better penetrate the tumor due to poor quality vessels in the tumor and are retained for a longer time at the tumor site because of the defective lymphatic vessel system in tumor microenvironment. EPR affects nanodrugs that are 20-100 nm in size A number of targets have been identified for potential treatment of brain malignancies by TKIs  Figure 3) Nanomedicine gives drugs the advantage of prolonged blood circulation time, which increases their chance to cross the BBB  resulted in the longest survival time.Wan et al. relied on enhanced EPR effect plus attraction of HSA NPs by SPARC (secreted protein acidic and rich in cysteine), as well as on beneficial properties of HSA that include preferential tumor uptake and binding to 60-kDa glycoprotein receptor on vascular endothelial cells et al. took up different approach - liposomes modified with transferrin and penetratin loaded with erlotinib and doxorubicin for treatment of glioblastoma To improve the transcytosis of lapatinib across the BBB, Wan Although research in brain-targeted nanomedicine is in full bloom, not many researchers focus on using nanoformulations for TKI treatment of brain cancer or metastases. However, these few promising examples show that TKIs could be used in this manner. We postulate that future investigations might focus on development of highly biocompatible nanovehicles of endogenous origin, able to utilize the inherent properties of BBB to mediate transcytosis of nanoformulations. In addition, nanovehicles for TKIs brain delivery might also be capable of inhibiting frequent brain-associated efflux mechanisms causing undesired transport of the payload to the extracellular region.Therapy that combines two or more anticancer agents is applied for two main reasons - to bypass/delay drug resistance or to enhance drug efficacy. Moreover, combination of drugs usually allows reducing the doses of individual drugs, and thus alleviates side effects. Main strategy would be to co-target compensatory signaling pathways with either TKIs or monoclonal antibodies. Some strategies are focusing on Heat shock protein 90 that is necessary for protein stabilization and plays a role in the folding of many RTKs Multidrug approach has been previously found beneficial in the control of HIV, showing that specific drug combinations were able to overcome treatment difficulties Use of drug combination in nanoformulation should be well devised, as there are some drugs that properly function only in specific order and timeframe. Their simultaneous release would cause severe side effects et al. showed this on castration-resistant prostate cancer cell lines with the combination of sorafenib and nilotinib et al. proved that crizotinib and dasatinib combination in micellar formulation shows enhanced anti-tumor efficacy against glioblastoma multiforme, possibly due to increased blood circulation and improved bioaccumulation of TKIs at the tumor site Combination therapy of two different TKIs has its benefits in targeting multiple TK signaling pathways, generating more potent anti-tumor effects and limiting possibilities of bypassing the inhibited pathway, thus avoiding or postponing the occurrence of resistance. Archibald etc.  et al. delivered chloroquine/gefitinib cocktail encapsulated in chitosan NPs to resistant hepatocellular carcinoma cells and managed to overcome said resistance through inhibition of the autophagic effects, evidenced by down-regulation of the LC3 II and LC3 I ratio et al. made polyamidoamine NPs functionalized with anti-EGFR aptamers for targeted co-delivery of erlotinib and survivin-shRNA with simultaneous systemic administration of chloroquine. Purpose of chloroquine in this case was mainly vascular normalization for smooth NPs delivery and endosomal escape, while therapeutic effects were mediated by erlotinib and shRNA down-regulation of survivin that inhibits drug-induced apoptosis There are records of using chloroquine in combination with nanoformulated TKIs based on its multiple mechanisms of action affecting tumor microenvironment. Chloroquine can inhibit late stage autophagy, activate tumor suppressor protein p53, normalize tumor vasculature, which decreases hypoxia, et al. developed a thermosensitive hydrogel for peritumoral use, containing a nanoformulated Pluronic F127-based chemotherapeutic agent paclitaxel with short release time and lapatinib microparticles with long-term release kinetics In vivo experiment showed that in comparison with oral administration of lapatinib, the gel administration performed comparably, although with much smaller amount of drug needed. In addition, by using the gel, steadier bioaccumulation of lapatinib in tumors together with lower undesired toxicity was achieved, and nanoformulated lapatinib exhibited good synergistic effects with paclitaxel.Interestingly, Hu et al. used sunitinib encapsulated in polymeric (anisamide-modified poly-lactic-glycolic-acid-PEG) micellar nanovehicle to support their tyrosinase-related protein 2 (Trp2) nanovaccine in melanoma therapy, with results showing attenuated tumor-associated immune suppression et al. combined their Toll-like receptor 7/8 agonist nanovaccine with free sunitinib and programmed death-ligand 1 (PD-L1) antibody, which, again, abrogated tumor-associated immune suppression and significantly decreased tumor weight in an in vivo experiment et al. also used PD-L1; however, they produced a nanobody acting as a targeting ligand of liposomal delivery system of gefitinib and simvastatin. Their goal, to overcome gefitinib resistance in T790M EGFR-mutated NSCLC, was achieved in an in vivo experiment In some cases, antibodies are used in combination with TKIs in nanoconstructs to target TK domains for precise delivery and for their anti-tumor activity in the form of competitive ligand binding et al. combined sorafenib with ursolic acid in mesoporous silica NPs coated with pH sensitive chitosan and lactobionic acid - targeting agent for asialoglycoprotein receptor overexpressed in hepatocellular carcinoma. They used ursolic acid for its reported ability to down-regulate VEGFR and EGFR There have been studies combining TKIs with natural substances that have multiple effects on human metabolism. For example, Zhao et al. combined sunitinib with curcumin in superparamagnetic iron oxide NPs coated with BSA et al. used sorafenib/curcumin cocktail encapsulated in directed self-assembled nanoparticles composed of PEG derivative of vitamin E succinate with similar results. Upon oral administration, significantly increased biodistribution of sorafenib and curcumin in gastrointestinal tract was found. The authors stated that this phenomenon is most likely due to the improvement of the affinity and accessibility of nanoformulations to the intestinal membrane that results in their enhanced absorption in the intestine Chen et al. co-delivered oxygen-releasing perfluorooctylbromide with erlotinib encapsulated in anti-EGFR aptamer-modified liposomal NPs to overcome hypoxia-induced resistance to chemotherapy. NPs with combination of drugs had significant reducing effect on tumor volume compared to NPs with erlotinib alone, which suggests good therapeutic synergy. The study highlights the importance of active targeting moiety on the surface of nanoformulation. Anti-EGFR aptamer enables specific ligand-receptor mediated accumulation of the payload enhancing efficiency of encapsulated drugs with concomitant inhibition of undesired side-effects in vivoHypoxia occurs in the center mass of solid tumors. It stimulates overexpression of HIF-1α that mediates signaling pathways leading to epithelial-mesenchymal transition, neovascularization, cell survival, metastasis and therapy resistance in situ that enables long-term monitoring, and therefore adaptation to modality of disease prognosis. This approach addresses pharmacokinetic variability of drug responses in the population of patients, hopefully resulting in a reduction in adverse event occurrence and in an improvement of treatment planning etc. Theranostic approach unites within itself the therapeutic and diagnostic aspects of medicine. In another words, it is the combination of therapy and pharmacokinetic imaging Figure 4). In case of using nanocarriers, targeting agent can be added, or solely the EPR effect of vehicles in the tumor tissue can be utilized. Targeting agents are typically small molecules, peptides, proteins or antibodies. The variety of labels used in theranostics corresponds to the selection of imaging techniques. For instance, gamma scintigraphy (positron emission or single-photon emission computed tomography) is suitable for visualization of radionuclides . Optical methods commonly detect fluorescent labels, quantum dots, carbon nanotubes or gold nanoshells. Magnetic resonance imaging (MRI) is a good method for para- or superparamagnetic metals, like gadolinium, iron and manganese or NPs from respective materials. Heavy elements, such as iodine, barium and sulphate are visualized by computed tomography, and last but not least, ultrasound can be used to view liposomes and perfluorocarbon nanodroplets Figure 5). Some approaches rely on internal cellular stimuli, like pH, redox potential or enzymes. Others trigger drug release with external stimuli, temperature, light, ultrasound and magnetic field. The stimuli combination concept boosts tumor cellular uptake and intracellular drug release while decreasing nonspecific cytotoxicity thanks to improved nanocarrier stability Theranostic agents typically consist of a therapeutic molecule and a traceable label -poly-(ethylene glycol)-poly(l-lysine)-diethylenetriamine glycol)-biotin] NPs with VEGFR targeting by anti-VEFGR antibody and gadolinium ions as MRI contrast agent. In vitro, NPs with anti-VEGFR antibody showed higher cellular uptake than NPs without the antibody. In vivo, NPs showed better anti-tumor efficacy than sorafenib alone with prolonged imaging time and signal enhancement compared to commercial contrast agent Magnevist®. In addition, NPs exhibited exceptional biocompatibility and did not alter organ histology. Overall, the devised NPs served as highly promising biocompatible, actively-targeted, multifunctional platform capable of facile dual loading with TKI and imaging agent et al. developed erlotinib delivery monitoring system via iron oxide NPs with MRI imaging and demonstrated the ability to estimate intake concentration of NPs into the tumor  In vivo experiments revealed that the erlotinib-conjugated NPs effectively inhibited progression of lung cancer. Ex vivo protein expression analysis revealed efficient suppression of antiapoptotic and activation of apoptotic proteins mediated by NF‑κB signaling.One of the earliest mentions of nano-TKI application in theranostics was made by Noh et al. harnessed the full potential of theranostic approach by fabricating indocyanine green-loaded mesoporous silica NPs lidded with zinc oxide quantum dots and coated with erlotinib-modified chitosan et al. constructed a complex nanodelivery system with multiple functions. Chitosan oligosaccharide was conjugated with IR780 cyanine fluorescent label and Black Hole Quencher for visualization of fluorescence triggered by NIR irradiation. This complex then self-assembled, encapsulating sorafenib and superparamagnetic iron oxide NPs that were intended for induction of ferroptosis characterized by a high rate of lipid peroxidation Zhang The few examples discussed above clearly demonstrate that TKIs-based chemotherapy might substantially benefit from involvement of nanoparticles with theranostic properties. Such structures could markedly improve the efficiency of anticancer therapy by real-time tracing of TKIs accumulation and continuous visualization of chemotherapy outcomes. Theranostic nanoformulations could also offer a reduction of the dose requirements of administered TKIs, which are frequently characterized by narrow therapeutic indices et al. summarize challenges in this field in their review in vivo phase, but we do not see these nanodrugs continue to clinical trials yet. For the advancement from in vivo to clinical trials, scale-up in nanodrug manufacturing must be achieved. That requires reproducibility and uniformity in production, drug stability and the possibility to involve automation in the production process, if the trials are successful. We see these requirements and hefty financial investment as the bottleneck of clinical transfer of today's nanomedicine. Especially TKI-nanoapplications are in the beginning phases of development and, although they have great potential, they still have a long way ahead of them.Nanotechnology offers unique properties in drug development. There has already been several nanomedicines approved by FDA et al. demonstrated the strengths of computational methods when they showed precise prediction of NP formulation and properties based on molecular structures of precursor molecules, producing NPs with selective targeting ability and impressive therapeutic impact Harnessing the potential of bioinformatics, artificial intelligence (AI) and deep learning could marginally increase the advancement in nanomedicine Figure 7). Tendencies to apply these methods are apparent today, even in clinical trials With growing number of public databases RNA interference is a potent therapeutic path especially suitable for undruggable targets. However, it faces multiple challenges, like systemic toxicity and obstacles with delivery to tissues other than kidney or liver Current focus on nanomedicines in cancer research stems from their unparalleled characteristics, starting with increased bioavailability that is especially important for poorly soluble drugs, prolonged blood circulation, passage through biological barriers, tumor targeting, improvement of drug efficacy, ability to effectively deliver multiple drugs at once and exploitation of their own physical attributes - optical, electrical or magnetic - to convey visualization, localization, additional therapeutic effect or controlled drug release. These features will be the foundation of future cutting-edge applications ranging from specific tissue targeting, real-time imaging and theranostic approach, hopefully leading to personalized cancer therapy.All in all, even though TKIs are fundamentally imperfect as any other type of cancer therapy, and there is a long way to reach clinical trials with currently researched TKIs-NPs combinations, there is a great potential in this union - potential in improving patients' quality of life and efficacy of TKI therapy. Attention should be paid to developing NPs with sound safety profiles that avoid damage caused by systemic exposure of the nanocarrier itself. NPs that will stand their ground in the clinical settings and that will be easy to handle by patients themselves, stable and prone to slight deviations from timely medication adherence.


Tumor cells demonstrate substantial plasticity in their genotypic and phenotypic characteristics. Epithelial-mesenchymal plasticity (EMP) can be characterized into dynamic intermediate states and can be orchestrated by many factors, either intercellularly via epigenetic reprograming, or extracellularly via growth factors, inflammation and/or hypoxia generated by the tumor stromal microenvironment. EMP has the capability to alter phenotype and produce heterogeneity, and thus by changing the whole cancer landscape can attenuate oncogenic signaling networks, invoke anti-apoptotic features, defend against chemotherapeutics and reprogram angiogenic and immune recognition functions. We discuss here the role of phenotypic plasticity in tumor initiation, progression and metastasis and provide an update of the modalities utilized for the molecular characterization of the EMT states and attributes of cellular behavior, including cellular metabolism, in the context of EMP. We also summarize recent findings in dynamic EMP studies that provide new insights into the phenotypic plasticity of EMP flux in cancer and propose therapeutic strategies to impede the metastatic outgrowth of phenotypically heterogeneous tumors. Epithelial–mesenchymal transition (EMT), in which epithelial cells undergo dynamic cellular transition from a sessile epithelial state to a motile mesenchymal state allowing the formation of new tissues, is considered one of the pivotal processes during embryogenesis and organogenesis . The proAfter invasion and spread, cancer recurrence at the metastatic site is thought to require the reverse process, termed mesenchymal to epithelial transition (MET) . The revin vivo and in vitro, while the drivers and transcriptional mediators of EMT are quite comprehensively documented  . Apart fEpigenetic modifications can also cause a shift of epithelial to mesenchymal state; for example, aberrant DNA CpG island methylation correlated with the repression of the miR-200 cluster, which promotes EMT and contributes to tumor progression . LSD1-deThe expression changes of various key molecular markers during EMT, are represented in Although much less studied than later tumor stages, a number of studies have made a connection between the linkage of EMT to stemness and tumor-initiating capacity . In somein vivo has remained a topic with various controversies  is a detection method that employs specific probes targeting different epithelial and mesenchymal genes to detect multiple transcripts simultaneously , which immunocaptures EpCAM-expressing CTCs for patient prognosis  and Mucin-1 (Muc1) across all CTCs compared with the primary xenograft tumors. in vitro and in vivo knockdown of Cdh1 in MDA- MB-468 breast cancer cells reduced proliferation, and this was also reported by Cdh1 increased invasion capacity while reducing cell survival, CTC number and metastasis spread in the breast cancer.Although the devices used to isolate CTCs have improved the quality and quantity assessment of CTCs, there are still limitations when studying CTCs. Over the past few years, use of the revolutionary single- cell RNA sequencing (scRNA-seq) has emerged to assess genome-wide expression profiles of isolated CTC populations and CTC clusters. +/partial-EMT+) were enriched after the first-line chemotherapy, implying that they were the most chemoresistant subpopulation, and had a favored prognostic value in patients with metastatic breast cancer. Another study has showed that EpCAMhigh CTCs were significantly associated with poor prognosis compared to EpCAMlow CTCs in patients with breast and prostate cancer  and SPARC (a collagen-binding glycoprotein related to ECM reorganization) were highly expressed in the CTCs. Although they stated that there was no intrinsic correlation between EMP state and stemness in their CTCs, other reports have shown expression of these genes were associated with Cdh1 reduction , which is an important enzyme of gluconeogenesis . The losFor lipid metabolism, higher expression levels of lipid synthesis enzymes such as ATP-citrate lyase (ACLY), stearoyl-CoA desaturase (SCD), fatty acid synthase (FASN) and HMG-CoA reductase, have been detected in more aggressive tumor cells . Jing etGrowth factors from the tumor microenvironment can also reprogram cancer cells from the Warburg-like metabolism to EMT-like metabolism. Activated PI3K/AKT/mTOR signaling due to growth factor stimulation can enhance the uptake of glucose and lipid, as well as the synthesis of FA and protein . The stuin vitro based molecular and functional assays and in vivo based cancer models. The in vitro assays routinely performed in EMP studies involve various molecular and functional assays. Molecular assays, using FACS and immunocytochemistry staining with microscopy analysis, relies on various validated EMP markers that are used to delineate the phenotypic state of cells  have been reported to remain after neoadjuvant chemotherapy and HER2 pharmacological inhibition, suggesting that they encode resistance (The presence of plasticity in tumor cells and resultant heterogeneity is one of the utmost challenges in targeting cancer on a whole . EMT andsistance . Many resistance , and revsistance . Therefosistance .In the first scenario to target EMP inducing stimuli, many different approaches have been utilized to inhibit different signaling pathways that contribute to the induction and maintenance of EMT, such as TGFβ/TGFβR, EGF/EGFR, FGF/FGFR, IGF/IGFR, IL-6/IL-6R, HGF/MET, PDGF/PDGFR, TNFα, Wnt and Notch signaling . Of all,Secondly, for therapies specifically targeting mesenchymal cells, different novel strategies such as EMP-targeting vaccines against transcription factors such as TWIST1 and Brachyury; nutraceuticals; and the repurposing of drugs such as metformin, salinomycin and resveratrol, have been extensively discussed in our previous review . Table 2For the third strategy, the detailed molecular knowledge of MET regulation will provide opportunities to curtail this event and prevent the development of metastasis, which is of high clinical relevance. Depending on the clinical scenario, MET-inducing/stabilizing factors may inhibit metastasis if they block the initial EMT stages that allow the dissemination, or promote the later stages of metastasis, which can cause some conflicting considerations . An emerThe crucial roles of EMT-MET during embryogenesis and organogenesis is hijacked during tumor progression and metastasis. The roles of various signaling cascades, intrinsic and extrinsic mechanisms, and regulators that contributes to EMP dynamics are reasonably well determined, but more refined studies and techniques need to be employed to recapitulate the MET behavior of cells while extravasating, seeding and colonizing at secondary niches. The intricacies associated with phenotypic plasticity, stemness and intratumoral heterogeneity further sheds light on several unresolved queries. The reliable features of cellular behavior relating to drug persistent states through the spectrum of EMT need to be verified. Is there a ubiquitous molecular feature of partial/hybrid EMT cells that can be identified and targeted across all different cancer context types? What actual mechanisms do cancer cells employ to intravasate from the primary sites, and how do EMT - MET programs cooperate to assist cancer cells through several stages of cancer progression? We are still lagging in obtaining a wider and more complete understanding of the contributions of EMP in cancer. The sophisticated developments in lineage tracing using confetti animal models and implementation of other novel technologies such as high-resolution intravital imaging, live cell imaging, inducible reporter systems and single-cell sequencing techniques will provide great avenues in the fields of plasticity and dynamics around EMP. Finally, it is imperative to determine how phenotypic plasticity can be exploited, as therapeutic interventions that push the conversion of cancer cells to fat cells or apoptosis, for example,  might beSB wrote the manuscript, developed the first draft, and contributed significantly toward development of the whole manuscript. PW contributed significantly in content material, figures and tables preparation and drafted metabolomics section. AT contributed in providing content material and drafted CTC section. ET refined, edited the manuscript, and approved the final version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The rapid progress in the field of stem cell research has laid strong foundations for their use in regenerative medicine applications of injured or diseased tissues. Growing evidences indicate that some observed therapeutic outcomes of stem cell-based therapy are due to paracrine effects rather than long-term engraftment and survival of transplanted cells. Given their ability to cross biological barriers and mediate intercellular information transfer of bioactive molecules, extracellular vesicles are being explored as potential cell-free therapeutic agents. In this review, we first discuss the state of the art of regenerative medicine and its current limitations and challenges, with particular attention on pluripotent stem cell-derived products to repair organs like the eye, heart, skeletal muscle and skin. We then focus on emerging beneficial roles of extracellular vesicles to alleviate these pathological conditions and address hurdles and operational issues of this acellular strategy. Finally, we discuss future directions and examine how careful integration of different approaches presented in this review could help to potentiate therapeutic results in preclinical models and their good manufacturing practice (GMP) implementation for future clinical trials. In the background of many diseases, the degeneration and/or dysfunction of a particular cell type affects tissues and organs, which will deteriorate over time and lose their functions. In this context, regenerative medicine offers new perspectives for pathologies that otherwise remain untreated ,2. HundrBesides the therapeutic effects of cells elicited by their direct presence, some of the observed benefits are mediated by their indirect actions and, as such, may not require them ,7. ExtraHerein, we first review the state of the art of cell therapy and its current limitations and challenges, with a particular focus on pluripotent stem cell-derived therapeutic products. In a second part, we elaborate on functions of EVs and discuss their interest in the field of “acellular therapies” for pathological conditions that affect the eye, skin, heart and skeletal muscle. Finally, we discuss the future prospects of these therapies and their combination to potentiate therapeutic outcomes.The source material for cell and tissue-based products is a major point to consider for regenerative medicine. A prerequisite for the broad application of cell products is the production of large quantities of cells, with consistency between different batches or manufacturing processes to ensure the same quality and therapeutic efficacy to each patient.This section will describe the numerous cell sources that may be used in cell therapy and their use in selected pathological conditions. In addition, we will discuss the transition from cell therapies formulated as a cell suspension to more complex tissue-engineered products.Various cell sources have been considered for cell therapy and regenerative medicine, including adult material from living donors or cadavers, fetal materials and pluripotent stem cell lines.Cell material from adult origin can be obtained directly from patients and purified/amplified in vitro  or epithelial cells from the skin). Such autologous approach prevents the risk of rejection. However, the manufacturing process and supply logistics of autologous cell-based therapy products are highly complicated and hinder their scale out. These limitations are particularly important when addressing widespread diseases that affect millions of patients like Age-related Macular Degeneration (AMD) or when the number of cells needed for therapy is important, as in the case of large burns. Alternatively, cells can be obtained from cadavers or, when possible, living donors. For all adult sources, donor variability—histocompatibility, age, genotype, etc.—drives cell product variability. In addition, adult cell sources have a reduced potential for expansion. This is especially true for terminally differentiated adult cell types  that hardly proliferate, or for organs for which there is limited access to endogenous stem cells.Another source of materials for cell therapy is aborted fetuses. Harvested cells retain a proliferation potential that is superior to that of adult cells. Although fetus stem/progenitor cells are restricted in their potentiality, giving rise to limited cell types, some of these cells are no longer present in adult tissues and could thus be used to generate specific cell types. Nevertheless, their access is highly variable around the world, depending on the abortion law and policy in place in each country. Indeed, in some countries, it is strictly prohibited; in others, abortion is prohibited after different gestational ages, which could be restrictive. The use of human fetal tissue also raises a number of ethical considerations. Besides these restrictions, the access to fetuses at a defined gestational age is complex, as highlighted for Huntington’s disease (HD) cell therapy as an example . ClinicaSince their first derivation, human pluripotent stem cells (hPSCs) have been considered a promising cell source for regenerative medicine. hPSCs are self-renewable and give rise to any cell type of the human body. They can be obtained from supernumerary in vitro fertilized embryos (human embryonic stem cells or hESCs) or after the conversion of adult primary cells to pluripotency by the overexpression of a cocktail of factors (human induced pluripotent stem cells or hiPSCs) ,14,15. hIn the following section, we highlight some recent therapeutic applications of hPSC-based cell therapy products.Different causes of visual impairment, such as AMD, Retinitis Pigmentosa (RP) and diabetic retinopathy , are chaIn the last decade, several protocols based on spontaneous differentiation have been developed to convert hPSCs into RPE cells ,18,19,20The therapeutic potential of these hPSC-RPE cells has been evaluated after injection as a cell suspension into the subretinal space between endogenous PRs and RPE/Bruch’s membrane of a rat model of RP. All these studies demonstrated the benefit of hPSC-RPE cells with a preservation of PRs and a restoration of the visual function ,28,29,30In the context of PR degeneration, hPSCs are able to differentiate into PRs  through Skin wounds, principally caused by traumas or thermal burns, can be self-repaired by the body ,54. The Occlusive dressings may be applied to the wound in order to improve healing by providing a mechanical protection and a moist and clean environment that favors re-epithelialization ,56,58. WhPSCs have been successfully differentiated into basal keratinocytes capable to form a pluristratified epidermis in vitro and in vivo in rodents ,61,62. HNevertheless, other characteristics of the skin are still missing . Different protocols based on hPSCs have recently been developed to obtain melanocytes ,68,69 anDespite advances in interventional and medication therapies, heart failure is the first cause of human death worldwide . The humVarious protocols have been developed to produce cardiomyocytes from hPSCs (hPSC-CMs) both from 3D or 2D cultures ,78,79,80The preclinical evaluation of hPSC-CM functionality has been conducted in rodents  and largHCN4 susceptible to trigger pacemaker currents [A major concern with the use of hPSC-CMs is the apparition of a ventricular arrhythmia probably due to immaturity and automaticity of the graft ,92. Indecurrents ,94. Exclcurrents . Overallcurrents ,95. In acurrents ,97. NeveMuscle regeneration involves the activation of PAX7 positive quiescent satellite cells that respond to tissue injury by proliferation and differentiation to give rise to MyoD positive progenitors called myoblasts (MBs) . MBs theUpon transplantation, freshly isolated rodent satellite cells are able to regenerate chemically injured skeletal muscles that were depleted by irradiation of endogenous satellite cells . The dys11-fold) [Protocols have been described allowing the conversion of hPSCs into myoblasts through cytokines or small molecules exposition, recapitulating in vivo developmental cues ,99. Brie11-fold) .To date, clinically compatible protocols are still missing . For volApproved in 2009 by the FDA, the first clinical trial led by Geron Corporation paved the way for the use of hESC-derived cells into the clinic . The comSince then, a number of indications were evaluated, including eye diseases, diabetes and ischemic hearts. A recent manually curated database identified 54 planned or initiated clinical trials based on hPSCs worldwide . StartinMost of these clinical trials focus on hPSC-derived RPE  to target macular degeneration, some forms of RP and Stargardt’s disease ,3. This The first autologous hiPSC clinical trial was also conducted with hiPSC-RPE epithelial sheets to target the wet form of AMD . The triIschemic heart disease is another critical indication with four clinical trials planned or already started . A complTo address type 1 diabetes, Viacyte launched four clinical trials based on hESC-derived beta-like cells delivered through subcutaneous implantation . In addiUp to now, all these clinical trials have demonstrated the safety of cell therapy products based on hPSC-derivatives. While the therapeutic application of these innovative cell products is still in its infancy, encouraging observations are also associated with some signs of efficacy. However, larger clinical trials are required to confirm the therapeutic efficacy of this strategy.Preclinical and first clinical studies have shed light on different issues and challenges. First, immune reaction to donor cells is a critical drawback. Autologous transplantation does not need an immunosuppressive regimen and favors an optimal cell survival. However, the cost of autologous cell therapy is prohibitive . The delOther approaches under investigation depend on genetic engineering to modulate immunogenicity of donor cells in order to generate a “universal” cell bank suitable to treat any patient, instead of multiple cell banks. For example, AAV (serotype 3B)-mediated knock-in of HLA-E at the beta-2-microglobulin (B2M) locus in B2M-/- PSCs leads to overexpression of HLA-E without surface expression of HLA-A, -B or -C. This prevents stimulation of allogenic T cells or natural killer (NK) cell mediated lysis . AnotherThe formulation of cell therapy is the object of intense research for achieving optimal functionality and integration within host surrounding tissues. When cells are injected as a suspension, it could affect their survival, retention into target organ or their functionality ,31,114. Finally, cell survival is not always required to achieve functional recovery of a specific organ, suggesting that a temporary paracrine effect is sufficient to stimulate endogenous regeneration in organs that retained this potency. This is shown in the case of ischemic hearts or through the action of MSCs in acute myocardial infarction, burned skins, liver or traumatic brain injuries and many other diseases ,133,134.Extracellular vesicles (EVs) form a heterogeneous group of double layered lipid membrane-enclosed vesicles, with distinct biophysical properties and functions both in physiology and under pathological conditions . They haEVs are categorized into three major classes based on their biogenesis: (i) Exosomes are formed by the endocytic pathway through invagination of the endosomal membrane, which ultimately forms multivesicular bodies MVBs; . Upon thClinicalTrials.gov website. Results are presented for each EV subpopulation in relation with the disease context and source of EVs . A phasRecent attention has focused on the potential interest of EVs as therapeutic tools for acellular regenerative medicine. Thirty interventional clinical trials are based on the use of EVs for therapeutic purpose . Only 16Increasing evidence suggests that EVs could recapitulate the beneficial effect of their parental cells in a number of applications. To draw a parallel with our previous section, we will focus here on the recent developments obtained with EVs as a therapeutic tool for diseases affecting the eye, skin, heart and skeletal muscle. We also explore how to integrate cell-based and acellular therapies to take advantages of these two approaches and potentiate therapeutic results B.Of note, studies described herein include, but are not limited to, PSCs and derivatives. Indeed, EVs from cells with well-described paracrine actions, for example, MSCs, have been shown to exert beneficial effects in various applications. Depending on the disease and affected tissue, the therapeutic potential of EVs should therefore be examined from a variety of cell sources.Therapeutic effects of EVs have been evaluated for RP, AMD, and following injuries to the eye. Most of these studies focused on the potential of EVs to limit inflammation that would otherwise trigger the death of retinal cells .As previously mentioned, different studies have started to reveal that the presence of cells might not be required to generate a beneficial effect. As an example, subretinal implantation of human neural progenitor cells preserved the vision of a model of RP through a paracrine effect . The samVEGF-A [A choroidal neovascularization (CNV) is characteristic in wet AMD and can be induced in vivo with laser injuries in rodent (laser-induced CNV model). Human umbilical cord blood MSC (hUCMSC)-derived EVs injected once intravitreally were sufficient to reduce vessel leakage and the development of CNV via downregulation of VEGF-A . PreservVEGF-A . In the VEGF-A . InteresVEGF-A . RetinalVEGF-A .Therefore, EVs are able to modulate retinal degeneration via the inflammatory response. This function is similar to transplanted cells, suggesting that the action of transplanted cells is likely through a paracrine effect . An important point raised by these studies is that EVs or their parental cells need to be delivered at an early stage before complete degeneration. As EVs may not need complex surgeries or immunosuppression, it could be envisioned as a first line of treatment to support retinal survival and delay the requirement for a cell-based intervention. Future preclinical studies need however to determine a delivery route that allows repeated injections for long-term efficiency.In the context of an advanced RPE cell degeneration as in late AMD, EVs may not be sufficient to recapitulate all RPE functions in order to preserve surrounding retina. Thus, endogenous RPE may be replaced through cell therapy A. A patcAnother potential EV therapy is related to PR transplantation studies for RP. Early studies showed the feasibility of transplanting post-mitotic PR precursors that achieved some degree of integration into the host mouse retina, ultimately resulting in partial visual function recovery ,158,159.Taking advantage of EV ability to carry nucleic acids, they could be used as vehicles in gene therapy strategies for RP . As an eMajor skin injuries, resulting from extensive burns, infection or trauma, require medical interventions to heal properly . TherapeEVs hold the potential to promote all phases of wound healing and facilitate skin regeneration . TrMMP3) expression of skin dermal fibroblasts through the activation of ERK/MAPK pathway [During the proliferative phase, re-epithelization, wound contraction and angiogenesis are essential processes for the restoration of normal tissue architecture. Early recruitment of resident keratinocytes and fibroblasts is particularly important as abnormalities in the intercellular epidermal-dermal crosstalk impairs the skin repair efficiency . In this pathway . As suchTransplantation of cellular skin substitutes have shown considerable potential to treat both acute and chronic wounds . ComplexAt present, one of the main obstacles in the treatment of skin wounds is achieving healing over time, particularly in patients with underlying skin disorders. Biomaterial-based wound dressings could be loaded with EVs to achieve sustained release to the wound sites ,193. ForOverall, all these proofs-of-concept experiments raised considerable interest of EVs for skin repair. Of interest, their delivery to skin wounds is relatively simple due to easy access and could be sustained over time by the use of biomaterial or repeated topical applications. However, additional preclinical studies are needed to evaluate the synergic effects of combined acellular and cellular strategies.EVs have been investigated as promising therapeutic options for various cardiac diseases such as ischemic heart diseases and myocardial infarctions. One of the main objectives is to promote vascular repair mechanisms to reduce myocardial injury that would lead to cell death and therefore improve cardiac functions.hESC-derived MSC conditioned medium (hESC-MSC-CoM), collected with clinically compatible processes, were shown to contain factors susceptible to modulate cardiovascular-related pathways . AdminisIt was also proposed that ESC-derived EVs could stimulate endogenous myocardial regeneration . Their dThe functionality of cells from the cardiac lineage is superior to MSCs in the different heart disease models ,200. TheIn order to maintain a sustained delivery, hPSC-CM-derived EVs were loaded into a collagen-based hydrogel patch . Such syOverall, recent results indicate that EVs recapitulate the beneficial effects of their parental cells in the treatment of heart diseases B. ImportSevere muscle injuries and genetic defects like muscular dystrophies cause myofiber death. Spontaneous reparation to regenerate skeletal myofibers do occur but are insufficient . A centrA mounting body of evidence suggests that EVs are actively produced by skeletal muscles cells and contribute to muscle repair and regeneration . For exaEVs are also evaluated as potential therapeutic agents to counteract muscle wasting and skeletal muscle dysfunction. Chronic kidney disease (CKD), which ultimately leads to end-stage renal failure, often leads to muscle wasting. Intramuscular injection of EV-encapsulated miR29, previously shown to have anti-fibrotic activity, could attenuate UUO-induced body weight loss and muscle atrophy . SimilarFinally, EVs could be used as efficient delivery tools of functional cargoes in vivo to restore expression of missing proteins in patients. For example, Gao et al. demonstrated that systemic administration of exosomes loaded with CP05-conjugated dystrophin splice increased dystrophin protein expression in dystrophin-deficient mdx mice with functional improvements .It has become clear that EVs enable intercellular signaling that facilitate myofiber regeneration, limit excessive ECM deposits and improve muscle functions. More strikingly, their systemic delivery improves muscle function in diseases like DMD or CDK. Future studies are now required to characterize their biogenesis, compositions and biological activities on recipient cells in both physiological and pathophysiological conditions to determine whether they might be envisioned for therapy .As discussed, some of the benefits observed with cell therapy are likely due to paracrine effects that can be recapitulated by EVs derived from these cells, rather than long-term engraftment and survival of transplanted cells . OverallDespite their promising roles, several challenges associated with EVs as acellular therapy products still need to be overcome.As products of cells, the identity and functions of EVs are directly correlated to their cell source. Consequently, it is essential to identify the most appropriate cell source for EV production . As illuAnother challenge is the development of robust procedures to isolate and purify EVs with high yield and purity while preserving their structure and activity . As withEVs are isolated from a variety of different sources, including body fluids with highly variable compositions  and cell culture media . ImportaTo purify EVs in a GMP-grade scalable manner, studies have employed ultrafiltration or tangential flow filtration (TFF) to concentrate EVs based on their size ,228,229.As with cells and other cell-derived products, maintenance of EV biological activity during storage is critical for their therapeutic use . StabiliCurrent legislation does not provide specific regulatory guidelines for EV-based therapies. In the European Union (EU) and United States of America, they enter in the framework of the biological medicinal products regulation under the definition of “biological medicine”  . The phaThree subcategories of EV products are possible, each with specific regulatory guidance: 1/native EVs from unmodified cells, 2/EVs from genetically manipulated cells which do not contain transgene products, and 3/EVs from genetically modified cells containing transgene products . In addiStem cell therapy and tissue engineering are intimately intertwined in the development of regenerative medicine products. In recent years, research has focused on the use of biocompatible scaffolds functionalized with EVs, with or without cells, to improve regenerative capacity of the grafted products. Regulatory strategies for advanced therapy medicinal products (ATMPs)-encompassing gene therapy, somatic cell therapy, and tissue-engineered products–could be applied to such complex EV-based products (DIRECTIVE 2001/83/EC). EVs produced by genetically modified cells with biologically active transgene products could also fall within this subcategory . Of noteAs outlined in this review, a number of key steps to develop regenerative therapies has been achieved. Preclinical evidences for many indications were the basis for first-in-man clinical trials. Main concerns related to the use of hPSCs were ruled out in clinical trials . So far, simplified cell therapy systems have been used with some promising therapeutic results. However, preclinical evidences highlight the need for more complex cell therapy products containing various cells and/or biomaterials, structured as the native tissue. In parallel, the roles played by EVs as intercellular effectors of paracrine signaling has led to a strong interest in their use as cell-free therapeutic products to stimulate endogenous factors that would limit cell death or improve tissue regeneration. Strikingly, as these two strategies could act at different levels depending on the pathological context, their complementarity opens new perspectives to maximize therapeutic outcomes. EVs could be substituted to cell therapy in a number of indications including heart diseases, simplifying treatment complexity. However, much preclinical work is still required to characterize optimal “acellular” and cellular therapies and their potential synergistic effects. In addition, definition of appropriate conditions of GMP production need to be elaborated and customized for each application. All these future improvements may help to achieve the ultimate goal of regenerative medicine: the replacement of a degenerated tissue.WH, CM and KB are inventors of a patent (FR3078712) related to medical devices for the preparation of retinal tissues for regenerative medicine. LM, CM and KB are inventors of a pending patent related to the automated differentiation of hPSC into RPE cells (EP3754014). LM and CB are inventors of a pending patent related to the automated differentiation of hPSC into keratinocytes (EP20305217.0). SD and CB are inventors of two pending patents related to the differentiation of hPSCs into keratinocytes (EP20305218.0) and fibroblasts (EP 20305214.7) and to the formation of a skin substitute composed of hPSCs derived cells (EP 20305213.9). AD and CB are inventors of a pending patent related to the differentiation of hPSCs into endothelial cells (EP 20305215.4).


Systemic sclerosis (SSc) is a multi-organ autoimmune disease with complex interactions between immune-mediated inflammatory processes and vascular pathology leading to small vessel obliteration, promoting uncontrolled fibrosis of skin and internal organs. Interstitial lung disease (ILD) is a common but highly variable manifestation of SSc and is associated with high morbidity and mortality. Treatment approaches have focused on immunosuppressive therapies, which have shown some efficacy on lung function. Recently, a large phase 3 trial showed that treatment with nintedanib was associated with a reduction in lung function decline. None of the conducted randomized clinical trials have so far shown convincing efficacy on other outcome measures including quality of life determined by patient reported outcomes. Little evidence is available for non-pharmacological treatment and supportive care specifically for SSc-ILD patients, including pulmonary rehabilitation, supplemental oxygen, symptom relief and adequate information. Improved management of SSc-ILD patients based on a holistic approach is necessary to support patients in maintaining as much quality of life as possible throughout the disease course and to improve long-term outcomes. Systemic sclerosis (SSc) is a multi-organ autoimmune disease with complex interactions between immune-mediated inflammatory processes, uncontrolled fibrosis of skin and internal organs and vascular pathology leading to small vessel obliteration and capillary loss . InterstIn 2017, the European League Against Rheumatism (EULAR) and the European Scleroderma trials and Research group (EUSTAR) updated their treatment recommendations for SSc 9). From real life data, however, it is known that treatment in daily clinical practise in patients with SSc-ILD vary and differ widely from these recommendations . From re. None ofIn this review, we aimed to elucidate current treatment options and supportive measures for SSc-ILD focusing on evidence-based data and how these treatments effect outcomes and quality of life in SSc patients. We also discuss evidence for non-pharmacological treatment options and other supporting therapies and their impact on QoL and patient reported symptoms. We identify missing evidence and discuss future perspectives on research priorities in SSc-ILD as a joint venture of ILD experts drawn from pulmonology and rheumatology.To date, there is no internationally established therapeutic regimen for SSc-ILD but current treatment approaches focus on immunosuppressant therapies . The decBiological therapeutics have also been assessed as treatment options for SSc-ILD but are facing challenges. For rituximab, a monoclonal anti-CD20 antibody depleting B-cells, randomized controlled trials (RCTs) are lacking. Limited evidence, however, may suggest an association with lung function improvements –19. One Another monoclonal antibody is tocilizumab, an anti-IL-6R antibody. Evidence for treatment effects on SSc-ILD comes from the phase II FaSScinate trial  and the Based on similarities in the clinical presentation and outcomes of idiopathic pulmonary fibrosis (IPF) and other fibrosing ILDs including SSc-ILD, the idea emerged that established drugs approved for IPF might also show similar effects in other progressive fibrosing ILDs . An uncoNintedanib for the treatment of SSc-ILD has been investigated in the Phase III Senscis trial . Here itIn contrast to the Senscis trial, the INBUILD trial included ILD patients with diseases other than IPF with progressive fibrosis, defined by a deterioration in two out of three of the following domains: FVC, high resolution computed tomography (HRCT) and symptoms over the past 24 months prior to study inclusion . The stuAnother treatment option discussed in the EULAR/EUSTAR recommendations is autologous hematopoietic stem cell transplantation (HSCT). It is suggested only for selected SSc patients at high risk of organ failure due to significant and rapid disease progression although clear definition of such phenotype is missing , 35.. ItThe only curative treatment option, similar to other ILDs, might be lung transplantation. However, this treatment possibility is also limited to selected patients. There exist concerns regarding the multi-organ involvement of SSc patients with frequent presence of reflux and esophageal dysfunction resulting in bronchiolitis obliterans syndrome after lung transplantation. Prior to lung transplantation, all patient with SSc should be assessed with esophageal manometry and pH monitoring. In individuals with abnormal reflux, fundoplication has been shown to potentially offer benefit in both survival and reduced rejection following lung transplantation , 38. It Outcomes of lung transplanted SSc-ILD patients were shown to be comparable to other ILD patients. Of note, there is an ongoing debate whether these results are applicable to a larger SSc population, as patients in these studies were highly selected .Very recently, evidence-based consensus statements for the identification and management of SSc-ILD were published. They included statements on treatment initiation, escalation as well as treatment drivers . Except It is of high importance to emphasize that treatment for ILD in SSc patients strongly depends on the presence of other organ manifestations and the disease course. All SSc patients should therefore be assessed for other organ complications by SSc experts and the choice of specific therapies should be based on a holistic approach Fig. .Fig. 2HAs there are several treatment options currently available for SSc-ILD, it is important to identify patients who are more likely to benefit from a specific drug with an acceptable toxicity profile. To date, available evidence is acquired by using these drugs as monotherapy. Another treatment concept, however, is combination regimens with either upfront combination or a sequential add-on of drugs. It is unknown whether these regimens will provide better outcomes with an acceptable toxicity profile. Additionally, we identified the following lack of evidence regarding the choices of drugs: how patient characteristics and patient preferences affect the choice of a drug; when to switch from one to another treatment and when to escalate treatment defined as either an increase in dose of existing therapy or the addition of a new therapy; what should be the appropriate duration of treatment and finally, how to define treatment success and/or failure in the individual patients.All SSc-ILD trials showing treatment efficacy involved SSc patients with established ILD at the time of inclusion , 16, 28.In order to truly evaluate the benefits of pharmacologic as well as non-pharmacologic treatments, reliable and valid outcome measures are needed, but very few have been validated in SSc-ILD. OMERACT (Outcome Measures in Rheumatology), a group of independent international health professionals and patient research partners, have strived to systematically identify and validate outcome measures in SSc and other connective tissue diseases (CTDs) based on the concepts of truth, discrimination and feasibility . To answLung physiology has been the preferred outcome measure so far. Pulmonary function tests with FVC, total lung capacity (TLC), and diffusing lung capacity for carbon monoxide (DLCO) have frequently been used in phase II and III RCTs in SSc Table . FVC is The extent of ILD assessed by HRCT has been used as secondary outcome in few studies. HSCT showed significantly reduced ILD extent in one study , whereasThe 6-min walk test as a functional assessment was used as the primary endpoint parameter in a trial investigating bosentan for SSc-ILD and as a secondary parameter comparing cyclophosphamide with rituximab and was found useful , 46. HowHospitalization, exacerbations and mortality have often been used as outcome measures in ILD trials but so far not as a primary outcome measure in any prospective SSc-ILD trial. Time to death or death were included as secondary outcomes in two trials without reaching statistical significance most likely due to the relative short study duration of 1–2 years , 46.There have been identified a number of circulating biomarkers for SSc-ILD including Krebs von den Lunge 6 (KL-6), surfactant protein D (SP-D) and serum chemokine (C-C motif) ligand 18 (CCL18), but none of them are fully validated and have not been used as outcome measures in SSc-ILD trials , 56.Quality of life assessment is increasingly requested by patients and by health authorities in research and as outcome parameters in RCTs. Despite increasing data about treatment effects on functional and clinical outcomes, data on QoL, disability and physical/mental function has traditionally been less well assessed. When considered in their totality, the results have not been very encouraging. QoL is commonly captured by patient related outcome measures (PROMs) which are based on reports directly from the patient and describe patients’ perception of their own health status or QoL. They may diverge from physicians’ needs and interests and often measure different perspectives than those captured by physiologic measures , 58. No Despite not reaching statistically significance, changes in some of the domains of these questionnaires could still represent clinically meaningful variation in patient’s perception and were therefore included in our review.HAQ-DI is the most commonly used disability index in RCTs, with a reported minimally clinical important difference (MCID) of 0.10–0.20 . In SLS MMF, despite its widespread use, has little evidence available to support an impact on QoL , 63.Tocilizumab, when tested in diffuse cutaneous SSc in the phase II FaSScinate trial, showed improvement in both HAQ-DI and FACIT with MCID reached, as well as in clinician and patient global VAS .Rituximab, in addition to background therapy regardless indication , determined a MCID decrease of HAQ-DI in a small RCT, which did not reach significance, comparing 1 year with baseline data . DespiteRegarding anti-fibrotic therapy, despite beneficial effects in arresting lung function decline by nintedanib, no significant improvement in any PROM has been reported so far. In the SENSCIS study, both change in SGRQ, HAQ-DI and FACIT-dyspnoea questionnaire at 52 weeks were assessed. Nintedanib did not reach MCID changes in HAQ-DI. Similarly, despite no MCID being available for SSc patients, the FACIT-Dyspnoea questionnaire and the SGRQ showed no significant change compared to placebo .Traditionally, trials on the treatment of ILD have focused on pharmacological therapies and little attention has been paid to non-pharmacological treatment, supporting therapies and holistic approaches Fig. . PatientPulmonary rehabilitation is often recommended for SSc-ILD, but the data to support this recommendation is scarce. A recent systematic review on SSc overall population and pulmonary rehabilitation concluded that exercise therapy is considered safe, but that no definite conclusion on its efficacy can be drawn . An RCT No specific guidance on the use of supplemental oxygen in SSc-ILD exists. In patients with ILD, supplemental oxygen is recommended if severe hypoxemia at rest is present, though there is no structured research to support these recommendations . In dailPalliative care comprises symptom management, supportive measures and end-of-life care; and aims to improve QoL throughout the entire disease course and a dignified death. To date, data on palliative care in SSc-ILD is lacking. Despite the paucity of studies on palliative measures in SSc-ILD, data from ILD and the respiratory field may give some guidance , 74. SymFor the relief of dyspnea often a combination of non-pharmacological and pharmacological measures may be needed. A longitudinal cohort study in patient with fibrosing ILD starting on long-term oxygen showed that opioids and low dose benzodiazepines could safely be used . The resCough is often a refractory symptom in fibrotic ILD and no evidence-based treatment recommendations exist to date. In the SLS II study, treatment with MMF or oral cyclophosphamide was associated with a decrease in reported cough frequency, but no effect on cough related QoL was found .Causes of fatigue and depression in ILD and SSc-ILD are often multifactorial , 65, 77.Besides supportive measures, preventive advice is given in line with recommendations to all ILD patients, mostly based on expert opinion and common sense. In the case of smoking, vaping or other substance abuse, patients should strongly be encouraged to stop. Influenza and pneumococcal vaccinations are offered to all patients to prevent infections that may trigger an exacerbation or worsening of disease.Current clinical practice, supported by treatment recommendations as discussed above, is to screen all SSc patients for ILD and initiate treatment of patients with SSc-ILD if apparent disease, defined by the presence of severe ILD on HRCT, restrictive lung disease or declining lung function. This clinical practice is partly influenced by the incomplete understanding of which patients are at risk of early progressive ILD, and the knowledge that only about 30% of SSc-ILD patients are progressive in the following year . It is however known that lower lung function, higher extent of ILD on HRCT and declining lung function, as well as other SSc specific risk factors as diffuse cutaneous SSc and anti-topoisomerase I antibodies, both associated with the presence of a severe ILD, is associated with a high mortality. On this background, it is apparent that early identification of patients at risk of progression, before loss of lung function and irreversible lung damage, is of high importance to initiate targeted treatment early and improve clinical outcome. In daily clinical practice SSc-ILD patients usually receive immunosuppressants. Nintedanib, the first targeted SSc-ILD treatment has been approved in many but not all countries already. To date, however, there is a lack of robust evidence to support the long-term efficacy of treatments or to guide their use. It seems tempting that novel treatment concepts should aim for prevention of progression to avoid irreversible organ damage. Further RCTs in different patient populations and head to head comparisons of the currently used drugs are needed to assess the treatment efficacies of available therapeutics for SSc-ILD patients. More research and RCTs on upfront combination and/or sequential use of immunosuppressive and nintedanib will hopefully be conducted in the future. Other targeted therapies that inhibit key pathways in the pathogenesis of SSc-ILD are also needed to expand the ILD treatment armamentarium and further improve patient outcomes.Many of the previously conducted RCTs in SSc have failed their primary outcome. The frequently used mRSS failed in recent clinical trials as a surrogate parameter for universal disease progression. Other clinical trial concepts i.e. time to worsening using worsening of organ involvement as a study endpoint with trial cohort enrichment for SSc patients at risk for overall disease worsening have been recently suggested and seem promising .Lastly, structured research on supportive measures and on palliative care in SSc-ILD is extremely limited to date and therefore much needed. Despite advances in the treatment SSc-ILD, no clear positive effect on QoL has been shown and many patients still progress and eventually die of their disease. Better measures are needed to support patients in maintaining as much QoL as possible throughout the disease course and thus holistic approaches will need more focus in the years to come.


We describe a 6-year-old boy with an asymptomatic linear eruption on the left index finger with mild erythema of the proximal nail fold, nail dystrophy, and subungual hyperkeratosis of the nail. A diagnosis of nail lichen striatus was made. The child was successfully treated with a topical corticosteroid. Because of its rarity, nail lichen striatus is often under-recognized. Physicians should be familiar with the nail involvement in individuals with lichen striatus so that an accurate diagnosis can be made and unnecessary investigations and treatment avoided. Nail lichen striatus is rare . There aA 6-year-old boy presented with a 10-month history of an asymptomatic linear eruption on the left index finger which gradually extended to the periungual area with resulting nail changes. His past health was unremarkable. There was no history of trauma for the affected areas. The family history was unremarkable.Physical examination revealed multiple tiny skin-colored papules along Blaschko lines on the left index finger, mild erythema of the proximal nail fold, and onychodystrophy and subungual hyperkeratosis of the nail Figures  and 2. TLichen striatus is a benign, acquired, asymptomatic, self-limited T-cell-mediated dermatosis that primarily occurs in children between 4 months and 15 years of age . The malInvolvement of the nail is rare and may occur before, after, or concurrent with the skin lesion of lichen striatus –3, 5. VeIn the present case, a diagnosis of nail lichen striatus was made based on onychodystrophy and subungual hyperkeratosis localized to one portion of the nail, single nail involvement, and presence of typical skin lesions of lichen striatus near the nail. Lichen striatus should be differentiated from cutaneous lichen planus and inflammatory linear verrucous epidermal nevus (ILVEN). Cutaneous lichen planus is characterized by 6 Ps: planar (flat-topped), purple (violaceous), polygonal, pruritic, papules/plaques that affect the skin. Lesions of lichen planus are often superimposed by lacy, reticular, white lines known as “Wickham striae.” Linear lichen planus is a distinct variant of lichen planus with papules in a linear distribution that may mimic lichen striatus. ILVEN is characterized by intensely pruritic, skin-colored to brown, hyperkeratotic papules linearly distributed along lines of Blaschko. The condition usually presents at birth or shortly thereafter, progresses slowly, and tends to be persistent. ILVEN often involves the lower limb or buttock and is usually unilateral.Because of the rarity of cases, there is no established treatment of nail lichen striatus. Some authors suggest watchful observation due to the benign and self-limited nature of nail lichen striatus . Other aWe describe a 6-year-old boy with nail lichen striatus who was successfully treated with a topical corticosteroid. Thus far, there is no established treatment of nail lichen striatus. This case serves as an example that the nail lichen striatus can be successfully treated with a topical corticosteroid. Because of its rarity, nail lichen striatus is often under-recognized. Physicians should be familiar with the nail involvement of lichen striatus so that an accurate diagnosis can be made and unnecessary investigations and inappropriate treatment avoided.