Congenital diaphragmatic hernia (CDH) is a relatively common and life-threatening birth defect, characterized by an abnormal opening in the primordial diaphragm that interferes with normal lung development. As a result, CDH is accompanied by immature and hypoplastic lungs, being the leading cause of morbidity and mortality in patients with this condition. In recent decades, various animal models have contributed novel insights into the pathogenic mechanisms underlying CDH and associated pulmonary hypoplasia. In particular, the generation of genetically modified mouse models, which show both diaphragm and lung abnormalities, has resulted in the discovery of multiple genes and signaling pathways involved in the pathogenesis of CDH. This article aims to offer an up-to-date overview on CDH-implicated transcription factors, molecules regulating cell migration and signal transduction as well as components contributing to the formation of extracellular matrix, whilst also discussing the significance of these genetic models for studying altered lung development with regard to the human situation. Congenital diaphragmatic hernia (CDH) represents a relatively common and life-threatening birth defect with an estimated global prevalence of 2.3 in 10,000 live births . Hence, A defect in the posterolateral diaphragm  is the most common type of CDH and comprises approximately 80–90% of all cases, with the majority being left-sided (85%), less often right-sided (10%), or bilaterally (<5%) . The duaThis article aims to offer a comprehensive overview of genetically modified mouse models of CDH, resultant candidate genes and signaling pathways, whilst also discussing new opportunities and limitations for studying altered lung development in relation to the human situation.A large variety of genetic factors have been found to play key roles during the pathogenesis of CDH and pulmonary hypoplasia. Currently, genetic causes are detected in about 30% of CDH patients –36. ThroNumerous transcription factors and transcriptional (co-)activators have been suggested in the development of the primordial diaphragm and lungs. Many of them are associated with retinoid or sonic hedgehog signaling pathways.retinoic acid receptors α and β (Rarα and Rarβ) have been demonstrated to generate offspring with posterolateral diaphragmatic defects identical to those observed in human patients , a membrane receptor that controls the cellular uptake of vitamin A and cellular retinoic acid binding protein 1 (CRABP1), which is located on chromosome 15, have been found to lead to a spectrum of developmental anomalies including CDH and hyperplastic lungs  and its derivates  are indispensable for various aspects of early embryogenesis. Over the years, several knockout models have indicated a role of the retinoid signaling pathway and its downstream targets in the pathogenesis of CDH . For inspatients –44, and patients , 46. Surficiency . Neverthpoplasia . Despitepoplasia , 42. Stiic lungs , 48.chick ovalbumin upstream promoter transcription factor II (COUP-TFII), a transcription factor that is affiliated with the nuclear steroid/thyroid hormone receptor superfamily, whose DNA-binding site has been shown to reduce the induction of retinoic acid receptors (COUP-TFII was recently renamed as nuclear receptor subfamily 2 group F (NR2F2), which is expressed in the diaphragm and lungs during early gestation , posterolateral diaphragmatic defects presumably arise because of the failure of the posthepatic mesenchymal plate to merge with the lateral body wall, thus enabling stomach and liver to protrude into the chest  null mutant mice die during mid-gestation, displaying posterolateral diaphragmatic defects and lung hypoplasia alongside urogenital abnormalities   , 58. Wt1n humans . More ren humans  and Clean humans  have repelopment . Surpristhe PPFs . Paris ethe PPFs  have devr spaces . Loss ofl of CDH .GLI-Kruppel family member 2 (Gli2) and Gli3 and are both members of a highly conserved morphogenetic family, belonging to the sonic hedgehog (Shh) signaling pathway  and pre-B-cell leukemia transcription factor 1 (Pbx1) were recently recognized as indispensable components of the Shh signaling pathway, functioning as regulators during early embryogenesis  have numerous developmental abnormalities including posterolateral defects in the diaphragm, pulmonary hypoplasia and liver herniation , formerly known as friend of GATA-binding protein 2 (FOG2), encodes another zinc finger-containing protein that regulates the transcriptional activity of GATA4, hereby controlling a number of developmental mechanisms in the forming diaphragm and lung  generated Fog2–/– offspring with bilateral hypoplastic lungs and a defective posterolateral diaphragm characteristic of CDH  displayed retrosternal diaphragmatic defects, dilated distal airways and thickened pulmonary mesenchyme  gene are accompanied with a significant risk of CDH and cardiovascular anomalies  is one of those, which is strongly expressed in the fetal diaphragm and in mesenchymal cells of the lung myogenic bHLH transcription factor R  and musculin (Msc), respectively  genes and the transcription factor sine oculis homebox 1 (Six1) form an important signaling network, which plays a central role during embryonic development  is a protein coding gene that is relatively conserved across various species  gene in mice resulted in offspring with poorly formed diaphragms and diaphragmatic defects, strikingly similar to the phenotype seen in humans with anterior CDH  CDH  in the mouse endothelium interferes with vascular development in the primordial diaphragm, resulting in hypoxia as well as diaphragmatic hypoplasia and central-type CDH. The observed phenotypes in these animals mirror the congenital anomalies seen in Slit3 knockout mice. In addition, implementation of a heterozygous mutation in the Robo4 gene, which encodes the receptor of Slit3, exacerbated the defect in vascular and diaphragmatic formation  gene exhibit not only posterolateral diaphragmatic defects, they also develop a spectrum of other comorbidities including cardiovascular anomalies, renal and urogenital malformations, facial clefts, lung hypoplasia and failure of alveogenesis (The Slit guidance ligand (Slit) family of proteins comprise a group of molecules with crucial functions in cell migration and adhesion through interaction with roundabout (Robo) receptors. ogenesis . Homozygsum) CDH , 106. Thchanisms –109. Forelopment , 110–114DH cases . Homozygnstances . More reeraction . Furthereraction  have notormation . Thus, togenesis , 119.fuzzy planar cell polarity protein (Fuz) that was associated with CDH, liver protrusion into the chest cavity and pulmonary hypoplasia with a single left lung lobe  gene encodes for a receptor tyrosine kinase that is necessary for the migration of muscle precursor cells into the forming diaphragm (fibroblast growth factor 10 (Fgf10) is crucial for early organogenesis of the lung  was recently identified in eight unrelated families, causing X-linked CDH and body wall defects. A genetically modified mouse model of this W499CPls3 variant resulted in perinatal death and reproduced the main features of the human phenotype, including diaphragmatic and body wall abnormalities . Today, several components of the ECM are known to be aberrant in CDH and associated lung defects.FREM1 deletion in a female infant with isolated left-sided CDH and a membranous sac, Beck et al. , an extracellular cuproenzyme , an integral part of microfibrils in elastic and non-elastic connective tissues , Fras1-related extracellular matrix 1 (Frem1) and Frem2 form a mutually stabilizing ternary complex in the ECM, which plays a critical role in cell adhesion and intercellular signaling , 126. Afk et al.  develope defects . More re defects  reportedroenzyme . In turnl tendon , 132. Ho tissues . A gene-ed edges . These hc cavity . Fibulinc cavity . Fbln4 nmphysema .Several other genes and enzymes responsible for signal transduction, intracellular signaling and apoptosis have been discovered in association with diaphragmatic defects in genetically modified mouse models.–/–ChAT mice showed liver herniation through the tendinous center of the diaphragm  has been reported to be implicated in various morphogenetic processes during embryonic development , 138. Iniaphragm , presumaficiency . Low-denficiency . ENU-indl cavity . Mitochol cavity . Mtpα–/–ficiency . WD repeficiency . Followificiency  noticed Over the years, experimental animal models of CDH have not only permitted us to investigate the pathogenesis of this relatively common but complex birth defect in more detail, they have also led to a better understanding of the molecular genetic basis of the underlying tissue defects. Therefore, animals with CDH in which this congenital anomaly develops naturally represent the ideal research models to study disease pathomechanisms and related lung abnormalities, as there is minimal interference to the animal before the examination. Furthermore, genetically modified animal models of CDH not only resemble the natural development of this malformation, they also provide new insights into the participating genes and signaling pathways, and how their modification can potentially change the course of this life-threatening condition. With the recent advent of novel molecular techniques including biomedical engineering and ENU mutagenesis screens, we hopefully may identify additional CDH-related mutations that are linked with abnormal diaphragm and lung development in other genetic mouse models –147.FF, UR, and PP critically revised the initial manuscript draft for important intellectual content and performed the literature search for the work. FF outlined and wrote the initial manuscript draft. All authors approved the final version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chemotaxis, regulated by oscillatory signals, drives critical processes in cancer metastasis. Crucial chemoattractant molecules in breast cancer, CXCL12 and EGF, drive the activation of ERK and Akt. Regulated by feedback and crosstalk mechanisms, oscillatory signals in ERK and Akt control resultant changes in cell morphology and chemotaxis. While commonly studied at the population scale, metastasis arises from small numbers of cells that successfully disseminate, underscoring the need to analyze processes that cancer cells use to connect oscillatory signaling to chemotaxis at single-cell resolution. Furthermore, little is known about how to successfully target fast-migrating cells to block metastasis. We investigated to what extent oscillatory networks in single cells associate with heterogeneous chemotactic responses and how targeted inhibitors block signaling processes in chemotaxis. We integrated live, single-cell imaging with time-dependent data processing to discover oscillatory signal processes defining heterogeneous chemotactic responses. We identified that short ERK and Akt waves, regulated by MEK-ERK and p38-MAPK signaling pathways, determine the heterogeneous random migration of cancer cells. By comparison, long ERK waves and the morphological changes regulated by MEK-ERK signaling, determine heterogeneous directed motion. This study indicates that treatments against chemotaxis in consider must interrupt oscillatory signaling. Chemokine CXCL12 and growth factor EGF are important signaling molecules from the microenvironment of a primary breast tumor and distant organs. These molecules contribute to the organ-specific chemotaxis of circulating tumor cells and subsequent metastasis in breast cancer and multiple other malignancies . CrucialChemotaxis plays an important role in the migration of cancer cells and subsequent metastasis toward chemotactic molecules such as CXCL12 and EGF. Chemotaxis and other cellular functions, such as proliferation, differentiation, and cell death, are regulated by signaling oscillations. Prior research into ERK oscillatory activity has revealed the dynamic nature and integrated feedback loops of signaling networks regulating migration ,20,21,22CXCL12–CXCR4 interaction elicits G protein signaling to activate Ras and PI3K and downstream ERK and Akt, which can also be activated by EGF-EGFR. Ras and PI3K drive protrusions of cell membranes, creating positive feedback for Ras activation ,20. Ras To answer these questions, we employed an integrated approach combining live, single-cell imaging and quantitative analysis to investigate key signaling pathways and chemotactic responses of MDA-MB-231 breast cancer cells. We implemented multiplexed fluorescence reporters with automated live-cell imaging and image processing to quantify oscillatory information in signaling, morphology, and chemotaxis. Then, we used a time-dependent data processing approach to quantify relationships of signaling, morphology, and chemotaxis within a heterogeneous cell population and across populations treated with targeted inhibitors. We also employed an independent approach termed Variational System Identification (VSI), which combines computation with mathematics to infer the two components of cell motion—random migration and directed motion. Concluding from our independent approaches, we found that amplitudes of short Akt and ERK waves, regulated by MEK-ERK and p38-MAPK signaling pathways, determine the heterogeneous random migration of cancer cells. By comparison, amplitudes of long Akt and ERK waves, the extent of cell elongation, and nuclear polarization determine the heterogeneous directional movement of cancer cells in a chemotactic gradient, which is regulated by MEK-ERK signaling. These data provide new insights into how single cells connect dynamics of signaling response to morphologic changes associated with chemotaxis.2. Prior to loading cells for chemotaxis experiments, we precultured MDA-MB-231 cells in FluoroBrite imaging media (Thermo Fisher Scientific), 10% FBS, 1% Pen/Strep, and 1% GlutaMAX for 2 days. We used previously engineered cells that have a stable expression of histone 2B fused to mCherry (H2B-mCherry), Akt-KTR (Aquamarine), ERK-KTR (mCitrine), and CXCR4 receptor for our experiments . EGBecause our results showed that long signaling waves and aspect ratios regulate the heterogeneity of directed motion, we next asked to what extent long signaling waves are related to the aspect ratio of cells during migration. When polarized cells reorientate and modulate their morphology during mesenchymal cell migration, nuclear reorientation is important in promoting directed motion . Since Ep < 0.0001, 99% confidence intervals shown in We isolated each aspect ratio peak of individual cells and compared them with the corresponding ERK signaling. All aspect ratio peaks were aligned to the same time point to facilitate the identification of time-dependent relationships between aspect ratio and long ERK signaling waves A. Comparp < 0.0001, 99% confidence intervals shown in To quantify the extent of nuclear polarization relative to ERK signaling and cell morphological change, we measured nuclear polarization as the distance of the nucleus with respect to the cell centroid. The amplitudes of long ERK waves and aspect ratio correlate with the degree of nucleus polarization. Quantile regressions between the 20th and 80th percentile illustrate that heterogeneous ERK and aspect ratio differences show a linear relationship with nuclear polarization  inhibitor, trametinib is a MEK inhibitor, and both are used clinically for cancer therapy. The inhibition of PI3K blocks downstream activation of Akt, while inhibition of MEK blocks the activation of downstream ERK. Kinase p38 has a context-dependent effect on mTORC1 ,40,41.We compared the single-cell data of MDA-MB-231 cancer cells preconditioned with either 100 nM alpelisib, 10 nM trametinib, and 10 µM SB203580 two hours prior to the addition of a CXCL12 gradient A. These We compared the effects of these compounds on various aspects of signaling and morphology amplitudes in relation to random migration and directed motion of cancer cells. We focused on amplitudes of short ERK and Akt waves relative to the heterogeneity of random migration. Despite similar reductions in mean Akt levels and no reduction in mean ERK levels B,C, the The present study investigated to what extent oscillatory signaling networks in cells are associated with heterogeneous chemotactic responses and the effects of targeted inhibitors. This single-cell chemotactic study in MDA-MB-231 cells led us to two main conclusions. First, the heterogeneity of cancer cell directed motion and random migration originates from the rate of change of kinase activities and cell morphology with oscillation period as the regulator. Second, the responsiveness of oscillatory signals and chemotaxis is regulated by the activities of MEK and p38 kinases. The fact that the heterogeneity of chemotactic response in cancer cells is determined by the oscillatory network and can be controlled by inhibition of signaling pathways suggests that potential treatment approaches for targeting fast-migrating cells should consider effects on oscillatory signaling and morphology.Three analyses provide evidence that the heterogeneity of cancer cell directed motion and random migration originate from the rate of change of signaling activities and cell morphology with oscillation period as the regulator. First, the migration speeds of individual cells are determined by the amplitude of Akt and ERK signaling waves and the morphological aspect ratio of each cell. Despite not referring to cell-to-cell heterogeneity, other previous investigations reinforce that ERK pulses are related to oncogenic transformation, collective migration, and polarization ,21,22. OThe second piece of evidence supports that the oscillation period is a regulator of the signaling and morphological amplitudes for directed motion and random migration heterogeneity. Depending on the oscillation periods of Akt and ERK signaling waves and morphological aspect ratio, cells have distinct levels of dependence on the CXCL12 gradient. Our current study shows that short Akt and ERK signaling waves with an oscillation period of 0.5–1 h contribute to random migration, whereas long signaling waves and morphological change with an oscillation period of 1.5–2 h tend to contribute to directed motion. This suggests that cell signaling networks regulate distinct modes of migration via different time scales, which was suggested previously . Random The third piece of evidence further supports that the rate of change of long ERK signaling waves and morphological aspect ratio with similar 1.5–2 h oscillation periods negatively correlate to determine the directed motion of cancer cells. Our current study shows a relationship between the extent of reduced long ERK signals and the extent of cell elongation. Cells reduce the long ERK signal and elongate at the same time and have a burst of migration speed with a delay of around 20 min. This evidence and previous investigations on short ERK pulses ,20 demonThree analyses provide evidence that activities of MEK and p38 kinases regulate the responsiveness of oscillatory signals and chemotaxis. First, the state of MEK kinase activity determines the responsiveness of both short and long signaling and morphological amplitudes with resultant impairment of cancer cell random migration and directed movement. Our study shows that the amplitudes of short and long ERK and Akt waves and aspect ratios reduce when cells are treated with a MEK inhibitor. A previous study showed that inhibitory phosphorylation of MEK is crucial for negative feedback and oscillatory ERK signaling and impacts signaling amplitudes . FurtherSecond, MEK inhibition also impacted nuclear polarization, which relates to the extent of ERK signal change, cell elongation, and directed motion acceleration. This study shows that nuclear polarization relates to single-cell changes in ERK signaling, cell elongation, and directed motion. This evidence supplements previous investigations that nuclear polarization promotes polarity and directed motion in cells ,50. HoweThird, the state of p38 kinase activity determines the responsiveness of only short Akt and ERK signaling amplitudes and blocks the random migration of cancer cells. Our study shows that the amplitudes of short ERK and Akt waves reduce when cells are treated with a p38 inhibitor. Recognizing that p38 may have context-dependent effects on mTorc1, our past work with these cells shows that p38 inhibits mTORC1 ,57. SincThis study assumes that live cell imaging reporters in cells capture all essential temporal information. However, the characterization of network dynamics is limited by imaging frequency and types of live-cell reporters. Our assumption is reasonable because the imaging frequency is five times faster than the signal dynamics that we studied. With recent advances in automated imaging techniques and live-cell reporters, the imaging frequency and the number of live-cell reporters can be increased in the future without causing phototoxicity or fluorescence crosstalk. This improvement will help reveal other mechanisms regulating heterogeneity in cancer cells, such as receptor dynamics, cytoskeletal structure, adhesion, and contractility. Cells also migrate differently in 2D versus 3D environments. We will need to confirm to what extent discoveries in this 2D chemotaxis study apply to a 3D environment. Our quantitative analysis approach focused on relationships and correlations among signaling, morphology, and migration and variations between migrating and non-migrating cells. Further studies are required to firmly establish causal relationships between these parameters. Future studies also are needed to explore pre-existing cell states and unique feedback delays in Ras/PI3K/ERK signaling and morphological networks regulating migration heterogeneity. Our study offers answers to processes in oscillatory signaling that associate with the heterogeneous responses of seemingly identical cancer cells with the same genetic mutation. In addition to the heterogeneity described here, heterogeneity also arises within a tumor and across different patients. While our study provides insights about the effective targeting of migrating cancer cells with constitutive activation of one major pathway, our findings need to be combined with other studies focusing on intratumor heterogeneity for effective prevention of metastasis.In summary, we have shown that the rate of change of ERK and Akt signaling activities and cell morphology determine the heterogeneity of cancer cell chemotaxis. We also demonstrated the effects of MEK and p38 kinases on random and directional cell movement. Our study suggests that future studies in signaling, morphology, and chemotaxis should consider the period and amplitude of oscillatory signaling outputs. Our finding paves the path towards better understanding of mechanisms regulating heterogeneous responsiveness in cancer cells and future identification of new interventions controlling cancer metastasis.


Studies on triple-negative breast cancer (TNBC) have recently confirmed the involvement of gamma-glutamyltransferase 1 (GGT1), whose enzyme activity expressed at the cell surface favors the cellular resupply of antioxidant glutathione (GSH) thus offering cancer cells protection against the prooxidant effects of CDDP. However, an additional well-established mechanism depends on GGT1-mediated matabolism of extracellular GSH. It was in fact shown that glycyl-cysteine – the dipeptide originated by GGT1-mediated GSH metabolism at the cell surface – can promptly form adducts with exogenous CDDP, thus hindering its access to the cell, interactions with DNA and overall cytotoxicity. Both mechanisms: mainainance of intracellular GSH levels  The cytotoxic effects of cisplatin (CDDP) are in the first place mediated through its binding to DNA with the formation of intra-strand DNA adducts, leading to the inhibition of DNA synthesis and cell growth as well as to up-regulation of pro-apoptotic/down-regulation of anti-apoptotic genes/proteins, resulting in the induction of both intrinsic and extrinsic pathways of apoptosis. CDDP also induces production of reactive oxygen species (ROS), lipid peroxidation and activation of p53 signaling and other signal transduction pathways . Indeed,Highly aggressivity, high recurrence rate and poor prognosis make the management of TNBC one of the hardest challenges in oncology. Treatment strategies over the last decades have been largely focused on chemotherapeutics such as taxanes and anthracyclines, but regimens based on CDDP – alone or in combination – have more recently taken the stage as a more versatile and promising tool against TNBC, especially in carriers of BRCA1 mutations or other deficiencies in the DNA repair systems making cells sensitive to clastogenic effects. Major difficulties are however represented by the frequent occurrence of CDDP resistance and the considerable heterogeneity of TNBC, leading to a wide variability in patients’ response to the drug. Several potential biomarkers are under investigation in order to identify patients most likely to benefit from CDDP, including homologous recombination repair deficiency, tumor infiltrating lymphocytes, TP53, cyclin-dependent kinase 2 expression, vascular endothelial growth factor and matrix metalloproteinase-9 –6.An interesting study has recently described a novel cellular/molecular mechanism leading to CDDP resistance of TNBC, highlighting a crucial role for the expression of gamma-glutamyltransferase 1 (GGT1). An interplay of cancer cells with tumor associated macrophages (TAMs) was in fact described, in which TGF-β1 released by TAMs induces TNBC cells to secrete hepatic leukemia factor (HLF), which in turn transactivates GGT1 . The expHowever, taking into account the complex set of effects produced by GGT1 enzyme activity , such inextracellular detoxication of CDDP, which adds to the antioxidant defense offered by intracellular GSH against CDDP-induced oxidative stress, and is likely a major factor concurring to prevent its cytotoxicity  is gratefully acknowledged.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The use of nanoparticles for developing vaccines has become a routine process for researchers and pharmaceutical companies. Gold nanoparticles (GNPs) are chemical inert, have low toxicity, and are easy to modify and functionalize, making them an attractive choice for nanovaccine development. GNPs are modified for diagnostics and detection of many pathogens. The biocompatibility and biodistribution properties of GNPs render them ideal for use in clinical settings. They have excellent immune modulatory and adjuvant properties. They have been used as the antigen carrier for the delivery system to a targeted site. Tagging them with antibodies can direct the drug or antigen-carrying GNPs to specific tissues or cells. The physicochemical properties of the GNP, together with its dynamic immune response based on its size, shape, surface charge, and optical properties, make it a suitable candidate for vaccine development. The clear outcome of modulating dendritic cells, T and B lymphocytes, which trigger cytokine release in the host, indicates GNPs’ efficiency in combating pathogens. The high titer of IgG and IgA antibody subtypes and their enhanced capacity to neutralize pathogens are reported in multiple studies on GNP-based vaccine development. The major focus of this review is to illustrate the role of GNPs in developing nanovaccines against multiple infectious agents, ranging from viruses to bacteria and parasites. Although the use of GNPs has its shortcomings and a low but detectable level of toxicity, their benefits warrant investing more thought and energy into the development of novel vaccine strategies. The development of vaccines and immunization programs against various kinds of diseases ranging from infections to cancer significantly progressed in the past few decades. One of the most important factors contributing to this growth is the advancement of nanotechnology. The use of nanoparticles in the development of vaccines is a major landmark step. One key step for the vaccine development process is the use of an optimal carrier or delivery system that can influence a potent immune response. The use of different types of nanoparticles and their roles in influencing the immune system has been studied in different disease models. Nanocarriers can be used as adjuvants in the preparation of new-age vaccines. The size, shape, route of administration, and antigen tagging mechanism on the nanoparticles are all critical in this ,2. In thAmong different nanoparticles already tried and used for the successful development of nanovaccines, the gold nanoparticle (GNP) is noteworthy. The chemical synthesis process of antigen tagging on the GNP and its formulations is easy, making it a suitable candidate in the nanovaccine manufacturing process . MetalliMultifunctional GNPs have been used by conjugating them with FDA-approved antimicrobial drugs and antibiotics in many studies ,13,14,15One key concern regarding the use of any foreign chemical such as gold in any form of treatment intervention is its possible harmful side effects. However, the administration of gold as an adjuvant or in a nanovaccine formulation has a high safety profile with few side effects ,37. LikeAlthough GNPs are widely used in the development of vaccines against multiple cancers, in this review, we focus on their use in vaccination against infectious agents. We discuss the characteristic features of gold nanoparticles that make them advantageous to use in vaccine development, including their shape, size, and generated immune response . We specGNPs are often used to develop a potent antigen carrier system for immunization ,35,36,37Precision in the nanocarrier delivery and penetration to the site of interest or the immune cells is a critical component. This facilitates the induction of the immune response genes, antigen processing, cytokine production, antibody secretion, and T cell stimulation for effective therapy or vaccine efficacy ,44,45,46GNPs are efficient in delivering antigens into the major antigen-presenting cells such as dendritic cells, facilitating the downstream immune response, cross-presentation, and CD8+ cytotoxic T cell response A,B 49]..49]. AloGNPs are biocompatible and inert. They are easily functionalized with a wide range of peptides and molecules and are also very stable ,57,58,59The GNP shape, size, charge, and conjugated materials all influence organ accumulation and blood clearance . ProgresThe size of the GNP, along with its shape, influences the immune system differently. This shape and size dependency of the adjuvant activity of GNPs is used to polarize the immune response in different scenarios to deliver the best outcome ,40,41,42Rod-shaped GNP-treated bone marrow dendritic cells (BMDCs) produce high levels of IL1b and IL18, whereas cube- and spherical-shaped GNPs result in the production of high levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFa), interleukin (IL)-6, IL17, and granulocyte-macrophage colony-stimulating factor (GM-CSF) . NiikuraChen et al. studied the impact of GNP sizes spanning from 2 to 50 nm and found that those between 8 and 12 nm are mostly drained nanoparticles . GNPs beListeria antigen were adoptively transferred to naïve animals, leading to the induction of natural killer cells, CD8 + T cells, and better Th1 response and vaccine efficacy than any other traditional immunization methods ..77]. Alt methods .DC from bone marrow, when stimulated by GNP, starts producing IL6, TNF-α, and interferon gamma (IFN-γ) . GNP canThe polarization and function of macrophages are reported to play a key role in different disease conditions . GNPs arGNPs are reported to have oxygen radical scavenging properties in the mouse macrophages as they reduce the reactive oxygen species (ROS) of GNPs in a dose-dependent manner in the presence of LPS treatment . The redNatural killer cells (NK) sourced from the lymphoid progenitor lineage play a crucial role in immune surveillance in the circulation. They release granzymes and perforins to induce infected cell lysis. GNPs have been researched to target NK cells by using the mechanism of NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This is the use and targeting of the NK cell receptors by antibody-tagged GNPs. This helps in the delivery and activation of NK cells .PEGylated polyamidoamine dendrimers entrapped within GNPs to transfect human ferritin heavy chain in the NK cells constitute a novel immunotherapy method . A modifGNP is a favored tool of virologists and has been used frequently in the development of the antiviral vaccination process.Human immunodeficiency virus (HIV) possesses an important cluster of mannose-rich glycans in its envelope glycoprotein called gp120, which is recognized by 2G12-like antibodies. Gold nanoparticles were synthesized with a monolayer coating of self-assembled oligomannosides (similar to gp120) and were capable of binding with 2G12 . GNPs atGNPs 2 nm in size coated with a synthetically prepared partial structure of multiple mannosidases ,92 proviRabbits were immunized intramuscularly with 50 μg of 2 nm glyconanoparticles coated with the V3β peptide of the HIV-1 gp120 protein. Post-immunization, they produced a high titer of neutralizing antibodies against HIV1 ,93. MoreHepatitis B virus surface antigen (HBsAg) DNA coated with GNP was injected into the epidermic cells employing a gene gun as a possible treatment measure . GNPs weIn vitro studies in RAW 264.7 macrophages with a gold nanocage conjugated with HBsAg showed better uptake and antigen processing with IL4 secretion . Recent Virus-like particles (VLP) were produced with 10 nm GNPs conjugated with CpG oligodeoxynucleotides (ODN) and core antigen of hepatitis B. Mice immunized four times with 50 μg conjugate (i.p.) showed a 200% increase in the antibody titer as compared to GNP-free administration. CD4 helper T cells and CD8 cytotoxic T cell population expanded with the higher secretion of IL-4 and IFN-γ, along with immunostimulation of both Th1 and Th2 responses .An interesting and effective means of delivery of hepatitis C virus DNA vaccine was proposed by a group that used plasmonic GNP activated by an electrical charge. This led to increased pore formation on the cell membrane and enhanced uptake of the DNA vaccine. The immunized mice group exhibited 100 times more gene expression as compared to the control group (without GNP use), with highly activated humoral and cell-mediated immunity being reported . E2 protGNPs 20, 40, and 80 nm in size were used to conjugate the serotype 2-derived domain III envelope glycoprotein of dengue virus (EDIII). The conjugate administered three times subcutaneously to BALB/C mice led to the production of serotype-specific concentrated neutralizing antibodies. The size and concentration of GNPs were manipulated to affect the levels of antibodies produced in the animals. Splenocyte proliferation, helper, and cytotoxic T cell expansion and activation with the increased synthesis of IL-4 and IFN-γ were observed in mice . GNP wasMuch work on the development of a vaccine against influenza by using the GNP has been carried out by Gill’s group. They took a highly conserved N-terminal conserved extracellular domain of influenza virus matrix protein 2 (M2e) peptide and conjugated it with a 12 nm GNP ,103. TheAnother group has shown that even after 15 months of vaccination with GNP/M2e+ CpG conjugate, the mice retained M2e-specific neutralizing antibody production and could survive the lethal H1N1 challenge. They suggested that the vaccinated mice could effectively retain the memory B cells specific to the M2e peptide used .Two intraperitoneal doses of M1 antigen of influenza virus conjugated with 15 nm GNP led to a higher titer of neutralizing antibody production along with the synthesis of IFN-γ and interleukins (ILs) 1β and 6. The activation of spleen lymphocytes and peritoneal macrophage respiration was also reported .Another study proposed the use of more than one antigen against influenza in the same vaccination dose. They prepared and administered GNPs conjugated with hemagglutinin and flagellin of the H3N2 influenza virus. This vaccination process generated stronger systemic and mucosal immunity and better protected the animals from the lethal influenza challenge, compared to when a single antigen conjugate was used ,108.Many other vaccination processes have been developed against viral pathogens. A few of them are highlighted in Expert opinion and future perspectives on Apart from the GNP-based influenza vaccine development ., most oGNP is used for designing and delivering antigens for immunization in a number of bacterial infections. In some cases, it also acts as an adjuvant. Antigenic fragments from bacterial sources are tagged along with the GNP to stimulate the immune response generated against them.Pseudomonas aeruginosa along with GNP and Freund’s adjuvant induces a better IgG response [Francisella tularensis were isolated and conjugated with 15 nm GNP to immunize the animals and obtain antitularemia sera rich in neutralizing antibodies [Yersinia pseudotuberculosis [A vaccine against the N terminal domain of the flagellin subunit of response . Two anttibodies . In anotrculosis .Y. pestis after coating it on 15 nm GNP. It helped elicit igG2a levels, interferon gamma, and Th1 cell activation [Salmonella typhimurium were also coated on GNPs and were reported to have better immunogenic properties in the clearance of the bacteria [Neisseria meningitidis were coated on GNP and reported to have better T cell activity, MHCII presentation, and immune properties [Another group studied the efficacy of the antibodies raised against F1 antigens of tivation . Similarbacteria . Non-immoperties .Several other uses of GNPs in the immunization process against bacterial infection are discussed in Expert opinion and future perspectives on Most of the animal studies discussed in Some parasitic infections are being studied where GNP plays a critical role in generating the immune response in the host to combat the infections. The following table describes a few of them.Expert opinion and future perspectives on Escherichia coli [Yersinia [Pseudomonas aeruginosa flagellin [Listeria monocytogenes [Streptococcus pneumoniae [Burkholderia mallei [Neisseria meningitides [In the previous three sections of this review, we discussed the successful laboratory implementation of GNP-based nanovaccines. It would be interesting to explore and carry out a comparative study of treatment with the antibody synthesized by using GNP-conjugated antigens side-by-side with the GNP nanovaccine formulations to determine which treatment works best in a particular infection. GNPs conjugated with the antigens, haptens, and adjuvants  of various pathogens have been used to obtain the antibodies. Some of these pathogens are dengue viruses , foot-anhia coli , YersiniYersinia , tetanusYersinia , Pseudomlagellin , Listeriytogenes , Streptoeumoniae , Burkhola mallei ,27, Neisngitides , tubercungitides ,30, and ngitides ,32.The use of the GNP shows some promising results in nanovaccine development technology. Still, it is not free from limitations. GNP, being a non-biodegradable agent, can easily be accumulated in vivo, which eventually might lead to certain side effects. Such non-porous and non-biodegradable properties might impair GNPs’ impact on the encapsulation and the timed or targeted release.Biosafety is a major concern when using any nanomaterial. A research group has shown that encapsulated GNP conjugated with fluorescein isothiocyanate (FITC) suppresses reactive oxygen species and cytokine secretion from the macrophages . GNP sizThe low penetration depth of GNPs due to the photothermal effect is a limiting factor to release the drugs or vaccinating agents into the depth required, leading to lessened immunoregulatory activities . SurfaceMoreover, there is still a lack of in-depth understanding about GNPs’ influence upon interaction with different cell types, especially after the modifications. Although reports suggest the formation of reactive oxygen species (ROS), oxidative stress and cell cycle impacts with induced DNA damage are also possible biological cellular responses ,136,137.Positively charged GNPs are reported to have a more toxic effect due to their propensity toward negatively charged DNA and cell membranes. However, both positively and negatively charged GNPs, and not neutrally charged, have been reported to have harmful impacts leading to mitochondrial stress ,140.In this review, we discussed the promising potential of the gold nanoparticle for prophylactic and therapeutic purposes. Advancements in the field of nanotechnology coupled with vaccine research have paved the way for successful nanovaccine development for combating many deadly infections. Being relatively safe to administer in humans, GNPs are widely used in the development of vaccines for many diseases, ranging from cancer to infections. Some of the vaccines developed against different cancer forms are in clinical trials and show promising outcomes. The summarized advantages and limitations of the GNP in its use in the vaccine development process are tabulated in Box 1AdvantagesBiocompatibleEasy synthesis processSize- and shape-dependent varied immune responseColloidal stabilityOptical propertiesEfficiency in molecule loading on the surfaceSurface functionalization flexibility and multi functionalization propertyCan be designed for targeted delivery and controlled release of drugsPhotothermal conversion potentialInherent adjuvant potentialUsage in imaging techniquesHigh binding affinity with wide range of moleculesHigher surface area to volume ratioLarge surface energy and chargeDisadvantages/LimitationsNon-biodegradableNon-porousLimited penetration depthAltered biodistribution profile upon surface modificationSurface functionalization-mediated toxicity and pharmacokinetics issuesLimited knowledge of impact on multiple cell typesClearance by macrophage phagocytosis system and renal pathwayAccumulation in cellular organelles such as mitochondria, lysosomes, etc., hampering normal cellular metabolism and ROS productionThe properties of gold nanoparticles and the ease of their usage and functionalization make them attractive to researchers. Attaching the isotopes or fluorochrome tags or optical probes with gold nanoparticles and targeting them to specific cells by attaching the antibodies or targeting ligands remarkably helped the advancement of optical imaging techniques, as well. Another success of using GNPs has been achieved in obtaining the antibodies for immunological identification of different pathogens in biosensor or microscopic methods. GNP antigen-conjugated vaccines are reported to protect animals from a lethal dose of virulent challenge with a 100% survival rate .There are certain concerns regarding the role of GNPs in the inhibition of Th1 response, which is crucial in combating many pathogenic infections. Only one study reported the enhancement of Th1 as well as Th17 immune response; the authors used the Listeria antigen along with the combination of Advax and 25 nm GNP adjuvants . Most ofTo improve prospects regarding the use of GNPs in vaccine development and in clinical settings, there is a pressing need to address certain issues. Firstly, there must be a large-scale production setup for GNPs with a high level of consistency. As we have addressed before, multiple variable factors such as charge, size, and shape all impact the cells in different ways. Thus, we need to be clear and cautious about each change that we are implementing. We have also noticed that most labs are working with GNP sizes ranging from 15 to 50 nm. Moreover, nanoshell-structured GNPs are most often used rather than other shapes such as nanocage, nanorods, nanocubes, etc. Therefore, we lack knowledge of the GNPs sized or shaped differently than as mentioned.Secondly, it is important to characterize with better clarity the GNPs’ impact upon interaction with immune cells. We believe a detailed investigation of immune cells in the presence of functionalized or empty GNPs will be helpful for answering various questions. Third, the biodistribution of the GNP must be evaluated in further detail, with special emphasis on the off-target cells and organs. Most studies highlight only the organs or cells or the disease pathogen and do not consider the possible accumulation of the nanomaterial in other organs. This leads to our fourth concern: nanotoxicology. As already discussed, the GNP is a non-biodegradable substance; thus, there may be a need to develop a synthesis or tagging method that can make it less toxic or better suited for clearance.Fifth, the protein coating formed outside the GNP surface upon in vivo introduction  is a problematic factor for the efficacy of the conjugated antigens on its surface. This bio-corona formation blocks the coating antigens and materials from interacting with in vivo cellular physiology. Sixth, as there is no specific dosage yet, the problem remains of replicating the success observed in animal experiments in early clinical trials. The standardization and normalization of the dose must be established.Seventh, a key important issue that needs to be addressed is how to fashion the GNP so that it can evade clearance by MPS or by renal excretion before its intended action. Studies have suggested coating GNPs with PEG, polyvinyl alcohol, poly (acrylic acid), or biomolecules such as glutathione or albumin to prevent bio-corona formation and MPS-based clearance and provide stability with relative less off-target toxicity ,141,142.Future work should address the impact of the combination strategies with GNP-based delivery along with photothermal therapy. It would also be interesting to explore the optical properties of GNPs in combination with thermal therapy in inflammatory responses. We expect that although some of the GNP-based nanoformulations discussed in this review might be translated into clinical settings, it is vital to address the multiple challenges associated with GNPs. Therefore, of paramount importance are the balanced testing and validation of their safety before establishing them in biomedical applications.


Canine melanoma is one of the most studied tumor of the dog, due to its high aggressiveness, especially in its mucosal  form, but also because its similarity with human melanoma. Canine and human mucosal melanoma show several common characteristics, in terms of histological features, biological behavior and genetic modifications –3. In boWe include in the following paragraphs a brief summary of the main findings reported by different authors in their 12 manuscripts that form the bulk of this Research Topic.Tarone et al.. A brief introduction on the importance of extracellular vesicles as valuable circulating carriers of easily accessible cancer biomarkers is also included, to discuss the potential of these novel approaches to improve the diagnosis and prognosis of canine MM.A perspective article is included in the present Research Topic, where authors underlined the relevance of translational research performed in last years on canine melanoma. In the paper, the most relevant knowledge on the tumor landscape of canine melanoma is reported. Furthermore, starting on the observation of the great achievements by immunotherapy in the treatment of human melanoma patients, the most promising immunotherapeutic approaches recently investigated, such as anti-cancer vaccination, are reviewed and critically discussed in the paper of Fonseca-Alves et al.). The success of this meeting, with more than 100 attendees, testifies the attention and interest on this Research Topic of the Brazilian oncology community.The current status of canine melanoma treatment and diagnostics in Brazil, reported from a “Colloquium on Canine Melanoma,” a meeting organized by the Brazilian Association of Veterinary Oncology (ABROVET) on December 2020, has been summarized in an opinion paper . In their study, Tsoi et al. investigated the IHC and RNA expression patterns of a set of additional markers, besides the well-known melanocyte markers Melan A, PNL2 and TRP-1 and−2, to investigate their potential usefulness for the challenging differentiation of spindloid oral MMs from soft tissue sarcomas (STS), when junctional activity and pigmentation are absent. Authors found that TYR, CD34, and CALD1 were the most discriminatory genes in differentiating between oral MM and STS, and should be evaluated in suspected amelanotic OMMs. The gene expression of H2AFZ and survivin in formalin-fixed, paraffin-embedded canine melanoma samples has been related to malignant histological features and malignant behavior, indicating H2AFZ as a new potentially useful prognostic biomarker and confirming survivin as a useful prognostic biomarker when evaluated also at the RNA levels . Leukotriene A4 hydrolase (LTA4H), and Fragile-X-mental retardation-related protein1 (FXR1), both reported as related to metastatic potential in different tumors, appeared to be highly expressed in oral melanoma, at both protein and mRNA level, but did not correlate to known histologic and immunohistochemical prognostic markers. However, their extensive expression in the investigated cases suggests that they may play a role in canine oral melanoma oncogenesis . Silveira et al. demonstrated COX-2 overexpression in both oral and cutaneous melanomas and observed that COX-2 expression correlates with established markers of poor prognosis. Porcellato et al. investigated the presence and significance of tumor-associated macrophages in canine melanocytic tumors, through the use of several IHC biomarkers , revealing an association of these markers with negative prognostic histologic features and a more aggressive biological behavior.A total of six articles that deal with the evaluation of new diagnostic and prognostic markers for canine MM have been published. Some of them have been already reviewed and included in the recently published review paper “g Group” . Global Studies on experimental therapeutic approaches exploiting immune-, gene- or targeted therapy were received and included in this Research Topic, showing interesting and encouraging results.in vitro models of canine melanoma. Silveira et al., by silencing Cox-2 in two melanoma cell lines, demonstrated that cellular proliferation, migration and invasion are COX-2 dependent, establishing that COX-2 is an essential driver of cellular proliferation, migration and invasion, and its expression correlated with malignant behavior in canine melanoma. Alpha-connexin carboxyl-terminal peptide (aCT1) and Bowman-birk protease inhibitor (BBI) treatment, alone or in combination, targeting Cx43, were investigated by Sato et al. on canine oral melanoma cell viability. Authors found that this dual treatment can be combined to achieve anticancer activity. The possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor  was investigated by Bongiovanni et al. as a potential anti-cancer therapy on four different canine oral melanoma cell lines. The selected drug was effective on three of the four tested cell lines, including a metastatic melanoma-derived one, indicating that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma.More than one group of researchers is currently engaged in the study and discovery of new targets for the development of new therapeutic approaches by the use of Sparger et al., revealing a different and unique T cell phenotype of both unstimulated and stimulated T cell populations in melanoma patients compared to healthy dogs. Takeuchi et al. demonstrated that the immunosuppressive cytokine transforming growth factor beta 1 (TGF-β1) is upregulated in vitro and in vivo in oral malignant melanoma and that its suppressive effect could be reverted in vitro by a decoy receptor, developed by the authors, exploring the potential application of this receptor in biologic therapy.The immunologic components of the immune environment and the mechanisms of evasion of the immune response by melanoma cells have been investigated in several papers on this Research Topic. The different T cell phenotypes and functions in melanoma tumor tissue and PBMCs of melanoma-affected patients were evaluated by Saellström et al. reported results on local AdCD40L treatment in dogs with different types of melanoma , indicating that this type of therapy is safe and could have beneficial effects in canine patients and potential valuable clinical translation to human patients.Included in the present Research Topic is also a study investigating the approach of gene therapy using adenoviruses encoding the immunomodulatory gene CD40L (AdCD40L). This strategy has already shown promise in clinical trials on human melanoma patients. Altogether, these manuscripts present new insights on the current researches on canine melanoma with the specific aim to improve our current diagnostic and therapeutic approaches to this highly aggressive and lethal disease of the dog, with a high translational potential to human mucosal melanoma.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


This Research Topic on skeletal muscle metabolism alterations in diseases associated with cachexia attempts to further elucidate how newly discovered molecular targets can influence muscle tissue structure and function. The skeletal apparatus, which accounts for approximately 30%–40% of total body weight , plays aIn this Research Topic, we collected four papers, three of which focus on cancer-derived cachexia and examine the role of several mitochondrial targets in influencing the metabolic profile during cancer progression.Mao et al. demonstrated that cachectic cancer patients exhibit reduced myofiber area, enlarged mitochondria with aberrant morphology and evidence of altered mitochondrial dynamics. Specifically, they observed upregulation of phosphorylated dynamin-related protein 1 (DRP1) at the Ser616 site, associated with markers of increased mitochondrial fission and consequent active fragmentation. The authors examined the relationship between DRP1, muscle degradation, and mitochondria using an in vitro model of cancer cachexia obtained by treating C2C12 myotubes with a conditioned C26 cell medium. They observed that inhibition of mitochondrial fission with Mdivi-1, a specific DRP1 inhibitor, counteracts muscle wasting by reducing protein degradation and improving mitochondrial function. While DRP1 regulates mitochondrial fission under normal physiological conditions, its overexpression in cancer patients with cachexia leads to promote myocellular mitochondrial dysfunction and myotube wasting.In an elegant original article originating from patient samples and functionally interrogated in cultured murine myotubes, Pin et al. The authors examined the effects of Mitoquinone Q (MitoQ), one of the most widely used antioxidants for mitochondria, on skeletal muscle wasting and metabolism in vitro and in vivo. Specifically, they found that MitoQ protects myotubes from atrophy in vitro by preventing increases in Atrogin-1 and Murf1 gene expression. In addition, they described MitoQ as a tool to prevent mitochondrial changes and improve the cachectic phenotype in C26 tumor-bearing mice. Specifically, MitoQ administration partially corrected skeletal muscle atrophy in male CD2F1 mice inoculated with C26 tumor cells. The authors demonstrated that MitoQ is an effective approach to improve mitochondrial function and metabolism by promoting a shift in fiber composition from glycolytic to oxidative, confirming the anticachectic properties of MitoQ in vivo. The authors suggest MitoQ could be administered concomitantly with chemotherapeutic agents routinely used in the clinic to counteract cancer-induced skeletal muscle atrophy. Whether MitoQ would be protective against chemotherapy-induced cachexia in the context of cancer or affect tumor response to chemotherapy remains to be determined in pre-clinical models.Mitochondrial alterations, as a target for cachexia therapy, are also the main object of original article by O’Connell et al. have developed an original article in an experimental model, focused on identifying molecular targets of cancer cachexia in the early stages with the goal of preventing refractory phase, which is characterized by patients becoming unresponsive to therapies and exhibiting uncontrolled weight loss. The unique feature of this study is the multiplatform strategy used  to profile progressive changes in the metabolome across the time course in C26 tumor-bearing mice. The authors found that the tumor progression induces significant differences in circulating amino acids, acylcarnitines, and lipoproteins prior to evident weight loss. These findings suggest that for patients, specific plasma metabolite biomarkers could serve as diagnostic predictors of cancer cachexia development or progression. Such a validated biomarker panel in patients would greatly facilitate drug development by identifying patients at risk of developing cachexia and monitoring response to therapy.The discovery of novel metabolic biomarkers involved in the onset and progression of cachexia is necessary to develop new therapies to treat patients and improve disease outcomes. In this context, Wen et al. addresses potential mechanisms driving the loss of skeletal muscle tissue in patients exposed to long-term controlled mechanical ventilation (CMV) in Intensive Care Units (ICUs). Specifically, CMV results in severe cachectic phenotype with impaired strength and movement, as well as alterations in other important skeletal muscle functions such as secretion of hormones, formation of metabolites, and reserve of amino acids. The authors conducted an in vivo study using Sprague-Dawley rats exposed to CMV for 5 days and then performed a metabolomic analysis of two respiratory muscles  and lung tissue. The authors showed a dramatic change in the metabolomic profile of lipids (Acyl Carnitine) and amino acids  with a specific signature for each tissue associated with increased protein breakdown in the two respiratory muscles, active inflammation in all tissues, attenuated energy production in the muscles, and enhance energy production in the lungs. This provides a pre-clinical basis for future studies in patients and for the ultimate discovery of biomarkers for early diagnosis to prevent the risk of secondary pulmonary complications and mortality.The original article by In conclusion, the articles published in this Research Topic illustrate novel insights and highlight the role of skeletal muscle metabolism as a potential candidate for exploring innovative therapeutic approaches for the treatment of diseases associated with cachexia.


Brain tumours are the most common solid tumour in children and the leading cause of cancer related death in children. Current treatments include surgery, chemotherapy and radiotherapy. The need for aggressive treatment means many survivors are left with permanent severe disability, physical, intellectual and social. Recent progress in immunotherapy, including genetically engineered T cells with chimeric antigen receptors (CARs) for treating cancer, may provide new avenues to improved outcomes for patients with paediatric brain cancer. In this review we discuss advances in CAR T cell immunotherapy, the major CAR T cell targets that are in clinical and pre-clinical development with a focus on paediatric brain tumours, the paediatric brain tumour microenvironment and strategies used to improve CAR T cell therapy for paediatric tumours. The mai domains . The bin domains . The co- domains .The application of CAR T cell therapy to treat CNS tumours is an emerging field. The challenges are many, including identifying suitable antigens expressed at sufficient levels and which are tumour specific or enriched, generating effective CARs, and determining the most effective methods of delivery across the blood-brain barrier. However, early reports of favourable individual responses have forged the way for significant developments in CAR T technology and suggest that many, if not all, of these challenges can be met , 9, 10. The predominant clinical experience using CAR T cell therapy for central nervous system cancer, has focused on glioblastoma (GBM), which is rare in children but the commonest primary brain cancer in adults, and one that is invariably lethal . TherefoThe rationale for CAR T targeting of IL13Rα2 has been reviewed elsewhere , 19, butGangliosides are glycolipids widely expressed, so are unlikley to be useful antigens for CAR T targeting. However, the disialoganglioside (GD2) has more restricted neuroectodermal expression, and is highly expressed in several cancers. From the paediatric cancer perspective, this was first reported in neuroblastoma, where a monclonal antibody generated against the LAN-1 neuroblastoma cell line was shown to recognise GD2 in neuroblastoma patient samples . ClinicaThe Human epidermal growth factor receptor (HER) family is comprised of four transmembrane growth factor receptors; EGFR (HER1), ERBB2 (HER2), ERBB3 (HER3) and ERBB4 (HER4). HER2 is an established immunotherapeutic target in breast and ovarian cancer. In paediatric maligancies, HER2 alterations – principally copy number gains and gene over expression - was observed in 6% of all tumours in a paediatric pan-cancer cohort, predominatly in CNS tumours including Diffuse Hemishperic Gliomas (DHG), medulloblastoma, ependymoma, and a subset of neuroblastoma .A phase I trial of HER2 specific CAR T cells in patients with HER2-positive GBM (NCT01109095) concluded that the therapy was safe, although the mean age of this cohort was 49 years, and only 6 paediatric patients were included. Of the 16 cases, eight showed an objective response, including one partial response (tumour regression) and seven patients with stable disease . Whilst + brain tumours, over the age of 3 years (NCT02442297). The delivery method, as for the IL13Rα2 studies, was either by direct injection into the tumour resection cavity or by intraventricular delivery. An ongoing trial currently recruiting patients between the ages of 1 and 26 with HER2+ brain tumours, will test HER2 CAR T cell therapy, delivered via an indwelling catheter in the tumour resection cavity or the ventricles (NCT03500991). Interim analysis of the first three patients, one with localised anaplastic astrocytoma and two with metastatic ependymoma, confirmed that treatment was feasible without dose-limiting toxicity, and that a local immune response was activated by the CAR T cells. Two patients progressed shortly after initiation of treatment  is another ErbB family member  being taSMARCB1  is, in a cancer context, an immune checkpoint factor which may dampen the adaptive immune response to tumours. There is considerable interest in the possibility that blocking monoclonal antibodies directed agains B7-H3 could enhance CD8+ and NK cell infiltration into tumours . InteresSMARCB1 , 38, alsSMARCB1 . B7-H3 CSMARCB1 . The curFactors common to all CAR T cell therapies may determine their efficacy, such as the antigen load and specificity in the target cells, the proliferative capacity of the CAR T cells, their persistence post delivery, the biological features of the transduced T cells and the nature of co-stimulatory signals included with the CAR , 40–43. RM) were observed deep within tumour cell-rich regions  is an inflammatory condition arising as a consequence of the activation of the infused CAR T by its target antigen. The inflammatory consequences include systemic symptoms such as fever, myalgia and rigors but may also prgress to more serious consequences include capilliary leak syndrome and consequent hypotension, circulatory collapse, and end-organ failure including neurotoxicity . Of the Beyond the identification of target antigens in paediatric brain tumours and the development of effective CARs, there are important therapeutic considerations to ensure effective trafficking of CAR T cells to the brain, either by direct inoculation to the tumour site or with the use of targeted combination therapy to overcome the immunosuppressive brain tumour microenvironment. Some of these therapeutic strategies are discussed below.Several strategies have been used to deliver CAR T cells across the BBB, most prominently by direct introduction into the tumour resection cavity or into the ventricular system of the brain.The direct innoculation of CAR T to the tumour site is an obvious strategy to bypass tumour-intrinsic or anatomical barriers, including the BBB, and immunosppressive niches which restrict traffiking of CAR T within tumours following systemic adminstration . MoreoveVascular endothelial growth factor A (VEGFA) is a proangiogenic growth factor that functions as a ligand for VEGF Receptor 2 (VEGFR2). Blocking VEGFA antibodies also have an immunomodulatory effect. Stickingly, VEGFA-blocking antibodies significantly delayed tumour progression in a melanoma model (B16 cells) when combined with a CAR T engineered to recognize a tumour specific antigen. Notably, without the CAR T, the antibody had no effect on tumour progression, suggesting the effect was not solely the result of an antiangiogenic effect . MoreoveThere is interest in understanding whether existing drugs which inhibit immuno-suppresive checkpoints can facilitate the entry of CAR T into CNS tumours. Checkpoint inhibitors have had virtually no clinical impact in paediatric maligancies with the sole, but important, exception of hypermutated cancers arising in the context of germline mutations of mismatch repair genes (Mismatch Repair Deficiency – MMRD), with or without Polymerase Proofreading deficiency (PPD). In chiildhood, a significant proportion of such patients present with gliomas . These tImmunotherapy in cancer treatment is having a renaissance, with checkpoint inhibitor therapy for melanoma and lung cancer, and CAR T cell therapy for B-cell malignancies showing promising results , 85, 86.All authors have contributed to the conception and writing of this review. All authors have read and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.PE acknowledges the support of Perpetual Trustees and the Samuel Nissen Foundation, as well as the Steven Walter Children’s Cancer Foundation. This research was supported by an Australian Government Research Training Program (RTP) Scholarship (CM). The Medical Research Future Fund through the Emerging Priorities and Consumer Driven Research scheme and the Australian Brain Cancer Mission/National Health & Medical Research Council/Lifting Clinical Trials and Registry Capacity (NHMRC MRF9500002) for salary support (CM).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A group of experts in dermatology, genetics, neuroscience, and regenerative medicine collaborated to summarize current knowledge on the defined factors contributing to cutaneous neurofibroma (cNF) development and to provide consensus recommendations for future research priorities to gain an improved understanding of the biology of cNF.The group members reviewed published and unpublished data on cNF and related diseases via literature search, defined a set of key topic areas deemed critical in cNF pathogenesis, and developed recommendations in a series of consensus meetings.Five specific topic areas were identified as being relevant to providing an enhanced understanding of the biology of cNF: (1) defining the human cells of origin; (2) understanding the role of the microenvironment, focusing on neurons, mast cells, and fibroblasts; (3) defining the genetic and molecular differences between the cNFs, focusing on size and number; (4) understanding if sex hormones are critical for cNF development or progression; and (5) identifying challenges in establishing in vitro and in vivo models representing human cNF.The complexity of cNF biology stems from its heterogeneity at multiple levels including genetic, spatial involvement, temporal development, and cellular composition. We propose a unified working model for cNF that builds a framework to address the key questions about cNF that, when answered, will provide the necessary understanding of cNF biology to allow meaningful development of therapies. NF1 (Neurofibromin 1) gene takes place, such as melanocytes (café-au-lait macules [CALM]), osteoblasts , and Schwann cells (cutaneous neurofibroma [cNF] and plexiform neurofibroma [pNF]). Importantly, the 2 main types of neurofibroma (cNF and pNF) differ widely clinically. cNF typically become apparent around puberty and increase in number with age. They reside exclusively in the dermis and never progress to malignancy. In contrast, pNF are often congenital, and progress predominantly during childhood and adolescence. Although pNF can involve the skin, they are generally deep tumors involving nerve plexuses below the dermis and they carry a risk of sarcomatous transformation. Despite knowing that the NF1 syndrome and its manifestations are caused by mutation of the NF1 tumor suppressor gene, there is little understanding about why the multiple manifestations of NF1 occur in some people but not others and with variable severity, even within families.The clinical spectrum of neurofibromatosis type 1 (NF1) is broad and dependent on in which cell type the biallelic inactivation of the NF1 in the Schwann cell lineage. cNF can vary widely in size (from millimeters up to few centimeters) and in numbers (from a few to thousands that can cover most of the skin surface) and can develop in virtually any location of the body. In addition to the important psychosocial consequence of cNF tumors, irritation, bleeding, pain, and superficial infections are unmet clinical needs. There are no preventative measures, and the only treatment options are surgical.The most common tumor in adults with NF1 is cNF, presumably resulting from the biallelic loss of NF1 gene and its mRNA are relatively large1 and difficult to manipulate into expression vectors. Therefore, the biochemistry of the different protein domains of NF1 (except for the GTPase-activating protein [GAP] domain) is not well-understood.2 Second, cNF are slow-growing, noncancerous tumors. Consequently, isolating and maintaining primary cultures from cNF is difficult. Third, the generation of robust 3D in vitro culture systems3 and in vivo models4 are notoriously challenging. In an effort to address these gaps, based on existing data from both cNF and pNF literature, this group sought to identify the key gaps in the understanding of cNF biology that require investigation in order to accelerate the development of effective therapies for these tumors.Researchers interested in understanding cNF biology face multiple critical challenges. First, the human A literature review was conducted including the terms NF1, cNF, dermal neurofibroma, pNF, genotype, phenotype, mast cell, macrophage, fibroblast, fibrosis, hormones, development, and animal models in an effort to identify all published literature that may be relevant to the general topics of cNF initiation, development, and progression. The working group was composed of experts in Schwann cell biology, genetics, dermatology, stem cell biology, fibrosis, regenerative medicine, and NF1 clinical care. Working group members reviewed published and unpublished data around the set of topic areas individually and as a group during a series of meetings facilitated over a 4-month period, prioritized key questions, and established consensus recommendations for each topic area.NF1 biallelic inactivation, regardless of the timing of this event. Identification and isolation of the human cells of origin would allow reconstruction of the biological steps leading to tumor initiation and progression ultimately leading to identification of targets that may be susceptible to therapeutic intervention.Theoretically, the cell of origin is the cell that first undergoes 5 proposed that cNF originate from human hair follicle stem cell–like neurofibroma precursor cells. However, patients with NF1 also develop cNF on the palms and soles where there are no hair follicles (4 observed pNF when Nf1−/− skin-derived precursor cells (SKPs), a population of neural crest–derived progenitors in the murine dermis, were implanted into the injured sciatic nerve of immune compromised mice. However, cNFs only developed when these cells were implanted in the skin of hormone-primed recipients or in a topically inducible model deleting Nf1 locally in the skin.4 Of note, biallelic inactivation of Nf1 in pNF models does not robustly lead to cNF, supporting a hypothesis that the cells of origin of cNF and pNF are different. Interestingly, Gresset et al.6 reported that the Schwann cells of the dermal nerve endings have a different origin compared to the majority of the other Schwann cells. Intriguingly, these neural crest–derived stem cells are reminiscent of the SKPs7 and suggest that they may be the embryonic source of the cNF cell of origin. In this sense, identifying mouse Cre lines that are expressed in the subpopulation of SKPs that give rise to cNF will be instrumental to our understanding of cNF biology. Other indirect evidence supporting the differential origin of cNF and pNF comes from experiments aiming to elucidate the contribution of skin or nerve trauma in the development of neurofibroma. Strikingly, cNF was never observed in an injured sciatic nerve–dependent pNF mouse model where deep skin incision was performed. This suggests that wounded small nerve endings do not behave like the sciatic nerve in their ability to promote neurofibroma. Directly assessing the effect of dermal nerve injury in existing4 or novel cNF mouse models will help clarify the role of injury in cNF formation. Importantly, the cell of origin of cNF may encompass melanocytes as skin hyperpigmentation completely covers some cNF (diffuse cNF). Some cNF present with redness or purplish hue, likely due to tumor vascularization  and are highly disorganized. Current data suggest that the most important cellular components for development and maintenance of cNF are the nerve, immune cells , and fibroblasts.8 Adameyko et al.9 described the migration of Schwann cell precursors (SCPs) along growing nerves, and demonstrated that the association with nerves influences the fate of the precursor cells. In the context of neurofibroma, Liao et al.3 demonstrated that murine Nf1-deficient SKPs more readily give rise to pNF when injected into nerve tissue, but they did not develop pNF when injected into non-nerve tissue. Further work is needed to clarify if human cNF may also require contact with nerves or factors such as Neuregulin 1 (NRG1) in the perineurial microenvironment for their development and maintenance ,14 explaining a second hallmark of human pNF: abundant collagen deposition.11 However, it is not clear to what extent these data can be extrapolated to human cNF. Clinical application of these findings via a Kit receptor inhibitor or mast cell stabilizer showed only modest results in clinic for pNF.16 One possibility is that the pNF mouse model is more dependent on the Schwann cell–mast cell axis than human pNF. Also, these models did not account for macrophages that are commonly found in cNF. Although infiltration of macrophages correlates with tumor progression, their exact role in neurofibroma tumorigenesis is not well-understood.17The immune cells that contribute to cNF are mast cells and macrophages. Mast cells are histamine-secreting granulocytic cells known for their involvement in allergic reactions. Mast cell infiltration is one of the histologic hallmarks of neurofibroma.ignaling .11 Speci18 Upon skin injury, the normal wound repair process includes inflammation followed by fibroblast proliferation, re-epithelization, and ECM remodeling. Fibrosis, defined as formation of excess connective tissue in an organ, occurs during the second phase of healing. High levels of collagen deposition, as well as the expression level of key components of the TGF-β pathway in activated fibroblasts, are surrogates often used to quantify the extent of fibrosis. No treatment is currently able to reverse fibrosis, but drugs inhibiting the TGF-β pathway are under investigation for this purpose.19Finally, fibroblasts are nonepithelial, noninflammatory, and nonvascular cells of the connective tissue responsible for ECM deposition and reorganization.20 However, initial attempts to verify the role of fibroblasts in human pNF by pharmacologically targeting fibroblast-producing collagen have had limited success.22 One explanation is that NAFs may not be the typical activated fibroblast cell characterized in major organ fibrosis and cancer contexts. Indeed, NAFs do not express fibroblast markers observed in other cutaneous disorders like dermatofibrosarcoma protuberans23 or keloid,24 and are negative for smooth muscle actin, a classic marker for activated fibroblasts.25 Alternatively, collagen-producing fibroblasts may not be functionally equivalent in cNF and pNF. Overall, multiple questions remain about the role of NAFs in cNF pathogenesis.Neurofibroma-associated fibroblasts (NAFs) are abundant in cNF, where up to 50% of human cNF dry weight is collagen and NAFs display abnormal collagen deposition, all hallmarks reminiscent of fibrosis.27 Indeed, nerve injury initially attracts immune cells such as macrophages 28 followed by mast cells (likely to increase capillary permeability).29 Specifically in the context of the peripheral nerve system, proliferating fibroblasts and Schwann cells are aligned in an ordered column  to bridge the gap between the distal and proximal nerve, allowing guidance for axonal regrowth.30 Finally, the ECM is slowly remodeled to eventually reach normal architecture. In the context of neurofibroma, one can envision that the high number and proliferative status of Schwann cells triggered by nerve injury and loss of axonal contact would significantly increase the likelihood of biallelic NF1 inactivation. How the ECM produced by fibroblasts and the recruited immune cells initially aiming at healing the nerve would then turn into a fibrotic microenvironment preventing the nerve to heal is currently unknown. What is increasingly clear is that for the best chance of effective therapeutics, prior to investigating treatments targeting various elements of the cNF microenvironment, a better understanding of the relative contributions of each of these various cells to tumorigenesis is needed. This will entail (1) identifying specific markers for each cell type described and their relative number and function in various phenotypes of cNFs31 and (2) the development of reliable in vitro and in vivo model systems that recapitulate human cNF progression to allow omission of specific cell types and enable pharmacologic/genetic interventions.6An interesting theory that bridges the hypotheses of fibrosis, neurofibroma formation, and a hyperactive immune response is that neurofibromas may develop in the setting of trauma.32 Of note, keratinocytes, melanocytes, and other cells from the skin structures  can be found at the margin of cNF but not within the tumor bulk. Finally, it is possible, but uncommon, to find abundant adipocytes or fat-like cells in neurofibromas.33Other cell types in the microenvironment of cNF include pericytes, and cellular components comprising the nerve perineurial barrier, but their role is unclear.NF1 gene mutations that result in a very low incidence of cNF or pNF  and the third results in a higher likelihood of early development and higher number of cNF (large NF1 gene deletions).36 It would be highly informative to validate the biochemical and biological consequences of mutated neurofibromin to better understand how these first 2 mutations “suppress” cNF development. Specifically, how do these changes alter 3D protein structure, influence sites for binding partners, or affect subcellular localization of neurofibromin? Even more intriguing is that this mild cutaneous phenotype is indistinguishable from Legius syndrome, which is caused by mutations in the Ras signaling regulator SPRED1,38 suggesting the major influence of this mutation is via the modulation of the Ras signaling pathway in melanocytes and other cells, but not significantly in Schwann cells. Further, these 2 mutations do not directly implicate the GAP domain of neurofibromin, so it is unclear how mutations at R1809 or c.2970-2972delAAT would affect the Ras signaling inhibitory capacity of neurofibromin. Elucidating the mechanism behind the lack of development of cNF in patients with mutations in NF1 R1809 or NF1 c.2970-2972delAAT could be groundbreaking for new therapeutic approaches for cNF.There is extensive interpatient and intrapatient heterogeneity in the presentation, degree of severity, and behavior over time of cNF. Efforts at defining genotype–phenotype correlations in NF1 in general and relative to cNF have largely been fruitless, with 3 notable exceptions. The first 2 exceptions are specific NF1 gene, which is associated with a high number of cNF and an increased risk for malignant peripheral nerve sheath tumor (MPNST). The genetically linked SUZ12 gene has been identified as an important modifier of NF1 in MPNST.39 It is unknown if SUZ12 or any other gene that is commonly inactivated with NF1 in the setting of these large gene deletions plays a role in determining the number, size, or timing of onset of cNF.In sharp contrast, around 5%–10% of NF1 patients have a large deletion encompassing the NF1 DNA mutations, the predicted functional consequence at the RNA and protein levels is unknown, contributing to the lack of genotype–phenotype correlation. By systematically annotating the RNA and protein effects due to different NF1 mutations in tissues and cells from NF1 patients,40–42 one can begin grouping together apparently unrelated mutations.44 An even better approach is systematic functional profiling of mutant neurofibromin. Scoring the effect on Ras signaling may help reclassify discordant results, generating new hypotheses about the role of neurofibromin outside its GAP domain and new mechanistic insights for cNF development.Outside of these examples, for the majority of NF1 are equivalent. In support of the latter hypothesis, the mutational landscape of cNF across 40 tumors from 11 patients revealed that the nature of the second hit mutation in NF1 is different in each tumor.45 It is currently unknown if there are different forms of cNF or if there are relatively few types of cNF that appear phenotypically distinct because they are observed at various stages along a growth continuum.46 A challenge is that cNF may not be visible at early stages and some patients report that cNF erupt over very short intervals. Hence, researchers investigating factors that explain the heterogeneity of cNF should be mindful that we do not yet have an adequate understanding of the natural history of cNF in humans.There is often wide variation of cNF tumor size and concentration on patients' bodies, suggesting that either tumor growth is under the influence of unidentified stochastic modifiers or not all somatic mutations (the second hit mutation) leading to biallelic loss of 46 One of the more pervasive hypotheses is that pregnancy or other periods of systemic hormonal shifts stimulate cNF appearance and growth. Dugoff and Sujansky47 retrospectively surveyed 105 women with NF1 who had a history of at least one pregnancy to assess perceived change in number or size of cNFs during prior pregnancies. More than half (64/105) of the women reported that new cNFs developed during pregnancy, but multiparous women did not have new tumors develop during all pregnancies. Lammert et al.48 distributed a survey to 59 women with NF1 who had used hormone contraceptives to evaluate the effect of exogenous estrogen and progesterone on the development or proliferation of cNFs while on these drugs. The majority (53/58) of women reported no change in cNFs, but 5 reported new cNFs and growth of existing ones. Although interesting, these studies are inconclusive as they suffer from their retrospective and subjective nature. Notably, Schwann cells derived from human neurofibromas express progesterone receptors, and they have elevated proliferation rates when exposed to progesterone in vitro.49 Further, Le et al.4 showed that implantation of Nf1−/− SKPs resulted in development of cNFs only in pregnant CMV-CreERT2;Nf1f/− mice, adding preclinical data to the working hypothesis of an association between sex hormone exposure and cNF development in female patients. However, Sbidian et al.50 analyzed prospective data collected between 2002 and 2005 from adults (mean age 36 ± 14 years) with NF1 to assess difference in growth and number of cNF between sexes and between nulliparous and parous women. Men were at higher risk of developing subcutaneous neurofibromas regardless of age, but there was no difference in the number of cNFs between men and women after the age of 40, regardless of the parous status of the women. Overall these data indicate that there may be a role of sex hormones in development of cNF; however, they are not causative or even dominant. Prospective epidemiologic case–control studies that address different phases of hormonal changes in women with concurrent assays of systemic hormonal levels  are important for understanding of the role of hormones at different phases of cNF development clinically.There have been many proposed factors that influence the development and growth of cNF.NF1 loss. Tumor development should be driven by specific changes in known pathways involved in human cNF, and tumor gene expression profiles should resemble those of human cNF. Second, biologically, cNFs develop through multistage progression . The cNF models should initiate from a small group of cells of origin that harbor NF1 loss, and the tumor histology and pathology should be similar to human cNF. In addition, tumor development should require similar microenvironmental factors as in human cNF. Third, therapeutically, new cNF models should be robust for the preclinical testing of inhibitors directed at pathways critical for cNF development that can lead to clinical trials in humans. To better understand the role of the tumor microenvironment on the development and proliferation of cNF, future studies should identify what extrinsic signals regulate cNF formation, which includes understanding the cell source of these signals, the timing of these signals, if biallelic inactivation in the cells is important, and elucidating the role of soluble signaling molecules, ECM, and cell contact.Developing cNF models will be critical to further elucidate their biology and to serve as preclinical models for therapeutic testing. However, there are many challenges in establishing in vitro and in vivo models of human cNF. First, genetically, cNF initiation and progression must associate with NF1/Nf1 is an early and necessary step in the development of neurofibromas. However, we do not know what further triggers or modifies neurofibroma growth to yield the wide range of intrapatient and interpatient variability that is seen. Assuming different cellular origins for the different phenotypic manifestations of the disease, it is possible that the different thresholds for functional NF1 from one cell type to another explain the variable phenotypes observed: melanocytes  < cNF cell of origin (cNF) < Schwann cell lineage (pNF). This hypothesis would explain why even mildly affected patients with NF1 have CALMS, why patients with severe pNF almost always carry CALMS and cNF, and why the phenotypic manifestations have different temporal onset. Each NF1 DNA mutation potentially has more or less consequence because it leaves neurofibromin with more or less residual function and some cell types are more dependent on NF1 function than others. Importantly, this theory does not exclude a role for the tumor microenvironment and more study is needed here.Analysis of NF1 patient tumors and mouse models has led to the conclusion that the loss of function of NF1 genotype and modifiers, cellular composition, and the effect of tumor microenvironment, as well as the effects of sex hormones. A top priority is to continue to gather large repositories of NF1-driven neurofibromas with annotated clinical data so that correlations can be delineated. Finally, analysis of these biospecimens should be expanded by including transcriptomic, proteomic, and epigenetic profiling through unified collaborative approaches to accelerate discovery most efficiently.As we progress in our quest to decipher the biology of cNF, it remains important to compare and contrast cNF and pNF in terms of cellular origin,