Primary immunodeficiency diseases (PIDs) are rare diseases that are characterized by genetic mutations that damage immunological function, defense, or both. Some of these rare diseases are caused by aberrations in the normal development of natural killer cells (NKs) or affect their lytic synapse. The pathogenesis of these types of diseases as well as the processes underlying target recognition by human NK cells is not well understood. Utilizing induced pluripotent stem cells (iPSCs) will aid in the study of human disorders, especially in the PIDs with defects in NK cells for PID disease modeling. This, together with genome editing technology, makes it possible for us to facilitate the discovery of future therapeutics and/or cell therapy treatments for these patients, because, to date, the only curative treatment available in the most severe cases is hematopoietic stem cell transplantation (HSCT). Recent progress in gene editing technology using CRISPR/Cas9 has significantly increased our capability to precisely modify target sites in the human genome. Among the many tools available for us to study human PIDs, disease- and patient-specific iPSCs together with gene editing offer unique and exceptional methodologies to gain deeper and more thorough understanding of these diseases as well as develop possible alternative treatment strategies. In this review, we will discuss some immunodeficiency disorders affecting NK cell function, such as classical NK deficiencies (CNKD), functional NK deficiencies (FNKD), and PIDs with involving NK cells as well as strategies to model and correct these diseases for further study and possible avenues for future therapies. Primary immunodeficiency disorders (PIDs) are rare diseases caused by genetic mutations that damage immunological function, defense, or both. PIDs refer to over 130 disorders that result from developmental and/or functional defects in one or more cell types of the immune system. PIDs are generally classified as disorders of adaptive immunity  or innate immunity . Some of these diseases affect natural killer (NK) cells .NK cells are lymphocytes of the innate immune system poised to deliver a response immediately after recognizing specific signals of “danger,” stress, or foreign origin. NK cells are initially defined as rapid cell-mediated cytotoxicity cells, though they also initiate a slower receptor-mediated apoptosis, and efficiently produce soluble mediators, such as cytokines, and provide contact-dependent co-stimulation. Hence, NK cells contribute to the regulation of immune responses, in the surveillance of stress and cancer cells, and in the defense against infections . While tNK cell effector functions happen after ligation of germline-encoded receptors and involve the secretion of cytolytic molecules. These molecules are contained in pre-formed lytic granules of resting human NK cells. As a result, the cytolytic process needs to be well controlled and may involve additional or enhanced mechanisms for controlling the secretion of lytic granule contents .Several NK cell deficiencies have been identified as being present in PIDs, including some characterized by an impediment in their development (classic NK deficiencies or CNKD) or by an aberrant formation of the lytic synapse . To date, only one FNKD has been shown to impair only NK cell function. Many studies on how the lytic synapse is formed have been performed with T cells from patients with PIDs. Though cytotoxic lymphocytes have critical roles in host defense and immune regulation, NK cell deficiency can add to the clinical phenotypes .In this review, we will focus on the most relevant PIDs with established functional defects in NK cells, such as CNKD  with mutThe pathogenesis and processes underlying target recognition by human NK cells of all the PIDs mentioned before are still not well understood. For this reason, the use of induced pluripotent stem cells (iPSCs), as a disease modeling tool, will help the study of human NK cell disorders with the final goal of elucidating the disease mechanisms to find novel gene and cell therapies for the treatment of those PIDs.Prof. Yamanaka in 2006 made the pivotal finding that somatic cells can be reprogrammed to pluripotent cells by introducing a set of transcription factors, named induced pluripotent stem cells (iPSCs) . This noSubstantial improvement has also been made in the development of other strategies to accomplish reprogramming. Generation of iPSCs was initially based on using four retroviral vectors . RecentlOne of the first reports of a PID patient-derived iPSC line was from a patient with an adenosine deaminase (ADA) deficit, which causes severe combined immunodeficiency (ADA-SCID) . While nFurther studies with PID-mutation-corrected iPSCs are needed to determine whether they are capable of differentiating into any target cell type as well as to gain more thorough knowledge of the mechanisms behind specific mutations . AltogetDuring the DNA replication process, after experimental manipulation by using endonucleases or after exposure to chemotherapy or ionizing radiation, DNA damage can occur. Luckily, DNA repair mechanisms are able to repair this damage, thus avoiding DNA mutations that can develop into disease. This repair process is the basis of gene editing therapies, which follow two main repair pathways for double-strand breaks (DSBs) in the DNA: non-homologous end joining (NHEJ) and homologous recombination (HR) .In the 1980s, pioneering scientists Prof. Capecchi, Smithies, and Evens discovered the HR pathway that repaired genes in mammalian cells. Later in 2007, they were awarded the Nobel Prize in Medicine for their findings in introducing gene modifications in mice models by using embryonic stem cells and HR-mediated gene editing. A few years later, Prof. Jasin enhanced gene targeting in mammalian cells using HR from yeast endonuclease I-SceI by using meganucleases. Since that time, next-generation gene editing tools have been used such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which all edit DNA in combination with the FokI endonuclease .E. coli. This was a clustered regularly interspaced short palindromic repeats (CRISPR) array, which is now one of the most commonly used gene editing technologies. Later, in 2005, the molecular mechanism was revealed, which showed that CRISPR arrays are transcribed into RNA to cleave and load into CRISPR-associated (Cas) proteins (Cas9) [In the late 1980s, a bizarre topology at the 3′ end of the alkaline phosphatase gene was revealed in s (Cas9) .For many years, scientists have been looking for a tool to induce or repair mutations in a targeted manner. Several techniques have been used in the past, such as engineered meganucleases, ZFNs, and TALENs, all with limited success, due to the fact that they are labor-intensive, expensive, or both. CRISPR-based methodologies together with RNA-guided nuclease activity meant that, theoretically, DSBs in eukaryotes can be induced, which was very hard to achieve before. In general, DSBs are repaired by DNA repair pathways, with NHEJ having the potential to induce indels—mutations caused by random insertion or deletion of nucleotides at the DSB site, whereas the HR repair pathway is more precise .The differences between engineered meganucleases, ZFNs, TALENs, and CRISPR/Cas9 nucleases are described in detail in Fig. Engineered meganucleases are derived from a huge family of natural homing endonucleases [ucleases , and somucleases , 47. HisZinc-finger nucleases (ZFNs) are artificial restriction enzymes in which a DNA-cleavage domain from the enzyme FokI is fused to a zinc-finger DNA-binding domain [g domain , 49. Theg domain . Though g domain .TAL effector nucleases (TALENs) are artificial proteins with a similar structure to ZFNs with the fusion of the enzyme FokI nuclease domain to an engineered DNA-binding domain. This DNA-binding domain is engineered by gathering serial TAL repeats [ repeats . Each re repeats . Thus, gCRISPR/Cas9 nucleases  originate from the immune system of bacteria and archea [d archea . The sped archea . The CasGene editing technology is a powerful tool currently being used in basic research, but the ultimate aim is to translate these tools to be applied in therapeutic treatments. Being able to use gene editing technology in the clinic stems from the possibility of treating monogenic diseases by developing a novel method to correct the disease-associated mutation , 57. TheCertainly, the use of gene editing tools in patient-specific iPS cells will aid in the development of future treatments aimed at correcting the point mutations in PIDs with defects in NK cells.. Since then, HSCT has been the standard care for SCID patients with great survival rates [The first time HSCT was used as a therapeutic option for treating PID in a severe combined immunodeficiency (SCID) patient was in 1965 , as show4al rates –62. Howeal rates . In ordeal rates . As a coal rates  al rates , 68, 69,The triumph of gene therapy in treating PIDs is a major advancement, though limitations in manufacturing disease-specific vectors remain a challenge . As thisCompared to lentiviral vectors, gene-specific editing technologies has become a tremendously promising tool, as it has the potential to physiologically regulate gene expression and prevent genome-wide vector integration. Some of the ongoing efforts are focused on developing sensitive techniques to detect genotoxicity derived from unintended effects of endonucleases (off-target effects).In the case of CRISPR-Cas9 approaches for HSC genome editing in PIDs, the design of the donor template is challenging and both the nature  and the functional effect of the mutation (gain of function versus loss of function) have to be taken into consideration .+ HSCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD) using a chemically modified 100-bp ssODN that resulted in production of 15–20% functional mature human myeloid and lymphoid cells for up to 5 months.Short donor templates (such as ssODN or linear or plasmid dsDNA donors) have been used to correct loss of function (LOF) mutations of a single or few nucleotides. For example, De Ravin and colleagues  could reIn contrast to small mutations, repair of large deletions or insertions is not possible with short donor templates and instead functional complementary DNA (cDNA) templates are inserted to target genes. Encouraging preclinical studies have been published using this approach for the treatment of X-SCID or X-CGD –81 and wHowever, one limitation to consider for the application of gene editing tools in a clinical setting might be the engraftment efficiency and HSC functionality of genetically modified cells due to cellular effects of the gene editing machinery. Indeed, global gene expression changes have been observed upon delivery of CRISPR-Cas9 machinery components into the cells. Immune response to viral infection, DNA damage response, apoptosis, and cell cycle processes have been reported as the most significantly enriched gene signatures . The actApart from CRISPR-Cas9, other genome editing tools have been used to modify genes in different cell types including HSCs. ZFN and TALEN techniques have been used to modify the IL2RG locus, which is responsible for SCID .Specifically in the case of PID with NK cell defects, a good example of the evolution of treatment approaches is seen with WAS. The first clinical trial with gene therapy in WAS patients was performed using gamma-retroviral vectors. Even if 9 of 10 patients showed partial or complete resolution of immunodeficiency, autoimmunity, and other malignancies, 7 of them developed acute leukemia. This study demonstrated that gene therapy for WAS can be effective, although it was essential to find an alternative to gamma-retroviruses given the high risk of leukemia after months or years . More reIn summary, thanks to a better understanding of stem cell biology, bone marrow transplantation, vector design, and genome editing, it is probable that gene therapy will become the gold standard of care for certain diseases in the future. In fact, its benefits have already been demonstrated for WAS, ADA, SCID, and X-CGD. In addition, a number of preclinical studies using targeted gene editing strategies show promise –81, 88 aDuring recent years, the genome editing field, together with cell reprogramming field, has shown tremendous progress. Currently, the first clinical trials with iPSC-derived NK cells (FT500) and gene-edited somatic cells have started. Here, we described the advantages of modeling and correcting PIDs with gene editing technologies while avoiding the use of viral vectors. However, further refinement of the genome editing tools is necessary if they are to be used in a clinical setting for PID treatment. Specifically, off-target mutagenesis has to be examined and the yield of gene-corrected HSCs or other blood cells (NK cells) needs to be improved so that an adequate number of cells for autologous transplantation and engraftment can be achieved. In spite of these issues, the influence of genome editing and stem cell therapies on modern medicine will be revolutionary for the PID field.


Prevalence of childhood obesity is progressively increasing, reaching worldwide levels of 5.6% in girls and of 7.8% in boys. Several evidences showed that obesity is a major preventable risk factor and disease modifier of some respiratory conditions such as asthma and Obstructive Sleep Apnea Syndrome (OSAS). Co-occurrence of asthma and obesity may be due to common pathogenetic factors including exposure to air pollutants and tobacco smoking, Western diet, and low Vitamin D levels. Lung growth and dysanapsis phenomenon in asthmatic obese children play a role in impaired respiratory function which appears to be different than in adults. Genes involved in both asthma and obesity have been identified, though a gene-by-environment interaction has not been properly investigated yet. The identification of modifiable environmental factors influencing gene expression through epigenetic mechanisms may change the natural history of both diseases. Another important pediatric respiratory condition associated with obesity is Sleep-Disordered Breathing (SDB), especially Obstructive Sleep Apnea Syndrome (OSAS). OSAS and obesity are linked by a bidirectional causality, where the effects of one affect the other. The factors most involved in the association between OSAS and obesity are oxidative stress, systemic inflammation, and gut microbiota. In OSAS pathogenesis, obesity’s role appears to be mainly due to mechanical factors leading to an increase of respiratory work at night-time. However, a causal link between obesity-related inflammatory state and OSAS pathogenesis still needs to be properly confirmed. To prevent obesity and its complications, family education and precocious lifestyle changes are critical. A healthy diet may lead to an improved quality of life in obese children suffering from respiratory diseases. The present review aimed to investigate the links between obesity, asthma and OSAS, focusing on the available evidence and looking for future research fields. Obesity in childhood represents a major global health challenge  mainly pThe prevalence of obesity shows a worrying increase of epidemic proportions. In the United States, 40% of adults and 19% of children have a condition of obesity .The 2018 Global Nutrition Report showed that 40 million children below 5 years of age and 330 million patients aged 5–19 years were overweight or obese in 2016 [Obesity is associated with several complications . It is aObesity alters osteo-cartilaginous structures leading to higher risk of forearm fractures following severe falls .Obesity is a major preventable risk factor and disease modifier of many respiratory conditions, mainly involved in wheeze, dyspnea, and orthopnea both in adults and children, affecting the prevalence and the severity of several lung diseases, which has been also testified during the SARS-CoV-2 pandemics ,15.Obese children have an increased relative risk of 1.29 (95% CI: 1.16–1.42) for asthma and obesity induces 23% to 27% of asthma cases in children [In a meta-analysis, Chen YC et al. reported that asthma incidence increases by a twofold risk in obese children, with higher susceptibility in males .Moreover, obese children are at risk for developing obstructive sleep apnea syndrome (OSAS) and obesity hypoventilation syndrome. Aspiration pneumonia, pulmonary thromboembolism, chronic obstructive pulmonary disease (COPD), and respiratory failure may occur later in life ,19,20.Several mechanisms have been suggested to explain the association between obesity and respiratory symptoms, including reduced compliance of respiratory system, higher airway resistance, decreased lung volumes, and expiratory flow rates ,22,23 anAuthors looked for relevant studies published up to October 2020 on MEDLINE (PubMed). They searched through selected reviews, original articles, textbooks, and organizations’ websites. To achieve the highest sensitivity, a combination of keywords and indexed terms were used including as keywords “asthma” and “overweight” or “obesity” or “obstructive sleep apnea syndrome” and “children”. Relevant articles were included in this review with the aim of highlighting the most relevant connection between obesity and two frequent respiratory chronic diseases, asthma, and obstructive sleep apnea syndrome.Asthma is an umbrella term that comprises heterogeneous conditions. Among the observable characteristics (phenotype) of asthma, those in obese children have been widely studied ,25,26,27In children, asthma and obesity can co-occur . FurtherHolguin et al.  identifiLate-onset phenotype identified obese patients ≥12 years of age, who were older at the time of diagnosis, often female, with a higher prevalence of severe asthma and a poorer disease control , increased use of high-dose inhaled corticosteroids, oral corticosteroids, antileukotrienics and anti-IgE therapy, low Forced Expiratory Volume in the 1st second (FEV1), and Forced Vital Capacity (FVC) predicted values, and a neutrophilic/paucigranulocitic inflammation (T2 low).The early-onset phenotype included patients <12 years of age, with a lower degree of female predominance and a similarity in asthma severity. Patients belonging to this subgroup required a greater use of health care resources (emergency visits and ICU admissions) and suffered from reduced quality of life, higher airway obstruction with marginally higher maximal FEV1 reversal and increased bronchial hyperresponsiveness. They also showed high IgE levels and positivity for skin prick test (T2 high). Both phenotypes lacked eosinophilia.Other clinical characteristics of obese asthmatics have been found. Obese asthmatics have more severe symptoms than non-obese asthmatics , greaterThis is even more evident in asthmatic obese black and Latino adolescents  who haveChildhood obesity is responsible for a more frequent emergency-department utilization for asthma exacerbations  and overAmong children hospitalized for acute asthma, obesity is associated with longer length of stay and higher risk of mechanical ventilation .Co-occurrence of asthma and obesity may be due to common pathogenetic factors .Common pathogenetic environmental factors include exposure to air pollutants ,44,45, tPRKCA, LEP, and ADRB3 [Asthma and obesity share some genetic factors. Some genes involved in both asthma and in the increase of Body Mass Index (BMI) have been identified such as nd ADRB3 ,50.An interesting study conducted by Wang et al. identified common single nucleotide polymorphisms.(SNPs) at 17q21 (an asthma-associated locus) associated with higher BMI only among asthmatic patients in two independent cohorts .CCL5), interleukin 2 receptor antagonist (IL2RA), and T-box transcription factor 21 (TBX21) genes involved in Th1 polarization that is observed in obese asthmatics [Rastogi et al. described differential patterns of DNA methylation in children as a function of their obesity and asthma status. In particular, they studied DNA methylation in peripheral blood mononuclear cells of eight asthmatic and obese children in comparison with a population of eight children with only asthma, obesity, or healthy controls. Children with both asthma and obesity had a distinctive appearance characterized by reduced methylation of the promoter of chemokine ligand type 5  .CHI3L1) gene inducible from a hyperlipidic diet and also related to the development of asthmatic disease. In particular, a case-control study has demonstrated that a hyperlipidic diet and exposure to aeroallergens increase the expression of the pulmonary Chitinase 3-like-1 (CHI3L1) gene, an evolutionarily conserved moiety involved in Th2 response. In addition, higher levels of CHI3L1 were found in serum both in patients with increased visceral adiposity and in patients with persistent asthma [Ahangari et al. found on mice and human subjects the expression of the Chitinase 3-like-1  associated to normal FEV1 levels .Studies on pediatric patients, despite few, appear to confirm the hypotheses that dysanapsis may play a role in airflow reduction in asthmatic obese.Jones et al. recruited almost 188 children suffering from asthma (61%) or overweight/obese (38%) finding an independent association of both conditions with a dysanaptic growth of airways, influenced more by overweight than asthma .Forno et al. conducted a huge study on more than 4000 children. Authors found a proportional increase of dysanapsis to BMI level and, interestingly, the presence of obesity-related dysanapsis lead to a more difficult control of symptoms and use of systemic corticosteroids in asthmatic children, leading to hypothesize that dysanapsis itself may lead to a reduced airway response to medications. Nevertheless, obese children in this cohort presented dysanapsis even in the absence of asthma features .Dysanapsis appears as an interesting pathogenic feature of asthma, especially in overweight children, whose role still needs to be further clarified.In obese subjects who develop asthma, obesity may be the cause of airway disease. Whereas in asthmatics who become obese, the overweight could influence asthma severity as comorbidity. Strunk et al. reported a worsening of airway obstruction in non-obese asthmatic children who later became obese. They experienced a significant decrease in FEV1/FVC and FEV1 and an inverse association between reduction of FEV1 percent predicted of 0.29 for each unit increase of BMI . This maObesity has been described to be detrimental to lung function. Obesity effects on lung function are different between adults and children.Recent results indicate a more pronounced decline of Total lung capacity (TLC) in obese adults compared to children with an inverse association between BMI and lung volumes resulting in a restrictive ventilatory deficit.Similarly, obesity was also associated with lower Residual volume (RV) among asthmatic and non-asthmatic adults ,61,62,63At variance, obesity in children is associated with normal/increased FEV1 and FVC and low FEV1/FVC and forced expiratory flow at 25–75% of FVC (FEF25–75%). This pattern indicates either an obstructive lung deficit or airway dysanapsis ,64 althoIn obese subjects the impairment of the elastic properties of the chest results in a reduction in Functional Residual Capacity (FRC) and in an increase airway resistance. In particular, the variation of the parenchymal retractive forces on the airways results in low-volume breathing and a reduction in FRC leads to smooth muscle discharge that over-responds to broncho-obstructive stimuli in the obese non-asthmatic adult but not in asthmatics . This fiFurther studies are needed to evaluate the impact of obesity on the variation of RV and FRC in children, as the data available to date are insufficient.Breathing at reduced lung volumes also increases airway hyperresponsiveness (AHR) which seems to have a direct correlation with the worsening of BMI ,69.Gender has an important modifier effect since obese girls tend to be more affected by onset of AHR than obese boys .Both mechanical (reduced expansion of chest) and inflammatory mechanisms (enhanced release of inflammatory cytokines by adipose tissue) may be involved in lung function decrease and in asthmatic symptoms .Regarding obesity-induced mechanical lung compression, abdominal adiposity can heighten diaphragm and diminish chest wall compliance reducing FRC and amplifying symptoms related to reduced airway caliber such as AHR, dyspnea, and wheeze. In agreement, in asthmatic adolescents, especially females, indicators of excess abdominal adiposity, such as high values of waist circumference and waist-to-height ratio, were observed .Adipose tissue produces proinflammatory cytokines (leptin), while there is a reduced release of anti-inflammatory adipokines, including adiponectin.Sideleva and colleagues recruited women undergoing bariatric surgery with a history of adult-onset asthma comparing them to women undergoing bariatric surgery with no evidence of asthma; subcutaneous fat, bronchoalveolar lavage (BAL) and brush biopsy were collected from each patient and were studied for inflammatory markers, including adipokines and cytokines. Inflammatory markers, especially in visceral adipose tissue, were found to be higher in patients with asthma, suggesting a central pathogenic role for visceral adipose tissue producing high levels of adipokines, able to affect airway reactivity .In asthmatics, leptin increases adipose tissue macrophage release  of IL6, Obesity is associated with increased neutrophils both in the circulation and adipose tissue vasculature. Higher neutrophilic airway inflammation has been linked with higher CRP and IL-6, lower testosterone and no oral contraceptive pill use . IncreasWhile immunologic features of childhood asthma are characterized by a white blood cells (WBC) polarization towards T-Helper 2 (TH2) cells, obese children with asthma have a polarization towards T-Helper 1 (TH1) cells and increased activation of circulating monocytes, suggesting obesity-associated asthma as a distinct nosologic entity compared to obesity-unrelated asthma . FurtherAn experimental model hypothesized a role for Surfactant Protein-A (SP-A), a crucial mediator in reducing tissue and lavage fluid eosinophilia in allergic mouse models. Obese asthmatic patients expressed significantly lower levels of this protein in bronchoalveolar lavage (BAL) compared to non-obese asthmatics and non-obese non-asthmatic patients. Authors suggested that in obese patients high local levels of TNF-α could reduce SP-A secretion, leading to the eosinophilic asthma phenotype in a subgroup of obese asthmatic subjects .Oxidative stress is an important feature of asthma in childhood . IncreasHowever, increased levels of oxidative stress have been found in obese patients with late-onset asthma and they have been associated with airway remodeling, impaired lung function and corticosteroid resistance. Nitric oxide synthase uncoupling, caused by dysfunction in the L-arginine/nitric oxide metabolism, and mitochondrial dysfunction could be two possible explanations of increased oxidative stress in obese asthmatics .A recent field of interest is the possible role of microbiome in obesity–asthma association. This finding may have several potential therapeutic implications considering the already proven effectiveness of probiotics in various diseases . ModificThe association between the gut microbiota and obesity-related asthma is supported by the evidence of microbiota influence on immune response via changes in IL-17A, an interleukin released by Th-17 cells crucial in the neutrophilic recruitment. It has been already demonstrated that germ free mice and those treated with antibiotics, have lower levels of intestinal Th-17 cells; the reverse phenomenon occurs in germ free mice colonized with filamentous bacteria ,87,88.Kim HY et al. demonstrated that wild-type mice on high-fat diet developed greater AHR compared with WT mice on control chow. On the contrary, this innate AHR was absent in IL-17A -/- mice both on high-fat diet and on a controlled diet .Th-17 cells are implicated in neutrophilic asthma and are increased in obese subjects; high levels of Th17 cells were found in the sputum of obese asthmatic subjects . In concThe role of obesity in asthma has also been underlined by studies on the effect of decreased body weight on asthma. After bariatric surgery, asthmatic obese adults undergo a significant improvement in asthma control, asthma quality of life, airways responsiveness to methacholine, and lung function .In children, weight reduction may lead to an improved asthma control. The reduction of weight can be mainly achieved by dietary advice and increased physical activity. In children dietary intervention has been shown to be effective in improving lung function and asthma control but not systemic and airway inflammation .Van Leeuwen et al. showed that weight loss was associated with improvement in exercise-induced bronchoconstriction and quality of life . HoweverFurthermore, overweight and obesity appear to potentially mislead the physician in the management of asthma. The strong relationship between asthma and gastro-esophageal reflux, a very common symptom in overweight/obese patients, affects the perception of symptoms by patients even in presence of satisfactory lung function. This leads to unnecessary use of emergency therapy (short-acting beta agonists) potentially producing a vicious cycle where SABA itself worsens the gastro-esophageal reflux which, in turn, worsens the personal perception of symptoms by patients . The treThe evidence that these patients present a higher percentage of neutrophils both in sputum and in blood, if compared to non-obese asthmatics, may nevertheless be helpful .Though still not justified in all cases of asthma, some evidences support the use of macrolides in difficult-to-control neutrophilic asthma . MacroliAnother important pediatric respiratory condition associated with overweight and obesity is Sleep-Disordered Breathing (SDB), especially Obstructive Sleep Apnea Syndrome (OSAS). OSAS is a recurrent, partial, or complete, obstruction of the upper airways during sleep, resulting in disruption of normal gas exchange and sleep fragmentation. This association has been widely demonstrated in both adults  and chilHence, they showed that the strength of the association seems to proportionally increase with age , in partA case control study was conducted to demonstrate that the risk of SDB was higher in obese children compared to normal population, and to understand the role of pharyngeal lymphoid tissue hypertrophy. Forty-six obese children (mean age 10.8 years) were compared with 44 healthy, normal weight children of the same age. A total of 34.8% of obese children reported snoring habitually more than 4 nights a week, compared to 15.9% of healthy controls. It was found that one-third of obese children had SDB and that both obesity and tonsillar hypertrophy were independent risk factors for the development of SDB .In addition, pediatric OSAS, like obesity, has been associated with increased cardiovascular morbidity . This coThe American Academy of Sleep Medicine (AASM) Manual for Scoring of Sleep and Associated Events recommends monitoring hypoventilation through diagnostic polysomnograms in children with suspected OSAS .Standard pediatric polysomnography offers a wide perspective on patient’s sleep patterns by recording multiple physiologic signals consisting of electroencephalogram, electrocardiogram, electromyogram, electro-oculogram, oro-nasal airflow, respiratory effort, and peripheral oxygen saturation.In 2002 the American Academy of Pediatrics prepared a technical report on the best diagnostic tests for sleep-disordered breathing; 2115 works were revised. This review concluded that overnight polysomnography (PSG) was the reference method for the diagnosis of OSAS, also in obese kids. On the other hand, they studied pulse oximetry’s effectiveness; it resulted specific but not sensitive enough .Nixon GM et al. tried to develop a scoring system in which pulse oximetry proved a diagnostic inferiority when compared to PSG . Similar2) Measurement during Pediatric Polysomnography. Their findings suggested that the levels of EtCO2 may contribute to the diagnosis similarly to the polysomnographic indices [Paruthi et al. analyzed the end-tidal Carbon Dioxide (EtCO indices .2 (TcCO2) appears to be a promising diagnostic tool in obese patients, showing a good correlation with arterial blood PaCO2 levels [2 changes has recently been evaluated also in children showing a good correlation with airways obstruction in subjects suspected of sleep disordered breathing [Transcutaneous monitoring of CO2 levels . TcCO2 creathing  and in creathing .The interaction between OSAS and obesity is very complex, and their coexistence may negatively influence the severity of both conditions, creating an interleaved vicious cycles. Systemic inflammation, oxidative stress, metabolic affection, and gut microbiota have a synergistic role in determining the pathogenesis of these two diseases .Obesity is mainly involved in the pathogenesis of OSAS by various mechanisms but at the same time, OSAS can cause obesity due to several factors such as reduced physical activity, insulin resistance and elevated ghrelin levels ,118,119.Furthermore, the abdominal adipose tissue is responsible for a lower diaphragmatic excursion and a reduction of the intrathoracic volume, especially in the supine position, increasing the respiratory work during sleep .Leptin is considered one of the main explanations of the association between obesity and OSAS, since obese subjects show resistance to leptin function. This hormone would act on central chemoreceptors causing an increase in triggering ventilation, but obese patients show a failure in this mechanism because of a resistance to leptin’s action .Leptin is a peptide hormone produced primarily in adipose tissue, that also influences energy homeostasis, metabolism, inflammation, and sympathetic nerve activity. Recent studies reported a positive correlation between blood leptin levels and OSAS severity ,123.Obesity is associated to an impaired adipose tissue function, which leads to adipocyte hypertrophy, hypoxia and activation of inflammatory processes within adipose tissue . In addiA Chinese case-control study from 2016 explored the possible pathogenic role for adipokines on a small sample of OSAS adult patients. All patients underwent PSG monitoring, and a venous blood sample was collected before and after PSG to test a defined group of adipokines . Multiple regression analyses showed that altered BMI and Apnea Hypopnea Index (AHI) were associated with high plasmatic levels of these adipokines in OSAS patients, finding a significant correlation between specific adipokines, OSAS severity and obesity .Many studies confirm that obesity and OSAS share similar dysregulation in metabolic process. Indeed, both conditions are characterized by oxidative stress, which influences their development and progression ,128.In particular, intermittent hypoxia-reoxygenation in OSAS patients is responsible for ATP depletion and xanthine oxidase activation, with consequent production of oxygen-derived free radicals  causing At the same time, oxidative stress and inflammation in obesity enhance OSAS.Moreover, an inflammatory state in obese children suffering from OSAS is attested by the upregulation of pro-inflammatory signaling pathways  which lead to greater expression of pro-atherogenic factors and endothelial dysfunctions .Promising data were found about the relationship between platelet activation and OSAS, with many authors hypothesizing platelet activation’s pathogenic role to be central in OSAS.Kurt OK et al. studied the correlation of some blood parameters, including Mean platelet volume (MPV) and platelet distribution width (PDW) to OSAS severity. They found an elevation of PDW in severe OSAS, suggesting a role for platelet activation as a marker of OSAS severity. Included patients presented mean BMI levels ranging from 28.4 (overweight) to 33.2 (obese) .In 2015, Akyol S et al. confirmed that MPV was associated with OSAS severity but also independently linked to high sensitivity C-reactive protein, a marker of systemic inflammation .A recent meta-analysis showed higher MPV values in pediatric patients suffering from SDB than in healthy children, revealing an increased platelet activity. They also demonstrated a reduction of the parameter after adenoidectomy and adenotonsillectomy .In recent years, human intestinal microbiota has emerged as a key factor for the development of both obesity and OSAS ,136. ManA high-fat diet might be responsible for a dysbiosis in the gut microbiota that could have a crucial role in the development of a low-grade inflammatory status ,140.Additionally, in OSAS, intermittent hypoxia and sleep deprivation further influence gut microbiota leading to a systemic inflammatory process which contributes to cardiovascular and metabolic morbidities .It is assumed that the so called mutual “organ crosstalk” between respiratory system, adipose tissue and intestine can also explain the complex relationship between OSAS and obesity.Among the therapeutic approaches to treat OSAS, the weight loss and lifestyle changes are the most effective . MedicalConfirming the strict interdependence between the two pathologies, many studies observed that the resolution of OSAS following adenotonsillectomy leads to improvement in metabolic disorders related to obesity such as dyslipidemia. OSAS resolution is associated to low-density lipoprotein (LDL) and apolipoprotein B (ApoB) decrease and to a high-density lipoprotein (HDL) increase .An Australian study analyzed polysomnographic results before and after bariatric surgery demonstrating an important decrease in the severity of OSAS after weight reduction .Andersen IG et al. conducted a prospective longitudinal study on 62 children and adolescents with BMI > 90th percentile with OSAS (defined by AHI > 2) who underwent sleep examinations and anthropometric assessments at baseline and after 6 and 12 months from baseline; they were subjected to an individualized obesity treatment regarding lifestyle changes.They observed a normalization of AHI (apnea-hypopnea index) in 38% of children after about 6 months of obesity treatment and in 44% one year after starting treatment. They also assessed that children with tonsillar hypertrophy achieved same effect as children without tonsillar hypertrophy ,147. Ta.Recent data on the spread of the new COVID 19 virus in the pediatric population seem to demonstrate the greater susceptibility of obese children to the development of a more severe respiratory disease than their normal-weight peers. Clinically, COVID 19 infection in pediatric age is lesser severe than in the adult population and occurs mainly with cough and fever, sore throat, myalgia, rhinorrhea, nasal obstruction, diarrhea, and vomiting. Obesity seems to increase susceptibility to infection and seems to worsen the prognosis; during the epidemic in Canada and in New York obesity was the most frequent comorbidity in children admitted to the ICU for COVID 19 infection; this association has been found in several studies on the adult population. A link between obesity and severity of COVID 19 respiratory infection probably lies not only in the causes listed above that predispose obese child to respiratory disease  also in the greater expression of the ACE2 receptor . The overexpression of the ACE2 receptor has been demonstrated in mouse models subjected to a hyperlipidemic diet and it is also present in obese subjects who take therapy with RAAS system inhibitors for cardiovascular comorbidities associated with obesity. However, we still have few data on the pediatric population and COVID 19 infection and further studies are needed .Though not overwhelming, evidences are convincing about the key role played by obesity in asthma and OSAS.Regarding the link between asthma and obesity, a gene-by-environment interaction should be thoroughly investigated. The discovery of modifiable environmental factors such as diet that can influence gene expression through epigenetic mechanisms inducing both obesity and asthma in predisposed subjects will be crucial to prevent the spread of these two conditions.Although the contribution of obesity in patients with OSAS appears to be due to mechanical factors, evidences strongly support the key role of inflammatory cytokines with a pro-inflammatory signaling pathways, a reduced effectiveness of leptin action combined with high adipokine levels. The role of platelet activation in OSAS asthmatic patients appears to be crucial but yet to be fully explained. At last, gut dysbiosis significantly affects the lung-gut axis leading to an impairment of lung function.Studies assessing the correlation between the pathogenic role of platelet activation and BMI levels may be useful for a deeper understanding of the issue, such as studies exploring the lung–gut axis in a detailed manner.In order to prevent obesity and the consequent complications, family education, and precocious changes of the lifestyle are essential. We believe that a healthy diet would lead to an overall improvement of the quality of life in obese children suffering from respiratory diseases.


MYC amplification, PIK3CA, and TP53 mutations, and extrinsic factors, such as hypoxia, oxidative stress, and acidosis, contribute to different metabolic reprogramming phenotypes in metastatic breast cancers. Understanding the metabolic mechanisms underlying breast cancer metastasis will provide important clues to develop novel therapeutic approaches for treatment of metastatic breast cancer.Breast cancer is one of the most common malignancy among women worldwide. Metastasis is mainly responsible for treatment failure and is the cause of most breast cancer deaths. The role of metabolism in the progression and metastasis of breast cancer is gradually being emphasized. However, the regulatory mechanisms that conduce to cancer metastasis by metabolic reprogramming in breast cancer have not been expounded. Breast cancer cells exhibit different metabolic phenotypes depending on their molecular subtypes and metastatic sites. Both intrinsic factors, such as  Breast cancer is the most common malignant tumor and the second capital reason for cancer death among women worldwide , 2. MetaRecently, increasing evidence point out that cancer is not only a genetic disease but also a metabolic disease, in which oncogenic signaling pathways participate in energy regulation and anabolism to support rapidly spreading tumors . In thisThis review addresses the current knowledge on the crosstalk between metabolic reprogramming and metastatic process in breast cancer. A better understanding of the metabolic mechanisms driving breast cancer metastasis may provide clues for discovering new anticancer therapeutics.In response to external growth signals, normal cells in a rapidly proliferating state activate assorted signaling pathways to suppress oxidative phosphorylation (OXPHOS), and advance glycolysis and anabolic metabolism for cell growth. Cancer cells is able to hijack this mechanism to meet developmental needs even if there are no external signals  Figure. MoreoveGlucose transport cross cell membrane through the glucose transporter proteins (GLUTs) and different GLUTs expression in breast cancers are related to dissimilar pathological grades and prognosis. GLUT1-5 and GLUT12 are functionally in breast cancer cells –37, and The pentose phosphate pathway (PPP) is another way of oxidative decomposition of glucose besides glycolysis and TCA cycle, which produces nicotinamide adenine dinucleotide phosphate (NADPH), ribose phosphate, fructose-6-phosphate (F6P) to make cancer cells satisfy their anabolic needs and respond to oxidative stress . Proteinc-MYC and RAS, can increase the cancer cell glutamine metabolic activity by upregulating glutamine transporters such as alanine-serine-cysteine transporter 2 (ASCT2) and enzymes participating in the conversion of glutamine-to-glutamate, such as glutaminase (GLS)-1 , and glutathione (GSH), participate in energy supply, supplement glucose metabolism and help cells resist oxidative stress to uphold proliferation and progression of tumor cells , 44. Som (GLS)-1 . For exaer cells . Notablyer cells . The gluer cells .de novo nucleotide biosynthesis, and methylation and reductive metabolism  to satisfy the augmented demand for membrane metabolism in favor of rapid growth and proliferation. The expression of fatty acid synthase (FASN), a key enzyme essential for the FAS, is elevated in breast cancer  and lipid metabolic programming also play significant parts in promoting breast cancer growth and progression . Cancer abolisms . Moreovet cancer , and itst cancer , suggestt cancer , 59. It t cancer . Sterol t cancer , 62. Andt cancer , 64. Undor cells . Inhibitor cells , 67.Tumor metastasis is a sequential multi-step process, which includes local invasion, intravasation, migration through the lymphatics or blood vessels, extravasation and colonization giving rise to the formation of metastases in distant organs . ParticuThe step one of the metastasis is that tumor cells break away from the tumor bed and migrate from the stroma into the bloodstream . In ordeAfter escaping from the original tumor site to blood circulation, breast tumor cells begin to migrate to remote organs. The first obstacle encountered by circulating tumor cells is the blood vessel wall, especially endothelial cells. In some organs, such as bone marrow and liver, microvessels are composed of sinuses with strong permeability, which make cancer cells easier to break through . WhereasAdjusting to new environment is another hurdle for circulating tumor cells (CTCs) to form metastasis. Disseminated cancer cells will spring up in targeted tissues and organs through a way that is significantly different from their origins. Cancer cells must acquire new capabilities, especially the ability to interact with cells in the ECM and new microenvironment. Tumor cells form a two-way connection with circumferent stroma in the early stage of invasion and after that, tumor-stroma interaction helps the tumor to develop toward metastasis .via the VEGFR-1/Flt-1 pathway, which is important for lung metastasis , a small number of cells with self-renewal and unlimited replication capabilities, have been shown in numerous cancer models to be involved in tumor development and metastatic dissemination. Moreover, the occurrence of BCSCs with the properties of stemness, EMT and drug resistibility, is the main cause for cancer recurrence and treatment failure . Multiplin vivo  and Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) analysis to study biochemical differences between metastasis tropisms in two TNBC cell lines and showed that bone metastasis tropism was characterized by the increase of amino acids and the decrease of mitochondrial signal, while high lipid and mitochondrial (cytochrome C and RNA) levels for lung metastasis . NETosisTP53 mutations are very common in breast cancers, especially in triple-negative and HER2-positive subtypes , the paramount stromal cells in breast tumor microenvironment, contribute to tumor progression through many mechanisms, such as releasing of assorted secretory proteins , direct interplaying with tumor cells, regulating immune-response, ECM remodeling, and inducing cancer metabolic reprogramming . Breast The “cancer-associated adipocytes (CAAs)” are generated by the transformation of tumor adjacent adipocytes . It has in vitro  pathway inhibitor, to taxane did not improve PFS compared with taxane alone  The side effects of such drugs limit their clinical effects as the optimal dose window is hard to be determined; 2) Due to the extremely complex signaling pathways in the regulation of normal cellular biology, inhibition of a specific signaling pathway will definitely have feedback activation or upregulation of other alternative signaling pathways, therefore causing ultimately treatment failure; 3) To specifically target related mutations involved in metabolic pathways is challenging. 4) Accurate screening of the beneficiaries is the key to improve the drug effect, and is an urgent problem to be solved in the future.MYC amplification, PIK3CA, and TP53 mutations, and extrinsic factors, metabolic stresses imposed by the microenvironment, such as hypoxia, oxidative stress, acidosis, contribute to different metabolic programming phenotypes in metastatic breast cancer. More importantly, interfering with tumor metabolism to control tumor progression is a very promising approach in cancer treatment, although it is full of challenges. More researches are required to further discover the related genes and molecular mechanisms involved in metabolism reprograming during cancer progression, so that they can be used for targeting therapy in clinical practice in the future. We also look forward to further advances in approaches to judge and quantify metabolic phenotypes in human breast cancers in vivo, including metabolomics, metabolic imaging and isotope tracing studies, so that clinical oncologists will develop treatment strategies by matching the treatment to the patient-specific tumor metabolic characteristics.Metabolic programming supports several steps of successful metastasis in breast cancer. Breast cancer cells exhibit different metabolic phenotypes in different metastasis sites. Both intrinsic factors, properties arising in the malignant cells, such as LW wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Zhejiang Provincial Natural Science Foundation of China (grant no. Y19H160283).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.


Advances in the use of targeted and immune therapies have revolutionized the clinical management of melanoma patients, prolonging significantly their overall and progression-free survival. However, both targeted and immune therapies suffer limitations due to genetic mutations and epigenetic modifications, which determine a great heterogeneity and phenotypic plasticity of melanoma cells. Acquired resistance of melanoma patients to inhibitors of BRAF (BRAFi) and MEK (MEKi), which block the mitogen-activated protein kinase (MAPK) pathway, limits their prolonged use. On the other hand, immune checkpoint inhibitors improve the outcomes of patients in only a subset of them and the molecular mechanisms underlying lack of responses are under investigation. There is growing evidence that altered expression levels of microRNAs (miRNA)s induce drug-resistance in tumor cells and that restoring normal expression of dysregulated miRNAs may re-establish drug sensitivity. However, the relationship between specific miRNA signatures and acquired resistance of melanoma to MAPK and immune checkpoint inhibitors is still limited and not fully elucidated. In this review, we provide an updated overview of how miRNAs induce resistance or restore melanoma cell sensitivity to mitogen-activated protein kinase inhibitors (MAPKi) as well as on the relationship existing between miRNAs and immune evasion by melanoma cell resistant to MAPKi. Melanoma represents one of the most aggressive skin cancers with a significantly increased incidence in the last decades ,2,3. CurIn the past years, either dabrafenib or vemurafenib BRAF inhibitors (BRAFi) showed encouraging response rates, although the duration of response appeared to be limited ,8. BRAF Using a variety of technical approaches such as chromosomal analysis, miRNA microarrays, miRNA qPCR arrays, and high-throughput small RNA sequencing platforms, microRNA (miRNA)s have been identified to function as oncogenes or tumor repressors genes. Oncogenic miRNAs (oncomiRs) are frequently overexpressed in cancers while tumor-suppressive miRNAs are down-regulated. It has been documented that miRNAs regulate more than 30% of human protein-coding genes  and contmiRNAs are short, single-stranded, non-coding nucleotide sequences with an average 22 nucleotides in length. They are transcribed as individual genes, from introns of coding genes (intronic miRNAs) or from regions between the clusters of genes (intergenic miRNAs) while clustered miRNAs are transcribed as polycistronic transcripts . miRNA gIt has been shown that chromosomal rearrangements, epigenetic regulation and disorders in miRNA biogenesis, result in increased or decreased expression of miRNAs in melanoma cells as compared to melanocytes ,35,36. FIn this review, we discuss the latest progress regarding mechanisms by which miRNAs regulate melanoma cell resistance to MAPKi and immune evasion. Furthermore, the potential predictive value of circulating miRNAs for monitoring melanoma responsiveness to targeted and immune therapies is debated.In recent years, by next-generation sequencing, the Cancer Genome Atlas provided the analysis on the somatic aberrations underlying melanoma genesis, identifying BRAF, RAS, and NF1 mutant genetic subtypes of cutaneous melanoma, all of them being able to deregulate the MAPK/ERK pathway, leading to uncontrolled cell growth . Over 50During the progressive development of drug resistance, several deregulated miRNAs have been shown to control both tumor cell growth and melanoma cell interactions with the tumor microenvironment. Some miRNAs provoke drug resistance while others restore drug sensitivity. In First, Liu and co-workers showed that miR-200c is a potential therapeutic target to restore melanoma cell sensitivity to BRAFi. They found that miR-200c reverts drug resistance to PLX4720 BRAF and U0126 MEK inhibitors by down-regulating the p16 transcriptional repressor BMI-1, which, in turn, inhibits melanoma cell growth and metastases in nude mice. Moreover, they found that miR-200c acts on ABC transporters, a superfamily of transmembrane proteins that mediate drug resistance in melanoma cells . The cliMelanoma cells are documented to release into the extracellular milieu different types of extracellular vesicles (EV)s, including oncosomes, ectosomes, exosomes, and melanosomes carrying protein and small RNAs cargos . CompariOverexpression of the Yes-associated protein (YAP) has been found to associate with resistance to anticancer therapies in solid tumors, including BRAFi resistant melanomas ,73. miR-There are several attempts to investigate the potential link between miRNAs expression profile and patients’ response to immune checkpoint inhibitors in order to verify at the same time their potential use for monitoring efficacy of immune checkpoint blockade and improving the outcomes of patients with advanced melanoma. Although few data regarding miRNAs and immune checkpoint inhibitors relationship are available in the literature, recent studies demonstrate that some miRNAs may regulate directly or indirectly the expression of immune checkpoints, acting on tumor cells or immune cells, respectively .Galore-Haskel and collaborators found higher levels of miR-222 in melanoma tissues from patients that were non-responders to ipilimumab when compared to responder patients, raising the possibility that miR-222 expression could be considered a valid biomarker for predicting responsiveness of melanoma patients to ipilimumab . These AmiRNAs are also involved in the regulation of immune cells within the tumor microenvironment, including cytotoxic, CD4 or γδ T lymphocytes, natural killer (NK), macrophages and myeloid-derived suppressor cells (MDSCs).+ T cells isolated from tumor-infiltrated lymph nodes and tumor tissues of melanoma patients and murine models. They found that miR-155 up-regulation within the tumors correlates with increased CD8+ T-cell infiltration while low expression of miR-155 targets in melanoma tumors associates with a prolonged overall survival. These findings allowed Authors to conclude that miR-155 could be considered a marker of responsiveness of CD8 T cells, as further demonstrated by its up-regulation after PD1 blockade [A direct involvement of tumor-suppressor miRNAs in the control of antitumor immune response through the regulation of immune checkpoints PD-1, PD-L1, and CTLA-4 has been ascertained in tumors of different origin . By a miblockade .Up-regulation of stress-induced ligands, including ULBP2, allows tumor cell recognition by immune cells trough the NKG2D receptor expressed on lymphocytes, Natural Killer cells, as well as cytotoxic, CD4 or γδ T cells . miR-34aThe activation of the MAPK pathway through BRAF mutations leads to downstream production of several cytokines that promote tumor growth and immune evasion with autocrine or paracrine mechanisms. Recent studies have documented that the MAPK signaling pathway may be considered as a potential molecular target for overcoming melanoma cell evasion of the immune surveillance . By actiTo date, there is an urgent need to develop new non-invasive methods for monitoring disease progression or resistance to treatments of melanoma patients. In this regard, liquid biopsy may be considered a non-invasive source of biomarkers, potentially useful for monitoring responsiveness of melanoma patients to targeted and immune therapies, although the strategies for these approaches are still under investigation. In the last decade, many efforts have been made to identify diagnostic and prognostic circulating miRNA biomarkers for melanoma. Circulating miRNAs have emerged as powerful biomarkers since they are highly stable in body fluids, which are protected against enzymatic degradation thanks to their association with RNA binding proteins (Argonaute-2 and nucleophosmin-1), with high- and low-density lipoproteins, or to their embedding in membrane vesicles such as exosomes ,41,42. AAs above described, numerous miRNAs have been shown to modulate melanoma sensitivity and resistance to MAPKi  and/or iFinally, Tengda L. and coworkers demonstrated that miR-532-5p and miR-106b, isolated from serous exosomes as well as from total serum, were able to discriminate patients with melanoma from healthy controls, metastatic patients from those with no metastasis, patients with stage I–II disease from those with stage III–IV, and patients treated with pembrolizumab from untreated ones .In melanoma, several miRNAs are deregulated because of epigenetic changes, impaired transcription, amplification, or deletion of miRNA genes as well as defects in the miRNA biogenesis machinery. It is currently accepted that distinct profiles of miRNA expression are detected at each step of melanoma development, and that an altered expression of miRNAs frequently correlates with poor prognosis and/or inadequate response to treatments. As recapitulated in this review, dysregulated miRNAs may induce and sustain or prevent melanoma cell resistance to BRAFi /MEKi and immune therapies by acting as oncogenes or tumor suppressors, respectively. A partial or complete reversion of melanoma cells resistance to BRAFi and MEKi may be achieved by restoring down-regulated miRNAs or silencing up-regulated miRNAs, suggesting that specific miRNAs or their antagonists may be considered for potential therapeutic applications to overcame melanoma cell resistance to BRAFi and MEKi. In this regard, miRNAs, especially those being part of the circulating transcriptome, may be useful as biomarkers for early melanoma response to treatments, but the strategies for these approaches are still under investigation. In melanoma, implications of microRNAs in the regulation of immune checkpoint blockade and controlling their expression for therapeutic purposes is the subject of intense ongoing research. Specific miRNA signatures associate with specific alterations of immune checkpoint pathways in the melanoma microenvironment while subsets of miRNAs directly regulate the transcription of immune checkpoints. Thus, miRNA could provide new biomarkers predicting patient response to immune checkpoint inhibition and it is reasonable to foresee that combining miRNAs with different immune checkpoint targets could mimic and possibly improve the effect of combined immune checkpoint blockade therapies. Activation of the MAPK pathway through BRAF mutations may be a potential molecular target for overcoming evasion of the immune surveillance by melanoma cells. MAPK cascade stimulates melanoma cells to secrete cytokines, chemokines and soluble growth factors that recruit immune and myeloid cells sustaining both tumor growth and immune evasion. In this contest, specific miRNAs or their antagonists may be considered for potential therapeutic use for restoring the effector function of immune cells. New approaches that look at simultaneous or sequential use of drugs targeting the MAPK pathway with immune checkpoint inhibitors are also a priority, with evidence suggesting that specific miRNAs may overcome melanoma growth and immune evasion. Thus, many questions regarding the best first- and second-line treatment and the best treatment sequence remain to be addressed.


Optoacoustic imaging (OAI), or photoacoustic imaging (PAI), has fundamentally influenced basic science by providing high-resolution visualization of biological mechanisms. With the introduction of multispectral optoacoustic tomography (MSOT), these technologies have now moved closer to clinical applications. MSOT utilizes short-pulsed near-infrared laser light to induce thermoelastic expansion in targeted tissues. This results in acoustic pressure waves, which are used to resolve specific endo- and exogenous chromophores. Especially in the pediatric population, this non-invasive imaging approach might hold fundamental advantages compared to conventional cross-sectional imaging modalities. As this technology allows the visualization of quantitative molecular tissue composition at high spatial resolution non-invasively in sufficient penetration depth, it paves the way to personalized medicine in pediatric diseases. Up to now, ultrasound has played a central role in pediatric diagnostics. Due to its neglectable risk profile and easy availability, it is the diagnostic tool of choice for a variety of different indications –4. As cuIn this regard, multispectral optoacoustic tomography (MSOT) is rapidly evolving as one promising key technology. MSOT utilizes short-pulsed laser light in the near- and extended near-infrared range (NIR/exNIR) for tissue illumination. As light in these wavelengths has fewer tissue absorption, it enables deeper tissue penetration. The NIR is typically represented in the wavelength between 700 and 900 m followed by extend NIR (exNIR)/NIR II window around 900–1600 nm. In OAI, this light is absorbed by tissue molecules and converted into acoustic pressure waves, which can be recorded and formed into optoacoustic images. In the NIR, endogenous absorbers such as deoxyhemoglobin, oxyhemoglobin, and melanin possess distinct absorption spectra, which gives them a unique signature in MSOT . In the R), 800  nm as measure for total hemoglobin , and 850  nm for oxygenated hemoglobin (HbO2).OAI has so far been used in a number of various clinical applications; selected studies are summarized in Table When examining vasculature, MSOT is capable of visualizing major blood vessels and microvasculature . As MSOTIn a consecutive study, the subcutaneous finger tissue of patients with systemic sclerosis (SSc) provided significantly lower MSOT values for oxygenated hemoglobin as well as total hemoglobin in comparison to healthy controls, reflecting microvascular dysfuntion in SSc as a possible marker of disease activity .www.euphoria2020.eu).Further investigations explored the quantification of hemoglobin in the intestinal wall of patients with Crohn’s disease (CD) with MSOT. These measures have been shown to be increased in patients with active CD, demonstrating robust correlation with endoscopic and microscopic intestinal inflammation . MSOT coOne of the main tissues examined with MSOT is breast tissue. In healthy breast tissue, hormone-related physiological changes of breast parenchyma were visualized with MSOT using only a three wavelengths illumination  . The autThe high resolution in OAI/PAI makes it possible to reveal more vessels than with conventional Doppler as shown in a photoacoustic/ultrasound dual imaging study of human thyroid cancers . While DIn near-filed applications such as non-melanoma skin cancers MSOT already successfully distinguished skin cancer from normal skin, being helpful for the visualization of lesion margins , 14. EveBesides these promising first clinical studies, OAI has similar limitations like ultrasound. Namely, penetration depth of up to several centimeters as well as air, thick bones, body fat, and body hair can significantly influence image quality in adults. Therefore, children and adolescents might be excellent candidates for MSOT imaging as their organs and/or muscles are closer to the body surface.Consequently, the first pediatric study showed that MSOT imaging is able to visualize the molecular composition of muscles in children. Muscular collagen content in children with Duchenne muscular dystrophy was increased as compared to healthy controls, suggesting non-invasive measured collagen content as a novel non-invasive biomarker for disease severity and potential monitoring tool for novel therapies . Given tFor translation into clinical practice, there are still several challenges to overcome: firstly, standardization of OAI imaging is required. It was already reported that preclinical OAI imaging systems consistently perform equal or even better  compared to other preclinical imaging modalities, which underlines their clinical potential . In agreMSOT is capable of visualizing endogenous chromophores such as hemoglobin, melanin, lipids, and recently collagens. Hemoglobin and collagens in particular display specific properties that are promising for monitoring the course of various diseases. Furthermore, the use of exogenous chromophores  as contrast agents for OAI imaging in pediatric patients is still unexplored. Therefore, the combination of endogenous and exogenous molecular information could pave the way for novel tailored theranostic approaches in pediatrics in the future.


Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. Its aggressive nature is attributed partly to its deeply invasive margins, its molecular and cellular heterogeneity, and uniquely tolerant site of origin—the brain. The immunosuppressive central nervous system (CNS) and GBM microenvironments are significant obstacles to generating an effective and long-lasting anti-tumoral response, as evidenced by this tumor’s reduced rate of treatment response and high probability of recurrence. Immunotherapy has revolutionized patients’ outcomes across many cancers and may open new avenues for patients with GBM. There is now a range of immunotherapeutic strategies being tested in patients with GBM that target both the innate and adaptive immune compartment. These strategies include antibodies that re-educate tumor macrophages, vaccines that introduce tumor-specific dendritic cells, checkpoint molecule inhibition, engineered T cells, and proteins that help T cells engage directly with tumor cells. Despite this, there is still much ground to be gained in improving the response rates of the various immunotherapies currently being trialed. Through historical and contemporary studies, we examine the fundamentals of CNS immunity that shape how to approach immune modulation in GBM, including the now revamped concept of CNS privilege. We also discuss the preclinical models used to study GBM progression and immunity. Lastly, we discuss the immunotherapeutic strategies currently being studied to help overcome the hurdles of the blood–brain barrier and the immunosuppressive tumor microenvironment. Glioblastoma (GBM), a World Health Organization (WHO) grade IV glioma, is the most common and malignant primary brain tumor in adults. Despite aggressive standard and targeted therapy, patients have a 14-month median survival following diagnosis. The survival timeline is slightly extended for the patients whose tumors express methylation of the promoter of the DNA repair gene, O-6-methylguanine-DNA methyltransferase (MGMT) ,3,4, driWhile most of these tumors arise de novo, about 10% arise secondarily from a low-grade glioma and are typically more likely to occur in younger patients ,6. GliomBeyond prognostication, molecular classification also helps refine systemic targeted therapies that can be used in conjunction with standard care treatment. The typical GBM treatment regimen includes tumor resection, radiotherapy with concurrent chemotherapy with temozolomide, followed by adjuvant temozolomide chemotherapy . While sInfiltration of blood-derived immune cells into the central nervous system (CNS) is typically limited to occasions when the blood–brain barrier (BBB) is disrupted, as is readily seen in glioblastoma using contrast-enhanced imaging . Due to In this review, we will give an overview of the dynamics of CNS immunity and discuss the seminal and contemporary studies that reveal the mechanisms by which these various immunotherapies can affect glioblastoma tumor immunity and progression. These strategies include chimeric antigen receptor T cells (CAR-T), bispecific T-cell engagers (BiTEs), dendritic cell (DC) vaccination, oncolytic virotherapy, checkpoint blockade, monoclonal antibodies, immune modulators, and lymphatic modulation.After taking up self or foreign (including tumor) antigen, DCs—the most effective professional antigen-presenting cells (APCs)—migrate from the tissue site to the accompanying draining lymph node (LN) via afferent lymphatic vessels (LVs). Once in the LN, these antigen-bearing DCs will present to naïve T lymphocytes (T cells) through major histocompatibility class type I (MHC I) or MHC type II (MHC II) complexes. Based on the type of antigen, the levels of co-stimulatory molecules expressed on the APC, and the milieu of the cytokine signals present at the time of priming, either a tolerogenic or an immunogenic T cell response will be initiated. Once activated, these T cells will expand and then recirculate back to the tissue site to reencounter their antigen, where they will then execute their effector functions . In addition to DCs, other professional APCs at the same tissue site, namely resident macrophages, also house the machinery to efficiently phagocytize and present antigen to T cells, thereby acting as essential shapers of immunity in situ.Unfortunately, tumors have evolved to evade this system of immune surveillance and activation, using several mechanisms that prevent robust and sustained T cell responses . FurtherThoughts on how immune surveillance and antigen presentation can occur in such a relatively immuno-privileged organ extended from studies examining fluid and solute homeostasis in the CNS. The brain is one of the most metabolically active organs, constitutively secreting cerebrospinal fluid (CSF) from the choroid plexus, and neurotransmitters, hormones, and trophic factors from neurons and glial cells. Furthermore, interstitial fluid (ISF) is continuously filtered through CNS capillaries, circulating throughout and washing over the brain parenchyma. As such, efficient methods are needed to transport and remove these fluids and metabolic waste from the CNS, in order to maintain a homeostatic balance.In the periphery, this trafficking of fluids, solutes, waste, as well as antigens and surveilling immune cells is achieved through the lymphatic system , which aWhile postulated and preliminarily demonstrated in the past , how we In summary, while relatively inert in the steady-state, it is clear that the brain is still capable of initiating immune activation when faced with an insult, as is seen following parenchymal damage, microbial infection, or autoimmune disease flares. This response is mediated by a direct connection between the CNS and the peripheral lymphoid organs, as the initiation of effector responses is lost if cervical LNs are ligated ,31,32. MUnderstanding the functional organization of CNS lymphatics and antigen drainage is one component of understanding how to approach reinvigorating CNS antitumoral immunity. Another element that we will discuss in the next sections is understanding the factors mediating the immunoregulatory nature of the CNS microenvironment.All of the major CNS cell types contribute to the overall anti-inflammatory microenvironment of the brain—a sensible evolutionary constraint given the high vulnerability of neurons to irreversible injury from uncontrolled inflammation. The first of these players are the cells that make up the BBB, the CNS vasculature composed of non-fenestrated endothelial cells (ECs) connected by tight junctions . PericytCompared to peripheral ECs, the BBB’s ECs have lower expression of adhesion molecules , preventThe BBB is often compromised to some degree at the lesion site, contributing to the increased blood-derived immune cells into the tumor ,52. AddiDue to its particularly important role in limiting CNS entry, targeting the BBB would be an essential means of enhancing drug or biologic penetration. The most studied method to enhance BBB penetrance is standard radiation therapy, which not only acts directly on tumor cells but also increases peripheral immune infiltration due to cytokine release from activated endothelial and stromal cells. An additional benefit of radiation therapy is that it can also induce tumor neoantigens, which contributes to the enhanced anti-tumoral responses of radiotherapy when combined with checkpoint blockade . Other mIf circulating T cells do end up passing the BBB, they will then encounter another challenge. Following extravasation, infiltrating leukocytes would enter into the perivascular space encountering the glia limitans, the final barrier before entering into the parenchyma that is composed of astrocytic end-feet surrounding the BBB ECs. The glia limitans constitutively expresses FasL  , a potenReceptor activator of nuclear factor kappa B ligand (RANKL) and other such factors secreted from tumor cells help to polarize non-transformed astrocytes, inducing them to produce pro-tumorigenic cytokines ) that enhance tumor growth and infiltration ,64. InteAnother of the key players of CNS immunity are microglia, the yolk-sac derived resident macrophages of the brain, whose survival is dependent on signaling through colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) either from CSF-1 or IL-34 binding ,70,71. MBoth animal and clinical studies have shown that GBM lesions are replete with both resident microglia and monocyte-derived macrophages (monoMacs), which also highly express CSF-1R. Together these TAMs have been shown to encompass up to 30% of the cells within the GBM TME. It is also well established that CSF-1R and tumor-associated macrophage (TAM) expression correlate with poorer patient outcomes across various cancers, including malignant gliomas . MacrophThis suggests that TAMs are a crucial target for GBM therapy and has led to the development and characterization of CSF-1R antibodies or small molecule inhibitors in various preclinical models. Blocking CSF-1R signaling can either deplete or “re-educate” TAMs ,79. ThisIt is now also becoming increasingly clear that macrophage ontogeny  might be an essential factor in their functional roles within tissues. This concept was nicely demonstrated in Bowman et al., one of the first to fate map macrophages in a glioma tumor model . This crCurrent and future work should be aimed at targeting the recruitment of monoMacs via C-C motif (CC) receptor 2 (CCR2) and CC ligand 2 (CCL2) inhibitors, key regulators of monoMac trafficking ,90. FurtThere are several models used to try to characterize the relevant microenvironmental factors mediating or hampering effective GBM tumor immunity. Some have argued that, in terms of tumor biology, the use of either human  or syngeneic mouse (GL261) cell lines is not an accurate representation. This is due in part to their propagation and differentiation in cell cultures. Furthermore, these lines tend to harbor mutations that are not often associated with patient GBM lesions . InsteadOrganoids are derived from the growth and aggregation of patient tumor-initiating cells. They allow for the 3D analysis of the TME and effectively recapitulate the hypoxic and necrotic variation found in human GBM . In addiGpnmb and Spp1, which are genes highly associated with human mesenchymal GBM tumors compared to other subtypes [While it is true that implantation of cell lines does not have exact fidelity to primary tumors, some are readily used pre-clinically, as they are still helpful in studying the importance of microenvironmental influence on GBM progression. This is particularly true for the mouse line GL261, due to its ease of scalability in experiments, presence of key histopathological characteristics of producing pseudo-palisading necrosis and microvascular proliferation. This cell line presents key aberrant signaling pathways , presence of cells that exhibit stem qualities , its “partial immunogenicity” that mimics well the “cold tumor” phenotype of human GBM, and most critically, its ability to be studied in immunocompetent in vivo systems ,96,97,98subtypes .Along these lines, another widely used preclinical system is the genetically engineered mouse model (GEMM). GEMMs use specific glioma-associated germ-line mutations in conjunction with breeding strategies to generate tumor-inducing events under tight spatial and temporal control . These sEven more complex models expressing key combinations of driver mutations that form these various GBM subtypes may soon emerge, as it is now clear that the expression of these mutations is formative in shaping the tumor immune landscape ,107,108.We have already established some of the main innate components contributing to the immunosuppressive nature of the GBM TME that need to be addressed to promote ideal anti-tumoral effector responses. Here, we now discuss some of the most promising methods for supporting the adaptive arm of the tumor immune response.It is well-known that there is a marked depletion (and aberrant function) of effector T cells in the blood and at the tumor site of patients with GBM, contributing to the overall immune suppression and evasion often seen in many cancers . InteresObservations of tumor regression in patients with concurrent viral infections have occurred for over a century ,112. SubAnother well-established method to enhance the quality of T cell priming and effector response is DC vaccination. As previously discussed, DCs are the most effective activators of T cell proliferation and effector function. Their presence is a requirement for anti-tumoral immunity, either with or without checkpoint blockade ,125. ManHowever, while promising in animal models, clinical trials of DC vaccines have yet to show a significant increase in overall survival . DespiteCheckpoint inhibition has changed the course of cancer therapeutics, particularly advanced metastatic melanoma . AlthougAnother route to activating a robust and antigen-specific adaptive response is to genetically engineer T cells whose sole targets are tumor antigens—as seen in CAR-T cell therapy. In GBM, this method has been used in a subset of patients with IL-13Rα and human epidermal growth factor receptor 2 (HER2) mutations; however it has been most extensively studied in the subpopulation of patients with EGFRvIII mutations ,143,144.Another form of T cell “re-education” and “re-direction” is through the use of BiTEs, which are two linked recombinant monoclonal antibodies that target both a T cell surface molecule (most often CD3) and a tumor-specific surface antigen ,147 – liLastly, another interesting method to boost T cell infiltration and enhance checkpoint response is the modulation of the CNS lymphatic vessels. As discussed previously, LVs are essential for fluid circulation and immune surveillance in any tissue. The recent discovery of functional LVs that drain into the deep and superficial cervical LNs within the CNS has significant implications for antigen flow and tumor response ,28. ThisDespite the wealth of knowledge gained regarding its molecular and cellular origins and its pathophysiology, effective GBM therapy remains elusive. However, recent advances made in the deep characterization of the TME have cleared new ground for assessing novel and effective treatments for this fatal disease. It has become more than apparent that the suppressive TME plays an enormous part in mitigating anti-tumoral immunity. Within the TME, the myeloid compartment is a major area of study, particularly given the enormous presence of immunosuppressive TAMs. This suppressive TME is further compounded by active sequestration of T cells from the blood and tissue, with the few effectors displaying an exhausted phenotype. Some of the more extensively studied exhaustion markers include inducible T cell co-stimulator (ICOS), lymphocyte activating 3 (LAG3), and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) . ExhaustThe ideal treatment strategy to generate robust anti-tumoral responses will likely come from the combination of a standard of care therapy along with a multi-pronged immunological approach: depleting/re-educating TAMs, boosting neoantigen expression with the use of radio- and chemotherapy, enhancing DC migration and activation in an antigen-specific manner, and finally enhancing T cell effector function—either by taking off their breaks with a checkpoint, engineering them with a specific antigenic target, or aiding their recognition and binding of tumor cells.As implied throughout this work, GBM therapy should quickly shift towards a “personalized” route, as we continue to stratify patients on both a molecular and microenvironmental level. Even more, continued immune and stromal cell monitoring of tumors—specifically in patients who do not respond to immunotherapy—will be crucial in revealing the high yield targets that need to be hit in order to overcome therapy resistance.


Cathepsins are the most abundant lysosomal proteases that are mainly found in acidic endo/lysosomal compartments where they play a vital role in intracellular protein degradation, energy metabolism, and immune responses among a host of other functions. The discovery that cathepsins are secreted and remain functionally active outside of the lysosome has caused a paradigm shift. Contemporary research has unraveled many versatile functions of cathepsins in extralysosomal locations including cytosol and extracellular space. Nevertheless, extracellular cathepsins are majorly upregulated in pathological states and are implicated in a wide range of diseases including cancer and cardiovascular diseases. Taking advantage of the differential expression of the cathepsins during pathological conditions, much research is focused on using cathepsins as diagnostic markers and therapeutic targets. A tailored therapeutic approach using selective cathepsin inhibitors is constantly emerging to be safe and efficient. Moreover, recent development of proteomic-based approaches for the identification of novel physiological substrates offers a major opportunity to understand the mechanism of cathepsin action. In this review, we summarize the available evidence regarding the role of cathepsins in health and disease, discuss their potential as biomarkers of disease progression, and shed light on the potential of extracellular cathepsin inhibitors as safe therapeutic tools. Lysosomes are intracellular membrane-bound organelles characterized by an acidic interior and harbor a variety of hydrolytic enzymes including lipases, proteases and glycosidases that participate in cellular catabolism ,2. The fAmong the variety of enzymes that lysosomes harbor, cathepsins are a family of lysosomal proteases with an astonishingly broad spectrum of functions. Cathepsins help in intracellular house-keeping where they for example, participate in the antigen processing during immune responses and degrade several proteases and chemokines to maintain cellular homeostasis . MammAlthough cathepsins exhibit some level of similarity in their proteolytic functions in physiological processes, consequences of cathepsin dysfunction are very diverse in terms of clinical symptoms. Deregulated cathepsin synthesis and activity has been associated with several diseases including the metabolic syndrome, cancer and inflammatory neurological diseases (reviewed in ). CathepThis review is divided into four parts. In the first part, we provide a basic overview of cathepsin expression and function within the endo/lysosomal compartments. In the next two parts, we emphasize on the mechanisms leading to the translocation of cathepsins into the cytosol and extracellular milieu respectively. Finally, this review further highlights the prospect of using highly specific, targeted cathepsin inhibitors for clinical implementation in various diseases.Cathepsins exhibit similarities in their cellular localization and biosynthesis with some differences in their expression pattern. Of all the lysosomal proteases, cathepsins L, B, and D are the most abundant with their lysosomal concentrations equivalent to 1 mM . CathepsCathepsins undergo transcriptional, translational, post-translational and epigenetic regulation. At transcriptional level, except for cathepsin D all other cathepsins show TATA-independent transcription initiation which defines the transcription initiation site. Cathepsin L, K, C, and B are known to require various transcription factors such as nuclear factor (NF-Y), specificity proteins, Sp1, Sp3 and erythroblast transformation-specific (Ets) family factors for transcription initiation and regulation ,29. Cath155–K173 to yield a double-chain mature cathepsin D [The primary structure of all cathepsins consists of a signal peptide, a propeptide, and a catalytically active mature functional enzyme. Cathepsins are synthesized as preprocathepsins in the endoplasmic reticulum (ER). A N- terminal signal peptide of 20–25 amino acids directs preprocathepsins into the ER lumen where they are cleaved co-translationally by a signal peptidase generating procathepsins, the less active zymogen forms. Simultaneously, N-linked glycosylation occurs within the ER, producing high levels of mannose in the procathepsins . Procathhepsin D .While most of the lysosomal enzymes are dependent on M6P for their transport, several studies have reported the existence of M6P-independent transport routes of cathepsins to reach endo/lysosomes with the aid of alternative receptors. One such alternative receptor protein is sortilin, a trans-membrane Golgi protein, which was shown to be involved in the sorting of cathepsin H and cathepsin D in COS-7 cells . In mousCathepsins carry out many proteolytic events in the compartments of the endocytic pathway thus contributing to the protein turn-over and normal metabolism of the cell. The pH inside the endo/lysosomal compartment favors cathepsin activity while it induces conformational changes in the substrates leading to their cleavage by cathepsins, and thereby helping cathepsins to successfully degrade the cargo transported to the endo/lysosomes ,47.Cathepsins display significant roles predominantly in the endosomes of immune cells. They are known to participate both in the innate and adaptive immune responses. During the innate immune responses, lysosomal cathepsins have been shown to cleave the ectodomains of Toll-like receptors (TLRs) 7 and 9 that are expressed on endo/lysosomal membranes, where TLRs recognize nucleic acids of phagocytosed microbes. The processed forms of TLR 7 and 9 then recruit the adaptor protein MyD88 leading to activation of TLR signaling pathways . During Cathepsins participate in autophagy, an essential catabolic process which delivers cytosolic constituents to lysosomes for their subsequent degradation in order to maintain cell homeostasis . As demoCathepsins regulate growth and development related processes by processing various hormones and growth factors. For instance, cathepsin B degrades a variety of substrates including insulin-like growth factor-1 (IGF-I), glucagon, pituitary hormone, thyroglobulin ,57. AddiInactivation or loss-of-function of cathepsins results in inappropriate degradation and abnormal accumulation of substrates in lysosomes leading to various diseases including lysosomal storage disorders (LSD). Further, defective proteolytic events by cathepsins can lead to several LSDs, namely, galactosialidosis  neuronalAs described in the previous section, proteolytic functions of cathepsins mainly occur in the endo/lysosomal compartments. However, most of the cathepsins are released into the extralysosomal locations such as cytosol, nucleus, and mitochondria where they perform crucial tasks. In the current section, we describe the mechanism of cathepsin translocation into different regions of the cell and their respective functions.Perhaps one of the most simplistic mechanisms though which cathepsins are translocated to cellular compartments other than lysosomes is by leaking outside of lysosomes. Lysosomes are surrounded by a limiting membrane, which is a phospholipid bilayer that is characterized by variety of integral membrane proteins, which together protect the organelle from the degradative enzymes . Damage Lysosomal destabilization is recognized feature during oxidative stress-mediated cell damage. The initial burst of reactive oxygen species (ROS) may be generated by different triggers inside or outside of the lysosomes. For example, ceramide accumulation or ingestion of heavy metals such as silica or asbestos is known to increase NAPDH oxidase in the lysosomes to generate ROS. The generated free radicals interact with free intralysosomal iron forming highly reactive hydroxyl radicals in a Fenton-type reaction . Such hyLysosome-targeting agents, referred to as lysosomotropic agents and lipid detergents cause LMP either by direct membrane lysis or by osmotic lysis of lysosomal membrane. Examples of agents that cause direct membrane lysis include O-methyl-serine dodecylamide hydrochloride (MSDH), N-dodecylimidazole, n-β-naphthylamide, and endogenous compounds such as lipofuscin. Due to their amphiphilic nature, these agents partition between the water phase and the phospholipid bilayer of lysosomal membrane. Further, accumulation of the lysosomotropic agents and molecules leads to thinning of the lysosomal bilayer and subsequent solubilization of lysosomal membrane (LM) (reviewed in ). LysosoBesides LMP, other mechanisms such as alternative translation or exon skipping can lead to extra lysosomal translocation of cathepsins. Translation initiation at a different start site produces cathepsins which are devoid of signal peptide, while exon skipping generates truncated cathepsins with modified signal sequences thus misrouting cathepsins from their biosynthetic pathway to locations such as cytosol, nucleus, and mitochondria. For instance, translational initiation at downstream AUG site is known to localize cathepsin L in nucleus  and alteAlthough cathepsins do not retain optimal activity at the neutral pH of the cytosol, their proteolytic activity is known to be preserved by substrate binding and acidification of the cytosol observed under certain conditions like apoptosis and cathepsins in the cytosol are known to mediate key physiological processes as described below.Apoptosis is a highly regulated, fundamental physiological process of cell death responsible for removal of damaged/aging cells by activation of caspase family of proteins. Apoptosis involves two different pathways; intrinsic pathway, also known as the mitochondria pathway, or the extrinsic pathway involving death ligands (reviewed in ). The miRecent studies have shown that cathepsins regulate programmed necrosis termed necroptosis. Necroptosis is initiated by various stimuli and requires the kinase activity of receptor-interacting serine/threonine kinase1 (Rip1). In macrophages, it has been reported that cathepsins B and S are known to cleave receptor-interacting protein 1 (Rip1) kinase and thus limit macrophage necroptosis .Cytosolic cathepsins are shown to be involved in mediating inflammatory responses via activation of inflammasomes. The NLRP3 inflammasome is a multi-protein complex which is activated upon bacterial infections, LMP or cellular damage. Upon activation, pro-caspase1 is converted to active caspase1 by autocatalysis. Active caspase1 then proceeds to cleave the cytokine precursors pro IL-1β and IL-18 into their mature secreted forms (reviewed in ). Gene kCathepsins in the nucleus are known to process transcription factors that control cell cycle progression thus facilitating cell proliferation and differentiation  to bridge the opposing charges on the membranes. Further, lysosomal docking and fusion are regulated by transcription factor EB (TFEB). EB modulates lysosomal exocytosis by triggering intracellular Ca2+ elevation through the endo/lysosomal cation-channel mucolipin 1 (MCOLN1) [The secretory pathway of lysosomes, known as lysosomal exocytosis, has been reported in many types of cells and is induced by various stimuli such as wound, cellular stress, cancer, or by signals from cytokines . The ind(MCOLN1) . Finally(MCOLN1)  elegantlAs described in the Cathepsins require acidic pH for their optimum activity in contrast to the neutral pH found in extracellular space. However, cathepsins are often secreted as less active procathepsins which are normally stable at neutral pH . For catECM is composed of fibrous proteins such as elastin, collagen, proteoglycans, and fibronectins that form the intricate meshwork for holding the cells embedded within the tissues Further, ECM undergoes constant remodeling and ECM components have several binding sites for growth factors that ultimately control cell adhesion, proliferation, migration, and polarity . ECM conBone remodeling is a continuous process that involves bone resorption and remodeling performed by specialized cells called osteoclasts. Further, bone and cartilage predominantly contain type 1 collagen. During bone resorption, osteoclasts attach to the surface of bone leading to the creation of an extracellular compartment which is isolated from general extracellular fluid (reviewed in ). NumeroCathepsins on the cell surface are known to be involved in plasma membrane repair. Cathepsin B released from keratinocytes attaches to cells surface where it is known to be involved in keratinocyte migration by degrading components of ECM during wound healing . AnotherNeurons and endocrine cells carry out cell–cell communication with the help of peptide neurotransmitters. Secretory vesicles in these cells provide regulated secretion of neurotransmitters, which are first synthesized as inactive prohormones. Proteolytic processing of the proproteins or prohormones occurs in the secretory vesicles by cathepsins . NumerouWhile ECM remodeling is an important physiological process, aberrant ECM dynamics can lead to uncontrolled cell proliferation, invasion, and differentiation leading to fatal pathologies including atherosclerosis, cancer, and tissue fibrosis . ExtraceCancer results from abnormal cell proliferation in the body. Increased or abnormal proteolytic activity of cathepsins is known to degrade ECM components facilitating migration and invasion of tumors leading to malignancy (reviewed in ). CathepElevated levels of extracellular cathepsins have been identified in various cancers such as breast, lung, colon, pancreas, skin, prostate, bladder, ovary, and head and neck . In addiLipoprotein accumulation and metabolism are important contributors to various diseases including cardiovascular diseases and obesity -associated disorders such as non-alcoholic steatohepatitis (NASH). Extracellular cathepsins are found to be involved in transport, efflux, and processing of lipoprotein molecules or their receptors (reviewed in ). For inThe mechanisms involved in the translocation of cathepsins and their respective site-specific functions are illustrated in Localization of cathepsins  governs several aspects of cathepsin function. A wealth of knowledge has been published on the differential expression and functional profiles of cathepsins in various pathologies making them potential diagnostic biomarkers and most desirable therapeutic targets.Cathepsin levels and activity have been found to be upregulated in sera and tumors of many cancer patients ,177. ForIn diseases caused by inactivation or loss-of-function of cathepsins, supplying functional cathepsins could be a valuable means to restore cellular function and ameliorate the disease. For instance, enzyme replacement therapy (ERT) replacing defective lysosomal cathepsin D by recombinant procathepsin D has been proven beneficial for CLN10 . HoweverIncreased understanding of the structure, differential expression and localization of cathepsins in various pathologies has opened a new avenue for the design of small molecule inhibitors of cathepsins with the hope of producing highly specific, targeted drugs for many diseases. Several small molecule inhibitors of cathepsin S and K are being tested in clinical trials . AdditioIn conclusion, cathepsins are a group of enzymes with distinct functions at different locations inside and outside of the cells. While many recent findings helped us to further understand these roles and establish the potential for targeting, many mechanical aspects of cathepsin action are still to be explored. Currently, the extracellular role of cathepsins have gained enormous attention in the biomedical field, establishing them as non-invasive diagnostic markers and pharmacological targets in immune disorders, cancer, osteoarthritis, and metabolic diseases. Many selective cathepsin inhibitors with limited side effects are being developed and have shown success in the preclinical animal models and are awaiting validation in the clinical setting. With the advent of new mass spectrometry technologies and systems biology approaches, the future research should focus to better understand the specific substrates of cathepsins in the physiological and pathological environment. Finally, the future of cathepsins in targeted drug delivery looks more promising than ever.