Chemokine signaling is crucial for tumorigenesis, proliferation, angiogenesis, tumor progression and metastasis in glioblastoma. Furthermore, chemokines have an impact on the overall survival of GBM patients that, with a median survival of 15 months, still lack effective treatment. Several chemokine signaling axes, for instance CXCR2/CXCL2/IL-8, CXCR4/CXCL12 and CCR2/CCL2, were investigated in detail and appear to be promising therapeutic targets. The aim was to review novel approaches that target chemokines or chemokine receptors in glioblastoma. Here, preclinical and clinical studies were used to clarify their significance for targeted treatments alone and in combination with other therapeutic applications.With a median patient survival of 15 months, glioblastoma (GBM) is still one of the deadliest malign tumors. Despite immense efforts, therapeutic regimens fail to prolong GBM patient overall survival due to various resistance mechanisms. Chemokine signaling as part of the tumor microenvironment plays a key role in gliomagenesis, proliferation, neovascularization, metastasis and tumor progression. In this review, we aimed to investigate novel therapeutic approaches targeting various chemokine axes, including CXCR2/CXCL2/IL-8, CXCR3/CXCL4/CXCL9/CXCL10, CXCR4/CXCR7/CXCL12, CXCR6/CXCL16, CCR2/CCL2, CCR5/CCL5 and CX3CR1/CX3CL1 in preclinical and clinical studies of GBM. We reviewed targeted therapies as single therapies, in combination with the standard of care, with antiangiogenic treatment as well as immunotherapy. We found that there are many antagonist-, antibody-, cell- and vaccine-based therapeutic approaches in preclinical and clinical studies. Furthermore, targeted therapies exerted their highest efficacy in combination with other established therapeutic applications. The novel chemokine-targeting therapies have mainly been examined in preclinical models. However, clinical applications are auspicious. Thus, it is crucial to broadly investigate the recently developed preclinical approaches. Promising preclinical applications should then be investigated in clinical studies to create new therapeutic regimens and to overcome therapy resistance to GBM treatment. IDHwt GBM, mutated IDH GBM and not otherwise specified GBM . IDHwt GBMs represent 90% of glioblastomas and are mainly primary GBMs that develop de novo in elderly patients. Only 10% of GBM patients exhibit an IDH mutation and correspond to secondary GBMs which originate from astrocytic tumors or oligodendrogliomas that occur in younger patients and have a better prognosis. Furthermore, GBMs were described by various molecular biomarkers besides IDH, including MGMT promoter methylation (O6-methylguanine DNA methyltransferase), chromosome 1p/19q deletion, TERT (telomerase reverse transcriptase) promoter mutation, TP53 (tumor protein P53) mutation, PTEN (phosphatase and tensin homolog) mutation, EGFR  and PDGFRA (platelet-derived growth factor receptor A) amplification. Especially, the MGMT promoter methylation is often used as a prognostic marker in GBM. A higher MGMT promoter methylation leads to a lower MGMT expression, supporting a better prognosis of the respective GBM patients [MGMT enzyme repairs the DNA damage caused during temozolomide (TMZ) therapy and therefore is responsible for drug resistance of glioblastoma cells to anticancer treatments [Glioblastoma (GBM) is the most common malignant brain tumor in adults ,2 and beeatments . The difThis highly vascularized tumor shows tremendous growth and depends on the formation of new blood vessels ,9,10. Ac+ cytotoxic T lymphocytes (CTLs), CD4+ T helper cells (Th1), regulatory T cells (Treg) and natural killer (NK) cells infiltrate glioma tissues [A special feature of GBM is the high infiltration of myeloid cells consisting of resident microglia and peripheral macrophages  which ma tissues . Thus, i tissues . Neverth tissues  and the  tissues ,24.Another cell population that occurs in glioblastoma tissues are glioma stem cells (GSCs). GSCs have the capability for self-renewal and differentiation to form a tumor . These GConsequently, despite new therapeutic approaches, including antiangiogenic treatment, tumor treating fields (TTF) and immunotherapies, OS has only marginally improved for GBM patients in recent years ,31,32,33Chemokines are small proteins known as the largest subfamily of cytokines . The namWithin the tumor, chemokines support many tumor-sustaining processes such as cell proliferation, tumor growth, angiogenesis and metastasis, and thus modify the tumor microenvironment ,49,55,61In this study, we aimed to review preclinical and clinical studies regarding the CXCR2/CXCL2/IL-8, CXCR4/CXCL12/CXCR7, CCR2/CCL2, as well as the less investigated CXCR3/CXCL4/CXCL9/CXCL10/CXCL11, CXCR6/CXCL16, CCR5/CCL5 and CX3CR1/CX3CL1 signaling pathways. We demonstrated different therapeutic approaches as targeted therapies, in combination with temozolomide  and radiotherapy, antiangiogenic agents as well as immunotherapies.Targeted therapies are an important step towards precision medicine and overcoming therapy resistance. While one therapy might help one patient, it might not help another one. The standard-of-care therapeutic regimens for GBM, established more than a decade ago, evidently fail to prolong patient OS for more than a few months ,4. ThereIL8, CXCL2, CXCR1 and CXCR2 are expressed by various GBM tumor cell lines as well as by tumor cells and endothelial cells in GBM patients [IL-8 reduced GBM cell invasiveness in vitro [Enhanced expression of CXCR2 and its ligands is associated with malignancy and recurrence in glioma ,67. IL8,patients ,61,68,69in vitro . There, in vitro .Mesenchymal stem cells (MSC) showed multipotent differentiation characteristics in vitro and can be isolated from the bone marrow, adipose tissue and the umbilical cord as described before . Due to Apart from blocking antibodies, antagonists and gene knockdown, Hübner et al. targeted a network of proinflammatory genes. In their innovative approach, using microRNA-93 (miR-93) which directly regulates glioma cell gene expression of HIF-1α and MAP3K2, cytokines and chemokines like IL-8 and also indirectly targets CXCL5 and COX-2, they showed various functions of this specific miRNA . Interes+ T cells and NK cells [CXCR3 signaling mediated by CXCL9, CXCL10, CXCL11 and CXCL4 plays a major role in inflammation and immunopathology as well as in prohibiting angiogenesis in gliomas ,39,79. CNK cells ,37, but NK cells ,82,83,84NK cells . AdditioNK cells  and CXCLNK cells ,81,82,87NK cells ,81,82,87NK cells . Neverth+ T lymphocytes express CXCR4 [The CXCR4/CXCR7/CXCL12 signaling pathway has been extensively studied in recent years and is one of the best-evaluated chemokine signaling pathways in the glioblastoma microenvironment. Among its various functions, CXCR4 signaling is linked to glioma cell invasion, proliferation, tumor progression and angiogenesis ,90,91,92ss CXCR4 ,97,98.AMD3100, also known as plerixafor, is an FDA-licensed CXCR4 antagonist which inhibited migration of glioma cells in vitro under normoxic and hypoxic conditions ,100. FurThere is a vast number of additional CXCR4 inhibitors including CPZ1344, AMD3465 and Peptide R that reduced tumor growth ,107,108 Apart from pharmacological approaches, some researchers focused on different microRNAs and how to best exploit their antitumoral functions in GBM. The expression of miR-9 directly targeted CXCR4 signaling in GBM; it functioned as a tumor suppressor by inhibiting the migratory capacity of GBM cells in vitro . AnotherA different approach that utilizes CXCR4 overexpression in glioma cells without directly interfering in the pathway was recently proposed by Gascon et al. . The useAnother pillar of novel therapeutic approaches is cell-based, specifically, stem cell-based therapy. There are conflicting data on the advantages and disadvantages of this specific therapeutic approach. Whereas some studies showed that migration of CXCR4-overexpressing human adipose-derived stem cells and human mesenchymal stem cells to the tumor resulted in reduced tumor volume and prolonged survival of tumor-bearing mice ,119, PavCCR5 downregulation significantly reduced tumor growth in a glioma xenograft mouse model [CCR5 deletion had no effect on the survival of tumor-bearing mice [+ T cells, however, a subset of activated CD8+ T cells has been shown to be enriched with CCR5 and is associated with disease progression [Another important signaling pathway in GBM is mediated by CCR5 and CCL5. Key functions of both the receptor and the ligand have been demonstrated. For instance, high expression of CCR5 and CCL5 correlates with a survival disadvantage of GBM patients . Furtherse model . Howevering mice . Blockining mice . In patiing mice . CCR5 ising mice . The immgression .CX3CL1 expression correlates with glioma grade and serves as a prognostic marker for glioma patient OS [CX3CL1 positively correlate with patient OS in glioma [Glioma cells express both CX3CR1 and CX3CL1 at mRNA and protein levels ,124. Furtient OS . Analysin glioma . Sciume n glioma  and NK cn glioma , which an glioma ,127, expn glioma ,126,128.A variety of these promising therapeutic approaches has been evaluated in detail in preclinical in vivo models and clinical phase I and II studies which will be discussed in the respective chapters. Firstly, we reviewed the combination with the standard-of-care therapy; secondly, the combination with antiangiogenic agents; and lastly, immunotherapeutic approaches.The current standard-of-care for GBM was established more than 15 years ago and is known as the Stupp protocol, which includes maximal resection and radio-chemotherapy followed by six cycles of temozolomide . Grand tThe CXCR4/CXCR7/CXCL12 signaling pathway plays an important role in proliferation, differentiation, cell survival and chemotaxis in glioblastoma ,89. NOX-These findings are emphasized by the effect of three alternative CXCR7 inhibitors CCX771, CCX662 and X7Ab which led to tumor regression ,84, blocDue to the groundbreaking achievements in preclinical studies with AMD3100 blocking CXCR4/CXCL12 signaling, its efficacy in the first clinical studies is being investigated. In 2013, a then 66-year-old male patient received AMD3100 as part of his first-line therapy . The patCXCL12 and CXCR4 which was enhanced by treatment with omeprazole [Other therapeutic approaches include unconventional therapies like omeprazole, a proton-pump inhibitor. Omeprazole inhibits GBM cell invasion and migration through acceleration of tumor-suppressive properties of the aryl hydrocarbon receptor (AhR) which is expressed in GBM in vitro and in a subcutaneous PDX glioma mouse model . AhR supeprazole . The effeprazole .188Re) could potentially reach disseminated tumor cells which often escape therapeutic approaches [Another very interesting approach of combining radiotherapy with CXCR4-targeted therapy was recently introduced by Séhédic et al. Their novel model of a nanocarrier-loaded combined lipophilic thiobenzoate complex of rhenium-188 in the core of a nanocapsule  in GBM. VEGF and its receptors are abundantly expressed in GBM . Angioge+ vessel-like structures, which account for a process called vascular mimicry (VM), in GBM patients [+ VM structures in vivo [CXCR2 and its ligands CXCL2 and IL-8 are overexpressed in GBM and associated with a reduced OS ,50,67. Apatients ,144. In patients , therefoSimilar effects were observed in an immunocompetent mouse model where the application of SB225002 led to reduced vessel density, inhibition of tumor growth, reduction in the tumor volume by about 50% and decreased infiltration of TAMs in vivo . FurtherVEGF expression [CXCR4 and CXCR7 alongside other proangiogenic molecules are overexpressed in GBM ,55,64. Cpression ,146, whipression . Severalpression . Additiopression .In an in vitro study with U87 glioblastoma cells, another important crosstalk was evaluated. There, the activation of CXCR4 by CXCL12 led to the protein expression of IL-8 . Nordy, Apart from its combined effect with the standard-of-care, NOX-A12 targeting CXCL12 exhibited antiangiogenic properties in a rat model . There, + myeloid cells, compared to vatalinib [As mentioned above, AMD3100, a well-known CXCR4 inhibitor, reduced vessel density and tumor growth in both rat and mouse glioma models ,149,150.atalinib . It is natalinib . Furtheratalinib . An addiatalinib . Vatalinatalinib  which coatalinib . Howeveratalinib . Althougatalinib ,154. NevOther CXCR4 antagonists, for instance, PRX177561 and POL5551, have not been extensively studied in combination with AAT. However, their efficacy seems to be comparable to AMD3100. PRX177561 reduced tumor cell proliferation and migration and induced apoptosis in vitro . FurtherThe combination of VEGF/VEGFR pathway inhibitors with CXCL12/CXCR4 inhibitors showed promising results, making this therapeutic concept an interesting approach that could increase patient survival. However, CXCR4 expression is variable. Therefore, as demonstrated by Dono et al., it is essential to stratify patients according to their CXCR4 expression, as only a subgroup of patients would benefit from anti-CXCR4 therapy .VEGFA gene expression [VEGF expression in human GBM cells and human endothelial cells significantly decreased in vitro [A very different approach was presented by Egorova et al. These authors developed a modular peptide “L1” which was labelled with a CXCR4 ligand to target CXCR4-expressing tumor and endothelial cells . Therefopression . With thin vitro . Furtherin vitro .Vegf, and the tumor vasculature was less leaky and more mature, underlining the importance of CCR2 signaling in GBM angiogenesis [+ monocytes, proliferation and vessel density in a GBM rat model [It is known that CCL2 attracts TAMs  which reogenesis . Using tat model . Althougat model . Furtherat model .+ T cells, NK cells, granulocytes and TAMs [Inflammatory stimuli are broadly mediated by the chemokine network. Chemokines are involved in autoimmunity, allergy, chronic inflammatory disease and cancer . Apart fand TAMs ,37,39,64and TAMs ,161,162.and TAMs ,37,39,64and TAMs ,164. In Fighting tumors with genetically engineered immune cells has been a challenging task in cancer research. A promising approach in cell-based tumor therapy is the use of chimeric antigen receptor (CAR) T cells which, upon migration to the tumor, fight it by activating multiple immunological responses . HoweverCxcr3ko mice prolonged the median survival of both groups but had no impact on tumor-infiltrating microglia and lymphocytes [CCL2 reduction and upregulation of CXCL10 [+ cytotoxic T lymphocytes, while the number of tumor-promoting MDSCs was decreased [CCL2, CXCL10 and CXCR3 [CCL2 and CXCR3, but not of CCR2 or CXCL10 [CCL2 [CXCL10 expression on gene expression levels; however, the used mouse models were different [CXCR3 is a pivotal receptor in neuroinflammatory and neurodegenerative diseases ,39,167. phocytes . Standarphocytes . COX-2 if CXCL10 ,171. Furecreased . Additioecreased ,164. Furecreased . As CTLsecreased . In anotnd CXCR3 . Furtherr CXCL10 . Moreove10 [CCL2 . These fifferent ,171,173.ifferent ,171,173.ifferent ,81,82,87CXCL10ko DCs, thus underlining the importance of CXCL10 in vaccine efficacy [Alternative approaches are cell-based therapies and vaccinations. Over a decade ago, Fujita et al. demonstrated the relevance of CXCL10 for DC-based vaccines in GBM . In theiefficacy . In the efficacy . This coefficacy . Therefoefficacy  and Yan efficacy . Anotherefficacy .De Waele et al. proposed a combination of poly(I:C), a synthetic analog of double-stranded RNA and inducer of interferons, with PD-L1 blockade . Anti-PD+ glioblastoma cells. Infusion of these modified NK cells hindered tumor growth and increased the median survival of subcutaneous tumor xenografts in mice [+/CD8+ ratio and additionally increased levels of circulating inflammatory antitumoral cytokines [It has been demonstrated that NK cells infiltrate gliomas to a lesser extent than other leukocytes under normal circumstances . Müller  in mice . Due to  in mice . Other i in mice ,127. The in mice . There,  in mice . Furtherytokines . Both imCcr2 knockdown led to a decreased number of TAMs in a GL261 mouse model [CCR2/CCL2 signaling is known for its proangiogenic and proinflammatory properties. CCL2 recruits regulatory T cells ,185, NK se model . Dexametse model . In a rese model . Furtherse model . A novelse model . Combinise model   mice to wildtype mice and found that Cxcr6ko mice had a distinct prolonged survival [The less investigated CXCR6/CXCL16 signaling pathway has recently shown to be a feasible target in GBM treatment. While CXCL16 is widely expressed by glioma and glioma stromal cells in vitro and in situ, CXCR6 has been shown to likely be restricted to highly proliferative glioma stem cells ,195. Viasurvival .The chemokine network is a crucial part of the tumor microenvironment in GBM, and chemokine signaling is involved in many tumor-promoting processes. Furthermore, targeting chemokine signaling is a novel therapeutic approach that has shown promising results in preclinical studies in vitro and in vivo. Nevertheless, to date, only agents targeting CXCR4/CXCR7/CXCL12 signaling have been evaluated in clinical studies, showing encouraging results. Thus, based on the promising preclinical studies, new chemokine-targeting therapies should be evaluated in clinical trials, assessing treatment efficacy, PFS and patient OS. As we have learned from previous studies, combining targeted therapies with the standard-of-care, established antiangiogenic treatment or immunotherapeutic approaches should be considered as most effective. These combined therapies could help improve patient OS and overcome therapy resistance in GBM treatment.


The left ventricular noncompaction cardiomyopathy (LVNC) is a rare subtype of cardiomyopathy associated with a high risk of heart failure (HF), thromboembolism, arrhythmia, and sudden cardiac death.The proband with overlap phenotypes of LVNC and hypertrophic cardiomyopathy (HCM) complicates atrial fibrillation (AF), ventricular tachycardia (VT), and HF due to the diffuse myocardial lesion, which were diagnosed by electrocardiogram, echocardiogram and cardiac magnetic resonance imaging. Peripheral blood was collected from the proband and his relatives. DNA was extracted from the peripheral blood of proband for high-throughput target capture sequencing. The Sanger sequence verified the variants. The protein was extracted from the skin of the proband and healthy volunteer. The expression difference of desmocollin2 was detected by Western blot.DSC2 gene encoding an important component of desmosomes was detected by targeted capture sequencing. The western blots showed that the expressing level of functional desmocollin2 protein (~ 94kd) was lower in the proband than that in the healthy volunteer, indicating that DSC2 p.K47Rfs*2 obviously reduced the functional desmocollin2 protein expression in the proband.The novel heterozygous truncated mutation (p.K47Rfs*2) of the DSC2 p.K47Rfs*2 remarkably and abnormally reduced the functional desmocollin2 expression, which may potentially induce the overlap phenotypes of LVNC and HCM, complicating AF, VT, and HF.The heterozygous  MYH7), protein-binding protein C myosin (MYBPC3), tropomyosin alfa (TPM1), myocardial actin (ACTC1), troponin T (TNNT2), and cardiac troponin I. The less common mutations of genes inducing LVNC include Z-band protein mutations, such as Cypher/ZASP cytoskeleton protein, alpha-distrobrevin (DTNA), calcium transport proteins, calsequestrin, phospholamban, membrane proteins (A/C lamina), para-zinc-finger transcription factor [The left ventricular noncompaction cardiomyopathy (LVNC) is a rare subtype of cardiomyopathy (CM) and is characterized by predominant left ventricular trabeculations with deep intertrabecular recesses and thinning of the compact epicardium . LVNC can factor , and mitAll participants signed informed consent. All procedures performed in the study involving human participants were in accordance with the Declaration of Helsinki and ethical standards and were approved by the Medicine Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (No. 2020K216-1).In accordance with previous studies , 9, LVNCImaging was acquired using the 1.5 T MR imaging system  by using a 6-channel phased-array coil. After scout and reference scans, functional and geometric assessments were achieved using ECG-gated, cine steady-state-free precession images in standard long-  and short-axis orientations with full ventricle coverage from basis to apex. Left and right ventricular outflow tract cine sequences were also obtained. Cine images with a temporal resolution of approximately 40 ms were obtained. Additional pulse sequences, including T1WI and T2WI of blood-suppressed double inversion recovery fast spin-echo, were acquired in a standard location before contrast administration. Then, the dynamic enhancement was performed during the administration of 0.2 mmol/kg contrast agent  with 50 frames. Late gadolinium-enhanced imaging was performed with 20 min delay and phase-sensitive inversion recovery sequence to detect myocardial scarring or fibrosis. CMRI analyses were performed using SigoVia (Siemens). The functional and geometric left ventricular indices, including left and right ventricular end-diastolic volumes, LVEF and right ventricular ejection fraction, indexed left ventricular compacted mass with papillary muscles, indexed left ventricular compacted and noncompacted mass, indexed left ventricular noncompacted mass, and their ratio was determined. In addition, the end-diastolic extent of compacted and noncompacted myocardium and their ratio were measured in one long axis geometry.http://sift.jcvi.org/www/), PolyPhen-2 , and MetaSVM [Skin biopsies were collected from the upper left arm of the proband and healthy volunteer. Protein was extracted from tissues. Peripheral blood was collected from the proband and his relatives. DNA was extracted  from the peripheral blood of proband for high-throughput target capture sequencing using Gene fragment capture chip  and MGIseq-2000 sequencer . The panel of common risk genes associated with CMs and arrhythmias Table  was dete MetaSVM . Variant MetaSVM  followin MetaSVM . Variant MetaSVM .Table 1SAccording to ACMG standards and guidelines, all variants have been reclassified for interpreting sequence variants as P, LP, VUS, LB, or B . The PM2DSC2 p.I520T was screened again using Sanger sequencing  in the other members of the family. The mutation of DSC2 p.K47Rfs*2 was screened again using the TA cloning technique in the other familial members. The purified PCR product was directly linked with pClone007 Versatile Simple Vector  by TA cloning technique and transformed into Escherichia coli (DH5α). The extracted plasmids were sequenced with ABI 3730XL . The primer of TA cloning sequencing was as follows: M13F-47:5′-CGCCAGGGTTTTCCCAGTCACGAC-3′. The primer designed with Primer Premier 5.0 was used and showed as follows: forward primer, 5′-AAGGCTATTAGAAAGCAGAC-3′; reverse primer 5′-ATATGACCCAGAAACAAGAA-3′.The variant of Tissues were homogenized on ice in the lysis buffer. After centrifugation and protein quantification, proteins were loaded onto SDS–PAGE gels and transferred onto nitrocellulose membranes. Membranes were incubated in 5% nonfat dry milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies. Rabbit anti-DSC2 and mouse anti-β-actin antibody were purchased from Abcam Inc . Antibodies were detected using 1:10,000 horseradish peroxidase-conjugated, donkey anti-rabbit, and donkey anti-mouse IgG . The Western blot luminol reagent was used to visualize bands corresponding to each antibody.4–V6  suffered from hypertension and stroke. He was persistently laid in bed for three years and subsequently died at the age of 67 years. I: 2 (mother of II: 1) died of SCD induced by acute myocardial infarction on the way to the hospital at 73 years old. We could not receive clinical information and blood samples of I: 1 and I: 2. The echocardiograms of II: 2 and II: 3 were normal. No characteristic change related to CMs was observed in the ECGs of II: 2 and II: 3  and DSC2 p.I520T (NM_004949: EX11/CDS11: c.1559T>C), which were located in chromosome 18. The local and 1000 Genomes Project database frequency of DSC2 p.K47Rfs*2 and p.I520T was less than 0.001. DSC2 p.K47Rfs*2 was not detected in gnomAD exomes combined population. Desmocollin2 encoded by the DSC2 gene was an important component for desmosome assembly. Abnormal desmocollin2 caused the dysfunction of cell–cell adhesions and intercellular gap junctions, failing to hold together the cardiomyocytes, fibrofatty myocardial replacement, cardiac conduction delay, mechanically ventricular dysfunction and arrhythmias [DSC2 p.K47Rfs*2 converted the 47th amino acid (AA) from lysine to arginine and led to a premature termination codon at the 48th AA, which was therefore considered as frameshift and truncated mutation. This phenomenon caused the interrupting protein synthesis at the cadherin pro domain and loss of protein structure, including extracellular domains 1–4, extracellular anchor, a transmembrane domain, intracellular anchor, intracellular cadherin-like sequence, resulting in truncated protein without normal function  was demonstrated to be benign or likely benign by Clinvar database and the CHEO Genetics Diagnostic Laboratory in Children’s Hospital of Eastern Ontario (https://www.ncbi.nlm.nih.gov/clinvar/variation/155783/). The MAF of DSC2 p.I520T was 0.02% and not fulfilled with the criteria of the vast majority of reported pathogenic variants (MAF < 0.01%). Additionally, according to the analysis of the UCSC Genome Browser, the amino acid site of DSC2 p.I520 is not conserved in several species  by target capture sequencing, and the rest of the family members were subjected to Sanger sequencing. The genetic screening revealed that the proband (II: 1) carried 10 exonic variants Table , includihythmias . Accordi by targeMYH7, MYBPC3, TPM1, TAZ, TTN, and NONO genes were the most common genes causing LVNC. LVNC caused by MYH7 and MYBPC3 mutations often complicated congenital heart diseases (CHD), such as atrial septal defect, ventricular septal defect, Ebstein, tetralogy of Fallot, patent ductus arteriosus, patent foramen ovale, and aortic hypoplasia. Some cases occurred with thromboembolism. Recently, LVNC induced by DES and DSP mutations was common and young-onset with HCM, DCM, ACM, myocarditis and HF, complicating ventricular and atrial tachycardias, and even needed therapy of heart transplantation in some cases. LVNC was associated with FBN1 mutation combined with DCM and Marfan syndrome. LVNC induced by HCN4 and EMD mutations complicated sick sinus syndrome (SSS), AF, atrial standstill, and interventricular block. Additionally, the LVNC induced by EMD mutation was combined with DCM. The LVNC caused by RYR2, KCNQ1, and KCNH2 mutations occurred with catecholamine-sensitive VT, long QT syndrome (LQTs), torsade de pointes, and ventricular fibrillation. In contrast, the LVNC result from SCN5A mutation complicated SSS, AF, LQTs, Wolff–Parkinson–White syndrome, VT, and atrioventricular block. Some genes led to LVNC and caused complex and critical clinical disease syndromes, such as Rubinstein–Taybi syndrome , Ehlers–Danlos syndrome , Emery–Dreifuss muscular dystrophy , Danon disease , intellectual disability syndrome , Sotos syndrome , LEOPARD syndrome , Coffin-Lowry syndrome , Cornelia de Lange syndrome , Barth syndrome , and Holt–Oram syndrome . Additionally, ARFGEF2, MIPEP, NONO, SH2B1, and TMEM70 led to LVNC complicating developmental delay. The deletion of one of these genes, including FKBP12, JARID2, NUMB, and PLZND1, induced LVNC by affecting the activity of the Notch signaling pathway in animal models, which had not been discovered in clinical cases until now. For these genes from Table ACTC1, ACTN2, DTNA, LDB3, MIB1, MYBPC3, MYH7, PRDM16, TNNT2, and TPM1 were reported to be associated with LVNC in the OMIM database  of MYH7, MYBPC3, TPM1, TAZ, TTN, and NONO genes are the most common pathogenic mutations in LVNC [th exon deletion of KCNQ1 induced mild Beckwith-Wiedemann and severe LQTs [LVNC is a rare primary CM and serves as a global cardiac disease induced by the arrest of normal embryogenesis of the endocardium and mesocardium that leads to prominent trabeculations and deep intertrabecular recesses within the LV wall communicating with the cavity, a thin compacted epicardial layer, and acquired ventricular wall remodeling . The inf in LVNC , 32, 33, in LVNC . These c in LVNC . Some LV in LVNC . Therefo in LVNC . Actuallere LQTs . NotablyFKBP12-deficient mouse model for LVNC [FKBP12-deficient hearts. The planar cell polarity (PCP) signaling is also an essential molecular mechanism of polarity establishment for epithelial cells in planes orthogonal to the apical-basal axis. The noncanonical Wnt signaling, which is independent of β-catenin, is critical for PCP signaling. Together, they serve as the Wnt/PCP signaling pathway, which is composed of core components , PCP regulator  and a large number of PCP effectors  [Two independent biological events leading to abnormal trabeculations and compaction, including hypertrabeculation and noncompaction, are demonstrated by the for LVNC , 39. Hypfor LVNC . The enhfor LVNC , 42. Thend PhoA) , 44. Thend PhoA) .DSC2 p.K47Rfs*2 is a truncated and loss-of-function mutation, likely to cause LVNC and HCM. Compared to the MAF (< 0.01%) of inherited arrhythmia and CMs in the population, the MAF of DSC2 p.I520T (0.02%) is relatively high. Moreover, DSC2 p.I520T is not conservative in multiple species. In addition, the ECG is the main tool to judge the early changes of CMs. Especially, the abnormal changes of ECG caused by CMs are often earlier than that of the cardiac structure illustrated by echocardiography [DSC2 p.I520T and without cardiac event showed normal ECG and echocardiography. Therefore, we speculated that DSC2 p.I520T might not be pathogenic. In fact, as an important component of the cardiac desmosome, desmocollin2 maintains the normal electromechanical connection among cardiomyocytes. The desmocollin2 abnormality would lead to various CMs (such as DCM and HCM), HF, complex arrhythmia and even SCD. Recent research and our literature review showed that the abnormal components of the cardiac desmosome, including DES and DSP genes, are associated with LVNC. Desmosome is the intracellular structure that anchors intermediate filament to the plasma membrane. Desmosome, adherent junction, and gap junction are the major components of the intercalated disc, which is vital for the cell–cell adhesion and electronic coupling of cardiomyocytes [DSG2) are the important familial members of cadherin. Desmocollin2 and desmoglein2 form the adhesive heterodimer, which provides a structural framework for desmosome assembly together with armadillo proteins  and the plakin family  [DES), also expressed in cardiac desmosomes, connects Z-bands, costameres, and nuclei with the cytoskeleton. The pathogenic mutations associated with desmosome proteins may cause desmosome dysfunction and remodel intercalated disc, leading to the disturbance of the mechanical–electrical coupling of cell–cell adhesion and further alteration of signaling pathways.In this study, the proband (II: 1) carried DSC2 p.K47Rfs*2 and p.I520T variants. According to the ACMG classification criteria, the iography . Whereasmyocytes . Desmocoencodes) . The desPKP2 lead to LVNC and rapid HF, whereas the heterozygous DES p.A337P  induces LVNC and skeletal myopathy [DSC2 variant  in a patient with sporadic LVNC, but whether the variant is the cause of LVNC remains unclear [DSC2 deletion in zebrafish induces the reduction of the desmosome plaque area, deficiency of desmosome extracellular electron-dense midlines, and disturbance of myocardial contractility [DSC2 p.Q554X (c.1660C>T) participates in the pathogenicity of familial ACM [DSC2 p.A897fs*900 shows an obvious reduction in desmosome binding to plakoglobin and plakophilin [DSC2 (c.2553delA) decreases the connexin 43 expression and lost plakoglobin signal [DSC2 activates the AKT/β-catenin pathway. AKT inhibition suppresses β-catenin-dependent transcription and proliferation, which are caused by DSC2 knockdown [DSC2 protein sequence at the cadherin pro is replaced by arginine, leading to premature codon termination. Consequently, DSC2 p.K47Rfs*2 remarkably and abnormally reduced the functional desmocollin2 expression, which may interfere with desmosome formation and the stability of the intermediated disc, and disturb the mechanical stress and electrical signal propagation of cardiomyocytes. Notably, the pathological and imaging changes induced by DSC2 p.K47Rfs*2 are observed in LV, resulting in the subendocardial thickening of the anterior lateral wall and interventricular septum, and thinning in part of the left ventricular wall. The ECG of the proband showed low voltage in the limb leads but no typical ST-T change, commonly suggesting diffuse myocardial lesion. The VT arises from the middle-posterior septum and lateral wall of LV. PVCs originate from the lateral wall and apex of LV and the left ventricular inflow tract, and this finding is consistent with the cardiac lesion extent. Additionally, trabeculae are observed with serious dysplasia and distributed in a rough and disordered pattern. An apparent slow blood flow is observed in the recess of trabeculae, which may increase the risk of thrombus. However, the mechanism of DSC2 p.K47Rfs*2 that induces overlap phenotypes of LVNC and HCM remains unknown. There is no research about the relationship among DSC2, Notch, and Wnt/PCP signaling pathways.Previous studies show that the homozygous deletion mutation of myopathy , 50. A r unclear . A previactility . Additiolial ACM . DSC2 p.kophilin . The truDSC2 p.K47Rfs*2 leading to abnormalities of the desmosome structure and function should be further explored. Second, DSC2 p.K47Rfs*2 is the main cause of LVNC and HCM but still cannot rule out DSC2 p.I520T, or other variants may increase the risk of LVNC and HCM phenotype penetrance. Third, the mechanism of how the DSC2 p.K47Rfs*2 induces overlap phenotypes needs further research. Fourth, the parents and grandparents of the patient (II: 1) could not be tracked because of death. The II: 1, II: 2 and II: 3 members declined to to carry out clinical examinations and genetic testing for their offspring. Therefore, long-term follow-up is needed in the future.The limitations of the present study are as follows. First, the molecular mechanism of DSC2 p.K47Rfs*2 mutation remarkably and abnormally reduced the functional desmocollin2 expression, which may consequently induce the overlap phenotypes of LVNC and HCM, complicating AF, VT, and HF. The LVNC may be one of the important phenotypes for DSC2 pathogenic mutations.The desmocollin2 was an important component of cardiac desmosome assembly, of which abnormality caused the dysfunction of cell–cell adhesions and intercellular gap junctions. The novel heterozygous


ICAM1, THY1, and CXCR4 significantly indicated adverse, while EPCAM indicated beneficial clinicopathological features of GC patients. The up-regulation of CXCR4 showed unfavorable prognostic significance, whereas EPCAM and TFRC showed the opposite. The six GCSC markers were all correlated with the infiltration and activation of distinct TIICs. Especially, ICAM1, THY1, and CXCR4 showed strongly positive correlations with tumor-associated macrophages. Besides, chemokine, Toll-like receptor, NF-kappa B, and HIF-1 signaling pathways might be involved in the regulation of GCSC markers on cancer development. This study proposed that GCSC markers might be promising targets of GC treatment to weaken cancer stem-like properties and strengthen anticancer immunity.The prognosis of patients with gastric cancer (GC) is still unsatisfying. Numerous markers of gastric cancer stem cells (GCSCs) have been identified and were thought to be related to cancer aggressiveness. However, the roles of GCSC markers in GC patients’ prognosis and immune infiltration remain unknown. Expression of GCSC markers was analyzed using Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA). Their associations with clinicopathological parameters were analyzed using UALCAN and LinkedOmics. Alternations and protein expression of GCSC markers were analyzed by cBioPortal and the Human Protein Atlas databases, respectively. The prognostic significance of GCSC markers was evaluated using Kaplan-Meier plotter. Correlations between the expression of GCSC markers and immune infiltration along with biomarkers of tumor-infiltrating immune cells (TIICs) were assessed combined Tumor Immune Estimation Resource and GEPIA. GeneMANIA was used to discover the interactive genes of GCSC markers, and enrichment analysis was performed using Database for Annotation, Visualization, and Integrated Discovery server. We identified six GCSC markers significantly up-expressed in GC, compared with normal stomach tissues. Among them, the overexpression of  Gastric cancer (GC) is the fifth most frequently diagnosed and the third leading cause of cancer mortality in the world . Due to CD44) was the first identified marker of human GCSC in 2009. After that, epithelial cell adhesion molecules (EPCAM), intercellular adhesion molecule 1 , aldehyde dehydrogenase 1 (ALDH1), CD90 (THY1), CD133 (PROM1), transferrin receptor protein 1 , signal transducer CD24 (CD24), octamer-binding transcription factor 4 (OCT4), sex determining region Y-box transcription factor 2 (SOX2), leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5 or GPR49), homeobox protein NANOG (NANOG), ATP-binding cassette subfamily B member 1 (ABCB1), ATP-binding cassette subfamily G member 2 (ABCG2), C-X-C motif chemokine receptor 4 (CXCR4), CD166 (ALCAM), doublecortin-like kinase (DCLK1), integrin alpha-6 , and RNA-binding protein Musashi homolog 1 (MSI1) were identified (Cancer stem cells (CSCs) are proposed to be a small population of the tumor mass with stem cell properties, which are believed to be responsible for generation, heterogeneity, and sustaining growth of cancer, as well as treatment resistance, recurrence, and metastasis . Abundanentified . The CSCAccumulating evidence proved that tumor-infiltrating immune cells (TIICs) in the tumor microenvironment (TME) play critical roles in immune escape and cancer progression, and could serve as prognostic biomarkers. For example, higher infiltrating density of CD4+, CD8+ T cells, and fewer FOXP3+ regulatory T cells (Tregs) could predict improved survival of postsurgical GC patients . Tumor-aIn this study, we comprehensively analyzed the expression, alternations, clinicopathological relevance, prognostic value, and correlations with immune infiltration of GCSC markers in GC. Moreover, the interactive genes of GCSC markers were discovered and their biological functions were elucidated to learn the potential regulatory mechanisms of GCSC markers on GC. The findings of our study will uncover the prognostic values of GCSC markers in GC patients, and provide novel insights into the relationships between GCSCs and cancer immunity.1 is an online server analyzing microarrays of 18,000 cancer samples integrating data from published literature, the Stanford Microarray Database, and the NCBI Gene Expression Omnibus (GEO)  . A meta-2 is a web portal to analyze transcriptome data, based on 9,736 tumors and 8,587 normal samples from the Cancer Genome Atlas (TCGA) and the GTEx projects projects . The exp3 and LinkedOmics  is a program aiming to map all the human proteins in cells, tissues, and organs, the resource of which is openly accessible at las.org/ . The pro6 is an online tool for survival analysis of 54 k genes in 21 types of cancers, the data sources of which include the GEO, European Genome-phenome Archive, and TCGA (Kaplan-Meier (KM) Plotterand TCGA . KM Plot7 is a web server for the investigation of tumor-immune interactions, which incorporates 10,897 samples across 32 kinds of cancers from TCGA (Tumor Immune Estimation Resource (TIMER)rom TCGA . The corrom TCGA .8 is a web tool for the investigation into associated genes for quired genes through analysis of physical and functional associations, such as co-expression, co-localization, and physical interaction , 95% confidence interval (CI), and P-value were generated. Spearman’s method was applied to evaluate the correlation of gene expression with immune infiltration level or biomarkers’ expression of TIICs. For all the analyses, P < 0.05 was considered statistically significant, and a false discovery rate (FDR) <0.05 was an additional criterion for functional enrichment analysis.The comparison of gene expression level in cancer and normal tissues by Oncomine and UALCAN platforms was conducted using student’s EPCAM, ICAM1, ALDH1, THY1, PROM1, TFRC, CD24, OCT4, SOX2, LGR5, NANOG, ABCB1, ABCG2, CXCR4, ALCAM, DCLK1, ITGA6, and MSI1 in GC and normal stomach tissues were analyzed using the Oncomine and GEPIA databases, respectively. It turned out that eight GCSC markers were significantly higher expressed in GC than normal stomach tissues in Oncomine  patients, compared with that in middle-aged (41–60 years old) patients (P < 0.05) (P < 0.05). In particular, ICAM1, THY1, and CXCR4 were higher expressed in GC patients in Stage II–IV, compared with that in Stage-I patients (P < 0.01) (P < 0.01) (EPCAM expression was observed in patients in advanced stages (Stage II–IV), compared with that in Stage-I patients. Besides, the expression of EPCAM, TFRC, and LGR5 was higher in Grade-2 tumors, compared with that in Grade-3 tumors (P < 0.05)  . General < 0.01) ; and the < 0.01) . In cont < 0.05) .ICAM1 (P = 2.73E-03), THY1 (P = 1.83E-06), and CXCR4 (P = 3.18E-05) were significantly associated with advanced T stages (EPCAM was linked with favorable N stages (P = 4.101E-02) and M stage (P = 4.60E-02). No significant finding of TFRC and LGR5 was identified here. All the above illustrated that the overexpression of ICAM1, THY1, and CXCR4 significantly implied adverse outcomes, whereas EPCAM implied more optimistic clinicopathological characteristics of GC patients.Moreover, the results from LinkedOmics showed elevated expression of T stages . OverexpEPCAM, ICAM1, THY1, TFRC, LGR5, and CXCR4 were altered in 31 (6.5%), 23 (4.8%), 23 (4.8%), 60 (12.6%), 50 (10.5%), and 17 (3.6%) of all samples, respectively  of GC samples; and mRNA overexpression occurred the most frequently. ectively .Following this, the expression of GCSC marker protein in normal stomach and GC tissues was verified using IHC data from the HPA database, except for CXCR4, whose data was still in preparation. As exhibited in EPCAM  and TFRC  had both better OS and RFS. Nevertheless, high expression of CXCR4  was associated with worse survival of GC patients. No significant prognostic indication was found for ICAM1, THY1, and LGR5.Wondering whether the expression of GCSC markers affects GC patients’ prognosis, survival analysis was performed using KM Plotter. As shown in EPCAM upregulation indicated both favorable OS and RFS of male GC patients and patients in advanced stage (Stage III) (P < 0.05). High-expression of TFRC was associated with better OS of male GC patients, and better RFS of GC patients in Stage II, patients with Grade 2 tumors, as well as females (P < 0.05). Overexpression of CXCR4 was correlated with both unfavorable OS and RFS of male patients; paradoxically, it implied better RFS of female patients (P < 0.05). Besides, no prognostic significance of ICAM1, THY1, and LGR5 was found in this part  was positively, while ICAM1 (P = 8.02E-05), THY1 (P = 1.95E-04), and CXCR4 (P = 6.38E-05) were negatively correlated to the tumor purity , but negatively correlated with that of MDSCs (P < 1.10E-04). Interestingly, the correlations between EPCAM expression and the infiltration of these TIICs was in an opposite way of ICAM1, THY1, and CXCR4 (P < 0.05). Furthermore, the expressions of TFRC (P = 3.16E-05) and LGR5 (P = 3.39E-02) were both positively correlated with the infiltration of MDSCs; but TFRC was negatively, while LGR5 was positively correlated with the infiltration of CD4+ T cells (P < 0.01). Notably, the correlation strength of ICAM1 expression with infiltration of DCs  and CD8+ T cells , THY1 with macrophages , also CXCR4 with DCs  were strong.The expression of ICAM1, THY1, and CXCR4 was positively, while EPCAM was negatively correlated with the expression of almost all biomarkers of B cells, T cells, neutrophils, macrophages, monocytes, and DCs (P < 0.05) , monocytes (CD86 and CD115), Tregs (CCR8 and TGFB1), exhausted T cells (TIM3), neutrophils (CD11b), TAM (IL10), and DCs (NRP1 and CD11c) (P < 9.0E-30). Until now, we could summarize GCSC markers that might modulate immunity through extensive modulation of immune infiltrates in the TME of GC.To further confirm the participation of TIICs, the correlations between the expression of GCSCs markers and biomarkers of TIICs in GC were analyzed using GEPIA. The biomarkers of all TIICs covered in the last step were investigated, along with monocytes, M1/M2 macrophages, subsets of T cells, including helper T cell (Th) 1, Th2, Th17, follicular helper T cell (Tfh), regulatory T cell (Tregs), and exhausted T cells. Consistent with the results from TIMER, the expression of  < 0.05) . NotablyEPCAM, ICAM1, THY1, LGR5, and CXCR4 were all components of the external cell membrane. Beyond that, EPCAM’s interactive genes participated in the maintenance of cell polarity, negative regulation of DNA damage response, protein transport, and positive regulation of apoptosis  and binding of RNA polymerase II and integrin (TFRC’s interactive genes were components of transferrin receptor complex and majored in iron ion homeostasis (LGR5’s interactive genes were involved with cellular ions homeostasis (CXCR4’s interactive genes regulated chemokine-mediated inflammatory response and G-protein coupled receptor kinase activity (To understand the biological functions of these GCSC markers, a gene interaction network was constructed for each gene using GeneMANIA. Twenty interactive genes were identified for each GCSC marker gene; thus, a gene interaction network was composed of a total of 21 genes . Subsequpoptosis . ICAM1’sintegrin . TFRC’s eostasis . LGR5’s eostasis , while Cactivity .Furthermore, all the significantly enriched KEGG pathway terms of the total six gene interaction networks were presented in Despite dramatic progress in diagnosis and treatment, the prognosis of GC patients remains poor, primarily blamed on the frequent treatment resistance, following relapse and metastasis. The CSCs are believed to contribute to the inefficacy of conventional therapies, for their quiescent nature, capabilities of anti-apoptosis, DNA damage repair, and diminished uptake and/or enhanced efflux of drugs . In thisEPCAM, ICAM1, THY1, TFRC, LGR5, and CXCR4 were identified significantly high-expressed and frequently altered in GC, compared with normal stomach samples. Among them, we that found upregulation of ICAM1, THY1, and CXCR4 was associated with pathological and histological advancement of GC patients, but only CXCR4 showed unfavorable prognostic significance. Consistent with the previous studies, CXCR4 had been widely reported as an unfavorable prognostic indicator of various cancers, including GC. CXCR4 was elevated in GC and was closely associated with cancer progression and metastasis (CXCR4 over-expression indicated better RFS of female GC patients, which needs further explorations with larger sample sizes. ICAM1 was ever identified as an unfavorable prognostic biomarker in earlier studies (THY1 expression was reported to relate with aggressive clinicopathological features of GC cases (ICAM1 and THY1 had no prognostic significance.From the start, six GCSC markers, tastasis . Surpris studies . High THGC cases , and theGC cases . HoweverEPCAM overexpression was an independent unfavorable prognostic factor and was linked with larger tumor size and lymph node metastasis in GC patients (EPCAM upregulation suggested more optimistic clinicopathological characteristics and outcomes of GC patients, even for the advanced-stage patients. In fact, the beneficial role of EPCAM in prognosis had been observed in ovarian cancer (TFRC high-expression implied better histological differentiation status and favorable prognosis of GC patients. Our findings could be supported by an earlier study that TFRC—cells represented high tumorigenicity, multipotency, invasiveness, and treatment resistance (LGR5 overexpression might contribute to the progressive clinical features and poor OS of GC patients (CXCR4 as unfavorable, whereas EPCAM and TFRC as favorable prognostic biomarkers of GC patients. Some findings inconsistent with the previous studies might lie in the intrinsic heterogeneity of cancer and different quantification methods. Therefore, more investigations with larger sample sizes are still required.The previous studies demonstrated that patients . Unexpecn cancer  and pancn cancer  before. sistance . Even thpatients , we had ICAM1, THY1, and CXCR4 was positively, while EPCAM was negatively correlated with the infiltration level and biomarkers expression of T cells, B cells, neutrophils, macrophages, and DCs. In particular, the expression of ICAM1, THY1, and CXCR4 exhibited strongly positive correlations with the biomarkers of M2 TAMs, TAM, monocytes, Tregs, and DCs. Furthermore, the expression of TFRC and LGR5 was positively, whereas ICAM1, THY1, and CXCR4 were negatively correlated with the infiltration of MDSCs.After that, we observed that the expression of GCSC markers was correlated with the infiltration and biomarkers expression of TIICs in the TME of GC. High expression of LGR5 expression in GC cells, which conferred poor prognosis (ICAM1, THY1, and CXCR4 might incline to promote immunosuppression, whereas EPCAM and TFRC tended to enhance immunity surveillance. The interplay between CSCs and the immune microenvironment is a promising breakthrough point for cancer therapy. Targeting CSCs can curtail cancer aggressiveness, and it is an attractive strategy to adjust anticancer immunity at the same time.It is acknowledged that CD8+ T cells are the main undertakers of anticancer immunity, a high infiltration level of which indicates better treatment response and prognosis . On the rognosis . Evidencrognosis . MDSCs crognosis . Furtherrognosis . DCs arerognosis . A studyrognosis . Whereasrognosis . Hereto,EPCAM’s interactive genes were responsible for cell polarity and regulation of DNA damage response. The interactive genes of ICAM1 and THY1 were both involved in cell adhesion, and the former also regulated cellular response to cytokine and mRNA translation. TFRC’s and LGR5’s interaction networks consistently partook in cellular ions homeostasis. And CXCR4 mainly acted as a chemokine-mediated inflammatory regulator. The KEGG enrichment analyses elucidated that signal pathways of chemokine, Toll-like receptor, cancer, NOD-like receptor, NF-kappa B, and HIF-1 were involved. Toll-like receptor, NOD-like receptor, and NF-kappa B pathways all play vital roles in cancer-related inflammation, which process proinflammatory cytokines engaging in cancer progression (At last, the results of GO functional annotation revealed that gression . HIF-1 sgression , and it gression .CXCR4 might serve as an unfavorable, while EPCAM and TFRC might be favorable prognostic biomarkers of GC patients. The GCSC markers might affect the disease outcomes by maintaining the stem cell-like properties of cancer and remodeling distinct TIICs in the TME, especially TAMs, Tregs, and DCs. Specifically, the processes of cell adhesion, mRNA translation, and inflammation, and signaling pathways of chemokine, Toll-like receptor, NF-kappa B, and HIF-1 might be involved. Our study suggested that GCSC markers were promising therapeutic targets to diminish cancer aggressivity and modulate anticancer immunity in GC. Despite this, there is still a long way to go for experimental and clinical validations.In conclusion, we found that elevated All the data that support the findings of this study have been provided in the article/TL designed the study, conducted bioinformatics analysis, and wrote the original manuscript. WP and PM studied the relevant literature. EZ refined the figures. LP provided comments and guidance of the manuscript. All authors have read, contributed to the revision of the manuscript, and approved the submitted version.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.


Ischemia/reperfusion (I/R) injury is associated with substantial clinical implications, including a wide range of organs such as the brain, kidneys, lungs, heart, and many others. I/R injury (IRI) occurs due to the tissue injury following the reestablishment of blood supply to ischemic tissues, leading to enhanced aseptic inflammation and stimulation of oxidative stress via reactive oxygen and nitrogen species (ROS/RNS). Since ROS causes membrane lipids’ peroxidation, triggers loss of membrane integrity, denaturation of proteins, DNA damage, and cell death, oxidative stress plays a critical part in I/R pathogenesis. Therefore, ROS regulation could be a promising therapeutic strategy for IRI. In this context, Nrf2 (NF-E2-related factor 2) is a transcription factor that regulates the expression of several factors involved in the cellular defense against oxidative stress and inflammation, including heme oxygenase-1 (HO-1). Numerous studies have shown the potential role of the Nrf2/HO-1 pathway in IRI; thus, we will review the molecular aspects of Nrf2/Kelch-like ECH-associated protein 1 (Keap1)/antioxidant response element (ARE) signaling pathway in I/R, and we will also highlight the recent insights into targeting this pathway as a promising therapeutic strategy for preventing IRI. IRI injury is a sequential catastrophic event with high morbidity and mortality, usually seen after thoracoabdominal surgeries, cardiopulmonary bypass, hemorrhagic, traumatic, or septic shock, severe burns, or after transplantation operations  characte2•−), oxygen, hydroxyl, and peroxyl radicals, nitric oxide and nitrogen dioxide, and non-radicals such as hydrogen peroxide (H2O2), hypochlorous acid, singlet oxygen, nitrous acid, nitroxyl anion, lipid peroxides’ aldehydes, and peroxynitrite [Oxidative stress is a pathological process caused by the imbalance between redox systems in favor of the oxidant systems. This imbalance usually originates either from the excessive production of ROS or the diminished ROS scavenging capacity ,4,5. ROSynitrite ,9,10,11.ynitrite ,9,10, ulynitrite ,9,10,11.ynitrite . Tissue destruction can be seen under excessive ROS exposure via lipid peroxidation in the cell membrane and organelle lipids, DNA oxidation, activation of matrix metalloproteinases, induced osmotic cell lysis, increased mitochondrial permeability, or via indirect mechanisms including free radical formation by the interaction with molecular oxygen, NO, free iron, or fatty acids or free iron. These mechanisms enhance the formation of oxidant-dependent pro-inflammatory mediators and upregulation of cytokine/chemokine and adhesion molecule expression during reperfusion . Indeed,Consequently, the myeloid differentiation primary response gene 88 (MyD88) and nuclear factor-κB (NF-κB) pathways are activated, followed by initiation of the inflammatory mediator release , which a2•− by superoxide dismutase (SOD), generating the formation of H2O2, which can be handled either by catalase or by glutathione peroxidase (GPx) and glutathione reductase [Other manifestations of I/R with similar pro-inflammatory features include cell death via necrosis or programmed cell death (apoptosis and autophagy). In normal conditions, autophagy recycles damage proteins and organelles after lysosomal degradation as a cytoprotective mechanism. However, ROS accumulation during I/R surpasses autophagic clearance capacity and eventually leads to cell death ,6. It haeductase ,11. The Nrf2/Keap1/ARE pathway regulates these aforementioned ARE-responsive antioxidant proteins ,24,25,26In this review, we will focus on the functions of the Nrf2/Keap1/ARE signaling pathway in IRI and the structure and regulation of this pathway’s elements, along with the potential relevance of this pathway as a promising therapeutic target in IRI.(low affinity) and ETGE (high affinity) motifs for the interaction with Keap1. Neh3 is responsible for the ARE activation. Neh4 and Neh5 are responsible for binding with different “cAMP response element-binding” (CREB) proteins and activate transcription. Neh6 regulates the protein stability by phosphorylation of serine residues or ubiquitination by b-TrCP. Neh7 is accountable for the RXRα  interaction and the disruption of the binding between CBP (CREB-binding protein) and the Neh4 and Neh5, as well as inhibition of the ARE sequence transcription. Nrf2 stays in its inactive form within cells via Keap1 unless there is a disturbance in redox homeostasis. Activated Nrf2 accumulates in the cell nucleus and binds to ARE on DNA and upregulates related genes’ transcription to reduce production or induce elimination of ROS [Nrf2, encoded by the NFE2L2 gene in humans and classified under the cap “n” collar (CNC) subfamily of basic-region leucine zipper (bZIP) transcription factors along with other five , is present in various cell types. It comprises 605-amino-acid-long protein with seven highly conserved Nrf2-ECH domains (Neh1-7), which serve as a different functional region ,24,25,26n of ROS ,25,26,31The Nrf2/Keap1/ARE pathway is a crucial mechanism to defend the cell against the chemical and oxidative stress caused by endogenous  or exogenous  factors by activating antioxidant genes in order to restore the redox balance ,26,32. TKeap1 is a cysteine-rich (27 cysteines), cytoplasmic, actin cytoskeleton-associated adapter zinc-metalloprotein of the Cul3/Rbx1 complex. It consists of five domains, and Keap1/Cul3 homodimerization is regulated by the N-terminal portion of the intervening region with the BTB domain. In contrast, Nrf2/Keap1 dimerization is regulated by the Kelch domain’s interaction and the C-terminal region of Keap1 with ETGE and DLG motifs of Nrf2’s Neh2 domain ,24,33,34Another mechanism concerning the separation of Nrf2 and Keap1 is modifying the several cysteines in Keap1 ,39. CystAs it is mentioned above, the Nrf2/Keap1 pathway is the master regulator of expression of the genes related to antioxidant proteins, phase I and II electrophile detoxification enzymes. It inhibits inflammation, recognizes the DNA damage, mediates glutathione homeostasis, proteasome function, and is also responsible for the transportation of toxic solutes and regulating the expression of detoxifying enzymes. These are accomplished by the interaction of Nrf2 with the antioxidant response element (ARE) ,33. Once the Nrf2/Keap1 complex is disrupted, Nrf2 dimerization with small Maf (sMaf) protein occurs to form Nrf2-sMaf heterodimers. sMaf proteins have a similar bZIP domain as Nrf2, which makes them Nrf-2-competitors binding to ARE. Nrf2-sMaf heterodimer formation ensures binding of Nrf2 to ARE due to the higher concentration of this heterodimer in the nucleus than the sMaf homodimers’ formation. Nrf2 binds to cis-regulatory ARE sequence (5′-RTGACnnnGC-3′) to control the basal and inducible expression of antioxidant and detoxifying genes under stressed conditions caused by xenobiotics, metals, and UV irradiation ,31,33,40Besides, ARE sequences are present in the regulatory regions of genes such as glutamate-cysteine ligase, NAD(P)H quinone oxidoreductase 1 (NQO1), heme-oxygenase-1 (HO-1), sulfiredoxin1 (SRXN1), heme-oxygenase (HO-1), glutathione S-transferase (GST), multidrug resistance-associated proteins (MRPs), and UDP-glucuronosyltransferase (UGT) . The HO In both normal and stressed conditions, redox balance can be maintained mainly at the transcription level via Nrf2/Keap1/ARE signaling. This pathway controls over 250 genes involved in antioxidant systems and detoxification, and cytoprotection ,33,43,44Nrf2 primarily regulates the transcription of the related genes of detoxifying and antioxidant enzymes, which function as ROS scavengers and electrophile neutralizers , glutathione peroxidase (GPx), glutathione reductase (GR), catalase), as well as induces glutathione synthase (indirectly). Besides, the activated Nrf-2/heme oxygenase-1 (HO-1) axis causes heme to degrade into bilirubin, an antioxidant substance ,26. Nrf2The Nrf2/Keap1/ARE signaling pathway has been established as the primary cellular defense mechanism against oxidative stress both in physiological conditions and in a wide array of disease models of various systems .As previously reviewed in the literature, although the reestablishment of blood flow resolves acute stroke during CNS ischemia, oxidative stress causes tissue damage and the prevention of complete recovery of cerebral functions during the reperfusion period . Hence, IRI of the kidneys caused by irregular renal blood flow following renal transplantation is a serious issue in which pro-inflammatory cytokines and chemokines, ROS, and mitochondrial dysfunction are involved . Here, NIntestines are among the most sensitive organs to I/R, and intestinal IRI can be seen in various scenarios following obstruction or strangulation of the intestines, thromboembolic incidents, shock, and intestinal transplantation. Intestinal IRI results in inflammation and loss of the intestinal barrier followed by bacterial translocation ,4,61.The protective effect of Lipoxin A4 was demonstrated in a rat model of intestinal IRI, where lipoxin A4 alleviated the IRIvia Keap1/Nrf2 pathway activation . SimilarEnd-stage respiratory diseases require lung transplantation, and IRI is a significant issue in primary graft dysfunction. Lung IRI (LIRI) also can be seen during cardiopulmonary bypass surgery, trauma, pulmonary thromboembolism, or cardiac arrest . It is rHepatic IRI often occurs due to hemorrhagic shock or surgical interventions such as hepatic resections and liver transplantation. Several studies proposed that hepatic IRI is mitigated by Nrf2/Keap1 complex via Keap1 signaling and induction of Nrf-2 to increase HO-1 expression, leading to boosted hepatocyte autophagy and subsequently discarded impaired mitochondria, and reduced ROS production ,70,71. A2 is reported to have cardioprotective properties during IRI through Nrf2 activation via the PGF2α receptor (FP) [Acute myocardial ischemia is caused by the occlusion of the coronary arteries, which restrains the heart’s blood flow, followed by cardiac muscle tissues’ necrosis ,73. Routtor (FP) . Myocardial IRI (MIRI) is a severe pathophysiological condition associated with complex mechanisms, including oxidative stress, cell apoptosis, and inflammatory responses ,6,7. OveZhang et al.  suggeste2O2 preconditioning alleviate the caspase-3 activity, increases the expression of Bcl2, and leads to the translocation of Nrf2 into the nucleus, which selectively increases the expression and activity of antioxidant enzymes to prevent MIRI-induced apoptosis [Tripterygium wilfordii Hook F, reportedly reduces I/R-induced myocardial infarction, inflammation, oxidative stress and improves the cardiac function in rats via its effect on the Nrf2/HO-1 defense pathway [Allium sativum) and its constituents, such as Allicin and Diallyl sulfide, were reported to activate Nrf2 and increase HO-1 and NQO1 expression through ERK/p38 signaling pathway activation, thus playing a protective role in the adaptation of diabetic cardiomyopathy [Plant polyphenols, such as flavonoids, chalcones, triterpenes, and proanthocyanidins, have cardioprotective, antiapoptotic, and anti-ischemic properties, which cause a decrease in the infarct size, arrhythmia score, and improvement in cardiac stunning primarily via the release of NO, the activation of the Nrf2 pathway and endogenous antioxidant defense system . Resverapoptosis . Triptol pathway . Aloin,  pathway . Recent myopathy . Anothermyopathy . New curmyopathy . Kaempfemyopathy  , a catalytic subunit of SWI2/SNF2-like chromatin remodeling complexes, is suggested to activate the Nrf2/HO-1 pathway during hepatic I/R. Recently, Ge et al.  showed tMyrica cerifera [Fisetin , a flavonoid found in various fruits ; Gastrodcerifera  and CDDOcerifera  were repAconiti Lateralis Radix Praeparata with anti-inflammatory properties, and it has been shown to regulate the Nrf2-HO-1-High mobility group box 1 protein (Hmgb1) axis and attenuate intestinal IRI in mice which appears as a novel therapy to protect intestinal IRI [Redox imbalance, nitric oxide, leukocyte adhesion molecule P-selectin, NF-κB, TNF-α, IL-1β, cellular adhesion molecules, and inducible nitric oxide synthase (iNOS) play a central role in intestinal IRI . Interleinal IRI .Radix Angelica sinensis, reportedly activates the Nrf2/HO-1 pathway, induces Nrf2 nuclear translocation, and upregulates HO-1 expression in a time- and concentration-dependent manner [Ischemic stroke is a worldwide debilitating clinical disorder and one of the most common causes of death. Reoxygenation of the ischemic brain causes IRI. Excessive ROS production is related to cerebral IRI in stroke. Recent studies are focused on the therapeutic potential of targeting the Nrf2/HO-1 pathway in brain injury after ischemic stroke . 11-Ketot manner . RS9, a t manner . Nomilint manner . Monomett manner . Singh et manner  recentlyt manner . Pterostt manner . Xu et at manner  recentlyt manner . Acute kidney injury (AKI) caused by ischemia-reperfusion also constitutes a major clinical issue. RTA-408, a new oleanane triterpenoid compound, induces Nrf2 and downstream antioxidant gene expression, protects the kidney from renal I/R by activating the Nrf2 antioxidant system and increasing the Nrf2 nucleus translocation and subsequently upregulating of downstream antioxidant genes, including GSH biosynthesis . HyperglI/R plays a significant role in the pathogenesis of numerous diseases, including liver, heart, kidney, brain, intestine, testis, eye, extremities, etc. . IschemiMany experimental studies have revealed that the protective role of the Nrf2 pathway in IRI I/R enhances Nrf2 dissociation from Keap1, facilitating Nrf2 translocation to the nucleus, binding to the ARE, and activating antioxidant and anti-inflammatory genes. In the present review, we focused on the Nrf2-ARE pathway as a target against oxidative stress-induced IRI and reported data on different bioactive compounds that demonstrated to be able to activate this pathway. Recent studies indicated that Nrf2 activation might play a vital role in preventing IRI. The modulation of the Nrf2-Keap1 pathway by combining different chemicals appears as a promising target for preventing and treating organ damages caused by IRI. In conclusion, combining different bioactive compounds, by promoting Nrf2-mediated protection against oxidative stress- and inflammation-derived injury, may represent new therapeutic strategies against IRI.


Mycosis fungoides (MF) is the most prevalent type of skin lymphoma. In its early stages, it has a favorable prognosis. However, in its late stages, it is associated with an increased risk of mortality. This systematic review aimed to identify the transcriptomic changes involved in MF pathogenesis and progression. A literature search was conducted using the database PubMed, followed by the extraction of 2245 genes which were further filtered to 150 recurrent genes that appeared in two or more publications. Categorization of these genes identified activated pathways involved in pathways such as cell cycle and proliferation, chromosomal instability, and DNA repair. We identified 15 genes implicated in MF progression, which were involved in cell proliferation, immune checkpoints, resistance to apoptosis, and immune response. In highlighting the discrepancies in the way MF transcriptomic data is obtained, further research can focus on not only unifying their approach but also focus on the 150 pertinent genes identified in this review. Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL) derived from CD4+ T-cells . MF initOmics approaches are widely used to elucidate the mechanisms of cancerogenesis and identify druggable targets for personalized treatment of cancer. Large open-access databases, such as The Cancer Genome Atlas (TCGA), allow exploration of the genome-wide expression of individual genes in different tissues and cancers from thousands of patients and aids in identifying several prognostic biomarkers ,6. HowevLeaving aside the low number of MF patients reported to the open-access databases, there are several barriers to the efficient analysis of the transcriptomic data. First, the clinical information is often insufficient. Some studies lump the data from different CTCLs and do not provide the clinical diagnosis, neglecting the fact that different CTCLs differ in their pathogenesis, clinical manifestations, and prognosis. Historically, Sézary syndrome (SS) was understood to be the more advanced form of MF; however, it is now recognized that these two diseases arise from two distinct cell types; SS from central memory T-cells and MF from effector memory T-cells . This biSecond, crude skin biopsies are commonly used to study transcriptomics, without taking into account that malignant lymphocytes may comprise only a tiny fraction (<10%) of the cells in the skin sample . Thus, mThe difficulties summarized above precluded formal meta-analyses of transcriptomic data in MF, necessitating manual data synthesis based on the binary assessment of the expression of single genes (upregulated vs downregulated) ,25. ThisWe searched PubMed using the phrase “mycosis fungoides AND (transcriptomics OR microarray OR transcript*)” to identify potentially relevant citations. Once duplicates were removed, we had 189 publications; after examining for relevance using our predetermined exclusion criteria, 10 of 189 publications relating to MF transcripts were selected for data extraction . StudiesTo determine the genetic profile of MF, 150 recurrent genes from the 10 papers were categorized based on the controls used and the type of lesion  . The majTOX, BLK, DEPDC, MYC, PLK1, and GTSE1) were upregulated and tumor suppressor genes  were downregulated. Other noteworthy categories included upregulation of genes involved in leukocyte migration and motility, and chromosomal instability. Furthermore, there was an upregulation of anti-apoptotic genes  and downregulation of a pro-apoptosis gene (NPHP3), highlighting a possible mechanism employed by MF cells to aid in their survival. Previous analyses of global expression patterns across different cancers revealed that approximately 40% of the protein-coding genes were expressed in all cancer types, the rest showing a more tumor type-restricted expression . The genCCNB1, CCNB2, and CCNF and a decrease in cyclin CCBNL2. Cyclin B1, encoded by CCNB1, is consistently overexpressed in many malignancies such as breast, esophageal, gastric cancer, and lung cancer [CCNB2) can induce aneuploidy through Aurora kinases A activation [Aberrant expression of cyclins is one of the most common disturbances in malignant cells. As highlighted in g cancer ,31,32,33g cancer , as has g cancer ,36,37. Otivation  and by itivation . CCNL2, which encodes Cyclin L2, the only cyclin decreased in MF, induces apoptosis by upregulating tumor suppressors such as p53 and downregulating pro-apoptosis mediators such as Bcl-2 [CCNL2 may serve as a growth advantage contributing to MF pathogenesis. Interestingly, as Bcl-2 . Thus, tCDKN3 and UBE2C) that negatively regulate cyclins were also observed , the mitotic error when a single kinetochore binds microtubules oriented toward both spindle poles [AURKA, the Aurora kinase involved in the regulation of the cell cycle progression [AURKB [KIF20A [BUB1 [CDC20 [CENPE [MAD2L1 [TRIP13 [EG5 [ECT2 [A recurrent feature of malignant cells is chromosomal instability (CIN), which results in a dramatic increase in DNA replication errors   and the le poles ,48. Incrgression , may caugression ,51,52. Tgression ,53. We on [AURKB ,53,54, K [KIF20A , BUB1 [40A [BUB1 , CDC20 [1 [CDC20 ,56, CENP0 [CENPE , MAD2L1  [MAD2L1 ,57, and  [TRIP13 ,59,60. FP13 [EG5  and ECT2G5 [ECT2  may distCXCL9, CCL18, CCR7, CD52, and MMP-9) involved in leukocyte migration and motility were upregulated in MF. Chemokines are a family of chemoattractant cytokines that enable efficient leukocyte transendothelial migration and are critical regulators of skin-selective T-cell homing and lymphoma dissemination. Depending on the stage of the disease, dysregulation and significant shifts in the expression of chemokine–chemokine receptor axis genes, such as CXCL9, CCL18, and CCR7, mediate the progression of MF from early, skin-limited infiltrates to an advanced, disseminated cancer with systemic manifestations [As illustrated in stations ,63. It iCXCL9, which preferentially attracts CD8 T-cells and T-helper 1 (Th1) cells that have the capacity to kill autologous malignant T-cells and represent an anti-tumor response in early disease [CXCL9 with concomitant increasing expression of T-helper 2 (Th2) associated chemokines such as CCL18. Specifically, studies have revealed elevated CCL18 mRNA levels in MF tumor lesions and serum samples, compared with controls from healthy individuals [In low-grade MF, benign cells within early MF skin lesions  express high levels of chemokines such as  disease ,64. Lateividuals . Higher ividuals . In addiividuals , suggestCCR7 in the dermis of tumor-stage MF [Previous microarray analyses have reported overexpression of the lymph node homing receptor stage MF . Immunohstage MF . As CCR7CD52 in lesional skin [MMP-9  at the RNA level in advanced MF [Beyond the rise of these well-known chemokine and chemokine receptors, studies have reported higher levels of the anti-adhesion molecule nal skin  as well anced MF ,69,70. MIL15) IL32, IL10, and SOCS3. These cytokines have been reported to play a pivotal role in suppressing cell-mediated anti-tumor responses while promoting a chronic pro-tumorigenic inflammatory microenvironment that fuels malignant T-cell proliferation. The cytokine IL-10 is elevated in malignant clones and is known to impair cellular anti-tumor response by virtue of its ability to inhibit the production of Th1 cytokine [IL10 mRNA, compared with patch and plaque stage MF [IL-15 and IL-32 with disease progression [As illustrated in cytokine . Studiesstage MF . Similargression ,74. It igression , while Igression . Finallygression . Taken tCD70, FYB, LCP2, KIR2DL3, TOX, and TIMP-1), immune checkpoints , resistance to apoptosis , and immune response   to the advanced stages (≥IB). We have extracted available data relevant to stage progression of MF and identified 15 genes which were categorized in the following way: cell proliferation (nd NKG7) . CD70, FYB, LCP2, KIR2DL3, TOX, and TIMP-1). Many of the genes differentially expressed between early and late-stage patients are relatively specific to the lymphocytes. CD70 is the TNF receptor ligand, which, upon its interaction with its receptor CD27, results in the activation of T-cell proliferation [CD70 in tumor cells from various hematological and solid malignancies is more commonly associated with poor prognoses [FYB and LCP2 are T-cell adaptor genes involved in T-cell proliferation and activation [KIR2DL3 has been described in Sezary syndrome and MF [As indicated in feration . Expressrognoses . Clinicarognoses . FYB andtivation ,82. Althtivation ,26,83,84e and MF , but itse and MF ,87. TOX (thymocyte selection associated HMG-box) encodes a protein that is highly expressed in the thymus due to its involvement in aiding the transition of immature CD4+CD8+ double-positive T-cells to lineage-specific CD4+ single positive T-cells [TOX in MF cell lines increased proliferation, migration, and invasion, further highlighting the oncological role of this gene [ T-cells . Upon le T-cells , with tuhis gene . Furtherhis gene ,91. TIMP-1 gene expression is clearly observed in MF skin biopsies [TIMP-1 is one of the tissue inhibitors of matrix metalloproteinases (MMP), the expression of which was previously linked with worse prognoses in melanoma , multiplbiopsies ,20,23,24MF is characterized by clonal expansion of malignant CD4+ T-cells that home to the epidermis . This prHAVCR2/TIM-3 and PDCD1 were not only increased with disease progression, but also displayed consistent levels of elevation in MF lesions (plaque and/or tumor) when compared to different controls , and its clinical significance in MF remains to be elucidated. However, clinical trials from other solid malignancies and lymphomas have indicated an enhanced response when TIM-3 blockade is used in conjunction with PD-1 inhibition . Future GTSF1 and PTPN7, were increased with MF progression  and propoptosis , while opoptosis . CCR4, IL10, IFNG, GNYL, and NKG7) were also upregulated in late-stage MF (CCR4) is responsible for the trafficking of T-cells to the skin and MF cells have increased expression for this receptor, thereby allowing their accumulation in the skin [IFNG), a Th1 cytokine, was also upregulated in tumors and increased with progression. IFN-γ is known to have both beneficial and harmful effects in the tumor environment, depending on its concentration [IFNG overexpression in MF is unclear.Lastly, the genes involved in immune response functions . Therefore, only descriptive data presentation was possible. Additionally, several of the studies included in our systematic review discussed treatments that patients had undergone around the time of sample collection, which serves as a significant confounding factor . Some ofOut of 2245 differentially expressed genes in MF found in the literature, we reduced the expression data set to only 150 genes which were reported in at least two independent studies. Most of those genes were associated with the already characterized pathways activated across many types of cancer: DNA repair and chromosomal stability, cell cycle and proliferation control, apoptosis, cell survival, and immune response. Also, in agreement with previous analyses of cancer transcriptomes, the majority of genes were transcriptionally activated, and only a few differentially expressed genes showed lower expression than the controls.The more interesting question from the clinical point of view is the functional importance of the differentially expressed genes with respect to prognosis and response to treatment. This question could not be answered directly because survival and stage progression data are rarely available. However, by comparing expression patterns in the early stages of the disease and advanced lesions, we could identify 15 genes which might have prognostic significance. This very limited set of genes is not unexpected because, across different cancers, only a few genes have prognostic significance . These potential prognostic genes seem to be more restricted to lymphoid tissue and, specifically, to T-lymphocyte function and development . This coCurrently, only a few treatments exist that specifically target proteins encoded by some of the 15 genes proposed to participate in MF progression. Of these treatments, only CCR4 inhibitor mogamulizumab is approved . PD-1 inProbably the largest deficiency which impedes the progress in biomarker development in MF is the lack of reproducible reporting of patient-level clinical data. Our systematic review has found a lack of consistency in the sampling, sample preparation methods, and evaluation of the results from the study. These differences, though significant, can still be accommodated to derive conclusive markers with sufficient sample numbers. The number of samples studied for transcriptomic evaluation is largely limited. Additionally, published studies do not include adequate background information such as stage, disease progression, patient characteristics, and survival information. This can be addressed by a rigorous peer review system that requires authors to provide sufficient patient information for other groups to corroborate similar findings. However, without the clinical information including the diagnosis, the stage of the disease, and the clinical description of the biopsied lesion, it is impossible to meaningfully correlate the genotype/transcriptome with the prognosis. Broad utilization of publicly accessible platforms such as TCGA or The Human Protein Atlas would accelerate the identification of prognostic genes relevant for MF.


Laboratory tests are an integral part of the overall diagnostic procedures for both physiological and pathological conditions. Ovariohysterectomy (OHE) in bitches is an invasive procedure, producing moderate to severe pain. The response to surgical stress is characterized by a series of neuroendocrine and metabolic changes that can be represented on hematological and biochemical profiles. The objective of this study was to evaluate the physiological response to surgical stress, predict the presence of possible post-surgical complications, and where appropriate, establish the appropriate treatment based on the hematochemical changes during and after OHE in healthy bitches. OHE in this study induced transient changes in certain hematological and biochemical parameters. Knowledge of the hematochemical changes in response to stress and trauma induced by OHE in healthy dogs would allow to predict possible post-surgical complications.The aim of this study was to monitor hematochemical changes during and after OHE in bitches. Twenty-four females were anesthetized with alfaxalone, midazolam, morphine and sevoflurane. Blood samples were taken before anesthesia (T0), at 30 (T1), and 60 min (T2), at 3 (T3), 6 (T4), 12 (T5), and 24 h (T6), and at 3 (T7) and 7 days (T8) from the start of surgery. Red blood cells (RBC) and packed cell volume (PCV) decreased significantly from T1 to T5 and hemoglobin (HB) concentration from T4 to T6. Both the white blood cell (WBC) and neutrophil (NFS) count increased significantly from T3 to T6, monocyte (MON) from T2 to T5, and eosinophil (EOS) at T5. Platelet (PLT) and plateletcrit (PCT) significantly decreased at T5 and increased from T6 to T8; platelet distribution width (PDW) increased significantly from T3 to T6. Creatine kinase (CK) activity increased significantly from T5 to T7. Glucose (GLU) concentrations increased significantly at T2 and P from T2 to T3. TG levels decreased from T2 to T4 and blood urea nitrogen (BUN) levels from T1 to T7, subsequently increasing until T8. Changes possibly resulting from stress and surgical trauma, as well as hemodilution and splenic storage, are due to anesthesia and surgery. In healthy bitches, these changes tend to gradually stabilize after the ending of OHE. A post-operative follow-up is essential to detect possible post-operative complications. Ovariohysterectomy (OHE) is one of the most common surgical contraceptive techniques used for population control in bitches. Other potential benefits of sterilization include prevention of inherited diseases, elimination of undesirable behaviors associated with hormonal cycling and if performed before 2½ years of age, a reduction in the risk of ovarian, uterine, and mammary neoplasia . Other pSurgical stress response during and after surgery involves a physiological reaction of the organism to restore homeostasis or counteract the stimulus . SomaticSurgical anesthesia is characterized by immobility, absence of consciousness, muscle relaxation and lack of pain . It has A) receptor and causes neuro-depression, muscular relaxation, chemical restraint and/or general anesthesia [Alfaxalone  is a synthetic neuroactive steroid molecule that modulates the gamma-aminobutyric acid A , yielding negative results in all cases. Only animals classified as ASA I were included in the study. None of the animals received medical treatment during the last 6 months prior to the study, nor were they diagnosed with active disease. During the study period, any animal received medication, and no animals had to be excluded.This study was conducted in the Veterinary Teaching Hospital of CEU-Cardenal Herrera University . A total of 24 client-owned adults, healthy, mixed-breed female dogs were included. A complete history of the animals, including breed, age, parity and stage of the oestrous cycle, was recorded. After being informed of the fundamentals of the study, the owners signed a consent form for their pets to be included. All animals underwent a pre-anesthetic evaluation to confirm that they were healthy. This evaluation included physical examination, complete hematological and biochemical analysis, thoracic X-rays, and electrocardiogram. In addition, all animals were subjected to a commercially available SNAP® 10 mg/mL; Dechra Veterinary Products SLU, Barcelona, Spain), applied at two different points to avoid patient discomfort, in combination with 0.1 mg/kg IM midazolam  and 0.2 mg/kg IM morphine . Induction consisted of alfaxalone administered through the cephalic catheter and titrated to effect until tracheal intubation was possible with an appropriately sized cuffed endotracheal tube. Maintenance was conducted with sevoflurane  vaporized in a mix of 60% oxygen and 40% air, as needed. During the surgical procedure, all animals received IV fluid therapy with Ringer-Lactate solution . Intraoperative monitoring consisted of body temperature, electrocardiogram, capnography, pulse oximetry, noninvasive blood pressure and oxygen and vapor concentrations. Intermittent positive pressure ventilation (VPPI) was applied when necessary to ensure normocapnia, and the volume was regulated to keep the end-tidal CO2 at normal levels (35–45 mmHg) [Animals were received and placed in individual accommodation 24 h before surgery with water and food ad libitum. The fasting time was 8 h of solids and 2 h of liquids. No animals received any medications prior to surgery. All the anesthetic procedures were performed by the same veterinary anesthesiologist. After placement of a cephalic 18 gauge 1” catheter, the anesthetic protocol consisted of premedication with 5 mg/kg IM alfaxalone (Alfaxan45 mmHg) . Post-op®, Normon laboratories, Madrid, Spain) [®, B. Braun VetCare SA, Barcelona, Spain). No blood loss was observed in any of the animals and no post-operative complications occurred.All surgeries were performed by the same surgeon with the help of an assistant. Thirty minutes before starting the surgery, a single dose of cefazolin was administered to all animals  . The sur, Spain) . A singl, Spain) . Subcuta®, Barcelona, Spain) and 1.0 mL in lithium heparin  for hematological and biochemical analysis, respectively. The anticoagulated samples in lithium heparin were centrifuged at 3000 rpm for 10 min  to obtain plasma. All samples were analyzed immediately after extraction.Blood samples were obtained from patients at different study times: before anesthesia (T0), at 30 min (T1), 60 min (T2), 3 h (T3), 6 h (T4), 12 h (T5), 24 h (T6), 3 days (T7) and 7 days (T8) after the anesthetic and surgical procedure. A total volume of 1.5 mL of blood was extracted from each animal, of which 0.5 mL was anticoagulated in EDTA , hemoglobin concentration , hematocrit , medium corpuscular volume , mean corpuscular hemoglobin , mean corpuscular hemoglobin concentration , erythrocyte distribution width , reticulocytes ; white blood cells , neutrophils , lymphocytes , eosinophils , monocytes , and platelet counts , plateletcrit , mean platelet volume  and platelet distribution width . The parameters studied in the serum biochemistry were: total plasma proteins , albumin , globulins  and glucose concentration ; alanine aminotransferase , alkaline phosphatase , aspartate aminotransferase , gamma glutamyl transferase  and creatine kinase  activities; total bilirubin , triglycerides , cholesterol , calcium , phosphorus  and sodium , chloride , potassium  as electrolytes concentrations. Hematological determinations were carried out with the cell counter CELL-DYN Emerald  and for the analysis of biochemical parameters, the automatic system Spin 200E was used .Both hematological and biochemical analyses were performed at different study times. The hematological parameters included were: red blood cell count  statistical software. To analyze the existence of significant differences between the times considered , the Kruskal–Wallis test was used. By means of the Mann–Whitney test, significant differences among times were assessed. Results are given as mean ± SD. A p < 0.05) and the HB concentration decreased from T4 to T6 , and the MON count increased significantly from T2 to T5 (p < 0.05). In addition, a significant increase in EOS count was seen at T5 , and the PDW significantly increased from T3 to T6 , respectively . The reqp < 0.05; p < 0.05; p concentration increased significantly from T2 to T3 , could potentially affect some of the hematochemical results reported here.In conclusion, conventional OHE in bitches using an alfaxalone-midazolam-morphine-sevoflurane protocol induces limited changes in certain hematological and biochemical parameters. Hematochemical analysis is a sensitive tool used to monitor the health status and surgical stress of the patient, as well as to detect any possible intra- or post-surgical complications.