Cervical cancer is one of the most common and lethal cancers among women worldwide. Treatment options are limited in patients with persistent, recurrent, or metastatic cervical cancer, with <20% of women living >5 years. Persistent human papillomavirus (HPV) infection has been implicated in almost all cases of cervical cancer. HPV infection not only causes normal cervical cells to transform into cancer cells, but also creates an immunosuppressive environment for cancer cells to evade the immune system. Recent clinical trials of drugs targeting the PD-(L)1 pathway have demonstrated improvement in overall survival in patients with cervical cancer, but only 20% to 30% of patients show overall survival benefit beyond 2 years, and resistance to these treatments remains common. Therefore, novel treatment strategies targeting HPV infection–associated factors are currently being evaluated in clinical trials. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human immunoglobulin G1 monoclonal antibody that blocks PD-L1. Early clinical trials of bintrafusp alfa have shown promising results in patients with advanced cervical cancer. With an estimated 604,000 new cases and 342,000 deaths globally in 2020, cervical cancer  is the fourth most commonly diagnosed cancer and the fourth leading cause of cancer-related death among women worldwide . In womeCervical cancer is divided into 2 main histological subtypes: squamous cell carcinoma, which accounts for 70% to 80% of cervical cancers, and adenocarcinoma, which accounts for 20% to 25% , 4. MostCancer stage at diagnosis determines treatment options and influences the length of survival . Rates vWomen with unresectable, locally advanced cervical cancer are frequently treated using chemoradiotherapy (CRT); however, CRT is associated with late toxicities that impact quality of life, and recurrence after CRT carries a very poor prognosis –16. WhilPersistent human papillomavirus (HPV) infection plays a crucial role in the pathogenesis of cervical cancers, particularly the oncogenic subtypes HPV16 and HPV18, which are responsible for 70% of cervical cancers , 20–23. HPV infection mediates cervical cancer pathogenesis through various pathways. During persistent infection, viral DNA is randomly integrated into the host genome, leading to cellular immortalization and eventually to malignant progression . The comHere, we discuss the current treatment options and the future of systemic treatment for patients with persistent , recurrent , or metastatic cervical cancer. We focus on the role of HPV infection, which has been implicated in 99% of all cases of cervical cancer, in the underlying disease biology of cervical cancer .P=0.0002) compared with a chemotherapy doublet alone  and median OS was not reached vs 16.3 months   approved pembrolizumab in combination with chemotherapy, with or without bevacizumab, as first-line treatment for patients with persistent, recurrent, or metastatic cervical cancer whose tumors express programmed death ligand 1  . This ap=0.0001) , 35. MedNo standard-of-care treatment for patients with recurrent or metastatic cervical cancer that progresses during or after platinum-containing chemotherapy is currently globally accepted , 26. SinGiven these data, while some advances in treatment have been made with anti-VEGF inhibitors and immune checkpoint inhibitors, much room exists for improving outcomes in patients with persistent, recurrent, or metastatic cervical cancer. Thus, a rational approach to identify new systemic therapies that address the underlying pathophysiology of the disease is needed.HPV infections that persist and progress to cervical cancer evade the immune system, in part by creating an immunosuppressive environment –44. DuriPreclinical studies have shown that HPV oncoproteins E6 and E7 can upregulate TGF-β1 promoter activity  and can A positive correlation between HPV infection and TGF-β expression was observed in the serum and saliva of patients with oral squamous cell carcinoma, a cancer type also known to be associated with HPV infection . RNA seqIncreased TGF-β activity has also been implicated in resistance to immunotherapy in urothelial and colorectal tumors , 71. EleWhile many novel strategies that address the biology of cervical cancer are being explored with the aim of improving outcomes in the first-line setting Figure 2Based on the efficacy demonstrated with pembrolizumab and the significant role of PD-L1 in cervical cancer, various anti–PD-(L)1 agents are currently under investigation as first-line therapies in cervical cancer 2. PhaseIn addition, several trials are examining novel combinations. One such novel combination includes the HPV vaccine ISA101, which initiates an immune response to HPV16 proteins E6 and E7 and presumably results in cells infected with HPV16 being recognized by the immune system . ISA101 The potential of anti–PD-(L)1 therapies in second and later lines of therapy for cervical cancer was demonstrated with the approval of pembrolizumab for patients with recurrent or metastatic cervical cancer who experience disease progression on or after chemotherapy and whose tumors express PD-L1. However, an unmet need remains because response rates with pembrolizumab were low and treatment eligibility is restricted based on PD-L1 expression. Several other anti–PD-(L)1 treatments and novel agents are being explored in this setting.Anti–PD-(L)1 therapies such as atezolizumab, durvalumab, nivolumab, and pembrolizumab have demonstrated strong efficacy in other tumor types but have limited efficacy as second-line treatment in cervical cancer, although safety profiles are consistent across tumor types. In small phase 1/2 trials in patients with cervical cancer, balstilimab, cemiplimab, nivolumab, pembrolizumab, and zimberelimab monotherapies have reported ORRs from 4% to 26.8%, and while median DORs were not reached in many trials, the median OS was approximately 1 year with most agents Table 1.P<0.001) was reported, and the trial was stopped early (P=0.00048); ORRs were 16.4% and 6.3%, respectively. Treatment-related AEs occurred in 56.7% of patients receiving cemiplimab, including 14.7% with grade 3/4 events; the most common treatment-emergent AEs were anemia (25.0%), nausea (18.3%), and fatigue (16.7%) 1 therapy can produce better outcomes than chemotherapy in this setting . This tred early , 96, 113 (16.7%) . HoweverIn the EMPOWER trial, cemiplimab resulted in a higher ORR in patients with squamous cell carcinoma than in those with adenocarcinoma (17.6% vs 12.3%). These results support a general trend that response rates in patients with adenocarcinoma are typically lower than those in patients with squamous cell carcinoma , 114. ThThe results to date demonstrate that while anti–PD-(L)1 therapies have efficacy as second- or later-line therapy for cervical cancer, not all patients benefit from therapy. Furthermore, resistance to anti–PD-(L)1 therapy remains a concern. Therefore, therapies that address mechanisms of resistance are needed to further improve outcomes in more patients.Studies of combinations of anti–PD-(L)1 agents with other checkpoint inhibitors , VEGF inhibitors , and HPV vaccines  reflect the importance of targeting multiple pathways to improve treatment outcomes 2. WhileTwo small phase 2 studies have evaluated combinations of anti–PD-(L)1 agents and VEGF inhibitors in patients with at least one prior systemic therapy , 103. DuAs previously described, in addition to PD-L1, TGF-β may play a significant and related but nonredundant role in the underlying pathophysiology of cervical cancer, providing a rationale for inhibiting both pathways simultaneously. Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the human TGF-β receptor II (TGF-βRII or TGF-β “trap”) fused via a flexible linker to the C-terminus of each heavy chain of an IgG1 antibody blocking PD-L1 (anti–PD-L1) –125. Binpost hoc pooled analysis of patients with recurrent or metastatic cervical cancer from the INTR@PID 001 study and a phase 2 single-center study (NCI 012), the ORR was 28.2%   and NCI 012 (NCT03427411), have evaluated bintrafusp alfa in cohorts of patients with pretreated recurrent or metastatic cervical cancer. In a cohort of 25 heavily pretreated patients with recurrent or metastatic cervical cancer from the phase 1 INTR@PID 001 clinical trial who had disease progression with prior platinum-containing chemotherapy, the ORR was 24.0% per investigator assessment. One patient had a delayed partial response after initial disease progression . In a po%-44.9%) . Respons%-44.9%) , 115 and%-44.9%) , 102. Ad%-44.9%) , 109.Bintrafusp alfa has also shown potential to treat other rare HPV-related tumors, such as anal, rectal, and vaginal cancer in INTR@PID 001 and NCI 012 trials; the overall ORR was 28.0% in patients with HPV-associated malignancies , 128. AnThese data indicate the potential benefit of bintrafusp alfa monotherapy in the postplatinum recurrent or metastatic setting. This is being explored further in a large phase 2 trial (NCT04246489). In addition, a phase 1 study is investigating the safety of bintrafusp alfa in combination with other anticancer therapies in patients with locally advanced or advanced cervical cancer (NCT04551950). Preliminary results from this study showed no new safety signals, and two partial responses among six patients who had a tumor assessment in patients with advanced cervical cancer .Treatment options are limited in patients with persistent, recurrent, or metastatic cervical cancer. Although several immunotherapies, including anti–PD-(L)1 agents, have shown clinical activity in persistent, recurrent, or metastatic cervical cancer, response rates are relatively low and vary based on PD-L1 expression and tumor histology. Agents that target immunosuppressive mechanisms within the TME associated with HPV infection, such as bintrafusp alfa, which simultaneously targets the TGF-β and PD-L1 pathways, are under investigation. Promising results were observed with bintrafusp alfa in phase 1/2 trials in patients with persistent, recurrent, or metastatic cervical cancer.Although bintrafusp alfa has shown promising results in patients with persistent, recurrent, or metastatic cervical cancer in phases 1-3, strategies for patient selection, including assessment of PD-L1 status, tumor histology, and TGF-β signature profile, are necessary. In the phase 1 INTR@PID 001 clinical trial, PD-L1 expression was detected by immunohistochemistry in a fraction of patients; a threshold of 1% was used to characterize tumors as PD-L1 positive (≥1%) or negative (<1%) . TreatmeThe TGF-β signature profile can identify patients with gynecologic cancers who are likely to benefit from immune checkpoint inhibitors . This apAll authors contributed to the conception and design of the manuscript and revised, read, and approved the submitted version.This work was funded by Merck (CrossRef Funder ID: 10.13039/100009945) and was previously part of an alliance between Merck and GlaxoSmithKline.KF reports institutional research funding from Merck and MSD. AO reports institutional research funding from AbbVie Deutschland, Abililty Pharmaceuticals, Advaxis, Aeterna Zentaris, Amgen SA, Aprea Therapeutics AB, Bristol Myers Squibb, Clovis Oncology, Eisai, Immunogen, Millennium Pharmaceuticals, MSD de España SA, Pharmamar SA, Regeneron Pharmaceuticals, Roche, and Tesaro; served on advisory boards for AstraZeneca Farmacéutica Spain SA, AstraZeneca KK, Clovis Oncology, Corcept Therapeutics, Deciphera Pharmaceuticals, Eisai, Merck, Roche, GlaxoSmithKline, Got It Consulting SL, Immunogen, KL Logistics, Medison Pharma, MSD de España, Mersana Therapeutics, Novocure GmbH, Pharmamar SA, prIME Oncology, Roche Farma, Shattuck Labs, Sutro Biopharma, Tesaro Bio GmbH, Tesaro Bio Spain SL, and Tesaro; and received travel or accommodation expenses from AstraZeneca, Pharmamar, and Roche. LR received honoraria from BluePrint Oncology, CurioScience, Physicians’ Education Research, and Products in Knowledge; reports consulting or advisory role for Agenus, AstraZeneca, Clovis Oncology, Merck, Genentech/Roche, GOG Foundation, MSD, Mersana, Myriad Genetics, Novartis, Rubius Therapeutics, and Seagen; participated in speakers bureau for AstraZeneca, MSD, and Tesaro; and reports institutional research funding from Aivita Biomedical, Akeso Biopharma, AstraZeneca, GEICO, Genentech/Roche, MSD, On Target Laboratories, Pfizer, and Tesaro. LO reports employment, at the time of the study, with EMD Serono Research & Development Institute, Inc, Billerica, MA, USA, an affiliate of Merck KGaA. CV reports employment with Merck.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The authors declare that this work was funded by Merck (CrossRef Funder ID: 10.13039/100009945) and was previously part of an alliance between Merck and GlaxoSmithKline. The funder had the following involvement with the study: two authors  were employees of EMD Serono Research & Development Institute, Inc, Billerica, MA, USA, an affiliate of Merck KGaA.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Wound healing (WH) and cancer seem to share common cellular and molecular processes that could work in a tight balance to maintain tissue homeostasis or, when unregulated, drive tumor progression. The “Cancer Hallmarks” comprise crucial biological properties that mediate the advancement of the disease and affect patient prognosis. These hallmarks have been proposed to overlap with essential features of the WH process. However, common hallmarks and proteins actively participating in both processes have yet to be described. In this work we identify 21 WH proteins strongly linked with solid tumors by integrated TCGA Pan-Cancer and multi-omics analyses. These proteins were associated with eight of the ten described cancer hallmarks, especially avoiding immune destruction. These results show that WH and cancer's common proteins are involved in the microenvironment modification of solid tissues and immune system regulation. This set of proteins, between WH and cancer, could represent key targets for developing therapies. Cancer cells differ from normal cells as they reproduce without control, wanting to prevail and survive through specific cancer hallmarks, such as resisting cell death and sustaining proliferative signaling that provides them with a selective advantage over normal cells3. Interestingly, cells in wounded areas proliferate for tissue repair and survival under the control of the organism in ways very similar to cancer hallmarks4. Identifying the overlapping survival and proliferative mechanisms between normal cells in wounds and cancer could be crucial for the development of new therapies that may lead to the prevention and inhibition of cancer progression.All eukaryotic cells share similar growth, proliferation, migration, and survival pathways. However, their mechanisms of control and differentiation are diverse and could be deregulated giving rise to disease. Mutations can accumulate in normal cells throughout a person’s lifetime, some of which may be silent, while others can alter key cellular functions and lead to cancer5. Back in the 1970s, Haddow raised the question of tumor production as a way of ‘overhealing’6. Similarly, Harold Dvorak published a work in 1986 entitled ‘Tumors: Wounds that do not heal’7. These contributed to the current concept that wound repair and cancer share cellular and molecular processes that are controlled in normal wound healing (e.g. self-limited process) but dysregulated in cancer (e.g. continuous activation of the pathways involved)8. Key and overlapping proteins between wound healing (WH) and cancer could be identified in both of these processes leading to the recognition of essential master proteins to stop cancer progression.The idea of molecular similarities between tumors and wounds has been in the literature for more than 150 years11. This process consists of four highly programmed and discrete yet overlapping phases: hemostasis, inflammation, proliferation, and remodeling13. These phases occur in a continuous and regulated manner. Hence, lack of control or lengthening of these processes driven by factors such as aberrancies in gene expression can lead to a delayed wound repair or non-healing wounds14. Such delayed processes, especially in the inflammatory response of chronic wounds, have been widely compared to the inflammation in cancer6.WH is a complex and evolved defense mechanism that integrates a cascade of cellular responses in the site of injury to restore tissue homeostasis, epidermal integrity, and the skin barrier function4.Hanahan and Weinbergs’ hallmarks of cancer highlight the key biological processes underlying the development, growth, and progression of tumors. Some of these cancer enabling characteristics coincide with similar mechanisms during the WH process, particularly the inflammatory and proliferative response. Furthermore, it has recently been proposed that the hallmarks of cancer are also the hallmarks of WH. However, not all cancer hallmarks have parallels in WH. Such is the case for enabling replicative immortality, genomic instability and mutation occurrenceNowadays, the scientific community still lacks information and has not reached a consensus on the set of specific genes and proteins that both activate the mechanisms of WH and are involved with cancer. The aim of the present work is to identify for the first time which WH proteins are enriched in the cancer hallmark phenotypes by using an integrated TCGA Pan-Cancer and multi-omics analyses in order to reveal novel therapeutic targets for anti-cancer therapy.http://www.geneontology.org)16, and the David Bioinformatics Resource (https://david.ncifcrf.gov/)17. On the other hand, to identify which WH proteins were already catalogued as cancer drivers, we retrieved 874 cancer driver proteins from the intOGen framework (https://www.intogen.org). This analysis helps to identify the mechanism of action of proteins across tumor types18. Then, we used The Catalogue of Somatic Mutations in Cancer (COSMIC) Cancer Gene Census (CGC) (https://cancer.sanger.ac.uk/census), that is an expert-curated description of the proteins driving cancer used in oncology research19. Both protein sets, the WH and the cancer, are provided in detail in the Supplementary Tables In order to identify WH proteins significantly enrolled in cancer, we analyzed two protein sets. On one hand, we retrieved 347 human proteins related to the “wound healing” term from the Gene Ontology (GO) database GO:0042060) , bladder urothelial carcinoma (BLCA), brain lower grade glioma (LGG), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical carcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), kidney chromophobe (KICH), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), mesothelioma (MESO), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), sarcoma (SARC), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (UCS), uterine corpus endometrial carcinoma (UCEC), and uveal melanoma (UVM).After identifying the set of WH-related proteins, we retrieved their genomic, transcriptomic and proteomic alterations in the Pan-Cancer Atlas (PCA) project which belongs to The Cancer Genome Atlas (TCGA) consortiumhttps://portal.gdc.cancer.gov/), and the cBioPortal database 23, the copy number variant (CNV) amplifications and CNV deep deletions were identified using GISTIC2.0, a computational approach that facilitates sensitive and confident localization of CNV in human cancers24; the inframe, truncating and missense driver mutations were identified through whole exome sequencing; the mRNA high and mRNA low alterations were analyzed through RNA sequencing V2 RSEM where the expression Z-scores of tumor samples were compared to the expression distribution of all log-transformed mRNA expression of adjacent normal samples in each cohort25; and the high and low protein expressions were measured by reverse-phase protein array (RPPA)26. Subsequently, we analyzed these genomic, transcriptomic and proteomic alterations belonging to WH genes/proteins of 10,711 individuals with 28 different cancer types.According to the Genomics Data Commons of the National Cancer Institute (P < 0.001). Lastly, we applied the Bonferroni correction test (P < 0.001) to perform a multiple comparison between 6) the whole TCGA Pan-Cancer alterations, and 7) the TCGA Pan-Cancer types.To generate the OncoPrint encompassing the most significantly altered WH genes/proteins we: (1) calculated the number of alterations per gene and per TCGA Pan-Cancer type; (2) normalized the frequency of alterations dividing the number of alterations per gene by the number of individuals per each cancer cohort; (3) calculated the mean frequency per gene and per alteration type considering all Pan-Cancer types; (4) identified the most altered WH genes/proteins taking into account as a cutoff the mean frequency of all genes/proteins; and (5) validated the most significantly altered WH genes/proteins comparing the alteration frequencies between the group of genes/proteins with the highest alteration frequencies (cutoff > mean frequency) versus the group of genes/proteins with the lowest alteration frequencies (cutoff < mean frequency) by using the Mann–Whitney U test (http://tumor.informatics.jax.org/%20mtbwi/pdxSearch.do) comprises 455 PDX models originating from 34 different primary sites27. The PDX models were genomically characterized to identify copy number variants, somatic mutations, and transcriptional profiles. Here, we analyzed the expression levels of the 347 WH genes taking into account Z-scores ≥ 2 as overexpressed genes and Z-scores ≤ − 2 as underexpressed genes in the PDX lineages. The gene expression is displayed as a Z-score which measures each gene’s model-specific expression in comparison with that gene in all models assayed by the same platform. Additionally, we have calculated a two-tailed P-value per each gene Z-score and have visualized the Z-score distribution of the transcriptional profile in a plot. Lastly, all overexpressed (Z-score ≥ 2) and underexpressed genes (Z-score ≤  − 2) with significant P-values (P < 0.05) encompassed the WH genes from the patient-derived xenograft approach.With the aim of generating a deeper understanding about the underexpression and overexpression of WH genes in a given tissue, we analyze their behavior in vivo by using bioinformatic resources. The Jackson Laboratory PDX resource  in the human proteome.In order to identify the most essential protein interactions, a WH protein–protein interactome (WH-PPi) network was created by using the human proteome of the Cytoscape StringApp, taking into account zero node addition and the highest confidence interactions (cutoff = 0.9) related to experiments, databases, and co-expressionP < 0.001) in order to determine a correlation between both groups of proteins.Regarding the cancer driver proteins encompassing the WH-PPi network, we compared the degree centrality between the cancer driver nodes and the wound healing nodes by using the Mann–Whitney U test (https://signor.uniroma2.it/CancerGeneNet/) is a resource that links proteins that are frequently altered in all cancer types to cancer hallmark phenotypes35. This bioinformatic tool, curated by SIGNOR36, is based on experimental information that allows to infer likely paths of causal interactions linking proteins to cancer phenotypes. According to Iannuccelli et al.35, the shortest paths from proteins to cancer phenotypes were programmatically implemented using the shortest path function of igraph R package, obtaining a distance score. Hence, we analyzed the distance score of shortest paths from wound healing proteins to cancer hallmark phenotypes. Positive regulations were calculated for tumor-promoting inflammation, inducing angiogenesis, cell differentiation, reprogramming of energy metabolism, activating invasion and metastasis, and sustaining proliferative signaling to better understand the signaling crosstalk between wound healing proteins and cancer hallmarks, and negative regulations were calculated for resisting cell death. Additionally, we performed the multiple comparison test as Bonferroni correction (P < 0.001) to compare the mean of the distance score of shortest paths across seven cancer hallmark phenotypes.CancerGenNet . The enrichment was analyzed using g:Profiler version e101_eg48_p14_baf17f0 (https://biit.cs.ut.ee/gprofiler/gost) to obtain significant annotations (Benjamini–Hochberg FDR q < 0.001) related GO biological processes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways, and Reactome signaling pathways40.The functional enrichment analysis gives curated signatures of protein sets generated from omics-scale experimentsP < 0.001 and a 95% confidence interval) to analyze: (1) significant differences of genomic, transcriptomic and proteomic alteration frequencies across alteration types , and (2) significant differences of genomic, transcriptomic and proteomic alteration frequencies across 28 TCGA Pan-Cancer types. We also validated the most significant WH genes/proteins that encompassed the OncoPrint comparing the alteration frequencies above and below the cutoff by using the Mann–Whitney U test (P < 0.001). The significance of gene expression in patient-derived xenografts was considered by using Z-scores and P-values. Therefore, genes with Z-score ≥ 2 and two-tailed P < 0.05 mean significant overexpression, and genes with Z-score ≤ − 2 and two-tailed P < 0.05 mean significant underexpression. The WH-PPi network takes into account the highest confidence interactions (cutoff = 0.9). We validated the nodes encompassing the WH-PPi network comparing the degree centrality of them with the cancer driver proteins by using the Mann–Whitney U test. Additionally, we calculated the mean of the distance score of the shortest paths across cancer hallmark phenotypes by using the Bonferroni correction as a multiple comparison test (P < 0.001). Lastly, the functional enrichment analysis of the key proteins in the signaling crossroad between cancer hallmarks and wound healing was performed using g:Profiler that determines the most significant GO: biological processes, KEGG signaling pathways, and Reactome signaling pathways with Benjamini–Hochberg FDR q < 0.001.We performed a multiple comparison test using the Bonferroni correction (significant level of P < 0.001) with alteration frequencies higher than the average (cutoff > 0.0075). Lastly, the top ten WH genes/proteins with the highest alteration frequencies were PTGER4, PRSS56, SCNN1G, SMAD3, PARD3, EGFR, EXT1, ERBB2, ARFGEF1, and F5.We have identified 212,459 genomic, transcriptomic and proteomic alterations in the 347 WH genes/proteins belonging to 10,711 individuals with 28 different TCGA Pan-Cancer types. Figure f mean of 0.0432 was mRNA high, followed by CNV amplification (0.0082), mRNA low (0.0028), CNV deep deletion (0.0027), protein high (0.0012), protein low (0.0007), driver mutations (0.0007), and fusion gene (0.0003). We performed the Bonferroni correction as a multiple comparison test to obtain significant alterations (P < 0.001) through the TCGA Pan-Cancer types. Therefore, we detected that mRNA high and CNV amplification were significantly altered (P < 0.001) across all genomic, transcriptomic, and proteomic alterations , followed by CHOL (0.075), ACC (0.070), LUSC (0.069), BLCA (0.068), CESC (0.067), UVM (0.066), LIHC (0.066), STAD (0.065), SARC (0.065), HNSC (0.064), PAAD (0.061), UCEC (0.061), UCS (0.061), KICH (0.061), COAD (0.060), TGCT (0.059), OV (0.059), PRAD (0.057), MESO (0.057), LUAD (0.057), BRCA (0.057), SKCM (0.056), THYM (0.051), LGG (0.050), KIRC (0.046), THCA (0.041), and GBM (0.020). Additionally, we performed the Bonferroni correction as a multiple comparison test to obtain the most significantly altered TCGA Pan-Cancer types (P < 0.001). For instance, ESCA was the most significantly altered (P < 0.001), and GBM was the less significantly altered (P < 0.001) TCGA Pan-Cancer type. Lastly, the complete information of Bonferroni correction results across TCGA Pan-Cancer types is detailed in Fig. Figure d GBM 0.00. Additi27. We analyzed the gene expression levels of 347 proteins related to the wound healing term (GO:0042060). Figure P-values. Of them, 33 were significantly overexpressed WH genes (P < 0.05), 47 were significantly underexpressed WH genes (P < 0.05), and 39 were both significantly overexpressed and underexpressed WH genes (P < 0.05). Regarding cancer drivers, we identified to CDKN1A, ERBB2, FLNA, MTOR, NF1, NFE2L2, SDC4, SMAD4, MYH9, NOTCH2, PTEN, GNA13, HIF1A, NACA, and GNAS as significantly expressed genes. Lastly, the overall analysis revealed 119 (34%) WH genes with significant expression in PDXs as detailed in the Supplementary Tables PDXs are in vivo models of human cancer types engrafted in mouse hosts for translational cancer research and therapy selection for individual patientsP > 0.05). Lastly, the overall analysis revealed 233 (67%) WH proteins with at least one high-confidence interaction (cutoff = 0.9) in the human proteome as detailed in the Supplementary Table Figure 35. On the one hand, Fig. P < 0.001) in comparison to the other cancer hallmarks. On the other hand, Fig. We analyzed the 347 WH proteins by using CancerGeneNet software to find the distance score of the shortest paths to cancer hallmark phenotypes according to Iannuccelli et al.q < 0.001 were wound healing , response to wound healing , response to stress , hemostasis , blood coagulation , platelet activation , among others. The most significant KEGG signaling pathways were pathways in cancer , proteoglycans in cancer , platelet activation , and endocrine resistance . Lastly, the most significant Reactome signaling pathways were platelet activation , hemostasis , and intrinsic pathway of fibrin clot formation   Fig. .Figure 62. On the one hand, six WH proteins of our study were already catalogued as key proteins in six hallmarks of cancer features according to the COSMIC and CGC databases19. NFE2L2, MTOR, and ERBB2 promotes change of cellular energetics; NOTCH2, MTOR, GNAS, and ERBB2 promotes proliferative signaling; MTOR promotes angiogenesis and NF1 suppresses angiogenesis; NOTCH2, NF1, and GNAS promotes suppression of growth; MTOR promotes invasion and metastasis and NF1 suppresses invasion and metastasis; and, NOTCH2, NFE2L2, and MTOR promotes escaping programmed cell death and NF1 suppresses escaping programmed cell death  sustaining proliferative signaling, (2) evading growth suppressors, (3) resisting cell death, (4) enabling replicative immortality, (5) inducing angiogenesis, (6) activating invasion and metastasis, 7) genome instability, (8) inflammation, (9) reprogramming of energy metabolism, and (10) evading immune destruction genome iath Fig. . On the 4. They proposed eight prospective hallmarks that might apply to both cancer and wound healing: (1) avoiding immune destruction, (2) wound-promoting inflammation, (3) activating invasion and migration, (4) inducing angiogenesis, (5) resisting cell death, (6) sustaining proliferative signaling, (7) evading growth suppressors, and 8) deregulating cellular energetics.Additionally, MacCarthy-Morrogh & Martin highlighted how tissue repair and cancer share cellular and molecular processes that are regulated in a wound but misregulated in cancerq < 0.001) between 20 WH proteins and hallmarks shared by cancer and wound healing. ANXA1, CDKN1A, FLNA, GNA13, HMGB1, KNG1, MTOR, NF1, NFE2L2, NOTCH2, YAP1, and VEGFA were involved in 8 significant annotations related to cell death ; ANXA1, CDKN1A, ERBB2, F2, FLNA, FZD6, GNA13, HMGB1, MTOR, NF1, NOTCH2, SDC4, YAP1, and VEGFA were involved in 6 significant annotations related to cell proliferation ; ANXA1, CDKN1A, CRK, ERBB2, F2, GNAS, MTOR, NOTCH2, YAP1, and VEGFA were involved in 2 significant annotations related to growth ; ACTG1, ANXA1, CDKN1A, CRK, ERBB2, F2, GNAS, HMGB1, MTOR, NFE2L2, NOTCH2, P2RY12, SDC4, YAP1, and VEGFA were involved in 5 significant annotations related to the immune system ; ANXA1, F2, GNAS, HMGB1, KNG1, NFE2L2, and NOTCH2 were involved in 1 significant annotation related to the inflammatory response ; ACTG1, ANXA1, CRK, F2, FLNA, GNA13, HMGB1, MTOR, NF1, NF2L2, P2RY12, SDC4, and VEGFA were involved in 10 significant annotations related to cell migration ; and ACTG1, ANXA1, ERBB2, FLNA, GNA13, HMGB1, MTOR, NF1, NF32L2, P2RY12, SDC4, and VEGFA were involved in 1 significant annotation related to angiogenesis  , NCI-60 and TCGA, showed similarities with our study regarding the mRNA high and CNV amplification frequency in solid tumors. Similarly, CNVs change the expression levels of genes located in the concerned genomic region52. However, a different set of genes were identified among studies due to the focus of our work in WH genes associated in cancer. Understanding the relationship between WH genes in cancer, their CNV (in germ cells), CNA (in somatic cells) and gene expression characteristics could help in the development of better diagnostic tools and therapeutics53.Among the alterations with the highest frequency mean, we found that mRNA high and CNV amplification were the most represented in the 347 WH genes/proteins analysed through the TCGA Pan-Cancer Atlas. Cancer is characterized by many genomic variations where a close correlation between CNVs and differential gene expression have a qualitative relationship with its downstream effect, especially for oncogenes and tumor suppressor genes4. We used these eight recently proposed wound healing hallmarks to classify groups of genes/proteins that participate in WH , ERBB2 , mTOR and VEGFA , HMGB1 , FLNA , and YAP1  promoting cell growth and metastasis when it is present in the cytoplasm and interacting with cell signalling molecules, or (2) inhibiting cell growth and preventing metastasis when it is acting in the nucleus and interacting with transcription factors79. In cancer, the inhibition of NF1 contributes to the generation of melanomagenesis by enhancing the activation of PI3K signaling, this inhibition favours angiogenesis, escaping apoptosis, migration, and cancer cell invasion83.In the tumor microenvironment and during WH, the physical interaction of fibroblasts with collagen regulate many cellular processes through the activity of FLNA. FLNA is an actin filament cross-linking protein that acts in the regulation of cell adhesion85. YAP1 has been associated with processes such as proliferation and the recruitment of M2 macrophages, myeloid-derived suppressor cells (MDSC), and regulatory T cells to suppress host effector T cells in the tumor microenvironment86. YAP1 activity in tumors results in cancer progression and drug resistance86. YAP1 contributes to cancer invasion and migration by promoting SNAI2 transcription through the transcription cofactor TEAD, in vivo and in vitro assays. YAP1 expression promotes cell proliferation, migration, and invasion, while silencing YAP1 significantly inhibits cell migration, invasion, and growth84. Given that YAP1 is hyperactive in many human cancers, it suggests that therapeutic targeting of YAP1 could regulate key processes in the tumor, thereby disrupting its survival mechanisms87.The YAP1 protein, a precursor of the activity of the YAP/TAZ signaling pathway, is essential for skin homeostasis and WH88. Similarly, ANXA1 promotes the development of blood vessels and metastasis with a deleterious effect in cancer, however positive effects during the WH process90.During the WH process, the inhibition of thrombospondin 1 (TSP-1) and 2 (TSP-2) by HRG promotes angiogenesis facilitaning wound closure91. Insufficient NFE2L2 expression impairs WH in a process associated with severe tissue damage and uncontrolled inflammation92. Inhibition of NFE2L2 in keratinocytes favors tumor development. In contrast, when NFE2L2 is functional, it plays an important role in reducing cellular stress, preventing DNA damage and cancerous mutations93. NFE2L2 expresses a bZIP protein with cytoprotective effects95, regulating genes involved in redox homeostasis97, this functioning in the stress defense in mammalian cells98. However, when NFE2L2 is overexpressed in cancer cells, it promotes their drug and radiotherapy resistance99. The CDKN1A gene was initially considered as a potential tumor suppressor. However, studies have reported that this gene could act as an oncogene due to its anti-apoptotic activities101. It is involved in p53-mediated inhibition of cell proliferation in response to DNA damage leading to cell cycle arrest at the G1/S checkpoint103. By overexpressing CDKN1A, wound fibroblasts enhance age-related healing, creating potential clinical avenues to promote wound healing in the elderly population104. Inhibition of this protein can act to enhance the regenerative response in several ways, altering DNA damage and checkpoint responses, leading to increased proliferation. It can reduce TGF-β signaling, which decreases scar formation and would alter differentiation patterns105.Among the least represented proteins, we found NFE2L2 and CDKN1A. NFE2L2 is activated after tissue damage and promotes wound repair protecting cells for the damaging effects of ROS production during inflammation106. These studies would allow therapeutic targeting and the development of specific pharmacological compounds that would stop cancer progression without affecting the healing process.This work identified 21 proteins in which eight have shown to be highly associated between WH and the cancer hallmarks. These proteins could represent critical nodes in the network of a controlled WH process or a deregulated interaction leading to tumor progression. Interestingly, the identified proteins were highly expressed only in solid tumors, as cancer cells must use them to modify the tissue microenvironment and induce immune cell regulation. This protein set could be specially needed to reorganize the wounded tissue and lead to regeneration. However, in the case of cancer, these proteins induce immune escape and survival. Putting in evidence these common proteins between WH and cancer brought light to those principally involved in sustained proliferation, invasion, angiogenesis, and others connected and overlapped between health and disease. Further interdisciplinary studies in vivo or in patients will support the in silico role of the identified and overlapped proteins. Interdisciplinarity is important as it brings together specialists in bioinformatics, health care professionals and technical experts leading to the development of better screening methods for early diagnosis and therapiesSupplementary Information.


A neuroimmune crosstalk is involved in somatic and visceral pathological pain including inflammatory and neuropathic components. Apart from microglia essential for spinal and supraspinal pain processing, the interaction of bone marrow-derived infiltrating macrophages and/or tissue-resident macrophages with the primary afferent neurons regulates pain signals in the peripheral tissue. Recent studies have uncovered previously unknown characteristics of tissue-resident macrophages, such as their origins and association with regulation of pain signals. Peripheral nerve macrophages and intestinal resident macrophages, in addition to adult monocyte-derived infiltrating macrophages, secrete a variety of mediators, such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, high mobility group box 1 and bone morphogenic protein 2 (BMP2), that regulate the excitability of the primary afferents. Neuron-derived mediators including neuropeptides, ATP and macrophage-colony stimulating factor regulate the activity or polarization of diverse macrophages. Thus, macrophages have multitasks in homeostatic conditions and participate in somatic and visceral pathological pain by interacting with neurons. Somatic or visceral nociceptive pain is an alarm reaction in response to tissue damage , abnormal muscle stresses, obstructions, high intraluminal pressure, inflammation, etc. Nociceptors sense the damaged or stimulated parts of the body, and signal the brain mostly via the spinal cord. Persistent or chronic pain that may remain unrelieved for a long time despite the disappearance of the causes, impairs the quality of life in patients, and is often resistant to existing analgesics such as non-steroidal anti-inflammatory drugs and narcotics . TherefoIn most tissues, two classes of macrophages, i.e., infiltrating and tissue-resident macrophages, are recognizable in health or disease. The former class of macrophages that infiltrate into tissues in response to injury or infection are derived from circulating monocytes that are produced by hematopoietic stem cells (HSCs) in the post-natal bone marrow. The latter class of macrophages are present in tissues under homeostatic conditions. The bone marrow-derived infiltrating macrophages and tissue-resident macrophages have common and different roles in health and/or disease, and both of them are considered to participate in processing or modulation of pain signals ,15. The It had been believed that tissue-resident macrophages were continuously repopulated by circulating monocytes that arose from progenitors in the adult bone marrow . HoweverTissue-resident macrophages play critical roles in the normal tissue homeostasis. On the other hand, in response to tissue injury or inflammatory insults, a number of circulating monocytes recruited from the bone marrow infiltrate the tissue and differentiate into macrophages. The recruited and resident macrophages proliferate and undergo phenotypic and functional changes in response to factors released from immune cells or neurons in the local tissue microenvironment ,25,26. M2 (PGE2) [2S)-forming enzyme, and CSE upregulation in activated macrophages increases generation of H2S and aggravates inflammation [2S causes functional upregulation of Cav3.2 T-type Ca2+ channels (a member of the low voltage-activated Ca2+ channel family) and activates transient receptor potential ankyrin 1 (TRPA1) channels in the sensory neurons, contributing to various intractable pain [2S mediates pathological pain by targeting Cav3.2 and TRPA1 (2+ mobilization and activation of cyclic AMP/protein kinase A (PKA), phospholipase C (PLC), protein kinase C (PKC), mitogen-activated protein (MAP) kinases (MAPK), nuclear factor-kappa B (NF-κB), Janus kinase (JAK)/signal transducers and activator of transcription (STAT) and Src, leading to neuronal sensitization or excitation [v3.2 channels, known as pro-nociceptive cation channels, contributing to pathological pain [A number of mediators released from M1 macrophages, including IL-1β ,30, IL-62 (PGE2) ,38,39, C2 (PGE2) , have be2 (PGE2) ,28,40 (F2 (PGE2) ,42,43,442 (PGE2) ,49,50,51ammation ,53. Inteble pain ,62,63,64nd TRPA1 B. M1 maccitation   3 inflammasome [4 receptors causes release of IL-1β and PGE2 from macrophages, participating in pathological pain [4 and/or P2X7 receptors [Some mediators released from the sensory neurons are capable of activating macrophages ,40,65,72nd edema  -induced type 1 diabetic rats, adult monocyte-derived macrophages accumulate in the sciatic nerves, contributing to painful diabetic neuropathy ,98. NeurChemotherapy-induced peripheral neuropathy (CIPN) frequently develops in cancer patients treated with anticancer agents, such as paclitaxel, oxaliplatin, vincristine, bortezomib, etc.  The impa+ channel, in SGCs, contributing to SGC activation [Neuron-glia-immune interaction in the spinal cord, DRG and enteric nervous system is now considered to play a role in the development of visceral hypersensitivity, which is associated with visceral pain accompanying inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and interstitial cystitis (IC)/bladder pain syndrome (BPS) . Interestivation  , which has been approved as a therapeutic medicine for treatment of disseminated intravascular coagulation (DIC) in Japan [Accumulating evidence for the macrophage–neuron crosstalk involved in pain regulation may provide a novel therapeutic avenue for patients with pathological pain. Among cytokines that induce polarization from M0 to M2 macrophages see A, IL-4, in Japan ,44. We hin Japan , CIPN [4in Japan ,45,108 ain Japan ,46,49 byin Japan .Tissue-resident macrophages, as well as bone marrow-derived infiltrating macrophages, interact with the primary sensory neurons in the peripheral tissues and DRG, and regulate not only inflammatory responses but also pain signals, as do microglial cells in the CNS. Recent studies have uncovered previously unknown characteristics of tissue-resident macrophages, such as their origins and association with regulation of pain signals. Particularly, peripheral nerve macrophages and intestinal resident macrophages including muscularis macrophages are considered to have multitasks in homeostatic conditions and participate in somatic and visceral pathological pain containing neuropathic and inflammatory components by interacting with neurons ,116,117.


Thyroid dysfunction  has been consistently linked to pregnancy adversity and abnormal fetal growth and development. Mood disorders such as anxiety, depression, and obsessive-compulsive disorder (OCD) are frequently diagnosed during pregnancy and at postpartum, and emerging evidence suggests association with impaired offspring neurodevelopment and growth. This study aimed to examine potential associations between thyroid function and mood symptoms during pregnancy and postpartum.This is a prospective study measuring thyroid hormones and assessing mood symptoms by employing specific questionnaires in the same cohort of 93 healthy pregnant women at the 24th (2nd trimester) and 36th (3rd trimester) gestational weeks and at the 1st postpartum week.Serum thyroid hormones, TSH, anti-TPO, and anti-Tg antibodies were measured at the 24th (2nd trimester) and 36th (3rd trimester) gestational weeks and at the 1st postpartum week. Specific validated questionnaires were employed at the same time-points to assess separately symptoms of anxiety , depression , and obsessive compulsive disorder (OCD) [Yale-Brown Obsessive Compulsive scale (Y-BOCS)].p < 0.010, r = −0.545) and positively with TSH  concentrations; GADI, PSWQ, EPDS and Y-BOCS scores correlated negatively with FT4 concentrations . At the 3rd trimester, BLUES score correlated positively with rT3 concentrations ; GADI, EPDS, and Y-BOCS scores correlated negatively with FT4 concentrations ; GADI, STAI-S, and Y-BOCS scores correlated positively with TSH concentrations . At postpartum, PSWQ, STAI-T, EPDS, and BDI scores correlated positively with rT3 concentrations , and BLUES score correlated positively with anti-TPO antibody concentrations .At the 2nd trimester, GADI score correlated negatively with FT3 (The reported findings demonstrate positive associations between low-normal thyroid function at the 2nd and 3rd trimesters of pregnancy and postpartum with anxiety, depression, and OCD scores. Mood disorders are common during pregnancy and postpartum and manifest as mild to severe depression, anxiety and obsessive-compulsive disorder (OCD), in previously healthy women . AccordiHomeostasis of thyroid hormones is challenged during pregnancy due to adaptive changes of the hypothalamic-pituitary thyroid (HPT) axis. In most pregnant women, the steep rise in serum concentrations of maternal thyroid-binding globulin, which follows the increase in estrogen concentrations, results in increased serum total triiodothyronine (TT3) and total tetraiodothyronine (TT4) concentrations. Free T3 (FT3) and free T4 (FT4) concentrations typically decrease after the 1st trimester, usually remaining within low-normal range . At postIn non-pregnant population, several reports have stressed the comorbidity of thyroid dysfunction and mood disorders: overt and subclinical hyperthyroidism have been associated with anxiety , mania , and depThis study investigated prospectively the possible associations between thyroid function and symptoms of anxiety, depression, and OCD  at the 2nd and 3rd trimesters of pregnancy and postpartum in the same cohort of healthy pregnant women.2.4 years old (mean ± SD), with normal weight or overweight, BMI 24.5 ± 1.1 kg/m2 (mean ± SD), were included in this study. The study has been approved by the ethics committee of our Institution, and all study participants gave their written informed consent.In an outpatient clinic of a University Obstetrics and Gynecology department, 121 primiparous Caucasian pregnant women were informed consecutively about this study and 105 of them accepted to participate by giving their informed consent. Patients were enrolled and followed up prospectively during the 2nd and 3rd trimesters of pregnancy and at postpartum (from 2015 to 2017). Inclusion criteria are as follows: clinical and biochemical euthyroidism according to TSH and FT4 concentrations , normal weight, or slightly overweight. Exclusion criteria are as follows: past or present history of diagnosed psychosis, depression, anxiety, and OCD based on medical history and screened with the Symptom Checklist (SCL-90-R) ; autoimmAll participants were seen in the outpatient clinic at the 2nd and 3rd trimesters of their pregnancy  and at the 1st postpartum week. At each visit, they underwent biochemical and psychometric tests. Biochemical evaluation was performed on serum obtained after an overnight fast. Blood samples were drawn at 8:00 a.m. for measurement of TT3, FT3, rT3, TT4, FT4, TSH, anti-thyroid peroxidase (anti-TPO), and anti-thyroglobulin (anti-Tg) antibodies and were stored immediately at −75°C for analysis. Psychometric evaluations were conducted by the same skilled psychiatrist (N.C.) with standardized structured instruments ; Penn State Worry Questionnaire (PSWQ); STAI-State Anxiety inventory (STAI-S); STAI-Trait Anxiety Inventory (STAI-T); Edinburgh Postnatal Depression Scale (EPDS); Stein-BLUES scale (BLUES); Beck’s Depression Inventory (BDI); Yale-Brown Obsessive Compulsive scale (Y-BOCS)]. There were no dropouts during the time period of this study.Serum TT3, FT3, rT3, TT4, FT4, TSH, anti-TPO, and anti-Tg antibody concentrations were measured by employing automated chemiluminescence immunoassay . For TT3, FT3, TT4, FT4, TSH, anti-TPO, and anti-Tg antibodies, the intra- and interassay coefficients of variation (CV) and the analytical sensitivity (AS) were, respectively, 5.5%, 7%, and 0.19 ng/ml; 9.1%, 9.4%, and 1.50 pmol/L; 4.6%, 6%, and 0.3 μg/dL; 5.9%, 6.4%, and 3.00 pmol/L; 3.8%, 4.6%, and 0.004 μUI/ml; 5.2%, 7.2%, and 10 U/ml; and 4.9%, 5.7%, and 20 U/L. Reverse T3 was measured using a radioimmunoassay  with intra- and interassay CV and AS at 8.54%, 6.21%, and 0.009 ng/ml, respectively. High and low concentrations were defined according to reference limits.This is a widely used, 90-item self-report questionnaire assessing a broad range of psychological and psychopathology symptoms . Each itThis is a self-report, 18-item, questionnaire employed to assess cognitive, somatic, and sleep symptoms in the context of generalized anxiety and panic disorder . Items aThis is a self-report, 16-item questionnaire employed to assess the trait of worry . Items aThis questionnaire consists of two separate 20-item questionnaires, one assessing state anxiety and the other trait anxiety . In the This is a widely employed, 10-item questionnaire assessing symptoms of depression and anxiety during perinatal period. Scores range from 0 to 80. Scores of 12 and greater are indicative of clinically significant symptoms of perinatal depression .This is a self-rating scale with 13 items which evaluates maternal blues. Scores range from 0 to 26, with greater scores denoting greater severity of maternal blues, while 8 is the clinical cut-off score, above which significant mood swings are suspected .This is a widely used, 21-item, self-report measure of depression . BDI iteThis is a self-report version of the Y-BOCS employed to assess obsessions and compulsions . It consClinical Endocrinology .The normality of the distributions was checked both graphically  and formally . Normally distributed variables are presented as mean ± SD, while non-normally as median (25th and 75th percentiles). All hormonal variables were normally distributed except for anti-TPO and anti-Tg antibodies. The quantitative results of the employed psychometric questionnaires exhibited non-normal distribution. To test the change of each variable during pregnancy, analysis of variance (ANOVA) and the non-parametric Wilcoxon tests were employed for variables with normal and non-normal distributions, respectively. To test the changes of hormonal and psychometric variables over the three time points of the study protocol during pregnancy and postpartum, ANOVA for repeated measures was employed. The previously reported TSH, FT4, FT3, TT4, TT3, rT3, anti-TPO, and anti-Tg antibody concentrations measured in peripheral maternal blood in the studied subjects at the three time points of the study protocol were employed in this study for correlation analyses according to authorization received from rinology . To testp = 0.042) from the 2nd to the 3d trimester of pregnancy. The rest of the applied questionnaires did not change significantly during pregnancy and postpartum. None of the participants developed a psychiatric illness during pregnancy although some women have scored in certain questionnaires above the respective cut-off points. Following assessment with the psychometric questionnaires during the 2nd and 3rd trimesters and at postpartum 27.8% (n = 25), 25.6% (n = 23), and 14.4% (n = 13) of the studied women, respectively, were considered possible candidates for anxiety while 11.1% (n = 10), 7.8% (n = 7), and 5.6% (n = 5) of the studied women, respectively, were considered possible candidates for depression.Serum TT3, TT4, FT3, FT4, TSH, anti-TPO, and anti-Tg antibodies mean concentrations at the 2nd and 3rd trimesters of pregnancy and postpartum and their differences at these three time points are reported in p < 0.010, r = −0.545); GADI, EPDS, PSWQ and Y-BOCS scores correlated negatively with FT4 concentrations ; GADI score correlated positively with TSH concentrations .Significant correlations among hormonal and psychometric variables at the 2nd trimester are presented in p = 0.000, r = 0.89); GADI, EPDS, and Y-BOCS scores correlated negatively with FT4 concentrations ; GADI, STAI-S, and Y-BOCS scores correlated positively with TSH concentrations .Significant correlations among hormonal and psychometric variables at the 3rd trimester are presented in p = 0.024, r = 0.478; p = 0.014, r = 0.527; p = 0.046, r = 0.44; p = 0.021, r = 0.556, respectively); BLUES score correlated positively with anti-TPO antibody concentrations ; Y-BOCS score correlated positively with TSH concentrations .Significant correlations among hormonal and psychometric variables at postpartum are presented in p = 0.006, p = 0.058, and p = 0.020, respectively) as presented in p < 0.001, p = 0.031, and p < 0.001, respectively).Longitudinal bivariate models showed a significant positive change of GADI, EPDS, and YBOCS scores (taken separately as dependent variables) with time and TSH taken together as independent variables , in these euthyroid women without history of mental illness, correlated negatively with circulating FT3 and FT4 and positively with TSH concentrations, while at postpartum anxiety scores  correlated positively with rT3 concentrations. These findings indicate a positive association between low-normal thyroid function with anxiety scoring during pregnancy and postpartum. In the past, the majority of studies in pregnancy and postpartum assessed concurrently depressive mood and anxiety with regard to thyroid function . In men via decreased central serotonin (5-HT) activity. Thyroid hormones modulate the serotonergic system while plasma 5-HT concentrations correlate positively with thyroid hormone concentrations  correlated negatively with circulating FT4 concentrations, while at the 3rd trimester depression scores (BLUES) correlated positively with rT3 concentrations. At postpartum, depression scores  correlated positively with rT3 concentrations while scores derived from the BLUES questionnaire showed a trend of positive correlation with anti-TPO concentrations. These findings indicate a positive association between low-normal thyroid function with depression scoring during pregnancy and postpartum. In the past, in the 3rd trimester of pregnancy, low-normal FT4 concentrations have been associated positively with depressive mood . At posttrations , 1996. Ltrations , 2013. Htrations . It is ntrations .Finally, in the present study, at the 2nd and 3d trimesters of pregnancy, OCD scores (Y-BOCS) correlated negatively with circulating FT4 concentrations while at the 3rd trimester and at post-partum, they correlated positively with TSH concentrations. To the best of our knowledge, this is the first report indicating a positive association between low-normal thyroid function with OCD scoring during pregnancy and postpartum. A meta-analysis revealed that women during pregnancy and postpartum had a 45 and a 138% increased risk, respectively, for developing OCD . The latThe simultaneous psychometric evaluation of anxiety, depression, and OCD in the same cohort of euthyroid pregnant women without history of mental illness at the 2nd and 3rd trimester of pregnancy as well as at postpartum is reported for the first time, to the best of our knowledge. The prospective design of the study and the separate rating of anxiety, depression, and OCD add to the consistency of the reported data. Limitations of this study are as follows: the studied women may not be representative of the general population because they were recruited from a tertiary care university hospital where a higher number of high risk pregnancies are attended; the lack of psychometric measurements during the 1st trimester of pregnancy; and the number of the studied women is relatively small.In this study, measures for depression, anxiety, and OCD improved in figures but not in a statistically significant way in the course of pregnancy and postpartum. Regarding anxiety, according to a published meta-analysis in 221,974 women from 34 countries, when gold standard diagnostic interviews were employed, the rates dropped from 18% in the 1st trimester to 15 and 15% in the 2nd and 3rd trimesters, respectively. Prevalence continued to drop at postpartum and ranged from 9.3 to 9.9% across 1st year . The repDepressive and anxiety disorders, during pregnancy and at postpartum, are complex clinical conditions with atypical features and symptoms, overlapping with “physiologic” mood changes. Maternal prenatal anxiety has been related to increased postnatal risk of neurodevelopmental disorders (separately and independently of depression) . FetusesThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Aretaieio University Hospital, National and Kapodistrian University of Athens. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer ST declared a past co-authorship with one of the authors GM to the handling editor.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As sphingolipids are constituents of the cell and vacuole membranes of eukaryotic cells, they are a critical component acquired from our daily diets. In the present review, we highlight the knowledge regarding how dietary sphingolipids affect our health, particularly our intestinal health. Animal- and plant-derived foods contain, respectively, sphingomyelin (SM) and glucosylceramide (GlcCer) as their representative sphingolipids, and the sphingoid base as a specific structure of sphingolipids also differs depending upon the source and class. For example, sphingosine is predominant among animal sphingolipids, and tri-hydroxy bases are present in free ceramide (Cer) from plants and fungi. Dietary sphingolipids exhibit low absorption ratios; however, they possess various functions. GlcCer facilitates improvements in intestinal impairments, lipid metabolisms, and skin disorders, and SM can exert both similar and different effects compared to those elicited by GlcCer. We discuss the digestion, absorption, metabolism, and function of sphingolipids while focused on the structure. Additionally, we also review old and new classes in the context of current advancements in analytical instruments. The intestine digests food materials and absorbs nutrients and water and is also deeply implicated in human health maintenance via the immune and nervous systems . HoweverSphingolipids are primarily located within the cell and vacuole membranes of most eukaryotes and some prokaryotes, and, based on this, we consume sphingolipids daily in our diets. Sphingolipids present in most foods primarily exist as complex sphingolipids that are composed of a sphingoid base (SB) with an amide-linked fatty acid  and a polar head group, rather than a free Cer . ThroughSaccharomyces cerevisiae) used for panary fermentation and sake brewing do not contain GlcCer, while yeasts contain Cer, IPC, and GIPC .,26.trans:24t,8t) ,28. Cer,in fungi . Sphingo,8t,10t) ,31,32,33A number of researchers have investigated the sphingolipid content in diets. The daily intake of sphingolipids from the American diet has been reported as 300–400 mg according to calculations of the sphingolipid content  in food materials . The daiAs mentioned above, foods contain various complex sphingolipids possessing different polar head groups and SBs. Complex sphingolipids are digested to generate SBs and then absorbed via the lymph, and absorbed SBs are partially resynthesized into complex sphingolipids, while they are predominantly metabolized into fatty acids ,32,39,40t4 from animals. Nelson previously reported that when SM with labeled d18:1t4 was administered to rats, approximately 8% and 20% of administered radioactivity levels were observed in lymph collected during a 24 h period and in feces collected for 4 days, respectively [t4 resulted in approximately 4% and 40% radioactivity found in lymph and feces, respectively [t4 were similar to those of SM with d18:1t4. Conversely, free d18:0 exhibited a much higher absorption ratio than did free d18:1t4; however, the majority of the d18:0 absorbed into the lymph was used for triglyceride synthesis, and, based on this, the level of d18:0 incorporated into sphingolipids was almost equal to that of d18:1t4. Sugawara et al. studied an intestinal tract in vitro model and reported that plant- and fungi-specific SBs  exhibited much lower absorption ratios as free SBs compared to those of the primary animal SB d18:1t4 due to the efflux of plant and fungal SBs by P-glycoprotein [t,8c4 as the major SB in rice and maize exhibited lower absorption into rat lymph than did d18:1t4 due to efflux. Among the polyunsaturated SBs , d18:2t,8c4 has been reported to be the SB that is most incorporated into generated sphingolipids in rat lymph [Oral administration of sphingolipids results in lower digestion and absorption compared to other lipids. Additionally, the digestive ratio depends upon the sphingolipid classes and the fatty acid composition, and the absorption ratio depends upon the SB structure. Glycosphingolipids exhibit lower digestion than do phosphosphingolipids, and plant- and fungi-specific SBs exhibit lower absorption than does SB d18:1ectively . In contectively ,47. The oprotein ,49,50, aat lymph .It has been reported that colon cancer alters sphingolipid metabolism . Due to N-palmitoylsphingomyelin; C16-d18:1t4 with phosphocholine), and synthetic dihydroSM (C16-d18:0 with phosphocholine), it was observed that all SMs suppressed DMH-induced ACF formation and that the effect of dihydroSM was greater than those of the other SMs [Schmelz et al. characterized the chemotherapeutic effects of SM in regard to colon cancer in detail. After an interval of 1 week following DMH treatment, feeding mice a diet containing 0.1% milk-derived SM for 4 weeks alleviated the formation of aberrant crypt foci (ACF), which are the earliest visible changes involved in the formation of colon cancer, in the colons of these mice . Low SM ther SMs . SM exhither SMs . Both app53 deficiency, wild-type and p53 mutant mice were treated with AOM to investigate the relationship between SM feeding and the tumor suppressor gene p53. The results indicated that a 0.1% SM diet for 4 weeks inhibited cell proliferation but did not induce apoptosis in the distal colon of both types of AOM-treated mice [p53 did not affect SM-inhibited cell proliferation.For aging rats (54 weeks of age) that were treated with AOM, daily administration of SM (35 mg/kg body weight) for 6 weeks after an interval of 6 weeks suppressed ACF formation in the proximal region; however, SM feeding did not affect the AOM-mediated decrease in NK cell activity . SM alsoted mice . FeedingDietary SM exerts chemopreventive and chemotherapeutic effects in the context of colon cancer. Upon SM intake, SM and the fragments that include Cer and SB can reach the large intestine . Continut4.Schmelz et al. also reported the chemotherapeutic effects of glycosphingolipids on colon cancer . DMH-indt,8c4 and a 0.1% diet of GlcCer from yeast (Saccharomyces kluyveri) that primarily contained the SB 9-Me d18:2t,8t4. All diets alleviated ACF formation, and the suppressive effects were essentially equal among the three groups. GlcCer bearing d18:2t,8c4 was found in the feces and colon mucosa of mice that were fed maize-derived GlcCer. Higher GlcCer concentrations within the diet resulted in higher GlcCer levels in the mouse feces. To clarify the suppressive mechanism of dietary GlcCer, DNA microarray and quantitative RT-PCR analyses were performed on the colon mucosa of mice that were fed 0.1% maize-derived GlcCer from 10 days prior to DMH treatment for 10 weeks [Soggy-1 mRNA, which suppressed the Wnt signaling pathway and decreased the expression of Ras-associated protein to induce the Ras pathway; however, no significant increases were observed in the expression of the genes, such as those of the caspase family, that induce apoptosis directly, thus suggesting that dietary GlcCer regulates cell proliferation and differentiation to prevent the development of ACF in the colon. Moreover, we investigated the effects of GlcCer feeding on colon inflammation following DMH treatment [We investigated the chemopreventive effects of GlcCer, and we also determined the SB structure . Ten day10 weeks . GlcCer reatment . Mice wet,8c4 for 5 weeks from 1 week prior to DMH treatment. GlcCer feeding suppressed the colonic formation of ACF and β-catenin-accumulated crypts. Moreover, GlcCer decreased the ratio of proliferation hallmarks in ACF and β-catenin-accumulated crypts but did not affect the ratio of apoptosis hallmarks in these crypts.When focusing on the chemopreventive mechanism of GlcCer, proliferation and apoptosis markers were examined in DMH-induced mutant crypts . DMH-tret,8t4 has also been reported to exert chemotherapeutic effects [adenomatous polyposis coli (APC) gene, GlcCer was found to affect spontaneous tumorigenesis. Min mice were fed 0.1% and 0.025% diets of soybean-derived GlcCer for 8 weeks. Dietary GlcCer intake reduced tumor proliferation in the small intestine in a dose-dependent manner and suppressed mRNA expression of the Wnt signaling pathway-related gene TCF4 and the angiogenesis-related gene HIF1-α in the intestinal mucosa.Dietary soybean-derived GlcCer that primarily contains the SB d18:2 effects . FeedingGlycosphingolipids exert suppressive effects on colon cancer regardless of their polar head groups and SB composition. These effects may result from anti-proliferation activities via anti-inflammatory effects. Epidemiological studies have demonstrated that colon inflammation increases the incidence of colon cancer and that long-term inflammation stress induces cell dedifferentiation ,3,4. GlyN-palmitoylsphingosine; C16-d18:1t4), milk-derived complex sphingolipids , and their combination (Cer/complex sphingolipids = 40/60) on intestinal cancer using Min mice with a truncated APC gene product, all of these diets at 0.1% for 8 weeks were demonstrated to suppress intestinal tumor formation. Their combination exhibited the highest efficacy, and Cer exerted a better effect on the small intestine and lesser effect on the colon compared to the effects of complex sphingolipids [When comparing the effects of dietary Cer (golipids . Additiogolipids . DietaryGrifola frondosa), a 10% combination of these ingredients (each 5%), and a 20% combination of these ingredients (each 10%) from 1 week prior to DMH treatment for 10 weeks [To examine the effects of food materials containing sphingolipids on DMH-treated mice, mice were fed diets containing 10% dried whole milk, 10% dried maitake mushroom  and its ethanol extract (RE), both of which contain the same level of GlcCer, can improve intestinal disease, and we also sought to determine if these effects depend upon the existence of GlcCer . Mice weSubsequently, to clarify whether changes in sphingolipid composition in foods subjected to fermentation affect intestinal protection, mice were fed a sake rice extract diet (0.9 g extracted from sake rice 150 g/kg diet) or a sake lees extract diet (42.1 g extracted from sake lees 150 g/kg diet) from 2 weeks prior to DMH treatment for 7 weeks . Sake leDietary sphingolipids are partially digested by intestinal enzymes and enteric bacteria into Cer and SB in the intestine. Exogenous Cer and SB induce apoptosis in various cancer cells, including colon cancer cells. Cer-bearing short-chain fatty acids (C2 to C8) are often used for experiments due to their cell permeability, while sphingolipids possessing long-chain fatty acids (C14 to C26) or hydroxy fatty acids are contained in foods.t4 and d18:0 as free SB and of C2-d18:1t4 as Cer induced apoptosis and arrested the cell cycle in the G2/M phase in human colon cancer cells (HT29 and HCT-116), while treatment with dihydroCer (C2-d18:0) did not induce apoptosis and cell cycle arrest [t4, t18:0, d18:1c8, d18:1t8, d18:2t,8c4, d18:2t,8t4, d18:3t,8t,10t4, 9-Me d18:2t,8t4, and 9-Me d18:3t,8 t,10t4), all of the SBs used in these experiments exhibited apoptotic effects on human colon cancer cells , but not in normal intestinal cells [t4 was enhanced in colon cancer cells  by treatment with P-glycoprotein inhibitors  [t4 with tamoxifen increased intracellular Cer levels, decreased GlcCer levels, and induced apoptosis in a manner that was independent of p53. Conversely, low levels of natural Cer possessing long-chain fatty acids from the bovine brain reduced cytosolic β-catenin levels in colon cancer cells (SW480), while C2-d18:1t4 did not reduce these levels [The addition of d18:1e arrest ,72. Compal cells ,74. SB irapamil) . Co-treae levels .Various SBs from foods exert similar levels of apoptotic induction in colon cancer cells ,73,74; hIBD is a form of refractory enteritis, and chronic colon inflammation increases the risk of the onset of colon cancer. Regulation of sphingolipid metabolism is an attractive therapeutic target for colitis diseases, including IBD. The colon mucosa of patients with IBD has been reported to increase the level of sphingosine kinase 1, an enzyme that phosphorylates SB to sphingosine1-phosphate (S1P) . ExperimDietary SM has been reported to suppress and to stimulate colon inflammation. In a study examining the suppressive effects, mice were fed 0.1% SM (origin-unknown) from 3 days prior to 2% DSS treatment for 1 week, and SM feeding suppressed body weight loss, colon injury, and colon myeloperoxidase (MPO) activity (an indicator of neutrophil invasion) in DSS-treated mice and promoted IgA secretion into the large intestine in non-DSS-treated mice . In contAnother study reported the effects of dietary SM on inflammation-related colon cancer induced by treatment with DSS in combination with AOM . Mice wit4, accelerates inflammation via apoptosis [Differences in the inflammation stage or SM structure may determine whether dietary SM exerts beneficial or unfavorable effects in the context of colon inflammation. Inflammation affects the intestinal barrier and sphingolipid metabolism, and Cer synthase deficiency and SMase inhibitors suppress colon inflammation in experimental colitis ,85. Addipoptosis ,87. In gpoptosis . C16-SM poptosis . Thus, eTo the best of our knowledge, there is only one report regarding the beneficial effects of dietary GlcCer on DSS-induced colitis . Mice weDietary ganglioside has been reported to protect against small intestinal inflammation induced by high-fat diet and lipopolysaccharide (LPS) treatment ,90. RatsPleurotus citrinopileatus). Mice were fed 1% or 5% mushroom extract diets 10 days prior to DSS treatment for 25 days. Colon samples on the 18th and 26th days from the switch to 1.5% DSS drinking water were used for cytokine analysis and histological analysis, respectively. Dietary mushroom extract suppressed DSS-induced body weight loss, colon length reduction, and spleen weight. Dietary mushroom extract ameliorated colon villi damage and increased the production of inflammation-related cytokines in a dose-dependent manner. Moreover, the mushroom extract was separated into two fractions containing polar lipids  and neutral lipids (Cer and free fatty acids) through the use of acetone. Mice were fed 1% polar lipid or neutral lipid diets from 1 week prior to DSS treatment for 20 days. On the 21st day after the switch to 1.5% DSS drinking water, dietary mushroom polar lipids improved DSS-induced colon villi damage, while dietary neutral lipids caused the damage to worsen. Using differentiated Caco-2 cells as an intestinal tract in vitro model, the polar lipid fraction suppressed LPS-induced apoptosis, while treatment with the neutral lipid fraction resulted in weaker suppression. Therefore, the consumption of mushroom glycosphingolipids is speculated to contribute to colitis prevention.The beneficial effects of a sphingolipid-rich fraction from mushrooms on DSS-induced colitis have been demonstrated ,93. The In experiments using bowel tissue obtained from infants requiring open bowel surgery for intestinal atresias, pretreatment with milk-derived ganglioside (primary species GD3) was reported to alleviate LPS-, hypoxia-, and combination-induced inflammation via suppression of inflammatory cytokine production and oxidative stress .c8), maize-derived GlcCer , and bovine brain-derived GalCer (primarily d18:1t4) suppressed cell injury due to inflammatory stress, and there was no observed difference in these suppressive effects among all complex sphingolipids studied [t4) treatment induced apoptosis in differentiated Caco-2 cells under non-stress conditions, and Cer treatment suppressed LPS-induced apoptosis (unpublished data). In contrast, GlcCer, SBs, and S1P were not observed to induce apoptosis under non-stress conditions, and they suppressed LPS-induced apoptosis. These results suggest that GlcCer is non-cytotoxic, accumulates on cell membranes, and is metabolized during inflammation to protect intestinal cells by maintaining sphingolipid levels in cells and producing S1P. Additionally, as highly polar sphingolipids containing GIPC from mushroom suppressed LPS-induced apoptosis to a greater degree compared to the observed suppression by Cer and GlcCer [Using differentiated Caco-2 cells as a normal human intestinal model, the protective mechanism of complex sphingolipids on inflammation stress was investigated in detail. The addition of TNF-α or LPS to induce inflammatory stress decreased cell viability through the induction of apoptosis. Wheat-derived GlcCer  and free SB (d18:1t4) also suppressed LPS-induced expression of TNF-α, while dihydroCer (C16-d18:0 and C24-d18:0) did not. In contrast, macrophages of SM synthase 2-deficient mice (deficiency of SM bearing very long-chain fatty acids) exhibited decreased sensitivity to thioglycolate and LPS [Milk-derived SM suppressed LPS-induced mRNA expression of TNF-α in RAW264.7 macrophages, while SM in combination with SMase inhibitor treatment did not . Cer  depending on the source, and, therefore, it is necessary to distinguish between the functions of food-derived and endogenous sphingolipids. There is a possibility that various functions of dietary sphingolipids are exerted by intestinal homeostasis mechanisms such as nutritional absorption, intestinal barrier function, and gut immunity. Further macro- and micro-studies will further clarify the roles of dietary sphingolipids in the near future.


Adipose tissue is widely recognized as an extremely active endocrine organ producing adipokines as leptin that bridge metabolism and the immune system. Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (PREP1) is a ubiquitous homeodomain transcription factor involved in the adipogenic differentiation and insulin-sensitivity processes. Leptin, as pleiotropic adipokine, and TGF-β, known to be expressed by primary pre-adipocytes [adipose-derived stem cells (ASCs)] and mature differentiated adipocytes, modulate inflammatory responses. We aimed to assess for the first time if leptin and TGF-β interfere with PREP1 expression in both ASCs and mature differentiated adipocytes. Human ASCs were isolated from subcutaneous adipose liposuction and, after expansion, fully differentiated to mature adipocytes. In both ASCs and adipocytes, leptin and TGF-β1 significantly decreased the expression of PREP1, alone and following concurrent Toll-like receptor 4 (TLR4) activation. Moreover, in adipocytes, but not in ASCs, leptin increased TLR4 and IL-33 expression, whereas TGF-β1 enhanced TLR4 and IL-6 expression. Taken together, we provide evidence for a direct regulation of PREP1 by leptin and TGF-β1 in ASCs and mature adipocytes. The effects of leptin and TGF-β1 on immune receptors and cytokines, however, are limited to mature adipocytes, suggesting that modulating immune responses depends on the differentiation of ASCs. Further studies are needed to fully understand the regulation of PREP1 expression and its potential for the development of new therapeutic approaches in obesity-related diseases. Adipokines, with inflammatory and anti-inflammatory activities, are known to modulate the immune system. Particularly, IL-6, IL-33, and leptin participate in the maintenance of the metabolic balance by increasing the insulin sensitivity and preserving adipose tissue homeostasis  was considered an energetic storage tissue only. Many years of experimental and clinical research have provided evidence for the crucial role of WAT in regulating, besides metabolism, the immune system . WAT doeeostasis . Leptin eostasis . Leptin eostasis .Interestingly, there is evidence that leptin can induce the perpetuation of inflammation by maintaining immune cell survival ; on the Leptin and IL-33 are both involved in basophil degranulation , whereasIt is well known that adipose tissue is an endocrine organ, but also an immune organ, as it is physiologically infiltrated by innate immune cells. The health and homeostasis of adipose tissue is important to contribute for maintaining a regular and physiological immune response. Adipose tissue exhibits important roles in regulating energy metabolism and insulin sensitivity . NeverthBoth leptin and insulin are of equal importance in the pathophysiology of obesity and type 2 diabetes T2DM; . On the Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (PREP1) plays a role in metabolic disorders. It is a homeodomain transcription factor from the three-amino acid loop extension (TALE) superclass of proteins that dimerizes with Pbx1, which is implicated in modulating embryonic development  and metaHowever, the precise mechanisms whereby adipocyte and various immune cells crosstalk with each other to aggravate obesity-induced adipose inflammation and metabolic dysregulation remain poorly defined. The present study was performed to establish the role of leptin and TGF-β in the regulation of PREP1 expression in both primary human ASCs and mature differentiated adipocytes. The expression of additional molecules involved in the interaction with the immune system was also investigated.Clostridium histolyticum cat no. 11088793001) was from Roche . Recombinant human insulin (cat no. 26360.01) and dexamethasone (cat no. 18660.01) were from Serva .1 Indomethacin (cat no. 70270) was from Cayman . Recombinant human leptin (398-LP) and recombinant human TGF-β1 (240-B) were purchased from R&D System . Monoclonal PREP1 (B-2): sc-25282 antibody, rabbit polyclonal TLR4 (H-80): sc-10741 antibody, HBD2 (FL-64): sc-20798 antibody, and goat polyclonal anti-leptin receptor (Ob-R M-18): sc-1834 antibody were from Santa Cruz Biotechnology, Inc. . Monoclonal CD284 (TLR4) PE (cat no 12-9917) antibody was from eBioscience . Monoclonal anti-CD34, anti-CD45, anti-CD73, anti-CD90, and anti-CD105 antibodies, pure or FITC/PE conjugate, were purchased from MACS, Miltenyi Biotec (Germany). Normal donkey , normal goat serum , and TRITC-conjugated donkey anti-rabbit IgG (711-025-152) were from Jackson Immunoresearch . Alexa Fluor® 488-conjugated goat anti-mouse IgG (A 11029) was from Thermo Fisher Scientific . TRIzol reagent was from Life Technologies , iScript cDNA Synthesis kit was from BioRad , and FAM-labeled probe and primers were from Applied Biosystems .Collagenase A (from 2 in complete proliferation medium: D-MEM cell growth medium high glucose (4.5 g/L D-glucose), 3.7 g/L NaHCO3, W/O sodium pyruvate, L-glutamine, and phenol red  with 1% antibiotics mixture , 1% L-glutamine 200 mM, 1% sodium pyruvate, and 20% FBS. The yield of ASCs (undifferentiated pre-adipocytes) obtained from liposuction sample after digestion was patient-dependent. Usually, we obtained approximately 2 × 105 cells in a single well of a 12-well plate or approximately 1 × 105 cells in a single well of a 24-well plate for an amount of approximately 500 or 250 mg of liposuction aspiration fluid adipose tissue, respectively. Seventy-two hours after plating, D-MEM cell growth medium 20% FBS was replaced with fresh D-MEM cell growth medium 10% FBS. This latter was changed every 3 days. At this point, there were two options: ASCs were allowed to reach between 80 and 90% confluence and induced to differentiate; otherwise, ASCs were directly stimulated as briefly described: ASCs were cultured in D-MEM cell growth medium 10% FBS in the presence and absence of recombinant human leptin (0.001–2.5 μM) and recombinant human TGF-β1 (40–1.600 pM) for 24 h. The best active concentrations were 0.5 μM for leptin and 400 pM for TGF-β1. For the TLR4 pathway activation, we used endotoxin, LPS  for 24 h. Cells and supernatants (stored at –80°C after centrifugation until ELISA test) were then collected for further analyses.Adipose tissue was collected by liposuction aspiration of subcutaneous fat from non-obese and non-diabetic anonymous subjects . All subjects were non-smokers and without hypertension, cardiovascular or metabolic disease, cancer, or chronic pulmonary diseases. They signed their informed consensus, and the Ethics Committee of Policlinico, Giaccone Hospital, Palermo, Italy (authorization reference number 12/2014) in agreement with the Helsinki Declaration approved the study. Every adipose sample was kept at room temperature (RT) for less than 24 h prior to use, and ASCs were isolated as previously described . Briefly5 ASCs per well seeded in a 24-well plate were at 80–90% confluence, we started to induce the adipogenic differentiation by adding 1 mL complete D-MEM differentiation medium. The medium was carefully changed twice per week. In 28 days, the cell differentiation process was completed. At this day, mature adipocytes either were tested for red oil staining (below described) or were cultured in complete D-MEM differentiation medium, in the presence and absence of recombinant human leptin and recombinant human TGF-β1 at the same conditions used for ASCs. Cells and supernatants (stored at –80°C after centrifugation until ELISA test) were then collected for further analyses.Adipose-derived stem cells display multipotent characteristics, meaning that despite they derive from an adult human tissue, they maintain the ability to differentiate into multilineage cell types  , leadingThe accumulation of neutral lipids was detected by staining the cells on a slide as described: cells, adherent in a plasticware plate, were fixed with 10% paraformaldehyde for 90 min at 37°C; afterward, cells were stained in a solution of Oil Red O solution (Oil Red O solution—0.5% in isopropanol (O1391) from Sigma-Aldrich, St. Louis, MO, United States) for 30 min at 37°C. The percentage of cells undergoing adipogenesis was calculated by image analyzer microscope .The multipotent ASCs cell differentiation process from mature differentiated adipocytes was also assessed by specific flow cytometric surface antigen expression analyses of both ASCs and mature differentiated adipocytes. The mesenchymal stromal cells express CD73, CD90, and CD105 and lack the expression (≤2% positive) of CD45, CD34, and other surface antigens . FurtherTo maintain the integrity of marker membrane, ASCs were seeded with proliferation medium on glass coverslips, previously sterilized with ethanol 70%, and allowed to attach for 72 h before stimulation or before adding differentiation medium. After stimulation of both ASCs and adipocytes, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and blocked in 0.1% Tween 20 in PBS (TPBS) containing 2% BSA and 3% normal goat serum for PREP1, CD34, CD73, and CD90 or 3% normal donkey serum for TLR4, for 1 h, before staining. Cells were then incubated with anti-PREP1, anti-CD34, anti-CD73, anti-CD90, and anti-TLR4  antibodies in TPBS containing 2% BSA and 1.5% normal goat or 1.5% normal donkey serum, respectively, for 2 h at RT. Incubation with the appropriate secondary antibodies Alexa Fluor 488 or TRITC-conjugated (1:200) in TPBS containing 2% BSA and 1.5% normal goat or 1.5% normal donkey serum, respectively, was performed for 30 min. Glass coverslips were mounted on slides in Vectashield  with 406-diamidine-2-phenylindole-dihydrochloride (DAPI) to visualize the nuclei. Staining of the samples was observed and evaluated with an Axioskop-2-Zeiss microscope/software. Control slides were prepared using an irrelevant antibody of the same isotype and at the same concentration as the specific primary, and no significant staining or fluorescence signals were detected.Ct–ΔΔ), and untreated cells were used as reference samples.The whole RNA was isolated from ASCs and from mature adipocytes using TRIzol reagent (Life Technologies) following the manufacturer’s instruction. Then, 1 μg of RNA was reverse-transcribed to cDNA, using iScript cDNA Synthesis kit (BioRad). Quantitative real-time PCR by TaqMan Assay to evaluate the gene expression of PREP1, leptin, leptin receptors, TGF-β1, TLR4, IL-6, and IL-33 was performed. Transcripts were carried out with StepOnePlus Real-time PCR System  using specific FAM-labeled probe and primers . Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  in each case . The resSupernatants were recovered, centrifuged, and stored at –80°C until assayed. IL-6, IL-8, IL-33, IL-1β, and TNF-α were measured with a commercially available ELISA Kit  following the manufacturer’s instructions.p-value < 0.05 was considered statistically significant.The analysis of results was performed from 3 to 7 independent biological replicates. The numbers of independent experiments are indicated in the figure legends. Analysis of variance (ANOVA) was used for testing differences between means. The possible association between categorical variables was evaluated by the accurate Fisher’s exact test. A Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy , whereas the expression of CD34 and CD45 markers was not significantly different between the two cytotypes and less than 2% (After the isolation from the liposuction sample (different devices), the undifferentiated multipotent ASCs were cultured and expanded by using specific proliferation culture medium . ASCs we Therapy . The oth than 2% . Figure ∗p = 0.0149 and ∗∗p = 0.0318, respectively) in both ASCs   in mature differentiated adipocytes    . Furtheripocytes , but notctively) . In adipctively) .in vitro    protein and multiple cytokines, namely, TNF-α, IL-1β, IL-6, IL-8, and IL-33. HBD2 protein expression was not significantly affected by either leptin or TGF-β1, on both ASCs and adipocytes , 8A. In ctively) , whereasctively) . Moreovectively) . On the ctively) , but IL-ctively) , 8C.in vitro model to investigate the mechanisms responsible for obesity in humans. Obesity is an epidemic disease characterized by a chronic low-grade state of inflammation; obesity and T2DM are both genetically defined and of absolute concern globally, in almost all countries in the world , deficit (lipodystrophy), and/or altered regional distribution of adipose tissue, are frequently associated with insulin resistance frameworks predisposed to T2DM . Leptin We demonstrate in this study that leptin, TGF-β1, or TLR4 activation, by decreasing PREP1 levels, can limit the adipogenesis and, at the same time, may maintain adipose tissue homeostasis. This latter concept is also important in understanding the different basal PREP1 expression levels in ASCs compared with mature differentiated adipocytes. PREP1 is a notable transcription factor involved in embryogenesis and first organ development. Its downregulation in zebrafish and its deletion in mice were associated with embryonic lethality . Based oInterestingly, IL-33 contributes to maintain the lean state through the activation of anti-inflammatory M2 macrophages, and in humans, low serum IL-33 levels are associated with high BMI .ob/ob demonstrated increased expression of TNF receptor genes, whereas IL-33, cholecystokinin, plasminogen activator, IL-1β, and serine peptidase inhibitor genes were downregulated  have suggested its efficacy in patients with T2DM  as leptiTo date, innovative hypoglycemic therapies have not only metabolic but also pleiotropic effects, directing the diabetologist no longer to the evaluation of glycemia but above all in the “tailored” management of the patient and the complications of the metabolic syndrome . The metHere, although the direct impact of leptin, TGF-β1, or TLR4 activation (by adding LPS) in the differentiation processes of ASCs was not assessed, we speculate that leptin, TGF-β1, or TLR4 activation reduced PREP1, a molecule with a known fundamental role in the adipocyte differentiation process, can also reduce adipogenesis. The impaired capacity of omental compared with subcutaneous adipose tissues for remodeling and expanding through hyperplasia is thought to be associated with visceral adiposity with insulin resistance and metabolic health. In this context, several experimental studies have been performed to test this hypothesis. In omental adipose tissue, local factors including inflammatory cytokines and members of the TGF-β superfamily inhibited adipogenesis and also hormonal factors, such as glucocorticoids regulate adipogenesis . Furthervia the NF-kB pathway in epidermal keratinocytes  the switch to an unhealthy adipose tissue characterized by pathological adipogenesis, insulin resistance, and infiltrating M1 macrophages producing the pro-inflammatory adipokines TNF-α and IL-1β. Leptin, TGF-β1, and TLR4 pathway through PREP1 reduction or increased IL-6 expression could be promising new biological markers and therapeutic targets in obesity-related diseases.This work is dedicated to the memory of Antonella Siena.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Ethic Committee of Policlinico-Giaccone Hospital-Palermo Italy (authorization reference number 12/2014) in agreement with the Helsinki Declaration approved the study. The patients/participants provided their written informed consent to participate in this study.AB: conception and design of the study, acquisition, interpretation, and analysis of data, and writing the manuscript. CD, SD, PD, VD’E, and PF: analysis and interpretation of the data. H-US and EP: analysis and interpretation of the data and revising critically the manuscript for important intellectual content for final approval of the version to be submitted. PC and FB: providing approval for publication of the content. AP: interpretation of the data and writing the manuscript. All authors contributed to the article 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.