LEF1, FZD3, SMAD3, ITGB6, and BMP4. The results provide valuable data for screening molecular markers involved in hair structure and facilitating enhanced selection of super-fine wool rabbits through gene-assisted selection.Wool fiber diameter (WFD) is one of the most important economic traits of wool, especially in Angora rabbits. Emerging studies have been conducted on sheep and goats. However, few studies of WFD have been reported in rabbits. In our study, the diameter of coarse and fine wool and their respective hair follicles in Wan strain Angora rabbits were analyzed, and it was found that the diameter of coarse wool and hair follicles was larger than that of fine wool and hair follicles. High-throughput sequencing analysis was performed using hair follicles from coarse and fine wool. Differentially expressed genes (DEGs), including transcription factors, keratin-associated protein (KAP) and keratin (KRT) families, and extracellular matrix (ECM)-related genes, were screened. Enrichment analysis showed that skin development, epidermal cell and keratinocyte differentiation, epithelium development, and Notch and ribosome signaling pathways were important in regulating hair structure. Gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) analysis further identified six important signaling pathways and functional DEGs, including LEF1, FZD3, SMAD3, ITGB6, and BMP4. Our findings provide valuable information for researching the molecular mechanisms regulating wool fiber and could facilitate enhanced selection of super-fine wool rabbits through gene-assisted selection in the future.Wool fiber diameter (WFD) is an important index of wool traits and the main determinant of wool quality and value. However, the genetic determinants of fiber diameter have not yet been fully elucidated. Here, coarse and fine wool of Wan strain Angora rabbits and their hair follicle traits were characterized. The results indicated significant differences in the diameters of wool fibers and their hair follicles. The RNA sequencing (RNA-Seq) technique was used to identify differences in gene expression in hair follicles between coarse and fine wool. In total, 2574 differentially expressed genes (DEGs) were found between the two hair follicle groups. Transcription factors, keratin-associated protein (KAP) and keratin (KRT) families, and ECM-related genes may control the structure of fine fibers in rabbits. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that skin development, epidermal cell and keratinocyte differentiation, epithelium development, and Notch and ribosome signaling pathways were significantly enriched, respectively. GSEA further filtered six important pathways and related core genes. PPI analysis also mined functional DEGs associated with hair structure, including  Rabbits are small domestic animals that provide not only meat and fur but also wool, which is a raw material  that is Wool fibers are formed by keratinocytes which derive from progenitor populations at the base of the hair follicle . Rabbit Dlx3), lymphatic enhancer factor 1 (Lef1), epidermal growth factor (EGF), fibroblast growth factor 5 (FGF5), insulin-like growth factor-1 (IGF-I), and Wnt, are involved in regulating hair follicle development [Hair follicles form the basis for hair growth. At least 20 different cells and tissues, including the dermal papilla, inner root sheath, outer root sheath, and hair matrix, are involved in the process of cell differentiation and hair follicle development . Dermal elopment ,24,25,26Gene expression profile analysis plays a vital role in the discovery and prediction of gene function. Gene expression profile analysis showed functional genes related to fur color determination using Solexa sequencing in rabbits . DiffereThe rabbits used in the experiments were obtained from the Animal Husbandry Institute of the Anhui Academy of Agriculture Sciences and raised in the same conditions, fed to appetite, and drinking water was supplied by an automatic water feeder.Fiber samples were obtained from three healthy, one-year-old Wan strain Angora rabbits. The wool fibers were collected from the back of the body by the roots and pulled out. Then, the hair follicles of fibers were cut off and placed in a 2.0 mL freezing tube and immediately frozen in liquid nitrogen. Fiber samples were used for diameter measurements. The wool fiber diameter was measured using W1Vnt software according to the manufacturer’s instructions.For sample collection, anesthesia through an ear vein injection of 0.7% pentobarbital sodium (6 mL/kg) was first administered. Then, skin tissue samples were collected from the backs of rabbits. Histological analysis was performed as previously described ,30.® Ultra™ RNA Library Prep Kit for Illumina® , quantified with a Qubit2.0 fluorometer. Library insert size was determined using an Agilent 2100 bioanalyzer, and sequenced using the Illumina NovaSeq 6000 platform .Total RNA was extracted from hair follicle samples using TRIzol reagent , following the manufacturer’s instructions. Then, the NanoPhotometer spectrophotometer  and Agilent 2100 bioanalyzer  were used to determine the RNA purity, concentration, and integrity. The high-quality RNA for cDNA library construction was obtained after RNA sample quality control. Then, six libraries for hair follicles of coarse and fine wool were constructed using the NEBNextfastp (version 0.19.7). The cleaned, high-quality reads from hair follicles of coarse and fine wool were aligned against the Oryctolagus cuniculus genome using HISAT2 [2|fold change| ≥1 and padj ≤ 0.05. GO terms  [http://www.genome.jp/kegg/, accessed on 2 August 2022) [The low-quality reads, adaptor sequences, and N reads were removed from raw sequencing reads using g HISAT2 . Unique-g HISAT2 . The valg HISAT2 . DESeq2 g HISAT2 . Differest 2022)  and KEGGst 2022)  were alshttp://www.broadinstitute.org/gsea/index.jsp, accessed on 8 November 2022). Enrichment scores of gene sets were calculated using full ranking, then the distribution of gene sets was obtained in the list. A NOM p-value < 0.05 and |NES| > 1 were considered to indicate significant enrichment.All genes in hair follicles of coarse and fine wool were applied using GSEA with GSEA software .The PPI network was constructed using the STRING (version 11.0)  online d−ΔΔCT method [GAPDH) gene was used as an internal control. The qRT-PCR analysis was conducted at least in triplicate.Nine candidate genes related to hair structure were selected from DEGs for validation by quantitative real-time PCR (qRT-PCR). The qRT-PCR was performed on an ABI 7500  using TransStart Green qRT-PCR SuperMix . The primer sequences for the qRT-PCR are in T method , and thet-test. p values < 0.05 were considered significant.The data were presented in terms of the mean ± standard deviation (SD) and were determined using GraphPad Prism 5. Significant differences were estimated using Student’s p < 0.01)  B. The mo2FoldChange| ≥ 1 and false discovery rate padj ≤ 0.05) were analyzed between the groups, comprising 1462 genes that were more highly expressed in hair follicles of coarse wool and 1112 genes that were more highly expressed in hair follicles of fine wool , and forkhead box N1 (FOXN1) were found to be highly expressed in the hair follicles of fine wool from Angora rabbits. A number of genes were identified as DEGs from the KRT and KAP families, including KRT71, KRT72, KRT73, KRT17, KRT25, KRT10, KRT82, KRT35, KRT85, KRT5, KRT32, KRT34, KRT39, KRT77, KRTAP6-1, KRTAP3-1, and KRTAP17-1  were differentially expressed between the two groups. The results suggested that transcription factors, KRT and KAP families, and ECM-related genes were key candidates in determining the structure of fine fibers in rabbits.To comprehensively understand the transcriptome profiles that could be involved in regulating fiber diameter differences, hair follicle transcriptomes of coarse and fine wool of Angora rabbits were examined. We obtained 45.8–47.4 million raw reads and 43.3–44.4 million high-quality clean reads, and Q30 was ≥93.50% . The totine wool . Hierarcine wool . TranscrRTAP17-1 . Of thesGO and KEGG were used to identify the potential functions of the DEGs. The GO terms and KEGG pathways with padj ≤ 0.05 were considered to be significantly enriched. In addition, 1545 and 1095 DEGs were involved in 3748 biological processes based on GO terms and 316 KEGG pathways, respectively. For GO term analysis, a comparison between hair follicles of coarse and fine wool showed that some biological processes, including skin development (50 DEGs), epidermis development (47 DEGs), regulation of epithelial cell proliferation (48 DEGs), epidermal cell differentiation (30 DEGs), keratinocyte differentiation (23 DEGs), epithelium development (118 DEGs), and developmental induction (11 DEGs) , were suLAMA1, COL4A6, COL1A1, ITGB6, HSPG2, COL4A1, and COL4A2), Wnt signaling , basal cell carcinoma , TGF-β signaling , Notch signaling , and Hippo signaling (STK3 and TGFBR1)  . Of the EGFR) had the most connections owing to its interactions with 30 genes in the network, followed by the vascular endothelial growth factor A (VEGFA) and fibronectin 1 (FN1), which had 20 connections. In addition, the DEGs with high degree values  were involved in ECM–receptor interaction; LEF1, FZD3, PLCB2, and SMAD3 were involved in Wnt signaling; LEF1, FZD3, PTCH2, GLI3, PTCH1, SHH, and BMP4 were involved in basal cell carcinoma; FST, BMPR2, SMAD7, ACVR1B, THBS1, BMP4, and SMAD3 were involved in TGF-β signaling; NOTCH2, MAML3, and DTX4 were involved in Notch signaling; and LEF1, DLG2, FZD3, BMPR2, SMAD7, YWHAZ, DLG4, YAP1, BMP4, and SMAD3 were involved in the Hippo signaling pathway. This suggests that the high-degree nodes are potential candidate genes regulating hair diameter.To further explore the mechanism of DEGs involved in regulating hair structure and establish the interaction between differential genes, a PPI network based on DEGs was constructed using the STRING database and Cytoscape software for functional association analysis . A totalKRT32, KRT72, KRT73, FABP5, FST, DLX3, LEF1, MSX2, and FOXN1 were selected and their expression patterns in the hair follicles of coarse and fine wool of Angora rabbits were examined using qRT-PCR  and Notch signaling and transcription factors such as ntiation ,42. FOXNhenotype ,44. DLX3ntiation . DLX3 an of SHFs . Four imKRTAP3-1 and KRTAP17-1 were in the high-sulfur and ultrahigh-sulfur group, respectively, and highly expressed in the hair follicles of fine wool. The results suggest that the proteins are important for the formation of fine wool. In addition, nine genes were selected for validation using qRT-PCR, and the results matched the RNA-Seq data, demonstrating the reliability of our sequencing data.KRTs and KAPs determining the quality of the fiber are the main structural proteins of wool fibers . KAPs maGO functional enrichment analysis showed that a large number of DEGs were involved in regulating skin development, epidermis development, epidermal cell differentiation, regulation of epithelial cell proliferation, keratinocyte differentiation, and epithelium development. The results indicated that DEGs are potential regulators of hair structure and diameter in rabbits. KEGG enrichment analysis is often used for gene function study. Interactions between the Wnt, Notch, Hedgehog, and BMP signaling pathways are involved in regulating normal hair follicle development . In the LAMA1, COL4A6, COL1A1, ITGB6, HSPG2, COL4A1, and COL4A2. The ITGB6 gene was expressed differently between the two groups, and it is a candidate gene for hairlessness and skin with few hair follicles [We used the GSEA approach  to compaollicles . The basollicles . TGF-β sollicles , and regollicles ,61. In sEGFR plays an important role in the control of epithelial hair layer development and hair shape [EGFR [VEGFA is paracrine factor that contributes to hair follicle growth and cell cycle regulation and stimulates the proliferation of dermal papilla cells [LEF1, FZD3, SMAD3, and BMP4 were important regulators of the Wnt signaling, basal cell carcinoma, and Hippo signaling pathways. NOTCH2, MAML3, and DTX4 were substantially enriched in Notch signaling. ITGB6 was also differentially regulated in ECM–receptor interaction between the two groups. These findings filter the crucial functional DEGs further.To further explore the function of DEGs, PPI analysis was performed, and hub nodes and genes with a high degree of connection involved in key pathways were filtered. ir shape . Mice dipe [EGFR . VEGFA ila cells ,65. The This study identified differences in the characterization of coarse and fine wool and their hair follicle traits, as well as the transcriptome profiles of hair follicles of coarse and fine wool from Wan strain Angora rabbits. The morphological and histological analysis showed that the diameters of coarse wool and hair follicles were substantially larger than those of fine wool. The mRNA profile analysis obtained potential functional genes and pathways regulating hair structure. Further studies are required to investigate the roles of candidate functional genes that regulate hair structure to improve super-fine wool rabbit breeding programs.


Natural product derivatives are essential in searching for compounds with important chemical, biological, and medical applications. Naphthoquinones are secondary metabolites found in plants and are used in traditional medicine to treat diverse human diseases. Considering this, the synthesis of naphthoquinone derivatives has been explored to contain compounds with potential biological activity. It has been reported that the chemical modification of naphthoquinones improves their pharmacological properties by introducing amines, amino acids, furan, pyran, pyrazole, triazole, indole, among other chemical groups. In this systematic review, we summarized the preparation of nitrogen naphthoquinones derivatives and discussed their biological effect associated with redox properties and other mechanisms. Preclinical evaluation of antibacterial and/or antitumoral naphthoquinones derivatives is included because cancer is a worldwide health problem, and there is a lack of effective drugs against multidrug-resistant bacteria. The information presented herein indicates that naphthoquinone derivatives could be considered for further studies to provide drugs efficient in treating cancer and multidrug-resistant bacteria. Natural products have played a major role in medicinal chemistry for several years. Numerous naturally occurring compounds are an important source of new drugs used to treat some human diseases. The remarkable structural diversity of natural products offers a broad field to discover new compounds with important applications in chemistry, biology, and medicine .Due to the extensive use of pharmaceutical compounds in medicinal treatment, different microorganisms have emerged with enhanced resistance. In addition, many of these compounds have shown strong and adverse secondary effects in humans. As a result, there has been an exponential increase in the efforts to synthesize novel antimicrobial and anticancer agents ,3,4,5. DBased on their structure, quinones have been classified as benzoquinones (containing one ring), naphthoquinones (containing two rings), or anthraquinones (containing three rings). They are extensively distributed in nature and can be found in plants, fungi, algae, and bacteria ,8. They 1, 2, lawsone 3, plumbagin 4, and lapachol 5 are natural NQs isolated from plants extensively used in traditional Indian medicine furan-4,9-dione derivatives via transition metal-free tandem formal [3+2] by a base-promoted alkynylation and subsequent intramolecular addition reaction with aryl bromoacetylenes . The synSome interesting molecules are chalcones, which possess various pharmacological properties and have been synthesized via microwave-assisted one-pot three-component reaction  56]..56].Moreover, 1,2,3-triazole and its derivatives have been reported as antimicrobial, anti-allergic, analgesic, anti-HIV, anti-inflammatory, anticancer, antimalarial, and antituberculosis agents. Hence, the synthesis of triazole-naphthoquinones derivatives has been studied. O-propargyl-naphthoquinone has been prepared in good yields from lawsone and propargyl bromide. It generates 1,2,3-triazole-1,4-naphthoquinone conjugates in moderate to good yields via click chemistry with alkyl and aryl azides . ValençaRecently, Kumari et al., 2022, reported the indole-fused nitrogen heterocycles by two-step methodology, directly modifying the NQ ring. This compound can be used as solid-state fluorescence material  60]..60].The nucleophilic substitution of 2,3-dichloro-1,4-naphthoquinone is a synthetic strategy for introducing nitrogen, oxygen, carbon, sulfur, and selenium nucleophiles at C2 and C3 positions .Furthermore, anilino-1,4-naphthoquinone derivatives are compounds with a particular focus due to several biological properties. In the last years, several derivatives have been reported. Campora et al. (2021) reported the synthesis of NQ derivatives bearing hydrophobic moieties as promising candidates for Alzheimer’s disease therapy . MahalapThe compound 2,3-dichloro-5-nitro-1,4-naphthoquinone was reacted with nucleophiles such as amines, piperazines, or morpholines to form new regioisomeric amino-naphthoquinone derivatives in the C2 and C3 positions . IntroduSynthesis of NQ derivatives containing phenoxy, aminobenzensulfonamide, carboxamide, carbamate moiety, an amine group, and a urea derivative has been reported  13,35,7,735,70.14 by adding protons under solution conditions.In several cases, the biological activity of NQ derivatives has been explained in terms of their physicochemical property to easily accept one or two electrons to generate a semiquinone or a dianion, respectively . In addiQuinone derivatives can interact with biological structures through several mechanisms. Firstly, quinones have a strong electrophilic character and can form covalent bonds. In solution, these molecules can easily undergo reversible oxidation–reduction reactions. Consequently, they can generate highly reactive oxygen species (ROS) and inhibit electron transport processes and different types of enzymes, such as topoisomerases. Since many quinones have a planar structure, they can function as DNA-intercalating agents ,10,11,71•–), hydroxyl radical (•OH), and hydrogen peroxide (H2O2). These latter chemical species can quickly diffuse through membranes, causing cytotoxicity. In addition, ROS quickly induce oxidative stress and apoptosis in cells since they cause damage to biomolecules, such as DNA, proteins, and lipids.At the cellular level, the biological activity of NQs has been associated with their redox properties  10]. Up. Up10]. The redox properties and reactivity of a given NQ can be modified by placing different substituents with electron acceptor or electron donor characters in the structure. Therefore, developing easy, fast, and efficient methods to synthesize novel NQ derivatives to find novel compounds with enhanced and adequate biological activity is essential.NQ molecules, either from natural sources or semisynthetic, exhibit antitumoral and antimicrobial effects in several biomodels, where some action mechanisms have been demonstrated, such as ROS imbalance, alteration of mitochondrial respiration in tumor cells and bacteria, DNA damage , inhibition of topoisomerase II enzyme, among others.2•-, peroxyl (RO2•), hydroxyl (HO2•), hydroperoxyl (HO•), and alkoxyl (RO•). In living organisms, the primary source of ROS is mitochondrial respiration, where electrons are transferred between protein complexes to produce energy. Some electrons will react with O2 to generate O2•-. The ROS levels are controlled by enzymes, including superoxide dismutase, catalase, glutathione peroxidase, and non-enzymatic antioxidants such as glutathione, vitamins C and E, and any other molecule that could quench free radicals. An imbalance of ROS levels leads to macromolecular damage associated with aging and chronic diseases  triphenylphosphonium bromide (MitoK3) 19 (50 0.3–0.5 µM) for human lung carcinoma cell lines A549 were more significant for the SkQN 18 compound than MitoK3 19 in mitochondria isolated from rat heart and liver mitochondria. Both compounds (5 to 25 µM) achieved access into the inner mitochondrial membrane, exerted uncoupling activity and hence inhibited ATP production. Additionally, at a concentration of 3 µM, SkQN 18 and MitoK3 19 induced mitochondrial permeability transition pore opening, an important trigger for apoptosis and necrosis  triphenylphosphonium bromide (SkQN) toK3) 19  that hydnecrosis .20 and 2-(octane-1-sulfinyl)-1,4-naphthoquinone (OQ) 21 on ATTC AGS gastric cancer cells lines. Both BQ and OQ (5 μM at 24 h) induced cell apoptosis (apoptotic rate between 40 and 60%). The same treatment with BQ 20 and QO 21 decreases the levels of Bcl-2, a protein that sequesters proforms of death/driving cysteine proteases (caspases), hence preventing apoptosis [20 and QO 21 increase the levels of biomarkers associated with apoptosis: Bcl-2-associated death promoter (BAD), cleaved-caspase-3 (cle-cas-3), and cleavage of poly(ADPribose) polymerase (cle-PAPR). The authors also reported the depletion in the Akt expression levels, suggesting that BQ 20 and QO 21 induced G2/M phase cell cycle arrest in the AGS cells, hence acting on the apoptotic process [Wang et al., 2019, studied by flow cytometry using Annexin V-FITC and propidium iodide, the mechanisms involved in the anticancer effect of 2-(butane-1-sulfinyl)-1,4-naphthoquinone (BQ) poptosis . Additio process .22, 23, 24 (50 µM) (−1) at 37 °C, pH 7.2 for 45 min. The current peaks related to redox activity for all three compounds decayed as dsDNA concentration increased. Controls showed that free NQ derivatives reduced more easily in the absence of dsDNA. Computational studies evidenced interactions by either covalent or non-covalent interaction of NQ and the DNA structure [25  interacting with bovine serum albumin (10 µM) resulted in higher binding constant values (105 M−1) than free NQ (103 M−1). Additionally, the interaction with CT-DNA (0–50 µM) showed binding interaction as follows: Cu2+ > Zn2+ > Ni2+ > Co2+ > free NQ [Due to the aromatic structure of NQs, they may also interact with DNA and proteins, altering temporally or permanently the biomolecule’s functions. Espinosa-Bustos et al., 2020, evaluated by cyclic voltammograms the interaction of modified 2-arylpiperidinyl-1,4-naphthoquinone compounds  (50 µM)  with dsDtructure . On the ture [25  with eit free NQ .26 (Staphylococcus aureus (MRSA) model. When MRSA was exposed to 26 , propidium iodide (a membrane-impermeable dye) uptake significantly increased, suggesting that the semisynthetic molecule can alter the bacteria cell membrane more effectively than vancomycin and lawsone. The authors linked the cell membrane alterations to intracellular ROS levels increasing  in MRSA. Moreover, the higher the concentration of 26 in the treatments, the lower the intracellular ion iron levels. The bacteria viability was rescued when an excess of Fe3+ was incorporated into the MRSA cultures, suggesting that NQ derivatives could chelate metal ions [NQs trigger similar mechanisms in microorganisms, such as bacteria. In the past years, the chemical modification of NQs has been conducted to enhance the selectivity and antibacterial action of NQs. This is the case of Song et al., 2020, report for a lawsone derivative 26 , which ctal ions .27, N--1,4-dihydronaphthalen-2-yl)amino)phenyl)sulfonyl)phenyl)-3,5-dinitrobenzamide 28, and 4-amino)benzenesulfonic acid 29  in E. coli to the treatment in the same bacteria with streptomycin (1.9 μg/mL), indicating that antibacterial activity of NQ derivatives is led by oxidative stress caused by ROS [Another study considered three NQ derivatives: N--1,4-dihydronaphthalen-2-yl)amino)phenyl)sulfonyl)phenyl)benzamide d 29 see  for antid by ROS .E. coli, while fluoro in position C4 decreased the antibacterial activity about three times. Removing fluoro groups and adding 3-sulfanylpropanoic acid reduce the MIC by about seven times [Candida krusei. The authors also discuss that a single OH in position C5 is essential in antibacterial activity, while methoxylation in C5 and/or C8 is inactive for yeasts [To understand the possible biological mechanistic action triggered by different NQ chemical structures, structure–activity relationship (SAR) and molecular docking studies are relevant. Firstly, SAR studies have made it possible to know that the position of substituents is critical for antibacterial activity. In a SAR study, Wellington et al., 2019, reported that the group fluoro in position C3 exhibited better MIC (31.3 μg/mL) against en times . Similarr yeasts .2, propionyl juglone, and 2-acetyl-8-methoxy-1,4-naphthoquinone  and induced high cytotoxicity of 31 in HepG2 and MCF-7 cancer cell lines, leading to upregulation of Caspase 3 biomarker, as described in the experiment in [33, hydroquinone 34, 1,2-naphthoquinone 35  results, where compounds , and 32  had a poiment in . In the inone 35 , 1,4-napne 35 . The findings pointed out that NQ derivatives require a planar aromatic region with substitution to form electrostatic interactions with Lys68 and Asp175 residues. A second interaction was revealed as a non-coplanar aromatic region with π–π stacking forces with His160 [Recently, a group of 2-amino-4h His160 .45, the 2-chloro-3-((4-nitrophenyl)amino)1,4-naphthoquinone 46, and the 2-chloro-3-((3-nitrophenyl)amino)1,4-naphthoquinone) 47 with the epidermal growth factor receptor (EGFR) tyrosine kinase where these compounds fit within the ATP of the EGFR by means of van der Waals, H-bonding, π–π, and alkyl interactions, with 2-chloro-3-((4-methyl-phenyl)amino)1,4-naphthoquinone as the compound with significant interactions and the highest EGFR inhibitory activity [Panupong Mahalapbutr et al., 2022, performed a molecular docking study to reveal the interaction of the 2-chloro-3-((4-methyl-phenyl)amino)1,4-naphthoquinone activity .Current experimental and computational evidence highlights several NQ derivatives as antitumoral and antibacterial potential drugs, with more than a single mechanism in eukaryotic and prokaryotic cells.The activity of a given substituted anilino naphthoquinone has been associated with its redox properties. Depending on the substituents in the aromatic ring, the amino group in the naphthoquinone can modulate its physicochemical properties and modify its biological activity and interactions with biomolecules ,36,37,38As shown in this review, the redox properties of NQs produce ROS as superoxide, hydroxyl radical, and hydrogen peroxide; ROS can trigger autophagic cell death, chemo-sensitive, apoptosis, and necrosis. Furthermore, NQ derivatives can induce cell membrane damage, generate intracellular chelation with metals as iron, and NQ derivatives that preserve the planar structure may intercalate with DNA. Thus, pieces of evidence in preclinical assays (mostly in vitro) highlight to NQs as potential antimicrobial and antitumoral drugs. However, to the knowledge of the authors, no clinical evaluations have been conducted, since NQs face some challenges: (1) low solubility and (2) potential cytotoxic effect over healthy eucaryotic cells. It seems that nitrogen NQ derivative compounds could face those disadvantages, and further studies, particularly rigorous toxicological evaluations, can reveal the new generation of NQs for commercial applications.


Despite the widespread use of ART and decreased HIV incidence, Kaposi Sarcoma (KS), predominantly recognized as an AIDS-associated malignancy, continues to be highly prevalent in sub-Saharan Africa, parts of the Mediterranean, and parts of South America. Currently, there are no preventative measures or curative therapies for KS. The treatment is primarily pleiotropic chemotherapy, but the outcomes are inconsistent. Using our previously published transcriptomic profiles from KS lesions, we have identified potential biomarkers, therapeutic targets, and cell lineage determinants that are upregulated in expression and associated with KSHV infection. The protein expression of four surface-associated glycoproteins, FLT4, KDR, UNC5A, and ADAM12, was validated in human KS lesions and KSHV-infected cell line-derived mouse xenografts. The co-localization of CD34 and Prox-1, which are distinct endothelial lineage markers, suggests that cells within KS lesions are of a mesenchymal or progenitor phenotype rather than of a singular endothelial lineage.In vitro L1T2 cultures showed evidence of only FLT4, KDR, and UNC5A, whereas mouse L1T2 xenografts recapitulated human KS cell surface expression profiles, with the exception of CD34 and Prox-1. In KS tumors, most LANA-positive cells co-expressed markers of vascular as well as lymphatic endothelial lineages, suggesting KS-associated dedifferentiation to a more mesenchymal/progenitor phenotype.HIV-associated epidemic Kaposi sarcoma (EpKS) remains one of the most prevalent cancers in sub-Saharan Africa despite the widespread uptake of anti-retroviral therapy and HIV-1 suppression. In an effort to define potential therapeutic targets against KS tumors, we analyzed previously published KS bulk tumor transcriptomics to identify cell surface biomarkers. In addition to upregulated gene expression (>6-fold) in the EpKS tumor microenvironment, biomarkers were selected for correlation with KSHV latency-associated nuclear antigen (LANA) expression. The cell surface glycoprotein genes identified were KDR, FLT4, ADAM12, UNC5A, ZP2, and OX40, as well as the endothelial lineage determinants Prox-1 and CD34. Each protein was evaluated for its expression and co-localization with KSHV LANA using multi-color immunofluorescence in KS tissues, KSHV-infected L1T2 cells, uninfected TIVE cells, and murine L1T2 tumor xenografts. Five surface glycoproteins  were associated with LANA-positive cells but were also detected in uninfected cells in the KS microenvironment.  Kaposi Sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi Sarcoma (KS), pulmonary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD), and KSHV-induced inflammatory cytokine syndrome (KICS), was discovered in 1994 by Chang and Moore . The incIn vitro findings from KSHV-infected primary endothelial cells linked the expression of FLT4 with an increased expression of the transcription factor Prox-1, a lineage-defining marker of lymphatic endothelial cells [KS is a highly vascularized cutaneous lesion characterized by the slit vasculature and spindle morphology of growth deregulated cells, which are seemingly of endothelial origin ,11. Preval cells ,16. An aal cells ,18. Thesin vivo KS tumor microenvironment characteristics. Consistent with observations from human KS lesions, tumors derived from L1T2 cells have variable percentages of KSHV-infected spindle cells throughout the tumor [Despite KSHV being discovered over 25 years ago, models of sustained infection are few, and those of tumorigenesis are even more limited. Fixed human tissues are not conducive for mechanistic studies or for evaluating biomarkers for therapeutic potential; thus, we are exploring the validity of KSHV-infectable telomerase immortalized vein endothelial cells (TIVE) and the long-term KSHV-infected L1T2 cell line ,20 in rein vitro, and cell line explants in NSG mice. We also explore the alternative endothelial cellular origins of KS lesions and demonstrate the extent to which explant KSHV-infected cell lines and explant models recapitulate the human KS microenvironment and expression patterns.We describe the identification of a panel of highly upregulated cell surface glycoproteins from data mining of our previous comparative human KS transcriptomics as well as the validation and expression of the identified panel in KS tissues, KSHV-infected cell lines Approval to conduct this study was obtained from the review boards of Tanzania National Institute for Medical Research, Ocean Road Cancer Institute, University of Zambia Biomedical Research Ethics Committee; the University of Nebraska-Lincoln (UNL); and the Louisiana State University Health Sciences Center—New Orleans IRBs. Sample collection for this study was as described in Lidenge et al. . InformeAll animal experiments for generating KS cell line xenograft models were reviewed and approved by the University of Nebraska-Lincoln (UNL) Institutional Animal Care and Use Committee (IACUC) protocol (ID 1812). All personnel who were involved with the mice studies underwent training in appropriate methods to accomplish these procedures.L1T2 cells, obtained from ATCC, were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and passaged every 3–4 days. At the onset of this project, L1T2 cells were available for purchase from ATCC but are no longer available despite having been demonstrated to maintain long-term KSHV infection and to induce tumors upon xenotransplantation into immune deficient mice . TIVE cenullNOD-scid IL2Rgamma (NSG) mice were acquired from Jackson Labs, Bar Harbor, ME, USA (Stock Number 005557). The animals were housed in negative pressure individually HEPA-filtered microisolator racked caging. Environmental controls in-cage and in-facility were monitored and maintained by a centralized computer network. All procedures were carried out in a Class II Type A2 biological safety cabinet. To maintain a liquid state, 100 µL of growth factor-depleted Matrigel  was mixed with 1 × 106 L1T2 cells  in 100 µL cold-PBS and injected subcutaneously into the flank of each irradiated NSG mouse. The mice were observed daily for palpable tumors. Tumor growth was followed by caliper measurements of volume  until sacrifice. The mice were anesthetized with isoflurane  during the xenograft transplantation process, and then tumor growth was monitored by a caliper. During euthanasia, the tumor and major organs such as the spleen, liver, and lung were excised; half of each tissue was frozen in liquid nitrogen immediately after dissection, and the other half was fixed in 10% neutral-buffered formalin for pathological and molecular analyses.The protocols for the preparation and xenografting of mice with human tissues or cell lines have been previously published . BrieflyFormalin-fixed, paraffin-embedded (FFPE), KS tumor biopsy specimens were sectioned into adjacent 6 μm-thick sections for protein detection, as described in Tso et al. . BrieflyDual IF was used to visualize protein colocalization. As described in the IHC protocol, adjacent slides were deparaffinized, followed by antigen retrieval in sodium citrate solution for 15 min at 98 °C, and then blocked for 30 min using Bloxall (Vector Labs). Primary antibodies were added, and the slides were incubated at 4 °C overnight. The primary antibodies used are as follows: rat anti-LANA , mouse-anti CD34 , rabbit-anti Prox-1 , rabbit-anti KDR , rabbit-anti FLT4 , rabbit-anti ADAM12 , rabbit-anti UNC5A , rabbit-anti OX40 , and rabbit-anti ZP2 . After washing, the slides were incubated with their corresponding secondary antibody , donkey anti-mouse AF488 , donkey anti-rabbit AF488 , donkey anti-rabbit AF647 ) for 2 h at RT. The slides were then counterstained with 300 nM DAPI for 30 min at RT and mounted using Fluoro-gel .IHC imaging was performed using the Motic EasyScan One. IF imaging and cell quantification were performed using the BZX Fluorescence Microscope and the Keyence Hybrid Cell Quantification software version 1.1.1.8.Pearson’s correlation coefficients were used to identify transcript correlations. Non-parametric Kruskal–Wallis with Dunn’s multiple comparisons tests were used in GraphPad Prism v9 to compare protein expression within KS tumors.Using the transcriptomic dataset described in Lidenge et al. (2020) , we idenAdditional notable upregulated transcripts encoding cell surface proteins were the metalloproteinase ADAM12 (7.19-fold upregulated), the netrin receptor UNC5A (16.89-fold upregulated), the tumor necrosis factor receptor OX40 (6.59-fold upregulated), and the major subunit of the zona pellucida sperm receptor ZP2 (7.71-fold upregulated). UNC5A, OX40, and ZP2 were highly correlated with KSHV LANA expression; however, ADAM12, despite the strong upregulation in KS versus uninvolved skin, showed no significant correlation with LANA and the KSHV latency program. This short list of transcriptomically implied cell surface markers of KS was evaluated for protein expression in human KS lesions, TIVE and L1T2 cell lines, and L1T2-derived mouse xenograft tumors to determine whether the proteins were KSHV-infection-associated or markers of the KS tumor microenvironment.It has been well demonstrated that transcript expression values calculated from bulk transcriptomics do not always accurately reflect protein abundance in tissues ,26. ThusHaving demonstrated the upregulated expression of several transcriptomically implicated cell surface proteins in regions of KS tissue harboring demonstrably KSHV-infected cells, we next sought to determine whether those proteins were directly co-localized with LANA  or, alternately, if they were upregulated cell markers in the tumor environment. We conducted dual immunofluorescence assays with each identified protein and LANA. The same antibody clones used for IHC were used for IF to minimize sensitivity differences based on epitope recognition between approaches. Representative images are shown in We next analyzed the frequency of the co-localization of Prox-1 and CD34, proteins which are normally part of distinct endothelial cell lineages and, therefore, are not typically expressed on the same cells. We evaluated 16–20 fields from 5 different lesions (N = 83) for staining patterns of Prox-1, CD34, and LANA expression. This revealed that 25% of tumor cells were CD34+/Prox-1+, 26% CD34+ only, and 4% Prox-1+ only A,E. We ain vivo (human) tumor microenvironment, we then tested the KSHV-infected cell line, L1T2, and its uninfected endothelial cell counterpart, TIVE. L1T2 is a heterogeneous cell line derived from KSHV-infected TIVE cells implanted into mice, as described in Roy et al. (2013), where it was reported that tumors reminiscent of KS develop and recapitulate the protein expression phenotypes of KS tumors [in vitro cultured L1T2 cells but not in the uninfected TIVE cells, which were uniformly negative  that comprise KS tumors. Here, in correlation with transcriptome data, we demonstrated the detectable protein expression of KDR, FLT4, UNC5A, ADAM12, CD34, and Prox-1 in the human KS tumor microenvironment. Despite significantly upregulated RNA expression in KS tumors, neither OX40 nor ZP2 proteins were detected. The absence of the former is consistent with its typical expression as a co-stimulatory molecule on T cells, which we have reported as largely absent in the vicinity of KSHV-infected cells in KS biopsies [The lack of biopsies . The faibiopsies . Thus, bbiopsies ,29.ADAM12, Prox-1 and CD34, were expressed at elevated levels in KS lesions versus normal skin. We compared the extent to which the KSHV-uninfected TIVE, KSHV-infected L1T2 cells, and the L1T2-derived mouse xenograft tissues recapitulated the KS tumor protein expression phenotypes. In contrast to KS lesions, ADAM12, Prox-1 and CD34 were not detectable in either the TIVE or L1T2 cell lines; however, ADAM12 was evident via IF in L1T2 xenografts. The metalloproteinase ADAM12 has been shown to be connected to hypoxia as well as T-cell mediated TGFβ activation, leading to cell death and sustained inflammation ,31. The de novo transformation model. Evidence from in vitro infections of vascular and lymphatic endothelial cells suggests that lymphatic endothelial cells (Prox-1+/CD34−) are more susceptible to KSHV infection and long-term episomal maintenance than vascular endothelial cells (Prox-1−/CD34+) [Despite some lineage marker data supporting both vascular and lymphatic endothelial origins of KS, this remains speculation due to inconsistency in systems and the lack of a −/CD34+) . However−/CD34+) ,34. An a−/CD34+) ,35. This−/CD34+) . The co-−/CD34+) . Howeverin vivo models that more completely reflect the human KS tumor environment since endothelial cell lines, the most time- and cost-effective choice, have been shown to only reflect about 10% of the transcriptomic changes seen in KS lesions [In vivo animal models may be more likely to recapitulate the KS microenvironment; however, tumor generation has proven difficult and inconsistent using both cell line and human KS tumor xenografts. Furthermore, while FLT4, KDR, UNC5A, and ADAM12 were detected in the L1T2 mouse xenografts in concert with human tumor results, we did not detect either endothelial cell marker (Prox-1 and CD34) in those grafts, which may indicate that the L1T2 cell line has lost its endothelial cell attributes in culture. Thus, as we have shown in the comparison of human transcriptomics to those from infected cell lines, protein expression profiles are also differential between KS patient tumors and cell lines/mouse models.There is much interest in generating  lesions . Here, win vivo models to ascertain the effects of therapy on KS tumorigenesis.The consistency in the transcriptomic expression of the selected markers indicated that our sample size is robust enough to be representative and generalizable. Consistency was also evident at the protein level, where the detectable markers were consistently found in all tissues evaluated, albeit with variation in the signal magnitude from field to field depending on the cellularity, the cell types, and the number of KSHV-infected cells. Additionally, many of these markers, namely, KDR, FLT4, and UNC5A, have been shown to be involved in other types of cancers, making these markers non-exclusive to KS tumorigenesis ,24,37. TWe show the robust expression of potential candidate therapeutic targets, KDR and FLT4, as well as newly identified biomarkers, UNC5A and ADAM12, expressed consistently in KS tissues. Our current study also highlights the importance of validating transcriptomic data at the protein level, since the OX40 and ZP2 glycoproteins were not detectable despite 6.59- and 7.71-fold upregulation in gene expression, respectively. We also showed, through the robust upregulation of the expression of KDR and FLT4 in L1T2 versus TIVE cells, that L1T2-derived mouse xenografts could be a model for studying pathways, such as VEGF activation in KSHV infection and neoplasia, but they cannot be used to resolve the endothelial origin of KS, as they lack appropriate lineage markers. Further evaluation in additional cell lines, lineages, and animal models is needed to identify the conditions that more completely recapitulate the human KS tumor microenvironment and the landscape of the cellular membrane proteins.


The tetraspanins (TSPANs) are a family of four-transmembrane proteins with 33 members in mammals. They are variably expressed on the cell surface, various intracellular organelles and vesicles in nearly all cell types. Different from the majority of cell membrane proteins, TSPANs do not have natural ligands. TSPANs typically organize laterally with other membrane proteins to form tetraspanin-enriched microdomains (TEMs) to influence cell adhesion, migration, invasion, survival and induce downstream signaling. Emerging evidence shows that TSPANs can regulate not only cancer cell growth, metastasis, stemness, drug resistance, but also biogenesis of extracellular vesicles (exosomes and migrasomes), and immunomicroenvironment. This review summarizes recent studies that have shown the versatile function of TSPANs in cancer development and progression, or the molecular mechanism of TSPANs. These findings support the potential of TSPANs as novel therapeutic targets against cancer. The tetraspanins (TSPANs) are a family of proteins with four transmembrane domains , two extracellular loops (ECL1 and ECL2), and one intracellular loop (ICL) . They foTSPANs are expressed on the surface of most nucleated cells and play important roles in cell proliferation, differentiation, adhesion, migration, and cell–cell crosstalk . Recent Moreover, the post-translational modifications at specific sites in TSPAN proteins are important for the protein–protein interaction and the subsequent downstream pathways. These modifications include N-glycosylations at the ECL2 domain, palmitoylation at the N- and C-terminal tails and ubiquitination at the N-terminal domain . For exaThe multiple possible combinations between different TSPANs and their interacting proteins could generate an enormous variability of biological function. In addition to the traditional role of TSPANs in cancer growth, invasion and metastasis, recent studies have revealed that these proteins also participate in cancer stemness, cell–cell communication, drug resistance and cancer immunology , 23–26 , and perinuclear membrane –35. High+ melanoma cells have elevated active MMP-3 and low TIMP-1 levels to promote keratinocyte-originated proMMP-9 activation process, collagen IV degradation and dermal colonization [TSPAN8 is reported to be highly expressed in colorectal cancer, pancreatic cancer tissues and melanoma –44. TSPAnization . Interesnization . The samTSPAN12 is highly expressed in both colorectal cancer and non-small lung cancer tissues –50. HighCD151 expression has been reported to be associated with advanced cancer stage, cancer invasiveness and poor prognosis in endometrial cancer, hepatocellular carcinoma, breast cancer and non-small cell lung cancer patients , 51–55. High TSPAN15 expression in esophageal squamous cell carcinoma tissues is significantly associated with lymph node and distant metastasis, and poor prognosis , 56. TSPCD81, CD9, TSPAN31, and TSPAN13 are also reported to facilitate cancer cell invasion and metastasis. Mice with C81 deficient develop fewer breast cancer metastases compared to their wild-type counterparts. The same group showed that a unique anti-human CD81 antibody (5A6) effectively halts invasion and metastasis of triple-negative breast cancer cell lines . CD9 is G12D-driven lung tumor initiation and malignant progression. Similar to the function in lung cancer cells, TSPAN6 binds to the EGFR and blocks EGFR-induced RAS activation, thus inhibiting EMT process and cell migration [On the other hand, some TSPAN family members, such as CD82, TSPAN6, TSPAN9 and CD63, exhibit tumor suppressor properties. For example, CD82 directly associates with EGFR and suppressed EGF-induced lamellipodial extensions and cell migration in non-small cell lung cancer cells. CD82 could specifically increase EGFR endocytosis after EGF stimulation but not the initial activation of EGFR . It alsoigration . The proigration . Finallyigration .Evidence has shown the context-dependent role of TSPAN7 in cancer. TSPAN7 is highly expressed in primary osteosarcomas and promote osteosarcoma cell growth, EMT process, and in vivo metastasis. Mechanistically, the authors demonstrated that TSPAN7 interacted with β1 integrin to activate FAK-Src-Ras-ERK1/2 signaling . TSPAN7 In summary, most TSPAN proteins  could promote cancer invasion and metastasis, while few members  have the opposite function. Meanwhile, the function of TSPAN7 in cancer invasion and metastasis is context-dependent.As mentioned above, several TSPAN family members could regulate the invasion and metastasis of cancer cells. Cell growth and proliferation are also essential for cancer metastasis or progression besides invasion. These biological processes should coordinate with each other to achieve cancer progression. We can see that TSPAN1, TSPAN12, TSPAN13, TSPAN6, TSPAN8, TSPAN9, CD151, CD63 can promote or inhibit both cancer cell invasion and proliferation. However, the function of TSPAN molecules seems to be context-dependent. For example, TSPAN31 has been reported to promote migration of hepatocellular carcinoma, but not affect cell proliferation . Even moOn the one hand, some TSPAN members could promote cancer cell growth. Pancreatic cancer is one of the most aggressive malignancy, with a 5-year survival rate of less than 5% . High TSOn the other hand, some other TSPAN members could inhibit cancer cell growth. TSPAN31 serves as a natural antisense transcript to inhibit CDK4 protein expression in human cervical cancer and hepatocellular carcinoma by targeting the 3ʹ-untranslated region of the CDK4 mRNA, thus suppressing cell proliferation , 82. TheAltogether, TSPAN1, TSPAN15, TSPAN29, and CD151 could support cancer cell growth, while TSPAN31, TSPAN6, CD9, CD37 could inhibit cancer growth.Stem cells are capable of both self-renewing and multilineage differentiating. Tumor heterogeneity is now recognized as a hallmark of tumors . Only a positive cells exhibit more resistance to chemotherapy drugs, higher migration potential, and stronger tumorigenicity [high pancreatic cancer cells have increased organoid formation capability and in vivo carcinogenesis. Mechanistically, CD9 enhances glutamine uptake in pancreatic cancer cells via promoting the plasma membrane localization of the glutamine transporter ASCT2 [+/CD38+ acute myelocytic leukemia stem cells and increased the phosphorylation of transcription factor STAT5 to transactivate IL-10 transcription [Several TSPAN members, such as CD9, TSPAN8, TSPAN3, CD82, CD81, and TSPAN1, have been implicated in the regulation of CSCs. CD9 has been reported to be specifically expressed on leukemia stem cells. CD9genicity . CD9 waser ASCT2 . TSPAN8 er ASCT2 . In addier ASCT2 . CD82 wacription . More recription . Furthercription .+/PDGFRAlow population present just below crypts is sufficient to expand intestinal stem cells in vitro and contribute to stemness maintenance in vivo via secreting the BMP antagonist Gremlin1 [CD63 and CD81 have also been reported to play important roles in stemness maintenance of non-malignant cells. CD63 could confer hematopoietic stem cells with more quiescent status, more robust self-renewal and myeloid differentiation abilities than those with negative/low CD63 expression. Knockout of CD63 in mice reduced the number of hematopoietic stem cells in bone marrow and CD63-deficient hematopoietic stem cells exhibit impaired quiescence and long-term repopulating capacity, and increased sensitivity to irradiation or 5-fluorouracil treatment. CD63 was found to interact with TGF-β receptors I and II to sustain TGF-β signaling activity . A CD81+Gremlin1 .Despite significant advances in cancer treatment, the development of resistance almost invariably emerges . MultiplCD9 mediates chemoresistance in acute myeloid leukemia  and smalTSPAN1, TSPAN3, TSPAN12, TSPAN31, CD82 and CD63 have also been reported to reduce chemosensitivity of cancer cells. TSPAN1 is found to be upregulated in cisplatin-resistant head and neck squamous cell carcinoma cells. TSPAN1 depletion reduces cell proliferation, induces apoptosis, decreases autophagy, sensitizes to chemotherapeutic agents and inhibits the phosphorylation of SRC signaling . MoreoveExtracellular vesicles (EV) are of utmost importance in intercellular communication under physiological and pathological conditions, allowing cells to exchange proteins, lipids, and genetic material . Exosomehigh expression pancreatic cancer cells remarkably promotes maturation, activation of endothelial cells and fibroblasts [TSPAN8 has been reported to promote EV production and the attachment to target cells. TSPAN8 mediated a several-fold increase in EV number in breast cancer cell culture and the circulation of tumour-bearing animals . Rat PDAroblasts . Tspan8-roblasts . TSPAN8-roblasts .In contrast, TSPAN6 can reduce EV production and the contents in EVs in cancer cells. TSPN6 acts as a suppressor of exosome release by facilitating the lysosomal degradation of SDC4 and syntenin in breast cancer cells . In addiCD151, TSPAN1, CD81, CD9 have also been implicated in the EV biogenesis. CD151 expression level in triple-negative breast cancer-derived serum exosomes is significantly higher than those from healthy subjects . CD151 fTSPAN proteins also regulate the formation of migrasomes . MigrasoImmunotherapy has revolutionized and rejuvenated cancer treatment. The immune system plays a pivotal role in immunosurveillance, as immune cells of the adaptive and innate immune systems infiltrate into the tumor microenvironment (TME) and modulate cancer progression. Due to their powerful tumor-killing capability, T cells are the focus of tumor immunology . AntigenCD9, CD82, CD37, CD151, CD63, and TSPAN5 have been revealed to interact with MHC complex in APCs. CD9 is reported to associated with MHC II molecules in dendritic cells (DCs) and B cells, which might facilitate the formation of MHC II multimers . DeletioMoreover, CD37, CD81, CD82, CD53 and TSPAN33 regulate the adhesion or migration of APCs. CD37 ablation impairs chemo-tactic migration and in vivo priming of adoptively transferred naive T cells of DCs via activating Rac-1 , 129. CDAlthough emerging evidence demonstrated that TSPAN proteins are important regulator of immune cells, there lacks direct evidence showing the function of TSPAN proteins in cancer immunology. Further work needs to be done to explore the potential functions. Interestingly, Daniel and colleagues identified that TSPAN8 can be used as a specific target candidate for chimeric antigen receptor T cells (CAR-T) against pancreatic cancer among 371 antigens. CAR-T cells specific for TSPAN8 can significantly decrease the tumor burden in a subcutaneous xenograft model .The TSPANs are a family of 33 four-transmembrane proteins in Homo sapiens. TSPANs are mainly expressed on the surface of most nucleated cells and play important roles in cell proliferation, differentiation, adhesion, migration, and cell–cell crosstalk. Recent studies have revealed that TSPANs are indispensable for cancer initiation and progression. TSPANs affect different biological processes mainly via interacting with different partner molecules to form TEMs, including integrins, EGFR, mtTGF-β, EWI2, ASCT2, LC3, PTCH1, P120 and others. We herein summarized the recent studies revealing the versatile role of TSPAN family members in cancer cell invasion, metastasis, proliferation, stemness maintenance, drug resistance, and EV biogenesis. However, other proteins with four transmembrane domains are not included in the TSPAN family, such as TM4SF5 .Most TSPAN proteins are reported to promote cancer invasion and metastasis, such as TSPAN1, TSPAN7, TSPAN8, TSPAN12, TSPAN15, CD151, CD81, CD9, TSPAN31, TSPAN13 and TSPAN9, while CD82, CD63 and TSPAN6 can inhibit cancer invasion or metastasis. The function of TSPAN7 is context-dependent. On the one hand, TSPAN7 can promote the invasion and metastasis of myeloma, non-small lung cancer, and osteosarcoma –74. ConvCSCs are a distinct population of cancer cells with the capabilities of self-renewal, drug-resistance, metastasis and tumorigenicity. TSPAN1, TSPAN8, TSPAN3, CD9, CD82 have been shown to enhance the stemness of cancer cells. CD63 and CD81 could contribute to the maintenance of hematopoietic stem cells and intestinal stem cells, respectively. Nevertheless, their role in CSCs remains to be explored. TSPAN1, TSPAN3, TSPAN12, CD9, CD81, TSPAN31 and CD82 can mediate chemoresistance in multi cancer types. Meanwhile, CD81 could enhance glioblastoma survival after radiation treatment . We thenThe immune system is critical in immunosurveillance against cancer. T cells are the focus of tumor immunology as they can efficiently kill cancer cells. The activation of T cells relies on the antigen presentation process . CD9, CDIncreasing studies have revealed the regulatory mechanism of TSPAN expression in cancer. The regulation of TSPAN expression can be divided into transcriptional regulation, post-transcriptional regulation, and post-translational regulation. (1) Several transcription factor, like SOX9 and β-cantenin, and promoter demethylase LSD1 have been reported to regulate TSPAN8 transcription. SOX9 could directly enhance the transcription of TSPAN8 expression in response to EGF stimulation , while βIn conclusion, emerging data has elucidated the critical role of TSPANs in cancer development. Although TSPANs have no natural ligands, they interact with other proteins to elicit their function on cancer cells, ranging from proliferation, apoptosis, migration, invasion, chemoresistance, stemness, to exosome biogenesis. Future work can be done in the following aspects: (1) Elucidate the function of other TSPAN members in carcinogenesis; (2) Demonstrate the upstream and downstream molecular mechanism of TSPANs; (3)Validate the therapeutic efficiency of TSPAN-based strategies, including developing specific antibodies, gene therapy, specific CAR-T cells and others; (4) Reveal the role of TSPANs in the uptake of exosomes; (5) Explore the role of TSPANs in cancer immunology or other stroma cells in the TME; (6) Resolve the structure of TSPAN proteins.


Tumor-associated lymph vessels and lymph node involvement are critical staging criteria in several cancers. In skin squamous cell carcinoma, lymph vessels play a role in cancer development and metastatic spread. However, their relationship with the cancer stem cell niche at early tumor stages remains unclear. To address this gap, we studied the lymph vessel localization at the cancer stem cell niche and observed an association from benign skin lesions to malignant stages of skin squamous cell carcinoma. By co-culturing lymphatic endothelial cells with cancer cell lines representing the initiation and promotion stages, and conducting RNA profiling, we observed a reciprocal induction of cell adhesion, immunity regulation, and vessel remodeling genes, suggesting dynamic interactions between lymphatic and cancer cells. Additionally, imaging analyses of the cultured cells revealed the establishment of heterotypic contacts between cancer cells and lymph endothelial cells, potentially contributing to the observed distribution and maintenance at the cancer stem cell niche, inducing downstream cellular responses. Our data provide evidence for an association of lymph vessels from the early stages of skin squamous cell carcinoma development, opening new avenues for better comprehending their involvement in cancer progression. Skin squamous cell carcinomas (SCC) are distressingly common and have the ability to metastasize . Whole-gIn recent years, several advances have provided insight into the composition and contribution of the complex milieu in which skin CSCs reside ,16,17. SDespite the relevance of LVs in skin tumorigenesis, whether LVs interact with the CSC niche remains elusive. Additionally, it is unknown whether such interactions only occur at the late SCC stages. Interestingly, the blockade of the lymphangiogenic factors VEGF-C and VEGF-D inhibits inflammatory skin carcinogenesis , supportIn the skin, dermal LVs distribute into two plexuses termed the upper and lower horizontal plexuses ,31. CapiThe fact that SCC can originate from HFSC ,9 suggesTo investigate the relative localization of LVs to CSCs, we conducted histopathological and immunofluorescence microscopy analyses (IFM) on mouse skin sections from benign papillomas and keratoacanthomas, along with SCC A–D obtaiTo evaluate the presence and distribution of LVs within mouse skin lesions, we conducted IFM analyses, utilizing markers for LVs  , cancer − positive CSCs rather than with all cancer cells, we quantified the relative distances between LV with SOX2+ cells, as well as between LV with K5+ SOX2− cells. We observed that the closest association occurred between LVs and CSCs presented as aggregated quantifications  genetically modified mice and control mice subjected to the two-stage DMBA/TPA skin carcinogenesis protocol. The genetic knockout of Wls in hair follicle Keratin 15+ cells results in reduced tumor growth, incidence, and reduced SOX2-positive CSC cell numbers in the skin . Si. Si45]. Seeking to address the direct role of lymphatic endothelial cells (LECs) in the CSC niche, we turned to in vitro studies due to the complexity of intercellular connections in skin tumors. We established co-cultures of LECs and skin carcinoma cell lines to allow indirect and direct intercellular communication A. For thWe evaluated the effect of co-culturing LECs and cancer cells on cell proliferation by IFM analyses using the phospho Histone H3 cell proliferation marker B,C. UponIcam1, Itga4, and Selp, whereas Itga2 was downregulated [Wisp1, Cyr61, Lgal3bp, Nid1, Lama4, Col5a3, and Tinagl1 [Wisp, Cyr61, and Lgal3bp have been described as playing roles in lymphangiogenesis or angiogenesis [Kdr and Flt4 (VEGFR2/VEGFR3) [Angpt2, Adm, and Mmp2 [Cxcl3 and Cxcl12 [We next conducted transcriptome analyses to gain further insight into the mechanisms underlying the connection between LECs and cancer cells at the CSC niche and their potential roles in tumorigenesis. For these studies, we used the HaCa4 cell line since it initiates a high number of tumors and progresses further into malignancy, thus, comprising a broader spectrum of tumorigenesis features ,51. We cegulated    and the and Mmp2 ,66,67,68d Cxcl12 , which wVcam1, Pdpn, Itga7, Sele, and Selp [Vcam1, Sele, and Pdpn could potentially associate with genes found in the cancer cell transcriptome, such as Itga4 and Lgal3bp [Hif1a and Adm [Il6, Cxcl1, Cxcl2, Cxcl3, Cxcl4, and Cxcl5 [Ccl2, Ccl7, Ccl9, Ccl20, Csf1, Csf2, Csf3, Il23a, and Tnfsf15, receptor Il1r1, and the transcription factors Nfkb1, Nfkb2, Nfkbia, and Nfkbiz [The transcriptome analyses of the LEC, when co-cultured with cancer cells, revealed a reciprocal modulation, with 191 genes deregulated, including 127 upregulated and 64 downregulated compared to controls . GO enriand Selp ,71,72 , as also detected in LECs stimulated with tumor-derived conditioned medium [Interestingly, we did not observe changes in cancer cell and LEC proliferation, suggesting that the LV–cancer cell crosstalk, in regulating this aspect of cell behavior, is indirect, possibly favored by other cell types or cues present at the niche. Indeed, our data show an increase in the expression of genes involved in immune response and chemotaxis  or previously donated by Robert Loewe from the Department of Dermatology, Medical University of Vienna . The ana/fl mice  were sub2. The cancer cell lines, even when cultured in a 10% FBS-containing medium, or in a normal calcium concentration, retain their CSC characteristics, being capable of initiating tumors that progress and maintain tumorigenicity upon transplantation into mice [The immortalized mouse keratinocyte cell line MCA3D ,52,53 annto mice ,51,53.2, inducing the expression of the SV40 large T antigen. The basal medium consisted of 40% DMEM , 40% Ham’s Nutrient F12 Medium , 0.2% heparin , and 0.2% VEGF . For functional assays, LECs were subjected to non-permissive conditions, cultured at 37 °C for 48–96 h without IFNγ. This approach facilitated the degradation of the SV40 large T antigen, thus enabling the cells to adopt a functional phenotype. Two different LEC clones were cultured and no discernible differences in the characteristics or behavior were observed and, thus, they were used equivalently during the study.Lymphatic endothelial cells (LECs), originally derived from the immortomouse strain, carry the Immortomouse transgene, derived from the H-2Kb-tsA58 transgenic mice. This transgene allowed the direct derivation of conditionally immortal LECs and enables inducible expression of a thermolabile large T antigen of the SV40 virus . LECs we®-TagRFP/GFP transgene  with 8 µg/mL polybrene .Regarding lentiviral infection, HaCa4 and LEC were transduced according to the manufacturer’s instructions with recombinant lentivirus harboring LifeActParaffin-embedded sections were deparaffinized, rehydrated, treated for antigen retrieval, and stained, as previously described . HematoxOptimal cutting temperature (OCT) compound embedded sections were fixed in 4% PFA for 10 min and the antibody stainings were performed as for paraffin-embedded sections. Cells plated on coverslips were fixed in 4% PFA for 10 min, permeabilized with 0.3% Triton X-100 PBS for 12 min, and blocked in 2% BSA for 1 h. Primary and secondary antibodies were incubated for 1 h or overnight and 1 h, respectively, at 4 °C in the blocking solution. Nuclei were stained with Hoechst. The following primary antibodies were used: anti-Keratin-5 , anti-SOX2 , anti-LYVE1 , anti-PROX1 , anti-VEGFR3 , anti-PODOPLANIN , and anti-phospho Histone H3 . The following secondary antibodies were used: anti-rat, anti-chicken, anti-goat, and anti-rabbit conjugated to AlexaFluor488 , to AlexaFluor 546 or to AlexaFluor 647 . Images were captured using a Nikon Eclipse Ni-E microscope  adapted with a Photometrics BSI Express camera and CFP plan objectives with the NIS-elements acquisition software .The acquired images were imported into the ImageJ software . Fixed-size frames and regions of interest (ROI) were used for each image. This standardization ensured consistent analysis of the same area across all samples, facilitating reliable comparisons.Thresholding: A uniform thresholding approach was employed to segment the ROIs containing lymphatic vessels (LVs), cancer stem cells (CSCs) expressing SOX2, and basal keratinocytes (K5). The threshold level was determined based on the distinctive characteristics of each marker and remained constant for all analyzed images. The peritumoral lymphatic vessel density quantification was performed using the Weidner technique , as desc+ LECs were sorted with an Aria II FACS sorter  excluding dead cells and doublets. For the sorting of co-cultured HaCa4 and LEC, and PDV and LECs, cells were stained as previously described  using thSamples were sequenced with the Beijing Genomics Institute sequencing (BGISEQ) platform. Raw reads were filtered with SOAPnuke (v1.5.2) software . FiltereTotal RNA was isolated using the RNeasy Kit  according to the manufacturer’s instructions. An amount of 1 µg RNA was used for cDNA synthesis using the Maxima First Strand cDNA synthesis KIT . Quantitative PCR assays were performed using 5 ng of cDNA as a template and the reactions were conducted using the GoTaq qPCR Master Mix . qRT-PCR was performed on QuantStudio™ 7 Flex Real-Time PCR . HPRT housekeeping gene was used for normalization. The gene-specific primer sets were used at a final concentration of 10 µM . All qRTLifeAct-RFP-mLECs were seeded and cultured to form a confluent monolayer. PDV cells were stained with CellTrace™ CFSE Cell Proliferation Kit  following the manufacturer’s instructions. Either PDV-CFSE or HaCa4-LifeAct-GFP were seeded on top of the monolayer and washed twice with PBS after 15, 30, 45, and 60 min, analyzing biological replicates for statistical analyses. 2 levels. Images were captured every 3 min over 13 h.LifeA-ctGFP-HaCa4 and LifeAct-RFP-mLECs were co-cultured or single-cultured in 8-well chamber slides  with 10 µM Hepes. Time-lapse experiments were performed with a Nikon Eclipse Ti2 microscope and the NIS-elements acquisition software  at 20× magnification, with controlled temperature and COp  <  0.05 as statistically significant.Statistical analyses were performed using Prism 9 (Graphpad) software. n refers to number of samples or biological replicates. Data represent mean  ±  SEM. Exact n numbers for each dataset are detailed in the Figure legends. The normality of the data was evaluated and the statistical significance was calculated by the two-tailed Mann–Whitney test, considering


The pathogenesis of osteoporosis involves multiple factors, among which alterations in the bone microenvironment play a crucial role in disrupting normal bone metabolic balance. Transient receptor potential vanilloid 5 (TRPV5), a member of the TRPV family, is an essential determinant of the bone microenvironment, acting at multiple levels to influence its properties. TRPV5 exerts a pivotal influence on bone through the regulation of calcium reabsorption and transportation while also responding to steroid hormones and agonists. Although the metabolic consequences of osteoporosis, such as loss of bone calcium, reduced mineralization capacity, and active osteoclasts, have received significant attention, this review focuses on the changes in the osteoporotic microenvironment and the specific effects of TRPV5 at various levels. Osteoporosis (OP) is a systemic skeletal disease primarily characterized by reduced bone mineral density (BMD), increased bone fragility, bone pain, and fracture susceptibility . RecogniIn recent years, researchers have increasingly focused on the “micro” aspects of osteoporosis research, exploring changes in the tissue microenvironment and downstream signal cascades , 7. Thes2+ icons, as well as its ubiquitous distribution throughout various tissues, including the bone, kidney, and placenta [2+, E2, 1,25-(OH)2D3, calcitonin, klotho, PTH, etc. [TRPV5, a member of the transient receptor potential vanilloid (TRPV) family is an ion channel situated on the cell membrane . Investiplacenta –14. Notaplacenta . ConsequTH, etc. –14. ThesTH, etc. . FurtherTH, etc. , 18. HenThe onset and progression of osteoporosis can be influenced by a myriad of factors, including age, gender, trauma, and medication . These fExamining the bone tissue level, alterations in the osteoporotic bone tissue microenvironment encompass the destruction of bone microarchitecture, reduction in bone mass, and modifications in mechanical stimulation among other changes.The skeleton, often refereed to as the as the calcium and phosphorus reservoir, is the body’s largest system for calcium and phosphorus deposition. It is composed of bone cells and bone matrix . The groFrom the perspective of bone loss, Type I postmenopausal osteoporosis and Type II senile osteoporosis (disuse osteoporosis) exhibit distinct mechanisms of bone loss . Type I Bone marrow adipose tissue can adversely impact bone formation, as bone marrow adipose cells and osteoblasts share a common precursor cell, resulting in a negative correlation between them and a decrease in the number of osteoblasts . The abnFrom a biomechanical perspective, the skeleton is a tissue capable of sensing changes in mechanical forces, continuously modulating external mechanical stimuli through the interaction of osteocytes, osteoclasts, and osteoblasts. Guillaume T. Charras et al. found that primary osteoblasts possess a non-selective stretch-activated cation channel with a conductivity of 15pS, and the opening of this cation channel is closely related to the mechanical force stimuli received . In someAdditionally, abnormal skeletal force lines are frequently associated with the onset of arthritis, chronic inflammation factors may provoke atypical responses in bone matrix, as well as intraosseous vascular growth and metabolism, leading to abnormal growth of bone joints, diaphysis, epiphyses, and periosteum. Wei Wang et al. showed that patients with lumbar disc herniation may experience lower limb pain, and long-term pain can cause abnormalities in lower limb force lines and lower limb kinematic disorders, leading to muscle and skeletal impairments .Therefore, changes in mechanical force stimulation can affect intraosseous structures, trabecular development, and vascular growth, acting on the bone tissue microenvironment. Insufficient mechanical stimuli have also become one of the pathogenic factors for type II osteoporosis. Furthermore, a recent study by Peng Hui Zhang showed that changes in the biomechanical microenvironment generated by mechanical loading can act on mesenchymal stem cells (MSCs) in three-dimensional scaffolds to promote the expression of osteogenic markers, enhance cell vitality, and inhibit inflammatory factors . As a reIn conclusion, changes in bone microstructure, bone loss, and bone marrow composition, as well as stimulation by the biomechanical microenvironment, can lead to alterations in skeletal growth and development and bone mass accumulation, involving dynamic changes in the bone tissue microenvironment at the tissue level.Bone tissue primarily comprises osteocytes, osteoblasts, osteoclasts, bone marrow-derived mesenchymal stem cells, and immune cells, among other components . These v43− and Ca2+ levels, intercellular communication with perivascular cells, remodeling of the surrounding environment, and secretion of relevant hormones, among others [Osteoblasts play a crucial role in bone formation, as they are responsible for the deposition of various bone minerals and type I collagen, and eventually differentiate into osteocytes. Osteocytes primarily serve functions such as mechanical force transmission, regulation of osteoblast activity, control of bone resorption, regulation of POg others . Multiplg others . In the g others . In the g others . RANKL ag others . In thisg others . Additiog others , indicatIn 1961, the direct external environment of osteocytes was defined as the Grenzscheide or limiting membrane, primarily composed of polysaccharides and extravascular fluid . This meNelson G et al. reported that senescent cells secrete found that aging cells secrete chemokines, inflammatory factors, and extracellular matrix proteins that generate a toxic microenvironment, affecting neighboring cells and facilitating the accumulation of aging cells and the development of tissue dysfunction . ExtraceImmune cells play a crucial role in the bone tissue microenvironment –58. StudRecently, new technologies have emerged for studying bone tissue microenvironment at the cellular level, with single-cell sequencing becoming a popular method for investigating bone tissue metabolic diseases. By extracting and sequencing from disease-affected areas, distinct heterogeneous cell subsets can be selected and compared with public data sets to identify interactions among various cell subsets during bone tissue diseases process. Associating different cell subsets with clinical indicators can help define the pathological state of related bone samples. The CyTOF (Cytometry by Time-Of-Flight) Mass Cytometry method enables high throughput, multi-omics single cell analysis, providing a technical means for detecting cellular changes and alteration in the surrounding microenvironment. Through microenvironment state analysis, sensitive cells exhibiting differential drug effects can be effectively screened, offering support for the identification of potential drug targets.In this study, we discuss the relationship between the TRPV family of ion channels and OP. Classical signaling pathways in OP, including Wnt/β-catenin, RANK/RANKL/OPG, TGF-β, PI3K/Akt, and Notch, have been widely studied and documented .+, K+, and Ca2+ ions [The TRPV ion channels, a subfamily of transient receptor potential vanilloid (TRPV) receptors, are ubiquitously distributed calcium ion receptor proteins present on the cell membranes of various tissues and organs in living organisms . Based oa2+ ions . However2+, pH, and chemical stimuli, predominantly inducing downstream pain responses. It has been demonstrated that pain associated with bone loss is intimately linked to TRPV1 [2+ oscillations governing bone metabolism [2+ ions, as well as its high sensitivity to extracellular pH, which directly influences its activity. Primarily distributed in bone tissue and renal cells, TRPV5 is crucial for regulating Ca2+ absorption within the body [2+sensitive ion channel is strictly regulated by Ca2+ and 1,25-(OH)2D3 [TRPV1, commonly referred to as the capsaicin receptor, exhibits sensitivity to Cato TRPV1 . TRPV2 ptabolism , 69. TRPtabolism . Furthertabolism . TRPV4 dtabolism . Researctabolism , has revthe body , 74. TRP-(OH)2D3 . A summa2+ uptake and transport. Research has demonstrated the indispensability of TRPV5 in epithelial calcium uptake, bone formation, and the regulation of calcium homeostasis, particularly in renal calcium reabsorption, with particular emphasis on renal calcium reabsorption and its intimate connection to bone homeostasis [2) functions as an endogenous regulator of TRPV5. Moiseenkova-Bell et al. demonstrated that PIP2 activates TRPV5 through the direct binding of the N-linker, S4–S5 linker, and S6 helix to TRPV5, enabling Ca2+ flow [2+ [As discussed earlier, the primary role of TRPV5 involves mediating Caeostasis , 110. Noeostasis . Dang eteostasis ; as illueostasis . Analysieostasis . Additioeostasis . Calciumeostasis . Simultaeostasis . Moreovea2+ flow . TRPV5'sflow [2+ . In the In recent years, an increasing number of researchers have focused on investigating the association between TRPV5 function and various tissue layers in osteoporosis. The objective of these investigations is to identify the functional targets of TRPV5 by examining its structure, function, and downstream signaling cascade. The outcomes of this research can offer valuable insights and guidance for the development of novel osteoporosis therapies, as well as for enhancing drug efficacy and absorption. Hoenderop JG have suggested that alterations in TRPV5 might be linked to age-related bone disorders, including osteoporosis , the rel−/− mice and discovered that these mice exhibited disordered bone resorption and decreased femur thickness compared to wild-type TRPV5+/+ mice [−/− mice [−/− mice, the number and volume of trabeculae decreased, the connectivity rate was suboptimal, and the thickness and volume of cortical bone diminished, with a significantly higher internal porosity compared to TRPV5+/+ mice [+/+ mice in young and middle-aged groups [−/− mice was related to and proportional to age, and the levels of young and middle-aged TRPV5−/− mice were significantly lower than those of the TRPV5+/+ group, suggesting an abnormal bone resorption rate [Eerden et al. reported that TRPV5 plays a significant role in bone resorption and morphological changes . Utilizi+/+ mice . The abs−/− mice . Althoug−/− mice , 116. TR+/+ mice . Quantitd groups . Urinaryion rate . The lacion rate .2D3, a conventional drug for osteoporosis treatment, can increase Ca2+ absorption but also inhibit TRPV5 expression in osteoclasts, leading to the inhibition of early osteoclast differentiation, activity, and bone loss [2+ and inhibited bone resorption [−/− mouse osteoclasts showed that the number of osteoclasts was approximately twice that of wild-type (WT) mice, and the number and size of osteoclast nuclei were significantly higher in TRPV5−/− mice [2) can increase TRPV5 expression in osteoclasts to inhibit osteoclast differentiation [2 may induce continuous Ca2+ oscillations by increasing TRPV5 expression. E2 exerts its biological activity mainly by binding with the estrogen receptor ER. Tianwen Ye et al. demonstrated that E2 can promote TRPV5 expression and stimulate osteoclast apoptosis by interacting with NF-κB when binding with ER [TRPV5 is expressed in various cells of bone tissue, including the bone matrix, osteoclasts, and chondrocytes . Osteoclone loss . TRPV5 isorption . In vitr−/− mice . However−/− mice . Fangjinntiation . E2 may  with ER . NF-κB c with ER .2+-dependent proteins, calmodulin, and TRPV5 in chondrocytes of osteoarthritis rats, substantiating the hypothesis that TRPV5 is a calmodulin-dependent channel [2+ influx, resulting in calcium overload and subsequent activation of calmodulin and CaMKII proteins. CaMKII proteins phosphorylate Beclin1, inhibiting autophagosome formation and consequently suppressing chondrocyte autophagy, which is also stimulated by associated inflammatory factors [Autophagy in chondrocytes is a self-protective mechanism and metabolic mode of cartilage tissue. TRPV5 is expressed in chondrocytes and upregulated during the progression of osteoarthritis. Ismail M. Hdud et al. used immunohistochemistry to identify TRPV5 distribution primarily in the superficial and middle chondrocytes . In a mo channel . The pri factors . Moreove factors . The upr factors .The presence of TRPV5 in osteoblasts is a matter of ongoing debate among researchers. Bram C. J. van der Eerden and Lieben L believe that TRPV5 is not expressed in osteoblasts, thus supporting the notion of absent of TRPV5 expression , 18. In 2CO3 in the body and elevates the H+ concentration, causing a decline in tissue pH and resulting in an acidic microenvironment. In untreated or severe COPD patients, hypercapnia and hypoxia can significantly impact bone metabolism, making patients more susceptible to developing osteoporosis [3− and tissue pH, leading to acidosis. Consequently, phosphate and carbonate in bone tissue are lost as buffer substances, and renal tubular epithelial cells exhibit reduced calcium reabsorption and increased urinary calcium excretion, which can contribute to osteoporosis [2+ and Mg2+ in mice with metabolic acidosis led to a decrease in the gene and protein abundance of TRPV5 and Calbindin-D28K in the kidney [2+ reabsorption was observed in TRPV5−/− mice, indicating that metabolic acidosis did not affect urinary calcium excretion in mice. Additionally, Mg2+ exerted an inhibitory effect on TRPV5 activity [The relationship between in vivo pH and osteoporosis has been a topic of interest among researchers for decades. In 1968, Pellegrino et al. suggested that inorganic salts in the bone system could function as a buffer matrix, and osteoporosis was thought to result from the absolute loss of bone minerals, which played a buffering role in absorbing acidic substances when the bone was “dissolved” . Howeveroporosis , 129.Preoporosis .Similarloporosis . A studye kidney . Howeveractivity . Thus, i+ Transporting V0 Subunit D2 (ATP6v0d2) [+ into the bone resorption area. Based on the unique acidic microenvironment surrounding osteoclasts, the team designed an osteoclast microenvironment-responsive nanoplatform, HA-MC/CaCO3/ZOL@PBAE-SA (HMCZP). PBAE, a pH-responsive polymer, can release ZOL in the acidic microenvironment of OP, thereby inhibiting osteoclast activity. Thus, several studied have confirmed the extracellular acidic microenvironment caused by osteoporosis. This acidic microenvironment simultaneously inhibits the expression of TRPV5 ion channels. The exact mechanism by which TRPV5 is affected by the acid–base environment is not entirely clear, but studies have reported the involvement of an acid-sensitive receptor on the basal side of cells in the proximal tubule of the kidney (PT) [+–H+ exchanger type 3 in PT [+ [+ influences the biological activity of TRPV5 by altering the conformation of TRPV5 [2 was prevented under low pH conditions to suppress TRPV5 activity, revealing a synergistic effect of pH and lipid co-factor in gating the channel. The transition of TRPV5 channel from open to closed conformation under low pH environment was captured using cryo-electron microscopy. These structural and molecular insights provide new understanding of the interplay between pH and TRPV5 gating.In recent years, numerous pH-responsive drugs have been developed to target local pH changes in osteoporotic tissues. Dou et al. reported the development of cerium bioactive nanoparticles with pH response, which can target the acidic extracellular microenvironment and inhibit the activity of mature osteoclasts (mOCs). During bone remodeling, the pH value of the bone resorption cavity can reach 3–4, with mOCs triggering the extracellular acidic microenvironment through ATPase HTP6v0d2) . Yi Hu eTP6v0d2) . Mature ney (PT) . When th 3 in PT . Yeh et in PT [+ . Additioin PT [+ . Therefoof TRPV5 . Moreoveof TRPV5 . When thof TRPV5 . Conversof TRPV5 . Furtherof TRPV5  found th+ modulates TRPV5 ion channel activity in bone cells in response to alterations in extracellular pH may offer promising opportunities for the innovation of drug delivery approaches and the identification of novel therapeutic targets for osteoporosis treatments.The multifaceted mechanisms through which H2+ gated channel, and its functionality is contingent on several proteins, including CaR, CaM, 80 k-H, S100A10-annexin II, and calbindin 28 K [2+ receptor that exhibits high sensitivity to plasma Ca2+ concentrations. Catalin N. Topala et al. discovered that CaR is predominantly located in the distal convoluted tubules (DCT) and connecting tubules (CNT) of the kidney [2+ concentrations. Patch clamp techniques demonstrated that CaR could stimulate the enhancement of TRPV5 activity , ubiquitin binding enzyme (E2), and ubiquitin ligase (E3). These enzymes interact with the target protein, specifically modifying it, and thereby regulating protein function . Tasaki nd Nedd4 .Protein post-translational modification (PTM) is a crucial mechanism that enables proteins to function . CovalenIn summary, the protein modification processes discussed above involve the coordinated interaction of numerous enzymes with protein molecules. Several protein factors, directly or indirectly related to osteoporosis, exert their effects on TRPV5. For a summary of these factors, please refer to Table 3, CaR, Klotho, PIP2, and others, through different pathways, which can enhance its activity and function [Nie et al. reported that TRPV5 is highly sensitive to calcium, and chemicals exhibiting specificity and potency may hold therapeutic significance for diseases related to imbalances in calcium homeostasis, such as hypercalcemia, renal calculi, and osteoporosis. TRPV5 can be activated by various agonists, including PTH, VDfunction . The dis2+ uptake, and enhances the activity of the TRPV5 channel by phosphorylating T709 residues via the PKA pathway [2+ and Mg2+ transport in the renal paracellular epithelium [2D3, and some Ca2+ and Mg2+ transport channels, including TRPV5, TRPM6, and calbindin-D9k, are significantly upregulated [Regarding agonists, the β-adrenergic receptor (β-AR) comprises β1-AR, β2-AR, and β3-AR, with β1-AR and β2-AR primarily distributed in the renal DCT2/CNT . The β1- pathway . Strepto pathway . Diabeti pathway . Claudinithelium . CLDN16-ithelium . Relatedegulated . Glucocoegulated . Dex admegulated .Econazole is a small molecule drug commonly used to treat skin antifungal infections and has been shown to inhibit TRPV5/6 in some studies , 159. In2+ influx, thereby inhibiting TRPV5 activity and activating chondrocyte autophagy [2+ by HEK293 cells. Plasmin-activated PAR-1-induced PKC-mediated phosphorylation of TRPV5 was found to affect the binding of calmodulin to TRPV5 and decrease the activity of the TRPV5 channel. It is speculated that urinary plasmin is an inhibitor of TRPV5 [To achieve higher specificity, researchers continuously screen new inhibitors. ZINC9155420 and ZINC17988990 bind to TRPV5 in a nonconductive conformation along the ionic conduction pathway. The binding sites of these inhibitors do not overlap with activators, indicating that their effects do not result from competitive binding. Instead, these inhibitors lock the channels and prevent agonists from reaching the activated state . Two newutophagy . In patiutophagy . PAR-1 cof TRPV5 .Osteoporosis (OP) as a systemic metabolic disease is influenced by multiple factors and can be explored from histological, cytological, and molecular biology perspectives. In recent years, the impact of the microenvironment on OP has become a research hotspot. Dolores M. Shoback's team posits that new OP treatment strategies will focus on targeted drugs that eliminate senescent cells in the bone tissue microenvironment . Moreove2+ at least at the mRNA level [2D3 in vitamin D-deficient mice can increase the expression of TRPV5 mRNA in the kidney by 3–4 fold [Research has shown that the absence of TRPV5 makes mice more susceptible to age-related osteoporosis . Pumroy NA level , 169. Th3–4 fold . FGF23, 3–4 fold , 172. ThOn the other hand, the process of TRPV5 exerting its protein function cannot be dissociated from various regulatory mechanisms, including transcription activation, intracellular transport, and post-translational modification . Chen etIn the future, the relationship between the osteoporosis microenvironment and TRPV5 will be more clearly elucidated, and drug development or disease diagnostic criteria centered on TRPV5 ion channels will provide more favorable conditions for the treatment of osteoporosis.


This study explored the use of IRDye800CW, conjugated to bevacizumab, with four commercially available NIRF laparoscopes optimised for indocyanine green (ICG). (2) Methods: A (lymph node) phantom was made from a calibration device for NIRF and tissue-mimicking material. Serial dilutions of bevacizumab-800CW were made and ICG functioned as a reference. System settings, working distance, and thickness of tissue-mimicking material were varied to assess visibility of the fluorescence signal and tissue penetration. Tests were performed with four laparoscopes: VISERA ELITE II, Olympus; IMAGE1 S™ 4U Rubina, KARL STORZ; ENDOCAM Logic 4K platform, Richard Wolf; da Vinci Xi, Intuitive Surgical. (3) Results: The lowest visible bevacizumab-800CW concentration ranged between 13–850 nM (8–512 times diluted stock solution) for all laparoscopes, but the tracer was not visible through 0.8 cm of tissue in all systems. In contrast, ICG was still visible at a concentration of 0.4 nM  and through 1.6–2.4 cm of tissue. Visibility and tissue penetration generally improved with a reduced working distance and manually adjusted system settings. (4) Conclusion: Depending on the application, bevacizumab-800CW might be sufficiently visible with current laparoscopes, but optimisation would widen applicability of tumour-targeted IRDye800CW tracers. Surgeons rely on visual and tactile feedback for intraoperative decision-making. However, many laparoscopic or robotic surgical systems lack tactile feedback. Image-guided surgery, such as near-infrared fluorescence (NIRF), aid surgical decision-making with additional visual feedback. There has been an increasing interest in NIRF and its new applications for fluorescence guided surgery. In oncological surgery, NIRF is primarily used to achieve tumour-free excision, identify occult lesions, debulking, and recognition of vital structures .Near-infrared light (650–950 nm for the first NIRF window) has superior tissue penetration compared to white light and has limited interference with human tissues ,3,4,5. FThe fluorophore IRDye800CW has an NH ester group for labelling proteins such as antibodies , making Our intention is to use targeted tracers for the detection of sentinel lymph nodes or lymph node metastases in patients with colon cancer during laparoscopic surgery ,34. SentClinical-grade bevacizumab-800CW was manufactured according to current Good Manufacturing Practice and supplied by the University Medical Center Groningen for use in this phantom study, as previously described . In summICG  was used as a fluorescent reference for bevacizumab-800CW. ICG was combined with 2% intralipid and PBS. Intralipid acted as human albumin in blood serum and was required for this phantom study, as ICG dissolved in water for injection or PBS has a different excitation and emission wavelength . The ICGThe molar concentration of ICG serial dilutions were a factor 950 higher than the bevacizumab-800CW concentrations, and 597 times higher than the molar concentration IRDye800CW due to the 1.6:1 dye to antibody ratio. Therefore, the first five concentrations of bevacizumab-800CW serial dilutions correspond approximately to the last five concentrations of ICG  Table A1Transparent polypropylene Eppendorf tubes were filled with the solutions of bevacizumab-800CW or ICG and placed in a CalibrationDisk  ,39. The Four commercially available laparoscopic systems for clinical use were tested: (1) VISERA ELITE II, Olympus; (2) ENDOCAM Logic 4K platform (short: ENDOCAM), Richard Wolf; (3) IMAGE1 S™ 4U Rubina (short: Rubina), KARL STORZ; and (4) da Vinci Xi, Intuitive Surgical . The VISImaging of phantoms, with different concentrations of bevacizumab-800CW and ICG, was performed with all four laparoscopic NIRF systems. Since 15 dilutions were used, imaging was performed in two steps by using the phantom of The acquired fluorescence images were independently assessed for visibility by two researchers (DJS and AJS). Visibility of individual Eppendorf tubes was scored as visible or invisible and disagreement was resolved through discussion or a third researcher (ECJ). Consensus judgement of visibility was used for analyses since the interobserver agreement was almost perfect (kappa = 0.88) .Segmentation of images was performed to evaluate the different image properties by three different parameters; target-to-background ), signal-to-noise ), and contrast-to-noise ) ratio. The fluorescence signal was expressed in arbitrary units (AU). Regions of interest were defined as the windows in the CalibrationDisk . TBR repSerial dilutions of bevacizumab-800CW (concentrations ranging from 0.42 to 6800 nM) and ICG (concentrations ranging from 394 nM to 6460 µM) were used to assessed visibility with different system settings and NIRF modes, totalling fourteen different scenarios with four laparoscopes for each fluorophore. Adjustment of brightness did not improve visibility of bevacizumab-800CW and ICG for the da Vinci Xi and ENDOCAM . All conHigh camera settings and monochromatic NIRF mode were used to assess the influence of working distance to the phantom . In geneThe lowest visible concentrations of bevacizumab-800CW for the da Vinci Xi, ENDOCAM, VISERA ELITE II, and Rubina system were 850, 850, 213 and 13 nM , respectively. In The TBR, SNR, and CNR of the lowest visible concentration bevacizumab-800CW were: (1) VISERA ELITE II 2.90, 1.94, 1.27; (2) ENDOCAM 0.86, 2.52, −0.41; (3) Rubina 1.75, 2.53, 1.08; (4) da Vinci Xi 1.44, 1.44, 0.44, respectively . PrimariTBR, SNR, and CNR were higher for the visible ICG concentrations compared to the bevacizumab-800CW concentrations, especially for the Rubina and da Vinci Xi e–h. HoweThe ratios found using the VISERA ELITE II a,b decreVisibility of bevacizumab-800CW and ICG decreased with the amount of tissue-mimicking material . High coImage-guided surgery and NIRF could aid surgeons in intra-operative decision-making. This study explored the use of IRDye800CW, conjugated to bevacizumab, in vitro with four commercially available NIRF laparoscopic surgical systems intended for use with ICG. All four laparoscopes were able to detect bevacizumab-800CW in the highest concentrations, relevant for lymph node metastases detection after local administration, even though optimal absorption and emission wavelengths of IRDye800CW are different from ICG ,38,52. D99mTc-tilmanocept-800CW was successfully used for sentinel lymph node detection with a laparoscope [In our study, laparoscopes were able to detect bevacizumab-800CW down to a concentration of 13–850 nM. In contrast, preclinical and clinical open-air camera systems, can detect ~0.05–15 nM bevacizumab-800CW in ideal conditions . Visibilaroscope . Intravearoscope . In comparoscope . Thus, taroscope ,59.There is a wide range of conditions that influence visibility of tracers. For example, reduced working distance improved visibility in our study like it did in another study that assessed bevacizumab-800CW with an endoscope (Xenon light source with ICG filters and Image1S H3-Z FI camera from KARL STORZ) , due to 99mTc-tilmanocept-800CW [99mTc-ICG [In this phantom study, tissue penetration of bevacizumab-800CW and ICG was up to 1.6 cm and 1.6–2.4 cm through tissue-mimicking material, respectively. In earlier phantom studies, ICG could be detected through 3.6 mm of beeswax  , 6 mm ofpt-800CW  and 99mT9mTc-ICG ,67,68, w9mTc-ICG .The lowest visible concentrations bevacizumab-800CW had a TBR ranging from 0.86 to 2.90, a SNR of 1.44 to 2.53, and a CNR of −0.41 to 1.27. Literature suggests that a TBR > 1.5 might be sufficient to discriminate fluorescent lesions and reduce false negatives in ideal situations. However, often a TBR > 2 is pursued for fluorescence-guided surgery and decision-making, and the TBR is also dependent on where it is measured . For exa111In-DTPA-trastuzumab-800CW and 68Ga-tilmanocept-800CW) were comparable [To our knowledge, this is the first study looking into the use of commercially available laparoscopes of key market players and the use of a tumour-targeted tracer without modification of hardware or software. For this study, IRDye800CW conjugated to only bevacizumab was used as a tumour-targeted tracer. However, we hypothesise that other antibodies conjugated to IRDye800CW will have similar results, since the same NHS ester group is used for conjugation. Furthermore, absorption and emission wavelengths of IRDye800CW and hybrid targeted IRDye800CW tracers . Tissue penetration of bevacizumab-800CW was up to 1.6 cm. In contrast, ICG was still visible after >16,000 times dilution and achieved tissue-penetration up to 2.4 cm. Depending on the application, visibility of bevacizumab-800CW might be sufficient for clinical use with current NIRF laparoscopes, but optimisation of sensitivity for IRDye800CW would widen the indication of tumour-targeted tracers in laparoscopic surgery. More future studies should focus on standardised testing of laparoscopes and their capabilities for detecting (new) NIRF tracers.