The purpose of this study was to determine whether YAP/TAZ activation in uveal melanoma (UM) and the susceptibility of melanoma cell lines to YAP/TAZ inhibition by verteporfin (VP) is related to the tumor's genetic background.Characteristics of 144 patients with enucleated UM were analyzed together with mRNA expression levels of YAP/TAZ-related genes . VP was administered to cell lines 92.1, OMM1, Mel270, XMP46, and MM28 (UM), CRMM1 and CRMM2 , and OCM3 (cutaneous melanoma). Viability, growth speed, and expression of YAP1-related proteins were assessed.YAP1 and WWTR1 correlated with the presence of monosomy 3  and BAP1-loss  in the primary UM; metastasis development correlated with higher expression of YAP1 (P = 0.05) and WWTR1 (P = 0.003). In Leiden data, downstream transcription factor TEAD4 was increased in cases with M3/BAP1-loss (P = 0.002 and P = 0.006) and related to metastasis (P = 0.004). UM cell lines 92.1, OMM1, and Mel270  and the fast-growing cell line OCM3 (BRAF-mutation) showed decreased proliferation after exposure to VP. Two slow-growing UM cell lines XMP46 and MM28  were not sensitive to VP, and neither were the two conjunctival melanoma cell lines (BRAF/NRAS-mutation).In TCGA data, high expression of High risk UM showed an increased expression of YAP/TAZ-related genes. Although most UM cell lines responded in vitro to VP, BAP1-negative and conjunctival melanoma cell lines did not. Not only the mutational background, but also cell growth rate is an important predictor of response to YAP/TAZ inhibition by VP. UM is known to have driver mutations in Tafazzin gene). YAP1 and TAZ can bind to TEAD proteins in the cell nucleus, allowing them to read DNA, and activate several genes that promote cell growth and proliferation .,Recent studies identified the importance of the YAP/TAZ pathway in oncology, for tumor growth and possible targeting.GNAQ/11 mutation,,,,GNAQ/11 mutation.,GNAQ/11 mutation is absent in CoM,,GNAQ/11 mutation (but harboring BRAF or NRAS mutations instead),,,,Approximately 90% of UM harbor a GNAQ/11 mutation) is altered by chromosome changes or other mutations, such as in BAP1, which is known to be related to adverse prognosis.,BAP1 as well as TAZ are located on chromosome 3. Hypothesizing a link between the genetic make-up of UM and YAP/TAZ activity, we wondered if UM cells lacking BAP1 expression are more susceptible to treatment with VP, and whether CoM cells are sensitive at all.Recently, it was reported that the YAP/TAZ pathway has little prognostic value for patient survival in UM.BRAF or NRAS mutation as a control. We show that the YAP/TAZ pathway has a higher activity in UM tissue with unfavorable genetic characteristics such as monosomy 3 (M3)/BAP1 loss. We confirm that VP inhibits growth of BAP1-positive UM cells in vitro, whereas it has limited effect on BAP1-negative cells and CoM, and observed that not only the genetic background, but other traits, such as cell growth rate, were major determinants of VP response.We set out to investigate whether mRNA expression of YAP1-related genes was related to clinical, histological, and genetic tumor characteristics in UM. Next, we studied the effect of YAP1-inhibition using VP without light activation on multiple UM cell lines with different genetic profiles (including cell lines with and without BAP1 expression), and included CoM cell lines with either a http://cancergenome.nih.gov/). The second set was comprised of 64 patients with UM who underwent primary enucleation at the Leiden University Medical Center (The Netherlands).Data from two independent sets of patients with UM were analyzed. The first set was comprised of 80 patients with UM from The Cancer Genome Atlas (TCGA) project .Messenger RNA was isolated from frozen tumor material for gene expression analysis using the RNeasy Mini Kit . The Illumina HT-12 version 4 chip was used to determine gene expression levels .,DNA was isolated for single-nucleotide polymorphism (SNP) analysis using the QIAmp DNA Mini kit . With the Affymetrix 250K_NSP microarray and Affymetrix Cytoscan HD chip , status of chromosome 3 was determined.The study was approved by the Biobank Committee of the Leiden University Medical Center . The tenets of the Declaration of Helsinki were followed.-pos, GNAQ-mut),GNA11-mut),GNAQ-mut),GNAQ-mut),GNA11-mut),BRAF-mut),NRAS-mut),BRAF-mut)Human uveal melanoma cell lines 92.1  supplemented with 10% fetal bovine serum  and 1% antibiotics . Cell lines XMP46 and MM28 were grown in IMDM medium  supplemented with 20% FBS and 2% antibiotics. Cell lines CRMM1 and CRMM2 were grown in F-12K medium  supplemented with 10% FBS and 1% antibiotics. Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. Cells were protected from light using aluminum foil, and the experiments were performed under dimmed lights.The investigated drug was liposomal verteporfin in phosphate buffered saline . As a control, PBS  was used. Drugs or controls were added to regular cell culture medium of the respective cell lines, in concentrations as described with the experimental designs.Cell viability was assessed using the Cell Counting Kit-8 . In this assay, a tetrazolium salt (WST-8) is reduced by dehydrogenase activity into a yellow/orange formazan dye. Light absorbance thereby reflects the activity of living cells. Cells were seeded in a 96-well plate at a density of 10,000 cells per well. The following day, various concentrations of VP were added. After 3 days, all wells were gently washed with fresh medium (to remove staining from VP) and the WST-8 salt was added according to the manufacturer's guideline. Light absorbance at 450 nm was measured using a microplate reader and normalized to control values. Experiments were performed in triplicate.Cells were seeded in 6-well plates at a density of 300,000 cells per well. The following day, culture medium was replaced by new medium with the addition of 1.25 ug/mL VP, 7.5 ug/mL VP or PBS. At days 2, 4, and 6, cell numbers were determined using the trypan blue (0.4%) dye exclusion method in an automated cell counter . Culture medium  was refreshed on days 2 and 4 for the remaining wells. Experiments were performed in triplicate.Cells were seeded in 6-well plates at a density of 800,000 cells per well. The following day, culture medium was replaced by new medium with the addition of 1.25 ug/mL VP, 7.5 ug/mL VP or PBS. After 24-hour incubation, cells were washed with ice-cold PBS and lysed with MPER with a protease and phosphatase inhibitor. Samples were sonicated for 15 seconds, and centrifugated for 20 minutes at 14,000 g in a precooled 4°C centrifuge. The supernatant was used for further analyses.Per lane, 20 ug of protein were loaded on a 4% to 12% Bis-Tris gel . After electrophoresis, the assay was transferred to a polyvinylidene difluoride (PVDF) membrane . Coomassie blue staining was used to ensure equal loading. The membrane was blocked for 1 hour at room temperature in 5% milk and incubated for 3 hours with the respective primary antibodies at a 1:1000 dilution. After washing, the membrane was incubated for 1.5 hours with the respective secondary antibodies at a 1:2000 dilution. Protein expression was visualized with the ECL technique (Amersham ECL Select). Antibodies were purchased from Cell Signaling Technology : YAP (4912S), TEAD1 (12292S), and c-Myc (9402S).U test  or the Jonckheere test for trends . The Spearman's rho was applied for analysis of correlations. Survival was analyzed with the Kaplan-Meier method and log-rank tests. When applicable, “high” and “low” expression of mRNA values was categorized based on the median expression values. Two-sided tests were reported, and P values < 0.05 were considered statistically significant.Data were analyzed using SPSS version 23. The applied statistical tests were the Mann-Whitney YAP1, WWTR1 (=TAZ), and TEAD1. In the Leiden dataset, probes were available for YAP1 and TEAD4, but not for the other YAP1-related genes.To study the activation of the YAP/TAZ pathway in human UM, we first analyzed the mRNA expression of YAP1-related genes in UM samples in two independent datasets. One set was comprised of material from 80 UM from the TCGA project, the other set of 64 UM from patients who underwent an enucleation in the LUMC (The Netherlands). In the TCGA dataset, probes were available for WWTR1 was associated with a greater largest basal diameter  and a mixed/epithelioid cell type (P = 0.002). Interestingly, a higher expression of YAP1 was noticed for lightly pigmented tumors in both data sets compared to highly-pigmented cases (TCGA: P = 0.006 and Leiden: P = 0.007).Both in the TCGA and Leiden datasets, expression of YAP1-related genes did not vary based on patient age, American Joint Commission on Cancer (AJCC) stage, or tumor prominence , 2. In tGNAQ/11, we examined the expression of mRNA in tumors with and without these mutations. In the TCGA dataset, tumors with either a GNAQ or GNA11 mutation (n = 72) did not differ in their expression of YAP1-related genes compared with tumors without these mutations  and WWTR1 . Gain of chromosome 8q related to a higher expression of WWTR1 in the TCGA data (P < 0.001) but a lower expression of TEAD1 (P = 0.025), whereas no association with 8q status were observed in the Leiden data. From these data, we conclude that the chromosome 3/BAP1 status of UM is related to the expression of YAP1-related genes, with a higher activity in the prognostically unfavorable cases.We then tested whether YAP1 activity relates to the genetic status of UM, such as monosomy 3 (M3)/BAP1-loss, or gain of chromosome 8q, two adverse prognostic factors. In the TCGA dataset, both M3 and BAP1-loss were associated with a higher expression of WWTR1 (P = 0.003) and a borderline insignificant higher expression of YAP1 (P = 0.050) compared to patients without metastases  but a higher expression of TEAD4 was , OMM1 (GNA11-mut), and Mel270 (GNAQ-mut) demonstrated more cell death with increased dosages of VP . Cell line OCM3 was sensitive to VP treatment at higher doses, with a remarkable drop in cell viability after treatment for 3 days with >2 ug/mL . Viability assays demonstrated a relative tolerance for VP at low dosages, whereas a dose-dependent decrease of viability tended to occur in both cell lines at dosages >4 ug/mL VP  against the doubling time (as determined with the cell growth experiments) of all cell lines. Three clusters of cells could be identified: (1) high doubling time and high LD50 ; (2) low doubling time and high LD50 ; and (3) low doubling time and low LD50 . Cluster 1 comprises the BAP1-negative UM cell lines (XMP46 and MM28). Cluster 2 comprises the CoM cell lines (CRMM1 and CRMM2). Cluster 3 comprises the other, BAP1-positive, UM cell lines  and melanoma cell line OCM3; .GNAQ/11 mutation may lower the threshold for VP sensitivity.It can be deduced that, in order to be susceptible to VP, cell lines need a certain amount of cell growth, and a To further understand the effects of VP on melanoma cells and the YAP/TAZ pathway in various cell lines, we performed Western Blot analyses of YAP, TEAD, and downstream target CMYC. Cell lines were cultured for 24 hours with a low dose liposomal VP in PBS (1.25 ug/mL), high dose liposomal VP in PBS (7.5 ug/mL), or control (PBS).GNAQ/11-mutant UM cell lines demonstrated a reduction of YAP, TEAD, and CMYC upon VP administration. This was similarly seen in cell line OCM3 (BRAF-mut) and to some extent in the NRAS-mutated cell line CRMM2. The rest of the cell lines  demonstrated little or no reduction of YAP, TEAD, or CMYC upon VP administration , and a cell line lacking GNAQ/11 mutations that did demonstrate decreased survival (OCM3). We noticed that the nonresponding cell lines had a slower growth rate compared to the responding ones, and we hypothesize that this may have been limiting the susceptibility for YAP1-inhibition.Our experiments showed that exposure to VP decreased cell viability in BAP1-positive UM cell lines harboring mutations in We expected that BAP1-negative UM cell lines would be more susceptible to YAP1-inhibition, because the YAP1 pathway was upregulated in BAP1-loss UM. Unexpectedly, these cell lines demonstrated very little response to VP; however, we noticed a remarkable slower growth rate compared to the BAP1-positive UM cell lines. An alternative explanation is that BAP1-loss results in YAP/TAZ pathway insensitivity, or that BAP1-loss causes a YAP1-independent growth disadvantage.GNAQ/11. We identified no convincing effect of VP in the two CoM cell lines with either a BRAF or NRAS mutation (CRMM1 and CRMM2), whereas the cutaneous melanoma cell line OCM3 did show a response to VP. Notably, the growth rate of OCM3 was higher than that of CRMM1 and CRMM2. In line with our findings, previous work by Yu et al. showed a limited, yet present, response to VP for cell line OCM3, with about a halving of cell count compared to control after 3 days of treatment with high-dose VP.GNAQ/11-mutation may be affected by VP.We also studied cell lines lacking a mutation in GNAQ/11 pathway and other pathways such as BAP1,As a treatment for UM, we concur with others reporting on the potential benefit of VP in preclinical models. Clinical experience shows that BAP1-mutated UM show more aggressive characteristics than BAP1-wildtype UM, however, which is opposite to the in vitro behavior of our cell line model with BAP1-loss. Unfortunately, no fast-growing UM cell line models lacking BAP1 are available. It would therefore still be interesting to test the susceptibility of BAP1-negative cells in vivo to VP treatment. It has been suggested that targeting the YAP1-pathway alone may not be the most effective route to attack UM, and that combined treatment aimed at the ,Being the first to study VP in CoM, our results are not supportive for VP as a single-agent therapy in this disease. This may resemble earlier work on cutaneous melanoma cell lines that demonstrated mixed responses to YAP/TAZ inhibition: whereas reduced cell growth and reduced YAP/TAZ protein levels were reported after VP,A strength of our study is the availability of data on mRNA expression and genetic status of a large number of UM cases. We were also able to test a broad panel of cell lines, representing various mutational backgrounds of UM and CoM. Some conflicting findings were observed between mRNA expression of YAP-related genes in the TCGA data and Leiden data. This may be due to differences in the study group, as UM in the Leiden cohort were somewhat smaller than those analyzed in the TCGA project, which may have influenced YAP1 activity.An interesting matter in cell line studies is whether cell lines mimic the traits of their original tumor type,,,Interestingly, the YAP/TAZ pathway has recently been linked to mechanisms of resistance against targeted therapy and escape against immunotherapy in cancer.Upregulation of the YAP/TAZ pathway has also been linked to several immune-suppressing effects, relevant for immunotherapy. YAP1 expression was positively associated with PD-L1 expression in samples of cutaneous melanoma, creating an escape for destruction by CD8+ T cells.Blocking the YAP/TAZ pathway may be beneficial to overcome MAPK-inhibitor resistance, as YAP/TAZ knockdown restored sensitivity to BRAF-inhibitors in previously resistant cutaneous melanoma cell lines,The true future application of YAP/TAZ inhibition (as with VP) may therefore possibly not be as a single-agent therapy to any type of melanoma, but as an additive to other (targeted or immuno-) therapies. This would be beneficial in the treatment of UM as well as CoM, mirroring the findings from cutaneous melanoma.Concluding, expression of YAP/TAZ-related genes correlated with tumor genetics in UM, with a higher activity in M3/BAP1-negative lesions. The prognostic value of YAP1-related gene expression on metastasis development was limited. Although most UM cell lines responded in vitro to VP, BAP1-negative UM cell lines and CoM cell lines did not. We find that not only the mutational background of the studied genes, but also cell growth rate is an important predictor of YAP/TAZ inhibition by VP. Our study implies a potential role for the YAP1 pathway as therapeutic target in UM, but finds a limited role for single-agent therapy in CoM. YAP1 inhibition may be used as a cotreatment with both targeted and immunotherapy, to overcome mechanisms of resistance and escape.


The present study proposes a 3-dimensional (3D) biomimetic collagen-based scaffold to mimic the tumor microenvironment and the crosstalk between the extracellular matrix (ECM) and cancer cells.Squamous cell carcinoma (SCC) represents the most common histotype of all head and neck malignancies and includes oropharyngeal squamous cell carcinoma (OSCC), a tumor associated with different clinical outcomes and linked to human papilloma virus (HPV) status. Translational research has few available We compared the phenotypic and genetic features of HPV-positive and HPV-negative OSCC cell lines cultured on common monolayer supports and on scaffolds. We also explored cancer cell adaptation to the 3D microenvironment and its impact on the efficacy of drugs tested on cell lines and primary cultures.HPV-positive and HPV-negative cell lines were successfully grown in the 3D model and displayed different collagen fiber organization. The 3D cultures induced an increased expression of markers related to epithelial–mesenchymal transition (EMT) and to matrix interactions and showed different migration behavior, as confirmed by zebrafish embryo xenografts. The expression of hypoxia-inducible factor 1α (1α) and glycolysis markers were indicative of the development of a hypoxic microenvironment inside the scaffold area. Furthermore, the 3D cultures activated drug-resistance signaling pathways in both cell lines and primary cultures.Our results suggest that collagen-based scaffolds could be a suitable model for the reproduction of the pathophysiological features of OSCCs. Moreover, 3D architecture appears capable of inducing drug-resistance processes that can be studied to better our understanding of the different clinical outcomes of HPV-positive and HPV-negative patients with OSCCs. Squamous cell carcinomas (SCCs) represent approximately 90% of all HNCs4. Although the primary risk factors are alcohol abuse and tobacco, human papilloma virus (HPV) has now been linked to the development of some tumor types5. HPV-positive SCCs arise mainly in the oropharynx and have different pathological and clinical characteristics with respect to HPV-negative tumors8. Oropharyngeal squamous cell carcinomas (OSCCs) show a more favorable clinical outcome than alcohol- and tobacco-related SCCs9. Standard treatment options for SCCs include radiation, surgery, chemotherapy, targeted therapies, and immunotherapy. In OSCCs, chemotherapy is often used in advanced stages of the disease. 5-fluorouracil (5-FU) and platinum-based drugs [cisplatin (CIS) and carboplatin] represent the most widely used chemotherapeutic agents, whereas taxol-based treatments are preferred in the metastatic setting10. The only targeted therapy approved for OSCCs, regardless of HPV status, is cetuximab (CETU), an epidermal growth factor receptor (EGFR)-targeting monoclonal antibody used alone or in combination with chemotherapy11. Immune checkpoint inhibitors show great promise for the treatment of HNCs, and several clinical trials and preclinical studies of anti-cytotoxic T-lymphocyte–associated antigen 4, anti-programmed death 1, anti-programmed death ligand 1, and novel immunotherapeutic drugs are currently ongoing to identify new treatment options12.Head and neck carcinomas (HNCs) represent the sixth most common non-skin cancer worldwide13. Like other cancers, SCCs are dynamic masses that remodel the 3-dimensional (3D) ECM structure and interact constantly with stromal components14. The ECM has a fiber network that provides physical support and is also involved in regulating numerous cellular processes15. For example, during cancer progression, the association between integrins and ECM components induces cell adhesion-mediated drug resistance16. Integrin signaling supports ECM biosynthesis, increases collagen fiber crosslinking, and sustains chemotaxis-driven cell invasion17. Given that the reproduction of these tumor microenvironment features is virtually impossible in monolayer models, 3D cultures have been developed in an attempt to mimic the architecture of the tumor niche20.Despite these innovative therapeutic approaches, disease relapse and drug resistance remain a challenge in OSCCs. Response to treatment is influenced by many factors, including interaction with the extracellular matrix (ECM)In the present study we investigated, as far as we know, for the first time, the impact of a 3D biomimetic collagen-based scaffold on the phenotypic and genotypic features of OSCC cells. We also evaluated the role of scaffold architecture in the response of cell lines and patient-derived primary cultures to standard treatments.21. Obtained scaffolds were immersed in 70% ethanol for 1 h for complete sterilization and followed by 3 washes of 5′ each in sterile Dulbecco phosphate-buffered saline .Collagen scaffolds were synthesized using chemicals purchased from Sigma-Aldrich . Bovine type I collagen (1 wt%) was suspended and precipitated to pH 5.5. The collagen was crosslinked with 1.4-butanediol diglycidyl ether (1 wt%) to control porosity and stabilize the collagen matrix. A freeze-drying process was used to generate the final monolithic scaffold. An established freezing and heating ramp yielded scaffolds with optimal levels of pore interconnectivity, porosity, and size (as previously reported)l-glutamine  and 10% of fetal bovine serum . UM-SCC6 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) High Medium (Euroclone) supplemented with 1% penicillin/streptomycin (PAA), 1% non-essential amino acids (Euroclone), and 10% FBS. Cells were counted and seeded in monolayer cultures or in collagen-based scaffolds13. The growth curves were generated on 2D and 3D cultures (concentration of 3.5 × 106 cells for scaffold) through MTT tests (Sigma-Aldrich) at 24 h, 72 h, and 5 days. All cell lines were maintained in a 5% CO2 atmosphere at 37 °C.UPCI:SCC090 OSCC cell line was purchased from American Type Culture Collection , and UM-SCC6 HPV-negative OSCC cell line was obtained from MD Millipore . UPCI:SCC090 cells were maintained in Eagle’s minimum essential medium  supplemented with 1% of For staining protocols, collagen-based scaffolds were embedded in paraffin, using cryomold (25 × 20 × 5 mm). To evaluate the culture’s morphological features, 5-µm-thick slides were used for staining. Slides were hydrated and stained with hematoxylin and eosin  or with phalloidin and 4′,6-diamidino-2-phenylindole .Hypoxia-inducible factor 1α (HIF-1α) expression was determined in 5-µm sections and on cells cytospinned onto glass slides with Ventana BenchMark ULTRA system  according to the manufacturer’s instructions. The samples were stained with rabbit monoclonal anti-HIF-1α antibody  and glucose transporter 1  . Counterstaining was performed with hematoxylin. Stained slides were analyzed using an inverted microscope . Images of phalloidin-stained samples images were acquired by a confocal microscope and analyzed with the NIS Elements software .21. 2D culture cells were collected by trypsinization and resuspended in TRIzol reagent (Invitrogen); meanwhile, 3D culture cells were processed with TRIzol directly on the scaffolds. All specimens were conserved at −80 °C. RNA (500 ng) was retro-transcribed to obtain cDNA using iScript cDNA Synthesis Kit . A 2× Taqman Universal PCR Master Mix and a 7,500 Real-Time PCR System  were used for the amplification of 2 µL of cDNA by real-time polymerase chain reaction assay. The following genes were analyzed: CDH1, vimentin (VIM), SNAIL, MMP2, MMP9, LOX, GAPDH, NT5E, FOXM1, FLT1, ABCA3, CXCR4, TP53, and TGFβ. ACTB and HPRT were used as housekeeping genes. The obtained data were normalized to the housekeeping genes with the delta-delta Ct (2-∆∆Ct) method.mRNA extraction and quantitative polymerase chain reaction (qPCR) assay were performed as previously described22. Before manipulation, zebrafish embryos were anesthetized in 0.02% tricaine solution (Sigma-Aldrich).Tg(fli1:EGFP) transgenic zebrafish strain was handled in compliance with local animal welfare regulations  and in conformity with the Directive 2010/63/EU. Fertilized eggs were collected by natural spawning and raised at 28 °C in embryo water with 0.1% methylene blue, according to Kimmel et al.5/µL. 300/500 cells were implanted in the sub-peridermal space of 48-hpf embryos after tricaine anesthetization. Embryos injected in the yolk sack or/and that show cancer cells in circulation were excluded. The 2 groups injected with 2D or 3D culture cells were incubated at 32 °C. At 24 h post-injection (hpi), the presence of circulating cells and micro-metastasis development was evaluated using a fluorescence stereomicroscope (Nikon SMZ 25 equipped with NIS Elements software).Tg(fli1:EGFP) transgenic zebrafish embryos were dechorionated at 48 h post-fertilization (hpf). Cells from 2D cultures were collected by trypsinization, whereas cells seeded on scaffolds were obtained after 2 mg/mL collagenase type I digestion  1:1 in DMEM High Medium for 15 min at 37 °C under stirring conditions. Cells were labeled with a red fluorescent dye  and resuspended in PBS at a concentration of 2.5 × 1023, 5-FU at 55.44 µg/mL24, CETU at 130 µg/mL25, and GEMCI at 15.83 µg/mL26. After 72 h of treatment, surviving cell fractions were measured using the MTT test (Sigma-Aldrich) following the manufacturer’s protocol27.Drug sensitivity assays were performed on 2D and 3D cultures. Two days after seeding, the cell lines were treated with plasmatic peak concentrations of CIS, 5-FU, CETU, and gemcitabine (GEMCI) in accordance with the pharmacokinetic/clinical data for each drug. CIS was administered at a concentration of 4.1 µg/mL3 as previously reported28. Fragments were incubated in a PBS solution of 2 mg/mL collagenase type I (Millipore Corporation) 1:1 in DMEM High Medium for 15 min at 37 °C and then at room temperature for a further 15 min. The suspension was then filtered with a 100-µm sterile mesh filter . Single-cell suspensions were seeded in monolayer or 3D cultures and maintained at 37 °C in a 5% CO2-humidified atmosphere. All the experiments were performed within 2 weeks and analyzed by an experienced pathologist. The present study was approved by the IRST-Area Vasta Romagna Ethics Committee  and performed according to Good Clinical Practice standards and with the principles laid down in the Declaration of Helsinki (1964).The study involved 2 patients affected by SCCs. Patients underwent surgical treatment after having signed informed written consent. Patient-derived primary cultures were obtained from surgical specimens. The tissue samples were analyzed by an experienced pathologist, and a section of cancer tissue was transported under sterile conditions to the Biosciences Laboratory of our institute (IRST IRCCS) within 45 min of removal. Tumor samples were washed twice with PBS and minced with surgical scalpels into fragments of approximately 0.5–1 mmn indicating the number of replicates. Two-tailed Student’s t-test was used to define the differences between groups, and P values < 0.05 were considered significant.Each experiment was repeated at least 3 times. Data are shown as mean ± standard deviation or mean ± standard error, as stated, with Figure 1A). Confocal microscopy imaging was used to study the distribution and proliferation in different regions of the scaffold area (core and edge) . The cells of both lines showed a high concentration in the scaffold core 24 h post-seeding: 75% for UM-SCC6 and 76.5% for UPCI:SCC090. However, cell distribution changed after 5 days’ culture, with images showing higher cell concentrations at the edge of the scaffold area in both cell lines (54.5% for UM-SCC6 and 69.55% for UPCI:SCC090).The proliferation curves of UM-SCC6 HPV-negative cells and UPCI:SCC090 HPV-positive cells were analyzed by MTT assay at different time points. Monolayer cultures grew rapidly and constantly for up to 5 days post-seeding. Conversely, scaffold cultures did not show an exponential growth phase at the same time points, maintaining a constant proliferation over time in culture . UM-SCC6 displayed a mesenchymal-like phenotype with homogeneous distribution along the collagen fibers, with low cell-to-cell contact. Conversely, UPCI:SCC090 showed a dense clustered organization with a higher mean cellularity. Confocal images reflected the migration tendency of UM-SCC6 and showed Indian filing, an aligned distribution over the collagen fibers that reflects an aggressiveness phenotype29. Conversely, UPCI:SCC090 images confirmed the tendency to grow in aggregates within the porous area and to display an epithelial-like morphology.The cell organization in the 3D scaffold matrix of the 2 cell lines was analyzed by H&E staining and confocal imaging (1G). UM-SCC6 showed a higher CDH1 (E-cadherin) expression after 72 h culture in the 3D model (P = 0.01). No change was detected between the different UPCI:SCC090 cultures. The expression of 2 epithelial–mesenchymal transition (EMT)-related markers, VIM and SNAIL, was also analyzed. In UM-SCC6 cells, VIM and SNAIL mRNA levels increased constantly since 72 h in 3D culture. UPCI:SCC090 expressed similar VIM levels in both culture systems, but SNAIL expression significantly increased in the scaffold microenvironment.Gene expression analysis on 2D and 3D cultures revealed different gene expression modulations in the 2 cell lines (MMP2 and MMP9), and lysyl oxidase (LOX) expression to understand how OSCC cells interact with collagen fibers32. Over time in culture, the 3D microenvironment induced the overexpression of both MMPs in UM-SCC6  . No modulation in MMP expression was observed in UPCI:SCC090 . Conversely, LOX was the most upregulated marker in both cell lines cultured in 3D, with expression levels 11.15- and 7.38-fold higher than those of monolayer cultures of UM-SCC6 and UPCI:SCC090, respectively .We analyzed 2 ECM-degrading metalloenzymes, metalloproteinase 2 and 9 , confirming, as expected, the more aggressive phenotype of the former. Cells injected in the perivitelline space migrated inside the vessels and formed metastases. As previously stated, HPV-negative cells showed higher in vitro aggressiveness than the HPV-positive line. This result was further confirmed by the higher number of metastatic embryos at 24 hpi. Both 3D cultures showed a higher number of migrated cells inside the circulation than their monolayer culture counterparts (P = 0.03 for UM-SCC6 and P = 0.05 for UPCI:SCC090) .We used the zebrafish 19. 3D culture systems have proven capable of reproducing the low oxygenation of the tumor environment, closely mimicking clinical practice/a real-world setting34. We evaluated whether the oxygenation pattern changed inside the scaffold structure by analyzing the expression of the hypoxic marker HIF-1α . Over time, the cell lines cultured in monolayers showed little or no HIF-1α positivity. Conversely, 3D cultures showed high immunohistochemical expression of HIF-1α. In particular, HPV-negative cells exhibited increased HIF-1α positivity , whereas HPV-positive 3D cultures showed decreased expression of the marker during the time in culture  (3C).Hypoxia is one of the consequences of the development of tumors, including OSCCs35. We determined the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an enzyme involved in glycolysis processes to evaluate glycolysis stimulation in the hypoxic core. Results revealed an increment in GAPDH mRNA in UPCI:SCC090 grown on scaffolds , whereas UM-SCC6 maintained the same expression levels in 2D and 3D cultures . We also measured the immunohistochemical expression of the glucose transporter (GLUT-1) to confirm glycolysis stimulation. Images displayed a higher membrane expression of GLUT-1 in both cell lines seeded in the 3D scaffold than in 2D monolayer cultures .In the event of oxygen deficiency, it is known that cancer cells take advantage of glycolysis as a source of energyP = 0.03; 5-FU, P = 0.0002; CETU, P = 0.0002; GEM, P = 0.0001) . A similar behavior was observed for UPCI:SCC090 , with the exception of a higher response to CETU in 2D cultures than in 3D models .Hypothesizing that 3D collagen-based scaffolds can successfully predict drug-induced cytotoxic effects, we treated 2D and 3D cultures with drugs used in clinical practice for OSCCs CIS, 5-FU, CETU, and GEM. The plasma peak concentration was used for all drugs. UM-SCC6 displayed a low response to each treatment when seeded on scaffolds . Findings showed higher expression levels of NT5E and FOXM1 in 3D HPV-positive cells  and lower levels in the HPV-negative cell line. The expression levels of FLT1 in UM-SCC6 3D cells were 1.63-fold higher than that in 2D cultures. Conversely, there was no overexpression of FLT1 in UPCI:SCC090 in scaffold cultures. qPCR analysis revealed increased ABCA3 expression in HPV-negative 3D cultures (4.3-fold higher than in monolayer cultures at 72 h), not observed in HPV-positive cells.We analyzed the expression of several genes involved in drug-resistance processes, including Table 1. mRNA extracted from healthy tissue (HN2 H and HN14 H) and cancer tissue (HN2 K and HN14 K) was used to detect genes involved in tumor pathological processes (5B). VIM, CDH1, CXCR4, TP53, TGFβ, MMP2, and MMP9 were valuated. HN2 and HN14 tumor samples expressed higher levels of VIM , CXCR4 , TP53 , and MMP9  than healthy tissue. CDH1 and TGFβ were downregulated in HN2 K and upregulated in HN14 K, whereas MMP2 displayed an inverse behavior.Patient-derived primary cultures were established from fresh surgery specimens obtained from 2 patients with SCCs. Patient characteristics are summarized in 36. H&E staining highlighted different morphology features in HN2 and HN14 primary cultures (5D). HN2 showed approximately 20% of cancer cells isolated or in small clusters characterized by prominent nuclei, clear central nucleolus and large eosinophilic cytoplasm. Conversely, HN14 cultures colonized the scaffold area with approximately 30% of malignant cells, isolated or aggregated in small clusters. The cells had a spindle morphology and eosinophilic cytoplasm, whereas the nuclei showed dispersed chromatin and a single, central, and clear nucleolus.Primary cultures preserved tumor stroma components, a key factor in maintaining the tumor microenvironment and the crosstalk between healthy and malignant cell populations5F). Drug resistance was induced in 3D primary cultures respect 2D counterparts  but was less pronounced than in UM-SCC6 and UPCI:SCC090 cell lines. 5-FU was the only treatment more effective in cells in 3D HN14 cultures than in 2D HN14 cultures.Cells seeded on scaffolds and on monolayer supports were treated with the same drugs used for cell lines  partially condition cell behavior and the reproducibility of the structure. Our aim was to understand whether these 3D cultures can successfully reproduce the different features of HPV-positive and HPV-negative OSCC cells.In the last few decades, new 33. Conversely, HPV-positive cells showed an epithelial-like phenotype, growing in clusters that occupied much of the pore surface. We previously studied the alterations induced by 3D scaffolds in 2 breast cancer cell lines with different levels of aggressiveness19. The cells associated with high invasiveness displayed the same Indian file organization as that of HPV-negative cells, whereas less aggressive breast cancer cells and HPV-positive cell lines had similar epithelial-like morphologies and cell distribution. Overall, these results suggest that collagen-based scaffolds are capable of reproducing morphology features driven by cell aggressiveness that cannot be recreated by monolayer supports.We demonstrated that the 3D structure affected cell growth dynamics and that the analyzed cell lines showed a different collagen fiber organization. Monolayer cultures displayed the typical proliferation rate characterized by an exponential growing phase, whereas 3D cultures showed different proliferation dynamics, resulting in a proliferation rate that remained virtually unaltered over time. Confocal imaging revealed a difference in the proliferation behavior between the core and the edge of the scaffold, e.g., 5 days after seeding, there was a higher proportion of cells from both cell lines in the edge than in the core area. The edge of the collagen matrix has closer contact with media nutrients and oxygen than the inner parts, and this microenvironment characteristic may partly explain the different proliferation rates between the 2 scaffold districts. Moreover, the cell distribution along the collagen fibers differed substantially in the 2 cell lines. HPV-negative cells showed a spindle-shaped mesenchymal phenotype and an aligned fiber distribution. These features are known as Indian filing, a morphology pattern often associated with aggressive invasive lobular breast carcinomaSNAIL and VIM, 2 genes associated with mesenchymal phenotype46. During the culture period, SNAIL expression increased in both cell lines, whereas VIM expression was only upregulated in UM-SCC6. The 2 cell lines interacted differently with the 3D microenvironment, activating EMT pathways when seeded on scaffolds. These data provide evidence of the ability of our 3D model to induce a more mesenchymal phenotype than 2D cultures.To better characterize our 3D model, we studied the influence of scaffolds on different pathways and processes involved in cancer progression. First, we investigated whether EMT processes play a role in the different behavior of the 2 cell lines. We analyzed the expression of 47. This is not normally possible with 2D in vitro models. We used our 3D device to mimic the hierarchically organized ECM structure. Collagen, which is present in all human tissues, stimulates intracellular signaling through the activation of cell receptors and provides physical support for cell migration and proliferation48. We thus evaluated the expression of genes involved in the remodeling of the ECM: MMP2, MMP9, and LOX. MMPs are a group of endopeptidases responsible for modifying extracellular components49. Their interaction with scaffold collagen fibers induced MMP overexpression in the HPV-negative OSCC cell line but not in HPV-positive UPCI:SCC090. Conversely, the expression of LOX, an enzyme involved in ECM remodeling through the crosslinking of collagen and elastin, increased over time in the 3D cultures of both cell lines50. The interaction of OSCC cells with collagen fibers allowed this crosslinking activity. ECM is the product of several proteins that differ from tissue to tissue and from tumor to tumor52. Type I collagen represents a key structural component of ECM, influencing tumor invasion and progression53. It is composed of a 2-chain heterotrimer of collagen type I α1 and collagen type I α2, encoded by COL1A1 and COL1A2, respectively54. We analyzed the TCGA and GTEx project databases using the Human Protein Atlas (https://www.proteinatlas.org/about) and GEPIA255  tools, identifying cancers expressing high levels of COL1A1 and COL1A2. Head and neck cancer, breast cancer, and sarcomas are all malignancies with increased type I collagen expression. Our model could thus represent a suitable tool to study the role of the ECM in tumors with a high type I collagen content, including head and neck cancers. It could also facilitate the in vitro evaluation of cancer cell–ECM interactions in OSCC pathophysiology, which standard in vitro models cannot reproduce. However, its reliability in other cancer types warrants further investigation.Then, our 3D model enabled us to study the interaction between cancer cells and the ECM, which affects several cancer processes56. GAPDH upregulation was observed in HPV-positive cells, and a high GLUT-1 expression was detected in both cell lines with respect to 2D monolayer cultures. These findings suggest that the hypoxic microenvironment could, in part, be responsible for phenotype alterations and drug resistance.We looked at the potential involvement of hypoxia signaling in our 3D model as the development of a hypoxic microenvironment is one of the causes of altered growth dynamics and phenotypes. HIF-1α expression was observed in 3D in the nuclei along the whole scaffold surface. The hypoxia phenotype was associated with a change in cancer cell metabolism, with a switch toward glycolysis as a means of adapting to low oxygen levelsin vivo systems for several applications18. The transgenic strain Tg(fli1:EGFP) has green fluorescent blood vessels and represents a suitable model for the study of cancer migration processes. As far as we know, this is the first time that OSCC cells grown on biomimetic scaffolds have been injected into zebrafish. At 24 hpi, a higher number of embryos showed diffuse micrometastases when injected with scaffold cells with respect to 2D culture cells. In agreement with our in vitro results, the scaffolds increased the migration capability of OSCC cells.We used the zebrafish embryo model to confirm the aggressiveness of the phenotype acquired by OSCC cell lines grown in 3D scaffolds. In recent years, the number of available transgenic zebrafish strains has increased, offering new 57 previously showed that the 3D structure of a polyester-based scaffold reduced the radiosensitivity of OSCC cell lines. Conversely, we investigated the sensitivity of our different biomimetic scaffold cultures to standard chemotherapeutic agents used to treat OSCC. Significant drug resistance to all treatments was observed in the 3D model compared with monolayer cultures, with the exception of CIS and CETU in UPCI:SCC090 cells. The cytotoxic effect of higher concentrations of CIS and CETU requires further investigation to elucidate the scaffold implication in the drug responses in these 2 conditions. HPV status is a strong and independent prognostic factor6. Patients with HPV-positive OSCC have a good prognosis, and their risk of death is half that of HPV-negative individuals58. Comparing the response to single treatments in monolayer cultures, the HPV-negative cell line exhibited higher sensitivity to 5-FU, CETU (P = 3.2 × 10−5), and GEM than the HPV-positive line UPCI:SCC090 . Conversely, HPV-positive cells in 3D scaffolds were more sensitive to drugs than HPV-negative cultures: CIS (P = 0.004), 5-FU, CETU (P = 0.002), and GEM (P = 0.006). Overall, these results suggest that the biomimetic scaffold is capable of reproducing the response to drug regimens seen in real-life HPV-positive and HPV-negative patients. However, further investigation is needed to confirm this potentially important finding.Zhang et al.NT5E, FOXM1, FLT1, and ABCA3, are modulated in 3D compared with standard monolayer cultures64. NT5E and FOXM1 genes encode for glycosylphosphatidylinositol-anchored cell surface protein and oncogenic transcription factor Forkhead box protein M1, respectively. FLT1 encodes for vascular endothelial growth factor receptor 1, a gene associated with anti-EGFR drug resistance50. ABCA3 is a member of the ATP-binding cassette family, which includes transporters driving chemotherapeutic agents outside the cell64. In our study, mRNA relative quantification highlighted a different expression pattern in the 2 culture systems. Both cell lines grown in 3D showed increased NT5E expression, albeit with different modulations, whereas only UPCI:SCC090 overexpressed FOXM1. Conversely, UM-SCC6 showed an upregulation of FLT1 and ABCA3 when seeded in scaffolds, but this was not observed in UPCI:SCC090. These findings suggest that collagen-based scaffolds are capable of activating drug-resistance mechanisms but via different pathways and depending on the cell type. For example, FLT1, which is associated with anti-EGFR drug resistance, was upregulated in HPV-negative UM-SCC6 and downregulated in HPV-positive UPCI:SCC090 3D cultures. CETU, an EGFR inhibitor, induced a different effect on UM-SCC6 2D and 3D systems, but not on the 2 UPCI:SCC090 culture types. Thus, CETU effects on our scaffold cultures match with the expression of FLT1.We assessed whether some of the mechanisms known to be involved in drug resistance, e.g., in vitro environment contributes to altering the original nature of the cell population. Ex vivo samples have the advantage of preserving many of the original features of the cancer lesion that are essential for the reproduction of the tumor microenvironment18. We used, for the first time, primary cultures and collagen-based scaffold devices to investigate the impact of chemotherapy treatments in head and neck cancers. The impact of drug treatments on 2D and 3D primary cultures was similar to that on cell lines. Primary cultures conserve part of the stromal populations and different subclones of patient tumor tissue. The crosstalk between healthy and malignant components plays a role in the induction of drug resistance18. In this scenario, the modest differences between primary cultures and cell lines might be ascribable to the presence of stromal cells. However, further analysis is needed to better understand the behavior of primary cultures in collagen-based scaffolds.We used patient-derived primary cultures to confirm the ability of collagen-based scaffolds to induce drug resistance. Immortalized cell lines are cells that have been manipulated to proliferate indefinitely, and a prolonged length of time in an in vitro and in vivo migration ability. Some of these features were also induced in HPV-positive UPCI:SCC090 cells, but with substantial differences. HPV-positive cells grew in clusters that filled up scaffold pores, acquired an epithelial-like phenotype, expressed low levels of MMPs, and showed less accentuated migration behavior than HPV-negative cells. Conversely, both cell lines on 3D scaffolds were less sensitive to treatments but activated the expression of different drug-resistance markers. In conclusion, the induction of migration properties and of EMT marker expression are 2 biological characteristics associated with cancer aggressiveness. Our results thus suggest that 3D scaffolds are capable of reproducing the different nature of HPV-related malignancies.The above findings indicate the substantial influence exerted by the 3D structure on the phenotype and genotype of cancer cells. The alterations seem to be different for each cancer cell type but might be oriented by the HPV status of the tumor. HPV-negative UM-SCC6 cells in 3D cultures acquired a spindle-shaped morphology and showed an aligned distribution of fibers, overexpresion of EMT-related genes, and enhanced in vitro systems.Our biomimetic scaffold proved to be a simple and cost-effective model to study the interactions between ECM and cancer cells. It is also the first study using zebrafish models to investigate the behavior of HPV-positive and HPV-negative OSCC cancer cells and to compare the aggressiveness induced by 3D collagen-based scaffold. The characterization of HPV-positive and HPV-negative cell lines in our 3D cultures underlined the different pathophysiological nature and drug sensitivity of these 2 tumor types. Overall, our results confirm the ability of 3D scaffolds to reproduce the microenvironment features of OSCC that are difficult to study with other Click here for additional data file.


AGA) gene, we show that these cell lines can be used as a model system for establishing potency assays for AAV9-based gene therapy approaches for human diseases.Recombinant adeno-associated viruses (AAV) have emerged as an important tool for gene therapy for human diseases. A prerequisite for clinical approval is an in vitro potency assay that can measure the transduction efficiency of each virus lot produced. The AAV serotypes are typical for gene therapy bind to different cell surface structures. The binding of AAV9 on the surface is mediated by terminal galactose residues present in the asparagine-linked carbohydrates in glycoproteins. However, such terminal galactose residues are rare in cultured cells. They are masked by sialic acid residues, which is an obstacle for the infection of many cell lines with AAV9 and the respective potency assays. The sialic acid residues can be removed by enzymatic digestion or chemical treatment. Still, such treatments are not practical for AAV9 potency assays since they may be difficult to standardize. In this study, we generated human cell lines (HEK293T and HeLa) that become permissive for AAV9 transduction after a knockout of the CMP–sialic acid transporter SLC35A1. Using the human aspartylglucosaminidase ( In the last years, an increased interest in generating recombinant adeno-associated virus (AAV)-based gene therapies for various human diseases has emerged. AAVs are DNA viruses of the Parvoviridae family, and they are non-pathogenic to humans. The AAV serotypes show different tissue tropisms due to using different cell surface structures as receptors. Thus, depending on the target tissue of the gene therapy approach, a suitable AAV serotype is selected that is capable of infecting the target organs. AAVs typically interact with a cell surface glycans, along with a co-receptor, to mediate cell surface binding and entry ,2. Cell AAV9 is one of the most favored vectors for gene therapy approaches, in particular those targeting the brain, heart, and muscle . This isExtensive quality control of the virus lots intended for clinical trials is required before the vector can be used in humans. Thus, establishing an in vitro potency assay that can be carried out in cell culture and providing quantitative data on the transduction efficiency by each virus lot is a prerequisite for approval of AAV-based gene therapy products. The low in vitro transduction efficiency of AAV9 has largely prevented using common human cell lines that proliferate rapidly, such as HEK293T and HeLa cells. Although strategies for enhancing AAV9 transduction by removing the surface sialic acid have been suggested, e.g., by enzymatic removal of sialic acid or treatment with various chemicals (see discussion), such approaches are frequently inconvenient for practical work and difficult to standardize ,17.AGA gene and the resulting lack of activity of the aspartylglucosaminidase (AGA) enzyme  assay. For the assessment, the cells were seeded onto 96-well plates at a density of 2.5 × 103 cells per well. On the day of the measurement, MTT  was added to a final concentration of 0.5 mg/mL. After 40 min incubation, the medium was removed. The cells were lysed in 100 µL lysis buffer (containing 5% formic acid and 95% isopropanol) per well. Then, absorbance recorded using a microplate reader at a wavelength of 550 nm (with a reference wavelength of 690 nm). Six replicas for each time point were included in each experiment, and 5 independent experiments were performed. The average absorbance measured for the parental cells at 24 h was set as 1. The corresponding normalized values (relative proliferation) for the other data points were calculated.The proliferation of the parental HEK293T and Hela and the respective p < 0.05 were considered significant (*), while values of p < 0.01 were considered very significant (**) and p < 0.001 extremely significant (***).All experiments were performed at least three times. Data are expressed as mean ± SD. Statistical comparisons between groups were made using Student’s t-tests, 1way or 2way ANOVA, as appropriate . Values of SLC35A1 gene by CRISPR/Cas9 technology in HEK293T cells. We produced single-cell clones containing a genomic insertion of 1 bp , as would be expected after loss of terminal sialic acid residues (Sambucus nigra lectin (SNA) that binds to sialic acid exhibited lower staining in SLC35A1 knockout cells than in the parental HEK293T cells . The resulting GFP fluorescence was measured using a plate reader and normalized to protein amount in the lysates (SLC35A1 knockout cells showed a significantly higher GFP fluorescence (average 10.5 times over the MOI range used) than the parental cells. Expression of the SLC35A1 gene product CMP–sialic acid transporter by transient transfection of an expression plasmid decreased the AAV9/GFP transduction efficiency  that carries the optimized human AGA gene coding for aspartylglucosaminidase (AGA) that is deficient in aspartylglucosaminuria (AGU). We have recently shown in an AGU mouse model that this vector shows a high potential for clinical translation [AGA and SLC35A1 in HEK293T cells by knocking out the SLC35A1 gene in our previously characterized AGA HEK293T knockout cells [SLC35A1 gene (AGA/SLC35A1 double knockout clones (clone 1 and 34) showed a significantly improved, dose-dependent transduction efficiency by AAV9/GFP than HEK293T cells  or vector DNA (LMNB2 gene). Direct comparison of the rescence A and therescence  increaseAGA/SLC35A1 double knockout cells with increasing MOI (2.5 × 104 to 5 × 105) of the AAV9/AGA virus resulted in a significantly increased AGA enzyme activity in a nonlinear and saturable fashion  and mRNA level (GAPDH mRNA). Compared to the glycolytic enzyme GAPDH, the mRNA level of the AGA transgene is very low  and endogenous AGA (about 2) as a reference. This may be because HEK293T cells are partially polyploid, and the copy number of the reference chromosomes may be different. Taken together, these data show that knockout of SLC35A1 in HEK293T cells results in significantly improved transduction and transgene expression mediated by AAV9-based vectors.Successful transduction was also shown by demonstrating an increased level of the codon-optimized very low B. HoweveAGA gene C. There SLC35A1 gene knockout results in improved transduction efficiencies also in further human cell lines, we produced knockout HeLa cell clones that showed a 1 bp genomic insertion in the SLC35A1 gene  Lec2 mutant cell line that is unable to transport CMP–sialic acid to Golgi lumen ,30, enzyAs compared to the parental CHO Pro5 cells, Lec2 cells can efficiently be infected with AAV9 . HoweverSLC35A1 gene knockout provides means for increasing the infection efficiency by AAV9 in cell lines that divide rapidly. This is due to the lack of sialic acid residues in the glycan determinants at the cell surface, providing improved access of AAV9 to its main receptors, terminal galactose residues [AGA knockout cell lines originally published in 2016 that have since been used for further studies [The human cell lines developed in the present study are based on a genetic knockout of the CMP–sialic acid transporter and thus exhibit the same defect in sialylation as the Lec2 cells. Functional ablation of the CMP–sialic acid transporter by residues . Our sys studies . The douSLC35A1 knockout cell lines that we have generated in this study do not require any further modifications or treatments before transduction by AAV9, making assays that are based on our cell lines easier to standardize and validate.Enzymatic removal of sialic acid by neuraminidase is a method that does not require any genetic modification of the target cells and thus facilitates using various cell lines for AAV transduction ,10,11. IRecently, Krotova and Aslanidi studied several AAV serotypes that carried the firefly luciferase (Luc) transgene . The autIn addition to potency assays, the cell lines generated in the present study may also be useful for in vitro assays that assess the presence of neutralizing antibodies against AAV in patients that are to be treated with gene therapy. Since a very large fraction of humans, estimated to be about 90% of adults, has had contact with naturally occurring, non-pathogenic AAV variants early in life, there is a risk of the prior existence of antibodies against AAV in patients to be treated . Not allSLC35A1 gene substantially increases the transduction efficiency of AAV9 in two human cell lines that are easy to culture. The SLC35A1 gene knockout did not result in a decreased viability of these cell lines, despite the sialylation defect, and additional genes can be knocked out in these cell lines without affecting viability. Therefore, by knocking out a gene of interest, e.g., the target gene for a gene therapy approach, it is possible to generate cell lines that do not express the endogenous protein and only express the transgene delivered by the AAV9 vector. This facilitates establishing potency assays without an endogenous background activity. Likely, the AAV9 tropism towards cultured human cell lines can generally be improved using SLC35A1 knockout, facilitating the generation of further cell lines beyond those tested in the present study that can be used for studies with AAV9.In this study, we have shown that knockout of the CMP–sialic acid transporter SLC35A1 knockout in cultured cells.Although our system may not be useful for viruses other than AAV9 that do not use the same receptor, it will certainly be of interest for studies using variants derived from AAV9, such as PHP.B and PHP.eB ,38. Alth


N-methyltransferase (NNMT) plays multiple roles in improving the aggressiveness of colorectal cancer (CRC) and enhancing resistance to 5-Fluorouracil (5-FU), making it an attractive therapeutic target. Curcumin (Cur) is a promising natural compound, exhibiting multiple antitumor effects and potentiating the effect of 5-FU. The aim of the present study is to explore the effect of Cur on attenuating NNMT-induced resistance to 5-FU in CRC. A panel of CRC cell lines with different NNMT expressions are used to characterize the effect of Cur. Herein, it is observed that Cur can depress the expression of NNMT and p-STAT3 in CRC cells. Furthermore, Cur can induce inhibition of cell proliferation, G2/M phase cell cycle arrest, and reactive oxygen species (ROS) generation, especially in high-NNMT-expression CRC cell lines. Cur can also re-sensitize high-NNMT-expression CRC cells to 5-FU both in vitro and in vivo. In summary, it is proposed that Cur can reverse NNMT-induced cell proliferation and 5-FU resistance through ROS generation and cell cycle arrest. Given that Cur has long been used, we suppose that Cur is a promising anticancer drug candidate with minimal side effects for human CRC therapy and can attenuate NNMT-induced resistance to 5-FU.Nicotinamide  Statistically, colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide . ChemothN-methyltransferase  was originally identified as an enzyme that catalyzes nicotinamide to 1-methylnicotinamide (1-MNA)  × 100%.Cell viability assay was detected using a Cell-Counting Kit-8  following the manufacturer’s instructions, Briefly, 5 × 10v/v) and horseradish peroxidase-conjugated secondary antibodies (1:5000 v/v). Signals were visualized and captured by Image Lab .Cells were lysed in RIPA lysis buffer  with protease inhibitors to extract total protein. Then, a 40 μg protein sample was separated by SDS-PAGE and transferred to a PVDF membrane , followed by immunoblotting with primary antibodies  and Annexin V-FITC/PI Apoptosis Detection kit  were used to detect apoptosis according to the manufacturer’s instructions, and signals were analyzed by flow cytometry , as previously described .Cell cycle was analyzed using a cell cycle staining kit  Biotech Co., Ltd., Hangzhou, China) according to the manufacturer’s instructions. Briefly, cells were harvested and washed with cold PBS twice, stained with binding buffer containing 50 μg/mL propidium iodide in the dark at room temperature for 30 min, and then immediately analyzed by flow cytometry .ROS was detected using an ROS assay kit  according to the manufacturer’s instructions and analyzed by flow cytometry , as previously described .2)/2.Six-week-old male BALB/c nude mice were purchased from Model Animal Research Center of Nanjing University and housed under pathogen-free conditions  with free access to food and water. All animals were acclimated for at least 5–7 days before treatment. The xenograft of CRC was constructed using SW480/NC and SW480/NNMT cell lines as previously described [Tumor tissues were fixed, embedded, and sliced according to a standard protocol. Apoptotic cells in tumor tissue sections was analyzed by TUNEL assay using an in situ Apoptosis Detection kit  following the manufacturer’s instructions, as previously described [t-test was used for two-group comparisons. The ANOVA and Bonferroni method were used for multiple comparisons among more than two groups. p value < 0.05 was considered statistically significant difference.GraphPad Prism v7.0 software was used for statistical analysis. All data are presented as the mean ± SD from three independent experiments. The two-sample 50 of Cur in the four cell lines is IC50 = 32.90 μM for HT-29/shNNMT, IC50 = 29.41 μM for HT-29/NC, IC50 = 13.69 μM for SW480/NNMT, and IC50 = 14.99 μM for SW480/NC  to co-treat with 5-FU, and the IC50 of 5-FU was calculated using the CCK-8 assay. 50 of 5-FU was dramatically reduced when combined with Cur: from 131.81 to 50.98 mg/L in HT-29/NC (fold decrease of approximately 2.59), from 54.79 to 29.95 mg/L in HT-29/shNNMT (fold decrease of approximately 1.83)  B,C, fromly 1.83) E,F. In s50 of HT-29/NC or HT-29/shNNMT, and 15 μM or 20 μM for SW480 cell lines, which was approximately IC50 of SW480/NNMT or SW480/NC. Following the treatment of cells with Cur, Western blot showed that NNMT was downregulated in HT-29 cell lines , to 22.79% (35 μM Cur), and to 24.95% (40 μM Cur) in HT-29/NC, and from 9.31 to 13.5% (30 μM Cur), to 17.97% (35 μM Cur), and to 21.53% (40 μM Cur) in HT-29/shNNMT A,B. DramThe expression of G2/M phase-related proteins was detected after cells were treated with Cur. Western blot shows that the cell cycle inhibitory protein p21 was upregulated, while the cyclin-dependent kinases CDK1 and CDK2 were downregulated in a Cur-dependent manner in HT-29/NC and HT-29/shNNMT cell lines A,B,D,F. 50 of 5-FU, combined with Cur (30 μM in HT-29 cells and 10μM in SW480 cells). Flow cytometry results show that 5-FU can dramatically induce G1/S phase arrest. The proportion of cells at the G1/S phase increased from 92.81 to 98.58% in HT-29/NC, from 93.83 to 98.23% in HT-29/shNNMT . NNMT is an enzyme of nicotinamide metabolism, and we have previously reported that NNMT could enhance resistance to 5-FU in CRC . TherefoGiven that NNMT is an enzyme that promotes cell proliferation and 5-FU resistance, inhibition of NNMT may be one of the ways to reduce 5-FU resistance. We have recently screened a natural products library and found that vanillin can decrease NNMT expression and attenuate NNMT-related resistance to 5-FU in CRC cells . CurcumiCur has been reported to exert multiple anticancer effects ,12,13,14In terms of the detection of cell cycles, it is found that Cur can induce more G2/M phase arrest in high-NNMT-expression cell lines than that in low ones, which is consistent with the CCK-8 assay results. Therefore, we deem that Cur reverse the NNMT-induced cell proliferation mainly through inducing cell cycle arrest rather than inducing cell apoptosis. Another important consideration is that subG1 changes is a marker of apoptosis and long exposures (at least 48 h) to some apoptosis-inducible drugs may lead to subG1 accumulation ,27,28. HGiven that NNMT has previously noted to promote the cell cycle via the active G1 phase in CRC cells , and higMoreover, it was found that Cur has a synergistic effect with 5-FU on inducing cell cycle arrest in CRC cells. 5-FU is a pyrimidine analogue and can cause cell cycle arrest at the G1/S phase by inhibiting thymidylate synthetase . In the ROS is one of the causes of cell cycle arrest, and previous studies have reported that Cur could induce ROS generation in CRC cells ,42. In tWe constructed a murine xenograft model using SW480/NC and SW480/NNMT cells to assess the synergistic effect of Cur on 5-FU in vivo. It was observed that Cur could enhance the inhibition of 5-FU in tumor proliferation in both SW480/NC and SW480/NNMT groups. Consistent with a previous study , tumors In the present study, it is demonstrated that Cur can reverse NNMT-induced resistance to 5-FU in vivo and in vitro, and the mechanism that involved in ROS generation and cell cycle arrest induced by Cur is partially demonstrated. Considering the fact that Cur has long been used, in this study, we propose Cur as a promising anticancer compound with minimal side effects for CRC adjuvant chemotherapy, particularly for CRC with high NNMT expression.


Type 2 diabetes is characterised by islet amyloid and toxic oligomers of islet amyloid polypeptide (IAPP). We posed the questions, (1) does IAPP toxicity induce an islet response comparable to that in humans with type 2 diabetes, and if so, (2) what are the key transcriptional drivers of this response?The islet transcriptome was evaluated in five groups of mice: beta cell specific transgenic for (1) human IAPP, (2) rodent IAPP, (3) human calpastatin, (4) human calpastatin and human IAPP, and (5) wild-type mice. RNA sequencing data was analysed by differential expression analysis and gene co-expression network analysis to establish the islet response to adaptation to an increased beta cell workload of soluble rodent IAPP, the islet response to increased expression of oligomeric human IAPP, and the extent to which the latter was rescued by suppression of calpain hyperactivation by calpastatin. Rank-rank hypergeometric overlap analysis was used to compare the transcriptome of islets from human or rodent IAPP transgenic mice vs humans with prediabetes or type 2 diabetes.The islet transcriptomes in humans with prediabetes and type 2 diabetes are remarkably similar. Beta cell overexpression of soluble rodent or oligomer-prone human IAPP induced changes in islet transcriptome present in prediabetes and type 2 diabetes, including decreased expression of genes that confer beta cell identity. Increased expression of human IAPP, but not rodent IAPP, induced islet inflammation present in prediabetes and type 2 diabetes in humans. Key mediators of the injury responses in islets transgenic for human IAPP or those from individuals with type 2 diabetes include STAT3, NF-κB, ESR1 and CTNNB1 by transcription factor analysis and COL3A1, NID1 and ZNF800 by gene regulatory network analysis.Beta cell injury mediated by IAPP is a plausible mechanism to contribute to islet inflammation and dedifferentiation in type 2 diabetes. Inhibition of IAPP toxicity is a potential therapeutic target in type 2 diabetes.The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-021-05569-2. The islet in type 2 diabetes is characterised by islet amyloid derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by beta cells that when misfolded and in aggregate form may contribute to beta cell failure –4. HumanGiven the known proximal role of misfolded protein stress in neurodegenerative diseases, and the connection of the risk factors for type 2 diabetes to misfolded protein stress, we hypothesised that hIAPP misfolded protein stress may be a proximal cause of the wide-ranging changes in islets in individuals with type 2 diabetes. Risk factors for type 2 diabetes include insulin resistance  and low 1c level of 42 to 47 mmol/mol [6% to 6.5%]) or type 2 diabetes to establish if the changes in the islet in type 2 diabetes are potentially attributable in part to hIAPP protein misfolding stress.In the present study we evaluated the islet transcriptome from a mouse model of beta cell hIAPP toxicity  before dIAPP (hIAPP) [Iapp (rIAPP), human calpastatin (hCAST), and both human IAPP and CAST (hIAPP:hCAST) on FVB background has been described elsewhere [Animal studies were approved by the University of California, Los Angeles (UCLA) Office of Animal Research Oversight. The transgenic mice homozygous for human  (hIAPP)  were orilsewhere , 16. Miclsewhere . Mice welsewhere . See ESMRNA samples from three mice per group were used for RNA-seq ; therefore 76 samples were included in this manuscript: 51 from normoglycaemic donors with HbA1c levels below 42 mmol/mol (<6%) , 15 prediabetic donors with HbA1c levels 42 to 47 mmol/mol (6% to 6.5%) , and ten donors with type 2 diabetes with HbA1c levels 48 mmol/mol (>6.5%) or higher . No additional information about the donors or islet morphology was available.RNA-seq data from human pancreatic islets were downloaded from the Gene Expression Omnibus (GEO) (GSE50244) . Data frThe RNA-seq data from mouse and human islets were subjected to differential expression analysis  to find To identify genes with coordinated expression in the form of co-expression modules that are related to diabetes development in mouse models, we utilised weighted gene co-expression network analysis  to two types of analysis. First, we carried out cell type marker enrichment analysis ESM  using Paχ2 like statistic  network analysis using the Enrichr tool  with thestic ESM .Statistical analysis was performed as described in the figure legends and There is striking concordance of the islet transcriptome between individuals with prediabetes and those with type 2 diabetes Fig. . Since ~Since by design neither the hIAPP or rIAPP mice had diabetes when islets were sampled, we further compared the changes in islet transcriptome in the mouse islets with those in human islets with prediabetes. There was again close concordance in transcriptome in both rIAPP and hIAPP islets with those from individuals with prediabetes Fig. . These fTo investigate beta cell adaptation to an increased workload of non-amyloidogenic rIAPP expression, we compared the transcriptome of islets from rIAPP vs WT mice. Gene expression was increased in WGCNA modules M8 , M9 , M13 (cell migration) and decreased in M7 (cell cycle) in rIAPP islets Fig. . Of the Since there is a considerable overlap in the changes in transcriptome in hIAPP and rIAPP mice Fig. , and hIABHLHA15 (MIST1), a potent endoplasmic reticulum (ER) stress-inducible transcriptional regulator upregulated by beta cell Ca2+ overload [Consistent with the comparable increase in IAPP expression , islets overload .Cd4, Cd68, Cd84), immune sensors , and macrophage genes  were upregulated in hIAPP islets and in type 2 diabetes. The top hub gene in the M1 module that is most highly activated in hIAPP islets is Cx3cr1 [The pronounced signal for inflammation in hIAPP, but not rIAPP islets is also present in human prediabetes and type 2 diabetes Fig. . Consists Cx3cr1 . This prCdk1, Cep55) or indirectly . In contrast, these genes are downregulated in islets from prediabetes and type 2 diabetes, which is perhaps consistent with epigenetic silencing of cell cycle genes in beta cells in adult humans compared with 9-week-old mice [Activation of cell replication is a key component of injury repair programmes. Cell cycle related genes are increased in hIAPP islets but decreased in rIAPP islets Fig. , likely old mice .Genes important for maintaining beta cell differentiation were decreased in islets of both rIAPP and hIAPP mice compared with islets from WT mice Fig. , a patte2+ signalling that results from nonselective ion channel activity of toxic oligomers [CAST) (hIAPP:hCAST), which inhibits calpain, delays or prevents diabetes in hIAPP transgenic mice [Calpain hyperactivation has been widely reported as a mediator of amyloidogenic protein induced cytotoxicity, presumably as a consequence of aberrant Caligomers . Concurrnic mice .CAST overexpression in hIAPP mice were those that mediate UPR and inflammation , NID1  and CXCL12  are plausible regulatory genes underlying stellate and endothelial cell contribution to islet pathogenesis. Moreover, module M7, which is enriched for beta cell markers


Then, zero-valent iron (ZVI) nanoparticles were synthesized in nanoscale lignin, producing a nZVI/n-lignin composite, via the borohydride reduction method. The use of nZVI/n-lignin for environmental remediation was tested by the removal of methylene blue in aqueous solutions at room temperature. The nZVI/n-lignin composite achieved a higher methylene blue removal ratio than that achieved by traditional nZVIs. Moreover, its excellent dispersibility in water and stability against oxidation in the air were observed. The functions of the nanoscale lignin in the composite material are (1) prevention of further growth and aggregation of the nZVI nanoparticles, (2) protection of nZVI from serious oxidation by H2O/O2, and (3) allowing better dispersibility of nZVI in aqueous solutions. These three functions are important for the field applications of nZVI/n-lignin, namely, to travel long distances before making contact with environmental pollutants. The present method for producing nZVI/n-lignin is straightforward, and the combination of nZVI and lignin is an efficient and environmentally friendly material for environmental applications.In the current study, lignin, an abundant natural polymer, was dissolved in ethylene glycol and acidic H Afterward, the iron ions trapped in the nanoscale lignin were reduced to nZVI by the borohydride method. By using the nanoscale lignin to stabilize the nZVI, the resulting composite, called nZVI/n-lignin, may possess a higher colloidal stability and better mobility than that of the bare nZVI and the EG-functionalized nZVI. Characterizations of nZVI/n-lignin were performed by transmission electron microscopy, X-ray photoelectron spectroscopy, and an X-ray diffractometer. The dispersibility in water and stability against oxidation of nZVI/n-lignin were compared with those of bare and EG-functionalized nZVIs. Finally, the effectiveness of nZVI/n-lignin for the removal of methylene blue in H2O was investigated, and the process was monitored via the characteristic UV–Vis absorption peaks of the cationic dye. This nZVI/n-lignin composite material could be employed for application in environmental remediation in the future.There is global awareness of limited resources, and so the development of sustainable materials for reuse and recycling is urgent . In thiso 70 wt% , EG was o 70 wt% ,27. Ther3·6H2O, ≧99%), Kraft lignin, ethylene glycol (EG), sodium borohydride , and ethanol were purchased from Sigma-Aldrich . Methylene blue solution (0.1%) was supplied by Choneye Pure Chemicals .Iron (III) chloride hexahydrate , X-ray diffractometer , and X-ray photoelectron spectroscopy . The XPS depth profile analyses were also conducted by argon ion etching using an energy level of 3000 eV for 1 min.The experimental procedure is shown in 2O was observed. Moreover, their stability against oxidation in air and water was verified. After the synthesis of materials, the three dried materials were stored in atmospheric conditions for 7 days and placed in water for 7 days. Then, the powders were measured by XRD to check the characteristic peaks of zero-valent iron and iron oxides.To verify the applicability of nZVI/n-lignin, the synthesized bare nZVI, EG-functionalized nZVI and nZVI/n-lignin were tested in the aqueous solutions for 7 days. The dispersibility of the two nZVIs and nZVI/n-lignin in H2O to give a total volume of 50 mL, with a concentration of 3 ppm. Forty milligrams each of the bare nZVI, EG-functionalized nZVI, and nZVI/n-lignin powders were added to the solution to start the degradation and adsorption of methylene blue dye. The absorption of the filtered solution after a certain duration was recorded by an UV–Vis spectrometer  to monitor the degradation and adsorption process of methylene blue. The absorption intensity of the methylene blue solution at the wavelength of 664 nm decreased as the reaction time increased.For the removal ratio of methylene blue, 0.5 mL methylene blue solution was added to DI-H2O containing FeCl3 (pH ~ 2), nanoscale lignin was formed, suspended in the H2O. Once NaBH4 was added, tiny black particles (nZVI/n-lignin) were formed instantly. After the synthesis for 30 min, the solution with nZVI/n-lignin was still turbid, as shown in 2O . The photoelectron peaks for Fe 2p of nZVI/n-lignin are less intensive than those for C 1s and O 1s, as shown in To investigate the composition and chemical states of nZVI/n-lignin, XPS measurements of nZVI/n-lignin are shown in 0 due to the formation of the composite with three-dimensional-branched lignin in the EG solution.To identify the presence of nZVI in the lignin, XRD measurements of the bare nZVI, EG-functionalized nZVI, and nZVI/n-lignin were also conducted. A strong peak of lignin was observed at the 2θ scan of 29.4° (not shown). As 2O. However, the solution with nZVI/n-lignin maintained its black color, which indicates that nZVI/n-lignin was still well dispersed and suspended in the H2O. The results for the solutions after standing for one day are shown in 2O/O2 to form iron oxide/hydroxide according to the following equations [0 + 2H2O → Fe2+ + H2 + 2OH−; (2) 2Fe0 + O2 + 2H2O → 2Fe2+ + 4OH−; (3) 6Fe2+ + O2 + 6H2O → 2Fe3O4(s) + 12H+; (4) Fe2+ + 2OH− → Fe(OH)2(s); (5) 6Fe(OH)2(s) + O2 → 2Fe3O4 + 6H2O. According to our observations, obvious precipitation of the solution with nZVI/n-lignin only happened after it had been standing for several days. The result shows that nZVI/n-lignin possesses better dispersibility in H2O than that of the bare nZVI and EG-functionalized nZVI, probably because ion nanoparticles imbed in nano-sized lignin to prevent the formation of a chain-like pattern.The dispersibility of the bare nZVI, EG-functionalized nZVI and nZVI/n-lignin in aqueous solutions over different durations was tested. After the synthesis and washing processes, the three samples were put into water and dispersed by an ultrasonicator. After sonication for a short time, optical images of the solutions were taken, as shown in quations ,32: (1) 2O for 7 days. The XRD analysis of the three aged materials placed in H2O for 7 days is shown in 3O4. These characteristic peaks in the X-ray diffraction indicate the crystal orientations of (311), (400), (331), (422), (511), and (440), respectively. The XRD results show the better stability against oxidation of nZVI/n-lignin and the oxidation reaction of nZVIs to iron oxide.After the synthesis, the three dried materials were stored in atmospheric conditions for 7 days. Then, the powders were analyzed by XRD. The XRD measurements of the three aged materials in atmospheric conditions are shown in 2O by nZVI/n-lignin was also investigated. In the presence of nZVIs, the removal mechanisms of methylene blue (MB) include reduction into colorless leuco-MB, precipitation as Fe(II)-MB, absorption as ZVI-MB, and degradation using hydroxyl radicals [2O had a strong absorption peak at the wavelength of 664 nm and a shoulder peak at around 615 nm from the dimer of methylene blue [2O are shown in Besides the synthesis, the dispersibility in water, and the antioxidation in the air of nZVI/n-lignin, the removal of methylene blue in Hradicals . Moreoveradicals ,34. The radicals . Thereforadicals . The remene blue . The UV–2O/O2 starts, which consumes the nZVI before it degrades the pollutants. The nZVI’s stability against H2O/O2 oxidation is of great importance. In our study, the applicability of bare nZVI, EG-functionalized nZVI, and nZVI/n-lignin was compared according to the removal ratio of methylene blue in aqueous solutions. After the addition of 40 mg each of bare nZVI, EG-functionalized nZVI, and nZVI/n-lignin to the methylene blue solution, their absorption rates were measured by UV–Vis spectra, as shown in In practical application, once the nZVI is added to ground water in the field for pollutant remediation, the oxidation reaction between nZVI and H2O/O2 are of great importance. The agglomeration of the nZVI results in reduced degradation activity and poor mobility. Strongly oxidized behavior results in the consumption of the nZVI before it makes contact with the pollutants away from the injection sites. In this study, the nanoscale lignin-protected nZVI (nZVI/n-lignin) was first proposed to solve the above-mentioned issues. According to the analysis of the TEM and XPS measurements, the structure of nZVI/n-lignin is nanoscale lignin surrounding nZVI particles, which consist of zero-valent iron as the core with a thin layer of defective iron oxide/hydroxide on the surface. The traditional chain-like pattern and large clusters of bare nZVI can be avoided in the case of nZVI/n-lignin probably because of the steric stabilization alleviating magnetic interactions. This feature, combined with the use of nano-sized lignin rather than the use of micro-sized lignin, makes nZVI/n-lignin possess much better dispersibility and mobility in H2O than those of bare nZVI. The structure of nano-lignin could provide more heterogeneous nucleation sites for the growth of ion nanoparticles. From their optical images, TEM, and XRD analysis, it was observed that nZVI/n-lignin possesses better stability against oxidation by H2O/O2 than those of bare and EG-functionalized nZVIs. The as-synthesized nZVI/n-lignin maintains its degradation ability even after it has been stored for several weeks in atmospheric conditions or several hours in H2O, while bare nZVI is quickly oxidized to form iron oxide/hydroxide in a much shorter time. Compared with nZVIs, the nZVI/n-lignin composite may have better mobility in ground water while maintaining its degradation ability toward pollutants far away from the injection sites. In summary, the presented synthetic method for nZVI/n-lignin involving a dissolution and precipitation approach with ethylene glycol/acidic H2O is easy to carry out, and it is environmentally friendly. It could be used as a universal approach for synthesizing nanoscale lignin-stabilized zero-valent metal and metal oxide nanoparticles for various applications.In our study, sustainable lignin biopolymer was employed for the synthesis of iron nanoparticles for the removal of environmental pollutants. nZVI is the most frequently studied material for the water treatment aspect of environmental engineering. In real field applications, the environmental pollutants in a contaminated area are far away from the nZVI injection sites. Consequently, the prevention of nZVI aggregation and the avoidance of strong oxidation by H