Unconventional Electrochemical Behaviors of Cu Underpotential Deposition in a Chloride-Based Deep Eutectic Solvent: High Underpotential Shift and Low Coverage.

The (5 × 5) Moiré pattern resulting from coadsorption of Cu atoms and chloride ions on the Au(111) electrode is one of the most classical structures for underpotential deposition (UPD) in electrochemical surface science. Although two models have been proposed to describe the pattern, the details of the structure remain ambiguous and controversial, leading to a question that remains to be answered. In this work, we investigate the UPD behaviors of Cu on the Au(111) electrode in a chloride-based deep eutectic solvent ethaline by in situ scanning tunneling microscopy (STM). Benefiting from the properties of the ultraconcentrated electrolyte, we directly image not only Cu but also Cl adlayers by finely tuning tunneling conditions. The structure is unambiguously determined for both Cu and Cl adlayers, where an incommensurate Cu layer is adsorbed on the Au(111) surface with a Cu coverage of 0.64, while the Cl coverage is 0.32 (only half of the expected value); i.e., the atomic arrangement of the observed (5 × 5) Moiré pattern in ethaline matches neither of the models proposed in the literature. Meanwhile, STM results confirm the origin of the cathodic peak in the cyclic voltammogram, which indicates that the underpotential shift of Cu UPD in ethaline indeed increases by ca. 0.40 V compared to its counterpart in a sulfuric acid solution, resulting in a significant deviation from the linear relation between the underpotential shift and the difference in work functions proposed in the literature. The unconventional electrochemical behaviors of Cu UPD reveal the specialty of both the bulk and the interface in the chloride-based deep eutectic solvent.

CREB3L1 promotes tumor growth and metastasis of anaplastic thyroid carcinoma by remodeling the tumor microenvironment.

Anaplastic thyroid carcinoma (ATC) is an extremely malignant type of endocrine cancer frequently accompanied by extrathyroidal extension or metastasis through mechanisms that remain elusive. We screened for the CREB3 transcription-factor family in a large cohort, consisting of four microarray datasets. This revealed that CREB3L1 was specifically up regulated in ATC tissues and negatively associated with overall survival of patients with thyroid cancer. Consistently, high expression of CREB3L1 was negatively correlated with progression-free survival in an independent cohort. CREB3L1 knockdown dramatically attenuated invasion of ATC cells, whereas overexpression of CREB3L1 facilitated the invasion of papillary thyroid carcinoma (PTC) cells. Loss of CREB3L1 inhibited metastasis and tumor growth of ATC xenografts in zebrafish and nude mouse model. Single-cell RNA-sequencing analysis revealed that CREB3L1 expression gradually increased during the neoplastic progression of a thyroid follicular epithelial cell to an ATC cell, accompanied by the activation of the extracellular matrix (ECM) signaling. CREB3L1 knockdown significantly decreased the expression of collagen subtypes in ATC cells and the fibrillar collagen in xenografts. Due to the loss of CREB3L1, ATC cells were unable to activate alpha-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs). After CREB3L1 knockdown, the presence of CAFs inhibited the growth of ATC spheroids and the metastasis of ATC cells. Further cytokine array screening showed that ATC cells activated α-SMA-positive CAFs through CREB3L1-mediated IL-1α production. Moreover, KPNA2 mediated the nuclear translocation of CREB3L1, thus allowing it to activate downstream ECM signaling. These results demonstrate that CREB3L1 maintains the CAF-like property of ATC cells by activating the ECM signaling, which remodels the tumor stromal microenvironment and drives the malignancy of ATC.

Systematic evaluation of the anti-tumor effect of Phellinus linteus polysaccharide in thyroid carcinoma in vitro.

BACKGROUND: Thyroid carcinoma (TC) is the most common malignant tumor of the endocrine system. Phellinus linteus polysaccharide (PLP) physiologically acts as a suitable anti-tumor molecule. In this study, for the first time, we systematically investigated the anti-tumor activity of PLP in TC, aiming to promote the application of PLP in TC treatment. METHODS AND RESULTS: TPC-1 and SW579 cells were treated with or without PLP. After treatment, MTT and EdU proliferation assays were performed to detect cell growth. Cell cycle was analyzed using flow cytometry. JC-1 staining was used to track change of mitochondrial membrane potential (MMP). Apoptotic cells were stained with annexin V-fluorescein isothiocyanate/propidium iodide and subsequently analyzed using flow cytometry. mCherry-GFP-LC3 was overexpressed in TC cells by lentiviral technology and the autophagosome was observed using confocal laser scanning microscope. Transwell migration and Matrigel invasion assays were performed to elucidate cell metastasis. Finally, underlying molecular mechanisms were investigated using RT-qPCR and western blotting. PLP inhibited cell growth in TC cells, which was attributable to the PLP-induced arrest at G0/G1 phase of cell cycle. PLP decreased the MMP, induced cell apoptosis, and promoted mitochondrial autophagy and endoplasmic reticulum autophagy. Furthermore, PLP may promote cell apoptosis via nuclear factor kappa-B pathway. In addition, PLP also inhibited cell migration and invasion through modulating the expression of epithelial-mesenchymal transition molecules such as E-cadherin, N-cadherin, and Vimentin CONCLUSIONS: PLP exhibits notable anti-tumor activity in TC cells and may be used in TC treatment.

Novel computer-aided reconstruction of soft tissue defects following resection of oral and oropharyngeal squamous cell carcinoma.

BACKGROUND: Reconstruction of soft tissue defects following surgical tumor resection is important for quality of life in cancer patients with oral and oropharyngeal squamous cell carcinoma (SCC). This study presents a novel computer-aided reconstruction of soft tissue (CARST) technology employed with these patients. METHODS: We first described the CARST technology in detail in a report of a 34-year-old male patient with locally invasive right-sided tongue SCC following a nearly total glossectomy and reported the postoperative outcomes. This digital technology was applied to construct a 3D model from CT images, which was used to delineate surgical resection boundaries and design a personalized reconstruction of the soft tissue defect. A nonuniform rational B-spline (NURBS) was generated and applied to transform the 3D model into a 2D flap-cutting guide printed out using a 3D printer. We then reported a case-series study on oral and oropharyngeal SCC patients who were randomly assigned to receive the CARST (n = 15) or a traditional soft tissue reconstruction (n = 15). Clinicopathological features and short- and long-term postoperative outcomes between the two groups were compared. RESULTS: The patient with the tongue SCC had a successful CARST following surgical tumor resection without any complications. His speech and swallowing functions recovered well after surgery and he experienced no significant changes to his appearance following recovery. There was no recurrence within a 3-year follow-up period. Results of the case-series study showed that the CARST group had significantly shorter operative and post-operation hospital-stay time, a higher flap utilization rate, and a trend of less and milder postoperative complications, and they experienced no significant difference in intraoperative blood loss and long-term outcomes compared to the traditional group. CONCLUSION: CARST is a safer and more efficient personalized technology of soft tissue reconstruction following surgical tumor resection in patients with oral and oropharyngeal SCC.

Effect of hydrogen bond donor molecules ethylene glycerol and lactic acid on electrochemical interfaces in choline chloride based-deep eutectic solvents.

Choline chloride (ChCl)-based-deep eutectic solvents (DESs) are widely used in electrochemical fields. In this work, the effect of two types of hydrogen bond donor (HBD) molecules, ethylene glycerol and lactic acid (LA), on electrochemical interfaces between the Au electrode and DESs has been investigated by employing voltammetry and electrochemical impedance spectroscopy. The anodic dissolution and passivation behaviors of the Au electrode are revealed in both ethaline and ChCl:LA. In ChCl:LA, the anodic dissolution of Au is slowed down, and the passivation film is relatively dense and stable due to the existence of the carboxyl group in HBD molecule LA. In the double layer region, the lifting and formation of Au(111) surface reconstruction and a disorder-order phase transition of the chloride ion adlayer were observed in the two DESs. Moreover, compared with ethaline, an extra pair of current peaks appears in ChCl-LA possibly due to the adsorption and desorption of LA on the Au(111) surface, which might imply the stronger interaction of LA with the Au electrode in ChCl:LA. HBD LA could even have marked an impact on the disorder-order phase transition of the chloride ion adlayer. The above results provide new insight into the significant effect of HBD molecules on the anodic dissolution and the passivation of the Au electrode and the electrochemical behaviors in the double layer region.

Advances in targeted therapy for anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is a highly malignant and aggressive thyroid malignancy with rapid onset and poor prognosis. There is no effective treatment for ATC yet. Molecular targeted therapy provides a new idea for ATC treatment. Tyrosine kinase inhibitor lenvatinib has potential in treating ATC patients with favorable efficacy in clinical trials. The effectiveness of the v-raf murine sarcoma viral oncogene homologue B1 () gene inhibitor dabrafenib in combination with trametinib for the treatment of positive ATC patients has been demonstrated in clinical trials. The has proposed dabrafenib in combination with trametinib as the preferred modality for the treatment of patients with positive ATC. The immune checkpoint inhibitor pembrolizumab can be applied to treat thyroid cancer with high tumor mutational load and may be considered as the preferred modality for the treatment of ATC patients with high programmed death ligand-1 expression. The mammalian target of rapamycin pathway inhibitors, peroxisome proliferators-activated receptor γ agonists, endothelial growth factor receptors-targeting monoclonal antibody cetuximab and novel vascular blocker fosbretabulin are still in the clinical research stage, which are expected to provide new directions for the development of novel targeted drugs. This article reviews the current research progress on targeted drugs for the treatment of ATC.

Genomic landscape of metastatic papillary thyroid carcinoma and novel biomarkers for predicting distant metastasis.

Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid gland, with a relatively high cure rate. Distant metastasis (DM) of PTC is uncommon, but when it occurs, it significantly decreases the survival of PTC patients. The molecular mechanisms of DM in PTC have not been systematically studied. We performed whole exome sequencing and GeneseeqPrime (425 genes) panel sequencing of the primary tumor, plasma and matched white blood cell samples from 20 PTC with DM and 46 PTC without DM. We identified somatic mutations, gene fusions and copy number alterations and analyzed their relationships with DM of PTC. BRAF-V600E was identified in 73% of PTC, followed by RET fusions (14%) in a mutually exclusive manner (P < 0.0001). We found that gene fusions (RET, ALK or NTRK1) (P < 0.01) and chromosome 22q loss (P < 0.01) were independently associated with DM in both univariate and multivariate analyses. A nomogram model consisting of chromosome 22q loss, gene fusions and three clinical variables was built for predicting DM in PTC (C-index = 0.89). The plasma circulating tumor DNA (ctDNA) detection rate in PTC was only 38.9%; however, it was significantly associated with the metastatic status (P = 0.04), tumor size (P = 0.001) and invasiveness (P = 0.01). In conclusion, gene fusions and chromosome 22q loss were independently associated with DM in PTC and could serve as molecular biomarkers for predicting DM. The ctDNA detection rate was low in non-DM PTC but significantly higher in PTC with DM.

Loss of PIM1 correlates with progression and prognosis of salivary adenoid cystic carcinoma (SACC).

BACKGROUND: Increasing evidence indicates that PIM1 is a potential prognostic marker and target for cancer treatment but its precise mechanisms of action remain to be determined in salivary adenoid cystic carcinoma (SACC). This study aims to decipher the prognostic and mechanistic role of PIM1 in progression of SACC cells and tumor tissues. METHODS: A SACC cell line (ACC-M) was transfected with shRNA plasmids targeting the PIM1 gene. The expression levels of PIM1, RUNX3 and p21 were measured by quantitative real-time PCR and western blot. Subcellular translocalization of RUNX3 and p21 proteins was assessed using immunofluorescence, and cell cycle phase was quantified using flow cytometry. A total of 97 SACC patients were retrospectively analyzed by clinicopathologic characteristics and survival outcomes. RESULTS: After down-regulation of PIM1 in ACC-M cells, RUNX3 and p21 proteins were translocated from cytoplasm to nucleus, with a decrease of p21 expression and increase of G0/G1 phase cells. PIM1 and RUNX3 levels show a distinct covariance. PIM1 is associated with T-status, lymph node involvement, nerve invasion, and distant metastasis in SACC tissues. Patients with low PIM1 level had a better outcome than those with higher PIM1 level. CONCLUSIONS: PIM1 is multifunctional in ACC-M cells and it serves as a neoteric therapeutic target and potential prognostic marker for SACC patients.

The synergic effect of BRAFV600E mutation and multifocality on central lymph node metastasis in unilateral papillary thyroid carcinoma.

The purpose of this study is to evaluate the potential synergic effect of BRAFV600E mutation and multifocality on central lymph nodes metastasis (CLNM) in the patients with unilateral papillary thyroid carcinoma (PTC). We enrolled 413 patients with unilateral PTCs who accepted prophylactic unilateral or bilateral central lymph node dissection (LND). Univariate and multivariate analyses were made to determine the association between related factors and CLNM. Then, all patients were divided into 4 groups based on their status of BRAFV600E mutation and multifocality. Relative excess risk of interaction (RERI), attributable proportion (AP) of interaction and synergy index (SI) were applied to evaluate the interactive effect of these two factors on CLNM. Results showed that BRAFV600E mutation and multifocality were independent risk factors for CLNM. A further study revealed that unilateral PTCs accompanying multifocality with BRAFV600E mutation had the highest incidence of CLNM compared with other subgroups. Besides, RERI was 4.323 (95% CI = 1.276-7.369), AP was 0.523 (95% CI = 0.364-0.682) and SI was 2.469 (95% CI = 1.607 to 3.794), indicating a significant additive interaction of BRAFV600E mutation and multifocality on CLNM. The present study has confirmed that BRAFV600E mutation and multifocality are risk factors for CLNM in unilateral PTC. Additionally, unilateral PTC patients accompanying multifocality with BRAFV600E mutation may have an increased risk of CLNM in clinically negative CLNM.

Antibacterial efficacy and cytotoxicity of low intensity direct current activated silver-titanium implant system prototype.

Silver-based devices activated by electric current are of interest in biomedicine because of their broad-spectrum antimicrobial activity. This study investigates the in vitro antibacterial efficacy and cytotoxicity of a low intensity direct current (LIDC)-activated silver-titanium implant system prototype designed for localized generation and delivery of silver ions at the implantation site. First, the antibacterial efficacy of the system was assessed against methicillin-resistant Staphylococcus aureus (MRSA) over 48 h at current levels of 3 and 6 µA in Mueller-Hinton broth. The cytotoxicity of the system was then evaluated over 48 h in two phases using an in vitro model with in which the activated electrodes were suspended in growth medium in a cell-seeded tissue culture plate. In phase-1, the system was tested on human osteosarcoma (MG-63) cell line and compared to titanium controls. In phase-2, the cytotoxicity characteristics were validated with normal human diploid osteoblast cells. The LIDC-activated system demonstrated high antimicrobial efficacy against MRSA, but was also toxic to human cells immediately surrounding the electrodes. The statistical analysis showed that the cytotoxicity was a result of the presence of silver, and the electric activation did not make it worse.

Effects of cathode design parameters on in vitro antimicrobial efficacy of electrically-activated silver-based iontophoretic system.

Post-operative infection is a major risk associated with implantable devices. Prior studies have demonstrated the effectiveness of ionic silver as an alternative to antibiotic-based infection prophylaxis and treatment. The focus of this study is on an electrically activated implant system engineered for active release of antimicrobial silver ions. The objective was to evaluate the effects of the cathode design, especially the cathode material, on the in vitro antimicrobial efficacy of the system. A modified Kirby-Bauer diffusion technique was used for the antimicrobial efficacy evaluations (24 h testing interval). In phase-1 of the study, a three-way ANOVA (n = 6, α = 0.05) was performed to determine the effects of cathode material (silver, titanium, and stainless steel), cathode surface area and electrode separation distance on the efficacy of the system against Staphylococcus aureus. The results show that within the design space tested, none of these parameters had a statistically significant effect on the antimicrobiality of the system (P > 0.15). Subsequently, one-way ANOVA (n = 6, α = 0.05) was conducted in phase-2 to validate the inference regarding the non-significance of the cathode material to the system efficacy using a broader spectrum of pathogens (methicillin-resistant S. aureus, Escherichia coli, Streptococcus agalactiae and Aspergillus flavus) responsible for osteomyelitis. The results confirmed the lack of statistical difference between efficacies of the three cathode material configurations against all pathogens tested (P > 0.58). Overall, the results demonstrate the ability to alter the cathode material and related design parameters in order to minimize the silver usage in the system without adversely affecting its antimicrobial efficacy.

Targeting SIGLEC15 as an emerging immunotherapy for anaplastic thyroid cancer.

Anaplastic thyroid carcinoma (ATC) is the most aggressive subtype of thyroid cancer with few effective therapies. Though immunotherapies such as targeting PD-1/PD-L1 axis have benefited patients with solid tumor, the druggable immune checkpoints are quite limited in ATC. In our study, we focused on the anti-tumor potential of sialic acid-binding Ig-like lectins (Siglecs) in ATC. Through screening by integrating microarray datasets including 216 thyroid-cancer tissues and single-cell RNA-sequencing, SIGLEC family members CD33, SIGLEC1, SIGLEC10 and SIGLEC15 were significantly overexpressed in ATC, among which SIGLEC15 increased highest and mainly expressed on cancer cells. SIGLEC15high ATC cells are characterized by high expression of serine protease PRSS23 and cancer stem cell marker CD44. Compared with SIGLEC15low cancer cells, SIGLEC15high ATC cells exhibited higher interaction frequency with tumor microenvironment cells. Further study showed that SIGLEC15high cancer cells mainly interacted with T cells by immunosuppressive signals such as MIF-TNFRSF14 and CXCL12-CXCR4. Notably, treatment of anti-SIGLEC15 antibody profoundly increased the cytotoxic ability of CD8+ T cells in a co-culture model and zebrafish-derived ATC xenografts. Consistently, administration of anti-SIGLEC15 antibody significantly inhibited tumor growth and prolonged mouse survival in an immunocompetent model of murine ATC, which was associated with increase of M1/M2, natural killer (NK) cells and CD8+ T cells, and decrease of myeloid-derived suppressor cells (MDSCs). SIGLEC15 inhibited T cell activation by reducing NFAT1, NFAT2, and NF-κB signals. Blocking SIGLEC15 increased the secretion of IFN-γ and IL-2 in vitro and in vivo. In conclusion, our finding demonstrates that SIGLEC15 is an emerging and promising target for immunotherapy in ATC.

Epigenetic inhibition of CTCF by HN1 promotes dedifferentiation and stemness of anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is one of the deadliest cancers, whose important malignant feature is dedifferentiation. Chromatin remodeling is critical for tumorigenesis and progression, while its roles and regulator in facilitating dedifferentiation of ATC had been poorly understood. In our study, an emerging function of hematological and neurological expressed 1 (HN1) in promoting dedifferentiation of ATC cells was uncovered. HN1 expression was negatively correlated with the thyroid differentiation markers both at mRNA and protein level. Knockdown of HN1 in ATC cells effectively upregulated the thyroid differentiation markers and impeded the sphere formation capacity, accompanying with the loss of cancer stemness. In contrast, overexpression of HN1 drove the gain of stemness and the loss of thyroid differentiation markers. Nude mouse and zebrafish xenograft models showed that inhibition of HN1 in ATC cells effectively hindered tumor growth due to the loss of cancer stemness. Further study showed that HN1 was negatively correlated with CTCF in an independent thyroid-cancer cohort, and inhibition of HN1 enhanced the expression of CTCF in ATC cells. Overexpression of CTCF significantly reversed the dedifferentiation phenotypes of ATC cells, whereas simultaneously inhibiting HN1 and CTCF was unable to recover the level of thyroid differentiation markers. The combination of ATAC-seq and ChIP-seq analysis confirmed that CTCF regulated genes relating with thyroid gland development through influencing their chromatin accessibility. HN1 inhibited the acetylation of H3K27 at the promoter of CTCF by recruiting HDAC2, thereby inhibiting the transcriptional activation of CTCF. These findings demonstrated an essential role of HN1 in regulating the chromatin accessibility of thyroid differentiation genes during ATC dedifferentiation.

Disrupting glycolysis and DNA repair in anaplastic thyroid cancer with nucleus-targeting platinum nanoclusters.

Cancer cells rely on aerobic glycolysis and DNA repair signals to drive tumor growth and develop drug resistance. Yet, fine-tuning aerobic glycolysis with the assist of nanotechnology, for example, dampening lactate dehydrogenase (LDH) for cancer cell metabolic reprograming remains to be investigated. Here we focus on anaplastic thyroid cancer (ATC) as an extremely malignant cancer with the high expression of LDH, and develop a pH-responsive and nucleus-targeting platinum nanocluster (Pt@TAT/sPEG) to simultaneously targets LDH and exacerbates DNA damage. Pt@TAT/sPEG effectively disrupts LDH activity, reducing lactate production and ATP levels, and meanwhile induces ROS production, DNA damage, and apoptosis in ATC tumor cells. We found Pt@TAT/sPEG also blocks nucleotide excision repair pathway and achieves effective tumor cell killing. In an orthotopic ATC xenograft model, Pt@TAT/sPEG demonstrates superior tumor growth suppression compared to Pt@sPEG and cisplatin. This nanostrategy offers a feasible approach to simultaneously inhibit glycolysis and DNA repair for metabolic reprogramming and enhanced tumor chemotherapy.

Biocompatibility analysis of an electrically-activated silver-based antibacterial surface system for medical device applications.

The costs associated with the treatment of medical device and surgical site infections are a major cause of concern in the global healthcare system. To prevent transmission of such infections, a prophylactic surface system that provides protracted release of antibacterial silver ions using low intensity direct electric current (LIDC; 28 µA system current at 6 V) activation has been recently developed. To ensure the safety for future in vivo studies and potential clinical applications, this study assessed the biocompatibility of the LIDC-activated interdigitated silver electrodes-based surface system; in vitro toxicity to human epidermal keratinocytes, human dermal fibroblasts, and normal human osteoblasts, and antibacterial efficacy against Staphylococcus aureus and Escherichia coli was evaluated. The study concluded that the technological applications of the surface system for medical devices and surgical tools, which contact human tissues for less than 1.5 h, are expected to be self-sterilizing without causing toxicity in vivo.

[Aberrant methylation of hMLH1 gene promoter in papillary thyroid cancer and its clinical significance].

OBJECTIVE: To investigate the aberrant promoter methylation of hMLH1 gene promoter and its clinical significance in papillary thyroid cancer (PTC). METHODS: methylation of hMLH1 gene promoter in the cancer tissue and matched tumor-adjacent normal tissue of 152 PTC patients were detected by real-time methylation specific PCR (qMSP). The relationship between the methylation of hMLH1 gene promoter and clinicopathological features was analyzed. RESULTS: The methylation rate of hMLH1 gene promoter in cancer tissues was 37.5% (57/152), of which 33 cases were totally methylated and 24 cases were partially methylated. The methylation rate of adjacent normal tissues was 5.3% (8/152)(all were partially methylated). The methylation rate of PTC tissues was significantly higher than that in the tumor-adjacent normal tissue (P < 0.01). The promoter methylation of hMLH1 gene in PTC was significantly correlated with age, size and number of the primary lesion, local invasion, T stage and lymph node metastasis (P < 0.05) , but not correlated with gender and clinical stage (P > 0.05). CONCLUSION: Promoter methylation of hMLH1 gene is a common molecular event in PTC tissue, and it is significantly correlated with the progression of PTC.

Molecular basis and targeted therapy in thyroid cancer: Progress and opportunities.

Thyroid cancer (TC) is the most prevalent endocrine malignant tumor. Surgery, chemotherapy, radiotherapy, and radioactive iodine (RAI) therapy are the standard TC treatment modalities. However, recurrence or tumor metastasis remains the main challenge in the management of anaplastic thyroid cancer (ATC) and radioiodine (RAI) radioactive iodine-refractory differentiated thyroid cancer (RR-DTC). Several multi-tyrosine kinase inhibitors (MKIs), or immune checkpoint inhibitors in combination with MKIs, have emerged as novel therapies for controlling the progression of DTC, medullary thyroid cancer (MTC), and ATC. Here, we discuss and summarize the molecular basis of TC, review molecularly targeted therapeutic drugs in clinical research, and explore potentially novel molecular therapeutic targets. We focused on the evaluation of current and recently emerging tyrosine kinase inhibitors approved for systemic therapy for TC, including lenvatinib, sorafenib and cabozantinib in DTC, vandetanib, cabozantinib, and RET-specific inhibitor (selpercatinib and pralsetinib) in MTC, combination dabrafenib with trametinib in ATC. In addition, we also discuss promising treatments that are in clinical trials and may be incorporated into clinical practice in the future, briefly describe the resistance mechanisms of targeted therapies, emphasizing that personalized medicine is critical to the design of second-line therapies.

IL2RA+VSIG4+ tumor-associated macrophage is a key subpopulation of the immunosuppressive microenvironment in anaplastic thyroid cancer.

Extensive infiltration of tumor-associated macrophages was correlated poor prognosis in anaplastic thyroid cancer (ATC). However, the heterogeneity and characteristics of the ATC-associated macrophages (ATAMs) in ATC remain far from clear. We combined single-cell RNA-sequencing analysis and gene expression microarray datasets to assess the molecular signature of ATAMs. Compared with normal thyroid-associated macrophages (NTAMs), 778 differentially expressed genes (DEGs) significantly changed in ATAMs compared with NTAMs. These DEGs were correlated with oxidative phosphorylation (M2 phenotype) and phagocytosis (M1 phenotype). Moreover, ATAMs highly expressed pro-tumor genes associated with angiogenesis, fibrosis, metalloprotease activity, and metastasis. Notably, we identified one ATC-specific subset, IL2RA+ VSIG4+ ATAMs, co-expressed M1 and M2 markers. The infiltration of IL2RA+ VSIG4+ ATAMs showed strong correlation with BRAF and RAS signaling, and its high infiltration was associated with favorable prognosis in thyroid-cancer patients. IL2RA+ VSIG4+ ATAMs were associated with increased tumor-infiltrating lymphocytes (B cells, CD8+ T cells, Tregs). IL2RA+ VSIG4+ ATAMs interacted with CD8+ T cells and Tregs through immune checkpoints (such as LGALS9_HAVCR2), cytokines (such as CXCL10_CXCR3), and receptors (such as CSF1R_CSF1), thereby forming an immunosuppressive microenvironment. Multiplex immunohistochemistry staining and coculture experiment confirmed that ATC cancer cells were able to induce the polarization of IL2RA+ VSIG4+ ATAMs. Besides, we identified several novel ATC-specific immune checkpoint genes including the immunosuppressive molecule VSIG4, LAIR1, and LILRB2. Expression of VSIG4 was also significantly correlated with tumor-infiltrating lymphocytes (B cells, CD8+ T cells, Tregs). In conclusion, our study revealed an ATC-specific ATAM subset with bifunctional phenotype, which provided a comprehensive insight to delineate the molecular characteristics of ATC-associated macrophages.

Identification and validation of subclusters of papillary thyroid carcinoma based on Human Phenotype Ontology.

Background: The increase in the diagnosis of papillary thyroid carcinoma (PTC) has prompted researchers to establish a diagnostic model and identify functional subclusters. The Human Phenotype Ontology (HPO) platform is widely available for differential diagnostics and phenotype-driven investigations based on next-generation sequence-variation data. However, a systematic and comprehensive study to identify and validate PTC subclusters based on HPO is lacking. Methods: We first used the HPO platform to identify the PTC subclusters. An enrichment analysis was then conducted to examine the key biological processes and pathways associated with the subclusters, and a gene mutation analysis of the subclusters was conducted. For each subcluster, the differentially expressed genes (DEGs) were selected and validated. Finally, a single-cell RNA-sequencing data set was used to verify the DEGs. Results: In our study, 489 PTC patients from The Cancer Genome Atlas (TCGA) were included. Our analysis demonstrated that distinct subclusters of PTC are associated with different survival times and have different functional enrichment, and that C-C motif chemokine ligand 21 (CCL21) and zinc finger CCHC-type containing 12 (ZCCHC12) were the common down- and upregulated genes, respectively, in the 4 subclusters. Additionally, 20 characteristic genes were identified in the 4 subclusters, some of which have previously been reported to have roles in PTC. Further, we found that these characteristic genes were mainly expressed in thyrocytes, endothelial cells, and fibroblasts, and were rarely expressed in immune cells. Conclusions: We first identified subclusters in PTC based on HPO and found that patients with distinct subclusters have different prognoses. We then identified and validated the characteristic genes in the 4 subclusters. These findings are expected to serve as a crucial reference that will improve our understanding of PTC heterogeneity and the use of novel targets.

ISG15 and ISGylation modulates cancer stem cell-like characteristics in promoting tumor growth of anaplastic thyroid carcinoma.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) was a rare and extremely malignant endocrine cancer with the distinct hallmark of high proportion of cancer stem cell-like characteristics. Therapies aiming to cancer stem-like cells (CSCs) were emerging as a new direction in cancer treatment, but targeting ATC CSCs remained challenging, mainly due to incomplete insights of the regulatory mechanism of CSCs. Here, we unveiled a novel role of ISG15 in the modulation of ATC CSCs. METHODS: The expression of ubiquitin-like proteins were detected by bioinformatics and immunohistochemistry. The correlation between ISG15 expression and tumor stem cells and malignant progression of ATC was analyzed by single-cell RNA sequence from the Gene Expression Omnibus. Flow cytometry combined with immunofluorescence were used to verify the enrichment of ISG15 and ISGyaltion in cancer stem cells. The effect and mechanism of ISG15 and KPNA2 on cancer stem cell-like characteristics of ATC cells were determined by molecular biology experiments. Mass spectrometry combined with immunoprecipitation to screen the substrates of ISG15 and validate its ISGylation modification. Nude mice and zebrafish xenograft models were utilized to demonstrate that ISG15 regulates stem cell characteristics and promotes malignant progression of ATC. RESULTS: We found that among several ubiquitin proteins, only ISG15 was aberrantly expressed in ATC and enriched in CSCs. Single-cell sequencing analysis revealed that abnormal expression of ISG15 were intensely associated with stemness and malignant cells in ATC. Inhibition of ISG15 expression dramatically attenuated clone and sphere formation of ATC cells, and facilitated its sensitivity to doxorubicin. Notably, overexpression of ISGylation, but not the non-ISGylation mutant, effectively reinforced cancer stem cell-like characteristics. Mechanistically, ISG15 mediated the ISGylation of KPNA2 and impeded its ubiquitination to promote stability, further maintaining cancer stem cell-like characteristics. Finally, depletion of ISG15 inhibited ATC growth and metastasis in xenografted mouse and zebrafish models. CONCLUSION: Our studies not only provided new insights into potential intervention strategies targeting ATC CSCs, but also uncovered the novel biological functions and mechanisms of ISG15 and ISGylation for maintaining ATC cancer stem cell-like characteristics.