Characterization of EpCAM in thyroid cancer biology by three-dimensional spheroids in vitro model.

BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy. Nowadays, undifferentiated thyroid cancers (UTCs) are still lethal, mostly due to the insurgence of therapy resistance and disease relapse. These events are believed to be caused by a subpopulation of cancer cells with stem-like phenotype and specific tumor-initiating abilities, known as tumor-initiating cells (TICs). A comprehensive understanding of how to isolate and target these cells is necessary. Here we provide insights into the role that the protein Epithelial Cell Adhesion Molecule (EpCAM), a known TICs marker for other solid tumors, may have in TC biology, thus considering EpCAM a potential marker of thyroid TICs in UTCs. METHODS: The characterization of EpCAM was accomplished through Western Blot and Immunofluorescence on patient-derived tissue samples, adherent cell cultures, and 3D sphere cultures of poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) cell lines. The frequency of tumor cells with putative tumor-initiating ability within the 3D cultures was assessed through extreme limiting dilution analysis (ELDA). EpCAM proteolytic cleavages were studied through treatments with different cleavages' inhibitors. To evaluate the involvement of EpCAM in inducing drug resistance, Vemurafenib (PLX-4032) treatments were assessed through MTT assay. RESULTS: Variable EpCAM expression pattern was observed in TC tissue samples, with increased cleavage in the more UTC. We demonstrated that EpCAM is subjected to an intense cleavage process in ATC-derived 3D tumor spheres and that the 3D model faithfully mimics what was observed in patient's samples. We also proved that the integrity of the protein appears to be crucial for the generation of 3D spheres, and its expression and cleavage in a 3D system could contribute to drug resistance in thyroid TICs. CONCLUSIONS: Our data provide novel information on the role of EpCAM expression and cleavage in the biology of thyroid TICs, and our 3D model reflects the variability of EpCAM cleavage observed in tissue samples. EpCAM evaluation could play a role in clinical decisions regarding patient therapy since its expression and cleavage may have a fundamental role in the switch to a drug-resistant phenotype of UTC cells.

Frequency-modulation stimulated Raman scattering microscopy with an acousto-optic tunable filter.

Stimulated Raman scattering (SRS) microscopy is increasingly employed for highly specific, label-free, and high-speed bioimaging. Despite its benefits, SRS is susceptible to spurious background signals caused by competing effects, which lower the possible imaging contrast and sensitivity. An efficient approach to suppress these undesired background signals is frequency-modulation (FM) SRS, which exploits the competing effects' weak spectral dependence compared to the SRS signal's high spectral specificity. We propose an FM-SRS scheme realized with an acousto-optic tunable filter, which presents a few advantages compared to other solutions presented in the literature. In particular, it can perform automated measurements from the fingerprint to the CH-stretching region of the vibrational spectrum without any manual adjustment of the optical setup. Moreover, it allows simple all-electronic control of the spectral separation and relative intensities of the pair of probed wavenumbers.

Fatigue behavior, failure mode, and stress distribution of occlusal veneers: influence of the prosthetic preparation cusp inclinations and the type of restorative material.

OBJECTIVE: To evaluate the effects of cusp inclination of the prosthetic preparation's occlusal surface and type of restorative material on the fatigue behavior, failure mode, and stress distribution of occlusal veneers. MATERIALS AND METHODS: Glass fiber-reinforced epoxy resin prosthetic preparations for occlusal veneers with three different occlusal surface cusp inclination degrees (0°, 15°, and 30°) were produced and assigned into six testing groups (n = 11) according to the cusp inclination (0°, 15°, or 30°) and type of restorative material (lithium disilicate-LD or resin composite-RC). Despite different substrate preparation cusp inclination degrees, the restorations were designed maintaining 30° inclination between the cusps at the occlusal surface and a thickness of 0.7 mm at the central groove region of the restorations to be machined in a CAD/CAM system. After cementation, the specimens were stored for about 7 days (under water at 37 °C), and subsequently submitted to a load to failure test (n = 2) and an intermittent cyclic fatigue test (n = 9) (initial load: 100 N; step size: 50 N; cycles/step: 10,000; loading frequency: 20 Hz; loading piston: 6-mm-diameter stainless steel) until observing cracks. The data were analyzed by two-way ANOVA, Kaplan-Meier, and Mantel-Cox post hoc tests. Finite element analysis (FEA) and fractographic analyses were performed. RESULTS: The fatigue performance of LD and RC occlusal veneers was evaluated based on different prosthetic preparation cusp inclinations. The 0° inclination showed the best fatigue performance for both materials (LD: 944N, RC: 861N), while the 15° and 30° inclinations had lower values (LD: 800N and 533N, RC: 739N and 717N, respectively). The study also found that for a 0° inclination, LD occlusal veneers performed better than RC ones (LD: 944 N > RC: 861N), while for a 30° inclination, RC occlusal veneers had better fatigue performance than LD ones (LD: 533N < RC: 717N). No significant difference was observed between the materials for a 15° inclination (LD: 800N = RC: 739N). The FEA results showed a higher tensile stress concentration on lithium disilicate than on resin composite occlusal veneers. All lithium disilicate occlusal veneers showed radial crack failures, while resin composite occlusal veneers showed Hertzian cone cracks and radial cracks combined. CONCLUSION: Considering mechanical perspective only, RC occlusal veneers should be indicated when prosthetic preparation cusps inclinations are 30°. When 0° prosthetic preparation cusps inclinations are observed, LD occlusal veneers will behave mechanically better. When a 15° cusp inclination is preserved, both restorative materials behave similarly.

Role of Epigenetic Therapy in the Modulation of Tumor Growth and Migration in Human Castration-Resistant Prostate Cancer Cells with Neuroendocrine Differentiation.

INTRODUCTION: Neuroendocrine transdifferentiation (NED) of prostate cancer (PC) cells is associated with the development of resistance to antiandrogen therapy and poor prognosis in patients with castration-resistant PC (CRPC). Many of the molecular events, involved in NED, appear to be mediated by epigenetic mechanisms. In this study, we evaluated the antitumor activity and epigenetic modulation of 2 epigenetic drugs, such as the demethylating agent 5-aza-2'-deoxycytidine (AZA) and the methyl donor S-adenosylmethionine (SAM), in 2 human CRPC cell lines with NED (DU-145 and PC-3). METHODS: The effects of AZA and SAM on cell viability, cell cycle, apoptosis, migration, and genome-wide DNA methylation profiling have been evaluated. RESULTS: Both drugs showed a prominent antitumor activity in DU-145 and PC-3 cells, through perturbation of cell cycle progression, induction of apoptosis, and inhibition of cell migration. AZA and SAM reversed NED in DU-145 and PC-3, respectively. Moreover, AZA treatment modified DNA methylation pattern in DU-145 cells, sustaining a pervasive hypomethylation of the genome, with a relevant effect on several pathways involved in the regulation of cell proliferation, apoptosis, and cell migration, in particular Wnt/ß-catenin. CONCLUSIONS: A relevant antitumor activity of these epigenetic drugs on CRPC cell lines with NED opens a new scenario in the therapy of this lethal variant of PC.

Fatigue behavior and stress distribution of molars restored with MOD inlays with and without deep margin elevation.

OBJECTIVES: This study evaluated the effect of deep margin elevation (DME) and restorative materials (leucite-reinforced glass-ceramics [C] vs. indirect resin composite [R]) on the fatigue behavior and stress distribution of maxillary molars with 2-mm deep proximal margins restored with MOD inlay. METHODS: Fifty-two extracted human third molars were randomly assigned into four groups (n = 13): C; DME + C; R; and DME + R. Inlays were fabricated in CAD-CAM and bonded to all teeth. The fatigue behavior was assessed with the stepwise stress test (10,000 cycles/step; step = 50 N; 20 Hz; initial load = 200 N). Fatigue failure loads and the number of cycles were analyzed with 2-way ANOVA and Tukey's test (p < 0.05) and Kaplan-Meier survival plots. The stress distribution was assessed with finite element analysis. The models were considered isotropic, linear, and homogeneous, and presented bonded contacts. A tripod axial load (400 N) was applied to the occlusal surface. The stress distribution was analyzed with the maximum principal stress criterion. RESULTS: For fatigue, there was no difference for DME factor (p > 0.05). For the material factor, the load and number of cycles for failure were statistically higher in the R groups (p < 0.05). The finite element analysis showed that resin composite inlays concentrated more stress in the tooth structure, while ceramic inlays concentrated more stress in the restoration. Non-reparable failures were more frequent in the resin composite inlays groups. CONCLUSIONS: DME was not negative for fatigue and biomechanical behaviors. Resin composite inlays were more resistant to the fatigue test, although the failure mode was more aggressive. CLINICAL SIGNIFICANCE: DME does not impair mechanical behavior. Resin composite inlays failed at higher loads but with a more aggressive failure mode.

AtezoTRIBE: a randomised phase II study of FOLFOXIRI plus bevacizumab alone or in combination with atezolizumab as initial therapy for patients with unresectable metastatic colorectal cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) reported remarkable achievements in several solid tumours. However, in metastatic colorectal cancer (mCRC) promising results are limited to patients with deficient mismatch repair/microsatellite instability-high (dMMR/MSI-high) tumours due to their immune-enriched microenvironment. Combining cytotoxic agents and bevacizumab in mCRC with proficient mismatch repair/microsatellite stability (pMMR/MSS) could make ICIs efficacious by increasing the exposure of neoantigens, especially with highly active chemotherapy regimens, inducing immunogenic cell death, increasing the tumoral infiltration of CD8+ T-cells and reducing tumour-associated myeloid-derived suppressor cells. VEGF-blockade also plays an immunomodulatory role by inhibiting the expansion of T regulatory lymphocytes. Consistently with this rationale, a phase Ib study combined the anti-PDL-1 atezolizumab with FOLFOX/bevacizumab as first-line treatment of mCRC, irrespective of microsatellite status, and reported interesting activity and efficacy results, without safety concerns. Phase III trials led to identify FOLFOXIRI plus bevacizumab as an upfront therapeutic option in selected mCRC patients. Drawing from these considerations, the combination of atezolizumab with an intensified upfront treatment (FOLFOXIRI) and bevacizumab could be worthy of investigation. METHODS: AtezoTRIBE is a prospective, open label, phase II, comparative trial in which initially unresectable and previously untreated mCRC patients, irrespective of microsatellite status, are randomized in a 1:2 ratio to receive up to 8 cycles of FOLFOXIRI/bevacizumab alone or in combination with atezolizumab, followed by maintenance with bevacizumab plus 5-fluoruracil/leucovorin with or without atezolizumab according to treatment arm until disease progression. The primary endpoint is PFS. Assuming a median PFS of 12 months for standard arm, 201 patients should be randomized in a 1:2 ratio to detect a hazard ratio of 0.66 in favour of the experimental arm. A safety run-in phase including the first 6 patients enrolled in the FOLFOXIRI/bevacizumab/atezolizumab arm was planned, and no unexpected adverse events or severe toxicities were highlighted by the Safety Monitoring Committee. DISCUSSION: The AtezoTRIBE study aims at assessing whether the addition of atezolizumab to an intensified chemotherapy plus bevacizumab might be an efficacious upfront strategy for the treatment of mCRC, irrespective of the microsatellite status. TRIAL REGISTRATION: AtezoTRIBE is registered at Clinicaltrials.gov ( NCT03721653 ), October 26th, 2018 and at EUDRACT (2017-000977-35), Februray 28th, 2017.

Therapeutic schemes in 177Lu and 90Y-PRRT: radiobiological considerations.

BACKGROUND: The purpose of this work is to implement a radiobiological model to compare different treatment schedules for Peptide Receptor Radionuclide Therapy (PRRT) with 177Lu and 90Y. The principal radiobiological quantities were studied as a function of radionuclides, fractionation schemes, activity distribution in kidneys and tumor radiosensitivity. METHODS: Clinical data were used to derive representative absorbed doses for several treatment schemes for 177Lu-PRRT and for 90Y-PRRT and considered as input data for the radiobiological model. Both uniform and non-uniform activity distributions were considered for kidneys and cortex; for tumors a possible uptake reduction after each cycle and inter-patient radiosensitivity variability were investigated. Normal-Tissue-Complication-Probability (NTCP) and Tumor-Control-Probability (TCP) were evaluated. RESULTS: Hyper-cycling has a limited advantage in terms of BED reduction on kidneys for 177Lu, while for 90Y the effect is sizable and helps in reducing the NTCP. For all 177Lu-schemes the renal toxicity risk is negligible while for some 90Y-schemes the NTCP is not null. In case of tumor uptake reduction with cycles the treatment efficacy is reduced with a BED loss up to 46%. The TCP decreases when assuming normally-distributed tumor radiosensitivity values. CONCLUSIONS: This paper discusses how the combination of dosimetry and radiobiological modeling may help in exploring the link between the treatment schedule and the potential clinical outcome. The results highlight the capability of model to reproduce the available clinical data and provide useful qualitative information. Further investigation on dose distribution and dose uptake reduction with accurate clinical data is needed to progress in this field.

Time-Activity data fitting in molecular Radiotherapy: Methodology and pitfalls.

Absorbed radiation doses are essential in assessing the effects, e.g. safety and efficacy, of radiopharmaceutical therapy (RPT). Patient-specific absorbed dose calculations in the target or the organ at risk require multiple inputs. These include the number of disintegrations in the organ, i.e. the time-integrated activities (TIAs) of the organs, as well as other parameters describing the process of radiation energy deposition in the target tissue (i.e. mean energy per disintegration, radiation dose constants, etc). TIAs are then estimated by incorporating the area under the radiopharmaceutical's time-activity curve (TAC), which can be obtained by quantitative measurements of the biokinetics in the patient (typically based on imaging data such as planar scintigraphy, SPECT/CT, PET/CT, or blood and urine samples). The process of TAC determination/calculation for RPT generally depends on the user, e.g., the chosen number and schedule of measured time points, the selection of the fit function, the error model for the data and the fit algorithm. These decisions can strongly affect the final TIA values and thus the accuracy of calculated absorbed doses. Despite the high clinical importance of the TIA values, there is currently no consensus on processing time-activity data or even a clear understanding of the influence of uncertainties and variations in personalised RPT dosimetry related to user-dependent TAC calculation. As a first step towards minimising site-dependent variability in RPT dosimetry, this work provides an overview of quality assurance and uncertainty management considerations of the TIA estimation.

Extra-nuclear TERT counteracts oxidative stress and promotes progression in papillary thyroid carcinoma.

The reactivation of TERT is associated with poor outcome in papillary thyroid cancer (PTC). Extra-telomeric functions of TERT were reported, with a protective role against oxidative stress (OS). The aim of the present study was to explore the extra-nuclear TERT localization in PTC and its role in cancer progression. TERT nuclear export under OS were analyzed in K1 PTC cell line. We investigated the role of different TERT localizations using specific TERT constructs that limit its localization to the nucleus or to the mitochondria. The effect of SRC kinase inhibitor PP2, which reduces TERT nuclear export, was investigated as well. Moreover, TERT localization was analyzed in 39 PTC tissues and correlated with the genetic profile and the level of OS, DNA damage and apoptosis in the tumors and with the clinical characteristics of the patients. We demonstrated that TERT is exported from the nucleus in response to OS induced either from H2O2 or the BRAF inhibitor PLX4720. We proved that extra-nuclear TERT reduces mitochondrial OS and induces mitochondrial fragmentation. Moreover, limiting mitochondrial TERT localization reduced proliferation, migration, AKT phosphorylation and glycolysis and increased DNA damage and p21 expression. Finally, in PTC tissues the fraction of mitochondrial/nuclear TERT resulted inversely correlated with OS and p21 expression and associated with tumor persistence. In conclusion, our data indicate that extra-nuclear TERT is involved in reducing the effect of excessive OS, thus promoting cancer cell survival. Extra-nuclear TERT may thus represent a marker of cancer progression and a possible therapeutic target in PTC.

Unusual causes of hyperthyrotropinemia and differential diagnosis of primary hypothyroidism: a revised diagnostic flowchart.

The clinical consequences of primary hypothyroidism include cardiovascular morbidity, increased mortality, and poor quality of life; therefore guidelines endorsed by several Scientific Societies recommend measuring circulating thyroid-stimulating hormone (TSH) in patients at risk. The assessment of serum TSH levels is also deemed to be the most robust and accurate biomarker during the management of replacement therapy in patients with a previous diagnosis of primary hypothyroidism. In line with a reflex TSH laboratory strategy, free thyroxine is measured only if the TSH falls outside specific cutoffs, in order to streamline investigations and save unjustified costs. This serum TSH-based approach to both diagnosis and monitoring has been widely accepted by several national and local health services; nevertheless, false-negative or -positive testing may occur, leading to inappropriate management or treatment. This review aims to describe several infrequent causes of increased circulating TSH, including analytical interferences, resistance to TSH, consumptive hypothyroidism, and refractoriness to levothyroxine replacement treatment. We propose a clinical flowchart to aid correct recognition of these various conditions, which represent important potential pitfalls in the diagnosis and treatment of primary hypothyroidism.

Safety injections of nuclear medicine radiotracers: towards a new modality for a real-time detection of extravasation events and 18F-FDG SUV data correction.

BACKGROUND: 18F-FDG PET/CT imaging allows to study oncological patients and their relative diagnosis through the standardised uptake value (SUV) evaluation. During radiopharmaceutical injection, an extravasation event may occur, making the SUV value less accurate and possibly leading to severe tissue damage. The study aimed to propose a new technique to monitor and manage these events, to provide an early evaluation and correction to the estimated SUV value through a SUV correction coefficient. METHODS: A cohort of 70 patients undergoing 18F- FDG PET/CT examinations was enrolled. Two portable detectors were secured on the patients' arms. The dose-rate (DR) time curves on the injected DRin and contralateral DRcon arm were acquired during the first 10 min of injection. Such data were processed to calculate the parameters ΔpinNOR = (DRinmax- DRinmean)/DRinmax and ΔRt = (DRin(t) - DRcon(t)), where DRinmax is the maximum DR value, DRinmean is the average DR value in the injected arm. OLINDA software allowed dosimetric estimation of the dose in the extravasation region. The estimated residual activity in the extravasation site allowed the evaluation of the SUV's correction value and to define an SUV correction coefficient. RESULTS: Four cases of extravasations were identified for which ΔRt [(390 ± 26) µSv/h], while ΔRt [(150 ± 22) µSv/h] for abnormal and ΔRt [(24 ± 11) µSv/h] for normal cases. The ΔpinNOR showed an average value of (0.44 ± 0.05) for extravasation cases and an average value of (0.91 ± 0.06) and (0.77 ± 0.23) in normal and abnormal classes, respectively. The percentage of SUV reduction (SUV%CR) ranges between 0.3% and 6%. The calculated self-tissue dose values range from 0.027 to 0.573 Gy, according to the segmentation modality. A similar correlation between the inverse of ΔpinNOR and the normalised ΔRt with the SUV correction coefficient was found. CONCLUSIONS: The proposed metrics allowed to characterised the extravasation events in the first few minutes after the injection, providing an early SUV correction when necessary. We also assume that the characterisation of the DR-time curve of the injection arm is sufficient for the detection of extravasation events. Further validation of these hypotheses and key metrics is recommended in larger cohorts.

The length of FOXE1 polyalanine tract in congenital hypothyroidism: Evidence for a pathogenic role from familial, molecular and cohort studies.

Introduction: FOXE1 is required for thyroid function and its homozygous mutations cause a rare syndromic form of congenital hypothyroidism (CH). FOXE1 has a polymorphic polyalanine tract whose involvement in thyroid pathology is controversial. Starting from genetic studies in a CH family, we explored the functional role and involvement of FOXE1 variations in a large CH population. Methods: We applied NGS screening to a large CH family and a cohort of 1752 individuals and validated these results by in silico modeling and in vitro experiments. Results: A new heterozygous FOXE1 variant segregated with 14-Alanine tract homozygosity in 5 CH siblings with athyreosis. The p.L107V variant demonstrated to significantly reduce the FOXE1 transcriptional activity. The 14-Alanine-FOXE1 displayed altered subcellular localization and significantly impaired synergy with other transcription factors, when compared with the more common 16-Alanine-FOXE1. The CH group with thyroid dysgenesis was largely and significantly enriched with the 14-Alanine-FOXE1 homozygosity. Discussion: We provide new evidence that disentangle the pathophysiological role of FOXE1 polyalanine tract, thereby significantly broadening the perspective on the role of FOXE1 in the complex pathogenesis of CH. FOXE1 should be therefore added to the group of polyalanine disease-associated transcription factors.

On the use of solid 133Ba sources as surrogate for liquid 131I in SPECT/CT calibration: a European multi-centre evaluation.

INTRODUCTION: Commissioning, calibration, and quality control procedures for nuclear medicine imaging systems are typically performed using hollow containers filled with radionuclide solutions. This leads to multiple sources of uncertainty, many of which can be overcome by using traceable, sealed, long-lived surrogate sources containing a radionuclide of comparable energies and emission probabilities. This study presents the results of a quantitative SPECT/CT imaging comparison exercise performed within the MRTDosimetry consortium to assess the feasibility of using 133Ba as a surrogate for 131I imaging. MATERIALS AND METHODS: Two sets of four traceable 133Ba sources were produced at two National Metrology Institutes and encapsulated in 3D-printed cylinders (volume range 1.68-107.4 mL). Corresponding hollow cylinders to be filled with liquid 131I and a mounting baseplate for repeatable positioning within a Jaszczak phantom were also produced. A quantitative SPECT/CT imaging comparison exercise was conducted between seven members of the consortium (eight SPECT/CT systems from two major vendors) based on a standardised protocol. Each site had to perform three measurements with the two sets of 133Ba sources and liquid 131I. RESULTS: As anticipated, the 131I pseudo-image calibration factors (cps/MBq) were higher than those for 133Ba for all reconstructions and systems. A site-specific cross-calibration reduced the performance differences between both radionuclides with respect to a cross-calibration based on the ratio of emission probabilities from a median of 12-1.5%. The site-specific cross-calibration method also showed agreement between 133Ba and 131I for all cylinder volumes, which highlights the potential use of 133Ba sources to calculate recovery coefficients for partial volume correction. CONCLUSION: This comparison exercise demonstrated that traceable solid 133Ba sources can be used as surrogate for liquid 131I imaging. The use of solid surrogate sources could solve the radiation protection problem inherent in the preparation of phantoms with 131I liquid activity solutions as well as reduce the measurement uncertainties in the activity. This is particularly relevant for stability measurements, which have to be carried out at regular intervals.

Alternatives for the treatment of local advanced disease: electrochemotherapy, limb perfusion, limb infusion, intralesional IL2. What is the role?

Nowadays, melanoma is one of the most common fatal malignancy of young adults. Incidence is increasing, but mortality rates from melanoma have remained stable. In-transit metastases from extremity or trunk melanoma are subcutaneous or cutaneous deposits of melanoma distant from the primary site, but not reaching the draining nodal basin. According to American Joint Committee on Cancer classification of stages based on Tumor, Node, Metastases classification stages IIIb and IIIc are considered local advanced disease and survival outcomes is quite poor, with 5-year survival rates of 24-54%. Loco-regional recurrence is an important risk factor for distant metastatic disease, either synchrone or metachrone. Therapy for this pattern of recurrence is limited and options vary based on the volume and site of disease. Definitive surgical resection remains the preferred therapeutic approach. However, when surgery cannot be performed with a reasonable cosmetic and functional outcome, other options must be utilized. Treatment options are classified as local, regional, or systemic. The choice of therapy depends on the number of lesions, their anatomic location, whether or not they are dermal or subcutaneous, the size, and the presence or absence of extra-regional disease.

Emerging Roles of DLK1 in the Stem Cell Niche and Cancer Stemness.

DLK1 is a maternally imprinted, paternally expressed gene coding for the transmembrane protein Delta-like homologue 1 (DLK1), a non-canonical NOTCH ligand with well-described roles during development, and tumor-supportive functions in several aggressive cancer forms. Here, we review the many functions of DLK1 as a regulator of stem cell pools and tissue differentiation in tissues such as brain, muscle, and liver. Furthermore, we review recent evidence supporting roles for DLK1 in the maintenance of aggressive stem cell characteristics of tumor cells, specifically focusing on central nervous system tumors, neuroblastoma, and hepatocellular carcinoma. We discuss NOTCH -dependent as well as NOTCH-independent functions of DLK1, and focus particularly on the complex pattern of DLK1 expression and cleavage that is finely regulated from a spatial and temporal perspective. Progress in recent years suggest differential functions of extracellular, soluble DLK1 as a paracrine stem cell niche-secreted factor, and has revealed a role for the intracellular domain of DLK1 in cell signaling and tumor stemness. A better understanding of DLK1 regulation and signaling may enable therapeutic targeting of cancer stemness by interfering with DLK1 release and/or intracellular signaling.

FAM83B is involved in thyroid cancer cell differentiation and migration.

FAM83B has been recently identified as an oncogene, but its role in thyroid cancers (TC) is still unclear. We examined the expression of FAM83B and its possible involvement in cell migration and differentiation, in neoplastic/normal thyroid tissues and in TC human cell lines. FAM83B expression in TC varies according to the tumor histotype, being significantly downregulated in more aggressive and metastatic tissues. FAM83B levels in cell lines recapitulate patients' samples variations, and its total and cytoplasmic levels decrease upon the induction of migration, together with an increase in its nuclear localization. Similar variations were detected in the primary tumor and in the metastatic tissues from a follicular TC. FAM83B knock down experiments confirmed its role in thyroid differentiation and cell migration, as demonstrated by the reduction of markers of thyroid differentiation and the increase of the mesenchymal marker vimentin. Moreover, the silencing of FAM83B significantly increased cells migration abilities, while not affecting the oncogenic RAS/MAPK/PI3K pathways. Our data indicate for the first time a role for FAM83B in TC cell differentiation and migration. Its expression is reduced in dedifferentiated tumors and its nuclear re-localization could favour distant migration, suggesting that FAM83B should be considered a possible diagnostic and prognostic biomarker.

Thyroid Cancer Stem-Like Cells: From Microenvironmental Niches to Therapeutic Strategies.

Thyroid cancer (TC) is the most common endocrine malignancy. Recent progress in thyroid cancer biology revealed a certain degree of intratumoral heterogeneity, highlighting the coexistence of cellular subpopulations with distinct proliferative capacities and differentiation abilities. Among those subpopulations, cancer stem-like cells (CSCs) are hypothesized to drive TC heterogeneity, contributing to its metastatic potential and therapy resistance. CSCs principally exist in tumor areas with specific microenvironmental conditions, the so-called stem cell niches. In particular, in thyroid cancer, CSCs' survival is enhanced in the hypoxic niche, the immune niche, and some areas with specific extracellular matrix composition. In this review, we summarize the current knowledge about thyroid CSCs, the tumoral niches that allow their survival, and the implications for TC therapy.

Thyrotropin Receptor p.N432D Retained Variant Is Degraded Through an Alternative Lysosomal/Autophagosomal Pathway and Can Be Functionally Rescued by Chemical Chaperones.

Background: Loss-of-function mutations of thyrotropin receptor (TSHR) are one of the main causes of congenital hypothyroidism. As for many disease-associated G-protein coupled receptors (GPCRs), these mutations often affect the correct trafficking and maturation of the receptor, thus impairing the expression on the cell surface. Several retained GPCR mutants are able to effectively bind their ligands and to transduce signals when they are forced to the cell surface by degradation inhibition or by treatment with chaperones. Despite the large number of well-characterized retained TSHR mutants, no attempts have been made for rescue. Further, little is known about TSHR degradation pathways. We hypothesize that, similar to other GPCRs, TSHR retained mutants may be at least partially functional if their maturation and membrane expression is facilitated by chaperones or degradation inhibitors. Methods: We performed in silico predictions of the functionality of known TSHR variants and compared the results with available in vitro data. Western blot, confocal microscopy, enzyme-linked immunosorbent assays, and dual luciferase assays were used to investigate the effects of degradation pathways inhibition and of chemical chaperone treatments on TSHR variants' maturation and functionality. Results: We found a high discordance rate between in silico predictions and in vitro data for retained TSHR variants, a fact indicative of a conserved potential to initiate signal transduction if these mutants were expressed on the cell surface. We show experimentally that some maturation defective TSHR mutants are able to effectively transduce Gs/cAMP signaling if their maturation and expression are enhanced by using chemical chaperones. Further, through the characterization of the intracellular retained p.N432D variant, we provide new insights on the TSHR degradation mechanism, as our results suggest that aggregation-prone mutant can be directed toward the autophagosomal pathway instead of the canonical proteasome system. Conclusions: Our study reveals alternative pathways for TSHR degradation. Retained TSHR variants can be functional when expressed on the cell surface membrane, thus opening the possibility of further studies on the pharmacological modulation of TSHR expression and functionality in patients in whom TSHR signaling is disrupted.

Current Therapeutic Strategies in BRAF-Mutant Metastatic Colorectal Cancer.

Around 8-12% of patients with advanced colon rectal cancer (CRC) present with BRAF alterations, in particular V600E mutation, which is associated with right-side, poorly differentiated and mucinous type tumors. The presence of BRAF mutation (BRAF-mt) has been identified as a hallmark of poor prognosis and treatment optimization in this patient subgroup is an important goal. Currently, the standard of care is an aggressive strategy involving triplet chemotherapy and anti-VEGF agents, but new therapeutic approaches are emerging. Very promising results have been obtained with targeted therapy combinations, such as anti-BRAF agents plus anti-EGFR agents. Furthermore, around 60% of BRAF-mt patients show a strong association with high microsatellite instability (MSI-H) and immune checkpoint inhibitors could represent the new standard of care for this subgroup. The focus of this review is to summarize current strategies for BRAF-mt CRC treatment and highlight new therapeutic options.

Mechanical Behavior of Different Restorative Materials and Onlay Preparation Designs in Endodontically Treated Molars.

This study evaluated the effect of the combination of three different onlay preparation designs and two restorative materials on the stress distribution, using 3D-finite element analysis. Six models of first lower molars were created according to three preparation designs: non-retentive (nRET), traditional with occlusal isthmus reduction (IST), and traditional without occlusal isthmus reduction (wIST); and according to two restorative materials: lithium-disilicate (LD) and nanoceramic resin (NR). A 600 N axial load was applied at the central fossa. All solids were considered isotropic, homogeneous, and linearly elastic. A static linear analysis was performed, and the Maximum Principal Stress (MPS) criteria were used to evaluate the results and compare the stress in MPa on the restoration, cement layer, and tooth structure (enamel and dentin). A novel statistical approach was used for quantitative analysis of the finite element analysis results. On restoration and cement layer, nRET showed a more homogeneous stress distribution, while the highest stress peaks were calculated for LD onlays (restoration: 69-110; cement layer: 10.2-13.3). On the tooth structure, the material had more influence, with better results for LD (27-38). It can be concluded that nRET design showed the best mechanical behavior compared to IST and wIST, with LD being more advantageous for tooth structure and NR for the restoration and cement layer.