Incidence of Postoperative Flare-Up After Single-Visit and Multi-visit Endodontic Therapy.

This article presents results obtained from a survey, including patients who underwent endodontic treatment by the single-visit or multi-visit method, after confirmation of the diagnosis of chronic apical periodontitis. OBJECTIVE: The aim of the survey was to obtain data from the studied patients on the frequency and the type of postoperative pain after treatment of chronic apical periodontitis, as well as whether there is a relation between gender, age, and postoperative pain. MATERIALS AND METHODS: A visual analog scale was used to study the intensity of postoperative pain in the treatment of teeth diagnosed with CPP, which are treated by one of two methods - single-visit or multi-visit method. The total number of surveyed patients is 71. The patients were examined and treated at the Dental Clinic "Imperial" in Varna, Bulgaria, in 2020. Thirty-one of them were treated by the single-visit method, and the remaining 40 by the multi-visit method with placement of a temporary dressing or sterile swab. RESULTS: A relatively large proportion (70%) of patients reported mild pain immediately after the root canal filling. A relatively large proportion (90.3%) of patients did not report pain one week after the root canal filling. The more frequent symptoms were observed in cases treated by the multi-visit method, after the application of a temporary dressing. Patients who reported taking analgesics were treated in the multi-visit method. More frequent pain symptoms with both methods of treatment were observed in men aged 36-60 years. CONCLUSION: Although exacerbation has been shown to have no significant effect on the outcome of endodontic treatment, it is highly undesirable. In the short term, the postoperative pain in patients treated by the multi-visit method through the use of intracanal medication is more pronounced. Patients receiving the single-visit treatment reported less postoperative pain.

NUMB as a Therapeutic Target for Melanoma.

The upregulation of the adaptor protein NUMB triggers melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Although NUMB acts as a tumor suppressor in various human cancer types, little is known about its role in melanoma. In this study, we investigated the role of NUMB in melanoma progression and its regulatory mechanism. Analysis of The Cancer Genome Atlas melanoma datasets revealed that high NUMB expression in melanoma tissues correlates with improved patient survival. Moreover, NUMB expression is downregulated in metastatic melanoma cells. NUMB knockdown significantly increased the invasion potential of melanoma cells in a three-dimensional collagen matrix in vitro and in the lungs of a mouse model in vivo; it also significantly upregulated the expression of the NOTCH target gene CCNE. Previous studies suggested that Wnt signaling increases NUMB expression. By mimicking Wnt stimulation through glycogen synthase kinase-3 inhibition, we increased NUMB expression in melanoma cells. Furthermore, a glycogen synthase kinase-3 inhibitor reduced the invasion of melanoma cells in a NUMB-dependent manner. Together, our results suggest that NUMB suppresses invasion and metastasis in melanoma, potentially through its regulation of the NOTCH‒CCNE axis and that the inhibitors that upregulate NUMB can exert therapeutic effects in melanoma.

Neural Crest-Like Stem Cell Transcriptome Analysis Identifies LPAR1 in Melanoma Progression and Therapy Resistance.

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell-like pathways hijacked by tumor cells. SIGNIFICANCE: This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.

Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34+ cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγnull (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3+ Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8+/Granz B+ T cells homeostasis are observed in tumor regions where FOXP3+ Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.

MSX1-Induced Neural Crest-Like Reprogramming Promotes Melanoma Progression.

Melanoma cells share many biological properties with neural crest stem cells. Here we show that the homeodomain transcription factor MSX1, which is significantly correlated with melanoma disease progression, reprograms melanocytes and melanoma cells toward a neural crest precursor-like state. MSX1-reprogrammed normal human melanocytes express the neural crest marker p75 and become multipotent. MSX1 induces a phenotypic switch in melanoma, which is characterized by an oncogenic transition from an E-cadherin-high nonmigratory state toward a ZEB1-high invasive state. ZEB1 up-regulation is responsible for the MSX1-induced migratory phenotype in melanoma cells. Depletion of MSX1 significantly inhibits melanoma metastasis in vivo. These results show that neural crest-like reprogramming achieved by a single factor is a critical process for melanoma progression.

Tumor-associated B-cells induce tumor heterogeneity and therapy resistance.

In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.

Targeting Notch enhances the efficacy of ERK inhibitors in BRAF-V600E melanoma.

The discovery of activating BRAF mutations in approximately 50% of melanomas has led to the development of MAPK pathway inhibitors, which have transformed melanoma therapy. However, not all BRAF-V600E melanomas respond to MAPK inhibition. Therefore, it is important to understand why tumors with the same oncogenic driver have variable responses to MAPK inhibitors. Here, we show that concurrent loss of PTEN and activation of the Notch pathway is associated with poor response to the ERK inhibitor SCH772984, and that co-inhibition of Notch and ERK decreased viability in BRAF-V600E melanomas. Additionally, patients with low PTEN and Notch activation had significantly shorter progression free survival when treated with BRAF inhibitors. Our studies provide a rationale to further develop combination strategies with Notch antagonists to maximize the efficacy of MAPK inhibition in melanoma. Our findings should prompt the evaluation of combinations co-targeting MAPK/ERK and Notch as a strategy to improve current therapies and warrant further evaluation of co-occurrence of aberrant PTEN and Notch activation as predictive markers of response to therapy.

UV-Induced Wnt7a in the Human Skin Microenvironment Specifies the Fate of Neural Crest-Like Cells via Suppression of Notch.

Multipotent stem cells with neural crest-like properties have been identified in the dermis of human skin. These neural crest stem cell (NCSC)-like cells display self-renewal capacity and differentiate into neural crest derivatives, including epidermal pigment-producing melanocytes. NCSC-like cells share many properties with aggressive melanoma cells, such as high migratory capabilities and expression of the neural crest markers. However, little is known about which intrinsic or extrinsic signals determine the proliferation or differentiation of these neural crest-like stem cells. Here we show that, in NCSC-like cells, Notch signaling is highly activated, similar to melanoma cells. Inhibition of Notch signaling reduced the proliferation of NCSC-like cells, induced cell death, and downregulated noncanonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells. In three-dimensional skin reconstructs, canonical Wnt signaling promoted the differentiation of NCSC-like cells into melanocytes. This differentiation was triggered by the endogenous Notch inhibitor Numb, which is upregulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes. Together, these data reveal a cross talk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells.