Case report: A patient with brachio-cervical inflammatory myopathy was misdiagnosed as flail arm syndrome.

Brachio-cervical inflammatory myopathy (BCIM) is a rare inflammatory myopathy characterized by dysphagia, bilateral upper limb atrophy, limb-girdle muscle weakness, and myositis-specific antibody (MSA) negativity. BCIM has a low incidence and is commonly associated with autoimmune diseases. We present a case report of a 55-year-old man with progressive upper limb weakness and atrophy, diagnosed with flail arm syndrome (FAS). The initial electromyography revealed extensive spontaneous muscle activity and increased duration of motor unit potentials (MUPs). During follow-up, evidence of myogenic damage was observed, as indicated by a decreased duration of MUPs in the right biceps muscle. Laboratory and genetic testing ruled out hereditary or acquired diseases. Negative serological antibodies for myasthenia gravis. Hereditary or acquired diseases were ruled out through laboratory and genetic testing. Whole-body muscle magnetic resonance imaging (MRI) showed extensive edema and fat replacement in the bilateral upper limbs, scapular, and central axis muscles, while the lower extremities were relatively mildly affected. Muscle biopsy revealed numerous foci of inflammatory cells distributed throughout the muscle bundle, with predominant CD20, CD138, and CD68 expression, accompanied by a light infiltration of CD3 and CD4 expression. The muscle weakness improved with the combination of oral prednisone (initially 60 mg/day, tapered) and methotrexate (5 mg/week) treatment.

Lipid droplet accumulation mediates macrophage survival and Treg recruitment via the CCL20/CCR6 axis in human hepatocellular carcinoma.

Metabolic changes play a crucial role in determining the status and function of macrophages, but how lipid reprogramming in macrophages contributes to tumor progression is not yet fully understood. Here, we investigated the phenotype, contribution, and regulatory mechanisms of lipid droplet (LD)-laden macrophages (LLMs) in hepatocellular carcinoma (HCC). Enriched LLMs were found in tumor tissues and were associated with disease progression in HCC patients. The LLMs displayed immunosuppressive phenotypes (with extensive expression of TREM2, PD-L1, CD206, and CD163) and attenuated the antitumor activities of CD8+ T cells. Mechanistically, tumor-induced reshuffling of cellular lipids and TNFα-mediated uptake of tumoral fatty acids contribute to the generation of triglycerides and LDs in macrophages. LDs prolong LLM survival and promote CCL20 secretion, which further recruits CCR6+ Tregs to HCC tissue. Inhibiting LLM formation by targeting DGAT1 and DGAT2, which catalyze the synthesis of triglycerides, significantly reduced Treg recruitment, and delayed tumor growth in a mouse hepatic tumor model. Our results reveal the suppressive phenotypes and mechanisms of LLM enrichment in HCC and suggest the therapeutic potential of targeting LLMs for HCC patients.

Retraction Notice to: circIFT80 Functions as a ceRNA of miR-1236-3p to Promote Colorectal Cancer Progression.

[This retracts the article DOI: 10.1016/j.omtn.2019.08.024.].

CD163+ macrophage density in perimysial connective tissue associated with prognosis in IMNM.

OBJECTIVE: The pathological features of immune-mediated necrotizing myopathy (IMNM) are dominated by the infiltration of macrophages. We aimed to perform a histopathologic semiquantitative analysis to investigate the relationship between macrophage markers and prognosis. METHODS: Semiquantitative analysis of histologic features was performed in 62 samples of IMNM. Independent risk factors were identified through univariate and multivariate regression analysis. Cluster analysis was performed using the partitioning around the medoids (PAM) method. Decision tree modeling was utilized to efficiently determine cluster labels for IMNM patients. The validity of the developmental cohort was assessed by accuracy in comparison with the validation cohort. RESULTS: The most enriched groups in patients with IMNM were macrophages expressing CD206 and CD163. In the multivariate logistic regression model, the high density of CD163+ macrophages in perimysial connective tissue increased the risk of unfavorable prognosis (p = 0.025, OR = 1.463, 95% CI: 1.049-2.041). In cluster analysis, patients in Cluster 1, with lower CD163+ macrophage density and inflammatory burden, had a more favorable prognosis. Conversely, patients in Cluster 3, which were enriched for CD163+ macrophages in the perimysial connective tissue, had the most severe clinical features and the worst prognosis. Correlations were found between the density of CD163+ macrophages in connective tissue and symptom duration (R2 = 0.166, p < 0.001), dysphagia (p = 0.004), cardiac involvement (p = 0.021), CK (R2 = 0.067, p = 0.042), CRP (R2 = 0.117, p < 0.001), and ESR (R2 = 0.171, p < 0.001). CONCLUSION: The density of CD163+ macrophages in perimysial connective tissue may serve as a potential marker for the prediction of IMNM prognosis.

Platycodon D protects human nasal epithelial cells from pyroptosis through the Nrf2/HO-1/ROS signaling cascade in chronic rhinosinusitis.

BACKGROUND: Pyroptosis has been demonstrated being closely associated with the inflammatory progression in chronic rhinosinusitis (CRS). However, platycodon D (PLD) has emerged as a key anti-inflammatory mediator in the inflammatory progression of various respiratory diseases. This study aims at investigating whether PLD could reduce inflammatory progression of CRS by inhibiting pyroptosis. METHODS: Nasal mucosal tissues from patients with CRS and the control group (simple nasal septal deviation) were analyzed for morphological difference using hematoxylin & eosin staining and for the expression of pyroptosis-related makers by immunofluorescence (IF). Human nasal epithelial cells (HNEpCs) were cultured and co-stimulated with lipopolysaccharide (LPS)/adenosine triphosphate (ATP) to construct an in vitro cellular model simulating CRS. After pretreatment with PLD, EthD-I staining, TUNEL staining, transmission electron microscopy (TEM), and GSDMD-NT detection were performed to evaluate pyroptosis markers. The NLRP3 inflammasome was detected by IF and western blotting (WB). Reactive oxygen species (ROS) were detected by H2DCFDA staining, and mitochondrial membrane potential was evaluated by JC-1 staining. Mitochondrial morphology and structure were observed using TEM. The Nrf2/HO-1 antioxidant signaling pathway was detected using WB. RESULTS: The nasal mucosa structure of patients with CRS exhibited significant damage, with a marked increase in the expression of pyroptosis-related proteins compared with the control group. LPS/ATP co-stimulation resulted in an increased expression of IL-18 and IL-1ß in HNEpCs, causing significant damage to nuclear and cell membranes, GSDMD-NT accumulation around the cell membrane, and intracellular NLRP3 inflammasome activation. Furthermore, it led to increased ROS expression, significantly decreased mitochondrial membrane potential, and damaged mitochondrial structure. However, pretreatment with PLD significantly reversed the aforementioned trends and activated the Nrf2/HO-1 antioxidant signaling pathway. CONCLUSIONS: The results of this study confirm that NLRP3-mediated pyroptosis plays a crucial role in the pathological process of nasal mucosal impairment in patients with CRS. PLD inhibits NLRP3-mediated pyroptosis, preventing inflammatory damage in HNEpCs of patients with CRS by activating the Nrf2/HO-1 antioxidant signaling pathway, which in turn reduces ROS production and ameliorates mitochondrial damage.

C/EBPα mediates the maturation and antitumor functions of macrophages in human hepatocellular carcinoma.

Recent studies have suggested that therapeutic upregulation of CCAAT/enhancer binding protein α (C/EBPα) prevents hepatocellular carcinoma (HCC) progression. However, the mechanisms underlying this outcome are not fully understood. In this study, we investigated the expression and functional roles of C/EBPα in human HCC, with a focus on monocytes/macrophages (Mφs). Paraffin-embedded tissues were used to visualize C/EBPα expression and analyze the prognostic value of C/EBPα+ monocytes/Mφs in HCC patients. The underlying regulatory mechanisms were examined using human monocyte-derived Mφs. The results showed that the expression of C/EBPα on monocytes/Mφs was significantly decreased in intra-tumor tissues compared to the corresponding peri-tumor tissues. C/EBPα+ monocytes/Mφs displayed well-differentiation and antitumor capacities, and the accumulation of these cells in tissue was associated with antitumor immune responses and predicted longer overall survival (OS) of HCC patients. Mechanistic studies demonstrated that C/EBPα was required for Mφ maturation and HLA-DR, CD169 and CD86 expression, which initiates antitumor cytotoxic T-cell responses; however, these effects were inhibited by monocyte autocrine IL-6- and IL-1ß-induced suppression of mTOR1 signaling. Reprogramming Mφs via the upregulation of C/EBPα may provide a novel strategy for cancer immunotherapy in patients with HCC.

Corrigendum: C-reactive protein is an indicator of the immunosuppressive microenvironment fostered by myeloid cells in hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fonc.2021.774823.].

Cholesterol Efflux Drives the Generation of Immunosuppressive Macrophages to Promote the Progression of Human Hepatocellular Carcinoma.

Cholesterol is often enriched in tumor microenvironment (TME); however, its impact on disease progression varies in different tissues and cells. Monocytes/macrophages (Mφ) are major components and regulators of the TME and play pivotal roles in tumor progression and therapeutic responses. We aimed to investigate the profile, effects, and regulatory mechanisms of Mφ cholesterol metabolism in the context of human hepatocellular carcinoma (HCC). Here, we found that patients with high serum levels of cholesterol had shorter survival times and lower response rates to anti-PD-1 treatment. However, the cholesterol content in tumor-infiltrating monocytes/Mφ was significantly lower than that in their counterparts in paired nontumor tissues. The expression of the cholesterol efflux transporter, ABCA1, was upregulated in tumor monocytes/Mφ, and ABCA1 upregulation positively associated with decreased cellular cholesterol content and increased serum cholesterol levels. Mechanistically, autocrine cytokines from tumor-treated monocytes increased LXRα and ABCA1 expression, which led to the generation of immature and immunosuppressive Mφ. Although exogenous cholesterol alone had little direct effect on Mφ, it did act synergistically with tumor-derived factors to promote ABCA1 expression in Mφ with more immunosuppressive features. Moreover, high numbers of ABCA1+ Mφ in HCC tumors associated with reduced CD8+ T-cell infiltration and predicted poor clinical outcome for patients. Our results revealed that dysregulated cholesterol homeostasis, due to the collaborative effects of tumors and exogenous cholesterol, drives the generation of immunosuppressive Mφ. The selective modulation of cholesterol metabolism in Mφ may represent a novel strategy for cancer treatment.

miR-210 Expression Is Strongly Hypoxia-Induced in Anaplastic Thyroid Cancer Cell Lines and Is Associated with Extracellular Vesicles and Argonaute-2.

Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2% O2). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia.

Deciphering the sequential changes of monocytes/macrophages in the progression of IDD with longitudinal approach using single-cell transcriptome.

Intervertebral disk degeneration (IDD) is a chronic inflammatory disease with intricate connections between immune infiltration and oxidative stress (OS). Complex cell niches exist in degenerative intervertebral disk (IVD) and interact with each other and regulate the disk homeostasis together. However, few studies have used longitudinal approach to describe the immune response of IDD progression. Here, we conducted conjoint analysis of bulk-RNA sequencing and single-cell sequencing, together with a series of techniques like weighted gene co-expression network analysis (WGCNA), immune infiltration analysis, and differential analysis, to systematically decipher the difference in OS-related functions of different cell populations within degenerative IVD tissues, and further depicted the longitudinal alterations of immune cells, especially monocytes/macrophages in the progression of IDD. The OS-related genes CYP1A1, MMP1, CCND1, and NQO1 are highly expressed and might be diagnostic biomarkers for the progression of IDD. Further landscape of IVD microenvironment showed distinct changes in cell proportions and characteristics at late degeneration compared to early degeneration of IDD. Monocytes/macrophages were classified into five distinct subpopulations with different roles. The trajectory lineage analysis revealed transcriptome alterations from effector monocytes/macrophages and regulatory macrophages to other subtypes during the evolution process and identified monocytes/macrophage subpopulations that had rapidly experienced the activation of inflammatory or anti-inflammatory responses. This study further proposed that personalized therapeutic strategies are needed to be formulated based on specific monocyte/macrophage subtypes and degenerative stages of IDD.

NudCD1 as a prognostic marker in colorectal cancer and its role in the upregulation of cellular spindle assembly checkpoint genes and LIS1 pathways.

OBJECTIVE: To investigate the role of NudCD1 in spindle assembly checkpoint regulation and in the prognosis of colorectal cancer.  METHODS: Immunohistochemical staining was used to detect in situ expression of NudCD1 in 100 colorectal cancer tissue samples. A chi-square test was used to analyse the correlation between the NudCD1 protein expression level of the cancer tissues and clinicopathological features. The Kaplan-Meier survival analysis was used to assess the correlation between the NudCD1 mRNA expression and the three-year survival of patients with colorectal cancer. The impact of NudCD1 on the development of colorectal cancer and the underlying molecular mechanisms were assessed by flow cytometry cell cycle and apoptosis assays after lentiviral overexpression of NudCD1 in two colorectal cancer cell lines. Quantitative real-time PCR was used to assess mRNA expression of the cellular spindle assembly checkpoint genes BUB1, BUBR1, MAD1, CDC20 and MPS1, as well as the downstream genes LIS1, DYNC1H1, and DYNLL1 in the NudC/LIS1/dynein pathway. RESULTS: Compared with normal intestinal tissue (8.00% with high expression), the expression of NudCD1 protein in colorectal cancer tissue was significantly higher (58.00% with high expression, P < 0.01). In addition, expression of NudCD1 significantly correlated with the degree of tumour differentiation and the TNM staging (P < 0.01), as well as the depth of invasion of the primary tumour and lymph node metastasis (P < 0.05). However, there was no correlation with gender, age, tumour site, gross type, tumour size or distant metastasis. The Kaplan-Meier survival analysis showed that patients with high NudCD1 expression in colorectal cancer tissues had a significantly shorter survival time than those with low expression of NudCD1 (P < 0.01). Compared with the transfection of the empty vector, colon cancer HT-29 cells with overexpressed NudCD1 had significantly increased mRNA levels of BUBR1, MPS1 and LIS1. The DNA synthesis phase (S phase) was significantly shorter in cells overexpressing NudCD1 than in the control group (43.83% ± 1.57%, P < 0.05), while there was no difference in apoptosis in the two groups. CONCLUSION: NudCD1 can serve as a valuable prognostic marker for colorectal cancer. It may be involved in the regulation of spindle-assembly checkpoint-gene expression and the LIS1 pathway of colorectal cancer cells.

A retrotransposon insertion in MUTL-HOMOLOG 1 affects wild rice seed set and cultivated rice crossover rate.

Wild rice (Oryza rufipogon) has a lower panicle seed setting rate (PSSR) and gamete fertility than domesticated rice (Oryza sativa), but the genetic mechanisms of this phenomenon remain unknown. Here, we cloned a null allele of OsMLH1, an ortholog of MutL-homolog 1 to yeast and mammals, from wild rice O. rufipogon W1943 and revealed a 5.4-kb retrotransposon insertion in OsMLH1 is responsible for the low PSSR in wild rice. In contrast to the wild-type, a near isogenic line NIL-mlh1 exhibits defective crossover (CO) formation during meiosis, resulting in reduced pollen viability, partial embryo lethality, and low PSSR. Except for the mutant of mismatch repair gene postmeiotic segregation 1 (Ospms1), all other MutL mutants from O. sativa indica subspecies displayed male and female semi-sterility similar to NIL-mlh1, but less severe than those from O. sativa japonica subspecies. MLH1 and MLH3 did not contribute in an additive fashion to fertility. Two types of MutL heterodimers, MLH1-PMS1 and MLH1-MLH3, were identified in rice, but only the latter functions in promoting meiotic CO formation. Compared to japonica varieties, indica cultivars had greater numbers of CO events per meiosis. Our results suggest that low fertility in wild rice may be caused by different gene defects, and indica and japonica subspecies have substantially different CO rates responsible for the discrepancy between the fertility of mlh1 and mlh3 mutants.

Biological characterization of the melanin biosynthesis gene Bcscd1 in the plant pathogenic fungus Botrytis cinerea.

The gray mold fungus Botrytis cinerea produces dark-colored conidia and sclerotia due to deposition of melanin on the cell wall of these structures. However, the role of melanin biosynthesis on development and function of conidia and sclerotia have not been well elucidated in this fungus. This study disrupted the melanin biosynthesis gene Bcscd1 (for scytalone dehydratase) in the wild type B05.10, and the resulting mutants were compared with B05.10 and complementary mutants (COM) for growth and development, virulence and response to biotic/abiotic stresses. Three disruption mutants were obtained, and they did not differ from B05.10 and COM in mycelial growth rate on potato dextrose agar, however, they formed brownish conidia and scleotia deficient in melanogenesis, whereas B05.10 and COM formed grayish conidia and black sclerotia with normal melanogenesis. The disruption mutants were as aggressive as B05.10 and COM in infection of tobacco leaves. TEM observation showed that the disruption mutant ΔScd1-85 formed numerous tiny grooves in the conidial cell wall, thereby causing uneven thickness in the cell wall. In contrast, B05.10 and COM rarely formed tiny grooves in their conidial cell wall with even thickness. Moreover, the sclerotial cortex cell wall of ΔScd1-85 lost rigidity and the cells became collapsed, whereas the sclerotial cortex cell wall of B05.10 and COM appeared rigid, and the cells appeared plump in shape. The disruption mutants were more sensitive than B05.10 and COM in response to chemical stresses (H2O2, NaCl, SDS, sorbitol) for conidial germination and sclerotial survival. The sclerotia of the disruption mutants were more susceptible than the sclerotia of B05.10 and COM to infection by the mycoparasite Trichoderma koningiopsis. These results confirmed previous studies about the effect of melanin production on pathogenicity of B. cinerea, and expanded our knowledge about the role of Bcscd1 in cell wall integrity and in response to biotic and abiotic stresses.

Notch signaling regulates vessel structure and function via Hspg2.

The abnormal structure of tumor blood vessels is an important reason for the low efficacy of anti-tumor drugs. Notch signaling is an evolutionarily highly conserved signaling pathway that plays an important role in vessel development. However, the role and mechanism of Notch signaling in the formation of vascular structure is not fully understood. In this study, we demonstrated that blocking Notch signaling in endothelial cells (ECs) leads to obstructed tumor blood vessel basement membrane formation and the reduction of blood perfusion, as well as blood-retinal barrier (BRB) and blood-brain barrier (BBB) destruction in healthy mice. Endothelial Notch overactivation exacerbates the increases in tumor blood vessel basement membrane and blood perfusion ratio, and promotes recruitment of retinal vascular smooth muscle cells in neonatal mice. Notch signaling also regulates the formation of adhesion junctions (AJs) in ECs. In addition, we confirmed that Notch signaling regulates the AJs of ECs by regulating the expression of downstream gene Hspg2. This research is of great theoretical and practical significance for understanding the mechanism of tumor vascular structure formation as well as the search for new targets for vascular-targeted therapy.

Retrospective analysis of cancer-specific gene expression panel for thyroid fine needle aspiration specimens.

BACKGROUND: While molecular testing is a promising strategy for preoperative assessment of cytologically indeterminate thyroid nodules, thyroid fine needle aspiration biopsy (FNA) presents unique challenges for molecular assays, including contaminating peripheral blood mononuclear cells (PBMC) and variable numbers of evaluable epithelial thyroid cells. Moreover, the newly recognized entity, noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), has added an additional challenge to the currently available molecular diagnostic platforms. New diagnostic tools are still needed to correctly distinguish benign and malignant thyroid nodules preoperatively. METHODS: Twenty-two transcript splice variants from 12 genes we previously identified as discriminating benign from malignant thyroid nodules were characterized in 80 frozen thyroid tumors from 8 histological subtypes. Isoforms detectable in PBMC were excluded, and the 5 most discriminating isoforms were further validated by real-time quantitative PCR (qPCR) on intraoperative FNA samples from 59 malignant tumors, 55 benign nodules, and 23 NIFTP samples. The qPCR threshold cycle values for each transcript were normalized to the thyrocyte-specific thyroid peroxidase isoform 1 (TPO1) and z-transformed. Receiver operating characteristic (ROC) analyses of the composite transcript scores were used to evaluate classification of thyroid FNAs by the 5-gene isoform expression panel. RESULTS: A molecular signature was developed by combining expression levels of specific isoforms of CDH3, FNDC4, HMGA2, KLK7, and PLAG1. FNAs containing at least 12-36 thyrocytes were sufficient for this assay. The 5-gene composite score achieved an area under the ROC curve (AUC) of 0.86 for distinguishing malignant from benign nodules, with a specificity of 91%, sensitivity of 75%, negative predictive value of 91%, and positive predictive value of 74%. CONCLUSION: Our newly developed 5-gene isoform expression panel distinguishes benign from malignant thyroid tumors and, may help distinguish benign from malignant thyroid nodules in the context of the new NIFTP subtype.

C-Reactive Protein Is an Indicator of the Immunosuppressive Microenvironment Fostered by Myeloid Cells in Hepatocellular Carcinoma.

BACKGROUND: C-reactive protein (CRP) is a widely used marker of systemic inflammation and predicts poor clinical outcomes in patients with hepatocellular carcinoma (HCC); however, its significance in the local immune response at the tumor site is not clear. METHODS: Serum CRP levels of 329 HCC patients were detected before resection. Paired paraffin-embedded tumor samples were used to quantify immune cell populations, such as CD11b+ myeloid cells, CD68+ macrophages (Mφs), CD15+ neutrophils, CD8+ T cells, and CD206+, CD204+, CD163+ and CD169+ Mφs, by immunohistochemistry. Enrichment scores for 34 types of immune cells based on transcriptome data from 24 HCC samples were calculated by xCell. Overall survival of patients was analyzed using the Kaplan-Meier method. RESULTS: Serum CRP levels were correlated with liver functions and tumor stages in patients with HCC. The densities of CD68+ tumor-associated macrophages (TAMs) and CD15+ tumor-associated neutrophils (TANs) were significantly higher in patients with elevated serum CRP levels than in those with low CRP levels (both p < 0.0001). Further analysis of TAM subtypes revealed that serum CRP levels were associated with CD204+ and CD163+ Mφ densities (p < 0.0001 and p = 0.0003, respectively). Moreover, transcriptome data showed that CRP expression was associated with the expression of myeloid cell infiltration-related genes in HCC tumors. The combination of serum CRP with TAMs or TANs in both the nontumor and intratumor regions could represent a powerful criterion for predicting patient prognoses. CONCLUSION: Serum CRP could serve as an indicator of an immunosuppressive TME in HCC, which could be of potential clinical application for treatment strategies targeting the TME.

Tumor-derived adenosine promotes macrophage proliferation in human hepatocellular carcinoma.

BACKGROUND & AIMS: Macrophages (Mϕ) represent a major component of tumor tissues and play an important role in both tumor progression and therapeutic response. Although tumor Mϕ are generally considered to be derived from circulating monocytes, emerging evidence indicates that tissue Mϕ pools can be maintained by self-renewal. We aimed to elucidate the contribution, phenotype, and regulatory mechanisms of proliferating Mϕ in human hepatocellular carcinoma (HCC). METHODS: Flow cytometry analyses were performed to examine the presence and phenotype of proliferating Mϕ in fresh HCC tissues. Dual immunofluorescence staining was applied to analyze the prognostic value of proliferating Mϕ. The underlying regulatory mechanisms were examined using human monocyte-derived Mϕ. RESULTS: Tumor-infiltrating Mϕ exhibited a significantly higher proliferative capacity than Mϕ in non-tumor tissues. A higher level of Mϕ proliferation was positively correlated with Mϕ density in the tumor and a poor prognosis in patients with HCC. Proliferating Mϕ were less differentiated (with increased CD206 expression) and were induced by the tumor cell-derived soluble small molecule, adenosine, but not proteins, lipids, or large peptides. Mechanistic studies demonstrated that autocrine granulocyte-macrophage colony-stimulating factor (GM-CSF) released by tumor-stimulated Mϕ could enhance A2A receptor expression on Mϕ and function synergistically with adenosine to elicit Mϕ proliferation in HCC. CONCLUSIONS: Local Mϕ proliferation is an important mechanism for Mϕ accumulation in HCC tissues. Tumor-derived adenosine functions synergistically with autocrine GM-CSF released from activated Mϕ, which promotes Mϕ proliferation. Thus, selective modulation of Mϕ accumulation at the source may provide a novel strategy for cancer therapy. LAY SUMMARY: Tumor-associated macrophages (TAMs) have been reported to play an essential role in both tumor progression and therapeutic response. A fundamental understanding of the mechanisms that regulate macrophage accumulation in tumors will undoubtedly lead to the development of strategies to target macrophages with high specificity and efficiency. The current study unveils a novel mechanism by which local proliferation is linked to macrophage accumulation in the tumor milieu, identifying potential targets for future immune-based anticancer therapies.

Prognostic value of tumor infiltrating lymphocytes combined with PD-L1 expression for patients with solitary colorectal cancer liver metastasis.

BACKGROUND: The aim of this study was to assess the prognostic value of CD8+ tumor infiltrating lymphocytes (TIL) combined with programmed cell death-ligand 1 (PD-L1) expression for patients with solitary colorectal cancer liver metastasis (SCLM) undergoing R0 resection. METHODS: Patients undergoing curative hepatectomy for SCLM were reviewed. Immunohistochemical multiplex technique was used for quantifying CD8+ TIL, and immunohistochemical staining was used for assessing PD-L1 expression. The tumor immune microenvironment (TIME) was classified as strong for high CD8+ TIL and low PD-L1, weak for low CD8+ TIL and high PD-L1, and mild for the rest. Recurrence-free survival (RFS) and overall survival (OS) was compared between these groups. RESULTS: Among the 94 patients included, a high CD8+ TIL and high PD-L1 expression was observed in 51 (54.3%) and 47 (50.0%) patients, respectively. Strong, mild, and weak TIME was observed in 24 (25.5%), 42 (44.7%), and 28 (29.8%) patients, respectively. Patients with a high CD8+ TIL had a significant longer RFS than patients with a low CD8+ TIL (3-year RFS rate, 71.6% vs. 55.3%, P=0.018). The 3-year RFS rate in the strong TIME group was significantly higher than that in the mild and weak TIME groups (89.5% vs. 71.7% and 28.8%, P<0.001), as was the 3-year rate of OS (93.8% vs. 81.8% and 61.6%, P<0.001). CD8+ TIL combined with PD-L1 expression showed better predicting accuracy for RFS than CD8+ TIL alone. CONCLUSIONS: The density of CD8+ TIL combined with PD-L1 expression in liver metastasis was a predictor of RFS for patients with SCLM undergoing R0 resection, and therefore can be used for guiding the postoperative treatment of these patients.

Characterization of Allele-Specific Regulation of Telomerase Reverse Transcriptase in Promoter Mutant Thyroid Cancer Cell Lines.

Background: Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation. Methods: Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA (GA binding protein transcription factor subunit alpha) and MYC, as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). Results: We found the mutant TERT promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated that the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, TERT exhibits MAE from the mutant allele only. Conclusions: In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous TERT mutations.