Laboratory Investigation最新文献

Solitary fibrous tumor (SFT) is a rare mesenchymal neoplasm which can arise at any anatomic site and is characterized by recurrent NAB2::STAT6 fusions and metastatic progression in 10-30%. The cell of origin has not been identified. Despite some progress in understanding the contribution of heterogeneous fusion types and secondary mutations to SFT biology, epigenetic alterations in extrameningeal SFT remain largely unexplored, and most sarcoma research to date has focused on the use of methylation profiling for tumor classification. We interrogated genome-wide DNA methylation in 79 SFTs to identify informative epigenetic changes. RNA-seq data from targeted panels and data from the Cancer Genome Atlas (TCGA) were used for orthogonal validation of selected findings. In unsupervised clustering analysis, the top 500 most variable CpGs segregated SFTs by primary anatomic site. Differentially methylated genes (DMGs) associated with primary SFT site included EGFR, TBX15, multiple HOX genes and their cofactors EBF1, EBF3, and PBX1, as well as RUNX1 and MEIS1. Of the 20 DMGs that were interrogated on the RNA-seq panel, twelve were significantly differentially expressed according to site. However, with the exception of TBX15, most of these also showed differential expression according to NAB2::STAT6 fusion type, suggesting that the fusion oncogene contributes to transcriptional regulation of these genes. Transcriptomic data confirmed an inverse correlation between gene methylation and the expression of TBX15 in both SFT and TCGA sarcomas. TBX15 also showed differential mRNA expression and 5' UTR methylation between tumors located in different anatomic sites in TCGA data. In all analyses, TBX15 methylation and mRNA expression retained the strongest association with tissue of origin in SFT and other sarcomas, suggesting a possible marker to distinguish metastatic tumors from new primaries without genomic profiling. Epigenetic signatures may further help to identify SFT progenitor cells at different anatomic sites.

The surge in demand for experimental monkeys has led to a rapid increase in their costs. Consequently, there is a growing need for a cost-effective model of Parkinson's disease (PD) that exhibits all core clinical and pathological phenotypes. Evolutionarily, tree shrews (Tupaia belangeri) are closer to primates in comparison to rodents and could be an ideal species for modeling PD. To develop a tree shrew PD model, we used the 1-methyl-4-phenylpyridinium (MPP⁺), a metabolite derived from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to induce lesions in dopaminergic neurons of the unilateral substantia nigra. The induced tree shrew model consistently exhibited and maintained all classic clinical manifestations of PD for a 5-month period. The symptoms included bradykinesia, rest tremor, postural instability, and about 50% individuals showed apomorphine-induced rotations, a classic phenotype of unilateral PD models. All these are closely resembled the ones observed in PD monkeys. Meanwhile, this model was also sensitive to L-dopa treatment with a dose dependent manner, which suggested that the motor deficits are dopamine dependent. Immunostaining showed a significant loss of dopaminergic neurons (approximately 95%) in the lesioned substantia nigra, which is a crucial PD pathological marker. Moreover, a control group of nigral saline injection did not show any motor deficits and pathological changes. Cytomorphological analysis revealed that the size of nigral dopaminergic neurons in tree shrews is much bigger than that of rodents and is close to that of macaques. The morphological similarity may be an important structural basis for the manifestation of the highly similar phenotypes between monkey and tree shrew PD models. Collectively, in this study we have successfully developed a PD model in a small animal species that faithfully recapitulated the classic clinical symptoms and key pathological indicators of PD monkeys, providing a novel and low-cost avenue for evaluation of PD treatments and underlying mechanisms.

An increasing number of studies have revealed a correlation between tertiary lymphoid structures (TLS) and the outcome of hepatocellular carcinoma (HCC). Nevertheless, the associations between the heterogeneity of cellular composition and the overall survival (OS) in HCC remain unexplored. Herein, we evaluated the cancer tissues from 150 HCC individuals treated at the Tumor Hospital Affiliated with Nantong University using multiplex immunofluorescence to determine the presence and characteristics of TLS and to investigate the relationship between intra-TLS immunologic activity, TLS maturation and intratumoral immune cell infiltration. Prognostic factors influencing the outcome were identified through both univariate and multivariate analyses. Additionally, the levels of cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1) and lymphocyte activation gene-3 (LAG3) were determined, as well as their relationship with TLS features. TLS was detected in 71 (47.3%) of the 150 HCC cases and was related to higher intratumoral infiltration levels of lymphocytes. Additionally, intra-TLS lymphocyte proliferation correlated with that of intratumoral lymphocytes, and the presence of TLS and a high proportion of mature TLS demonstrated a significant correlation with better prognosis (p=0.013 and p=0.03, respectively). Among TLS-positive tumors, a high proportion of B-cells expressing activation-induced cytidine deaminase (AID) and a high proportion of CD8⁺ T-cells expressing CD45RO were significantly related to improved OS (p=0.01 and p<0.001, respectively). Comparatively, a high proportion of CD21⁺CD20⁺ B-cells demonstrated a significant correlation with poorer OS (p<0.002). A markedly reduced number of CTLA4⁺ cells in the stromal regions in TLS-negative tumors was observed compared to TLS-positive tumors (p=0.01). These findings reveal a correlation between TLS presence and improved OS in HCC patients. However, TLS exhibited significant variation in maturation state, T and B cell proliferation, and expression of markers related to B and T cell function. Notably, these characteristics were also found to possess prognostic significance, indicating that certain TLS might hinder tumor immunity by inhibiting immune cells while others may foster antigen-driven immune responses, likely influenced by the composition and functional status of intra-TLS lymphocytes.

Osteosarcoma, predominantly affecting children and adolescents, is a highly aggressive bone cancer with a 5-year survival rate of 65-70%. The spatial dynamics between TAM and other cellular subtypes, including T cells, osteoblasts and osteoclasts, are critical for understanding the complexities of the osteosarcoma tumor microenvironment (TME) and can provide insights into potential immunotherapeutic strategies. Our study employs a pioneering approach that combines deep learning-based digital image analysis with multiplex fluorescence immunohistochemistry (mfIHC) to accurately implement cell detection, segmentation, and fluorescence intensity measurements for in-depth study of the TME. We introduce a novel algorithm for TAM/osteoclast differentiation, crucial for accurate characterization of cellular composition. Our findings reveal distinct heterogeneity in cell composition and spatial orchestration between PD-1 (-/+) and PD-L1 (-/+) patients, highlighting the role of T-cell functionality in this context. Furthermore, our analysis demonstrates the efficacy of nivolumab in suppressing tumor growth and enhancing lymphocyte infiltration without altering the M1/M2 TAM ratio. This study provides critical insights into the spatial orchestration of cellular subtypes within the PD-1/PD-L1 defined osteosarcoma TME. By leveraging advanced mfIHC and artificial intelligence, we underscore the critical role of TAMs and T-cell interactions, proposing new therapeutic avenues focusing on TAM repolarization and targeted immunotherapies, thus underscoring the study's potential impact on improving osteosarcoma treatment.

Circulating tumour cells and liquid biopsy-based biomarkers might one day play a crucial role in the treatment decision process for patients of several cancer entities. However, clinical studies on liquid biopsy approaches revealed distinct detection rates and thus, different risk scoring for patients. This study delves into the comparison of two utilised reverse transcription (RT) enzymes, namely SuperScript™ IV VILO™ (VILO) and Sensiscript (SS), aiming to understand their impact on biomarker detection rates. Prostate cancer cell lines were used to assess detection limits, followed by an investigation of biomarker status in clinical liquid biopsy samples of distinct tumour entities. Our findings highlight the superior reverse transcription efficacy of VILO over SS, commonly used in studies employing the AdnaTest platform. The enhanced efficacy of VILO results in a significantly higher number of patients positive for biomarkers. Clinically, the use of a less sensitive enzyme system may lead to the misclassification of genuinely biomarker-positive patients, potentially altering their prognosis due to inadequate clinical monitoring or inappropriate treatment strategies.

Alcoholic liver disease (ALD) caused by chronic alcohol abuse involves complex processes from steatosis to fibrosis, cirrhosis, and hepatocellular carcinoma, posing a global health issue. Bromodomain protein 4 (BRD4) typically serves as a "reader" modulating the functions of transcription factors involved in various biological processes and disease progression. However, the specific mechanisms underlying alcoholic liver injury remain unclear. Here, we detected aberrant BRD4 expression in the alcohol-induced ALD mouse model of chronic and binge ethanol feeding developed by the National Institute on Alcohol Abuse and Alcoholism (NIAAA model), consistent with the in vitro results in Aml-12 mouse hepatocytes. Blocking and inhibiting BRD4 restored the impaired autophagic flux and lysosomal functions in alcohol-treated Aml-12 cells, whereas BRD4 overexpression reduced the expression levels of autophagy marker and lysosomal genes. Furthermore, mouse BRD4 knockdown, mediated by a short hairpin RNA carried by the adeno-associated virus serotype 8, significantly attenuated the alcohol-induced hepatocyte damage, including lipid deposition and inflammatory cell infiltration. Mechanistically, BRD4 overexpression in alcoholic liver injury inhibited the expression of sirtuin (SIRT)-1 in Aml-12 cells. Chromatin immunoprecipitation and dual-luciferase reporter assays revealed that BRD4 functions as a transcription factor and suppressor, actively binding to the SIRT1 promoter region and inhibiting its transcription. SIRT1 activated autophagy, which was suppressed in alcoholic liver injury via Beclin1 deacetylation. In conclusion, our study revealed that BRD4 negatively regulated the SIRT1/Beclin1 axis and that its deficiency alleviated alcohol-induced liver injury in mice, thus providing a new strategy for ALD treatment.

Alcohol-related liver disease (ALD) affects ∼30% of heavy drinkers and is characterized by liver steatosis, fibrosis, and steatohepatitis. The aggregation of keratins 8 (KRT8) and 18 (KRT18) plays a key role in the formation of Mallory-Denk bodies, a hallmark of ALD. Circulating levels of KRT18 fragments are elevated during ALD, and several KRT8/18 genetic variants have been linked to an increased risk of liver disease. In this study, we explored the relationship between the histologic features of ALD and genetic variants of KRT8/18 in 106 severe patients with ALD from the Hôpitaux Universitaires de Genève. We found a significant over-representation of several KRT8 (rs2070910, rs137898974, rs1065306) and KRT18 (rs17120866, rs1492241) variants located in the noncoding regions of these genes. Increased circulating level of keratins 18 fragments were associated with rs17120866 and alcoholic hepatitis. The combination of several KRT18 variants appeared associated with a poorer prognosis. These results highlight the possible role of KRT18 variants in ALD.