Tissue Samples Of Patients Research Articles

Detection of NRAS Mutation in Kaposiform Lymphangiomatosis Using Cell-free DNA from Plasma

Kaposiform lymphangiomatosis is a rare lymphatic anomaly that occurs in children and is associated with life-threatening complications. The somatic mutation NRAS Q61R (c.182A>G) has been identified in lesional tissue samples of patients with kaposiform lymphangiomatosis, and can be used to differentiate this condition from other complex lymphatic anomalies and to guide treatment. Tissue sampling in this lymphatic anomaly is inherently risky due to its involvement of thoracic and abdominal organs and associated coagulopathy. Analysis of cell-free DNA isolated from blood plasma presents a promising noninvasive diagnostic strategy. Here, we describe the use of this approach using a commercial assay.

A New Risk Score Model Based on Pyrogenic Signatures for the Prediction of Survival and Immune Microenvironment Features in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) accounts for about 40-50% of all lung cancer cases with poor prognoses. Pyroptosis plays important roles in tumor development and anti-tumor processes. This study aims to investigate the prognostic value of pyroptosis-related genes in survival and tumor immune microenvironment (TIME) in LUAD. Three datasets were collected, of which 59 normal samples and 513 LUAD samples as experimental group, 163 LUAD samples for validation analysis, and 43 non-small cell lung cancer (NSCLC) samples included in the immunotherapy cohort. A total of 33 pyrolysis-related genes were included in univariate Cox regression analysis. Five pyroptosis-related genes, including NLRC4, NLRP1, NOD1, PLCG1 and CASP9 were screened using Lasso to construct a pyroptosis-related risk score model. Functional enrichment and immune microenvironment analyses were performed. Another 5 tissue samples of LUAD patients were collected for qRT-PCR validation. According to the median risk score, the samples were divided into high-risk group and low-risk group, and the immune cell infiltration in the low-risk group was significantly higher than that in the high-risk group. Then, a nomogram was established based on clinical characteristics and risk score, which demonstrated high accuracy in 1-year OS. The risk score was significantly correlated with overall survival, immune-cell infiltration, and tumor mutation burden (TMB). qRT-PCR results showed that the expression of pyroptosis-related genes in tissues of LUAD patients was consistent with the trend in the experimental group. The risk score model may predict the overall survival of LUAD patients with good accuracy. Our results also demonstrate effectiveness in evaluating the response to immunosuppressive therapy, and may help improve the overall prognosis and treatment outcome of LUAD.

To evaluate an association between prognostic significance of Moesin with histopathological grading of oral squamous cell carcinoma: A systematic review.

The aim of the present Aetiology/Risk type and Prognostic type of systematic review is to evaluate the value of Moesin as a biomarker of invasiveness in Oral Squamous Cell Carcinoma patients and to review/assess the available evidence regarding the prospective prognostic association between Moesin and histopathological grading of OSCC to enhance the quality of life and survival rate of oral cancer patients. A systematic wide-range literature search was performed by authors (BS, KS, and DK) till October 2022 using both, electronic search media and manual search by hand, searching appropriate journals as per the focussed guiding question and inclusion/exclusion criteria. Major databases such as Scopus, EMBASE, Web of Science, Cochrane central register for controlled trials, PubMed & Google Scholar were conducted by two calibrated reviewers independently to gauge the association between the prognostic significance of Moesin with histopathological grading of oral squamous cell carcinoma. As this study is based on tissue samples of oral squamous cell carcinoma patients, all the selected studies were mostly, cross-sectional studies, and retrospective in nature. The studies were integrated with this review to gauge the association between the prognostic significance of Moesin with histopathological grading of oral squamous cell carcinoma (OSCC). The review included a total of 7 studies with tissue samples of 645 cases. The prime outcome was to assess the immunoexpression of Moesin among the different histopathological grades i.e well-differentiated SCC, moderately differentiated SCC, and poorly differentiated SCC and the subordinate outcome was to consider the extent of strong immunoexpression characteristics (cytoplasmic, membranous and mixed type) in different grades of OSCC as well as to correlate with morbidity, mortality, and/or 5 years or 10 years survival rate. The results were analyzed and presented narratively using the Critical Appraisal Tools developed by the University Of Oxford; Risk of Bias - Cochrane Risk of Bias tool - RoB 2.0, and GRADE-pro (Grading of Recommendations, Assessment, Development, and Evaluations) which rates the features of the evidence as high, moderate, low and very low. The risk of mortality expressed in terms of Hazard ratio has been elicited as a 1.37 times higher rate of mortality in the advanced histopathological stages of the OSCC cases. As the sample size of this review was insignificant, therefore, the authors have incorporated hazard ratios of some other studies of carcinomas in diverse sites in the body to give a flavor of prognostic outcomes of Moesin. It was observed that Moesin expression in Breast cancer and UADT carcinomas have a higher mortality rate as compared to OSCC and lung carcinoma cases and this decree strengthens our conviction that Moesin expression in the cytoplasm of advanced histopathological stages of cancer can be assumed as a sign of poor prognosis in all carcinomas including OSCC patients. A sample of seven studies is inadequate as definite evidence for claiming that Moesin is a strong biomarker of invasiveness in OSCC cases and more clinical trials need to be conducted on the prognostic efficacy of Moesin expression in the various histopathological grades of OSCC cases.

Comparing Tribbles Homolog 3 (TRIB3) Protein Expression Levels with Clinicopathological Characteristics and Survival Among Neuroblastoma Patients.

Tribbles Homolog 3 (TRIB3) is a member of the pseudokinase family of tribbles and acts as an adaptor protein to regulate different cellular processes. Upregulation of TRIB3 expression was shown either as a favorable or an adverse prognostic factor in various adult malignancies. However, TRIB3 expression has not been examined in pediatric cancers. Neuroblastoma is the most common malignant solid tumor of childhood, which affects mostly children under 5 years old. Risk stratification of patients defined by International Neuroblastoma Risk Group was used to determine prognosis and treatment of the disease. This study aimed to examine the relationship between TRIB3 protein expression levels and clinicopathological features and survival of patients. TRIB3 protein expression was analyzed using immunohistochemical staining on formalin-fixed paraffin-embedded tissue samples of neuroblastoma patients (n = 56). Survival analyses were performed with Kaplan-Meier method and log-rank tests. Association between TRIB3 expression and clinicopathological characteristics were analyzed with Spearman's correlation. Of the patients, 32.1% were in the low-risk group, 21.4% in the medium-risk group, and 46.4% in the high-risk group. Survival analysis was performed in the entire neuroblastoma patient group and sub-risk groups of neuroblastoma patients. In the entire patient group, there was no significant difference in overall survival (P = .202) and event-free survival (P = .172) between TRIB3-positive and -negative patients. However, when survival analyses were performed in each risk group, TRIB3 expression was significantly associated with higher overall survival (P = .034) and event-free survival (P = .032) in low-risk group neuroblastoma patients. Nevertheless, no association was found between TRIB3 expression and overall survival (P = .799) and event-free survival (P = .448) in high-risk neuroblastoma patients. Furthermore, a significant correlation was identified between 1p36 loss-of-heterozygosity and TRIB3 expression (P = .030). However, TRIB3 expression did not correlate with other clinicopathological features. TRIB3 expression is a potential predictive biomarker for low-risk neuroblastoma patients.

Detection of mutations and fusions in lung adenocarcinoma using ion torrent sequenc-ing technology

Introduction. Adenocarcinomas are the most common lung tumors and are often diagnosed in advanced stages when the tumor has a polyclonal form with a wide variety of genetic alterations and activated mutational processes. Comprehensive analysis of mutational status from FFPE tissue samples in such patients can provide a therapeutic perspective and contribute considerably to clinical decisions thereby increasing the overall survival rate. 
 Material and methods. 22 genes were analyzed for mutations and 4 genes for fusion transcripts using two Ion AmpliSeq panels. DNA was isolated from paraffin-embedded tissue sections while RNA analysis was performed using two types of samples: paraffin blocks and fresh tumor tissue. Key variant detection and data analysis was performed using next platforms: Ion Reporter, ONCOMINE, R language. 
 Results. The study of 30 tumor samples allowed the detection of 147 mutations and 4 fusions in 19 genes, and the therapeutically actionable variants were associated with different drugs clinically approved or in the phase of clinical trials. The most genetic variants were identified in the TP53, EGFR and NOTCH1 genes with a prevalence of over 50% in the TP53 gene, while all 4 detected fusions (one fusion per sample) represent the association of the ALK gene with other partners: EML4(13)-ALK(20) – present in 2 samples; EML4(6)-ALK(20); and an ALK fusion with an unknown partner gene. 
 Conclusions. Analyzing the mutational status of tumor samples from patients with lung adenocarcinoma it has been ascertained the therapeutic utility of gene panel sequencing covering point mutations, INDELs and SNVs, as well as gene fusions, using FFPE tissue as primary material. This is valid for both targeted monotherapies and combined therapies.

An Analytical Approach of Micro-RNA Interaction Study in Ovarian Cancer

A gynecological malignancy with a high mortality rate and a variety of forms, ovarian cancer is challenging to identify early and prevent. Numerous studies have examined the expression profile of microRNAs (miRNAs) in tissue and serum samples of patients to discover pertinent biomarkers for ovarian cancer. Functional tests also demonstrated that a number of miRNAs had oncogenic or suppressive effects. Despite the invention of various biological markers, mostly mRNA and protein, ovarian cancer mortality remains a problem due to late detection, which is related to low specificities and sensitivities. Recent advancements in expression biology have turned towards this compulsive direction for cancer diagnosis and prognosis; focusing on identification and development of specific and sensitive biomarkers, such as microRNAs (miRNAs). A number of miRNAs, including miR-23b, miR- 146b, miR-200-a/b/c, miR-630, miR-31 influence the epithelial-mesenchymal transition pathway, modulating ovarian cancer cell invasiveness. MiRNAs have such a broad range of functions in ovarian cancer that they have been recommended as potential treatment strategies in the future. In this review, we discuss the role of various micro RNAs and the genes they target in the pathogenesis of ovarian cancer, as well as the potential applications of these molecules as biomarkers.

Sex-based Dysregulation of Inflammation-related Genes in Periodontitis.

Periodontitis is a chronic inflammatory condition affecting a large population all over the world. This condition is linked with abnormal expression of numerous genes. We measured levels of CYFIP1, KDR, RABGGTA, RABGGTB and FOXD2 in gingival tissue and circulation of people with periodontitis and healthy controls. KDR was more expressed in tissue samples of female patients compared with female controls (Ratio of mean expression (RME) =4.16, P=0.02). However, this gene was less expressed in the blood of female patients compared with female control subjects (RME=0.12, P=0.04). RABGGTB was less expressed in the blood of male patients compared with male controls (RME=0.20, P=0.02). Finally, FOXD2 was less expressed in total blood samples compared with total controls (RME=0.3, P<0.001) and in blood samples of female patients compared with female control subjects (RME=0.02, P<0.001). RABGGTA had the best area under curve (AUC) value in differentiation of patients' tissues from normal tissues (AUC=0.60, sensitivity=0.37, specificity=0.92). In distinction of abnormal blood samples from controls, FOXD2 had the best performance (AUC=0.85, sensitivity=0.66, specificity=0.91). In brief, we demonstrated a sex-dependent dysregulation of KDR, RABGGTBand FOXD2 genes in circulation or tissue of patients with periodontitis.

MICROBIOLOGICAL ANALYSIS OF ABDOMINAL CAVITY EXUDATE, BLOOD AND AFFECTED TISSUES SAMPLES FROM PATIENTS WITH INTRA-ABDOMINAL ABSCESSES IN COMPLICATED INFECTION OF ABDOMINAL CAVITY.

The aim: To conduct an analysis of the results of a microbiological examination of biological samples taken from patients with intra-abdominal abscesses. Materials and methods: Material for microbiological examination was collected from 60 patients during surgery and transported to laboratory at the same day. Isolation and identification of microbial pure cultures were performed by standard microbiological methods. Statistical analysis was performed using Statistica software. Results: Analyzing the microbiological research results indicated importance of the sample collecting time (first or repeated surgery). In pa¬tient's blood taken during first surgery it was found a statistically significant predominance of no growth of microflora. In abdominal cav¬ity exudates anaerobic cultures increased statistically significantly in repeated surgery. It was noted that in samples taken during first sur¬gery mixed pathogens were represented mainly by facultative anaerobic cocci, then in repeated surgery anaerobic microorganisms were predominant. Examination of liver abscess content found that monoculture was isolated in 85.7 %. Blood and affected tissue samples in such patients were sterile. Investigation of samples from patients with multiple abdominal cavity abscesses revealed anaerobic microorganisms in 16.7 %. Blood samples of that patients in 40 % were sterile. Conclusions: An analysis showed that in appendicular abscesses content gram-negatives were predominant. Gram-positive bacteria dominated in paravesical abscesses with 65 % isolates from gallbladder and 66.7 % from the affected tissue samples. In liver abscesses gram-positive cocci were isolated in 57.1 %. In multiple abdominal abscesses due to bowel perforation rod-shaped microflora was predominant (76 %) and represented by either obligate aerobes or obligate and facultative anaerobes.

Global and promoter specific hypermethylation of tumor suppressor genes P16, SOCS1, and SHP1 in oral squamous cell carcinoma and oral submucous fibrosis.

Aberrant methylation pattern leads to altered gene expression, that is, involved in the transformation of various cancers, including oral squamous cell carcinoma (OSCC). In the present study, an attempt has been made to examine the association of global and promoter-specific methylation of tumor suppressor genes in patients with OSCC and oral submucous fibrosis (OSMF). Promoter-specific methylation of tumor suppressor genes P16, SOCS1, and SHP1 had been studied earlier for their aberrant methylation patterns in other cancers; however, these studies were mainly conducted in-vitro or in animal models, and as such, only a few studies are available on human samples. In the present study evaluation of promoter-specific methylation of genes P16, SOCS1, and SHP1 in 76 patients' blood and tissue samples was done and compared with methylation of 35 healthy control samples using qPCR. Further, these samples were analyzed for global methylation patterns using ELISA. The results have shown a significant decreasing trend of promoter methylation (OSCC > OSMF > Controls); the methylation indices (MI) were significantly higher in OSCC than in the controls. The median MI of three genes for OSCC were P16MI (0.96), SHP1MI (0.79), and SOCS1 (0.80). Similarly, median MIs for OSMF were P16MI (0.18), SHP1 MI (0.19), and SOCS1 MI (0.5) against controls with MI (0) for each of the three genes. The global methylation %mC values were 1.9, 0.5, and 0.1, respectively. The values of MI and %mC were found to correlate with various risk factors such as tobacco, smoking, and alcohol consumption, which are positively involved in OSMF pathogenesis followed by oral cancer progression. Further, the methylation trend in tissue was reflected in blood samples, proving a window for methylation load to be used as a lesser invasive biomarker. The sensitivity and specificity of methylation load were also found reasonable. Therefore, the current study suggests that there may be a role of global and promoter-specific methylation load in the transition of OSMF to OSCC.

The Role of the Notch Signaling Pathway in the Pathogenesis of Lung Diseases of Non-infectious Etiology

A review of current literature data on the significance of the Notch signaling pathway in the mechanisms of the development of diseases of the respiratory system – chronic obstructive pulmonary disease (COPD), bronchial asthma (BA) and lung cancer is presented. In studies of lung tissue samples of patients with COPD and lung tissues of mice, it was found that activation of the Notch signaling pathway promotes metaplasia and increases the functional activity of goblet cells, protects epithelial cells from apoptosis and oxidative stress. Suppression of the Notch−Jagged1/Jagged2 pathway is associated with the transdifferentiation of club-shaped cells into ciliated ones. In patients with AD, the Notch signaling pathway promotes differentiation of Th2 lymphocytes. In the ovalbumin-induced bronchial asthma model, the Notch cascade increases the imbalance of Th17/Treg lymphocyte populations, the production of IL-4, IL-5, IL-13, IL-17, the formation of allergen-specific IgE, eosinophilic infiltration and metaplasia of goblet-shaped epithelial cells of the respiratory tract. A decrease in the concentration of IgE, Th2-type cytokines (IL-4, IL‑5, IL-13), an increase in the number of Treg cells and the level of TGFß in bronchoalveolar lavage in mice with asthma, mediated by the introduction of dendritic cells expressing the ligands DLL1 and Jagged1, indicates the protective role of the Notch signaling pathway. On samples of tumor tissue and cell lines of non-small cell lung cancer, it was found that an increase in the expression of Notch-1 and Notch-3 mRNA is associated with increased proliferative activity, malignant cell transformation, a high risk of metastasis to lymph nodes and an unfavorable prognosis of the disease. In the samples of tumor tissue of small cell lung cancer, an increase in the expression of the Notch ligand DLK1 signaling inhibitor gene, the Ascl1 transcription factor gene and lysine-specific histone demethylase 1 (LSD1) was recorded. Suppression of LSD1 activity is accompanied by reactivation of signaling via Notch-1 receptor and subsequent inhibition of the transcription factor Ascl1, which induces the initial stages of tumor transformation.

Detection of gene mutation related to non-small cell lung cancer using next-generation sequencing

To date, lung cancer is one of the most well-understood cancers in terms of molecular mechanisms, with high incidence and mortality rates in the population. Detecting lung cancer-related gene mutations plays a vital role in offering targeted therapy, thereby improving the progression-free survival and overall survival rates of patients. This study aims to identify some clinical features and factors related to non-small cell lung cancer and detect some types of gene mutations related to non-small cell lung cancer by new-generation sequencing techniques on biopsy tissue samples of patients. 40 patients with non-small cell lung cancer who were tested to detect gene mutations in biopsy tissue samples by next-generation sequencing. Results showed the majority of patients were male (70%), over 60 years old (65%), in the late stage (100%), and have not received any treatment (85%). 26/40 patients have detected mutations, in which mutations are most frequently in EGFR (27.5%) and KRAS (20.0%), followed by ALK (12.5%), BRAF (5.0%), did not detect mutations on NRAS, ROS1, and PIK3CA. The rate of mutations related to the target drug susceptibility was quite high, no resistant mutants were detected.

Evaluation of Cellular miR-122 Expression in Association with the Presence of Varicella-Zoster Virus among Central Nervous System Tumors

Background: Brain tumors are all primary central nervous system (CNS) tumors with unclear etiologies and viral infections, especially human herpesviruses, which have emerged as a hot topic for comprehensive research. Objectives: The present study aimed at assessing the molecular epidemiology of varicella-zoster virus (VZV) and its association with microRNA 122 (miR-122) expression in CNS tumor samples. Methods: Fresh frozen tissue samples were collected from 60 CNS tumor patients and 45 healthy controls. A nested PCR assay was performed to detect the VZV-DNA. Subsequently, the expression level of miR-122 was evaluated in the CNS tumor tissue samples of patients and the brain tissue samples were obtained from healthy controls, using a real-time PCR assay. Results: Of 60 patients with CNS tumors, 29 were men and 31 were women. VZV-DNA was detected in 13.3% of the CNS tumor tissue specimens. There was no statistically significant association between the presence of VZV-DNA and different types of CNS tumors (P &gt; 0.05). Furthermore, the expression level of miR-122 was significantly downregulated in the CNS tumor tissue samples obtained from the patients compared with those of the healthy controls (P &lt; 0.05). Additionally, the expression level of miR-122 was significantly lower in the VZV-positive tumor samples as compared with those of the VZV-negative tumor samples and the healthy controls. Conclusions: Although VZV plays no direct role in the development of CNS tumors, the virus may affect the biology of CNS tumors by decreasing the expression levels of miR-122, which consequently leads to an increased risk of malignancy. However, the experimental data are not conclusive enough; so, further investigations are needed.

Candidate MicroRNA Biomarkers in Lupus Nephritis: A Meta-analysis of Profiling Studies in Kidney, Blood and Urine Samples.

Lupus nephritis (LN) is a kidney disease caused by systemic lupus erythematosus in which kidneys are attacked by the immune system. So far, various investigations have reported altered miRNA expression profiles in LN patients and different miRNAs have been introduced as biomarkers and/or therapeutic targets in LN. The aim of this study was to introduce a consensus panel of potential miRNA biomarkers by performing a meta-analysis of miRNA profiles in the LN patients. A comprehensive literature review approach was performed to find LN-related miRNA expression profiles in renal tissues, blood, and urine samples. After selecting the eligible studies and performing the data extraction, meta-analysis was done based on the vote-counting rank strategy as well as meta-analysis of p-values. The meta-miRNAs and their related genes were subjected to functional enrichment analyses and network construction. The results of the meta-analysis of 41 studies were three lists of consensus miRNAs with altered expression profiles in the various tissue samples of LN patients (meta-analysis of p-values < 0.05). Of the 13 studies on kidney tissue, the meta-miRNAs were let-7a, miR-198, let-7e, miR-145, and miR-26a. In addition, meta-miRNAs of miR-199a, miR-21, miR-423, miR-1260b, miR-589, miR-150, miR-155, miR-146a, and miR-183 from 21 studies on blood samples, and miR-146a, miR-204, miR-30c, miR-3201, and miR-1273e from 11 studies on urine samples can be considered as non-invasive biomarker panels for LN. Functional enrichment analysis on the meta-miRNA lists confirmed the involvement of their target genes in nephropathy-related signaling pathways. Using a meta-analytical approach, our study proposes three meta-miRNA panels that could be the target of further research to assess their potential as therapeutic targets/biomarkers in LN disease.

Genetic locus copy number variation level and minimally invasive assessment of radiation therapy efficiency in rectal cancer patients

Aim. The screening of molecular genetic markers for a minimally invasive assessment of the radiation therapy effectiveness for rectum malignant tumors.Materials and methods. The study was carried out in 4 stages: 1) bioinformatic analysis of TCGA (The Cancer Ge- nome Atlas) databases using the GISTIC algorithm; 2) validation of bioinformatics analysis data in a model experiment on cell culture; 3) study of genes copy number features validated in a model experiment in patients with different responses to radiation therapy; 4) determination of the gene copy number in cell-free DNA in patientswith different responses to radiation therapy. 100 patients with rectum adenocarcinoma (G1–2), as well as 30 apparently healthy individuals, took part in the work. Radiotherapy was carried out according to the standard protocol (single focal dose 2.4 Gy, total focal dose 54 Gy) on a Novalis TX linear accelerator. The relative copy number of genetic loci was determined by real-time quantitative polymerase chain reaction.Results. Bioinformatic analysis revealed 32 candidate genetic loci. Validation of these markers on irradiated HT-29 cells showed that the copy number of BRCA2, H2AX, CASP9 and RBBP8 genes was increased, while the copy number of BCL2 gene was reduced relative to intact cells. In 74 patients with a partial response to radiation therapy, an increase in the copy number of BRCA2, H2AX, RBBP8 and BCL2 was found, which positively correlated with the copy number of these genes in blood plasma cell-free DNA.Conclusion. The application of an integrated approach based on TCGA database bioinformatic analysis, radiation therapy modeling in cell culture and validation of the identified markers on tissue and blood samples of patients with rectal adenocarcinoma revealed RBBP8, BRCA2, H2AX and BCL2 genes copy number association with the preoperative radiation therapy effectiveness.

The ctDNA-based postoperative molecular residual disease status in different subtypes of early-stage breast cancer.

Breast cancer is a highly heterogeneous disease. Early-stage, non-metastatic breast cancer is considered curable after definitive treatment. Early detection of tumor recurrence and metastasis through sensitive biomarkers is helpful for guiding clinical decision-making and early intervention in second-line treatment, which could improve patient prognosis and survival. In this real-world study, we retrospectively analyzed 82 patients with stages I to III breast cancer who had been analyzed by molecular residual disease (MRD) assay. A total of 82 tumor tissues and 224 peripheral blood samples were collected and detected by next-generation sequencing (NGS) based on a 1,021-gene panel in this study. MRD positivity was detected in 18 of 82 patients (22.0%). The hormone receptor-/human epidermal growth factor receptor 2+ (HR-/HER2+) subgroup had the highest postoperative MRD detection rate at 30.8% (4/13). The BRCA2 and SLX4 genes were significantly enriched in all patients in the MRD positive group and FGFR1 amplification was significantly enriched in the MRD negative group with HR+/HER2-. The number of single nucleotide variants (SNVs) in tissue samples of MRD-positive patients was higher than that of MRD-negative patients (11.94 vs. 8.50 SNVs/sample). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that there was a similar biological function of the tumor-mutated genes in the 2 MRD status groups. This real-world study confirmed that patient samples of primary tumor tissue with different MRD status and molecular subtypes had differential genetic features, which may be used to predict patients at high risk for recurrence.

Extracellular vesicles derived from cancer-associated fibroblasts carry tumor-promotive microRNA-1228-3p to enhance the resistance of hepatocellular carcinoma cells to sorafenib.

Cancer-associated fibroblasts (CAFs)-derived extracellular vesicles (EVs) can promote tumor progression by delivering microRNA (miRNA). Whether EVs could transfer miR-1228-3p into hepatocellular carcinoma (HCC) cells to affect chemoresistance was discussed in this study. Normal fibroblasts (NFs) and CAFs were isolated from tissue samples of HCC patients. We assessed the functions of HCC cells after co-culturing with NFs and CAFs. miR-1228-3p gain-of-function experiments were conducted. Next, functional assays were carried out to determine the binding of miR-1228-3p to placenta associated 8 (PLAC8). In vivo models were constructed for validation. CAFs-derived EVs exerted promoting effect on proliferative, migrating, invading potential of HCC cells and their resistance to sorafenib. PLAC8 was demonstrated as a direct target of miR-1228-3p. By targeting PLAC8, miR-1228-3p activated the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. In addition, the transfer of miR-1228-3p from CAFs-derived EVs into HCC cells boosted chemoresistance of HCC cells, which was reversed by restoring PLAC8. All in all, CAF-EV-carried miR-1228-3p strengthens the chemoresistance of HCC through activating PLAC8-mediated PI3K/AKT pathway. This finding contributes to the development of EV-based therapies overcoming the chemoresistance of HCC.

Cancer-associated fibroblasts-derived exosomes from chemoresistant patients regulate cisplatin resistance and angiogenesis by delivering VEGFA in colorectal cancer.

The purpose of this study was to investigate the effect of chemoresistant cancer-associated fibroblasts (R-CAFs) against cisplatin (DDP) on colorectal cancer (CRC) progression. First, clinical tissue samples of chemoresistant or chemosensitive CRC patients were collected to isolate R-CAFs or chemosensitive CAFs (S-CAFs), respectively. HT29 cells or HUVECs were co-cultured with R-CAFs by transwell device. Then the proliferation and apoptosis of HT29 cells were detected with Cell Counting Kit-8 (CCK-8) and flow cytometry. Transwell assay and tube formation assay was used to detect the migration and angiogenesis of HUVECs. In addition, a colorectal cancer transplantation model was established subcutaneously in nude mice by injecting stably transfected HT29 cells and exosomes from different CAF groups, and then the tumor volume and weight were measured and recorded. Hematoxylin and eosin staining, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining were performed to characterize the histopathological characteristics and apoptosis level of tumor tissues, respectively. S-CAFs and R-CAFs were isolated successfully. HT29 cell co-culture with R-CAFs significantly affected the proliferation and apoptosis of HT29 cells. Exosomes derived from R-CAFs (R-CAFs-Exo) were delivered to HT29 cells, which could induce viability, suppress apoptosis and accelerate the angiogenesis of CRC. In addition, VEGFA was highly expressed in R-CAFs-Exo, which might indicate that R-CAFs could transmit VEGFA through exosomes. Overexpressed VEGFA in R-CAFs apparently regulates the viability, apoptosis, DDP resistance, and angiogenesis of CRC. In-vivo experiments confirmed that R-CAFs-Exo promoted the progression of CRC and DDP resistance by delivering VEGFA . R-CAFs-derived exosomes promote the viability, apoptosis, DDP resistance, and angiogenesis of CRC by delivering VEGFA .

The diagnostic and prognostic potential of the EGFR/MUC4/MMP9 axis in glioma patients

Glioblastoma is the most aggressive form of brain cancer, presenting poor prognosis despite current advances in treatment. There is therefore an urgent need for novel biomarkers and therapeutic targets. Interactions between mucin 4 (MUC4) and the epidermal growth factor receptor (EGFR) are involved in carcinogenesis, and may lead to matrix metalloproteinase-9 (MMP9) overexpression, exacerbating cancer cell invasiveness. In this study, the role of MUC4, MMP9, and EGFR in the progression and clinical outcome of glioma patients was investigated. Immunohistochemistry (IHC) and immunofluorescence (IF) in fixed tissue samples of glioma patients were used to evaluate the expression and localization of EGFR, MMP9, and MUC4. Kaplan–Meier survival analysis was also performed to test the prognostic utility of the proteins for glioma patients. The protein levels were assessed with enzyme-linked immunosorbent assay (ELISA) in serum of glioma patients, to further investigate their potential as non-invasive serum biomarkers. We demonstrated that MUC4 and MMP9 are both significantly upregulated during glioma progression. Moreover, MUC4 is co-expressed with MMP9 and EGFR in the proliferative microvasculature of glioblastoma, suggesting a potential role for MUC4 in microvascular proliferation and angiogenesis. The combined high expression of MUC4/MMP9, and MUC4/MMP9/EGFR was associated with poor overall survival (OS). Finally, MMP9 mean protein level was significantly higher in the serum of glioblastoma compared with grade III glioma patients, whereas MUC4 mean protein level was minimally elevated in higher glioma grades (III and IV) compared with control. Our results suggest that MUC4, along with MMP9, might account for glioblastoma progression, representing potential therapeutic targets, and suggesting the ‘MUC4/MMP9/EGFR axis’ may play a vital role in glioblastoma diagnostics.

Multi-Omics Profiling of Skin Biopsies of Patients with Sclerodermatous Graft-Vs-Host Disease Suggests Therapeutic Potential of Targeting Don't Eat Me Signals

Introduction: Chronic graft-vs-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HCT). This debilitating, chronic condition is characterized by inflammation, cell-mediated and humoral immunity, and ultimately tissue fibrosis. There is currently little or no understanding of the molecular pathogenesis of chronic cGVHD resulting in poor effective treatment strategies. Sclerodermatous GVHD (sclGVHD) is a highly morbid form of cGVHD associated with poor prognosis and low sensitivity to immune suppressive therapy. Understanding the underlying pathophysiology of sclGVHD and identifying the keyfibroticpathways involved iscriticalfor translation into targeted therapies that improve patient care. Methods: To further our understanding of the underlying pathophysiology of sclGVHD we used single cell RNA sequencing analyses on 5 fresh patient biopsy specimens and healthy controls. Human studies were carried out using: i) Patient skin biopsy samples (n =45) and matching them with 5 normal matched skin biopsy controls. These patient tissue samples were used to perform spatial gene expression analyses (10X Vizium) and identify the unique molecular profiles in each SclGVHD sample. ii) ATAC-sequencing analyses was performed on primary dermal fibroblasts from patients with SclGVHD to assess chromatin accessibility patterns genome wide in SclGVHD iii) TMA and tissues from patients were used to validate targets using immunofluorescent staining. iv) Animal studies were conducted to evaluate the therapeutic potential of an immune therapy-based treatment approach with a combined anti-fibrotic/anti-inflammatory strategy, and effectiveness of the treatment was determined with CyTOF studies. Results: To investigate cGVHD immunopathogenesis, normal-appearing skin, lesioned skin, and circulating immune cells from chronic patients were analyzed via single-cell RNA sequencing (figure A). A distinct transcriptional signature, the upregulation of clusters 11, 3, 1 and 17 in Scl cGVHD and the downregulation of clusters 6, 5, 9, 8 was observed. To validate these results in the context of tissue architecture we integrated spatial RNA sequencing on a tissue microarray assembled with 45 skin biopsies of SclGVHD patients to demonstrate a tight correlation between JUN activation and immune checkpoint upregulation (figure B).Hierarchical clustering of ATAC-seq signals revealed that the chromatin landscape was greatly remodeled after JUN deletion (figure C).We blocked CD47 and IL6 in the SclGVHD mouse model to evaluate therapeutic efficacy. We found a significant decrease of dermal thickness and collagen deposition in SclGVHD mice treated with blocking antibodies against IL6 and CD47 but not in untreated mice (figure D). We also detected a noticeable decrease in the expression of CD47, pJUN and IL6 in the treated group (figure E). Analyses of skin tissues from sclerotic lesions in mice using CyTOF revealed different cell populations in the skin clustered in spatially distinct groups. Finally, an increase in total leukocyte as well as a significant decrease in the fibroblast populations was observed (figure F). Conclusion: Our findings are significant because we profile skin biopsies matched with peripheral blood from patients with Scl GVHD with a multiomics approach and identify a unique molecular signature in patients with SclGVHD thus providing a rationale for subsequent combined interventions targeting innate immunity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Omics Analyses Identify the TIM-3 Ligand Galectin-9 As Novel Immunotherapy Target for Chronic Lymphocytic Leukemia

Failure of response to immunotherapy including checkpoint inhibitors or CAR-T-cell therapy in chronic lymphocytic leukemia (CLL) has been linked to dysfunctional effector T cells. Using single-cell analyses, we performed an in-depth characterization of the T-cell compartment in blood and tissue samples of CLL patients and mouse models to gain insights into the spectrum of phenotypes and transcriptional programs of T cells and the underlying mechanisms of their development. By mass cytometry (CyTOF) using 35 antibodies, we characterized T cells in blood (n=8), bone marrow (n=3), and lymph nodes (n=21) of CLL patients, as well as reactive lymph nodes of non-tumor patients (n=13). Integrative analyses of all data sets allowed us to identify and quantify 15 clusters of CD4+ and 14 clusters of CD8+ T cells. First, our data showed that T cells in blood and bone marrow are similar, but clearly distinct from lymph node derived cells. Second, we observed an accumulation of several regulatory T-cell subsets, as well as T cells harboring an exhausted and dysfunctional phenotype in the lymph node samples, and a positively correlated abundance of these T-cell subsets. Third, an increased frequency of regulatory and exhausted T cells was detected in CLL in comparison to non-malignant lymph nodes. We further performed single-cell RNA-sequencing generating transcriptome and T-cell receptor (TCR) data of T cells from lymph nodes of CLL patients (n=5) and spleen samples of the Eµ-TCL1 mouse model (n=3). These data confirmed the presence of several exhausted T-cell clusters in CLL lymph nodes and spleens of Eµ-TCL1 mice, and allowed for the identification and transcriptional characterization of terminally exhausted T cells and their precursor state (Figure A). By integrating single-cell TCR data, a clonal expansion mainly of the precursor exhausted T cells was observed, suggesting their reactivity for CLL cells. Finally, we used the single-cell transcriptome data for interactome analyses and identified both known and novel ligand-receptor-interactions between CLL and different T-cell clusters with a presumable function in survival and growth support of CLL cells, but also in suppression of T cells. Among the latter, we focused on galectin-9 which is expressed by CLL cells in patients and mice, and known as ligand for the immunoregulatory receptor TIM-3. We treated mice that had developed CLL after injection of Eµ-TCL1 leukemic cells with galectin-9 blocking antibodies and showed that this treatment slowed down disease development (Figure B). Altogether, our study provides a detailed characterization of the T-cell compartment in CLL that helps to understand T-cell exhaustion and suggests the TIM-3 ligand galectin-9 as novel target for immunotherapy in CLL. MZ, EM, JP and MS are co-senior authors of this study. Figure: A) UMAP of single-cell transcriptome data of T cells from lymph node samples of CLL patients (n=5) displaying a precursor exhaustion score described by Guo et al. in Nat.Med. in 2018. B) Mice that have developed CLL-like disease after adoptive transfer (AT) of Eµ-TCL1 tumor cells were treated with blocking anti-galectin-9 (α-GAL9) or isotype antibodies. CLL cell counts were monitored in blood over time. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal