Tissue Microarray Technology Research Articles

Expression of histone methyltransferase WHSC1 in invasive breast cancer and its correlation with clinical and pathological data

BackgroundThe WHSC1 protein facilitates specific dimethylation of histone H3 at the K36 position, enhancing gene transcription and expression. Studies have confirmed its high expression in diverse malignant tumors. We aimed to identify novel molecular markers to assess the biological behavior of breast cancer cells. MethodsWe conducted a comprehensive analysis of mRNA expression in breast cancer and adjacent tissues based on TCGA data. We enrolled 141 breast cancer patients treated at the First Affiliated Hospital of Fujian Medical University between 2012 and 2016. Patient clinical information and pathological specimens were obtained. We utilized tissue microarray (TMA) technology. We employed the chi-square test for between-group comparisons, with p < 0.05 indicating statistical significance. Furthermore, we analyzed the associations between WHSC1 expression and clinical or pathological data. ResultsWHSC1 mRNA expression was significantly higher in breast cancer tissues than in adjacent tissues (p < 0.001). Moreover, high WHSC1 protein expression in breast cancer was associated with several important clinical parameters, such as pathological type (p = 0.007), high Ki67 expression(Ki67＞20 %) (p < 0.001), lymph node metastasis (p < 0.001), T stage (p = 0.011), N stage (p < 0.001), postoperative pathological stage (p < 0.001), premenopausal status (p = 0.004), and positive HER2 status (p < 0.001). Multivariate regression analysis showed that high WHSC1 expression, elevated Ki67 levels, and positive HER2 status were independent risk factors for axillary lymph node metastasis in breast cancer patients. ConclusionWHSC1 protein expression is upregulated in breast cancer patients and represents an independent risk factor influencing axillary lymph node metastasis, highlighting its potential significance as a strong candidate biomarker.

Evaluation of Ki67 Biomarker as a Prognostic Marker in Breast Invasive Ductal Carcinoma in Khartoum State in Sudan

Introduction: Worldwide, breast cancer is the most prevalent cancer among women to be diagnosed, and it is the primary cause of cancer-related mortality, coming in second only to lung cancer. High levels of Ki67, a nuclear marker of cell proliferation, in breast cancer are linked to worse outcomes. Methods and materials: This retrospective cross-sectional laboratory investigation aimed to examine Ki67 expression as a prognostic predictor in invasive ductal carcinoma (IDC) utilizing manual tissue microarrays (MTMAs) technology. The study was done from June 2018 to July 2019 at the Elrahman Health Centre in Khartoum, Sudan, using thirty-five paraffin block samples collected from patients previously diagnosed with invasive ductal carcinoma (IDC). The study population ranged in age from 31 to 71 years. Results: The study found that 94.3% (n=33/35) of the tissues were positive for the Ki67 antigen, while 5.7% (n=2/35) were negative. Age and score correlation is (P=0.047), and a favorable prognosis could be the cause of the two unfavorable results. Conclusion: This study highlights the importance of the Ki67 biomarker as a prognostic indicator in invasive ductal carcinoma (IDC) of the breast. High levels of Ki67 expression (94.3%) were associated with more aggressive tumors and poorer prognostic outcomes. However, there was no significant correlation between Ki67 scores and patient age, indicating age does not influence the prognostic value of Ki67 in this cohort.

BOC targets SMO to regulate the Hedgehog pathway and promote proliferation, migration, and invasion of glioma cells

The purpose of this study was to investigate the effects of BOC on glioblastoma cells and its underlying mechanisms. In vitro, BOC-knockdown was performed in glioma cell lines. CCK-8 and Transwell were used to assess the impact of BOC on the viability, invasion, and migration of gliobma cells. RNA-seq technology was employed to analyze the differential gene expression between BOC-knockdown glioma cells and the control group, and qRT-PCR was used to validate the expression of downstream differential genes. SMO-overexpression was performed to investigate the effects of SMO on glioma cells. A BOC-knockdown mouse subcutaneous tumor model was to verify the effects of BOC on mouse tumors. Tissue microarray technology was used to detect the expression of BOC and SMO in samples of normal human brain tissue and glioma tissue. In vitro, BOC-knockdown inhibited the viability, invasion, and migration of glioma cells, as well as downregulated the expression of downstream differential genes SMO, EGFR, HRAS, and MRAS. Conversely, SMO-overexpression upregulated the viability, invasion, and migration abilities of BOC-knockdown cells. In vivo, BOC-knockdown suppressed tumor growth in mice and downregulated the expression of downstream differential genes SMO, EGFR, HRAS, and MRAS. Tissue microarray results showed that both BOC and SMO were highly expressed in glioma tissues. BOC is aberrantly overexpressed in glioma patients and promotes glioma development. Mechanistically, BOC activates the Hedgehog (Hh) and RAS signaling pathways by upregulating the expression of SMO, EGFR, HRAS, and MRAS, thereby facilitating the Proliferation, invasion and migration of glioma cells.

Constructing high-density tissue microarrays with a novel method and a self-made tissue-arraying instrument.

Tissue microarrays (TMAs), also called tissue chips, contain hundreds to thousands of tissue cores obtained from different tissue donor blocks. By using TMA technology, a molecular marker, such as protein, RNA or DNA, can be simultaneously examined in hundreds of different specimens under the same experimental conditions. A growing number of previous studies have introduced different methods for constructing TMAs. Many authors tried to use various methods to implant more tissue cores in a single recipient block, and most of these methods involved reducing the diameter of the tissue cores and/or the spacing between adjacent tissue cores. However, when creating TMAs, it is difficult to reduce the distance between tissue cores to zero except with extremely expensive automatic TMA arrayers. Here, we introduce a novel method to construct a high-density TMA that does not have spacing between the tissue cores. We also introduce a method for preparing a self-made tissue-arraying instrument. With this method and the tissue-arraying instrument, we successfully created a TMA containing 126 tissue cores that were 2mm in diameter. H&E staining and immunohistochemical staining were performed on the sections cut from the TMA without any tissue spot loss. This method is easy to operate, and the materials for creating the tissue-arraying instrument are inexpensive and can be purchased anywhere. Therefore, this method can be applied in all laboratories.

Prognostic Value and Immune Infiltration of HPV-Related Genes in the Immune Microenvironment of Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma.

Mounting evidence has highlighted the immune environment as a critical feature in the development of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). However, the relationship between the clinical characteristics of the immune environment and CESC remain unclear. Therefore, the aim of this study was to further characterize the relationship between the tumor and immune microenvironment and the clinical features of CESC using a variety of bioinformatic methods. Expression profiles (303 CESCs and three control samples) and relevant clinical data were obtained from The Cancer Genome Atlas. We divided CESC cases into different subtypes and performed a differential gene expression analysis. In addition, gene ontology (GO) and gene set enrichment analysis (GSEA) were performed to identify potential molecular mechanisms. Furthermore, data from 115 CESC patients from East Hospital were used to help identify the relationship between the protein expressions of key genes and disease-free survival using tissue microarray technology. Cases of CESC (n = 303) were divided into five subtypes (C1-C5) based on their expression profiles. A total of 69 cross-validated differentially expressed immune-related genes were identified. Subtype C4 demonstrated a downregulation of the immune profile, lower tumor immune/stroma scores, and worse prognosis. In contrast, the C1 subtype showed an upregulation of the immune profile, higher tumor immune/stroma scores, and better prognosis. A GO analysis suggested that changes in CESC were primarily enriched nuclear division, chromatin binding, and condensed chromosomes. In addition, GSEA demonstrated that cellular senescence, the p53 signaling pathway, and viral carcinogenesis are critical features of CESC. Moreover, high FOXO3 and low IGF-1 protein expression were closely correlated with decreased clinical prognosis. In summary, our findings provide novel insight into the relationship between the immune microenvironment and CESC. As such, our results may provide guidance for developing potential immunotherapeutic targets and biomarkers for CESC.

Assessment of HER2/neu among Female with Invasive Ductal Breast Carcinoma, Using Manual Tissue Microarray Technology in Khartoum State, Sudan

The human epidermal growth factor receptor-2 (HER2), which is expressed in large amount in 20%-30% of breast cancer patients, is associated with disease recurrences, shortened disease survival, and bad prognosis. The aim of this study is to evaluate the expression of (Her2/neu) receptor as a diagnostic and prognostic marker in Invasive Ductal Breast cancer in Sudanese female patients. The study was performed on 35 archival paraffin blocks previously diagnosed as invasive ductal breast cancer (IDC), then a second diagnosis was done using IHC staining (Her2/neu). The obtained information is arranged in a standard master sheet, then analyzed using a statistical package for social science (SPSS) version 20 computer program. The age 30-45years was the most frequent age group 48.6% (17/35), followed by 46-60 years 40% (14/35). The majority of samples were grade III 74.3% (26/35) and the samples of grade II were 25.7% (9/35). the majority of TMA results were positive for Her2/neu receptor 65.7% (23/35) and the negative results were 34.3% (12/35) the majority of TMA score were (+) 31.4% (11/35), followed by (++) 17.2% (6/35), then (+++) 11.4% (4/35), then (++++) 5.7% (2/35), and the negative result were, 34.3% (12/35). There was a significant relation between H&amp;E diagnosis with TMA result for Her2/ neu diagnosis that the P-value (0.0002). There was an insignificance relation between H&amp;E diagnosis of grades with TMA results for Her2/ neu diagnosis that the P-value (0.08). In conclusion, HER2 is over-expressed in 65.7% of the patient while internationally it is over-expressed in 15-30%. This provides valuable prognostic and predictive implications in order to choose the suitable treatment for the patients.

The Application of the Tissue Microarray (TMA) Technology to Analyze Cerebral Organoids.

"Multi-Omics" technologies have contributed greatly to the understanding of various diseases by enabling researchers to accurately and rapidly investigate the molecular circuitry that connects cellular systems. The tissue-engineered, three-dimensional (3D), in vitro disease model "organoid" integrates the "omics" results in a model system, elucidating the complex links between genotype and phenotype. These 3D structures have been used to model cancer, infectious disease, toxicity, and neurological disorders. Here, we describe the advantage of using the tissue microarray (TMA) technology to analyze human-induced pluripotent stem cell-derived cerebral organoids. Compared with the conventional processing of individual samples, sectioning and staining of TMA slides are faster and can be automated, decreasing labor and reagent costs. The TMA technology faithfully captures cell morphology variations and detects specific biomarkers. The use of this technology can scale up organoid research results in at least two ways: (1) in the number of specimens that can be analyzed simultaneously and (2) in the number of consecutive sections that can be produced for analysis with different probes and antibodies.

Preparation, construction and high-throughput automated analysis of human brain tissue microarrays for neurodegenerative disease drug development.

A major challenge in the treatment of neurodegenerative disorders is the translation of effective therapies from the lab to the clinic. One approach to improve this process is the use of human brain tissue microarray (HBTMA) technology to aid in the discovery and validation of drug targets for brain disorders. In this protocol we describe a platform for the production of high-quality HBTMAs that can be used for drug target discovery and validation. We provide examples of the use of this platform and describe detailed protocols for HBTMA design, construction and use for both protein and mRNA detection. This platform requires less tissue and reagents than single-slide approaches, greatly increasing throughput and capacity, enabling samples to be compared in a more consistent way. It takes 4 d to construct a 60 core HBTMA. Immunohistochemistry and in situ hybridization take a further 2 d. Imaging of each HBTMA slide takes 15 min, with subsequent high-content analysis taking 30 min-2 h.

Atractylenolide-I Suppresses Tumorigenesis of Breast Cancer by Inhibiting Toll-Like Receptor 4-Mediated Nuclear Factor-κB Signaling Pathway.

Background: Toll-like receptor 4 (TLR4) is an essential sensor related to tumorigenesis, and overexpression of TLR4 in human tumors often correlates with poor prognosis. Atractylenolide‐I (AT-I), a novel TLR4-antagonizing agent, is a major bioactive component from Rhizoma Atractylodes Macrocephalae. Emerging evidence suggests that AT-I exerts anti-tumor effects on various cancers such as colorectal cancer, bladder cancer and melanoma. Nevertheless, the effects of AT-I on mammary tumorigenesis remain unclear. Methods: In order to ascertain the correlation of TLR4/NF-κB pathway with breast cancer, the expression of TLR4 and NF-κB in normal breast tissues and cancer tissues with different TNM-stages was detected by human tissue microarray and immunohistochemistry technology. The effects of AT-I on tumorigenesis were investigated by cell viability, colony formation, apoptosis, migration and invasion assays in two breast cancer cells (MCF-7 and MDA-MB-231), and N-Nitroso-N-methylurea induced rat breast cancer models were developed to evaluate the anti-tumor effects of AT-I in vivo. The possible underlying mechanisms were further explored by western blot and ELISA assays after a series of LPS treatment and TLR4 knockdown experiments. Results: We found that TLR4 and NF-κB were significantly up-regulated in breast cancer tissues, and was correlated with advanced TNM-stages. AT-I could inhibit TLR4 mediated NF-κB signaling pathway and decrease NF-κB-regulated cytokines in breast cancer cells, thus inhibiting cell proliferation, migration and invasion, and inducing apoptosis of breast cancer cells. Furthermore, AT-I could inhibit N-Nitroso-N-methylurea-induced rat mammary tumor progression through TLR4/NF-κB pathway. Conclusion: Our findings demonstrated that TLR4 and NF-κB were over expressed in breast cancer, and AT-I could suppress tumorigenesis of breast cancer via inhibiting TLR4-mediated NF-κB signaling pathway.

Human epidermal growth factor receptor 2 fluorescence in situ hybridization and P57KIP2 immunohistochemistry using tissue microarray: Improving histopathological subtyping of hydatidiform mole

IntroductionTrophoblastic neoplasia is detected in approximately 25% of complete hydatidiform moles (CMs) and 0.5% of partial hydatidiform moles (PMs). Hydatidiform mole (HM) subtyping is important to properly monitor and predict patient outcomes. Ploidy studies generally involve diploid CMs and triploid PMs. P57KIP2, expressed in the maternal genome, is usually not detected in CM. We determined whether HER2 FISH and p57 immunostaining contributed to the histopathological classification of HMs. MethodsThis retrospective cohort study focused on patients diagnosed with HM by histopathological examination who were followed up at a trophoblastic disease center from 2002 to 2017. Pathological samples of 108 products of conception were reviewed and reclassified according to detailed criteria. Tissue microarray technology (TMA) was used for p57 KIP2 immunostaining and HER2 FISH analysis. ResultsHistopathological review showed 57 (53%) CMs, 47 (43%) PMs and 4 (4%) inconclusive cases. P57 immunostaining revealed 59 (55%) negative and 22 (20%) positive specimens, and 27 (25%) were inadequate for analysis. FISH HER2 detected 68 (63%) diploid and 33 (30%) triploid cases; two (2%) had oncogene amplification. The three strategies led to a diagnostic change in 28 samples (26%). The final diagnosis was CM in 75 cases (70%) and PM in 30 (28%); three cases remained inconclusive. DiscussionTMA is a cost-saving method that allows the simultaneous study of large case series. The combination of histopathology, HER2 FISH and p57 tests can be useful for accurately differentiating CM and PM, thus providing additional information on disease prognosis.

Jab1 promotes gastric cancer tumorigenesis via non-ubiquitin proteasomal degradation of p14ARF.

Jab1 has been reported to regulate various proteins in signal transduction pathways and be implicated in carcinogenesis or tumor progression. However, the precise role and molecular mechanism of Jab1 in gastric tumorigenesis have not yet been fully elucidated. Jab1 staining in gastric cancer tissues and paired non-cancerous tissues was measured using tissue microarray (TMA) technology. The impact of Jab1 on tumor growth in vivo was analyzed using xenotransplantation experiments in Balb/c mice. The expression of Jab1 and p14ARF in gastric cancer cells was analyzed by western blot and confocal immunofluorescence. CCK-8 and cell cycle experiment were used to evaluate the cell proliferation. Ubiquitination assay was performed to validate whether ubiquitination is involved in Jab1-mediated p14ARF degradation. The expression level of protein p14ARF was inversely correlated with the protein level of Jab1. Then, we investigated the mechanism that how Jab1 induced p14ARF depletion. Mechanistic studies showed that Jab1 induced ubiquitin-independent proteasomal p14ARF degradation in gastric cancer cells. Our data demonstrated that Jab1 protein was a vital upstream negative modulation factor of p14ARF, and Jab1 could promote cell proliferation and tumor growth via inhibiting the expression of p14ARF in vivo and in vitro. Moreover, silencing Jab1 protein expression declined tumor growth and further increased the apoptosis rate of gastric cancer cells. In further studies of gastric cancer specimens, we found the increased level of Jab1 protein shortened the overall survival. Jab1 is upstream of p14ARF and promote gastric cancer cell proliferation in vitro and in vivo. Furthermore, Jab1 decreased the expression of p14ARF though ubiquitination independent proteasomal degradation. Therefore, the connection of Jab1 and p14ARF may provide new methods for the treatment of gastric cancer.

Cyclin D1 Gene Numerical Imbalances in Laryngeal Squamous Cell Carcinoma: A Tissue Microarray Grid Based Analysis.

Background:Deregulation of critical proteins involved in cell cycle stability, such as cyclins, is a frequent genetic event in the development and progression of solid malignancies. Concerning laryngeal squamous cell carcinoma (LSCC), cyclin D1 oncogenic transformation leads to an aberrant protein expression and seems to affect the biological behaviour of the neoplasm. The aim of this study was to determine the correlation of cyclin D1 numerical imbalances with the corresponding protein expression levels in LSCCs. Material and Method:Using tissue microarray (TMA) technology, fifty (n=50) histologically confirmed primary LSSCs were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) analyses were applied. Concerning the screening process in CISH slides, a novel real-time reference and calibration grid platform was implemented. Results:Protein overexpression was observed in 22/50 (44%) cases; whereas, gene amplification was seen in 13/50 (26%) cases (p=0.02). Combined protein/ gene deregulation was associated with the stage of malignancy (p= 0.0014, p=0.001), whereas overall protein expression was strongly correlated with the grade of tumour (p= 0.001). Conclusion: Cyclin D1 gene amplification led to aberrant protein expression in LSCCs and it was also correlated with an aggressive biological behaviour. To best of our knowledge, this study was the first described grid based CISH analysis under conventional bright field microscopy for detecting gene numerical imbalances while providing a novel and accurate description for screening-mapping process in the entire slide area.

"Analysis of immunohistochemical expression of epidermal growth factor receptor in oral squamous cell carcinoma using tissue microarray technology and whole sections:" A comparative study.

Background:Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers. Epidermal growth factor receptor (EGFR) dysregulation is associated with essentially all of the key features of cancer. Tissue microarrays (TMAs) allow the simultaneous analysis of many tumours using small-diameter cores sampled from larger blocks of tissue. Hence present study was taken up to validate TMA technology.Aims and Objectives:To analyse and compare the immunohistochemical (IHC) expression of EGFR in OSCC using TMA technology and in whole tissue sections.Materials and Methods:Study included 34 cases of OSCC.Three tissue cores, each 1 mm in diameter were placed into a recipient paraffin block using a precision microarray instrument finally containing 102 spots. EGFR expression was analysed. Agreement between whole sections and TMA scores was analysed using Cohen's weighted Kappa.Results:EGFR expression was seen in 61.8% of whole section cases. In TMA out of 102 cores 75.50 % of the disks were confirmed to represent an adequate amount of tumor tissue. In TMA 48.5% cases showed EGFR expression.The EGFR expression of whole.Conclusion:Some OSCC express high EGFR and this expression may be an independent Wfactor of certain clinico-pathological variables. TMA may be used as an adjunct with conventional method of evaluation of OSCC especially in larger sample sized studies keeping in mind its limitations.

CSN6 promotes the cell migration of breast cancer cells by positively regulating Snail1 stability.

Background: CSN6, a subunit of the highly conserved constitutive photomorphogenesis 9 (COP9) signalosome (CSN), has been reported to be implicated in tumor progression in various kinds of malignant tumors. However, the mechanism underlying CSN6 in the tumor development of breast cancer has not yet been fully elucidated.Methods: CSN6 staining in breast cancer tissues and paracancerous tissues was measured by tissue microarray (TMA) technology. The metastatic effect of CSN6 was measured by cell migration assay. Co-immunoprecipitation study was used to show the interaction between the protein CSN6 and Snail1. Ubiquitination assay was performed to validate whether ubiquitination is involved in the upregulation of Snail1 by CSN6. The impact of CSN6 on tumor metastasis in vivo was analyzed using xenotransplantation experiments in BALB/c mice.Results: Here, we demonstrated that CSN6 expression was dramatically increased in breast cancer tissues compared with paired adjacent cancerous tissues. CSN6 promoted the cell migration and wound healing abilities in breast cancer cell lines. Also we showed that CSN6 associates with Snail1 and enhances Snail1 protein level by inhibiting the ubiquitin-mediated degradation of Snail1. Thus, CSN6 is involved in positively regulating the stability of Snail1. We further proved that CSN6 protein level was positively correlated with the Snail1 expression in xenograft model.Conclusion: These findings provide new insight into applicability of using the CSN6-Snail1 axis as a potential therapeutic target in breast cancer.

Immunohistochemical analysis of the expression of cancer-associated fibroblast markers in esophageal cancer with and without neoadjuvant therapy.

Esophageal carcinoma (EC) is one of the most aggressive human malignancies with high rates of resistance to conventional anticancer treatment. Cancer-associated fibroblasts (CAFs) are an important part of the tumor microenvironment and associated with tumor progression. COL11A1, SPARC, and CD90 have been identified as rather specific CAF markers, with COL11A1 expression particularly shown to influence response to chemotherapy. We investigated the impact of CAFs in esophageal cancer with a special focus on response to neoadjuvant treatment (nTX). Two collections of esophageal carcinomas were investigated: 164 cases treated with primary resection and 256 cases receiving nTX before resection. The expression of CAF markers was determined using next-generation tissue microarray (ngTMA®) technology and immunohistochemistry. The presence of COL11A1 and SPARC in fibroblasts within both primary resected cases and nTX-treated cases was associated with unfavorable clinicopathological variables such as higher (y)pT category and lymphatic invasion (p<0.001 each). The presence of COL11A1-positive CAFs was associated with worse overall survival in primary resected cases (HR: 2.162, p = 0.004, CI 95% 1.275-3.686). While in tumors showing regression after nTX, COL11A1-positive CAFs were detected less frequently, SPARC-positive CAFs were enriched after nTX, in both responding and non-responding patients (p < 0.001). Our results support the concept of CAFs as an important factor of tumor promotion and maintenance in EC. The population of CAFs increases with tumor progression and decreases, partly depending on the subtype, after regression following nTX. CAFs may serve as potential target for future therapeutic approaches for these highly aggressive tumors.

Expression and significance of c-kit and epithelial-mesenchymal transition (EMT) molecules in thymic epithelial tumors (TETs).

To investigate the expression and significance of c-kit and epithelial-mesenchymal transition (EMT) molecules (E-cadherin, N-cadherin, Twist, Snail) in thymic epithelial tumors (TETs). The tissue microarray technology and immunohistochemistry MaxVisionTM-use kit were used to detect the expression of c-kit and EMT molecular markers in 150 cases of paraffin sections of TET tissue and analysis the correlation between c-kit and EMT molecules and explore the malignancy and the relationship of clinicopathological parameters between c-kit, EMT molecules and TETs. The expression difference of c-kit and EMT molecular markers (E-cadherin, N-cadherin, Snail, Twist) in TETs subtypes was statistically significant (P<0.01) and their positive expression rate of thymic carcinoma was significantly higher than that in thymoma, and the difference was statistically significant, respectively (P<0.01). There is a negative correlation between the expression of c-kit and E-cadherin as well as a positive correlation between the expression level of c-kit, N-cadherin, Twist, and Snail. Furthermore, E-cadherin was negatively correlated with N-cadherin, Twist, and Snail while N-cadherin expression was positively correlated with Twist, Snail. Five indicators (c-kit, E-cadherin, N-cadherin, Twist, and Snail) may determine the malignancy of TETs, especially for distinguishing thymoma and thymic carcinoma.

Morphological Changes in the Cells of the Ductal Adenocarcinoma of the Pancreas at Epithelial-Mesenchymal Transition

The aim of the research was to study the expression of marker genes for the epithelial-mesenchymal transition in the ductal adenocarcinoma of the pancreas.Material and methods. Surgical material from 44 patients with ductal adenocarcinoma of the pancreas was subjected to morphological analysis with molecular genetic research. Total RNA was detected in the detected sections of the anaplastic component using the RNeasy Mini Kit (Qiagen, Germany). 5 marker genes were used for molecular genetic studies of the epithelial-mesenchymal transition (EMT): ZEB1, ZEB2, CDH1, VIM, SNAIL1 (SLUG). Gene expression was measured in triplicate using an EvaGreen intercalating dye. On serial paraffin sections using tissue microarrays technology, immunohistochemical detection of p63, smooth muscle actin, total cytokeratin, cytokeratin 7, vimentin, E-cadherin (Ventana) was performed.Results. As a result of the study, a positive reaction with mesenchymal markers (vimentin, p63, smooth muscle actin) was detected in the cells of the anaplastic component, in contrast to the glandular component. In a molecular study of the anaplastic component, changes in gene expression were characterized as EMT-positive.Conclusion. The heterogeneity of ductal cancer is manifested in the appearance of an anaplastic (sarcomalike) component, in which the ability of epithelial tumor cells to acquire the property of mesenchymal cells that do not require stroma and have aggressive malignant potential that affects the survival of patients is traced.

In situ measurement of miR-138 expression in oral squamous cell carcinoma tissue supports the role of this microRNA as a tumor suppressor.

Oral squamous cell carcinoma is the eighth most common cancer worldwide with a relatively high rate of metastasis (~40%). Previously, we showed that microRNA-138 serves as a functional tumor suppressor and plays an important role in oral squamous cell carcinoma metastasis. However, to date, microRNA-138 expression has not been examined in this tumor tissue. Herein, we demonstrated that microRNA-138 expression is downregulated in metastatic oral squamous cell carcinoma specimens using tissue microarray technology with in situ hybridization. The study included 254 oral squamous cell carcinoma patients from two centers (160 from the Chengdu center and 90 from the Guangzhou center) and four healthy volunteers. Multivariate analysis showed that microRNA-138 expression was independent of tumor stage, age, gender, smoking, and alcohol consumption in oral squamous cell carcinoma patients. Interestingly, patients that expressed lower levels of microRNA-138 (determined by in situ hybridization) were more prone to regional lymph node metastasis and exhibited poorer outcomes. These findings support the role of microRNA-138 as a tumor suppressor in oral squamous cell carcinoma. In summary, the expression level of microRNA-138 is negatively correlated with oral squamous cell carcinoma metastasis; the lower the expression of microRNA-138, the higher the rate of metastasis and the poorer the prognosis of the patients. Therefore, our study confirms that microRNA-138 serves as a tumor suppressor and plays a functional role in oral squamous cell carcinoma tumor metastasis; microRNA-138 constitutes a promising prognosis biomarker and therapeutic target for oral squamous cell carcinoma with metastasis potential.

Abstract 1112: CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1

Background: Breast cancer is one of the leading causes of cancer death among women and the pathological status of axillary lymph nodes is an important predictor of prognosis. However, the mechanism involved in this early stage of metastasis remains largely unknown. Methods: We used microarray analysis to carry out differential genomics analyses between matched pairs of sentinel lymph node metastatic tissues and breast primary tumors. We found 182 up-regulated genes in metastatic tissues (fold change≥1.5). We used CRISPR/Cas9 gene editing system to knockout these genes, each with 6 unique sgRNAs, and screened in vivo by transplanting loss-of-function cell pool into immunocompromised mice. MAGeCK was used to analyze the screening. Immunohistochemistry was performed to examine protein expressions in 212 cases of breast cancer tissues using tissue microarray (TMA) technology. Gene and protein expressions were monitored by qPCR and western blot. The relationship between coiled-coil domain containing 102B (CCDC102B) and receptor for activated C kinase 1 (RACK1) was examined using Co-IP, mass spectrometry, nuclear protein extraction assay and dual-luciferase reporter assay. Functional studies were detected in vitro by cell proliferation, wound-healing and migration, and in vivo by lung metastasis in immunocompromised mice injected CCDC102B-overexpressed cells into tail vein. The primary biological functions and pathways related to CCDC102B were enriched by RNA sequencing. The regulation of NF-κB signaling by CCDC102B and RACK1 was detected using dual-luciferase reporter assay and western blot. Results: We identified CCDC102B through screening and found that it was significantly up-regulated in breast cancer patients with poor prognosis. The expression of CCDC102B were independent predictors in both DFS and OS ( P =0.009; P =0.014). CCDC102B expression was found to promote the proliferation and migration of breast cancer cells MDA-MB-231 and BT549, and lung metastasis in mice. Moreover, we found that overexpression of CCDC102B decreased RACK1 expression whereas knockdown of CCDC102B increased RACK1 expression at both protein and mRNA levels, and the aggressive behavior of CCDC102B in breast cancer cells could be reversed by the expression of RACK1. According to gene set enrichment analysis (GSEA), CCDC102B was correlated with significant enrichment focused on NF-κB pathway. The mechanistic study revealed that gain of CCDC102B resulted in decreased expression of RACK1, which increased levels of epithelial mesenchymal transition (EMT) and phosphorylated Rel-A (p65), leading to the activation of NF-κB pathway and progression of breast cancer metastasis. Conclusion: Taken together, our findings suggest that CCDC102B is strongly associated with negative clinical outcomes and may serve as a potential metastasis promotor by regulating the activation of NF-κB pathway via RACK1. Citation Format: Jing Si, Rong Guo. CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1112.

Abstract 1112: CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1

Abstract Background: Breast cancer is one of the leading causes of cancer death among women and the pathological status of axillary lymph nodes is an important predictor of prognosis. However, the mechanism involved in this early stage of metastasis remains largely unknown. Methods: We used microarray analysis to carry out differential genomics analyses between matched pairs of sentinel lymph node metastatic tissues and breast primary tumors. We found 182 up-regulated genes in metastatic tissues (fold change≥1.5). We used CRISPR/Cas9 gene editing system to knockout these genes, each with 6 unique sgRNAs, and screened in vivo by transplanting loss-of-function cell pool into immunocompromised mice. MAGeCK was used to analyze the screening. Immunohistochemistry was performed to examine protein expressions in 212 cases of breast cancer tissues using tissue microarray (TMA) technology. Gene and protein expressions were monitored by qPCR and western blot. The relationship between coiled-coil domain containing 102B (CCDC102B) and receptor for activated C kinase 1 (RACK1) was examined using Co-IP, mass spectrometry, nuclear protein extraction assay and dual-luciferase reporter assay. Functional studies were detected in vitro by cell proliferation, wound-healing and migration, and in vivo by lung metastasis in immunocompromised mice injected CCDC102B-overexpressed cells into tail vein. The primary biological functions and pathways related to CCDC102B were enriched by RNA sequencing. The regulation of NF-κB signaling by CCDC102B and RACK1 was detected using dual-luciferase reporter assay and western blot. Results: We identified CCDC102B through screening and found that it was significantly up-regulated in breast cancer patients with poor prognosis. The expression of CCDC102B were independent predictors in both DFS and OS (P=0.009; P=0.014). CCDC102B expression was found to promote the proliferation and migration of breast cancer cells MDA-MB-231 and BT549, and lung metastasis in mice. Moreover, we found that overexpression of CCDC102B decreased RACK1 expression whereas knockdown of CCDC102B increased RACK1 expression at both protein and mRNA levels, and the aggressive behavior of CCDC102B in breast cancer cells could be reversed by the expression of RACK1. According to gene set enrichment analysis (GSEA), CCDC102B was correlated with significant enrichment focused on NF-κB pathway. The mechanistic study revealed that gain of CCDC102B resulted in decreased expression of RACK1, which increased levels of epithelial mesenchymal transition (EMT) and phosphorylated Rel-A (p65), leading to the activation of NF-κB pathway and progression of breast cancer metastasis. Conclusion: Taken together, our findings suggest that CCDC102B is strongly associated with negative clinical outcomes and may serve as a potential metastasis promotor by regulating the activation of NF-κB pathway via RACK1. Citation Format: Jing Si, Rong Guo. CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1112.