Colorectal Adenocarcinoma Tissues Research Articles

Expression and significance of cystine transporter SLC7A11/xCT in early colorectal cancer specimens from endoscopic submucosal dissection.

The SLC7A11/xCT cystine transporter is intricately linked with ferroptosis. By mediating intracellular cystine flux, it regulates oxidative stress within neoplastic cells, thereby curtailing ferroptosis and influencing the emergence of colorectal cancer. This study aimed to gauge the SLC7A11/xCT expression across various tumorigenic stages in early colorectal adenocarcinoma tissues, shedding light on its specific role in the genesis of these early malignancies. Sixty specimens that underwent endoscopic submucosal dissection (ESD) resection with pathologic diagnosis of colorectal adenocarcinoma were collected. SLC7A11/xCT expression was pinpointed using immunohistochemistry, and correlations with the patients' clinical-pathological features were drawn. Additionally, a comprehensive bioinformatics assessment was undertaken to discern differential SLC7A11/xCT expressions across a spectrum of cancers. Immunohistochemical assessments unveiled a pronounced cytoplasmic SLC7A11/xCT expression, manifesting as a brownish-yellow hue, particularly in nascent colorectal cancer samples. Its expression was discernibly correlated with both patient gender and adenocarcinoma differentiation grade (P<0.05). Nevertheless, factors such as patient age, tumor localization, infiltration depth, diameter, adjacent adenoma histology, its major axis, and dysplasia degree bore no statistical significance with SLC7A11/xCT levels (P>0.05). Bioinformatics insights pointed to an upregulated SLC7A11/xCT expression across diverse malignancies, inclusive of colon adenocarcinoma, esophageal cancer, acute myeloid leukemia, lung squamous cell carcinoma, colorectal cancer, and endometrial cancer (P<0.05). Elevated SLC7A11/xCT expression marks early colorectal adenocarcinoma, with the intensity of this expression being intertwined with the patient's gender and the tumor's differentiation grade. It is postulated that colorectal cancer cells might amplify SLC7A11/xCT to stymie ferroptosis, thus fostering neoplastic proliferation, metastasis, and cellular stemness.

Colorectal cancer image recognition algorithm based on improved transformer

Aiming at the problems of the complex background of colorectal cancer tissue cell images and the difficulty of detection caused by the low differentiation of cancer cell regions, a deep learning method is used to detect the cancer cell regions. By integrating the skip feedback connection structure into U-Net and combining it with the Swin Transformer for feature extraction, we improve the multi-level feature extraction capabilities of the model. This algorithm enables end-to-end recognition of colorectal adenocarcinoma tissue images and achieves an accuracy of 95.8% on the NCT-CRC-HE-100K dataset, demonstrating its potential to significantly support colorectal cancer detection and treatment.

Identification of homer protein homolog 3 as a prognostic marker of colon adenocarcinoma

BackgroundHomer protein homolog 3 (HOMER3), a factor implicated in both physiological and pathological processes, has been studied extensively to determine the relationship between its expression level and the prognosis of various malignancies. However, the significance and clinicopathological role of HOMER3 in colorectal adenocarcinoma remain unclear. MethodsIn this study, bioinformatics techniques were used to find the correlation between high HOMER3 expression levels and clinicopathological features of colorectal adenocarcinoma (COAD) patients. ResultsCellular experiments confirmed the differential expression of HOMER3 in tumor cells compared to normal cells. HOMER3 overexpression was significantly associated with COAD staging and carcinoembryonic antigen (CEA) levels. Patients with high HOMER3 expression levels have a poor prognosis. HOMER3 expression levels can be distinguished more accurately between tumor and non-tumor tissues (AUC = 0.634). The HOMER3 gene variation rate in COAD tissue was 0.7 %. Moreover, 16 of the 22 DNA methylation sites in HOMER3 were associated with COAD prognosis. Our findings confirmed that HOMER3 was positively correlated with immune cell infiltration and immune checkpoints (PD-1, CTLA-4, LMTK3, and LAG3) in COAD, Specifically, we will clearly state that while there is statistical significance, the actual strength of the correlations is weak. During KEGG enrichment analysis, HOMER3 was enriched along with DLG4 and SHANK1 in glutamatergic synapses. Additionally, upstream microRNAs that could bind to HOMER3 were predicted. These findings suggest that HOMER3 might be involved in COAD development and immune regulation. ConclusionsHOMER3 acts as a potential biomarker that can facilitate innovative developments in the diagnosis and prognostic assessment of COAD.

NIFK, a Potential Prognostic and Immunological Biomarker in Pan-- Cancer Analysis, Significantly Regulates Proliferation and Metastasis of Colorectal Cancer.

As a binding protein of Ki67, NIFK plays an important role in the mitosis of cells and is closely related to the progression of specific types of tumors. However, there is still a lack of systematic analysis of NIFK in pan-cancer and insufficient research to explore its role in human tumors. We systematically evaluated the pan-cancer expression and mutation of NIFK in human cancers using data from The Cancer Genome Atlas (TCGA) through large-scale bioinformatics analysis. In addition, we explored the pan-cancer immunological characteristics of NIFK, especially in colorectal adenocarcinoma (COAD). Furthermore, we used single-cell sequencing to analyze the expression of NIFK in different cells of COAD tissues and performed GO, KEGG, and gene set enrichment analysis of NIFK in COAD. Lastly, we evaluated the effects of NIFK knockdown on the colorectal cancer cell lines in in vitro experiment. We found that NIFK was overexpressed in almost all types of tumors and showed significant prognostic efficacy. Additionally, correlations between NIFK and specific immune features, such as immune cell infiltration, immune checkpoint genes, TMB, and MSI, suggest that NIFK may be used to guide immunotherapy. Subsequently, it was found that the expression of NIFK was significantly upregulated in tumor cells through single-cell sequencing analysis, and the NIFK gene was closely associated with tumor progression and immune therapy response. Finally, we further elucidated the role of NIFK in colorectal cancer and found that downregulation of NIFK expression could inhibit the proliferation, migration, and invasion ability of colorectal cancer cells. The results of this study demonstrated that NIFK, as a member of the pan-cancer genes, will serve as a biomarker and a potential therapeutic target for a range of cancer types, providing new insight into precision medicine.

Investigation of Trpa1 and Trpc1 Immunreactivities in Colon Adenocarcinomas.

As the normal colon epithelium differentiates into adenoma, invasive cancer and metastatic cancer, the cell acquires new characteristics such as apoptosis, proliferation, differentiation, invasion and metastasis. Many mechanisms are effective in acquiring these qualities. One of these is the regulation of the functioning of ion channels. This study aimed to examine TRPA1 and TRPC1 expression in colorectal adenocarcinomas showing different degrees of differentiation. We examined the biopsy specimens of 60 patients diagnosed with colorectal adenocarcinomas, including those of patients with well-differentiated (n = 20), moderately differentiated (n = 20) and poorly differentiated (n = 20) carcinomas. Moreover, 20 biopsy specimens of individuals with normal colonic mucosa were examined. Histoscores were calculated for TRPA1 and TRPC1 based on the extent of diffusion and intensity of immunoreactivity, and these scores were compared statistically. A statistically significant increase in both TRPA1 and TRPC1 immunoreactivity was observed in low-grade and high-grade colon adenocarcinomas compared to the control group (p<0.001). A statistically significant decrease in both TRPA1 and TRPC1 immunoreactivity was observed in high-grade colon adenocarcinomas compared to low-grade colon adenocarcinomas (p<0.001). TRPA1 and TRPC1 immunoreactivites are increased in colorectal adenocarcinoma tissue compared with the healthy tissue. Furthermore, the immunoreactivity decreases as the grade of cancer increases.

Claudin18.2 expression and its clinicopathological feature in adenocarcinoma from various parts

AimsTo clarify claudin18.2 expression and its clinicopathological features in various cancers, especially in lung adenocarcinoma.MethodsImmunohistochemistry staining and fluorescence in situ hybridisation (FISH) were performed to detect claudin18.2 expression and CLDN18...

Abstract 3496: EpigenPlot: A tool for the gene-level methylation analysis of colorectal tumors

Abstract Introduction. Aberrant DNA methylation patterns are frequent in colorectal adenocarcinoma (CRC) tissues. The investigation of this layer of epigenetic regulation holds the promise of a better understanding of tumour progression and the identification of novel biomarker candidates. Purpose. Our goal was to collect and analyse publicly available DNA methylation data of colorectal samples and create a user-friendly tool for exploring and visualizing the CRC methylome. Methods. We used Illumina Infinium Methylation Arrays, which is a popular platform for genome-wide methylation analysis of large cohorts. Gene Expression Omnibus database (GEO) and TCGA-COAD (The Cancer Genome Atlas Colon Adenocarcinoma) datasets including normal colorectal mucosa, adenoma, and adenocarcinoma samples were systematically collected and filtered. Differentially methylated regions were identified using the Kruskal-Wallis test. Genes with significant methylation changes were further analysed using ROC (Receiver Operator Characteristic) analysis. Finally, we established a web application using the shiny R package. Results. We assembled a database including methylation data of 2,586 samples. Adenoma and adenocarcinoma samples were globally hypomethylated. Hypermethylation (p &amp;lt; 0.05, Δβ &amp;gt;= 0.2) in adenocarcinoma samples was predominantly present in regions near proximal promoters. Out of the 74 previously proposed biomarkers examined, 50 had a cross-validated AUC of over 0.8. FDA-approved biomarkers BMP3, NDRG4, and SEPT9 have shown the highest performance in the TSS200 region (cvAUC = 0.8, 0.83, and 0.76, respectively). Genes with the highest performance were ITGA4 (5’UTR, first exon: cvAUC = 0.9), MDFI (5’UTR: cvAUC = 0.9), CNRIP1 (5’UTR, first exon: cvAUC = 0.89). The established web application can visualize methylation at CpG sites and gene regions, and KEGG pathway genes. The platform is available at https://epigenplot.com. Conclusion. We collected and analysed a sizeable database of colorectal tissue data. Gene regions with the best performance include previously proposed and novel diagnostic biomarker candidates. The web platform provides an tool for the efficient exploration of the assembled data. Citation Format: Dalma Müller, Balazs Gyorffy. EpigenPlot: A tool for the gene-level methylation analysis of colorectal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3496.

Comprehensive analysis of bulk and single-cell RNA sequencing data reveals Schlafen-5 (SLFN5) as a novel prognosis and immunity.

Recent advancements have elucidated the multifaceted roles of the Schlafen (SLFN) family, including SLFN5, SLFN11, SLFN12, SLFN13, and SLFN14, which are implicated in immunological responses. However, little is known about the roles of this gene family in relation to malignancy development. The current study aimed to explore the diagnostic and prognostic potential of Schlafen family genes in colorectal adenocarcinoma (COAD) through bioinformatics analysis. Leveraging advanced bioinformatics tools of bulk RNA-sequencing and single-cell sequencing, we conducted in-depth analyses of gene expressions, functional enrichment, and survival patterns of patients with colorectal cancer compared to normal tissue. Among Schlafen family genes, the transcription levels of SLFN5 in COAD tissues were significantly elevated and correlated with poor survival outcomes. Furthermore, SLFN5 regulated the immune response via Janus kinase (JAK)/signal transduction and activator of transcription (STAT)/interferon (IFN)-alpha/beta signaling. These chemokines in inflammation are associated with diabetes and metabolism, suggesting their involvement in altered cellular energetics for COAD progress. In addition, an immune cell deconvolution analysis indicated a correlation between SLFN5 expression and immune-related cell populations, such as regulatory T cells (Tregs). These findings highlighted the potential clinical significance of SLFN5 in COAD and provided insights into its involvement in the tumor microenvironment and immune regulation. Meanwhile, the drug discovery data of SFLN5 with potential targeted small molecules suggested its therapeutic potential for COAD. Collectively, the current research demonstrated that SFLN5 play crucial roles in tumor development and serve as a prospective biomarker for COAD.

Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

Expression and correlation of COX-2 and NUCB1 in colorectal adenocarcinoma.

To investigate the expression and correlation of COX-2 and NUCB1 in colorectal adenocarcinoma and adjacent tissues. The expression of COX-2 and NUCB1 and their effects on prognosis were predicted using bioinformatics. Immunohistochemistry was used to identify the expression of two molecules in 56 cases of colorectal adenocarcinoma and the surrounding tissues. The expression of two molecules and their association with clinicopathological variables were examined using the chi-square test. The association between COX-2 and NUCB1 was investigated using the Spearman correlation test. The STRING database revealed that COX-2 and NUCB1 were strongly linked. According to the UALCAN and HPA database, COX-2 was upregulated while NUCB1 was downregulated in colorectal adenocarcinoma, both at the protein and gene levels. The OS times for COX-2 and NUCB1 high expression, however, exhibited the same patterns. The rate of positive COX-2 immunohistochemical staining in cancer tissues was 69.64% (39/56), which was significantly higher than the rate in healthy tissues 28.57% (16/56). NUCB1 was expressed positively in cancer tissues at a rate of 64.29% (36/56) compared to just 19.64% (11/56) in neighboring tissues. The positive expression levels of COX-2 and NUCB1 were both closely related to clinical stage, differentiation degree, and lymphatic metastases (P < 0.05). In colorectal cancer, COX-2 and NUCB1 expression were significantly correlated (rs = 0.6312, P < 0.001). Both COX-2 and NUCB1 are overexpressed and significantly associated in colorectal adenocarcinoma.

Forkhead Box P4 promotes the proliferation of cells in colorectal adenocarcinoma

Abstract Objectives Forkhead Box P4 (FOXP4) is a transcription factor that promotes tumor formation and progression. However, studies on its roles in colorectal adenocarcinoma (CRAC) and cell proliferation regulation are few to date. This work investigates the expression of FOXP4 in CRAC, explores the characteristic of FOXP4 in different clinicopathological features, and analyzes its regulation of cell proliferation. Methods The GEPIA database was used to predict the trend of FOXP4 expression in colon cancer and normal mucosa. Tumor tissue and normal paracancerous mucosal tissue were sampled from 64 cases diagnosed with CRAC and who were receiving radical surgery at Tianjin Hospital from January 2017 and December 2022. FOXP4 and proliferating cell nuclear antigen (PCNA) were detected by the immunohistochemistry EnVision method. The colon cancer cell lines SW480, HCT15, and SW620 and the normal colon cell line NCM460 were selected, and expression of FOXP4 was detected by the Western blot method. The siRNA-FOXP4 plasmid was synthesized and transfected with SW480 and HCT15 cell lines, respectively, to establish si-FOXP4 groups, and empty vector transfection group (NC-FOXP4) and blank control group (NC) was set up. The expression levels of FOXP4 and PCNA were detected by the Western blot method, while the cell proliferation activity was assessed using CCK-8. Normally distributed quantitative data were compared between two and more groups with ANOVA (SNK-based pairwise comparison), while intergroup enumeration data comparisons were performed through χ 2 test and assessed through linear correlation analysis. Results GEPIA-based prediction shows a potential rise in FOXP4 expression in colon cancer. The rate of positive FOXP4 expression is significantly higher in CRAC tissue than in normal mucosa (p&lt;0.05). The difference in FOXP4 is statistically significant in the comparison of maximum tumor diameter and depth of invasion in CRAC (p&lt;0.05) but not in the comparison of gender, age, degree of differentiation, tumor focus, tumor embolism, and lymph node metastasis (p&gt;0.05). The expression levels of FOXP4 and PCNA in CRAC are positively correlated (p&lt;0.05). FOXP4 expression is significantly higher in cell lines SW480, HCT15, and SW620 than in cell line NCM460. The cell proliferation activity and PCNA expression are significantly lower in si-FOXP4 group than in NC-FOXP4 and NC groups for cell lines SW480 and HCT15. Conclusions FOXP4 is highly expressed and has a proliferative effect on tumor cells in CRAC.

Expression of eIF6 and its relationship with cell proliferation in colorectal adenocarcinoma

Abstract Objectives Eukaryotic translation initiation factors (eIFs) are genes related to tumor formation. They selectively translate some mRNAs, regulate protein synthesis, promote cell proliferation, and effectively control the progression of some tumors. As a member of this family, eukaryotic translation initiation factor 6 (eIF6) plays a crucial role in tumor formation and progression. Exploring the expression characteristics of eIF6 and proliferating cell nuclear antigen (PCNA), a proliferation-associated factor, in colorectal adenocarcinoma (CRAC) and analyzing their correlation and clinical significance can provide a theoretical basis for the study of CRAC and objective biological indicators for the clinical judgment of tumor formation and progression. Methods The GEPIA database was used to predict the trend of eIF6 and PCNA in CRAC. Sixty-four patients diagnosed with CRAC and undergoing surgical treatment were selected from January 2017 to November 2022. CRAC tissues and normal mucous tissues (&gt;3 cm away from the tumor margin) were retained. The expression of eIF6 and PCNA was detected by immunohistochemistry (IHC). Colon cancer cell lines SW480, HCT15, and SW620 and normal colon cell line NCM460 were selected, and the expression of eFI6 and PCNA in these cells was detected by Western blot. Results A trend of elevated expression of eIF6 and PCNA was predicted in CRAC by the GEPIA database. The expression levels of eIF6 (65.63 % vs. 9.38 %, X2=43.2000, p&lt;0.0001) and PCNA (84.38 % vs. 46.88 %, X2=19.9481, p&lt;0.0001) were higher in CRAC tissues than that in normal mucosa as indicated by IHC. Significant difference in eIF6 and PCNA expression was found among different maxim tumor diameters and depths of infiltration in colon adenocarcinoma (p&lt;0.05). No statistical difference in eIF6 and PCNA expression was observed among different genders, ages, degrees of differentiation, LNMets, intravascular cancer thrombosis, TNM stages, and numbers of lymph node metastases (p&gt;0.05). A positive correlation was found between eIF6 and PCNA in CRAC (X2=5.05, r=0.77, p=0.0283). Their expression was significantly higher in SW480, HCT15, and SW620 than in NCM460 (p&lt;0.05). Conclusions eIF6 is highly expressed in CRAC, participates in tumor formation and progression, and has a positive correlation with PCNA.

GFAT1: A Potential Prognostic Biomarker in Colorectal Cancer

Introduction: There is an increasing demand for additional techniques to diagnose and treat cancer including CRC or colorectal cancer effectively. Utilizing antibodies as biomarker could contribute to accurate diagnosis of cancer due to its high specificity and sensitivity. One of the etiologies of CRC progression was proposed as the alterations of hexosamine biosynthetic pathway which could subsequently influence the rate-limiting enzyme, glutamine-fructose-6-phosphate aminotransferase (GFAT1). These increased enzymatic activities resulted in an elevation of glucose uptake that provides nutrients facilitating the progression of cancer cells. Therefore, we attempted to determine the potential of GFAT1 as the biomarker for CRC by correlating its expression with clinicopathological features of the patients. Methods: A total of 132 10% formalin-fixed paraffin embedded tissue were retrieved. Immunohistochemistry (IHC) was performed on the tissue sections and digital images were subsequently acquired. All the images were automatedly analyzed using IHC Profiler. GFAT1 immunoreactivity in colorectal tissues was calculated using an adapted H-score formula. Clinicopathological features of the patients were statistically correlated with the status of GFAT1. Results: Colorectal adenocarcinoma tissues had the significantly highest GFAT1 H-scores with the mean of 103.18 compared to adenoma and non-tumor tissues. There have been no significant associations between clinicopathological characteristics of the patients and the status of GFAT1 except for tumor size. Conclusion: Immunoreactivity of GFAT1 was significantly different between non-tumorous tissues and adenocarcinoma as well as between adenoma and adenocarcinoma tissues. GFAT1 could serve as one of the prognostic biomarkers or useful targets.

Identification of Epithelial FTL Expression as an Important Predictor for Prognosis of Colon Adenocarcinoma

Ferroptosis has been shown to be a non-self apoptotic, iron ion-catalysed, controlled cell death process involving cellular lipid oxides. The gnes that may affect ferroptosis may be candidate biomarkers. But the value and predictive value of ferroptosis-related genes in colorectal adenocarcinoma (COAD) are not fully understood. Therefore, we analyzed the expression status in COAD and healthy colon tissue using GEO, GEPIA and HPA databases. The role of predictive and genetic alterations of ferroptosis-related genes were investigated using cBioPortal, HPA, PrognoScan, STRING, GeneMANIA and LinkedOmics databases. Then, immunohistochemistry staining was performed to detect the both epithelial and stromal expression of FTL in COAD. Using FerrDb and GEO databases, the transcript levels of XBP1, TSC22D3 and YWHAE were identified to correlate with advanced tumor stage, FTL, DUSP1, TSC22D3 and GDF15 were related to poor prognosis of COAD. As immunohistochemistry staining shown that FTL was significantly up-regulated in COAD tissues. Cox survival analysis and KM survival analysis indicated that high epithelial FTL levels is associated with a poor prognosis in COAD.

Exploration of an Prognostic Signature Related to Endoplasmic Reticulum Stress in Colorectal Adenocarcinoma and Their Response Targeting Immunotherapy.

Background: Endoplasmic reticulum (ER) stress plays a pro-apoptotic role in colorectal adenocarcinoma (COAD). This study aimed to develop a novel ER-stress-related prognostic risk model for COAD and provide support for COAD cohorts with different risk score responses to immune checkpoint inhibitor therapies. Methods: TCGA-COAD and GSE39582 were included in this prospective study. Univariate and multivariate Cox analyses were performed to identify prognostic ER stress-related genes (ERSGs). Accordingly, the immune infiltration landscape and immunotherapy response in different risk groups were assessed. Finally, the expression of prognostic genes in 10 normal and 10 COAD tissue samples was verified using reverse transcription-quantitative polymerase chain reaction. Results: Eight prognostic genes were selected to establish an ERSG-based signature in the training set of the TCGA-COAD cohort. The accuracy of this was confirmed using a testing set of TCGA-COAD and GSE39582 cohorts. Gene set variation analysis indicated that differential functionality in high-low-risk groups was related to immune-related pathways. Corresponding to this, CD36, TIMP1, and PTGIS were significantly associated with 19 immune cells with distinct proportions between the different risk groups, such as central memory CD4T cells and central memory CD8T cells. Moreover, the risk score was considered effective for predicting the clinical response to immunotherapy, and the immunotherapy response was significantly and negatively correlated with the risk score of individuals with COAD. Furthermore, the immune checkpoint inhibitor treatment was less effective in the high-risk group, where the expression levels of PD-L1 and tumor immune dysfunction and exclusion scores in the high-risk group were significantly increased. Finally, the experimental results demonstrated that the expression trends of prognostic genes in clinical samples were consistent with the results from public databases. Conclusion: Our study established a novel risk signature to predict the COAD prognosis of patients and provide theoretical support for the clinical treatment of COAD.

DNA Extracted from Cytologic Slides Is a Valuable Source for PCR-Based Molecular Tests

Introduction: DNA extracted from cytologic samples is occasionally used for various molecular tests. The aim of this study was to evaluate DNA extracted from differently prepared cytologic slides that can be used for PCR-based molecular tests. Methods: For each 23 cases of papillary thyroid carcinoma or colorectal adenocarcinoma tissues, six touch-imprinted cytological slides were prepared (group 1∼3), and remnant tissues were blocked for FFPE tissue (group 4). Cytologic slides were grouped by preparation methods: air-dried slides (group 1), fixed slides (group 2), and stained slides (group 3). Fixed slides were classified as 95% ethanol fixed (group 2A) and CytoRich Red Preservative solution fixed (group 2B). Stained slides were divided in 3 ways: Giemsa, Pap, and H&E stained (group 3A, 3B, and 3C, respectively). DNA extracted from each group was evaluated for concentration, 260/280 ratio, DNA Integrity Number (DIN) value, and mutation. Results: DNA concentration was highest in group 1 and lowest in group 2B. DIN value was highest in group 2A and lowest in group 2B. A mutation of BRAF or KRAS genes was detected in 18 FFPE tissue samples. Matched DNA extracts from groups 1, 2A, and 3 produced results consistent with FFPE tissue results, while mutation testing was successful for only four samples of DNA from group 2B. Conclusion: The mutation tests worked well for most samples except CytoRich Red Preservative-fixed slides. This study indicates that stained and unstained cytologic slides are a suitable source of PCR-based molecular tests as long as they are fixed in ethanol or stored for a short time in an air-dried condition.

Enrichment of Prevotella intermedia in human colorectal cancer and its additive effects with Fusobacterium nucleatum on the malignant transformation of colorectal adenomas

BackgroundOwing to the heterogeneity of microbiota among individuals and populations, only Fusobacterium nucleatum and Bacteroides fragilis have been reported to be enriched in colorectal cancer (CRC) in multiple studies. Thus, the discovery of additional bacteria contributing to CRC development in various populations can be expected. We aimed to identify bacteria associated with the progression of colorectal adenoma to carcinoma and determine the contribution of these bacteria to malignant transformation in patients of Han Chinese origin.MethodsMicrobiota composition was determined through 16S rRNA V3–V4 amplicon sequencing of autologous adenocarcinomas, adenomatous polyps, and non-neoplastic colon tissue samples (referred to as “tri-part samples”) in patients with CRC. Enriched taxa in adenocarcinoma tissues were identified through pairwise comparison. The abundance of candidate bacteria was quantified through genomic quantitative polymerase chain reaction (qPCR) in tissue samples from 116 patients. Associations of candidate bacteria with clinicopathological features and genomic and genetic alterations were evaluated through odds ratio tests. Additionally, the effects of candidate bacteria on CRC cell proliferation, migration, and invasion were evaluated through the co-culture of CRC cells with bacterial cells or with conditioned media from bacteria.ResultsPrevotella intermedia was overrepresented in adenocarcinomas compared with paired adenomatous polyps. Furthermore, co-abundance of P. intermedia and F. nucleatum was observed in tumor tissues. More notably, the coexistence of these two bacteria in adenocarcinomas was associated with lymph node involvement and distant metastasis. These two bacteria also exerted additive effects on the enhancement of the migration and invasion abilities of CRC cells. Finally, conditioned media from P. intermedia promoted the migration and invasion of CRC cells.ConclusionThis report is the first to demonstrate that P. intermedia is enriched in colorectal adenocarcinoma tissues and enhances the migration and invasion abilities of CRC cells. Moreover, P. intermedia and F. nucleatum exert additive effects on the malignant transformation of colorectal adenomas into carcinomas. These findings can be used to identify patients at a high risk of malignant transformation of colorectal adenomas or metastasis of CRC, and they can accordingly be provided optimal clinical management.

Overexpression of LncRNA MNX1-AS1/PPFIA4 Activates AKT/HIF-1α Signal Pathway to Promote Stemness of Colorectal Adenocarcinoma Cells.

Purpose The purpose of this study was to explore the role of the lncRNA MNX1-AS1 and its related downstream signaling pathways in colorectal adenocarcinoma (COAD). Methods COAD tissues and cells were prepared and treated with sh-MNX1-AS1, pcDNA-MNX1-AS1, sh-PPFIA4, LY29004, and their controls. CCK8 and colony formation assays were undertaken for evaluating cell proliferation. Tumor cell migratory ability was detected by transwell assay. Apoptosis detection was processed by YO-PRO-1/PI Staining. The regulated relationship between lncRNA MNX1-AS1 and PPFIA4 was confirmed by RIP-ChIP assay. Q-PCR was applied to detect genes related to tumor cell stemness, proliferation, migration, and apoptosis in each group. Finally, a xenograft tumor model was constructed to verify the result in vivo. Results COAD patients with high expression of the lncRNA MNX1-AS1 have poor prognosis. LncRNA MNX1-AS1 promotes the stemness of COAD cells. PPFIA4 mediates lncRNA MNX1-AS1 expression and affects COAD cell stemness. LncRNA MNX1-AS1 accelerates proliferation and migration, while it suppresses apoptosis. LncRNA MNX1-AS1/PPFIA4 accelerates tumor growth in COAD model. LncRNA MNX1-AS1/PPFIA4 activates the downstream AKT/HIF-1α signaling pathway to promote COAD development. LY29004 significantly inhibits the tumorigenic ability of lncRNA MNX1-AS1 and PPFIA4. Conclusion LncRNA MNX1-AS1/PPFIA4 activates AKT/HIF-1α signal pathway to promote the stemness of COAD cells, which could be a new target for COAD treatment.

Expression and significance of miR-34 with PI3K, AKT and mTOR proteins in colorectal adenocarcinoma tissues

This research was carried out to investigate the expression of miR-34a, miR-34b and p-PI3K, p-AKT, and mTOR proteins in colorectal adenocarcinoma and corresponding distal cutaneous normal mucosal tissues and their relationship with the clinicopathological parameters of colorectal adenocarcinoma as well as the correlation between miR-34a, miR-34b and PI3K/AKT/mTOR signaling pathway. The expression of p-PI3K, p-AKT, and mTOR proteins in 67 colorectal adenocarcinomas and the corresponding distal cut-off normal mucosa were assayed by immunohistochemistry. Their relationship with clinicopathological parameters and the correlation of the three proteins were evaluated. The expression of miR-34a and miR-34b in colorectal adenocarcinoma and the corresponding distal cutaneous normal mucosa was detected by applying real-time quantitative PCR. The correlation between colorectal adenocarcinoma tissue miR-34a, miR-34b and p-PI3K, p-AKT, and mTOR proteins, respectively, was analyzed. Results showed that the expression of p-PI3K, p-AKT and mTOR proteins in colorectal adenocarcinoma tissues was higher than that in the corresponding distal cutaneous normal mucosa (P=0.000), and there was a positive correlation between the expression of the three proteins in colorectal adenocarcinoma tissues. The expression of p-PI3K and p-AKT protein in colorectal adenocarcinoma tissues were correlated with tumor size, differentiation degree, infiltration degree, lymph node metastasis and TNM stage (P<0.05). The expression of mTOR protein was related to tumor size and differentiation degree (P<0.05). The relative expression of miR-34a and miR-34b in colorectal adenocarcinoma tissues was less than that in the corresponding distal cutaneous normal mucosa (P<0.05), and the expression of miR-34a and miR-34b was positively correlated. The expression of miR-34a and miR-34b in colorectal adenocarcinoma tissues was negatively correlated with the expression of p-PI3K, p-AKT and mTOR proteins. In conclusion, the PI3K/AKT/mTOR signaling pathway may promote colorectal adenocarcinoma and differentially participate in differentiation, infiltration and lymph node metastasis. Also, miR-34a and miR-34b may inhibit colorectal adenocarcinoma. Importantly, miR-34a and miR-34b may affect the development and progression of colorectal adenocarcinoma by regulating PI3K/AKT/mTOR signaling pathway.

Infiltration and Significance of CD103+CD8+ T Cells in Gastrointestinal Adenocarcinoma.

Immunohistochemical staining is a common technique to study tissue morphology and in situ protein expression. With the rise of immunotherapy, more and more markers in the tumor microenvironment have been discovered. In this paper, the multiplex immunofluorescence staining method was used to stain paraffin sections of 35 cases of gastric adenocarcinoma tissue and 35 cases of colorectal adenocarcinoma tissue and its adjacent tissue. The infiltration of CD8+ T cells and CD103+CD8+ T cells in the slices was detected by using the slice scanner to form the map and CaseViewer2.0 software analysis. The experimental results in this paper showed that compared with adjacent tissues, the infiltration degree of CD8+ T cells in gastric adenocarcinoma tissue was significantly lower (Z = 2.244, P = 0.025, P < 0.05), and the infiltration degree of cells was significantly lower (Z = 2.785, P = 0.005, P < 0.05). In gastric adenocarcinoma tissue, the infiltration of CD8+ T cells was correlated with the tumor diameter of gastric adenocarcinoma (P = 0.002). There was a statistically significant difference. The degree of CD103+CD8+ T cell infiltration was related to the survival of patients with gastric adenocarcinoma (P = 0.002) and the degree of tumor differentiation (P = 0.004). The infiltration of CD103+CD8+ T cells in gastric cancer tissue and colorectal cancer tissue is related to the survival of patients. CD103+CD8+ T cells are thought to play a role in the prognosis of gastric adenocarcinoma and colorectal adenocarcinoma. Experiments have shown that the more the CD103+CD8+ T cells, the better the prognosis of gastric adenocarcinoma and colorectal adenocarcinoma, so the prognosis of gastric adenocarcinoma and colorectal adenocarcinoma can be improved by increasing CD103+CD8+ T cells.