Colorectal Cancer Tissue Specimens Research Articles

Abstract 2352: Development of an AI-based algorithm to quantify eosinophils in H&E images from colorectal cancer (CRC) tissue sections guided by biomarker staining using multiplex immunofluorescence imaging

Abstract Background: Eosinophils are innate immune granulocytes that migrate to areas of inflammation to combat against infection and disease. Best known for their detrimental role in asthma and allergic disorders, there is growing interest in the involvement of eosinophils in cancer. Eosinophils are routinely observed in the tumor microenvironment (TME) and, depending on the cancer type, have been shown to drive other immune cells to either suppress or promote tumor growth - In colorectal cancer (CRC), eosinophil infiltration into the TME has been linked to a favorable prognosis. However, the behavior of eosinophils and their effect on associated immune mediators in the TME remains poorly understood. Currently, eosinophils are primarily identified from H&E stained tissue sections based on morphological features by a pathologist, but it can be challenging to reliably and efficiently identify all eosinophils based on morphology alone. Here, we present a novel image analysis workflow that established an AI-based cell classifier which can accurately quantify eosinophils in H&E stained CRC tissue sections by leveraging biomarker staining of eosinophils using multiplex immunofluorescence (mIF) imaging in conjunction with the morphological characteristics of eosinophils observed by H&E. Methods: CRC tissue sections were labeled with a 5plex panel of eosinophil-specific markers and imaged by mIF using the PhenoImager HT platform (Akoya). Biomarker fluorescence was then quenched, sections stained by H&E, and reimaged using the PhenoImager. mIF and H&E images were then imported into the HALO® platform for algorithm development. Halo AI cell classification of eosinophils was trained on the morphological features of eosinophil staining in H&E images, guided by eosinophil-specific labeling in the 5plex mIF images. H&E staining was also performed on a serial section of each specimen. Morphological identification of eosinophils in H&E images was performed by a pathologist. Results: Correlation analysis was performed to evaluate the relationship, per high power field (HPF), between manual eosinophil counts by a pathologist and AI algorithm derived eosinophils counts. The results showed the two methods are highly correlated, demonstrating reliable algorithm performance. Conclusion: The AI-based eosinophil detection algorithm established here enables high-throughput analysis and quantification of eosinophils from H&E stained CRC tissue specimens and facilitates morphology diagnosis. Citation Format: Arezoo Hanifi, Elizabeth Blain, James Hargrove, Jeff Lock, Nam Tran, Vladislav Chizhevsky, Qingyan Au. Development of an AI-based algorithm to quantify eosinophils in H&E images from colorectal cancer (CRC) tissue sections guided by biomarker staining using multiplex immunofluorescence imaging [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 2352.

Expression of IZUMO2 in colorectal cancer in association with clinicopathological features

IZUMO2 belongs to the testis-expressed IZUMO family of proteins, which are characterized by an N-terminal IZUMO domain. Based on integrated analysis of expression profiles and matched DNA methylation data from a public database, IZUMO2 represents a prognosis-related methylation-driven gene in colorectal cancer. However, it remains unclear whether IZUMO2 protein expression is suppressed or overexpressed in colorectal cancer cells. In this study, we aimed to elucidate the expression of the IZUMO2 protein in colorectal cancer, with a focus on the clinicopathological features. Sixty-four colorectal cancer tissue specimens were immunohistochemically stained using specific antibodies against IZUMO2. IZUMO2 immunoreactivity was detected at the invasion front in 30 of the 64 colorectal cancer samples. Kaplan–Meier analysis demonstrated that patients with IZUMO2 immunoreactivity had a relatively shorter overall and progression-free survival (log-rank test, P = 0.046 and 0.019, respectively). IZUMO2 immunoreactivity served as an independent factor predictive of poor progression-free survival in colorectal cancer (P = 0.025) as determined via the Cox proportional hazard regression model. Moreover, IZUMO2 immunoreactivity represented an independent factor for poor overall survival (P = 0.035) and progression-free survival (P = 0.013) in patients with colon cancer. The present findings suggest that IZUMO2 is expressed in many colorectal cancers, especially at the cancer invasion front, and may represent an indicator of poor prognosis in colorectal cancer.

Tectorigenin Inhibits Glycolysis-induced Cell Growth and Proliferation by Modulating LncRNA CCAT2/miR-145 Pathway in Colorectal Cancer.

Colorectal cancer (CRC) places a heavy burden on global health. Tectorigenin (Tec) is a type of flavonoid-based compound obtained from the Chinese medical herb Leopard Lily Rhizome. It was found to exhibit remarkable anti-tumor properties in previous studies. However, the effect and molecular mechanisms of Tec in colorectal cancer have not been reported. The objective of this study was to explore the action of Tec in proliferation and glycolysis in CRC and the potential mechanism with regard to the long non-coding RNA (lncRNA) CCAT2/micro RNA-145(miR-145) pathway in vitro and in vivo. The anti-tumor effect of Tec in CRC was examined in cell and animal studies, applying Cell Counting Kit-8 (CCK-8) assay as well as xenograft model experiments. Assay kits were utilized to detect glucose consumption and lactate production in the supernatant of cells and animal serum. The expression of the glycolysis-related proteins was assessed by Western Blotting, and levels of lncRNA CCAT2 and miR-145 in CRC tissue specimens and cells were assessed by realtime quantitative PCR (RT-qPCR). Tec significantly suppressed cell glycolysis and proliferative rate in CRC cells. It could decrease lncRNA CCAT2 in CRC cells but increase the expression of miR-145. LncRNA CCAT2 overexpression or inhibition of miR-145 could abolish the inhibitive effects of Tec on the proliferation and glycolysis of CRC cells. The miR-145 mimic rescued the increased cell viability and glycolysis levels caused by lncRNA CCAT2 overexpression. Tec significantly inhibited the growth and glycolysis of CRC xenograft tumor. The expression of lncRNA CCAT2 decreased while the expression of miR-145 increased after Tec treatment in vivo. Tec can inhibit the proliferation and glycolysis of CRC cells through the lncRNA CCAT2/miR-145 axis. Altogether, the potential targets discovered in this research are of great significance for CRC treatment and new drug development.

5-Fluorouracil resistant CRC cells derived exosomes promote cancer-associated fibroblasts secreting more CXCL12.

Background: Chemoresistance is a key reason for treatment failure in colorectal cancer (CRC) patients. The tumor microenvironment of chemoresistant CRC is distinctly immunosuppressive, although the underlying mechanisms are unclear. Methods: The CRC data sets GSE69657 and GSE62080 were downloaded from the GEO database, and the correlation between TRPC5 and FAP expression was analyzed by Pearson method. The in-situ expression of transient receptor potential channel 5 (TRPC5) and fibroblast activation protein (FAP) in the CRC tissues was examined by immunohistochemistry. TRPC5 expression levels in the HCT8 and HCT116 cell lines and the corresponding 5-fluorouracil (5-FU)-resistant cell lines (HCT8R and HCT116R) were analyzed by western blotting and RT-PCR. Exosomes were isolated from the HCT8R and HCT116R cells and incubated with colorectal normal fibroblasts (NFs), and cancer-associated fibroblasts (CAFs)markers were detected. NFs were also incubated with exosomes isolated from TRPC5-knockdown HCT8R cells, and the changes in intracellular Ca2+ levels and C-X-C motif chemokine ligand 12 (CXCL12) secretion were analyzed. Results: TRPC5 and FAP expression showed positive correlation in the datasets. Immunostaining of CRC tissue specimens further revealed that high TRPC5 and FAP expressions were significantly associated with worse tumor regression. Furthermore, chemoresistant CRC cells expressed higher levels of TRPC5 compared to the chemosensitive cells, and knocking down TRPC5 reversed chemoresistance. Exosomes derived from CRC cells induced the transformation of NFs to CAFs. However, TRPC5-exosomes derived from chemoresistant CRC cells can promote CAFs to secrete more CXCL12. Conclusion: Chemoresistant CRC cells can induce CAFs activation and promote CXCL12 secretion through exosomal TRPC5.

ERO1α promotes the proliferation and inhibits apoptosis of colorectal cancer cells by regulating the PI3K/AKT pathway.

Endoplasmic reticulum oxidoreductin 1α (ERO1α) is an oxidase that exists in the endoplasmic reticulum and plays an important role in regulating oxidized protein folding and tumor malignant progression. However, the specific role and mechanism of ERO1α in the progression of colorectal cancer (CRC) have not yet been fully elucidated. In this study, 280 specimens of CRC tissues and adjacent noncancerous tissues were collected to detect the expression of ERO1α and analyze the clinical significance. ERO1α was stably knocked-down in RKO and HT29 CRC cells to investigate its function and mechanism in vitro and in vivo. We found that ERO1α was remarkably upregulated in CRC tissues and high ERO1α expression is associated with N stage and poor prognosis of CRC patients. ERO1α knockdown in CRC cells significantly inhibited the proliferation and induced apoptosis while inactivating the PI3K/AKT pathway. Rescue assays revealed that AKT activator 740Y-P could reverse the effects on proliferation and apoptosis of ERO1α knockdown in CRC cells. In vivo tumorigenicity assay also confirmed that ERO1α knockdown suppressed tumor growth. Taken together, our findings demonstrated ERO1α promotes the proliferation and inhibits apoptosis of CRC cells by regulating the PI3K/AKT pathway. High expression of ERO1α is associated with poor prognosis in CRC patients, and ERO1α could be a potential therapeutic target for CRC.

Study the Bacterial Activity Isolated from Colon and Rectal Cancer Biopsy in Cell Lines Culture

Colorectal cancer (CRC) is the most common disease and cause of death globally. The aim of the study is investigation and detection of some bacterial interfering with CRC occurrence and progression. The study conducted between September 2022 till February 2023, a total of 50 specimens were collected from confirmed CRC patients. In addition, 50 stool specimens were collected from Healthy volunteers, considers as control group. Isolation and identification of bacteria in all collected specimens were done by using cultural and differential media (blood agar, macconkey agar and Pfizer agar), as well as the VITEK- 2 compact system. The bacterial species, in the specimens of control were ( Escherichia coli 50 (86.20%), Klebsiella Pneumonia 3(5.17%), Salmonella typhi 2(3.44%), Staphylococcus aureus 1(1.72%), Proteus mirabilis 1(1.72%) and Pseudomonas aeruginosa 1(1.72%), while in the specimens of CRC and polyp were (Escherichia coli 30(38.69%), Streptococcus uberis 6(7.79%), Enterobacter cloacae 4(5.19%), Proteus mirabilis 11(14.28), Streptococcus constellatus pharyneis 1(1.29%), Micrococcus luteus 1(1.29%), Staphlococcus pseudintermedius 1(1.29%), Streptococcus thoraltensis 1(1.29%), Citrobacter freundii 1(1.29%), Streptoccus mutans 1(1.29%), Enterococcus faecium 5(6.49%), Enterococcus faecalis 4(5.19%), Granulicatella elegans 1(1.29%), Enterococcus gallinarum 2(2.59%), Serratia marcescens 1(1.29%), Streptococcus sangunis 1(1.29%), Staphylococcus lentus 1(1.29%), Comamons testosteroni 1(1.29%), Morganella morganii 1(1.29%), Pseudomonas aeruginosa 1(1.29%), Klebsiella pneumonia 2(2.59%). The bacteria which has been shown to be associated and more abundance in the specimens of CRC tissues are Escherichia.coli 30(38.96%), Streptococcus uberis 6(7.79%), Enterobacter cloacae 4(5.19%), Enterococcus faecium 5(6.49%), Enterococcus faecalis 4(5.19%). Cell-line culture techniques for the five species showed a cellular viability, sequentially Streptocccus uberis (16.12%), Enterococcus faecium (16.39%), Entreococcus faecalis (9.48%), Enterobacter cloacae (15.11%) and Escherichia coli (17.61%). The results statistically studied by using SPSS, which showed excellent or (highly) significant (p-value is in the range of 0.001).

Study the Bacterial Activity Isolated from Colon and Rectal Cancer Biopsy in Cell Lines Culture

Colorectal cancer (CRC) is the most common disease and cause of death globally. The aim of the study is investigation and detection of some bacterial interfering with CRC occurrence and progression. The study conducted between September 2022 till February 2023, a total of 50 specimens were collected from confirmed CRC patients. In addition, 50 stool specimens were collected from Healthy volunteers, considers as control group. Isolation and identification of bacteria in all collected specimens were done by using cultural and differential media (blood agar, macconkey agar and Pfizer agar), as well as the VITEK- 2 compact system. The bacterial species, in the specimens of control were ( Escherichia coli 50 (86.20%), Klebsiella Pneumonia 3(5.17%), Salmonella typhi 2(3.44%), Staphylococcus aureus 1(1.72%), Proteus mirabilis 1(1.72%) and Pseudomonas aeruginosa 1(1.72%), while in the specimens of CRC and polyp were (Escherichia coli 30(38.69%), Streptococcus uberis 6(7.79%), Enterobacter cloacae 4(5.19%), Proteus mirabilis 11(14.28), Streptococcus constellatus pharyneis 1(1.29%), Micrococcus luteus 1(1.29%), Staphlococcus pseudintermedius 1(1.29%), Streptococcus thoraltensis 1(1.29%), Citrobacter freundii 1(1.29%), Streptoccus mutans 1(1.29%), Enterococcus faecium 5(6.49%), Enterococcus faecalis 4(5.19%), Granulicatella elegans 1(1.29%), Enterococcus gallinarum 2(2.59%), Serratia marcescens 1(1.29%), Streptococcus sangunis 1(1.29%), Staphylococcus lentus 1(1.29%), Comamons testosteroni 1(1.29%), Morganella morganii 1(1.29%), Pseudomonas aeruginosa 1(1.29%), Klebsiella pneumonia 2(2.59%). The bacteria which has been shown to be associated and more abundance in the specimens of CRC tissues are Escherichia.coli 30(38.96%), Streptococcus uberis 6(7.79%), Enterobacter cloacae 4(5.19%), Enterococcus faecium 5(6.49%), Enterococcus faecalis 4(5.19%). Cell-line culture techniques for the five species showed a cellular viability, sequentially Streptocccus uberis (16.12%), Enterococcus faecium (16.39%), Entreococcus faecalis (9.48%), Enterobacter cloacae (15.11%) and Escherichia coli (17.61%). The results statistically studied by using SPSS, which showed excellent or (highly) significant (p-value is in the range of 0.001).

High Wnt2 Expression Confers Poor Prognosis in Colorectal Cancer, and Represents a Novel Therapeutic Target in BRAF-Mutated Colorectal Cancer.

Background and Objectives: We aimed to investigate the role of Wnt2 expression in colorectal cancer (CRC) prognosis and evaluate its potential as a therapeutic target in BRAF-mutated CRC. Materials and Methods: Exactly 136 samples of formalin-fixed paraffin-embedded CRC tissue specimens were obtained from patients who underwent surgical resection for CRC. The gene mutation status of the samples was detected using fluorescence PCR. Wnt2 expression was detected using immunohistochemistry. Survival curves with high Wnt2 expression and BRAF mutations were compared using the Kaplan-Meier method. A nomogram was constructed to determine the estimated overall survival probability. We also predicted the 3-year and 5-year survival rates for patients with high Wnt2 expression and BRAF mutations. In total, 50 samples of BRAF-mutated CRC were collected and detected Wnt2 expression by immunohistochemistry. The Chi-squared test was used to analyze the association between Wnt2 expression and BRAF-mutated CRC. Results: High Wnt2 expression and BRAF mutations are associated with poor prognosis of CRC. Multivariate survival analyses indicated that high Wnt2 expression and BRAF mutations are significant independent predictors of CRC prognosis. Furthermore, high Wnt2 expression was significantly associated with BRAF-mutated CRC, and Wnt2 may be a potential therapeutic target for BRAF-mutated CRC. Conclusions: High Wnt2 expression confers poor prognosis in colorectal cancer and represents a novel therapeutic target in BRAF-mutated CRC.

Multiregion single cell analysis reveals a novel subtype of cancer-associated fibroblasts located in the hypoxic tumor microenvironment in colorectal cancer

Background: The tumor microenvironment (TME) plays a critical role in shaping tumor progression and determining the outcome of the therapeutic response. In this study, we aimed to generate a comprehensive cellular landscape of the colorectal cancer (CRC) TME. Methods: We generated a comprehensive single-cell atlas by collecting CRC cases that have been uploaded to the online database and conducting an in-depth secondary analysis. We then carried out spatial transcriptomic sequencing and multiple immunohistochemical analyses to verify the results of the single-cell analysis. Moreover, we applied our findings to the TCGA database and used tissue microarray (TMA) on CRC tissue specimens to validate clinical prognosis. FindingsWe re-analyzed the transcriptomes of 23785 cells, revealing a pattern of cell heterogeneity in the tumor region, leading-edge region, and non-tumor region. A subtype of COL11A1+INHBA+ tumor-resident cancer-associated fibroblasts (CAFs) was identified, and marker genes, transcription factors, and tissue-specific expression differences were noted and suggested to have potential roles in promoting cancer. We further confirmed that COL11A1+INHBA+ tumor-resident CAFs are mainly located in the hypoxic TME and we propose that they interact with CD44+ CRC cells via INHBA. Elevation of INHBA in CRC is associated with a poor prognosis. Interpretation: Our results demonstrated a single cell landscape of CRC in different regions and identified in hypoxic TME a special subtype of CAFs producing INHBA, which promotes CRC development and correlates with poor prognosis. This special subtype of CAFs is a candidate target for translational research.

Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage.

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.

CircCAPRIN1 interacts with STAT2 to promote tumor progression and lipid synthesis via upregulating ACC1 expression in colorectal cancer.

Circular RNAs (circRNAs) generated by back-splicing of precursor mRNAs (pre-mRNAs) are often aberrantly expressed in cancer cells. Accumulating evidence has revealed that circRNAs play a critical role in the progression of several cancers, including colorectal cancer (CRC). However, the current understandings of the emerging functions of circRNAs in CRC lipid metabolism and the underlying molecular mechanisms are still limited. Here, we aimed to explore the role of circCAPRIN1 in regulating CRC lipid metabolism and tumorigenesis. circRNA microarray was performed with three pairs of tumor and non-tumor tissues from CRC patients. The expression of circRNAs were determined by quantitative PCR (qPCR) and in situ hybridization (ISH). The endogenous levels of circRNAs in CRC cells were manipulated by transfection with lentiviruses overexpressing or silencing circRNAs. The regulatory roles of circRNAs in the occurrence of CRC were investigated both in vitro and in vivo using gene expression array, RNA pull-down/mass spectrometry, RNA immunoprecipitation assay, luciferase reporter assay, chromatin immunoprecipitation analysis, and fluorescence in situ hybridization (FISH). Among circRNAs, circCAPRIN1 was most significantly upregulated in CRC tissue specimens. circCAPRIN1 expression was positively correlated with the clinical stage and unfavorable prognosis of CRC patients. Downregulation of circCAPRIN1 suppressed proliferation, migration, and epithelial-mesenchymal transition of CRC cells, while circCAPRIN1 overexpression had opposite effects. RNA sequencing and gene ontology analysis indicated that circCAPRIN1 upregulated the expressions of genes involved in CRC lipid metabolism. Moreover, circCAPRIN1 promoted lipid synthesis by enhancing Acetyl-CoA carboxylase 1 (ACC1) expression. Further mechanistic assays demonstrated that circCAPRIN1 directly bound signal transducer and activator of transcription 2 (STAT2) to activate ACC1 transcription, thus regulating lipid metabolism and facilitating CRC tumorigenesis. These findings revealed the oncogenic role and mechanism of circCAPRIN1 in CRC. circCAPRIN1 interacted with STAT2 to promote CRC tumor progression and lipid synthesis by enhancing the expression of ACC1. circCAPRIN1 may be considered as a novel potential diagnostic and therapeutic target for CRC patients.

Downregulation of iNOS/NO Promotes Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

iNOS downregulation-induced pathway networks mediate the EMT program and metastasis. As an EMT inducer, the reduced-iNOS may serve as a potential therapeutic target for patients with colorectal cancer.

Log P analyzation-based discovery of GSH activated biotin-tagged fluorescence probe for selective colorectal cancer imaging

Targeted activatable fluorescent probes could provide an effective approach for colorectal cancer imaging. In this study, F1 was found as an effective targeted activatable fluorescent probe based on log P analysis. In vitro experiments demonstrated that the initial fluorescence of the developed probe F1 was initially well quenched, and the fluorescence increased after the probe interacted with glutathione. Cell imaging results showed that the probe had good cell permeability and selectivity. Remarkably, F1 displayed enhanced tumor tissue fluorescence in MC-38 tumor-bearing mice. Notably, it showed selectivity in imaging clinical specimens of human colorectal cancer tissues. Accordingly, this study shows that log P analysis can facilitate the developing efficient of biotin-tagged activatable probes, and the identified F1 has a good potential in clinical colorectal cancer diagnosis.

Hsa_circ_0001550 facilitates colorectal cancer progression through mediating microRNA-4262/nuclear casein kinase and cyclin-dependent kinase substrate 1 cascade.

BackgroundCircular RNAs (circRNAs) play important roles in various malignancies, such as colorectal cancer (CRC). However, the function of hsa_circ_0001550 in CRC remains to be elucidated.MethodsThe expression levels of hsa_circ_0001550, microRNA (miR)‐4262, and nuclear casein kinase and cyclin‐dependent kinase substrate 1 (NUCKS1) were determined by real‐time qPCR. Cell biological behaviors were evaluated via colony formation assay, transwell assay, flow cytometry, and sphere formation assays. The target relationship was validated via dual‐luciferase reporter and RNA pull‐down assays. Protein expression was analyzed by western blot. Xenograft tumor model was adopted to evaluate hsa_circ_0001550 function in vivo.ResultsHsa_circ_0001550 enrichment was enhanced in CRC tissue specimens and cell lines. Hsa_circ_0001550 absence hindered CRC cell proliferation, metastasis, stemness, and caused apoptosis. Hsa_circ_0001550 targeted miR‐4262, and hsa_circ_0001550 absence‐caused impacts were diminished by anti‐miR‐4262. MiR‐4262 targeted NUCKS1. Hsa_circ_0001550 had positive regulation on NUCKS1 expression. NUCKS1 overexpression overturned the influences of hsa_circ_0001550 silencingon CRC cell progression. Hsa_circ_0001550 interference notably blocked in vivo xenograft tumor growth.ConclusionHsa_circ_0001550 facilitated CRC progression by binding to miR‐4262 to positively regulate NUCKS1 abundance.

METTL27 is a prognostic biomarker of colon cancer and associated with immune invasion

To investigate the expression and gene function of methyltransferase-like protein 27 (METTL27) in colon cancer, its association with immune infiltration and its prognostic significance. We analyzed the expression levels of METTL27 in 33 cancers using R language and identified METTL27 as a differential gene in colon cancer. The related signaling pathways of METTL27 were analyzed by gene functional annotation and enrichment. SsGSEA algorithm was used to analyze immune infiltration, and logistic analysis was used to evaluate the correlation between METTL27 expression and clinicopathological features of the patients. Kaplan-meier analysis, univariate and multivariate Cox regression analysis were performed to construct a nomogram for evaluating the correlation between METTL27 expression and clinical prognosis. The expression level of METTL27 was further verified in colorectal cancer cell lines and 16 clinical specimens of colorectal cancer tissues using qPCR and Western blotting. METTL27 was highly expressed in 21 cancers, and its expression was significantly higher in colon cancer than in adjacent tissues (P < 0.001). METTL27-related genes were identified by differential analysis, and functional annotation revealed that METTL27 was significantly enriched in transmembrane transport and lipid metabolism, and 5 related signaling pathways were identified by GSEA. METTL27 expression was negatively correlated with different T helper cells and central memory T cells (P < 0.001). The patients with a high METTL27 mRNA expression had a poor survival outcome. Cox regression analysis showed that METTL27 expression was an independent prognostic factor of the overall survival. The expression level of METTL27 was significantly higher in the colorectal cancer cell line than in normal cells (P < 0.05). METTL27 is overexpressed in colon cancer and is associated with a poor prognosis of the patients. A high expression of METTL27 showed is associated less T cell immune infiltration, suggesting the potential of METTL27 as a prognostic marker of colon cancer.

ActivinA promotes cell proliferation, invasion and migration and predicts poor prognosis in patients with colorectal cancer.

Activin A is a member of the transforming growth factor-β superfamily of cytokines and displays various pathophysiological activities, including regulation of muscle catabolism and atrophy. Activin A expression is upregulated in several human cancer types and in certain pathologies, its expression is associated with poor prognosis. In the present study, activin A expression was assessed in colorectal cancer (CRC) tissue specimens from 157 patients with primary CRC and the relationship between activin A levels and clinicopathological characteristics, including skeletal muscle mass, and prognosis, was determined. Furthermore, the effects of knockdown of endogenous or exposure to exogenous activin A on the malignant behavior of human CRC cell lines were investigated in vitro. The results indicated that activin A mRNA was significantly upregulated in CRC tumor tissues compared with normal intestinal epithelium. High activin A expression was significantly associated with shorter cancer-specific survival (P=0.047) and overall survival (P=0.014). According to a multivariate analysis, tumor activin A levels were an independent prognostic factor for overall survival (P=0.001). However, activin A mRNA levels were not associated with the skeletal muscle index. The in vitro experiments demonstrated that exposure to exogenous activin A increased the proliferation, invasion and migration of CRC cell lines, whereas knockdown of endogenous activin A had the opposite effects. In conclusion, activin A is an autocrine and paracrine regulator of CRC cell proliferation and high tumor expression of activin A is associated with poor prognosis in patients with CRC.

Delta-like ligand 4 level in colorectal cancer is associated with tumor aggressiveness and clinical outcome.

The Notch signaling regulates numerous cell growth, differentiation, and death. However, the expression pattern of its ligand Delta-like 4 (DLL4) in tumors is still uncertain. In the present study, we examined DLL4 expression in colorectal cancer as well as assessed its role as a prognostic indicator in the present study. DLL4 expression was examined by immunohistochemistry in 289 surgically resected specimens of colorectal cancer and adjacent normal tissues. The relationship between DLL4 expression and clinicopathological characteristics was analyzed. The association of DLL4 expression with the patients' overall survival rate was assessed by Kaplan-Meier and Cox proportional-hazards regression. Increased DLL4 level was detected in colorectal cancer compared with that of normal tissues. Elevated DLL4 level in colorectal cancer was associated with increased body mass index of patients. Moreover, increased DLL4 level was also found to be correlated with tumor invasion, metastases and unfavorable clinical outcom of patients. DLL4 level is increased in colorectal cancer, especially in patients with increased body mass index, indicating potential involvement of obesity-related tumorigenesis and development. It might also serve as a novel molecular marker to predicate outcome of patients.

Chosen Antioxidant Enzymes GPx4 and GPx8 in Human Colorectal Carcinoma: Study of the Slovak Population.

Background and Objectives: Nowadays colorectal carcinoma (CRC) is one of the most common causes of death in patients with malignant neoplasms worldwide. Our work aimed to determine the possible involvement of glutathione peroxidases 4 and 8 (GPx4 and GPx8) in this specific tumor process. Materials and Methods: The expression of GPx4 and GPx8 in 58 specimens of human colorectal cancer tissues and normal tissues was detected by the indirect immunohistochemical method under a light microscope. Statistical analysis was done by Chi-squared test. Histological findings were compared with data such as gender, age, tumor grade, histotype and lymph nodes alteration. Results: In all specimens of healthy tissue the presence of both, GPx4 and GPx8, was detected in the cytoplasm of epithelial cells. On the other hand, a positive immunohistochemical reaction against GPx4 only in 41.4% and against GPx8 only in 29.3% of human colorectal adenocarcinoma specimens were observed. Any significant difference between the presence of GPx and the age, the gender of the patient, tumor grade, histotype of cancer and the lesion of regional lymph nodes has not been detected. Conclusions: Our foundation could mean, that GPx4 and GPx8 have no important role in CRC pathogenesis, but the loss of these enzymes probably indicates a serious pathological process ongoing in the large intestine. To our knowledge, this is the first paper describing GPx8 presence in human colorectal carcinoma.

Evaluation of intestinal cancer by 1H HR-MAS NMR spectroscopy: A new diagnostic tool?

Previous studies have demonstrated that cancer cells harbor unique metabolic characteristics relative to healthy counterparts. The current study is a prospective ex-vivo HR-MAS NMR analysis of malignant colorectal cancer (CRC) tissue specimens and its corresponding benign tissues.To assess the HR-MAS Spectroscope qualitatively &amp; to analyze significant difference between the normal, benign and malignant intestinal mucosa.Between November 2013and January 2016, 36 consecutive patients with confirmed CRC were recruited to a prospective observational study. Fresh tissue samples were obtained from center of tumor and 5 cm from tumor margin from surgical resection specimens. Samples were run in duplicate where tissue volume permitted to compensate for anticipated sample heterogeneity. Typically, the sample was packed into a 4 mm ZrO2 rotor of 50 μl capacity; a volume of 20μl of D2O having 0.03% TSP was used as a chemical shift reference. The sample-rotor-setup was then transferred into the HR-MAS NMR probe for analysis.A total of 36 spectra were acquired (center of tumor, n = 18; 5 cm from tumor margin, n = 18). The malignant clustering occurs due to increased Val (0.90ppm), Lac (1.34ppm), Ala(1.48ppm) levels of acetate (1.90ppm), glutamate (2.35ppm), taurine (3.23 ppm), choline containing compounds (3.20-3.22ppm), glycine (3.56ppm), lactate (4.12ppm) and α-H of Leu, Ileu, Val, Lys, Ala (3.76-3.79ppm . In addition unique metabolic profiles were observed for tumors of differing T-stage. The information gathered from clustering in PCA had highly suggested that malignancy induces metabolic perturbations at cellular levels.HR-MAS NMR profiling demonstrates cancer-specific metabolic signatures in CRC and reveals metabolic differences between benign and malignant tumors. In addition, this approach reveals that tumor metabolism undergoes modification during local tumor advancement, offering potential in future staging and therapeutic approaches.

LncRNA ENSG00000254615 Modulates Proliferation and 5-FU Resistance by Regulating p21 and Cyclin D1 in Colorectal Cancer

5-Fluorouracil (5-FU) resistance is an urgent problem of colorectal cancer (CRC) chemotherapy that needs to be resolved. To investigate 5-FU-associated lncRNAs for CRC might be of great significant. LncRNA ENSG00000254615 was detected by RNA-sequencing. ENSG00000254615 were detected highly expressed in 5-FU-sensitive CRC cells and tissue specimens, and inhibited cell proliferation and attenuated 5-FU resistance in vitro and in vivo. Furthermore, ENSG00000254615 participated in the regulation of p21 and Cyclin D1. Taken together, we proposed that ENSG00000254615 inhibits proliferation and attenuates 5-FU resistance of CRC by regulating p21 and Cyclin D1 expression.