Fusobacterium nucleatum and Bacteroides fragilis detection in colorectal tumours: Optimal target site and correlation with total bacterial load

Background Mucosal infiltration by certain bacterial species may contribute to the development and progression of colorectal cancer (CRC). There is considerable variation in reported detection rates in human CRC samples and the extent to which bacterial infiltration varies across regions of the primary tumour is unknown. This study aimed to determine if there is an optimal site for bacterial detection within CRC tumours. Methods Presence of target bacterial species was assessed by quantitative real-time PCR (qPCR) in 42 human CRC tumours. Abundance in primary tumour regions, normal epithelium and at metastatic sites was investigated in an expanded cohort of 51 patients. Species presence/absence was confirmed by diversity profiling in five patients. Correlation with total bacterial load and clinicopathological features was assessed. Results Fusobacterium nucleatum and Bacteroides fragilis were detected in tumours from 43% and 24% of patients, respectively (17% positive for both species). The optimal detection site was the tumour luminal surface (TLS). Patients testing positive at the TLS frequently tested negative at other sites, including central tumour and invasive margin. F. nucleatum was detected at a higher frequency in tumour versus normal epithelium (p < 0.01) and was associated with more advanced disease (p = 0.01). Detection of both species correlated with total bacterial load. However, corroboration of qPCR results via diversity profiling suggests detection of these species may indicate a specific microbial signature. Conclusions This study supports a role for F. nucleatum in CRC development. Presence of F. nucleatum and B. fragilis varies across primary tumour regions, with the TLS representing the optimal site for bacterial detection.

FNDC3B and BPGM Are Involved in Human Papillomavirus-Mediated Carcinogenesis of Cervical Cancer

Human papillomavirus (HPV)-mediated cervical carcinogenesis is a multistep progressing from persistent infection, precancerous lesion to cervical cancer (CCa). Although molecular alterations driven by viral oncoproteins are necessary in cervical carcinogenesis, the key regulators behind the multistep process remain not well understood. It is pivotal to identify the key genes involved in the process for early diagnosis and treatment of this disease. Here we analyzed the mRNA expression profiles in cervical samples including normal, cervical intraepithelial neoplasia (CIN), and CCa. A co-expression network was constructed using weighted gene co-expression network analysis (WGCNA) to reveal the crucial modules in the dynamic process from HPV infection to CCa development. Furthermore, the differentially expressed genes (DEGs) that could distinguish all stages of progression of CCa were screened. The key genes involved in HPV-CCa were identified. It was found that the genes involved in DNA replication/repair and cell cycle were upregulated in CIN compared with normal control, and sustained in CCa, accompanied by substantial metabolic shifts. We found that upregulated fibronectin type III domain-containing 3B (FNDC3B) and downregulated bisphosphoglycerate mutase (BPGM) could differentiate all stages of CCa progression. In patients with CCa, a higher expression of FNDC3B or lower expression of BPGM was closely correlated with a shorter overall survival (OS) and disease-free survival (DFS). A receiver operating characteristic (ROC) analysis of CIN and CCa showed that FNDC3B had the highest sensitivity and specificity for predicting CCa development. Taken together, the current data showed that FNDC3B and BPGM were key genes involved in HPV-mediated transformation from normal epithelium to precancerous lesions and CCa.

673 (Re-) Solving the biology of colorectal cancer onset and progression to improve treatment and prevention

BackgroundColorectal cancer (CRC) development is accompanied by the gradual accumulation of genetic alterations in epithelial cells of the colon and rectum.1 2 The paradigm of the adenoma-carcinoma sequence was originally centered around cancer cells; however, it is now clear that the tumor microenvironment plays a substantial role in cancer progression and patient outcome.3 In recent years, technologies have evolved rapidly, allowing the multiplexed quantification of gene expression while preserving spatial context.4 Furthermore, some spatial transcriptomic technologies also allow the parallel interrogation of different cell populations in the tumor microenvironment. Here, we performed digital spatial profiling on early-stage CRC samples to elucidate the biological processes that are at the basis of malignant transformation and to identify novel therapeutic targets and (immune) biomarkers.MethodsEndoscopically resected early-stage CRC samples were obtained at Leiden University Medical Center. In total, 144 areas of illumination were interrogated with GeoMx digital spatial profiling using the Cancer Transcriptome Atlas (>1,800 genes). In each of eight samples, nine regions of interest with different levels of cancer progression were selected, including normal epithelium, transition areas, low-, and high-grade dysplasia, and invasive carcinoma (figure 1A). We segmented each region based on cytokeratin and vimentin protein expression (figure 1B). Immunohistochemical detection was performed on these samples and 26 additional samples to validate targets associated with disease progression.ResultsDigital spatial profiling allowed us to dissect transcriptional alterations in epithelial and stromal fractions between different regions from healthy tissue, different degrees of dysplasia, and cancer. Gene expression data revealed a clear separation of profiled areas by histologic category. Interestingly, gene expression features in the stromal compartment provided a better data-driven separation of histologic categories than the epithelial fraction (figure 1C). Substantial changes in immune-related pathways were identified, including differential expression of specific immunomodulators. We validated the expression of several candidate biomarkers/targets that demonstrated consistent alterations from normal tissue to cancer by immunohistochemistry. Several proteins were identified that could clearly discriminate benign from malignant tissue.ConclusionsWe here demonstrated the unique biological insights that are provided by spatial examination of early-stage CRC by digital spatial profiling. We identified specific genes that were altered during CRC tumorigenesis, in epithelial and stromal/immune fractions. Furthermore, our results indicate an essential role for innate immunity in colorectal cancer onset and progression. The genes identified by this approach could potentially serve as novel biomarkers and targets for early interception or prevention of CRC development.AcknowledgementsThis work was supported by the European Research Council (ERC) Starting grant awarded to Dr. Noel F. de Miranda and the Stichting Management Apothekers en de Gezondheidszorg (STIMAG) Research grant awarded to Jessica Roelands.Trial RegistrationN/AReferencesFearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61(5):759–767. doi: 10.1016/0092-8674(90)90186-I.Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194(4260):23–28. doi: 10.1126/science.959840.Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646–674. doi: 10.1016/j.cell.2011.02.013.Merritt CR, et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat Biotechnol 2020;38(5):586–599. doi: 10.1038/s41587-020-0472-9.Ethics ApprovalThis study was approved by the METC Leiden-Den Haag-Delft (protocol B20.039). Patient samples were anonymised and handled according to the medical ethical guidelines described in the Code of Conduct for Proper Secondary Use of Human Tissue of the Dutch Federation of Biomedical Scientific Societies.Abstract 673 Figure 1Transcriptional alterations in early-stage colorectal cancer. Digital spatial profiling defines transcriptional alterations in early-stage colorectal cancer. (A) Schematic representation of an early-stage CRC sample containing regions with different levels of cancer progression, including normal epithelium, transition areas, low-, and high-grade dysplasia, and invasive carcinoma. (B) Segmentation based immunofluorescent labelling with antibodies directed against PanCK and Vimentin in one of the early-stage CRC samples. Artificial overlay of implemented segmentation is indicated for each ROI, visualizing Vimentin+ (pink) and PanCK+ (orange) segments. Inset: higher magnification of an individual ROI. (C) Dimension reduction of expression of all quantified genes by t-Distributed Stochastic Neighbor Embedding (tSNE). tSNE plots are annotated by segment (left), and histological region (right).

When Activator and Inhibitor of PPARα Do the Same: Consequence for Differentiation of Human Intestinal Cells

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-dependent transcription factor that plays a role in various processes including differentiation of several cell types. We investigated the role of PPARα in the differentiation of intestinal cells using HT-29 and Caco2 cell lines as a model as well as human normal colon and colorectal carcinoma tissues. We detected a significant increase in PPARα expression in differentiated HT-29 cells as well as in normal surface colon epithelium where differentiated cells are localised. Thus, it seems that PPARα may play a role in differentiation of intestinal cells. Interestingly, we found that both PPARα activators (fenofibrate and WY-14643) as well as its inhibitor (GW6471) regulated proliferation and differentiation of HT-29 cells in vitro in the same way. Both compounds led to a decrease in proliferation accompanied by a significant increase in expression of villin, intestinal alkaline phosphatase (differentiation markers). Moreover, the same trend in villin expression was observed in Caco2 cells. Furthermore, villin expression was independent of subcellular localisation of PPARα. In addition, we found similar levels of PPARα expression in colorectal carcinomas in comparison to adjacent normal epithelium. All these findings support the hypothesis that differentiation of intestinal epithelium is PPARα-independent.

A local tumor microenvironment acquired super-enhancer induces an oncogenic driver for efficient growth under oxidative conditions in colorectal carcinoma

Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating its etiology via local tumor microenvironment acquisition. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor.

Organoids and Their Use in Modeling Gut Epithelial Cell Lineage Differentiation and Barrier Properties During Intestinal Diseases

Maintenance of intestinal epithelium homeostasis is a complex process because of the multicellular and molecular composition of the gastrointestinal wall and the involvement of surrounding interactive signals. The complex nature of this intestinal barrier system poses challenges in the detailed mechanistic understanding of intestinal morphogenesis and the onset of several gut pathologies, including intestinal inflammatory disorders, food allergies, and cancer. For several years, the gut scientific community has explored different alternatives in research involving animals and in vitro models consisting of cultured monolayers derived from the immortalized or cancerous origin cell lines. The recent ability to recapitulate intestinal epithelial dynamics from mini-gut cultures has proven to be a promising step in the field of scientific research and biomedicine. The organoids can be grown as two- or three-dimensional structures, and are derived from adult or pluripotent stem cells that ultimately establish an intestinal epithelium that is composed of all differentiated cell types present in the normal epithelium. In this review, we summarize the different origins and recent use of organoids in modeling intestinal epithelial differentiation and barrier properties.

PROLIFERATIVE POTENTIALITY OF AMELOBLASTOMA BY EXPRESSION OF Ki-67

Ameloblastoma is a type of Odontogenic tumor, commonly encountered in orofacial region. It is very aggressive in nature with potential features of recurrence. Ki -67 is already a proven proliferative marker, which is used in our study to assess the proliferative potentiality of Ameloblastoma. Incisional biopsy was done from 10 cases of Ameloblastoma and 5 normal cases as control, at the department of Oral pathology and Microbiology, BIDSH, Patna. Each specimen was processed and two sections were prepared and stained with hematoxylin and eosin and Ki-67 immunohistochemical marker. Positive stained cells with with Ki-67 in ameloblastoma along with normal epithelium were counted from four randomly selected areas and were quantied using a microscope at 400x magnication. Positive correlation seen with Ki-67 positive cells and percentages, which indicates the value of one variable increases, the other also increases. The overall conclusion is that Ki-67 expression can be used as a prognostic marker in Ameloblastoma.