Poster H2 - Specificity of Claudin 8, α-SMA, and Anti-LRRN4 as Markers for Mesothelial Cells in Ovarian Cancer Progression

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Daniel Ibrahim

Researcher at Tumor Microenvironment Engineering Lab
University of Pittsburgh
SEWICKLEY, Pennsylvania, United States

Primary Investigator(s)

IZ

Ioannis Zervantonakis

PI for Tumor Microenvironment Engineering Lab
University of Pittsburgh, United States

Introduction: Ovarian cancer is one of the most lethal gynecologic cancers, often diagnosed late due to subtle symptoms and a lack of effective screening. Mesothelial cells, which line the peritoneal cavity, play a crucial role in cancer progression by interacting with ovarian cancer cells and promoting tumor growth. Despite their importance, better diagnostic tools and therapies targeting these interactions are needed. This research investigates the specificity of antibodies for mesothelial cells to improve microfluidic device design for more accurate diagnostics. By staining for Claudin 8 and anti-LRRN4, their potential as reliable markers was assessed, focusing on cell adhesion, signaling, and tight junctions. Additionally, α-SMA staining was performed to explore mesothelial-to-mesenchymal transition. Positive stains would indicate strong marker specificity, while negative results would suggest the need for alternative markers

Materials and

Methods: The methodology involved several steps to identify and analyze mesothelial cells from primary cancer patient xenografts. Paraffin-embedded tissue sections were deparaffinized, followed by antigen retrieval to enhance primary antibody binding. Hematoxylin and Eosin (H&E) staining confirmed the presence of tumors and relevant organs. Primary antibodies, diluted at 1:250, were applied and incubated overnight at 4°C. Secondary antibodies, diluted at 1:500, were then introduced for fluorescence detection. Hoechst stain, diluted at 1:1000, was used to counterstain nuclei by binding to DAPI. The stained slides were imaged using a fluorescence microscope, and the images were analyzed with Cell Profiler software to identify mesothelial cells based on specific markers. To ensure reliability, the experiments were repeated with different cancer patients across multiple trials.

Results, Conclusions, and Discussions: The immunofluorescence staining experiments revealed significant differences in the mean intensity of Claudin 8, α-SMA, and Anti-LRRN4 between mesothelial cells (MC) and the cytoplasm (CY), with highly significant p-values (Fig 1). This strong statistical significance underscores the reliability of these markers in distinguishing mesothelial cells from other cell types. Figure 1A illustrates the anatomical and cellular context of mesothelial cells within the peritoneal cavity. They function as a protective barrier, secrete lubricating fluid to reduce friction between organs, and regulate selective permeability to maintain homeostasis. In the context of ovarian cancer, during mesothelial to mesenchymal transition (MMT), mesothelial cells become more cuboidal, break cell–cell junctions, separate and expose underlying basement membrane and ECM{Mutsaers, 2015 #2}.
Claudin 8, a tight junction protein crucial for maintaining cell-to-cell adhesion and barrier functions, showed a significant difference in staining intensity between mesothelial cells and the cytoplasm indicating its specificity and role in forming selective permeability barriers in the peritoneal cavity.
The detection of alpha-smooth muscle actin (α-SMA), in mesothelial cells (not a common marker for mesothelial cells) could suggest MMT. Marked differences in α-SMA staining intensity between mesothelial cells and the cytoplasm further highlights the dynamic nature of mesothelial cells in the tumor microenvironment.
Leucine-rich repeat neuronal protein 4 (LRRN4), involved in cell signaling and adhesion, also displayed a substantial difference in staining intensity between mesothelial cells and the cytoplasm. This further supports its specificity as a mesothelial cell marker and its potential role in MMT.

Acknowledgements (Optional):