The Role of E-Cadherin and microRNA on FAK Inhibitor Response in Malignant Pleural Mesothelioma (MPM).

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with limited effective treatment options. Focal adhesion kinase (FAK) inhibitors have been shown to efficiently suppress MPM cell growth initially, with limited utility in the current clinical setting. In this study, we utilised a large collection of MPM cell lines and MPM tissue samples to study the role of E-cadherin (CDH1) and microRNA on the efficacy of FAK inhibitors in MPM. The immunohistochemistry (IHC) results showed that the majority of MPM FFPE samples exhibited either the absence of, or very low, E-cadherin protein expression in MPM tissue. We showed that MPM cells with high CDH1 mRNA levels exhibited resistance to the FAK inhibitor PND-1186. In summary, MPM cells that did not express CDH1 mRNA were sensitive to PND-1186, and MPM cells that retained CDH1 mRNA were resistant. A cell cycle analysis showed that PND-1186 induced cell cycle disruption by inducing the G2/M arrest of MPM cells. A protein-protein interaction study showed that EGFR is linked to the FAK pathway, and a target scan of the microRNAs revealed that microRNAs (miR-17, miR221, miR-222, miR137, and miR148) interact with EGFR 3'UTR. Transfection of MPM cells with these microRNAs sensitised the CHD1-expressing FAK-inhibitor-resistant MPM cells to the FAK inhibitor.

Genomic Deletion of BAP1 and CDKN2A Are Useful Markers for Quality Control of Malignant Pleural Mesothelioma (MPM) Primary Cultures.

Malignant pleural mesothelioma (MPM) is a deadly cancer that is caused by asbestos exposure and that has limited treatment options. The current standard of MPM diagnosis requires the testing of multiple immunohistochemical (IHC) markers on formalin-fixed paraffin-embedded tissue to differentiate MPM from other lung malignancies. To date, no single biomarker exists for definitive diagnosis of MPM due to the lack of specificity and sensitivity; therefore, there is ongoing research and development in order to identify alternative biomarkers for this purpose. In this study, we utilized primary MPM cell lines and tested the expression of clinically used biomarker panels, including CK8/18, Calretinin, CK 5/6, CD141, HBME-1, WT-1, D2-40, EMA, CEA, TAG72, BG8, CD15, TTF-1, BAP1, and Ber-Ep4. The genomic alteration of CDNK2A and BAP1 is common in MPM and has potential diagnostic value. Changes in CDKN2A and BAP1 genomic expression were confirmed in MPM samples in the current study using Fluorescence In situ Hybridization (FISH) analysis or copy number variation (CNV) analysis with digital droplet PCR (ddPCR). To determine whether MPM tissue and cell lines were comparable in terms of molecular alterations, IHC marker expression was analyzed in both sample types. The percentage of MPM biomarker levels showed variation between original tissue and matched cells established in culture. Genomic deletions of BAP1 and CDKN2A, however, showed consistent levels between the two. The data from this study suggest that genomic deletion analysis may provide more accurate biomarker options for MPM diagnosis.

3-Dimensional mesothelioma spheroids provide closer to natural pathophysiological tumor microenvironment for drug response studies.

Traditional studies using cancer cell lines are often performed on a two-dimensional (2D) cell culture model with a low success rate of translating to Phase I or Phase II clinical studies. In comparison, with the advent of developments three-dimensional (3D) cell culture has been championed as the latest cellular model system that better mimics in vivo conditions and pathological conditions such as cancer. In comparison to biospecimens taken from in vivo tissue, the details of gene expression of 3D culture models are largely undefined, especially in mesothelioma - an aggressive cancer with very limited effective treatment options. In this study, we examined the veracity of the 3D mesothelioma cell culture model to study cell-to-cell interaction, gene expression and drug response from 3D cell culture, and compared them to 2D cell and tumor samples. We confirmed via SEM analysis that 3D cells grown using the spheroid methods expressed highly interconnected cell-to-cell junctions. The 3D spheroids were revealed to be an improved mini-tumor model as indicated by the TEM visualization of cell junctions and microvilli, features not seen in the 2D models. Growing 3D cell models using decellularized lung scaffold provided a platform for cell growth and infiltration for all cell types including primary cell lines. The most time-effective method was growing cells in spheroids using low-adhesive U-bottom plates. However, not every cell type grew into a 3D model using the the other methods of hanging drop or poly-HEMA. Cells grown in 3D showed more resistance to chemotherapeutic drugs, exhibiting reduced apoptosis. 3D cells stained with H&E showed cell-to-cell interactions and internal architecture that better represent that of in vivo patient tumors when compared to 2D cells. IHC staining revealed increased protein expression in 3D spheroids compared to 2D culture. Lastly, cells grown in 3D showed very different microRNA expression when compared to that of 2D counterparts. In conclusion, 3D cell models, regardless of which method is used. Showed a more realistic tumor microenvironment for architecture, gene expression and drug response, when compared to 2D cell models, and thus are superior preclinical cancer models.