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Yuen Yee Cheng
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MA 19 - Mesothelioma: Bench to Bedside (ID 680)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Mesothelioma
- Presentations: 1
- Moderators:Dean A Fennell, Hedy Lee Kindler
- Coordinates: 10/18/2017, 11:00 - 12:30, Room 315
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MA 19.06 - Multiple Mechanisms Contribute to Downregulation of Tumour Suppressor microRNAs in Malignant Pleural Mesothelioma (ID 9745)
11:35 - 11:40 | Author(s): Yuen Yee Cheng
- Abstract
- Presentation
Background:
Malignant pleural mesothelioma (MPM) is a disease with an almost invariably fatal diagnosis with limited therapeutic options. Characteristic patterns of deregulated microRNA expression have been demonstrated in MPM, and many downregulated microRNAs have been shown to have tumour suppressor activity. However, apart from silencing of miR-34b/c by promoter hypermethylation and co-deletion of miR-31 with the CDKN2A locus, the mechanisms responsible for downregulation of other tumour suppressor miRNAs such as miR-16 are yet to be elucidated.
Method:
Tumour samples (n=60) were from MPM patients undergoing extrapleural pneumonectomy, and samples of pleura (n=23) collected from patients undergoing cardiac surgery were used as normal controls. MPM cells lines were obtained from the ATCC. Expression levels of mature microRNAs in MPM tumour samples and cell lines, and pri-miRs and miRNA host genes in cell lines, were determined by RT-qPCR. Copy number variation (CNV) was analysed by droplet digital PCR (ddPCR), and methylation was inferred by miRNA expression following decitabine treatment. MYC was analysed by Western blot, and expression modulated by siRNAs.
Result:
Analysis of microRNA expression in tumour samples revealed a consistent and significant downregulation of miR-15a (4-fold, P<0.01), 15b (10-fold, P<0.01), 16 (22-fold, P<0.05), 34a (1.6-fold, P<0.05), 34b (1.8-fold, P<0.01), 34c (2.3-fold, P<0.0001) and 193a (3.1-fold, P<0.001) compared with normal pleura. Copy number variation analysis showed evidence of heterozygous loss for miR-193a (4 of 5 cell lines) and miR-15a/16-1 (2 of 5), but no change in miR-15b/16-2. Treating cell lines with the demethylating agent decitabine resulted in dramatic upregulation only in the case of miR-34c. RNAi-mediated knockdown of c-MYC led to upregulation of miR-15b and 16, and to a lesser extent miR-15a, as well as a consistent increase in the miR-15b/16-2 host gene SMC4 and the miR-15a/16-1 host gene DLEU2. Analysing the expression of these microRNAs in the tumour samples revealed a strong correlation between miR-15b and 16 (R[2]=0.793) and miR-34b and 34c (R[2]=0.753), but not between others.
Conclusion:
Our data suggest that a combination of deletion, hypermethylation and transcriptional regulation contribute to the downregulation of miR-15a/b, 16, 34a/b/c and 193a. In MPM, unlike other cancers, the downregulation of miR-15a/16-1, miR-15b/16-2 appears to be due to transcriptional changes rather than deletion or promoter hypermethylation. MYC appears to contribute to miR-16 downregulation primarily via control of SMC4 and the miR-15b/16-2 locus, suggesting that the transcriptional control of miR-16 expression by c-Myc contributes to the malignant phenotype of MPM.
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-078 - Establishing Malignant Pleural Mesothelioma Primary Cell Lines Using the 3D Spheroid Method Produces a Model with Better Tumour Architecture (ID 10456)
09:30 - 09:30 | Presenting Author(s): Yuen Yee Cheng
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive malignancy with no effective treatment options. Poor prognosis and drug resistance are the main challenges of this deadly disease. There is also no simple distinctive diagnosis tool for identification of MPM. Better diagnostic markers may also provide better biological information for newer treatment option development. In this study we have established primary MPM cell lines and characterised them with current biomarkers. Our ultimately goal is to use these cell lines for better identification of diagnostic biomarkers.
Method:
MPM cell lines were either established from tissue specimens or pleural effusion from patients with pathologically confirmed MPM. Cells were cultured in standard (2D) and spheroid (3D) versions for characterisation. Cells prepared in 2D and 3D were stained with H&E and analysed with a diagnostic biomarker panel (CK-8/18, Calretinin, CK5/6, CD141, WT-1, D2-40, EMA, CEA, Tag-72, BG8, CD15, TTF-1 and BAP1). Scoring and comments were provided by pathologists experienced in MPM diagnosis (KL, SK). Established cell lines were also analysed for ploidy (flow cytometry) and interphase (fluorescent microscope) for chromosome number. PBMC from healthy donor was used as a control diploid.
Result:
We successfully established nine cell lines from MPM patient specimens. The original tumour histological sub-types were: three epithelioid, four biphasic, one desmoplastic and one not otherwise specified. Cells grown in 3D with H&E staining revealed better tumour architecture, cell-cell contacts and morphology when compared to cells grown in standard 2D culture. Mesothelioma positive markers were more distinctive and intense in biphasic cell lines grown 3D culture. Other sub-types showed similar staining when grown in both formats. Results from ploidy showed no distinctive difference between sub-types, however, 5 out of 9 cell lines established had tetraploid chromosome content.
Conclusion:
Cells grown in 3D provide more tumour architecture when compared with 2D cells. 3D cells also provide more intensity and greater percentage of positive MPM markers
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P3.02-079 - A 3D Spheroid Culture Represents an Improved in Vitro Model of Malignant Plural Mesothelioma (MPM) (ID 10472)
09:30 - 09:30 | Presenting Author(s): Yuen Yee Cheng
- Abstract
Background:
Most biologists rely on cell culture in the two-dimensional (2D) format for studying tumour context which does not accurately reflect the in vivo state. 3D cell culture techniques provide cell-to-cell interactions that better mimic pathological conditions such as cancer. Malignant pleural mesothelioma (MPM) is a deadly cancer with no effective treatment and is highly drug resistance. Our study has addressed this problem by growing cells in 3D thus creating an environment that is more closely mimics the realistic tumour state for molecular and cell effect studies.
Method:
MPM cell lines were grown in conventional 2D, our newly optimised 3D format, hanging drop and poly-HEMA methods. Cells were analysed their structure by light, scanning electron (SEM) and transmission electron (TEM) microscopy. Drug infiltration was confirmed by intravital-microscopy. Cell proliferationswith with different dose of drugs (Cisplatin and Gemcitabine) were analysed in 2D and 3D cells. Cells grown as truly spherical spheroids and their 2D counterparts were harvested for tumour suppressor analysis. 16 previously reported tumour suppressors (ANK1, MIB1, RGS22, TNIL, GMC, SVIL, ATG4D, HOXB4, SCLC25A13, CHST11, ATG4D, GTF2A1, KIAA1361, PDZD2, WDR1 and TMSB15B) and 3 oncogenes (YAP1, ABCG2 and YB1) were analysed.
Result:
The 3D spheroids represent an improved 'mini-tumour' model as indicated by the visualization of cell junctions under TEM. The 3D spheroids (11 MPM lines) formed using our method also provided perfect spherical shape and revealed healthy and surface morphology by SEM analysis. We showed in our model drugs was able to penetrate from outside to centre of the spheroids. However, in our hands, the hanging drop and the poly-HEMA versions did not always produce spherical 3D cells. Cells grown in our 3D model display greater drug resistance when compared with 2D cells. Most tumour suppressor biomarkers we analysed showed down-regulation of mRNA expression level compared with cells in 2D. These tumour suppressor genes were host genes of microRNAs (e.g. MIB1 for miR-1). Most of them are frequently down-regulated in MPM. Our 3D model also showed up-regulation of genes that contribute to drug resistance such as Hif1a, YAP1, ABCG2 and YB1.
Conclusion:
3D cells grown with the newly optimised method provide a better mimic of more realistic MPM evidence by the more resistance of MPM cells to cisplatin and gemcitabine when compared to cells grown in 2D. MPM cells grown in 3D also down-regulated of tumour suppressors and up-regulation of drug resistance genes when compared to 2D cells.
