Analysis of Gene Expression in 3D Spheroids Highlights a Survival Role for ASS1 in Mesothelioma

Our plain-English summary of Analysis of Gene Expression in 3D Spheroids Highlights a Survival Role for ASS1 in Mesothelioma, by D. Barbone, L. Van Dam, C. Follo, et al (PloS One, 2016).

The study

• Authors: D. Barbone, L. Van Dam, C. Follo, P.V. Jithesh, S.D. Zhang, W.G. Richards, R. Bueno, D.A. Fennell, V.C. Broaddus
• Journal: PloS One
• Published: 2016;11(3):e0150044
• Cited by: 33 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

To investigate the underlying causes of chemoresistance in malignant pleural mesothelioma, we have studied mesothelioma cell lines as 3D spheroids, which acquire increased chemoresistance compared to 2D monolayers. We asked whether the gene expression of 3D spheroids would reveal mechanisms of resistance. To address this, we measured gene expression of three mesothelioma cell lines, M28, REN and VAMT, grown as 2D monolayers and 3D spheroids. A total of 209 genes were differentially expressed in common by the three cell lines in 3D (138 upregulated and 71 downregulated), although a clear resistance pathway was not apparent. We then compared the list of 3D genes with two publicly available datasets of gene expression of 56 pleural mesotheliomas compared to normal tissues. Interestingly, only three genes were increased in both 3D spheroids and human tumors: argininosuccinate synthase 1 (ASS1), annexin A4 (ANXA4) and major vault protein (MVP); of these, ASS1 was the only consistently upregulated of the three genes by qRT-PCR. To measure ASS1 protein expression, we stained 2 sets of tissue microarrays (TMA): one with 88 pleural mesothelioma samples and the other with additional 88 pleural mesotheliomas paired with matched normal tissues. Of the 176 tumors represented on the two TMAs, ASS1 was expressed in 87 (50%; staining greater than 1 up to 3+). For the paired samples, ASS1 expression in mesothelioma was significantly greater than in the normal tissues. Reduction of ASS1 expression by siRNA significantly sensitized mesothelioma spheroids to the pro-apoptotic effects of bortezomib and of cisplatin plus pemetrexed. Although mesothelioma is considered by many to be an ASS1-deficient tumor, our results show that ASS1 is elevated at the mRNA and protein levels in mesothelioma 3D spheroids and in human pleural mesotheliomas. We also have uncovered a survival role for ASS1, which may be amenable to targeting to undermine mesothelioma multicellular resistance.

In plain English

This was a laboratory study of mesothelioma cells and tumor tissue. It did not study asbestos exposure. The authors grew three mesothelioma cell lines (M28, REN and VAMT) both as flat layers (2D) and as three-dimensional cell clusters called spheroids (3D). In all three lines, 209 genes changed in 3D: 138 went up and 71 went down. The authors compared these with public data on 56 mesotheliomas and 14 normal tissues. Three genes were raised in both, but only ASS1 was confirmed as raised in all three cell lines by a second test. ASS1 protein rose in 3D in four of six cell lines, but not in normal mesothelial cells. Among 176 pleural mesotheliomas, 87 (50%) had moderate to high ASS1 staining; the other 89 had low or none. In 88 tumors paired with normal tissue from the same patients, ASS1 was significantly higher in the tumors. In spheroids of three cell lines, lowering ASS1 increased cell death, both on its own and with bortezomib or with cisplatin plus pemetrexed. The authors concluded that ASS1 overexpression in mesothelioma merits attention.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study: gene expression microarray comparing mesothelioma cell lines grown as 2D monolayers and 3D spheroids, cross-referenced with public tumor datasets, followed by protein tests, immunohistochemistry on tumor tissue microarrays, and gene-silencing (siRNA) experiments
• Who was studied: Human mesothelioma cell lines M28 and REN (epithelial) and VAMT (sarcomatous), plus SARC, JMN and MSTO-211H. Normal human mesothelial cells. Tissue samples from 176 pleural mesotheliomas on two tissue microarrays: 88 chemonaive tumors, and a different 88 tumors with matching normal tissue from Brigham and Women's Hospital patients.
• Exposure: No asbestos exposure was studied. The conditions compared were growth as 3D spheroids versus 2D monolayers and reduction of ASS1 by siRNA. Spheroids were treated with bortezomib (25 nM) or with cisplatin (200 μM) plus pemetrexed (10 μM).
• Main results:
  • In spheroids compared with monolayers, 209 genes were differentially expressed in common by all three cell lines: 138 upregulated and 71 downregulated.
  • The 3D gene list was cross-referenced with two public datasets covering 56 mesotheliomas and 14 normal tissue samples. Three genes (ASS1, ANXA4, MVP) were raised in both. On qRT-PCR, only ASS1 was confirmed upregulated in all three cell lines.
  • ASS1 protein was increased in 3D compared with 2D in four of six cell lines. JMN and MSTO-211H lacked ASS1 altogether. Normal mesothelial cells did not upregulate ASS1 in 3D.
  • In the first set of 88 mesotheliomas, 8 samples showed no ASS1 staining (reported by the article as 10%).
  • In the 88 tumors with matched normal tissue from the same patients, ASS1 was significantly higher in mesothelioma (p less than 0.0001).
  • Across all 176 tumors, 87 showed moderate to high ASS1 expression (staining greater than 1 up to 3+). The other 89 showed low expression (0 to 1+).
  • ASS1 expression did not differ significantly between epithelioid (46) and non-epithelioid (42; sarcomatous and biphasic) mesotheliomas (p = 0.5662).
  • In spheroids of the three cell lines that expressed ASS1 (M28, SARC, VAMT), reducing ASS1 by siRNA raised the baseline rate of cell death (apoptosis) and the response to chemotherapy. It had no effect in ASS1-deficient MSTO spheroids.
• The authors' conclusion: The authors concluded that, although mesothelioma has been described as ASS1-deficient, there appear to be mesotheliomas with ASS1 upregulation. They found that ASS1 had a survival role in 3D spheroids, and they said ASS1 overexpression merits attention.
• Limitations the authors state:
  • The authors stated that the significance of ASS1 in mesothelioma biology is not yet known.
  • The authors noted that differences from an earlier report of ASS1 protein levels in mesothelioma could be due to different antibodies or technique.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, Pleural Mesothelioma, Cisplatin, Pemetrexed (Alimta).

Related studies

• Vorinostat eliminates multicellular resistance of mesothelioma 3D spheroids via restoration of Noxa expression — D. Barbone, P. Cheung, et al. (2012)
• Changes in global gene expression associated with 3D structure of tumors: an ex vivo matrix-free mesothelioma spheroid model — H. Kim, Y. Phung, et al. (2012)
• The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance — D. Barbone, J.A. Ryan, et al. (2011)
• Narciclasine as a Novel Treatment for Lung Cancer and Malignant Pleural Mesothelioma: Insights from 3D Tumor Spheroid Models — S. Jiang, S. Berkcan, et al. (2025)
• 2-Deoxy-glucose Enhances the Effect of Cisplatin and Pemetrexed in Reducing Malignant Pleural Mesothelioma Cell Proliferation But Not Spheroid Growth — I. Gerogianni, E. Pitaraki, et al. (2019)
• Rac1-mediated mitochondrial H2O2 generation regulates MMP-9 gene expression in macrophages via inhibition of SP-1 and AP-1 — S. Murthy, A. Ryan, et al. (2010)

Browse more research in PloS One, or return to medical research.