Changes in expression of mesothelial BBS genes in 2D and 3D after lithium chloride and ammonium sulphate induction of primary cilium disturbance: a pilot study

Our plain-English summary of Changes in expression of mesothelial BBS genes in 2D and 3D after lithium chloride and ammonium sulphate induction of primary cilium disturbance: a pilot study, by E. Rouka, R.M. Jagirdar, I. Sarrigeorgiou, et al (Pharmacological Reports, 2023).

The study

• Authors: E. Rouka, R.M. Jagirdar, I. Sarrigeorgiou, E. Pitaraki, S.I. Sinis, C. Varsamas, E.D. Papazoglou, O.S. Kotsiou, P. Lymberi, A. Giannou, C. Hatzoglou, K.I. Gourgoulianis, S.G. Zarogiannis
• Journal: Pharmacological Reports
• Published: 2023;75(5):1230-1239
• Cited by: 5 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Malignant pleural mesothelioma (MPM), a rare and aggressive pleural tumor, has significant histological and molecular heterogeneity. Primary Cilium (PC), an organelle of emerging importance in malignancies, has been scarcely investigated in MPM. A critical molecular complex for the PC function is the BBSome and here we aimed at assessing its expression patterns in ordinary 2D and spheroid 3D cell cultures.

Methods: A human benign mesothelial cell line (MeT-5A), MPM cell lines (M14K, epithelioid MPM; MSTO, biphasic MPM), and primary MPM cells (pMPM) were used. Primers specific for the human BBS1, 2, 4, 5, 7, 9, 18 transcripts were designed, and quantitative real-time PCR (qRT-PCR) was done with β-actin as the gene of reference. The relative gene expression across 2D and 3D cultures was analyzed by the expression factor (mean of 1/ΔCt values). With the 2-∆∆Ct method the gene expression fold changes were assessed from qRT-PCR data. Molecular changes using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) were also determined.

Results: PC was present in all cells used in the study at approximately 15% of the observed area. BBSome transcripts were differentially expressed in different dimensions of cell culture (2D vs. 3D) in all cell lines and pMPM. Treatment with AS and LC affected the expression of the ciliary BBS2 and BBS18 genes in the benign as well as in the MPM cells.

Conclusions: These data indicate distinct BBSome molecular profiles in human benign and MPM cells cultured in 2D and 3D dimensions and support the notion that PC genes should be investigated as potential MPM therapeutic targets.

© 2023. The Author(s).

In plain English

This pilot study grew cells in dishes. It did not study asbestos exposure or measure health outcomes in people. It measured seven genes linked to the primary cilium, a small structure on cells. The authors used benign mesothelial cells (MeT-5A), two malignant pleural mesothelioma cell lines (M14K, epithelioid; MSTO, biphasic) and mesothelioma cells from one patient's pleural fluid. Cells were grown flat (2D) or as clumps called spheroids (3D). Cilia were seen in 14% to 18% of cells, with no significant difference between cell types. Some gene activity differed between 2D and 3D. In MSTO cells, BBS2, BBS5 and BBS18 were significantly lower in 3D. In the patient's cells, BBS1, BBS2 and BBS18 were significantly higher in 3D. Ammonium sulfate and lithium chloride, drugs that alter cilia, changed BBS2 or BBS18 activity in some cell types. In the patient's cells, drugs and culture dimension had no significant overall effect, but the two drugs differed significantly from each other for BBS2 in 2D. The authors concluded that this gene activity depends on culture dimension and is drug-sensitive. They noted they did not measure the proteins.

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 (in vitro) cell-culture pilot study measuring gene expression by qRT-PCR and imaging primary cilia by confocal microscopy
• Who was studied: Cultured cells: the benign mesothelial cell line MeT-5A, the malignant pleural mesothelioma cell lines M14K (epithelioid) and MSTO (biphasic), and primary MPM cells (pMPM) isolated from the pleural effusion of one patient with epithelioid MPM at the University Hospital of Larissa, Greece. The patient gave informed consent and the study had ethics approval
• Exposure: No asbestos exposure was studied. Cells were grown in 2D layers or 3D spheroids and treated for 24 hours with ammonium sulphate (30 Mm, a deciliating agent) or lithium chloride (50 Mm, a cilium-elongating agent)
• Main results:
  • Primary cilia were seen in 14% of MeT-5A, 16% of M14K and 14% of MSTO cells per unit area, and in 18% of pMPM cells per unit area
  • The proportion of cells with cilia did not differ significantly among the four cell types (chi-square test, p = 0.9775)
  • In MeT-5A benign mesothelial cells, BBS5 expression was absent in 2D cultures and was detected in 3D spheroids
  • In M14K cells, BBS9 expression differed significantly between 3D and 2D
  • In MSTO cells, BBS2, BBS5 and BBS18 expression was significantly lower in 3D spheroids
  • In the patient-derived pMPM cells, BBS1, BBS2 and BBS18 expression was significantly higher in 3D cultures
  • BBS2 expression in MeT-5A cells was significantly reduced in 3D after ammonium sulfate and lithium chloride. It was unchanged in M14K and pMPM cells. In MSTO cells, ammonium sulfate significantly increased it in 2D
  • Ammonium sulfate significantly increased BBS18 expression in 2D in MeT-5A and MSTO cells
  • In M14K cells, neither drug significantly changed BBS18 expression in 2D or 3D
  • In pMPM cells, neither drug treatment nor culture dimension significantly altered BBS2 or BBS18 expression overall
  • In pMPM cells, the effects of ammonium sulfate and lithium chloride on BBS2 expression in 2D differed significantly from each other (p = 0.0229)
• The authors' conclusion: The authors concluded that the relative expression of cilium-associated BBS genes depends on whether cells are grown in 2D or 3D and is sensitive to drugs that modulate the primary cilium. They added that the primary cilium should be further investigated as a therapeutic target in malignant pleural mesothelioma
• Limitations the authors state:
  • The study measured gene activity only and gave no data on BBS protein levels

Topics

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Mesothelial Cells, Epithelioid, Biphasic Mesothelioma.

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