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

Our plain-English summary of 3-Dimensional mesothelioma spheroids provide closer to natural pathophysiological tumor microenvironment for drug response studies, by H. Shi, E.M. Rath, R.C.Y. Lin, et al (Frontiers in Oncology, 2022).

The study

• Authors: H. Shi, E.M. Rath, R.C.Y. Lin, K.H. Sarun, C.J. Clarke, B.C. McCaughan, H. Ke, A. Linton, K. Lee, S. Klebe, J. Maitz, K. Song, Y. Wang, S. Kao, Y.Y. Cheng
• Journal: Frontiers in Oncology
• Published: 2022;12:973576
• Cited by: 17 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

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.

Copyright © 2022 Shi, Rath, Lin, Sarun, Clarke, McCaughan, Ke, Linton, Lee, Klebe, Maitz, Song, Wang, Kao and Cheng.

In plain English

This laboratory study grew mesothelioma cells in dishes. It did not study asbestos exposure. The authors compared growing cells as three-dimensional (3D) clumps, called spheroids, with ordinary flat (2D) culture. They used mesothelioma cell lines (H28, H226, MSTO), a non-cancer mesothelial line (MeT-5A) and primary (patient-derived) cells. They tested hanging-drop, poly-HEMA and low-adhesive plate methods, and a pig-lung scaffold with its cells removed. Under electron microscopy, low-adhesive spheroids showed tight cell junctions and microvilli. After the chemotherapy drugs cisplatin and gemcitabine, spheroids averaged 60% and 80% cell viability respectively, compared with 20% for 2D cells, and 3D MSTO cells had fewer dead cells. Four of nine primary cell lines failed to form spheroids. 3D cells showed higher staining for several mesothelioma markers and different microRNA patterns. Primary MM05 cells grew on the pig-lung scaffold over 14 days. The authors concluded that 3D cultures are more appropriate preclinical models for drug screening and biomarker discovery. They noted that laboratory drug tests have limits compared with living animals.

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 experiment comparing methods of growing mesothelioma cells in 3D with standard 2D culture
• Who was studied: No people were studied. The study used cultured cells: three mesothelioma (MPM) cell lines (H28, H226, MSTO) and the immortalised mesothelial cell line MeT-5A from ATCC; primary mesothelioma cell line MM05 (Brisbane); and primary mesothelioma cells 2175, 1187 and 1157 established by the ADRI laboratory in Sydney. Nine primary cell lines from the ADRI biobank were tried as spheroids.
• Exposure: No asbestos or fiber exposure was studied. The comparison was between culture methods: 2D culture versus 3D methods (hanging drop, poly-HEMA, low-adhesive plates, and a decellularized porcine lung scaffold). Cells were treated with the chemotherapy drugs cisplatin and gemcitabine for 72 hours in the viability tests.
• Main results:
  • Poly-HEMA and low-adhesive methods formed uniform spheroids of H28 and MSTO cells after 24 hours. The hanging-drop method formed MSTO spheroids after 48 hours but failed for H28.
  • Low-adhesive spheroids showed tight junctions between cells and microvilli under transmission electron microscopy.
  • In mesothelioma cell lines, 3D spheroids averaged 60% cell viability after cisplatin and 80% after gemcitabine, compared with 20% viability in 2D cultured cells.
  • Drug (doxorubicin) penetrated low-adhesive cultured MSTO spheroids.
  • MSTO cells grown in 3D had on average 36% fewer dead cells after cisplatin and 12% fewer after gemcitabine than MSTO cells grown in 2D.
  • Four of nine primary mesothelioma cell lines (1170, 1180, 1137 and 1843) failed to form spheroids by the low-adhesive method.
  • Staining for mesothelioma markers (including BAP1, EMA, CD141, WT1, D2-40, Calretinin and CK-6) showed increased protein expression in 3D spheroids of primary cells 2175, 1187 or 1157 compared with 2D. For example, in 2175, EMA was under 5% in 2D and 80% in 3D (Table 1).
  • Comparing 3D with 2D, 18 microRNAs were differentially expressed. This used an unadjusted p value below 0.05.
  • Primary mesothelioma cells (MM05) grew on the decellularized porcine lung scaffold, outside and inside, over 14 days after seeding.
• The authors' conclusion: The authors concluded that 3D culture systems are more appropriate preclinical cancer models for drug screening and biomarker discovery than 2D models. They found low-adhesive U-bottom plates the most time-effective method.
• Limitations the authors state:
  • Laboratory drug tests have limits compared with living-animal models. These include difficulty capturing interactions between cell types and translating lab drug concentrations into doses in the body.
  • Further studies in animals injected with cells grown in 2D or 3D are needed.
  • Not every primary mesothelioma cell culture formed spheroids with the scaffold-free methods.
  • The authors list disadvantages of the porcine scaffold: it is expensive, difficult and time-consuming to make, and hard to use at large scale.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Tumor Microenvironment, Apoptosis.

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