Matrigel-based organoid culture of malignant mesothelioma reproduces cisplatin sensitivity through CTR1

Our plain-English summary of Matrigel-based organoid culture of malignant mesothelioma reproduces cisplatin sensitivity through CTR1, by F. Ito, K. Kato, I. Yanatori, et al (BMC Cancer, 2023).

The study

• Authors: F. Ito, K. Kato, I. Yanatori, Y. Maeda, T. Murohara, S. Toyokuni
• Journal: BMC Cancer
• Published: 2023;23(1):487
• Cited by: 14 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Organoids are a three-dimensional (3D) culture system that simulate actual organs. Therefore, tumor organoids are expected to predict precise response to chemotherapy in patients. However, to date, few studies have studied the drug responses in organoids of malignant mesothelioma (MM). The poor prognosis of MM emphasizes the importance of establishing a protocol for generating MM-organoid for research and clinical use. Here, we established murine MM organoids from p53+/- or wild-type C57BL/6 strain by intraperitoneal injection either with crocidolite or carbon nanotube. Established MM-organoids proliferated in Matrigel as spheroids. Subcutaneous injection assays revealed that the MM-organoids mimicked actual tissue architecture and maintained the original histological features of the primary MM. RNA sequencing and pathway analyses revealed that the significant expressional differences between the 2D- and 3D-culture systems were observed in receptor tyrosine kinases, including IGF1R and EGFR, glycosylation and cholesterol/steroid metabolism. MM-organoids exhibited a more sensitive response to cisplatin through stable plasma membrane localization of a major cisplatin transporter, copper transporter 1/Slc31A1 (Ctr1) in comparison to 2D-cultures, presumably through glycosylation and lipidation. The Matrigel culture system facilitated the localization of CTR1 on the plasma membrane, which simulated the original MMs and the subcutaneous xenografts. These results suggest that the newly developed protocol for MM-organoids is useful to study strategies to overcome chemotherapy resistance to cisplatin.

© 2023. The Author(s).

In plain English

This laboratory study combined mouse experiments with cell and organoid culture. The authors induced malignant mesothelioma (MM) in p53+/- and wild-type C57BL/6 mice by single intraperitoneal injection of 3.0 mg crocidolite or 1.5 mg of 50 nm multi-walled carbon nanotube (NT-50), with vehicle controls, then grew organoids in Matrigel from the MM tissue or ascites. MM incidence was 42.9% in crocidolite-treated p53+/- mice, and organoids were established and propagated from 7 of the 11 MM cases. Most organoids were spherical, one line (m12) budded, and growth required EGF. Two lines chosen as typical mouse MM, m5 and m107, were injected subcutaneously into nude mice; these implants expressed WT1 and mesothelin and grew significantly faster than the matching 2D cell lines. MM organoids were significantly more sensitive to cisplatin than the corresponding 2D lines, although Slc31a1 (Ctr1) mRNA levels did not differ significantly; CTR1 was cytosolic in 2D culture but at the plasma membrane on Matrigel, and the glycosylation inhibitor tunicamycin blocked this membrane localisation. The authors concluded they had established a novel Matrigel-based protocol for mouse MM organoids.

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: Experimental animal and cell-culture study: asbestos/carbon-nanotube induction of mesothelioma in mice, establishment of tumour-derived organoids, xenografting, RNA sequencing and cisplatin sensitivity testing
• Who was studied: Male C57BL/6 mice, p53-heterozygous knockout (p53+/-) and wild-type, aged 6–8 weeks; 11 mesotheliomas arose, from which 7 organoid lines were established; immunodeficient KSN/Slc nu/nu nude mice were used for subcutaneous injection; Nagoya University, approval dated 2022/03/18
• Exposure: Single intraperitoneal injection of 3.0 mg crocidolite asbestos (UICC) or 1.5 mg multi-walled carbon nanotube (NT-50, 50 nm diameter); vehicle (0.5% bovine serum albumin/saline) in controls; in vitro exposure of cells and organoids to cisplatin
• Main results:
  • Mesothelioma incidence was 42.9% in crocidolite-treated p53+/- mice (6 of 14 per Table 2)
  • Organoids were established and propagated from 7 of the 11 mesothelioma cases, starting from MM tissue or ascites
  • Most organoids formed a spherical pattern; only one organoid line (m12) formed a budding pattern
  • Carbon nanotube-treated mice mainly died of ileus from peritonitis, whereas mesothelioma was judged the main cause of death in crocidolite-treated mice
  • Growth factor dependence differed between organoid lines: m12 grew independently of all tested factors except EGF, while m5 required both EGF and R-spondin1
  • Subcutaneous injection used only the m5 and m107 lines, selected because they expressed WT1 and mesothelin; Table 3 lists other lines negative for one or both markers (m12 and m60 MSLN- and WT1-negative, m54 WT1-negative)
  • In these implants (assessed at day 36), the organoid-derived tumours grew significantly faster than tumours from the 2D cell lines, and expressed WT1 and mesothelin
  • MM organoids (3D) were significantly more sensitive to cisplatin than the matching 2D cell lines
  • Slc31a1 (Ctr1) mRNA levels did not differ significantly between organoids and 2D cell lines; a difference in CTR1 band number and band shift on immunoblot suggested differing glycosylation or lipidation
  • In cells expressing flag-tagged mouse CTR1, CTR1 was cytosolic in standard 2D culture but localised to the plasma membrane when cells were plated on a Matrigel layer
  • The N-glycosylation inhibitor tunicamycin shifted the CTR1 band from 30–50 kDa to 25 kDa and blocked plasma membrane localisation, whereas the lipidation inhibitor 2-bromopalmitate did not cause the band shift
  • RNA sequencing separated organoid and 2D cultures into distinct clusters, with differences involving receptor tyrosine kinases such as IGFR and EGFR
• The authors' conclusion: The authors concluded that they had established a novel Matrigel-based protocol for generating mouse MM organoids and organoid lines, which they consider useful for future mesothelioma studies and as a possible pre-clinical model for drug discovery
• Limitations the authors state:
  • The authors state that the gene expression comparison may have been influenced by the different culture media and additives used for 2D and 3D cultures
  • The authors state the organoid cultures did not retain CD68-positive macrophages and possibly other immune cells, so further studies are needed for pre-clinical immunotherapy testing
  • The authors state the 1.5 mg NT-50 dose used may have been excessive, causing peritonitis and ileus
  • The authors state that attempts to establish organoids from non-immortalised normal mesothelial cells failed to proliferate and could not be passaged

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, Cisplatin, Chemotherapy, Prognosis, Crocidolite, EGFR.

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