Introduction
Materials and methods
Materials
Cell experiments
MM carcinogenesis model in mice
Isolation of MM cells
Organoid culture of MM cells
Pathological analysis
Immunofluorescence staining
Cloning of mCTR1
Primer | Primer orientation | Nucleotide sequence |
---|---|---|
N-terminal Flag mCTR1 | Forward | 5'-GAATTCGCCACCATGGACTACAAAGACGATGACGACAAGAACCA-3' |
Reverse | 5'-GGATCCTCAATGGCAGTGCTCTGTGA-3' | |
mCTR1 | Forward | 5'-GAATTCGCCACCATGAACCATATGGGGATGAACCATATGG-3' |
Reverse | 5'-GGATCCTCAATGGCAGTGCTCTGTGA-3' | |
N23D | Forward | 5'-ATTACCATGCCACCTCACCACCAC-3' |
Reverse | 5'-GTCGTCGTCCGTGTGGTTCATACCCATATG-3' | |
N23Q | Forward | 5'-ATTACCATGCCACCTCACCACCAC-3' |
Reverse | 5'-CTGGTCGTCCGTGTGGTTCATACCCATATG-3' |
Cell proliferation and drug sensitivity assays
Tumorigenicity assay
RNA sequencing and data analysis
Immunoblot analysis
Statistical analyses
Results
Establishment of organoids from mouse MM
Mouse genotype | Material | Total number | Mesothelioma incidence | Established organoids | ||
---|---|---|---|---|---|---|
Number | % | Number | % | |||
P53 +/- | Crocidolite | 14 | 6 | 42.9 | 5 | 83.3 |
NT50 | 10(3) | 3 | 42.9 | 1 | 33.3 | |
NT | 8 | 0 | 0.0 | 0 | ND | |
P53 + / + | Crocidolite | 10(1) | 1 | 11.1 | 1 | 100.0 |
NT50 | 4(2) | 1 | 50.0 | 0 | 0 | |
NT | 7 | 0 | 0.0 | 0 | ND |
Organoid ID | Material | mouse genotype | Mesothelioma incidence | Histology | ||
---|---|---|---|---|---|---|
Days | Subtype | MSLN | WT1 | |||
m5 | Crocidolite | P53 +/- | 211 | Biphasic | + | + |
m12 | Crocidolite | P53 +/- | 156 | Biphasic | - | - |
m28 | Crocidolite | P53 +/- | 221 | Sarcomatoid | + | + |
m54 | Crocidolite | P53 +/- | 173 | Sarcomatoid | + | - |
m60 | Crocidolite | P53 +/- | 194 | Biphasic | - | - |
m67 | Crocidolite | P53 + / + | 139 | ND(Ascites) | ND(Ascites) | ND(Ascites) |
m107 | NT50 | P53 +/- | 174 | Sarcomatoid | + | + |