Establishment of a cell line from a Japanese patient useful for generating an in vivo model of malignant pleural mesothelioma

Abstract

Malignant pleural mesothelioma is a refractory tumor with increasing incidence. In the present study, we established six mesothelioma cell lines possessing two allele deletions of the p16 INK4A gene and one allele deletion of the neurofibromatosis type 2 gene, MM16, MM21, MM26, MM35, MM46 and MM56, from pleural effusion fluids or surgically resected tumors of Japanese patients. MM21, MM26 and MM46 cells failed to develop tumors in BALB ⁄ cnude mice following subcutaneous inoculation. MM16 and MM35 cells slowly generated tumors at the site of subcutaneous inoculation in BALB ⁄ c-nude mice, but lost the expression of mesothelioma-related markers such as calretinin, D2-40 and Wilms' tumor 1 in the subcutaneous tumors. On the other hand, MM56 cells rapidly generated tumors with the expression of calretinin and D2-40 in BALB ⁄ c-nude mice following subcutaneous inoculation. In addition, orthotopic implantation of MM56 cells into BALB ⁄ c-nude mice developed diffusely growing thoracic tumors by 3 weeks after implantation. Pleural effusions were observed in these mice 4 weeks after implantation. Thoracic tumors invaded aggressively into the chest wall 5 weeks after implantation and often metastasized into the lung, rib, peritoneum and pericardial cavity. On the pleural surface, MM56 cells were growing as single or multiple cell layers with the reactive mesothelium of recipient mice. These results indicate that MM56 cells can behave in a manner characteristic of human malignant pleural mesothelioma in the thoracic cavity of BALB ⁄ c-nude mice. The in vivo model using MM56 cells may be useful for studying the biological behavior of malignant pleural mesothelioma and developing its diagnostic and therapeutic strategies. (Cancer Sci 2011; 102: 648-655) M alignant pleural mesothelioma (MPM), considered to be closely associated with asbestos exposure, is an aggressive tumor arising from mesothelial cells on the serosal surfaces of the thoracic cavity. Malignant pleural mesothelioma was once a rare disease, but its incidence is dramatically increasing worldwide. In Japan, it is expected to peak around 2025 as a result of widespread use of asbestos.(1) Malignant pleural mesothelioma is often diagnosed at an advanced stage and known to be resistant to conventional therapies. As a result it is associated with poor prognosis, with the median survival in the range of 9-17 months after the first diagnosis.(2) It is therefore important to establish a means for investigating the behaviors of MPM, leading to the development of early diagnosis and effective therapies.Cell lines and animal models of human tumors are useful for studying the characteristics of tumors. Several MPM cell lines have been established (3)(4)(5) and animal models have been produced by inoculation of MPM cells or surgically resected MPM tissues into immunodeficient mice or rats.(6-9) Orthotopic implantation models are considered to be the most useful for studying the characteristics of MPM in vivo, (10)(11)(12) but most require a long period to develop MPM aft...

Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma

“…Melting curve analysis was also performed for non-specific products. The copy number was determined by the relative standard curve method using the RPS6 gene (9p22) (15) as the reference gene according to the previous reports (9,15), and the calculated numbers were evaluated as follows: ≥0.8 as no deletion, >0.5 and <0.8 as a one-allele deletion, and ≤0.5 as a two-allele deletion.…”

Frequent deletion of 3p21.1 region carrying semaphorin 3G and aberrant expression of the genes participating in semaphorin signaling in the epithelioid type of malignant mesothelioma cells

“…Adherent cells were expanded by several passages for several weeks or months. These cells were used as MM primary cell cultures in this report and were used to establish six MM cell lines (33). Similarly, primary cell cultures were prepared also from pleural effusions of two RM patients.…”

Establishment and molecular characterization of cell lines from Japanese patients with malignant pleural mesothelioma

“…Copy number loss of the P16 and NF2 genes in the genomic DNA of MPM cells was analyzed by qPCR and determined by the comparative threshold cycle method using the RPS6 gene as a control and the genomic DNA of MeT-5A, which are human pleural mesothelial cells, as a reference, as described previously (12). In the comparative threshold cycle method, a calculated value of 1.0 indicates no deletion, a value of 0.5 indicates a one-allele deletion, and a value of 0 indicates a two-allele deletion.…”

“…For example, >200 lung cancer cell lines have been established and used for research, which has led to the discovery of intriguing properties that are linked with treatment, such as EGFR-activating mutations and EML-ALK fusion genes (9). In contrast, a relatively small number of MPM cell lines have been established compared with those for lung cancer (12,17), and the number of cell lines that have been deposited in publicly open banks is limited. Thus, we plan to deposit them with the molecular profiles that we determined in a public cell bank for the convenience of other researchers.…”