- Top of page
- Materials and Methods
- Disclosure Statement
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 p16INK4A 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/c-nude 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)
Malignant 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(3–5) 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,(10–12) but most require a long period to develop MPM after implantation and often they have not reproduced the biological features of MPM well. In the pathological diagnosis of MPM, the distinction between MPM and reactive mesothelium (RM) is challenging because of the similar morphology and lack of reliable discriminating markers.(13,14) This problem may be resolved by establishing an experimental system that allows analysis of the morphological and immunohistological differences of MPM and RM on the pleural surface.
In the present study, we established six cell lines of MPM and found that one of them, termed MM56, exhibited the ability to reproduce the characteristic features of human MPM in BALB/c-nude mice. The in vivo model using MM56 cells might be useful for studying the biological behaviors of MPM and developing its diagnostic and therapeutic approaches.
- Top of page
- Materials and Methods
- Disclosure Statement
In the present study we have established six mesothelioma cell lines, MM16, MM21, MM26, MM35, MM46 and MM56, from pleural effusion fluids or surgically resected tumors of untreated Japanese patients with MPM. The MM56 cell line was derived from a soft tumor of MPM consisting of polygonal cells partially with tubulopapillary or microcystic structures and expressing calretinin and D2-40. In addition, pleural effusion cytology of this MPM showed a cellular arrangement of sheets or mirror ball-like cell clusters. The MM56 cells formed subcutaneous tumors soft and rich in fluids and positive for calretinin and D2-40 in BALB/c-nude mice, indicating that the MM56 cells retain characteristic features of human MPM in BALB/c-nude mice. In addition, MM56 cells diffusely grew on the surface of parietal pleura with pleural effusions containing mirror ball-like cell clusters, invaded into the musculature of the chest wall and often metastasized into the lung, rib, peritoneum and pericardial cavity during 3–7 weeks after orthotopic implantation. Because MM56 cells could reproduce various stages of MPM from the earliest phases in situ to the advanced phases with metastasis within a short period in the BALB/c-nude mice, the MM56 orthotopic implantation model can be regarded as an excellent animal model for studying the biological behavior of human MPM in vivo and developing its diagnostic and therapeutic strategies.
Proliferation of atypical mesothelial cells on the pleural surface is considered as mesothelioma in situ, but it could only be diagnosed when accompanied by adjacent or subsequent invasive MPM.(16–19) Discrimination between early stage MPM and RM has been problematic due to morphological similarities and the lack of reliable discriminating markers.(13,14) On the other hand, we have recently reported that CD146 is a useful marker to discriminate MPM from RM in pleural effusion cytology.(15) In the MM56 orthotopic implantation model, the coexistence of MPM and RM on the pleural surface is reproduced, and immunostaining with anti-CD146 antibody is able to identify MM56-derived MPM cells. Therefore, the MM56 orthotopic implantation model should be useful in detecting in situ lesions and identifying additional markers that discriminate between early stage MPM and RM.
The MM35 cells were less useful, because they slowly produced subcutaneous tumors in BALB/c-nude mice and thoracic tumors in only two of five mice 30 weeks after inoculation in an orthotopic implantation model. However, cytological analysis of the pleural effusion fluids in the recipient mice revealed that the MM35 cells formed papillary cell clusters, one of the cytological features of MPM.(20,21) Isolation of sublines with high tumorigenicity and MPM characteristics from pleural effusion fluids of recipient mice may improve the usefulness of MM35 cells.
Most human MPM grow diffusely in the thoracic cavity and a few proliferate as a nodule. The former is classified as diffuse MPM and the latter is classified as localized MPM. In addition, some MPM develop as multiple nodules in the thoracic cavity. Biopsy tissues of MPM, from which the MM16 cell line was established, were histologically biphasic mesothelioma growing as multiple nodules, but MPM at autopsy was sarcomatoid mesothelioma with a loss of expression of the mesothelioma-related markers. The MM16 cell line kept the expression of the mesothelioma-related markers, calretinin, D2-40, WT-1 and CK5/6, but MM16 subcutaneous and thoracic tumors were negative for these markers. Orthotopic inoculation of MM16 cells generated tumor nodules, but not diffusely growing tumors along the pleura, in the thoracic cavity of the BALB/c-nude mice. The MM16 subcutaneous and thoracic implantation models might be useful to clarify the mechanism(s) by which MPM grows as nodules and changes the phenotype.
It has been reported that WT-1, CAM5.2 and AE1/AE3 are expressed in more than 80% of sarcomatoid mesothelioma and the combined use of these markers provides the highest sensitivity in the differentiation of sarcomatoid mesothelioma from true sarcoma.(22) Consistent with this report, the MM46 cell line established from the sarcomatoid mesothelioma was positive for WT-1, CAM5.2 and AE1/AE3. Although sarcomatoid mesothelioma is highly malignant in human MPM, the MM46 cells failed to develop tumors in the BALB/c-nude mice. Previously, Usami et al.(4) established an epithelioid mesothelioma cell line, Y-MESO-8A, and a sarcomatoid mesothelioma cell line, Y-MESO-8D, from a biphasic mesothelioma consisting of epithelioid and sarcomatoid components, and reported that Y-MESO-8A cells, but not Y-MESO-8D cells, had the potential for developing a subcutaneous tumor in BALB/c-nude mice. Therefore, in sarcomatoid mesothelioma, it is unlikely that a correlation is observed between malignancy in human and tumorigenicity in nude mice. The MM46 cells may be useful to study the characteristic features of sarcomatoid mesothelioma.
The p16INK4A gene is the most frequently inactivated tumor suppressor gene in human MPM, and ∼90% of MPM possesses deletions of the p16INK4A gene.(23) Abnormalities of the p16INK4A gene take part in the dysregulation of the cell cycle leading to malignant transformation of mesothelial cells. The NF2 tumor suppressor gene is also frequently mutated in MPM. Recently, Yokoyama et al.(24) reported that mutation of the NF2 gene promotes MPM proliferation through activation of YAP1, a transcriptional coactivator functionally inhibited by Merlin, a product of the NF2 gene. In all six cell lines established in the present study, two allele deletions of the p16INK4A gene and one allele deletion of the NF2 gene were found, which supports the theory that abnormalities of the p16INK4A and the NF2 genes participate in the development of MPM. On the other hand, there is a possibility that abnormalities other than the p16INK4A and the NF2 genes are related to the difference of tumorigenicity in nude mice among these six cell lines.
Novel biological therapies for MPM are under investigation.(2) Effects of various inhibitors against angiogenic factor, tyrosine kinase, ribonuclease, histone deacetylase and proteasome on MPM patients have been studied without much success. Mesothelin and CD26, mesothelioma-associated cell surface antigens, have been reported to be an immunotherapeutic target.(25–27) Recently, CD146 has been identified as the surface antigen recognized by an internalizing single chain antibody that can deliver liposome-encapsulated small molecule drugs into the cytoplasm of mesothelioma cells.(28) Our earlier study showed that CD146 was expressed in MPM but not RM,(15) supporting the theory that CD146 can be used as an effective immunotherapeutic target of MPM. The MM56 subcutaneous and orthotopic implantation models may serve as suitable in vivo systems to evaluate this possibility and develop preclinical strategies.