Novel orthotopic implantation model of human malignant pleural mesothelioma (EHMES-10 cells) highly expressing vascular endothelial growth factor and its receptor

Abstract

Malignant pleural mesothelioma (MPM) is closely related to exposure to asbestos, and a rapid increase in the number of MPM patients in Japan is estimated in the years 2010-2050. The purpose of the present study was to establish a clinically relevant animal model that shows human patient-like progression of MPM. Here, we demonstrate that a human MPM cell line (EHMES-10) inoculated orthotopically (thoracic cavity) into severe combined immunodeficiency (SCID) mice produces highly vascularized thoracic tumors with pleural dissemination and bloody pleural effusions by 5 weeks, suggesting a patient-like progression of this cell line after orthotopic inoculation. EHMES-10 cells overexpressed vascular endothelial growth factor (VEGF), a molecule responsible for malignant effusions, and its receptor. Treatment with cisplatin, but not gemcitabine, significantly inhibited the production of pleural effusions, but it was not effective for thoracic tumors, consistent with chemotherapy refractory characteristics of MPM in patients. Our patient-like orthotopic model using EHMES-10 cells overexpressing VEGF and its receptor may be useful for examining the molecular pathogenesis of MPM and may contribute to the development of novel treatment strategies for MPM.

Figure 1

Morphology and proliferation of EHMES‐10 cells in vitro. (A) Culture of EHMES‐10 cells. Bars indicate 100 µm. (B) EHMES‐10 (closed circles) were cultured in 10‐cm dishes in MEM with 10% fetal bovine serum. The number of cells was counted daily. Values are the mean ± SD of triplicate cultures. Data shown are representative of three independent experiments with similar results.

Figure 2

Thoracic tumors and bloody pleural effusions produced by orthotopically implanted EHMES‐10 cells in severe combined immunodeficiency (SCID) mice. EHMES‐10 cells (3 × 10⁶) were inoculated into the thoracic cavity of SCID mice. (A) thoracic cavity on day 28. White arrow and arrow heads indicate thoracic tumor and bloody pleural effusion, respectively. (B) Thoracic cavity on day 28. White arrows indicate tumor rind encasing the right lung. White arrow heads indicate the compressed left lung. (C) Pleural‐based masses grown diffusely in the thoracic cavity (white arrow). (D) Bloody pleural effusion aspirated in the syringe. (E) Tumor cells in the bloody pleural effusion produced by EHMES‐10 cells (hematoxylin–oesin [H&E] staining, ×400). H&E staining of pleural‐based masses at low (F) and high (G) magnifications. Note: solid sheet‐like arrangement of large tumor cells was observed. Those masses diffusely invaded the parietal pleura. Bars indicate 10 mm (A–C) and 100 µm (E–G).

Figure 3

Survival of severe combined immunodeficiency (SCID) mice inoculated with EHMES‐10 cells. EHMES‐10 cells (3 × 10⁶) were inoculated into the thoracic cavity of SCID mice. When the mice became moribund, they were killed. Survival was therefore determined up to the day the mice were killed.

Figure 4

Expression of vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) by EHMES‐10 cells. (A) Tumor cells (2 × 10⁵) were cultured for 48 h in 2 mL MEM with 10% fetal bovine serum. The culture supernatants were harvested and their VEGF level was measured by enzyme‐linked immunosorbent assay. Bars are the SD of duplicate cultures. EHMES‐10 cells produced VEGF at levels as high as in lung adenocarcinoma PC14PE6 cells. (B) The levels of VEGF isoforms and VEGFR were determined by reverse transcription–polymerase chain reaction. HMVEC were used as a positive control VEGFR expression. Data shown are representative of three independent experiments with similar results.

Figure 5

Histological examinations of thoracic tumors produced by EHMES‐10 cells. (A) vascular endothelial growth factor (VEGF); and (B) CD31 (panendothelial marker). Note: thoracic tumors produced by EHMES‐10 cells expressed VEGF protein. The tumors were highly vascularized when determined by staining for CD31 (×200). Bars indicate 100 µm.

Figure 6

Sensitivity of EHMES‐10 cells to chemotherapeutic agents in vitro. EHMES‐10 (○) and PC14PE6 (•) (2 × 10³) were cultured in 96‐well plates with or without various concentrations of cisplatin (CDDP), gemcitabine (GEM), docetaxel (DOC) or vinorelbine (VNR) for 3 days. Growth of tumor cells was determined by MTT assay. Bars are the SD of triplicate cultures. Data shown are representative of four independent experiments with similar results.