Effect Of Itraconazole On The Lymphangiogenesis In A Mouse Model Of Mglignant Pleural Effusion

Objective and backgroundNon-small-cell lung cancer (NSCLC) is the leading cause of cancer mortality in the world. More than15%of NSCLC patients present with malignant pleural effusion (MPE) when diagnosed, and with the progression of the disease,50%of the patients will have MPE. MPE is a poor prognostic marker for lung cancer patients, and our understanding of the causes and mechanisms that result in MPE has been inadequate until now. It is generally believed that tumor-induced malignant pleural effusion is generated by the following several ways:1. Drainage disorders of the lymphatic system, such effusion often serous or chylous, not bloody.2. Pleural metastatic disease or primary tumor associated with inflammation-induced high capillary permeability, such effusion often bloody.3. The hypoproteinemia caused by malnutrition often result in exudative pleural effusion of cancer patients.4. Pleural effusion caused by loss of tumor thrombus in patients with pulmonary embolism.5. The irritation or injury of pleural caused by treatment of intrathoracic tumors.6. Some scholars found that vascular endothelial growth factor (VEGF) plays a very important role in generating of malignant pleural effusion.Some means of treatment for malignant tumors are in progressing, however, overall the treatment for malignant pleural effusion are limited, and the effect of the treatment is not satisfactory. Patients with malignant coIn1627, Asellius found the lymphatic system for the first time. After centuries of research, the understanding of the structure and function of the lymphatic gradually deepened. Compared with capillaries, the lumen of lymphatic capillary relatively large and irregular, and the wall of lymphatic capillary is thinner. The lymphatic capillary is only constituted by endothelial cells, and its basement membrane is not continuous, also the endothelial gap is opening. Reticular fibers and collagen tissue connected to the extracellular cell matrix. The overstretching endothelial cells allow the wall of lymphatic capillary becomes thinner and thereby forming a unique high permeability of lymphatic vessels. The lymphangiogenesis and lymph node metastasis of malignant tumor are associated with high expression of vascular endothelial growth factor C (VEGF-C). The specific tyrosine kinase receptor VEGFR-3(Vascular endothelial growth factor receptor-3, VEGFR-3) of VEGF-C is mainly expressed in epithelial cells of the lymphatic vessels and VEGF-C mainly binding with VEGFR-3to induce intratumoral and peritumoral lymphangiogenesis and lymph node metastasis, which is the metastasis ways of majority tumor.In pleural metastatic tumors, VEGF-C secreted by tumor cells resulting in increased tumor lymphangiogenesis and regional lymph node metastasis. Compared with the normal lymphatic, the nascent lymphatic bureaucratic is easier to rupture, leading to leakage of lymph and to form the malignant pleural effusion. Cancer cell through lymph into the lymphatic circulation, gathered into a group and clogged lymphatic vessels, finally caused disorder of lymphatic drainage. On the other hand, because of the tortuous bureaucratic of lymphangiogenesis tube and the large gap between endothelial cells, the easier transfer of tumor cells along the lymphatic vessels, lymph leakage and bureaucratic. They are two important reasons for forming malignant pleural effusion. The present study confirmed the key pathological process of many diseases involving lymphangiogenesis, especially the spread of the malignant tumors. Lymphatic vessels as transfer channel for many malignant tumors, but the exact mechanism is not clear so far.A number of tests have been confirmed that itraconazole (ITCZ) has the ability of inhibiting vascular endothelial cell and in Matrigel metastatic tumor model itraconazole could inhibit angiogenesis and tumor growth. As we all know, tumor angiogenesis and lymphangiogenesis has a similar approach, and both participate in the formation of malignant pleural effusion. There are few reports about whether itraconazole can inhibit lymphangiogenesis or lymph node metastasis and achieve the purpose of treatment with malignant pleural effusion. This study was designed to create a tumor-bearing nude mouse model of malignant pleural effusion by pleural injection of tumor cells. Different doses of itraconazole injected into the pleural cavity for treating mice with malignant pleural effusion. We also established a control group to observe the role of itraconazole in treating for malignant pleural effusion and its effect on tumor lymph node metastasis for providing a theoretical basis for lymphangiogenesis and lymph node metastasis in MPE treatment in the future.Materials and methodsPart I establishment of malignant pleural effusion mice model.The GFP-LLC (green fluorescent protein-lewis lung cancer)cell line was purchased from the Cell Bank of Chinese Academy of Sciences(Shanghai, China), In this study, GFP-LLC (green fluorescent protein-Lewis lung cancer) lung cancer cells, whichever is good growth state of suspension to the phosphate buffer solution (Phosphate buffer solution, PBS), adjusting the concentration to1×10 6/50μl to build a nude mouse model of malignant pleural effusion, by injecting lung cancer cells into the pleural cavity of mouse. After inoculation of cells were observed daily life signs, tumor growth in mice. Four days after Inoculation of tumor cells,2mice were killed every3days, after dissection in near infrared fluorescence microscopy and eye state observation for the tumor foci in mice, and measure the volume of pleural effusion. From the seventh day after inoculation of tumor cells, every7days, mice were given a CT scan of the chest for observing mice pleural effusion.Part II Effect of itraconazole on the lymphangiogenesis in a mouse model of mglignant pleural effusion30BALB/c sex-matched nude mice (6-8weeks old, weight of18-24g) were randomly divided into3groups, with10rats in each group. The malignant mice model was made according to the method before. Animals in each group were treated with either hydroxypropyl β-cyclodextrin, L-ITCZ or H-ITCZ. The mice were treated with itraconazole at a dose of25mg/kg or8mg/kg four times, with a3-day interval between doses, while the control group (hydroxypropyl β-cyclodextrin group, H-0-c group) was similarly treated with50ml of hydroxypropyl β-cyclodextrin(130mg/ml). Fourteen days after injection of the LLC cells, the mice were scanned by computed tomography (CT, Siemens Somatom Sensation16,120kVp,93μA) to observe the pleural effusion. The mice were anesthetized, as described previously, during the imaging session, and the scanned images were transferred in real-time to a multi-functional image post-processing workstation (Syngo MMWP CT workplace VA30A).Three days after the final drug treatment, the mice were anesthetized and sacrificed. Pleural effusion was gently aspirated using a1ml syringe, and the volume was measured. The pleural cavity of the mice was opened to observe the distribution of tumor lesions. Pleural tumors were found scattered on visceral and parietal pleural surfaces as well as on mediastinal and pulmonary hilar lymph nodes. The total number of tumor lesions was independently counted by two investigators, the numbers were compared and discrepancies were resolved by consensus. Tumors from the parietal pleura were acquired and fixed in10%formalin for24hours and then incubated in70%ethanol for3days; the samples were then embedded in paraffin and finally stained with hematoxylin-eosin. For cytological testing, the pleural effusion from the control group was centrifuged, and the cells in the effusion were applied to slides, air dried, fixed in methanol for10seconds and stained with modified Wright’s Giemsa stain. Pleural effusion was gently aspirated using a1ml syringe, and the cell-free supernatants were collected and stored at-80℃.An enzyme-linked immunosorbent assay was performed on the MPE supernatant; the concentration of VEGF-C was measured using commercial ELISA kits (R&D, USA) according to the manufactures protocol. The absorbance at450nm was determined using a microplate reader (Bio-Rad, Hercules, USA). Lymphangiogenesis was tested by IHC staining. Tumors from the pleura were fixed in10%formalin for24hours and embedded in paraffin after dehydration. Two consecutive slides from each tissue were stained for lymphatic micro vessel density (LMVD) and VEGF-C analysis. Briefly, tissues mounted on slides were incubated with a mouse monoclonal D2-40antibody or a polyclonal goat anti-mouse VEGF-C antibody at4℃overnight. A positive reaction was defined as a brown stain using3,3-diamino-benzidine (DAB)(Vector Laboratories), and the nucleus was restained with hematoxylin. Staining for D2-40was used to evaluate the LMVD, and stained vessels were quantitated at a200x magnification. The mean number of vessels was defined as the LMVD. Positive staining of VEGF-C expression was evaluated based on previous studies. The scores from two independent investigators were compared, and inconsistencies were resolved by consensus.ResultsPart I malignant pleural effusion mice model can be established by injecting cancer cells into pleural cavityFour days after the cells injected, a small amount of fluorescent in mice pleural cavity can be seen in NIR fluorescence imaging system. With the prolongation of mice inoculated with tumor, the volume of MPE, the number of tumor nodules gradually increased. Mouse chest CT scan results suggest:seventh days, fourteenth days and twenty-first days of MPE formation rates were28%,74%and81%. The average survival time was16.7days. All pleural effusion were bloody, not solidification. The volume of pleural effusion reached a peak at sixteenth days after GFP-LLC cell injection.Part II itraconazole can inhibit lymphangiogenesis in nude mice with MPEA CT scan confirmed that pleural effusion was more visible in mice treated with H-β-C, unilateral pleural effusion was observed in the L-ITCZ group, and no obvious pleural effusion was observed in the H-ITCZ group. Pleural effusion from the three groups appeared to be hemorrhagic and did not coagulate in vitro. The mean volume of pleural effusion in the H-β-C, L-ITCZ and H-ITCZ groups was575.76±32.61ml,445.64±27.75ml, and311.19±27.20ml, respectively. The volume of pleural effusion was reduced in the H-ITCZ group and was significantly different from that in the H-β-C group (P<0.01) and the L-ITCZ group (P<0.01). Similarly, the L-ITCZ group also displayed a significant reduction in the volume of pleural effusion compared to the H-β-C group (P<0.01). The numbers of tumor foci in the H-β-C, L-ITCZ and H-ITCZ groups were27.40±3.92,21.40±3.37and11.55±3.28, respectively, and the number of tumor foci in the H-ITCZ group were notably reduced compared to those of the H-β-C group (P<0.001) and the L-ITCZ (P<0.001) group. Observation of the anatomy of the thoracic cavity revealed the presence of MPEs. To avoid the influence of drugs, tumors located on the parietal pleura of the H-β-C-treated mice were removed and observed following histological stainiThe results of the staining confirmed the presence of adenocarcinoma cells in the tumors, while the cytological preparation from the pleural effusion of the H-β-C group revealed LLC cells with large nuclei and visible nucleoli. Among the known lymphangiogenic factors, VEGF-C has been reported to act as an important lymphatic-specific growth factor. Therefore, we hypothesized that itraconazole might suppress lymphangiogenesis by inhibiting VEGF-C expression by LLC cells; thus, we evaluated the expression of VEGF-C in the MPEs secreted by LLC cells using an ELISA. As expected, VEGF-C expression was reduced in the supernatant of pleural effusions that were treated with H-ITCZ. Compared with the control group, VEGF-C in the supernatants of the H-ITCZ treated group was significantly lower than that of the two other groups (P<0.001). To validate the precise expression of LMVD and VEGF-C in mouse tumors, we stained a series of lung cancer sections using a published IHC technique. While staining was restricted to the cytoplasm with no nuclear staining observed, LMVD and VEGF-C were detected within the stroma, and their expression was mainly restricted to the cancerous regions with almost no expression in the normal tumor tissue. The mean LMVD values in the H-β-C, L-ITCZ and H-ITCZ groups were24.1±2.51,20.5±1.58and14.1±1.45, respectively. LMVD was significantly reduced in the H-ITCZ-treated group compared with the H-P-C group (P<0.001) and the L-ITCZ group (P<0.001). Additionally, a statistically significant difference was observed between the two other groups (P<0.01). VEGF-C expression in the H-β-C group, L-ITCZ group and H-ITCZ group was65%,47%,36%, respectively. A significant decrease was observed in the H-ITCZ-treated group compared with the two control groups treated with H-β-C (P<0.001) or L-ITCZ (P<0.01), and there was also a significant difference between the control groups (P<0.05).Conclusions:Our results showed that itraconazole suppressed lymphangiogenesis and MPE intuitively. Taken together, the results of our study allowed us to substantiate the therapeutic effect of itraconazole on lymphangiogenesis and MPE through an established MPE mouse model, ensuring the intra-pleural injection of LLC cells. Based on our results, itraconazole proved to be a potent inhibitor of tumor lymphangiogenesis and MPE. Our results provide an apparent theoretical basis for the effectiveness of itraconazole on MPE treatment, and large randomized clinical studies are required for further confirming these results.