Studies On The Relationships Among Lymphangiogenesis, Expression Of VEGF-C With Cervical Lymph Node Metastasis In Laryngeal Cell Squamous Carcinomas

IntroductionThe laryngeal carcinoma is a common malignant tumor in head and neck, specially in Northern China. The metastasis to cervical glands of laryngeal cancer cells occurs primarily through lymphatic system and the extent of lymph node involvement is the most important prognostic factor of laryngeal cancer. The condition of cervical glands have very significant impact on the recovery rate and the survival rate of laryngeal cancer patients. But duing to the lack of recognition to metastasis mechanisms of solid tumor, laryngeal cancer metastasis were badly controlled so that many patients have died of lymph node metastasis. Accordingly, the studies on metastasis mechanisms have very important significance to treatment of laryngeal cancer.The vascular system was very important during the growth and metastasis of tumor though it was complicated. Angiogenesis and lymphomagenesis is considered essential for development and metastasis of tumor. From Folkman founding that angiogenesis was related to the growth and metastasis of tumor, the blood vessel density (BVD) of tumor have been reported to influence the clinical outcomes of patients in a variety of solid malignant tumor. As the tunnel of lymph circulation, lymphomagenesis is undoubtedly more important. but the study of tumor lymph vessels were fell behind because of the limitation of methods. Moreover, there are a lot of dispute about tumor lymph vessels. Since 5'- Nase -ALPase double staining were devised, it has allowed easy differentiation between blood vessels and lymphatic vessels under light microscopy . We used 5'- Nase - ALPase double staining assey to demonstrate and distinguish lymphatic vesselsfrom blood vessels so as to study the structure and distribution of lymphatic vessels.The changes of tumor lymphatic vessels were influenced by lymphatic vessel regulatory factors. With vascular endothelial growth factor - C ( VEGF - C) , the first lymphangiogenic factor, was identified by Joukov in 1996, the spreading process of cancer cells via lymphatic vessels was studied widely. VEGF - C is a new member of the VEGF family, which is homophylic with VEGF - A and mainly improve chematropism and proliferation of lymphatic vessel endothelial cell and drive lymphomagenesis so that was called lymphatic vessel endothelial cell growth factor. For the past few years, many studies all found that VEGF - C was overexpressed and related with lymphatic metastasis in lots kind of tumors , such as prostatic cancerNcolon cancerxbreast cancer x thyroid cancer .Jung cancer%oral cancer,and so on. But the recent literatures reports that VEGF -C induces lymphomagenesis only relative specificly, and also promote tumor metastasis through driving angiogenesis.Nowadays, the studies about laryngeal carcinoma metastasis mechanism mainly focu on the ability of tumor cell proliferation xoncogenes or anti — onco-genes,etc. There are rarely studies about metastasis tunnels, especially about lymphatic vessels. Further,we measured the density of lymphatic vessels in different tissues of human laryngeal carcinomas;At the same time, the immunohis-tochemical staining assay and RT - PCR technique were used to study expression of vscular endothelial growth factor C (VEGF - C) at the level of translation and transcription respectively. In conclusion, we investigated the relationships among lymph vessel density ? expression of VEGF - C and cervical lymph node metastasis in laryngeal carcinoma.Materials and MethodsMaterials:The subjects were 46 patients who were diagnosed as laryngeal cancer and operated with laryngectomy in department of otorhinolaryngology, the First Affiliated Hospital,China Medical University and the NO. 463 Hospital of PLA,dur-ing July 2002 to April 2005. The group included 28 men and 18 women,their average age was 62. 13 8. 90 years old (range,40 -77 years);According to the tumor position,30 cases are supraglottic carcinoma and 16 cases are glottic carcinoma and according to 1992 UICC TNM Staging criteria,6 cases are Tl stage, 20 cases are T2 stage, 13 cases are T3 stage and 7 cases are T4 stage;Among them,, 21 cases were No at presentation and 25 cases had metastatic spred to the cervical lymph node (N + ). Tumor were greaded as well differentiated (20 cases) , moderately differentiated (10 cases) , or poorly differentiated( 16 cases ). None of the patients was received chemiotherapy or radiotherapy before operation. Each patients'primary tumor were diagnosed as squamous cell carcinoma by pathological exam after operation. If cervical lymph node is metastasized or not were decided by postoperative pathological findings about lymph node of radical or functional neck dissection samples.Immediately (within one hour) after resection of primary tumor, The samples were cut in the center, periphery (less than 1. 0 cm outside primary tumor edge ) and normal regions ( more than 2. 0 cm outside primary tumor edge ) of the each postoperative tissue of the laryngeal cancer, and these samples were put in Alpdose ducts and quick -freezed by liquid nitrogen ,then stored at -80T!oEmpirical method(1)5' - Nase - ALPase enzymohistochemistry double staining;10 — jxm sections of defrosted tissue were cut using cryostat, then were fixed for 5 minutes in 4T1 cold propanone. On completion of the fixing procedure , the sections were washed for 5 minutes thoroughly in distilled water. After wathing, the sections were immersed for 30 minutes at 371 in the 5' - Nase substrate solution (Tris - maleate buffer 20ml>adenosine 5' mono - phosphate 20mgx2. 5% MgSO45mlx2% Pb ( NO3) 23mlN saccharose 3gxlevamisole 30mg% distilled water 22ml) , washed for 15 minutes thoroughly in distilled water,immersed for development for 1 -2 minutes in 1% ammonium sulfide,and washed for 15 minutes in distilled water. A part of sections were mountinged, others were immersed for 45 minutes at 37 °C in the ALPase substrate solution (naphthol AS - MX phosphate 20mgNfast blue BB 20mgNN,N - dimethylformamide lml, Tris - HCL buffer 20 ml), and washed in distilled water. After mounting the specimens, each speci-men was observered under the light microscope to find lymphatic vessels and blood vessels. Then the lymphatic vessels and blood vessels densities of objective areas were measured using MetaMorph/DP10/BX41 micro - image analysis system. The vessels densities were assessed blindly by two investigators. After the areas of the highest vascularization ( hotspots) were chosen under low power (100 magnification) , vessels counts were done in the five fields at high power (200 magnification). The average counts of the five fields were recorded and the mean of the two investigators findings was used for the final LVD.(2) Immunohistochemistry for expression of VEGF - C protein:The immunohistochemical test were performed by routine SABC method. 5 - (xmserial cryostat sections were cut onto the slide which had been treated with Poly - L - Lysine. After fixed in pure acetone for 30 minutes at ordinary temperature, the sections were washed in distilled water. Then placed in a solution of absolute methanol and 2% hydrogen peroxide for 30minutes. They were subsequently washed in distilled water three times, and treated with normal rabbit blocking serum for 20 minutes at ordinary temperature for blocking of nonspecific reaction. The slide were then incubated overnight at 4^ in a humidified chamber with anti - VEGF - C antibody diluted 1: 200. After the overningt treatment the slide were rinsed three times (two minutes every time) in PBS, and then were performed using biotinylated goat anti - rabbit IgG for 20 minutes at 37 °C , rinsed three times (two minutes every time) in PBS and incubated for 20 minutes at 37X, with Strept Avidin - Biotin Complex, rinsed four times(five minutes every time) in PBS, and immunostaining was performed by incubating the slides in diaminobenzidine (DAB) solution for about 5 minutes, After chromogen development , the slides were washed in distilled water, counterstained by hematoxylin , dehydrated with alcohol and xylene and mounted with neutral gum. Negative control were performed in all cases by omitting the first antibody. Only cases in whose slide immunoreaction of VEGF - C was obvious were scored as positive .(3) RT - PCR for expression of VEGF - C mRNA:According to manufacturer' s instructions, total RNAs were extracted from samples using Total RNA Extraction System (II) reagen, then the concentration and purity of RNA were detected by DNA/RNA detector, the prepared RNAswere reverse - transcribed according to manufacturer * s instructions. 20ul reverse transcription solution including: 25mM MgS044ul, 2 x Bca 1st Buffer lOul.RNase Free H20 0.5ul,10mM dNTP Mixture lul.RNase Inhibitor0.5ul, BcaBEST Polymerase (22U/ul) 1 ul, Oligo dT Primer 1 ul, RNA 2uL The Reaction was performed by the condition;lmin at 651;5 min at 30T!;30min at 65t;5min at 98TI;5min at 5Tl.The cDNA samples were incubated at 94X. for 3 min to inactivate the reverse transcriptase and then chilled, the samples were amplified by addition of 25 ul of PCR mixture (10 x buffer 2. 5ul, lOmM dNTPs 2ul, sterile purified water 17.1 ill,Ex Taq 0.2ul,Primerl 0. lulfPrimer2 0. lul, cDNA 3ul). The amplification was performed for 45s at 94*t , 1 min at 52*C and lmin at 72t for 35 cycles, followed by 7min at 72X1.Specific primers for VEGF - C gene,targeting an 152bp fragment,were:forward primer, 5'-CCA TTA TTA GAC GTT CCC TG - 3',reverse primer, 5'-TGT TGA GTC ATC TCC AGC AT-3';As internal standards, theft -actin gene were used. Primer pair specific to the (3 - actin gene targeting a 690bp fragment, were;forward primer,5'- CAC CCT GTG CTG CTC ACC GAG GCC -3', reverse primer, 5'- CCA CAC AGA TGA CTT GCG CTC AGG -3: The kit RNA sample were used as positive control and the negative control were prepared without RNA amplification. The product were electrophoresed on 2% agarose gel and then were observed under ultraviolet lamp. The gray scale of VEGF - C mR-NA and fj - actin mRNA were scanned and measured using Gelatum image analysis system and VEGF - C/{3 - actin were calculated as relative value.3. StatisticsStatistical analysis was carried out using SPSS 12. 0 for Windows sofeware package. The Chi - square test was used to analyze numeration data. The measurement data are presented as means ± standard deviation and were respectively analysed using independent - sample t test, paired t — test, ONE - WAY ANO-VA or Spearman correlation analysis according to different statistical requirement (the homogeneity test for variance was used by F test) ,value with a P of < 0.05 were considered to be statistically significance.Results1. Not only intratumoral lymphatic vessel density (ILVD) and but also per-itumoral lymphatic vessel density (PLVD) was significantly higher than that of the normal tissue (P <0.01);The morphous of tumor lymphatic vessels was obviously various in different areas.2. LVD (no matter in the intratumoral tissues or peritumoral tissues ) were significantly higher in the group with positive lymph nodes than that with negative lymph nodes (P < 0. 01). The lympgatic vessels of the group with positive lymph nodes mainly were appeared with obviously dilated cavity and a few had ill - defined lumina.3. There were obviously difference in LVD (no matter in the intratumoral tissues or peritumoral tissues ) between supraglottic larynegal cancer group and glottic larynegal cancer group N between T,₂ stages and T3₄ stages and among the Gl ,G2 and G3 groups( P <0. 01).4. Immunohistostaining of VEGF - C were different obviously in different tissues of the laryngeal cancers. VEGF - C protein expressions were all observed in the center,periphery and normal region samples of the postoperative tissues. The intratumoral VEGF - C positive rate (60. 87% ) was significantly higher than that of peritumoral (30.43 % ) and normal samples (8.70 % ) .5. It was noted that intratumoral VEGF — C protein expression was cottelat-ed well with PLVD and ILVD ( P < 0. 01) , and Peritumoral VEGF - C protein expression-was cottelated well with PLVD and ILVD(0.01