Study On The Mechanism Of VEGF-D/VEGFR-3 Signal Pathway To Stimulate Lymphatic Formation And Lymph Metastasis In Supraglottic Squamous Cell Carcinoma

IntroductionLaryngeal carcinoma is a very common malignant tumor in head and neck. Its morbility is on the second stage of head and neck malignant tumors and 93% -99% pathologic type of laryngeal carcinoma is laryngeal squamous cell carcinoma. The epidemiology data showed that the morbilities and types of laryngeal carcinoma have great difference in different areas in our country. In Northeast area, the morbility is the highest and the supraglottic carcinoma is much common. Supraglottic carcinoma has poor prognosis and expresses high incidence of cervical lymph node metastasis. Since most supraglottic carcinoma died from metastasis, cervical lymph metastasis has great influence on the prognosis of supraglottic carcinoma and the state of cervical lymph node has great influence on the cure rate and surviving rate. It is reported that the 5 - year surviving rate of N + is lower than that of NO. Therefore, It is significant for diagnosing,preventing , treating lymph metastasis of supraglottic carcinoma and improving the surviving rate and quality to study the mechanism of cervical lymph metastasis in supraglottic carcinoma.It is the initial phase that tumor cells drain into the local lymph nodes through lymphatics, so the lymphatic is the essential way for lymph metastasis. On the early grow stage of tumor especially metastasis tumor, most of its nutrition materials come from diffusion and expansion of micro lymph node and newlymphatics. The cell factors secreted by lymphatics also participate in the composition of cell microenvironment. The aberration of their function and structure will let to the change of cell microenvironment and promote the evolution of tumor cells which will make the tumor have more pathogenicity x metastasis potency and more energy to escape the immune attack. Because of the domain of research methods, the researches about lymphatics are very rare. Along with the funding of lymphatic endothelial markers, the research about lymphatics becomes a new hot recently. Up to now, the reports about the rule of lymphatic formation and the relationship between lymphatic formation and clinical pathological factors in supraglottic carcinoma are still rare and the conclusions are also different.VEGFR - 3 ( vascular endothelial growth factor receptor - 3) , also called flt4 ( fins - like tyrosine kinase 4) , is the first identified lymph endothelial marker, which can be used to calculate lymphatics. VEGFR - 3 also is one of several proteins which control lymphatic formation and development. VEGF - D (vascular endothelial growth factor - D) identified by Achen through computer homology search in 1998 is a new member of VEGF family. Apart from VEGF -C, VEGF - D is another lymphatic endothelial stimulation factor. It is reported that tumor cells paracrine VEGF - D precusor which forms the mature type through proteolytic to play its biological effect which can activate its receptor VEGFR - 3 to induce lymphatics formation and motivate metastasis. The mechanism VEGF - D promote lymph metastasis may be that VEGF - D motivates lymphatic endothelial proliferation and dilation which aggrandizes the touch surface between tumor cells and lymphatic endothelials and the permeability of vessels. VEGFR -3 is the special receptor of VEGF - D. VEGF - D forms VEGF - D/ VEGFR - 3 signal passway through activating and combining with VEGFR - 3 to model lymphatic formation and facilitate lymph metastasis. This signal way may be an important step for tumor lymphatic proliferation and dilation which provide a way for lymph metastasis. It is reported that the inhibitor of VEGFR - 3 or the neutralizing antibody can block this signal way so as to inhibit the lymphatic formation in animal models. Moreover, It will be helpful for blocking the metastasis intervened by VEGF - D to identify the protease which changes the VEGF -D precusor to mature type. At present, it is not clear about the mechanism that VEGF - D/ VEGFR - 3 signal pathway model lymphatic formation and metastasis. It is not reported about this signal way in supraglottic carcinoma. Therefore, VEGFR - 3 immunohistochemistry method and automatic image analysis technique were applied to mark lymphatics and observe the lymphatic formation rule and the relationship between the lymphatic formation and clinical pathological factors in supraglottic squamous carcinoma from central tumor to normal mucosa. Immunohistochemistry method was applied to examine VEGF - D protein expression rule from central tumor to normal mucosa. Reverse transcription polymerase chain reaction method was applied to examine the mRNA expression level from tumor joint area to normal mucosa. As a result, we explore the mechanism of VEGF - D/VEGFR - 3 signal payhway to stimulate lymphatic formation and lymph metastasis in supraglottic squamous cell carcinoma from protein and mRNA level respectively so as to provide theoretical basis for the clinical using of the medicine which block VEGF - D/VEGFR -3 signal pathway % choosing more scientific treatment and giving more accurate prognosis.Materials and Methods—N Materials:1.50 cases, diagnosed as supragllotic squamous cell carcinoma and operated with laryngectomy and neck dissection at the same time without any other treatment before the operation, were collected from the departement of head and neck surgery of Iiaoning province cancer hospital from July 2002 to October 2004. The group included 33 male and 17 female, their average age was 60.7 -year (from 42 - year to 82 - year). According to 1997 UICC TNM Staging criteria , 29 cases were Tl - 2 stage and 21 cases were T3 - 4 stage, 25 cases were NO at presentation and 25 cases had metastasized to the cervical lymph node (N + ). According to the differentiating degree, 22 cases were greaded as well differentiated , 20 cases moderately differentiated and 8 cases poorly differentiated.2. RT - PCR samples;Immediately (within half hour) after resection of primary tumor, the samples were cut out 0.5 cm from carcinoma edge to tumor,0.5 cm from carcinoma edge to para - tumor and the normal mucosa off the tumor edge more than 2 cm respectively. All samples were put into the Quick -freezed ducts and frozen by liquid nitrogen , then stored in - SOX, deep freeze.3. Immunohistochemistry samples;Border on the RT - PCR samples, the samples were cut out from the tumor edge to tumor and paratumor 0. 5cm (marked as joint area) , in the centrial tumor off the tumor edge 1. 0cm(marked as centrial tumor) and the normal mucosa off the tumor edge more than 2 cm respectively. All samples were put into the 10% formaldehyde, then made into wax lump and 6 - jxm sections.4. The main reagents:RT - PCR agents: Total RNA Extraction System (II) reagent boxes were produced by Henan Huamei Biological Corp. The RT - PCR reagent boxes were produced by Dab'an Takara Biological Cop. The DNA Marker was produced by Dalian Boruide Biological Corp. The primers were produced by Shanghai Boya Biological Corp.Immunohistochemistry reagents;DAB reagent kit and polyclozal rabbit anti human against VEGFR - D and VEGFR - 3 antibodies were produced by Wuhan Boside Biological Corp.Hx Empirical method;1. Immunohistochemistry staining:Routine SABC ( strep avidin - biotin complex) method was applied to mark lymph endothelial cell with polyclozal rabbit anti human against VEGFR - 3 antibody. The positive was defined as brown to yellow staining in cytoplasm and cell membrane. The ducts without erythrocyte in them, circled with brown -yellow one layer endothelial cell without basic membranex smooth musle cell, were identified as lymphatics. The lymphatic formation rule from central tumor to normal mucosa was observed with this method.Routine SABC method was applied to examine the VEGF - D expression from central tumor to joint area to normal mucosa with polyclozal rabbit anti human against VEGFR - D antibody. The positive was difined as brown to yellow staining in cytoplasm. Semiquantitative AB was adoped to describe the results of VEGF-D.2. RT - PCR for the expression of VEGF - D and VEGFR - 3 mRNA in joint tumor xparatumor and normal mucosa;With Total RNA Extraction System(II) reagent, total RNAs were extracted from samples , wash - dried with ethonal then preserved under - 20^! in RNase - Free Water according to manufacturer' s instructions, then the concentration and purity of RNA were detected by DNA/RNA detector.According to manufacturer's instructions, the cDNA was formed. Reactionsystem;RNA2jxl, 2 x buffer 10uJ, 25mMMgSO44ni, dNTPs(lOmM) lui,AMV(22U/jxl) lfjbl, Oligo dT 15(50mM) 1 julI,RNase Inhibitor 0.5 ul, RNase-Free dH2O0.5u>l. Reaction condition;65*C 1 min, 30^ 5 min, uniform ac-celetate to 65 T! withinl5 min, 98t 5 min, 5X1 5min.VEGF - D PCR reaction. System;cDNA3ui, RNase - Free dH2O 17. ljxl , 10xbuffer2.5uJ, dNTPs(2.5mM)2uJ, Taq -E(5U/uJ)0.2uJ, VEGF-D -F 0. ljxl, VEGF - D -R 0. ljxl. Reaction condition: 94°C 3min, 35 cycles (94°C 45seconds—>50. 5X1 lmin—?720.05) whileas the difference of VEGF - D expression in tumor, paratumor and normal mucosa is significant(P <0.01). Under different clinical pathological factors(lymph node state, clinical T stage, tumor differentiation type ,age and gender) , only the difference of positive rate between NO and N + is significant (P <0.05). The VEGF - D positive rate of N + group is higher than that of NO group significantly. Other clinical pathological factors have not obvious influence on the positive rate (P >0.05). The correlation between VEGF - D protein expression and LVD marked with VEGFR - 3 in jointarea is significant (r =0.212). The correlation between VEGF - D protein expression in carcinoma or para - carcinoma and LVD marked with VEGFR - 3 in joint area is insignificant respectively.HsFrom the molecular biology point of view and on the reverse transcription level, the highest expression area of VEGF - D is carcinoma tissue, then is para-tumor, then is normal mucosa and the differences are significant(t^9. 629 ,P =0.000) in joint area. The relative value of VEGF - D/p - actin were 1.448 ± 0.357 x 1.028 ± 0.314 and 0.418 ± 0. 207 in joint tumor, para - tumor and normal mucosa respectively and the relative value decreased gradually from tumor to normal mucosa. The levels in carcinoma and para - tumor of N + group are higher than that of NO group and the differences are significant (t^2.377, P^0.021) , but the difference in normal tissue are insignificant (t = 1. 113,P = 0.271). In joint area, the VEGF - D protein expression also is that the positive rate in carcinoma is higher than that in para - tumor than in normal mucosa and the differences also are significant (\2^7. 853 , P$0.005). The correlation between VEGF — D mRNA and protein expression from joint tumor to para -tumor to normal mucosa is significant! r^0. 411 ). The correlation between VEGF - D and LVD marked with VEGFR -3 in joint area is significant! r =0. 230). The correlation between VEGF - D mRNA expression in carcinoma or para - carcinoma and LVD marked with VEGFR - 3 in joint area is insignificant respectively.BKIn joint area, the highest expression of VEGFR -3 mRNA is carcinoma ,then is para -tumor, then is normal mucosa and the differences are significant (tS*9.588,P = 0.000). The relative values of VEGFR - 3/0 - actin were 1. 425 ±0.3014.024 ±0. 337 and 0.406 ±0. 273 in tumor, paratumor and normal mucosa respectively and the relative value decreased gradually from tumor to normal mucosa. The levels in carcinoma and para - tumor of N + group are higher than these of NO group and the differences are significant (t^2.335 ,P ^0.024), but the difference in normal tissue is insignificant(t = 1.900,P =0. 063). The correlation between VEGFR - 3 mRNA expression and LVD marked with VEGFR - 3 in joint area is significant! r = 0.243). The correlation between VEGFR - 3 mRNA expression in carcinoma or para - carcinoma and LVDmarked with VEGFR - 3 in joint area is insignificant respectively.3IxThe correlation between VEGFR -3 and VEGF - D mRNA expression in joint carcinoma and para - tumor is significant (r = 0. 288). The correlation between VEGF - D and VEGFR - 3 mRNA expression in carcinoma or para -carcinoma in joint area are insignificant respectively.Conclusions— x There is lymphatic formation and expansion in joint area of supraglottic squamous carcinoma and those new lymphatics play an important role in the process of tumor growth and lymph metastasis.HxIn supraglottic squamous carcinoma, both tumor and para - tumor tissue can secrete VEGF - D so as to express high level VEGF - D mRNA and protein. VEGF - D can motivate microlymphatic formation and cervical lymph metastasis through self - secrete and paracrine in joint area. But the VEGF - D quantity secreted by para - tumor cell is less than that secreted by tumor cells.H>The mechanism that VEGF -D motivate lymph metastasis in tumor and para - tumor of supraglottic squamous carcinoma may be that tumor cells and para - tumor cells secrete VEGF - D precusor through self - secrete and paracrine which forms the mature type through proteolytic to play its biological role in activating its receptor VEGFR - 3 to induce lymphatics formation % expansion and motivate metastasis.HsThe LVD and VEGF - D/VEGFR -3 expression (protein or mRNA level ) in joint area of supraglottic squamous carcinoma may become a new target for forecasting metastasis and assessing prognosis.3LNThe medicines that block any step of VEGF - D/VEGFR - 3 so as to inhibit lymphatic formation and lymph metastasis are still in the empirical period. This kind of anti - lymph metastasis medicine may become a new method for preventing and treating larynx carcinoma metastasis.an % It is very useful and workable in clinical working to calculate lamphatics with VEGFR - 3 antibody. Examining VEGF - D and VEGFR - 3 expression with immunohistochemistry and RT - PCR methods let us deeply research themechanism that VEGF - D/ VEGFR - 3 signal pathway motivate lymphatic formation and lymph metastasis from the morphology and molecular biology points of view and provide theoretical basis and feasible method for using this kind of medicine which block VEGF - D/VEGFR - 3 signal way in clinical work.