Expression of Lymphangiogenic Factors and Evidence of Intratumoral Lymphangiogenesis in Pancreatic Endocrine Tumors
2004; Elsevier BV; Volume: 165; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63379-2
ISSN1525-2191
AutoresBence Sipos, Wolfram Klapper, Marie‐Luise Kruse, Holger Kalthoff, Dontscho Kerjaschki, Günter Klöppel,
Tópico(s)Lymphatic Disorders and Treatments
ResumoLymphangiogenesis is thought to promote the progression of malignant tumors. Because the lymphangiogenic factors vascular endothelial factor (VEGF)-C and -D are expressed in endocrine cells, we investigated their expression in pancreatic endocrine tumors (PETs) and correlated these data and intratumoral lymph vessel density (iLVD) with clinicopathological features. Lymph vessels were identified with anti-podoplanin antiserum and with podoplanin/proliferating cell nuclear antigen double labeling. PETs (n = 104) were investigated by immunohistochemical staining for VEGF, basic fibroblast growth factor, and VEGF-C expression. VEGF-C and VEGF-D mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. PETs showed higher iLVD than normal pancreata, but iLVD did not discriminate between benign and malignant PETs. In PETs proliferating lymph vessels were identified. High iLVD was associated with lymph vessel invasion and it was more frequent in angioinvasive/metastatic tumors than in grossly invasive tumors. VEGF-C expression correlated with iLVD as well as with glucagon and pancreatic polypeptide expression. PETs show intratumoral lymphangiogenesis, which is associated with VEGF-C expression in tumor cells. The association between iLVD and lymph vessel invasion and angioinvasive/metastatic features in PETs suggests that lymphangiogenesis may promote malignant progression of PETs. PET is the first human tumor entity in which VEGF-C-related intratumoral lymphangiogenesis has been demonstrated. Lymphangiogenesis is thought to promote the progression of malignant tumors. Because the lymphangiogenic factors vascular endothelial factor (VEGF)-C and -D are expressed in endocrine cells, we investigated their expression in pancreatic endocrine tumors (PETs) and correlated these data and intratumoral lymph vessel density (iLVD) with clinicopathological features. Lymph vessels were identified with anti-podoplanin antiserum and with podoplanin/proliferating cell nuclear antigen double labeling. PETs (n = 104) were investigated by immunohistochemical staining for VEGF, basic fibroblast growth factor, and VEGF-C expression. VEGF-C and VEGF-D mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. PETs showed higher iLVD than normal pancreata, but iLVD did not discriminate between benign and malignant PETs. In PETs proliferating lymph vessels were identified. High iLVD was associated with lymph vessel invasion and it was more frequent in angioinvasive/metastatic tumors than in grossly invasive tumors. VEGF-C expression correlated with iLVD as well as with glucagon and pancreatic polypeptide expression. PETs show intratumoral lymphangiogenesis, which is associated with VEGF-C expression in tumor cells. The association between iLVD and lymph vessel invasion and angioinvasive/metastatic features in PETs suggests that lymphangiogenesis may promote malignant progression of PETs. PET is the first human tumor entity in which VEGF-C-related intratumoral lymphangiogenesis has been demonstrated. The biological behavior of pancreatic endocrine tumors (PET) is difficult to predict on the basis of histological criteria. In the absence of clear signs of malignancy, such as invasion of adjacent organs, angioinvasion, or metastasis, the prognosis remains uncertain. Because most human carcinomas metastasize via lymphatic invasion, lymph node metastasis is a key prognostic factor for the clinical outcome. By which mechanism tumor cells spread through the lymph vessels is unknown. One proposed mechanism is the induction of new lymph vessels by tumor or inflammatory cells, facilitating lymphangioinvasion. Intratumoral lymph vessels have been detected in head and neck squamous cell carcinomas1Beasley NJ Prevo R Banerji S Leek RD Moore J van Trappen P Cox G Harris AL Jackson DG Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer.Cancer Res. 2002; 62: 1315-1320PubMed Google Scholar, 2Maula SM Luukkaa M Grenman R Jackson D Jalkanen S Ristamaki R Intratumoral lymphatics are essential for the metastatic spread and prognosis in squamous cell carcinomas of the head and neck region.Cancer Res. 2003; 63: 1920-1926PubMed Google Scholar and in cutaneous melanomas.3Straume O Jackson DG Akslen LA Independent prognostic impact of lymphatic vessel density and presence of low-grade lymphangiogenesis in cutaneous melanoma.Clin Cancer Res. 2003; 9: 250-256PubMed Google Scholar, 4Dadras SS Paul T Bertoncini J Brown LF Muzikansky A Jackson DG Ellwanger U Garbe C Mihm MC Detmar M Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival.Am J Pathol. 2003; 162: 1951-1960Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar In squamous cell carcinomas intratumoral lymph vessel density (iLVD) has been shown to correlate with lymph node metastasis, whereas the results for melanomas are inconsistent. 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VEGF-C was identified in pituitary cells, pancreatic α-cells, adrenal medullary cells, and serotonin-producing cells of the gastrointestinal tract, whereas VEGF-D was detected in the cortex of the adrenal gland and in gastrin-producing cells.28Partanen TA Arola J Saaristo A Jussila L Ora A Miettinen M Stacker SA Achen MG Alitalo K VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues.FASEB J. 2000; 14: 2087-2096Crossref PubMed Scopus (286) Google Scholar Because malignant PETs frequently exhibit lymphatic invasion and lymph node metastases, we investigated whether these tumors are capable of inducing lymphangiogenesis that may promote tumor progression. On the basis of the distribution pattern of lymphangiogenic factors in normal endocrine cells, we hypothesized that PETs may express VEGF-C or VEGF-D and that this expression is related to lymphangiogenesis and biological features. Formalin- or Bouin-fixed paraffin-embedded tissue blocks from 111 PETs from the period 1972 to 2002 were investigated. They were retrieved from the consultation files of the Department of Pathology of the University of Kiel. Only cases with complete patient records, including gender, age, clinical manifestation, size and localization of tumor, and presence of metastasis were considered. Table 1 summarizes the most important clinicopathological characteristics of the investigated PETs. All benign functionally active PETs were insulinomas. Two of them arose in patients with multiple endocrine neoplasia type I. The malignant functionally active PETs consisted of 11 insulinomas, 7 gastrinomas, 4 glucagonomas, 2 VIPomas, and 1 ACTH-producing tumor. Clinical symptoms and elevated serum hormone levels defined functionally active PETs. PETs that were ≤2 cm in diameter and showed no signs of invasive growth or metastasis were considered benign. Malignant PETs showed blood or lymph vessel invasion (detected with endothelial cell-specific immunostains), tumor infiltration of adjacent organs, or histologically verified lymph node or blood-borne metastases. To increase the number of glucagon-expressing tumors, seven glucagon-expressing PETs (one glucagonoma, six functionally inactive) that lacked signs of malignancy but were larger than 2 cm were also included in the study. Immunohistochemically, all tumors expressed synaptophysin and/or chromogranin and were well differentiated according to the World Health Organization classification.29Solcia E Kloppel G Sobin LH Histological Typing of Endocrine Tumours. Springer, Berlin2000: 56-60Google ScholarTable 1Clinicopathological Features of 111 PETsGenderAge (years)Tumor size*Maximum diameter in cm.No. of tumorsFMMedianSDRangeMedianSDRangeFunctionally active Benign3219134819.617–881.50.420.5–2 Malignant2514115115.224–7942.462–11Functionally inactive Benign7526114.835–7521.670.7–6†This group contains one large cystic tumor (6 cm) that had already been noted 10 years before the surgical resection and showed no signs of malignant transformation. Malignant4023175714.23–7553.032–12Probably malignant‡This group was included only to test the correlation between glucagon expression, VEGF-C expression, and LVD.7525014.717–5953.62.2–12Total11166455416.263–8833.040.5–12* Maximum diameter in cm.† This group contains one large cystic tumor (6 cm) that had already been noted 10 years before the surgical resection and showed no signs of malignant transformation.‡ This group was included only to test the correlation between glucagon expression, VEGF-C expression, and LVD. Open table in a new tab Freshly cryoconserved tumor tissue from six pancreatic PETs served as source of reference RNA for the quantitative polymerase chain reaction (PCR) investigations. These tumors were collected according to the protocol approved by the ethics committee of the University of Kiel Hospitals (permission number, 110/99). This protocol prescribed signed informed consent by all patients. Normal pancreatic tissues removed from 12 individuals who died of suicide were obtained from the Department of Forensic Medicine of Semmelweis University (Budapest, Hungary). The pancreata were removed within 1 hour after death with the permission of the local Ethical Commission for Scientific Research (permission number, 140-1/1996) and immediately fixed in 4% buffered formalin. Histological examination of the pancreas tissues revealed no signs of pancreatic disease. During the forensic autopsy no signs of organic disease affecting the pancreas were seen. Tumor tissues were fixed in formalin or in Bouin's fixative and routinely processed for paraffin sectioning. Three-μm-thin paraffin sections were deparaffinized, rehydrated, and immunohistochemical stains were performed according to routine methods. Before application of the primary antibody, blocking with nonimmune serum was performed for 20 minutes. Table 2 lists the positive controls, demasking methods, antibodies, and detection systems. For negative controls, the primary antibodies were omitted or the primary anti-basic fibroblast growth factor antibody was incubated with specific blocking peptide in 10-fold molar excess before the staining. For the VEGF-D staining, the primary antibody was replaced with control mouse IgG2a (DAKO Cytomation, Glostrup, Denmark).Table 2Antibodies, Conditions, and Controls for Immunohistochemical ReactionsAntigenPrimary antibody clone/codeSourceConcentration/dilutionAntigen demaskingSecondary antibodyDetection systemPositive controlSynaptophysinRabbit/A0010Dakocytomation, Glostrup, Denmark6 μg/mlPressure cooker, TEC buffer 3 minutesRabbit Vectastain peroxidase kit; Vector Laboratories, Burlingame, CAPancreasChromograninLK2H10/E001LINARIS, Wertheim-Bettingen, Germany1:2–Mouse Vectastain peroxidase kit, Vector LaboratoriesPancreasInsulinHB125/02911Biogenex, San Ramon, CA1:40–Mouse Vectastain peroxidase kit, Vector LaboratoriesPancreasGlucagonRabbit/039PBiogenex1:60–Rabbit Vectastain peroxidase kit, Vector LaboratoriesPancreasPancreatic polypeptideRabbitR.E. Chance, Indianapolis, IN1:5000–Rabbit Vectastain peroxidase kit, Vector LaboratoriesPancreasSomatostatinRabbit/A0566DAKO1:200–Rabbit Vectastain peroxidase kit, Vector LaboratoriesPancreasGastrinRabbitPaesel, Frankfurt, Germany1:3000–Rabbit Vectastain peroxidase kit, Vector LaboratoriesDuodenumVasoactive intestinal peptideRabbit/18-0080ZYMED, San Francisco, CA1:10–Rabbit Vectastain peroxidase kit, Vector LaboratoriesFunctionally active VIPomaCD34QBEND10/0786Immunotech, Marseille, France1 μg/ml–Mouse Vectastain peroxidase kit, Vector LaboratoriesEndothelial cellsPodoplaninRabbit321:2000Pressure cooker, TEC buffer, 3 minutesRabbit Envision kit, DAKOLymphangiomVEGFGoat/AF293 NAR&D Systems, Wiesbaden, Germany10 μg/mlPressure cooker, TEC buffer, 3 minutesBiotin conjugated anti-goat IgG, 705-065-147; Jackson Laboratories, West Grove, PAABC Elite Kit, Vector LaboratoriesFetal kidneyVEGF-CGoat/AF752R&D Systems0.5 μg/mlPressure cooker, Glycol containing solution,*Biologo, Kiel, Germany. 5 minutesBiotin conjugated anti-goat IgG, 705-065-147; Jackson LaboratoriesABC Elite Kit, Vector Laboratoriesα-Cells in Langerhans-isletsVEGF-DMouse/MAB 286R&D Systems15 μg/ml, overnightPressure cooker, Glycol containing solution,*Biologo, Kiel, Germany. 5 minutesMouse Envision kit, DakoCytomationGastrin-producing cells of stomachbFGFRabbit/sc-79Santa Cruz Biotechnology, Santa Cruz, CA, USA1 μg/mlPressure cooker, TEC buffer, 3 minutesRabbit Vectastain peroxidase kit, Vector LaboratoriesChronic pancreatitis* Biologo, Kiel, Germany. Open table in a new tab Expression of hormones, VEGF, VEGF-C, VEGF-D, and bFGF was semiquantitatively evaluated as mild ( 50%) by one investigator (B.S.) without knowledge of the other tumor features. Hormone expression was carefully analyzed in consecutive sections to avoid misinterpretation of entrapped pancreatic islets. iLVD and peritumoral lymph vessel density (pLVD) were determined by the hot spot method.30Weidner N Semple JP Welch WR Folkman J Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma.N Engl J Med. 1991; 324: 1-8Crossref PubMed Scopus (5298) Google Scholar All areas (0.75 mm2) with high lymph vessel density stained with anti-podoplanin antiserum were photographed with a Nikon Coolpix 990 digital camera mounted on an Axioskop 50 microscope (Zeiss, Oberkochen, Germany). Intratumoral hot spots in all PETs and peritumoral hot spots (areas in the fibrous capsule of the tumor or in peritumoral tissues without close relationship to tumor cells) in 80 PETs were evaluated. The localization of the 349 digitalized intratumoral hot spots was recorded as peripheral (within 4 mm of the tumor border) or central. Podoplanin-positive vessels with or without lumen were counted with Scion Image Software (Scion Corp., Frederick, MD). Vessels were considered to be invaded if vascular spaces lined by podoplanin-positive (lymph vessel) or CD34-positive (blood vessel) endothelial cells contained definite tumor cell complexes. LVD was determined with the same method in 12 normal pancreata (one section from the head, body, and tail from each pancreas) and peripancreatic fat tissues (one section from each pancreas). Overall 244 hot spots were analyzed in normal pancreatic and peripancreatic tissue. Tissue sections were incubated in 1% sodium borohydride in phosphate-buffered saline for 30 minutes to reduce autofluorescence and subsequently stained with 1% Sudan Black B (Sigma, Taufkirchen, Germany) for 10 minutes. Nonspecific binding sites were blocked in blocking buffer containing 0.1% bovine serum albumin and 0.2% glycine in Tris-buffered saline for 1 hour at room temperature. Incubation of antibodies was performed overnight in Tris-buffered saline. Primary antibody dilutions were as follows: rabbit anti-podoplanin antiserum, 1:400; monoclonal anti-proliferating-cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, Santa Cruz, CA), 1:300; mouse monoclonal anti-glucagon antibody, 1:30; and goat anti-VEGF-C antibody, 1:50. After extensive washing in Tris-buffered saline, secondary antibodies were incubated for 1 hour at 37°C (goat anti-rabbit Alexa Fluor 488, 1:1000; goat anti-mouse Alexa Fluor 546, 1:1000; donkey anti-goat Alexa Fluor 488, 1:1000; all from Molecular Probes, Eugene, OR). Sections were counterstained for nuclei using Hoechst A33528 dye at a concentration of 0.025 mg/ml. After extensive washing in Tris-buffered saline, sections were mounted using ProLong anti-fade mounting medium (Molecular Probes). Laser-scanning analysis was performed using a Zeiss LSM510 confocal laser-scanning microscope (Carl Zeiss Jena, Jena, Germany). All recordings were done using multitracking with ×400 original magnification and pinhole diameter set at 1.0 Airy unit. For colorometric double labeling, PCNA immunostaining was developed with alkaline phosphatase-conjugated secondary antibody and podoplanin staining was developed with peroxidase-conjugated secondary antibody using an Envision double-labeling kit (DAKO Cytomation) according to the manufacturer's instructions. The rate of the double-labeled vessels was determined by counting the nuclei of intratumoral podoplanin-positive microvessels (100 to 350 nuclei in each tumor). Paraffin blocks from 29 formalin-fixed PETs, which consisted of >90% tumor tissue were selected for quantitative PCR. Immunohistochemically, 10 tumors showed no or low VEGF-C expression; 19 tumors revealed moderate or strong VEGF-C positivity. Ten 5-μm-thin sections were cut into tubes, deparaffinized, washed in ethanol, air-dried, and resuspended in 80 μl of 60 mg/ml Proteinase K (Sigma) plus 720 μl of digestion buffer as described by Godfrey and colleagues31Godfrey TE Kim SH Chavira M Ruff DW Warren RS Gray JW Jensen RH Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction.J Mol Diagn. 2000; 2: 84-91Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar overnight at 55°C in a shaker. RNA extraction was performed with an RNAeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To remove remnants of genomic DNA, extracted RNA was treated with RNase-free DNase Set (Qiagen) for 20 minutes. The RNA concentration was determined by spectrophotometer (Spectramax 250; Molecular Devices, Sunnyvale, CA) at 260 nm. The purity of the RNA extracts was monitored by OD 260/280 measurement. RNA extracts were aliquoted and stored at −20°C. The cryoconserved tissues were cut into pieces, transferred to tubes, homogenized by pellet pestles, pushed through a 21-gauge needle, and subjected to RNA extraction with the same method. Primers were designed to span introns in the genomic DNA. Primers and probes for quantitative PCR are listed in Table 3. The levels of VEGF-C and VEGF-D mRNA were quantified by real-time reverse transcriptase (RT)-PCR using an ABI Prism 7000 sequence detection system (Applied Biosystem, Foster City, CA). One-step RT-PCR (QuantiTect Probe RT-PCR, Qiagen) was performed using 100 ng of RNA, 10 pmol of forward and reverse primer, respectively, and 3 pmol of probe in each reaction. The mRNA for VEGF-C and VEGF-D and the control GAPDH were each amplified in separate tubes. VEGF-C and VEGF-D quantification was performed in triplicate and in duplicate, respectively. DNA for the PCR standard was obtained from RNA from fresh PET tissue by RT-PCR using the same primers as for the quantitative RT-PCR. Standard curves were constructed with 10-fold serial dilutions of gel-purified DNA. After performing a linear regression analysis for the standard dilutions the values for the experimental samples were extrapolated and expressed as corresponding attomol of the standard DNA. The values in attomol for VEGF-C and VEGF-D were divided by the value for GAPDH.Table 3Oligonucleotide Primers and Probes Used for Quantitative PCRTarget5′ Primer3′ PrimerProbeSize of amplicon (bp)Annealing temperatureVEGF-C15′-TCAAGGACAGAAGAGACTATAAAATTTGC5′-ACTCCAAACTCCTTCCCCACAT6FAM-ATACACACCTCCCGTGGCATGCATTGT-TAMRA13760VEGF-D5′-TACTCTTCCCCAGCTCACTG5′-TACTCTTCCCCAGCTCACTG6FAM-CAAAGAACTCAGTGCAGCCCTAGAGAAACGTTAMRA11660GAPDH315′-ATTCCACCCATGGCAAATTC5′-TGGGATTTCCATTGATGACAAGVIC-CAAGCTTCCCGTTCTCAGCC-TAMRA7260 Open table in a new tab The association between LVDs (continuous variable) and clinicopathological features or cytokine expression was analyzed with the Mann-Whitney U-test and the Kruskal-Wallis H-test. Correlations between VEGF-C expression and other tumor parameters were determined with Fisher's exact
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