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[Cancer Research 64, 8630-8638, December 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Loss of Neural Cell Adhesion Molecule Induces Tumor Metastasis by Up-regulating Lymphangiogenesis

Ivana Crnic1, Karin Strittmatter1, Ugo Cavallaro1,2, Lucie Kopfstein1, Lotta Jussila3, Kari Alitalo3 and Gerhard Christofori1

1 Institute of Biochemistry and Genetics, Department of Clinical-Biological Sciences, University of Basel, Basel, Switzerland; 2 Istituto Federazione Italiana Ricerca Cancro di Oncologia Molecolare, Milano, Italy; and 3 Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reduced expression of neural cell adhesion molecule (NCAM) has been implicated in the progression to tumor malignancy in cancer patients. Previously, we have shown that the loss of NCAM function causes the formation of lymph node metastasis in a transgenic mouse model of pancreatic ß cell carcinogenesis (Rip1Tag2). Here we show that tumors of NCAM-deficient Rip1Tag2 transgenic mice exhibit up-regulated expression of the lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and -D (17% in wild-type versus 60% in NCAM-deficient Rip1Tag2 mice) and, with it, increased lymphangiogenesis (0% in wild-type versus 19% in NCAM-deficient Rip1Tag2 mice). Repression of VEGF-C and -D function by adenoviral expression of a soluble form of their cognate receptor, VEGF receptor-3, results in reduced tumor lymphangiogenesis (56% versus 28% in control versus treated mice) and lymph node metastasis (36% versus 8% in control versus treated mice). The results indicate that the loss of NCAM function causes lymph node metastasis via VEGF-C- and VEGF-D-mediated lymphangiogenesis. These results also establish Rip1Tag2;NCAM-deficient mice as a unique model for stochastic, endogenous tumor lymphangiogenesis and lymph node metastasis in immunocompetent mice.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neural cell adhesion molecule (NCAM, CD56) is a Ca2+-independent cell adhesion molecule, which mediates homotypic and heterotypic cell-cell and cell-matrix adhesion (1, 2, 3) . Its role in neurite outgrowth, axon guidance, and long-term potentiation has been investigated in great detail (4) . However, NCAM is also expressed in many other cell types, including epithelial cells of various organs, muscle, and pancreatic ß cells. On the basis of alternative splicing of the COOH-terminal domains, NCAM isoforms are grouped into three main classes: a glycosylphosphatidyl inositol-linked Mr 120,000 isoform (NCAM120) and transmembrane Mr 140,000 and Mr 180,000 isoforms (NCAM140 and NCAM180, respectively; refs. 1, 2, 3 ). NCAM140 and 180 are predominantly expressed during embryonic development, whereas NCAM120 is found in many different adult tissues (5 , 6) . In various cancer types, the expression of NCAM shifts from the Mr 120,000 isoform to the Mr 140,000 and Mr 180,000 isoforms (7) . Moreover, an overall decrease in the NCAM level has been observed in a subset of tumors. For example, in colon carcinoma, pancreatic cancer, and astrocytoma, NCAM expression is markedly down-regulated, and the loss of NCAM correlates with poor prognosis (8, 9, 10, 11) . In contrast, in neuroblastoma and certain neuroendocrine tumors, cancer progression correlates with increased NCAM expression and extensive polysialylation (12, 13, 14, 15, 16) .

Although NCAM has been extensively studied as a cell-cell adhesion molecule (2 , 17) , recent reports highlight the role of NCAM in signal transduction. For example, in primary neurons or PC12 cells NCAM is able to activate fibroblast growth factor receptor, thereby stimulating classical fibroblast growth factor receptor signal transduction pathways and neurite outgrowth (18, 19, 20) . Previously, we have studied the functional role of NCAM during tumor progression in a transgenic mouse model of pancreatic ß cell tumorigenesis (Rip1Tag2). In Rip1Tag2 transgenic mice, SV40 T antigen (Tag) is expressed under the control of the rat insulin promoter (Rip1), resulting in the development of ß cell tumors in a multistage tumor progression pathway (21) . Although Rip1Tag2 transgenic mice usually do not develop any metastases, on ablation of NCAM function, metastases to the regional lymph nodes of the pancreas are observed (22) . We have previously shown that, similar to NCAM-mediated outgrowth in neuronal cells, in ß tumor cells, NCAM associates with fibroblast growth factor receptor and stimulates classical fibroblast growth factor receptor signal transduction cascades, which lead to the activation of ß1 integrin function and cell-substrate adhesion (23) . Yet, how the loss of NCAM may cause the metastatic dissemination of ß tumor cells to regional lymph nodes has remained elusive.

In the past few years, several major players in the regulation of lymphangiogenesis, the formation of new lymphatic vessels, have been identified. Most importantly, vascular endothelial growth factor (VEGF)-C and -D, members of the VEGF family of growth factors, have been characterized as the critical players in the induction of lymphangiogenesis in physiology and disease (24, 25, 26) . VEGF-C and VEGF-D bind preferentially to VEGF receptor (VEGFR)-3 (Flt-4), which in the adult is predominantly expressed on lymphatic endothelial cells. Fully processed VEGF-C and -D bind with high affinity also to VEGFR-2 (27, 28, 29) . The lymphangiogenic capability of VEGF-C and -D has been shown in many different experimental settings. For example, forced expression of VEGF-C or -D in tumor cell lines or in transgenic mouse models of tumorigenesis results in up-regulated lymphangiogenesis and in the formation of lymph node metastasis (24) . Conversely, a soluble form of VEGFR-3 blocks lymphangiogenesis, by binding and sequestering VEGF-C and -D (30 , 31) . Up-regulated expression of VEGF-C, and to a lesser extent of VEGF-D, also highly correlates with lymphangiogenesis and lymph node metastasis in cancer patients (24 , 32) . The recent discovery of a number of novel markers for lymphatic endothelium such as VEGFR-3 (Flt-4; refs. 33 , 34 ), LYVE-1 (35 , 36) , podoplanin (37) , and the transcription factor Prox-1 (38) has facilitated studies on the molecular mechanisms of lymphangiogenesis.

Here we report that the lymph node metastases, which are observed in NCAM-deficient Rip1Tag2 transgenic mice, are caused by up-regulated expression of VEGF-C and -D and, with it, increased tumor lymphangiogenesis. Interference with lymphangiogenesis in these mice by adenoviral expression of soluble VEGFR-3 results in a reduction of tumor lymphangiogenesis and of lymph node metastasis, indicating that VEGF-C–driven tumor lymphangiogenesis is a metastatic route for NCAM-deficient ß tumor cells. Moreover, the results establish NCAM-deficient Rip1Tag2 transgenic mice as a unique animal model for stochastic tumor lymphangiogenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Adenoviruses.
For the expression of soluble VEGFR-3, a cDNA fragment encoding the first three immunoglobulin homology domains of the extracellular portion of mouse VEGFR-3 (Flt-4; amino acids 1–332) was amplified by reverse transcription-PCR from RNA extracted from embryonic day 10.5 mice with the primer pair 5'-CCA TCG ATG AGA ATG CAG CCG GGC GCT GCG-3'/5'-CGC CGG ATA TCA CTG ATG AAG GGC TTT TCG TGC AC-3' and ligated in frame to a cDNA construct encoding the mouse immunoglobulin Fc-domain (39) . Recombinant E1/E3 defective adenovirus expressing soluble VEGFR-3 (AdsFlt-4) was generated with homologous recombination in E. coli as described previously (ref. 40 ; see supplementary information). Recombinant adenovirus expressing enhanced green fluorescent protein (AdEGFP) was used as control (41) .

Cell Culture.
Murine fibroblastoid cells (L cells) were cultured in DMEM supplemented with 10% FCS (Invitrogen, Carlsbad, CA), 2 mmol/L glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin sulfate. Cells were transfected with pCDNA-sFlt–4 with LipofectAMINE (Invitrogen), and conditioned medium (48 hours conditioning) was concentrated on Centricon YM-3 columns (Millipore, Billerica, MA).

Immunoblotting.
Forty microliters of concentrated conditioned media and 10 µL of mice sera were resolved by 15% and 10% SDS-PAGE, respectively, and transferred to Immobilon-P membranes (Millipore). Membranes were probed with 1 µg/mL antibody specific for mouse immunoglobulin heavy chains (Pierce, Rockford, IL) or with 0.2 µg/mL antibody specific for mouse Flt-4 (R&D Systems, Minneapolis, MN). Immunostained proteins were visualized with the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s recommendations.

Adenoviral Gene Delivery in Mice.
Two hundred microliters of HEPES-buffered saline containing 5 x 109 adenoviral particles (AdsFlt-4 or control AdEGFP) were injected into the tail vein (i.v.) of Rip1Tag2;NCAM-deficient mice, 3 times (day 1, 10, and 20), starting at 9 to 10 weeks of age. Glucose (5% w/v) was added to the drinking water at 10 weeks of age to counteract hypoglycemia, which results from insulinoma development. Mice were sacrificed 4 weeks after the first injection, and tumor incidence and diameters were determined. Tumor volumes were calculated from the tumor diameter by assuming a spherical shape of the tumors, and total tumor volumes per mouse were determined.

Histology and Immunohistochemistry.
Pancreata were fixed overnight in 4% paraformaldehyde in PBS, dehydrated, and embedded in paraffin. Alternatively, freshly isolated tissue was snap frozen in liquid nitrogen and then embedded in OCT (Tissue Tek, Redding, CA). Five-micron sections were cut for immunohistochemical analysis. For BrdUrd labeling, mice were injected i.p. with 100 µg BrdUrd (Sigma, St. Louis, MO) per gram of body weight 2 hours before sacrifice. For immunohistochemistry on paraffin sections, the following antibodies were used: goat antihuman VEGF-C (C-20, Santa Cruz Biotechnology, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit antimouse LYVE-1 (a gift from David Jackson, Oxford, United Kingdom), hamster antimouse podoplanin (8.1.1; Developmental Studies Hybridoma Bank, Iowa University, Iowa City, Iowa), rabbit antihuman VEGF-D (a gift from Herbert Weich, German Research Center for Biotechnology, Braunschweig, Germany), biotinylated mouse anti-BrdUrd (Zymed, South San Francisco, CA), and guinea pig anti-insulin (DakoCytomation, Glostrup, Denmark). Rabbit antimouse Prox1 (42) , rat antimouse CD-31 (PharMingen, Franklin Lakes, NJ), and double-stainings with these antibodies were performed on fresh frozen sections fixed for 10 minutes at –20°C in methanol. Apoptotic cells were visualized with the In Situ Cell Death Detection Kit, POD (Roche, Basel, Switzerland). All biotinylated secondary antibodies for immunohistochemistry (Vector, Burlingame, CA) were used at a 1:200 dilution, and positive staining was visualized with the ABC horseradish peroxidase kit (Vector) and DAB Peroxidase Substrate (Sigma) according to the manufacturers’ recommendations. For the analysis of tissue morphology, slides were slightly counterstained with hematoxylin. For immunofluorescence analysis, Alexa Fluor 568 and Alexa Fluor 488-labeled secondary antibodies diluted 1:400 were used (Molecular Probes, Eugene, OR). The 6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. To ensure specificity of the antibodies used, pancreatic sections from normal control mice or sections from Rip1Tag2 mice without primary antibodies were used as negative controls. Pancreatic sections from Rip1Tag2; Rip1VEGF-C transgenic mice were included as positive controls (43) .

For quantitation of blood vessel density, proliferation (BrdUrd incorporation), and apoptosis [terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) reaction], the numbers of vascular profiles positive for CD-31 or the number of nuclei staining positive for BrdUrd or the TUNEL reaction were determined in 10 comparable fields per section at 400 x magnification, and the mean of positive cells ± SD/field was calculated. Lymphangiogenesis was quantified as described in the legend to Table 1Citation .


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  		Table 1 VEGF-C expression, tumor lymphangiogenesis, and metastasis in Rip1Tag2;NCAM-deficient mice

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reduced NCAM Expression Correlates with Higher VEGF-C Expression.
As we have previously reported, the loss of NCAM function in the Rip1Tag2 transgenic mouse model of ß cell tumorigenesis results in a marked disaggregation of primary ß cell tumors (23) and in the formation of metastasis to the regional draining lymph nodes of the pancreas (ref. 22 ; supplementary Fig. S1). A comparable induction of lymph node metastasis has been observed on forced expression of VEGF-C in the Rip1Tag2 mouse model (43) . This intriguing similarity motivated us to investigate whether VEGF-C and VEGF-D play a role in lymph node metastasis in NCAM-deficient Rip1Tag2 mice. NCAM is a haploinsufficient gene, manifested, for example, by the lack of significant difference in the phenotype of heterozygous (NCAM+/–) versus homozygous (NCAM–/–) knockout mice (17 , 44 , 45) . Similarly, NCAM+/– and NCAM–/– Rip1Tag2 mice also do not exhibit any detectable differences in tumor progression and in the incidence of lymph node metastasis (22) . Therefore, we used Rip1Tag2;NCAM+/– and Rip1Tag2;NCAM–/– mice in the same experimental groups and, for the purpose of clarity, refer to both of them as Rip1Tag2;NCAM-deficient mice.

First, we determined VEGF-C expression in ß cell tumors of Rip1Tag2 and Rip1Tag2;NCAM-deficient mice by immunohistochemical staining with specific antibodies against VEGF-C. These experiments revealed a significant up-regulation of VEGF-C protein expression in NCAM-deficient tumors (Fig. 1, B, C, and FCitation ; Table 1Citation ). Although only a very small subset of wild-type ß cell tumors expressed VEGF-C, it was detected in more than half of the NCAM-deficient tumors analyzed (Table 1)Citation . VEGF-C staining in ß tumor cells seemed granular and was predominantly found in the perinuclear area of tumor cells (Fig. 1, A–C and FCitation ; data not shown). VEGF-C expression seemed to be independent of the tumor stage, as it was detectable in benign tumors (adenomas) as well as in malignant tumors (carcinomas; Fig. 1, B, F, and GCitation ; data not shown). Smaller adenoma expressed VEGF-C with higher incidence and more uniformly (Fig. 1B)Citation than larger tumors, where VEGF-C expression was limited to specific areas within the tumor (Fig. 1GCitation ; and data not shown) or single scattered cells (Fig. 1C)Citation . Simultaneous immunofluorescence staining for insulin and VEGF-C revealed that VEGF-C is expressed exclusively by insulin-expressing ß tumor cells, confirming that VEGF-C is produced by ß tumor cells and not by other cell types. On the other hand, not all of the insulin-expressing cells expressed VEGF-C (Fig. 1, G–I)Citation .



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  		Fig. 1. VEGF-C and VEGF-D expression in Rip1Tag2;NCAM-deficient tumors. Immunohistochemical analysis of pancreata from Rip1Tag2;NCAM+/+ and Rip1Tag2;NCAM-deficient mice, as indicated. Immunohistochemical analysis with antibodies against VEGF-C (A–C) reveals that tumors of wild-type Rip1Tag2 mice only rarely express VEGF-C (A; see also Table 1Citation ), whereas in small adenoma of NCAM-deficient Rip1Tag2 mice, VEGF-C (gray) is often expressed by a majority of the tumor cells within the tumor (B). In larger tumors, VEGF-C is expressed by a subset of tumor cells scattered within the tumor (C, arrows). Immunohistochemical analysis with antibodies against VEGF-D (D and E) reveals that VEGF-D (gray) is often expressed by a subset of tumor cells (D, arrows) or only a few cells scattered within a tumor (E, arrows). Immunohistochemical analysis with antibodies against VEGF-D (E) and VEGF-C (F) on serial sections reveals that most of the ß cells of a Rip1Tag2;NCAM–/– tumor express VEGF-C (F), whereas only a few cells of the same tumor express VEGF-D (E). G–I, visualization of VEGF-C (G, red) and insulin (H, green) expression by immunofluorescence microscopy in a Rip1Tag2;NCAM–/– tumor. Overlay of the VEGF-C and insulin staining visualizes cells that coexpress VEGF-C and insulin (I, yellow) and cells that do not express VEGF-C (green), both displayed at higher magnification in the inset. VEGF-C expressing cells (red) that do not express insulin are not observed. Light red staining marked by arrowheads originates from nonspecific staining of erythrocytes in blood capillaries. Scale bar, 50 µm. (T, tumor; E, exocrine)

 
Immunohistochemical analysis of VEGF-D expression in NCAM-deficient Rip1Tag2 tumors revealed an even more variable expression pattern: whereas in some tumors a high proportion of tumor cells were expressing VEGF-D (Fig. 1D)Citation , only single, scattered VEGF-D expressing cells were found in other tumors (Fig. 1E)Citation . A comparison of VEGF-D and VEGF-C distribution on serial sections revealed that VEGF-D was frequently found to be expressed by a small subset of VEGF-C–expressing tumor cells, as exemplified in Fig. 1Citation , panels E and F. Tumors of NCAM-expressing Rip1Tag2 mice were all negative for VEGF-D expression (data not shown).

Taken together, these results show that the loss of NCAM expression leads to the up-regulation of VEGF-C and -D expression in tumors of Rip1Tag2 mice. The lack of a clear correlation between VEGF-C and -D expression and tumor stage suggests that the expression of VEGF-C and -D may involve stochastic events that are not directly affected by tumor progression.

Reduced NCAM Expression Correlates with Tumor Lymphangiogenesis.
Because Rip1Tag2;NCAM-deficient tumors exhibited increased expression of VEGF-C and -D, we verified whether this resulted in the induction of lymphangiogenesis. Immunohistochemical staining with antibodies against LYVE-1 and VEGFR-3 (Flt-4) revealed a significant increase of lymphatic vessel density in Rip1Tag2;NCAM-deficient mice (Fig. 2Citation ; Table 1Citation ). In normal control mice, lymphatic vessels were present only in the exocrine pancreas and were not in significant contact with islets of Langerhans (43) . In Rip1Tag2 mice, the majority of lymphatic vessels were also not in significant contact with ß cell tumors (Fig. 2A)Citation . In contrast, almost half of the NCAM-deficient tumors were either completely or partially surrounded by lymphatic vessels (Fig. 2B, C and FCitation ; Fig. 3Citation ; Table 1Citation ). In accordance with previously published results, the highest percentage of tumors surrounded by lymphatic vessels was observed in Rip1Tag2;Rip1VEGF-C mice (Table 1)Citation , which we have used as a reference in these experiments (ref. 43 ; data not shown).



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  		Fig. 2. Tumor lymphangiogenesis in NCAM-deficient tumors of Rip1Tag2 mice. Immunohistochemical staining of histologic sections of pancreata from Rip1Tag2 and Rip1Tag2;NCAM-deficient mice with antibodies against LYVE-1 and VEGFR-3 (Flt-4), as indicated. Arrowheads indicate lymphatic vessels visualized by LYVE-1 or VEGFR-3 antibodies. In Rip1Tag2 mice LYVE-1 antibodies decorate lymphatic vessels in the exocrine pancreas (A), whereas tumors of Rip1Tag2;NCAM-deficient mice are completely or partially surrounded by lymphatic vessels (B–F). Intratumoral lymphatic vessels of Rip1Tag2;NCAM-deficient tumors seem thin and collapsed and are usually localized at the tumor periphery (D). Clusters of ß tumor cells are frequently detected in lymphatic vessels surrounding the primary tumor (E) or in its proximity (F, arrows). Scale bar, 50 µm. (T, tumor; E, exocrine pancreas)

 


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  		Fig. 3. Specificity of lymphatic and blood endothelial cell markers. Immunofluorescence staining of histologic sections from pancreata of Rip1Tag2;NCAM-deficient mice with antibodies against podoplanin (PODO), LYVE-1, Prox-1, and CD31, as indicated. Nuclei are stained with DAPI. A, double-immunofluorescence staining reveals significant colocalization of the two different lymphatic endothelial markers podoplanin (red) and LYVE-1 (green) in lymphatic endothelium surrounding the NCAM–/– tumors. The arrow indicates a cluster of tumor cells within a lymphatic vessel surrounding the tumor. B, double-immunofluorescence staining with antibodies against podoplanin (red) and Prox-1 (green) reveals significant colocalization of nuclear Prox-1 (arrows) with lymphatic endothelium in the vicinity of a NCAM-deficient Rip1Tag2 tumor. Dashed lines indicate the tumor edge. Double-immunofluorescence staining with antibodies against LYVE-1 (green)/CD31 (red; C) and podoplanin (red)/CD31 (green; D) reveals that both LYVE-1 and podoplanin (PODO) antibodies specifically stain lymphatic vessels. Blood vessels strongly express CD31 and are negative for LYVE-1 and podoplanin. Weak CD31 expression is also observed on lymphatic vessels. Scale bar, 100 µm. (T, tumor; E, exocrine pancreas)

 
A small subset of Rip1Tag2;NCAM-deficient and Rip1Tag2;Rip1VEGF-C tumors (9% and 12%, respectively) contained intratumoral structures that reacted positively with antibodies against LYVE-1 (Fig. 2DCitation ; data not shown) and podoplanin (data not shown). The majority of intratumoral lymphatic vessels was "collapsed," although noncollapsed vessels with open lumen were sometimes observed within tumors (data not shown). Notably, the occurrence of intratumoral lymphatics did not significantly correlate with the incidence of lymph node metastasis.

In addition to LYVE-1, we used antibodies against another specific marker for lymphatic endothelial cells, podoplanin, which confirmed the increased lymphatic vessel density in NCAM-deficient tumors (Fig. 3, A, B, and D)Citation . Immunofluorescence costaining for podoplanin and LYVE-1 revealed a significant colocalization in most of the vessels stained (Fig. 3A)Citation . However, a few minor but significant differences in specificity could be observed. For example, colocalization of the two markers was best on vessels surrounding NCAM-deficient tumors, whereas some vessels in the exocrine pancreas were only weakly stained by the podoplanin antibodies (Fig. 3A)Citation . Prox-1 was also specifically expressed in lymphatic vessels of Rip1Tag2;NCAM-deficient tumors, where it colocalized with podoplanin (Fig. 2B)Citation . Antibodies against VEGFR-3 (Flt-4) decorated some intratumoral blood capillaries in addition to lymphatic vessels (data not shown), and, therefore, they were not the antibody of choice to study lymphangiogenesis in NCAM-deficient Rip1Tag2 mice and possibly in other systems.

To confirm the specificity of LYVE-1 and podoplanin as markers of lymphatic endothelium, we performed a costaining for LYVE-1 and for the blood vessel endothelial marker CD31 in tumors from Rip1Tag2;NCAM-deficient mice. As expected, antibodies against CD31 strongly decorated blood vessels and capillaries, whereas LYVE-1–positive lymphatic vessels showed only very weak staining for CD31 (Fig. 3C)Citation . Podoplanin/CD31 double fluorescence analysis in Rip1Tag2;NCAM-deficient tumors also revealed only a weak signal for CD31 on podoplanin-positive lymphatic vessels (Fig. 3D)Citation .

As it has been reported for Rip1Tag2;Rip1VEGF-C mice (43) , tumor cell clusters were frequently found in the lumen of LYVE-1/podoplanin-positive lymphatic vessels in Rip1Tag2;NCAM-deficient mice (arrows in Fig. 2, E and FCitation , and Fig. 3ACitation ), suggesting that lymphatic vessels provide a metastatic route for ß tumor cells in Rip1Tag2;NCAM-deficient mice.

Lymphangiogenesis Is a Rate-Limiting Step in ß Cell Lymph Node Metastasis.
In the Rip1Tag2 tumor model, both VEGF-C overexpression and NCAM deficiency resulted in the formation of lymph node metastasis (22 , 43) . To determine whether up-regulated lymphangiogenesis is a rate-limiting event in the formation of lymph node metastasis, we crossed Rip1Tag2;NCAM-deficient mice with Rip1Tag;Rip1VEGF-C mice and examined the incidence of lymph node metastasis by histopathological analysis of pancreata from these mice. In these separate breeding experiments, 20% of the Rip1Tag2;NCAM-deficient mice (n = 20), 37.5% of Rip1Tag2;Rip1VEGF-C mice (n = 8), and 80% of the composite Rip1Tag2;Rip1VEGF-C;NCAM-deficient mice (n = 10) exhibited metastasis to the regional lymph nodes of the pancreas. These results indicate that the induction of lymphangiogenesis in Rip1Tag2;NCAM-deficient mice is indeed a rate-limiting step for the metastatic dissemination of ß tumor cells to the regional lymph nodes. However, because the loss of NCAM function potentiated VEGF-C–dependent lymph node metastasis in Rip1Tag2;Rip1VEGF-C;NCAM-deficient composite mice, an additional mechanism, that involves the loss of NCAM function but is distinct from VEGF-C/D–mediated lymphangiogenesis, may be involved as well. This conclusion is also supported by the fact that not all of the NCAM-deficient mice that exhibited increased lymphatic vessel density surrounding their tumors developed lymph node metastasis (Table 2)Citation . Conversely, there were also subsets of mice displaying lymph node metastasis in the absence of lymphangiogenesis (Table 2)Citation .


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  		Table 2 Correlation between lymphangiogenesis and lymph node metastasis

 
The results presented above suggest that NCAM-deficiency leads to lymph node metastasis, at least in part, through VEGF-C– and VEGF-D–mediated lymphangiogenesis. To test this hypothesis, we used a soluble form of VEGFR-3 (sFlt-4) to interfere with the function of VEGF-C and -D, and potentially with tumor lymphangiogenesis and metastasis, in Rip1Tag2;NCAM-deficient mice. To systemically express high levels of sFlt-4, we generated a recombinant, replication-defective adenovirus encoding for the ligand-binding, extracellular domain of the receptor (the first three immunoglobulin domains of mouse Flt-4) with its COOH-terminus fused to a mouse immunoglobulin heavy chain (Fc) under the control of the cytomegalovirus enhancer/promoter. Intact expression of the recombinant protein was confirmed in vitro by analyzing conditioned medium of L cells transfected with an expression construct encoding sFlt-4 (Fig. 4A)Citation . Recombinant adenoviruses either expressing sFlt-4 (AdsFlt-4) or EGFP as control (AdEGFP) were then injected into the tail vein of Rip1Tag2;NCAM-deficient mice starting at 9 weeks of age, with a bolus of 5 x 109 viral particles administered at day 1, 10, and 20. From previous experiments, it was known that i.v. application of recombinant adenovirus exclusively infects hepatocytes in the liver, which in turn produce and secrete soluble proteins into the blood stream (39) . The presence of sFlt-4 in the sera of mice injected i.v. with purified recombinant adenoviral particles was confirmed by immunoblotting indicating that sFlt-4 was systemically present in these mice at high levels (ranging between ~50 µg/mL and 300 µg/mL; Fig. 4, B and CCitation ).



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  		Fig. 4. Expression of recombinant soluble VEGFR-3 (sFlt-4). A, detection of sFlt-4 in the conditioned medium of L cells transfected with an expression construct encoding a soluble version of VEGFR-3 fused to a mouse immunoglobulin Fc domain (sFlt-4; Lanes 1 and 3) or with empty vector (mock; Lanes 2 and 4). Soluble Flt-4 is visualized by immunoblotting with antibodies against mouse immunoglobulin Fc (Lanes 1 and 2) and against Flt-4 (Lanes 3 and 4). B, detection of soluble VEGFR-3 (sFlt-4) in the sera of one AdEGFP-injected mouse (Lane 1) and four AdsFlt-4–injected mice (Lanes 2 to 5) by immunoblotting with antibodies against mouse Flt-4. A reproduction of the Ponceau S staining is shown at the bottom of the panel to ensure equal loading of protein. C. Recombinant soluble VEGFR-3 purchased from R&D Systems (rsVEGFR-3; Lanes 1 to 3, 6.7 ng, 2.2 ng, and 0.7 ng, respectively) was used as control to quantitate serum sFlt-4 levels (AdsFlt-4; Lanes 4 to 6, 10 µL of 1 of 100, 1 of 300 and 1 of 900 serum dilution from a single mouse). Note that purchased soluble VEGFR-3 contains a longer extracellular portion of VEGFR-3 than the adenoviral soluble VEGFR-3 construct (745 versus 332 amino acids, respectively; see Material and Methods).

 
Immunohistochemical staining for podoplanin in adenovirus- injected Rip1Tag2;NCAM-deficient mice revealed a significant reduction in the number of tumors associated with lymphatic vessels on systemic expression of sFlt-4 as compared with EGFP (P < 0.001, z-test; Table 3Citation ).


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  		Table 3 Soluble Flt-4 represses tumor lymphangiogenesis and lymph node metastasis in Rip1Tag2;NCAM-deficient mice

 
Analysis of the incidence of lymph node metastasis revealed a reduction in the number of mice with lymph node metastasis on treatment with AdsFlt-4 as compared with AdEGFP (Table 3)Citation . Notably, the one and only lymph node metastasis that has been detected in a AdsFlt-4-treated Rip1Tag2;NCAM-deficient mouse exhibited a highly malignant, anaplastic phenotype, and most likely has originated from a primary tumor of similar malignancy (supplementary Fig. S2). Because these experiments had to be performed at the very late stages of Rip1Tag2 tumor development, and for ethical reasons, the number of experimental mice had to be kept at a minimum. As a result, the number of mice in this experiment is rather low and, although a clear trend is apparent, the reduction of lymph node metastasis on AdsFlt-4 treatment is not statistically significant (P = 0.115, two-tailed Fisher exact test; Table 3Citation ). Besides the changes in the incidence of lymph node metastasis, no significant differences were observed for tumor incidences and volumes, tumor cell proliferation (BrdUrd-incorporation), apoptosis (TUNEL assay), and tumor angiogenesis (CD31 staining; Table 4Citation ).


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  		Table 4 Systemic expression of sFlt-4 does not affect general tumor growth in Rip1Tag2;NCAM-deficient mice

 
Together, the results indicate that interfering with the function of VEGF-C and -D during tumor development in Rip1Tag2;NCAM-deficient mice reduces lymphangiogenesis and, to a lesser extent, lymph node metastasis but does not affect tumor cell proliferation, tumor cell apoptosis, or blood vessel angiogenesis. Thus, our data raise the possibility that, at least in the Rip1Tag2 transgenic mouse model, the loss of NCAM induces lymph node metastasis by up-regulated expression of VEGF-C and VEGF-D, which in turn induces lymphangiogenesis.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
On the basis of changes in gene expression, alternative splicing, and differential polysialylation, the cell-cell adhesion molecule NCAM has been functionally implicated in tumor progression in a variety of cancer types (7) . However, the cellular and molecular mechanisms by which changes in NCAM function may affect tumor progression have remained elusive. A first glimpse into such mechanisms has come from experiments with Rip1Tag2 transgenic mice, where the loss of NCAM contributes to the metastatic dissemination of ß tumor cells to regional lymph nodes (22) . Here, we set out to identify and characterize the molecular mechanisms connecting the loss of NCAM function with lymph node metastasis. We show that, on loss of NCAM function, ß cell tumors of Rip1Tag2 transgenic mice express increased levels of the lymphangiogenic factors VEGF-C and VEGF-D. As a result, NCAM-deficient tumors exhibit up-regulated lymphangiogenesis, which in turn provides tumor cells with a route for metastatic dissemination to local lymph nodes.

Immunohistochemical analysis with antibodies against VEGF-C and -D, and against a number of well-established markers for lymphatic vessels, revealed that in a significant proportion of NCAM-deficient Rip1Tag2 tumors, both the expression of VEGF-C and -D and lymphangiogenesis are significantly up-regulated (Table 1)Citation . However, VEGF-D expression by ß tumor cells of NCAM-deficient Rip1Tag2 mice was less frequent as compared with the expression of VEGF-C (Fig. 1, D–F)Citation , implicating that VEGF-C is the major lymphangiogenic player in Rip1Tag2;NCAM-deficient mice.

Interestingly, tumors for which the diameter is partially (<50%) surrounded by lymphatic vessels were equally represented in NCAM-deficient as well as NCAM-expressing Rip1Tag2 tumors (Table 1)Citation . Such a significant interaction between ß cell tumors and lymphatic vessels can be explained by the fact that the mouse pancreas contains a rich network of lymphatic vessels, which is frequently found in the vicinity of islets of Langerhans (46) . Hence, it is conceivable that outgrowing ß cell tumors may have a high chance of becoming partially surrounded by pre-existing lymphatic vessels of the pancreas. In contrast, during active tumor lymphangiogenesis, for example induced by up-regulated VEGF-C and -D expression in NCAM-deficient tumors or by transgenic expression of VEGF-C in Rip1Tag2;Rip1VEGF-C tumors, most of the tumor perimeter (>50%) is surrounded by lymphatic vessels (ref. 43 ; Table 1Citation ). Thus, Rip1Tag2;NCAM-deficient mice represent a model for stochastic, endogenous tumor lymphangiogenesis, a useful tool for future investigations.

Combining the lymphangiogenic effect of forced expression of VEGF-C in Rip1VEGF-C transgenic mice with the lymphangiogenesis observed in NCAM-deficient mice in composite Rip1VEGF-C;NCAM-deficient;Rip1Tag2 mice resulted in an increase of lymph node metastasis that was more than additive. This result indicates that the expression of VEGF-C and -D is a rate limiting event in the formation of lymph node metastases, but that additional mechanisms, triggered by the ablation of NCAM function, are also involved in this process in Rip1Tag2 mice. Variations in the genetic background of the different composite mouse lines may also contribute: the incidence of lymph node metastasis observed in NCAM-deficient mice varies with different genetic backgrounds (50% in ref. 22 , 47% and 37%, respectively, when Rip1Tag2 were excusivley crossed to NCAM knockout mice, and 20% when Rip1Tag2 mice were crossed to NCAM-deficient and Rip1VEGF-C transgenic mice). A correlation analysis between lymphangiogenesis and lymph node metastasis within single mice revealed that in a subset of NCAM-deficient mice, lymph node metastasis developed in the absence of active tumor lymphangiogenesis (Table 2)Citation . On the other hand, the finding that some mice exhibited ongoing lymphangiogenesis in their tumors and did not develop lymph node metastasis may be explained by the fact that some of the metastases were missed by the histologic analysis or that the mice were sacrificed before the formation of metastasis. Likewise, not all of the Rip1VEGF-C transgenic mice that exhibited active lymphangiogenesis developed lymph node metastasis (ref. 43 ; Table 2Citation ).

Systemic expression of a soluble form of VEGFR-3 (sFlt-4) by adenoviral gene delivery reduced the percentage of tumors surrounded by lymphatic vessels. Such a repression of tumor lymphangiogenesis by sFlt-4 is in accordance with previous reports on experimental tumors in chicken, mouse, and rat (31 , 47 , 48) . Here we show for the first time that sFlt-4 is also able to reduce tumor lymphangiogenesis caused by the up-regulation of VEGF-C and -D in endogenously growing tumors not involving tumor transplantation. Soluble Flt-4 in the circulation of Rip1Tag2;NCAM-deficient mice also reduced the incidence of lymph node metastasis from 36% (4 of 11) in the control group of mice to 8% (1 of 12; Table 3Citation ), raising the possibility that loss-of-NCAM–mediated induction of lymphangiogenesis contributes to the formation of lymph node metastasis.

In contrast to the significant reduction of lymphatic vessel density, blood vessels were not affected by the adenoviral expression of sFlt4 in Rip1Tag2;NCAM-deficient mice (Table 4)Citation . This result indicates that the treatment specifically repressed lymphangiogenesis and not tumor angiogenesis, a selectivity that has been previously reported in various experimental models (48 , 49) .

There has been much controversy about the functionality of intratumoral lymphatic vessels and their importance for metastatic tumor dissemination (reviewed in ref. 32 ). Although we clearly observed intratumoral lymphatic vessels in Rip1Tag2;NCAM-deficient mice, their occurrence did not correlate with the incidence of lymph node metastasis. Moreover, the lumina of these intratumoral lymphatic vessels were usually collapsed. Finally, sFlt-4 reduced only lymphatic vessels surrounding tumors and not intratumoral lymphatic vessels (data not shown). Together, these observations support the notion that intratumoral lymphatics detected in Rip1Tag2 tumors are not functional.

The correlation between the loss of NCAM function and the gain of VEGF-C and -D expression in Rip1Tag2 tumors raises a number of questions about the cellular and molecular mechanisms involved in this process. First, is the loss of NCAM directly affecting VEGF-C and -D gene expression? To address this issue, we have investigated VEGF-C expression in ß tumor cell lines derived from tumors of wild-type and NCAM-deficient Rip1Tag2 mice (23) . The results indicate that in cultured cells VEGF-C expression is not directly affected by changes in NCAM expression (data not shown). Hence, we speculate that up-regulated VEGF-C expression is caused by mechanisms that depend on the tissue or tumor context in Rip1Tag2 mice in vivo.

One obvious phenotypic difference between wild-type and NCAM-deficient Rip1Tag2 tumors is the dramatic tissue disaggregation of NCAM-deficient tumors. In previous work, we have shown that the loss of NCAM results in a failure to activate ß1 integrin via fibroblast growth factor receptor signaling (23) , a likely cause for tumor tissue disaggregation. It is conceivable that such changes in cell-matrix adhesion may be the cause for the up-regulation of VEGF-C and -D expression, for example, by changing the interstitial fluid pressure within the tumor or by causing major fluid leakages and increased tissue fluid volume. In fact, many of the ß cell tumors of Rip1Tag2;NCAM-deficient mice exhibit hemorrhages filled with lymphatic fluid (23) , an observation that initially motivated us to investigate lymphangiogenesis in these tumors.

Alternatively, tissue disaggregation may lead to an inflammatory response which in turn could affect lymphangiogenesis. Such mechanism is attractive, because human and mouse VEGF-C promoters contain conserved binding sites for nuclear factor {kappa}B (50) , a transcription factor which plays a central role in inflammation and which has often been correlated with cancer (51) . Notably, the proinflammatory cytokines interleukin-1 and tumor necrosis factor-{alpha} are able to activate both nuclear factor {kappa}B transcriptional activity and VEGF-C gene expression (52) . Moreover, heregulin-ß1 increases VEGF-C expression through nuclear factor {kappa}B/p38 mitogen-activated protein kinase signaling pathways in human breast cancer cells (53) . Hence, it will be important to investigate whether NCAM-deficient tumors exhibit certain inflammatory responses, which type of inflammatory cells have infiltrated in these tumors, and which cytokines could possibly be responsible for the up-regulation of VEGF-C and -D in NCAM-deficient ß tumor cells. Finally, future studies will also have to address whether changes in NCAM expression also correlate with up-regulated lymphangiogenesis and lymph node metastasis in cancer patients.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. Matt Cotten, Michael Detmar, David Jackson, Dontscho Kerjaschki, and Herbert Weich for reagents, advice, and scientific input and Terhi Kärpänen for help with soluble receptor determinations. We thank Joachim Niedermeyer for the generation and analysis of Rip1VEGF-C;NCAM-deficient;Rip1Tag2 composite mice, Michaela Herzig for help with cloning, Edelgard Kux, Ursula Schmieder, and Helena Antoniadis for excellent technical assistance, and Miguel Cabrita and Birgit Schaffhauser for critical comments on the manuscript. The monoclonal antibody against mouse podoplanin was obtained from the Developmental Studies Hybridoma Bank under the auspices of the National Institute of Child Health and Human Development.


   FOOTNOTES
 
Grant support: Boehringer Ingelheim Inc., Austrian Industrial Research Promotion Fund, Swiss National Science Foundation (I. Crnic, U. Cavallaro, and G. Christofori), Human Frontier Science Program (R6P0231/2001-M), European Union (QLC1-CT-2001-01172, and QLK3-CT-2002-02059), and NIH Grant HD37243 (L. Jussila and K. Alitalo).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: U. Cavallaro is currently in IFOM-Federazione Italiana Ricerca Cancro Institute of Molecular Oncology, Milano, Italy. Supplementary data for this article can be found at Cancer Research Online at http://cancerres.aacrjournals.org.

Requests for reprints: Gerhard Christofori, Institute of Biochemistry and Genetics, Department of Clinical-Biological Sciences, University of Basel, Mattenstrasse 28, CH–4058 Basel, Switzerland. E-mail: gerhard.christofori{at}unibas.ch

Received 7/15/04. Revised 9/ 2/04. Accepted 9/23/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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