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[Cancer Research 64, 146-151, January 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Inhibition of Tumorigenicity and Metastasis of Human Melanoma Cells by Anti-Cathepsin L Single Chain Variable Fragment

Nathalie Rousselet1, Lisa Mills2, Didier Jean1, Carmen Tellez2, Menashe Bar-Eli2 and Raymond Frade1

1Immunochimie des Régulations Cellulaires et des Interactions Virales, INSERM U.354, Genopole, Evry, France, and 2Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We demonstrated previously that the switch from nonmetastatic to highly metastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L form. This cysteine proteinase was identified on the basis of its property to cleave human C3, the third component of complement. In an attempt to control procathepsin L secretion, we have recently generated an anti-cathepsin L single chain variable fragment (ScFv) from an anti-cathepsin L monoclonal antibody generated against recombinant cathepsin L. We herein selected clones stably transfected with this anti-cathepsin L ScFv and analyzed them for changes in tumor growth and metastasis. We show that in stably transfected clones, anti-cathepsin L ScFv strongly inhibited the secretion of procathepsin L without modifying the intracellular amount or processing pattern of cathepsin L forms. Confocal analysis demonstrated colocalization of endogenous cathepsin L and anti-cathepsin L ScFv. In addition, expression of this ScFv strongly inhibited generation of tumor and metastasis by these human melanoma clones in nude mice. In vivo, the anti-cathepsin L ScFv-transfected cells produced tumors with decreased vascularization (angiogenesis) concomitant with increased apoptosis of tumor cells. Matrigel assay also demonstrated that melanoma invasiveness was completely abolished. Thus, this is the first demonstration that anti-cathepsin L ScFv could be used to inhibit the tumorigenic and metastatic phenotype of human melanoma, depending on procathepsin L secretion, and could therefore be used as a molecular tool in a therapeutic cellular approach.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The switch of human melanoma phenotype from nonmetastatic to highly metastatic is related to different molecular mechanisms that have not yet been elucidated (1 , 2) . Among these events, the increase of human melanoma resistance to the immune surveillance system should be taken into account. Therefore, the molecules involved in such resistance need to be identified. We have previously undertaken to identify proteinases, which cleaved human C3, the third component of complement, and might be involved in this process (3) . Indeed, because C3 is the pivotal component of this humoral immune system, its degradation leads to inhibition of complement pathways and to generation of C3 fragments, which are involved in a large spectrum of biological functions and regulations (4) . This led us to identify in highly metastatic human melanoma cells, a p41 procathepsin L, which cleaves human C3 and contributes to their resistance to complement-mediated cell lysis (3 , 5) . Procathepsin L is the proenzyme of cathepsin L, a papain-type cysteine proteinase (6, 7, 8) , whose gene is localized on human chromosome 9q21–22 (9) . Cathepsin L is successively translated as preprocathepsin L, transferred through the Golgi apparatus as procathepsin L, and then stored in lysosomes as mature cathepsin L (10) . Cathepsin L acts as endopeptidase, which degrades a wide range of intracellular cytoplasmic and nuclear proteins (11 , 12) and extracellular proteins, thus modifying the functions of these proteins. From our data (3 , 5 , 13 , 14) and data from other studies (10 , 15 , 16) , it became clear that high tumorigenic and metastatic properties of human melanoma cells are associated with overexpression and increase of procathepsin L secretion. On regulation of cathepsin L expression, it was shown that antisense inhibition of cathepsin L mRNA also decreased tumor growth of murine myeloma (17) . Because only few and partial data existed on the regulation of human procathepsin L expression, we recently determined the sequence of the 5'-flanking region of the human cathepsin L gene up to 3263 bp upstream from the translation start site and localized the major transcription initiation site (18) . We demonstrated the presence of three mRNA splice variants, which differed in their 5'-untranslated ends. We also identified regulatory sites crucial for cathepsin L promoter activity between -1489 and -1646 bp: in this region, two GC boxes and a CCAAT motif were involved in specific DNA-protein interactions, with three transcription factors [Sp1 or Sp3 and NF-Y, respectively (18) ].

Furthermore, data strongly supported that the increase in procathepsin L secretion by human melanoma cells acts to increase their tumorigenicity and to switch their phenotype from nonmetastatic to highly metastatic. Indeed, pretreatment of tumorigenic and highly metastatic human melanoma cells with anti-cathepsin L antibody, which neutralizes cathepsin L proteinase activity, strongly inhibited the tumorigenicity and significantly decreased the metastatic potential of human melanoma cells in nude mice (13 , 14 , 19) . Cathepsin L secreted by an endothelial tumor cell line was shown to generate endostatin from collagen XVIII, suggesting that cathepsin L could play a role in angiogenesis (20) . These results pointed out the need to design new molecular tools to inhibit procathepsin L secretion in human melanoma cells. To that end, we recently generated an antihuman cathepsin L single chain variable fragment (ScFv), from an anti-cathepsin L monoclonal antibody (moAb) directed against recombinant cathepsin L, used as immunogen (21) . The nucleotide and amino acid sequences of this anti-cathepsin L ScFv were determined. We demonstrated that transient transfection of anti-cathepsin L ScFv in human melanoma cells could inhibit procathepsin L secretion (21) .

We herein selected human melanoma clones stably transfected with this anti-cathepsin L ScFv. Our data demonstrate that expression of this ScFv in human melanoma cells totally inhibited the tumorigenic and metastatic properties of these clones. This inhibition is correlated to the inhibition of procathepsin L secretion, whereas the amount and ratio of intracellular cathepsin L forms were not modified. In addition, anti-cathepsin L ScFv-transfected cells produced less angiogenic and smaller tumors. The invasiveness of these clones, as tested in a Matrigel assay, was strongly inhibited.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Culture Conditions.
The highly tumorigenic and metastatic human melanoma A375SM cell line was established from nude mice lung metastases produced by the A375-P human cell line isolated from a lymph node metastasis (22 , 23) . Melanoma cells were grown as described previously (21) . Transfected cells (A375SM neo1, A375SM neo2, A375SM ScFv1, and A375SM ScFv2) were grown in the same medium with G418 (Invitrogen) at 1.2 mg/ml.

Stable Transfection of Melanoma Cells.
The 3D8 ScFv cDNA was cloned in pCMV/myc/ER (Invitrogen) to generate pCMV/myc/ER-3D8ScFv, as described previously (21) , to allow expression and retention in the endoplasmic reticulum of melanoma cells. A375SM melanoma cells were seeded in 6-well tissue culture plates, cultured for 24 h, and transfected by using 5 µl of Lipofectin reagent (Invitrogen) and 1.5 µg of pCMV/myc/ER-3D8ScFv expression vector or control pCMV/myc/ER vector. Transfections were carried out according to the manufacturer’s instructions. At 16 h after transfection, the medium was changed to complete culture medium. Cells were selected after 48 h with culture medium containing G418 at 1.2 mg/ml. G418-resistant colonies were isolated and established in culture.

Conditioned Media and Cell Extract Preparations.
Conditioned media and cell extracts were prepared as described previously by incubating A375SM cells for 24 h in serum-free culture medium (3 , 21) . The concentration of solubilized proteins was determined by the bicinchoninic acid protein assay reagent (Pierce).

Reverse Transcription-PCR.
Total RNA was isolated from melanoma cells using TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions. Total RNA (1 µg) was then reverse transcribed by using oligo(dT)15 primer and avian myeloblastosis virus reverse transcriptase in presence of RNasin RNase inhibitor (Promega). Using DyNAzyme II DNA polymerase (Finnzyme), cDNA was used for PCR amplification with specific primers: 5'-ACGCGTCGACATGGCCCAGGTGAAGCTGCAGCAGTC-3' and 3'-ATAAGAATGCGGCCGCCCGTTTGATTTCCAGCTTGGTGCCAGCACC-5' for 3D8 ScFv; 5'-GCGGATCCATGTATGAGGCCCCCAGATCT-3' and 3'-GCGGATCCGCACAGTGGGGTAGCTGGCTG-5' for cathepsin L; and 5'-GTCTTCACCACCATGGAGAAGGCT-3' and 5'-CATGCCAGTGAGCTTCCCGTTCA-3' for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR reaction was carried out in a GeneAmp PCR system 9700 (Applied Biosystems) with an initial denaturation step (3 min at 94°C); 22 cycles of denaturation (30 s at 94°C), annealing (30 s at 48°C), and extension (30 s at 72°C); and an extension time of 3 min at 72°C. PCR products were analyzed on a 3% agarose gel containing ethidium bromide.

Immunoblotting Analysis.
Immunoblot assays were performed as described previously (24) , using anti-cathepsin L moAb (clone 3D8) prepared in our laboratory (21) , anti-GAPDH moAb (moAb374; Chemicon International), or anti-myc tag moAb (clone 9E10; Upstate Biotechnology). Bound antibodies were detected with peroxidase-linked goat antimouse immunoglobulin antibody (Dako) and the enhanced chemiluminescence system (Amersham Biosciences).

Confocal Microscopy Analysis.
Cells exponentially growing on glass slides were fixed in cold 100% methanol for 5 min and then permeabilized in PBS containing 0.2% (v/v) Triton X-100 for 10 min at room temperature. After saturation in PBS containing 0.2% BSA for 45 min, cells were incubated with sheep anti-cathepsin L polyclonal antibody at 10 µg/ml (Biogenesis) and mouse anti-myc tag moAb at 5 µg/ml (clone 9E10; Upstate Biotechnology) for 1 h at room temperature. After washing, the cells were incubated with Alexa Fluor 488 donkey antisheep IgG (H + L) and Alexa Fluor 546 goat antimouse IgG (H + L) at 2 µg/ml (Molecular Probes) for 1 h at room temperature. Washed cells were embedded in Prolong Antifade mounting medium (Molecular Probes). Computer-assisted image analysis of fluorescence was performed using a confocal microscopy scanning laser microscope (Leica TCS).

Animals.
Male athymic BALB/c nude mice were purchased from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). The mice were housed in laminar flow cabinets under specific pathogen-free conditions and used at 8 weeks of age. Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and the NIH.

In Vivo Tumor Growth and Metastasis.
Preparation of tumor cells for inoculation, production of s.c. tumors, and monitoring of s.c. tumor growth were performed as described previously (13 , 14 , 25) , The mice were killed 2 months after injection, and tumors were processed for H&E staining.

Experimental Lung Metastasis.
Injections of tumor cells into the lateral tail vein of nude mice were also performed as previously described (13 , 14 , 25) . The mice were killed after 60 days and the lungs were removed and prepared as previously described (13 , 14 , 25) . The number of surface tumor nodules was counted under a dissecting microscope.

Invasion Assay through Matrigel.
Invasion of A375SM cells was measured as described previously (25) . The data were expressed as the average number (±SE) of cells from 10 fields that migrated to the lower surface of the filter from each of three experiments performed.

Terminal Deoxynucleotidyl Transferase-Mediated Nick End Labeling.
Tissue sections and the slides were prepared as described previously (25) . Slides were then stained with hematoxylin.

Immunohistochemistry.
For CD31 staining, sections of frozen tissues and slides were prepared from tumor xenografts as described previously (25) . Slides were analyzed using Optimas 6.5 imaging software. Microvessel density (MVD) was determined by examining 8 fields/slide and 3 slides/group. Representative pictures were taken after analyzing all of the data.

Statistical Analysis.
The in vitro data were analyzed for significance by using Student’s t test (two-tailed), and the in vivo data were analyzed by using the Mann-Whitney test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Human Melanoma Clones Stably Transfected with Anti-Cathepsin L ScFv.
Our previous observation that increased procathepsin L secretion by human melanoma cells switched their phenotype from nonmetastatic to highly metastatic (14 , 19) led us to generate and characterize an anti-cathepsin L ScFv. We recently demonstrated that, when transiently transfected in human melanoma cells, this ScFv inhibited procathepsin L secretion (21) . Thus, next we examined the effect of anti-cathepsin L ScFv expression on in vivo tumor growth and metastasis of human melanoma cells.

Highly metastatic A375SM melanoma cells were stably transfected with the anti-cathepsin L 3D8 ScFv expression vector. Expression of this ScFv in the transfected cells was analyzed by reverse transcription-PCR using specific primers (Fig. 1A)Citation . Among the isolated clones, we selected two positive clones, one with a low 3D8 ScFv expression, named A375SM ScFv1, and one with a higher 3D8 ScFv expression, named A375SM ScFv2 (Fig. 1ACitation , top panel, Lanes 4 and 5). As a control, we used A375SM parental cells (Fig. 1ACitation , top panel, Lane 1), and we also prepared and selected, using the same experimental conditions, two other A375SM clones stably transfected with the empty vector and named A375SM neo1 and A375SM neo2 (Fig. 1ACitation , top panel, Lanes 2 and 3). To verify the integrity of DNA samples, GAPDH cDNA was amplified from the same cDNA (Fig. 1ACitation , bottom panel, Lanes 1–5). Furthermore, expression of the 3D8 ScFv protein was measured in total cell extracts by Western blot using anti-myc tag moAb (Fig. 1B)Citation . The 3D8 ScFv protein, characterized as expected by an apparent molecular weight of 30,000, was present at a 3x higher level in cell extracts of A375SM ScFv2 (Fig. 1BCitation , top panel, Lane 5), than in cell extracts of A375SM ScFv1 (Fig. 1Citation B, top panel, Lane 4) and, as expected, totally absent in cell extracts of A375SM parental, neo1 and neo2 cells (Fig. 1BCitation , top panel, Lanes 1–3). In all these A375SM cells, GAPDH protein was constant, as measured by Western blot using anti-GAPDH moAb (Fig. 1BCitation , bottom panel, Lanes 1–5). In addition, using the anti-myc tag moAb described above, we found that 3D8 ScFv protein was not secreted in medium conditioned from A375SM ScFv1 and A375SM ScFv2 (data not shown).



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  		Fig. 1. Single chain variable fragment (ScFv) expression in stably transfected A375SM melanoma cells. cDNA (A) and total cell extracts (B) were prepared from A375SM parental (Lane 1), A375SM neo1 (Lane 2), A375SM neo2 (Lane 3), A375SM ScFv1 (Lane 4), and A375SM ScFv2 (Lane 5). In A, expression of ScFv cDNA was analyzed by reverse transcription-PCR. Glyceraldehyde-3-phosphate dehydrogenase cDNA was also amplified from the same cDNA. In B, total cell extracts were subjected to SDS-PAGE and analyzed by Western blot with anti-myc tag and anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibodies.

 
Colocalization of Anti-Cathepsin L ScFv with Endogenous Cathepsin L.
Immunofluorescence studies were performed on A375SM cells transfected with 3D8 ScFv to analyze subcellular localization of the ScFv protein and endogenous procathepsin L. For this purpose, A375SM ScFv-transfected cells were stained with the anti-myc tag moAb to detect 3D8 ScFv or with anti-cathepsin L moAb. Representative confocal analysis of the staining obtained with A375SM ScFv2 is shown in Fig. 2Citation , bottom panels. We observed colocalization of endogenous cathepsin L (Fig. 2ACitation , green color) and 3D8 ScFv (Fig. 2BCitation , red color); indeed, both chromophores mainly superimposed in the same image, resulting in the yellow color (Fig. 2C)Citation . In contrast, A375SM neo2 cells subjected to the same confocal analysis did not present any positive staining with anti-myc tag moAb (Fig. 2BCitation , top panel).



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  		Fig. 2. Confocal analysis of anti-cathepsin L single chain variable fragment (ScFv) and endogenous cathepsin L subcellular localization. Immunofluorescence staining of A375SM neo2 (top panels) and A375SM ScFv2 (bottom panels) cells with anti-cathepsin L antibody (A) and anti-myc tag monoclonal antibody, to detect tagged anti-cathepsin L ScFv (B), is shown. In C, both stainings were superimposed to demonstrate the colocalization of anti-cathepsin L 3D8 ScFv and cathepsin L proteins. Size bars are indicated.

 
Anti-Cathepsin L ScFv Expression Inhibits Procathepsin L Secretion in Stably Transfected Clones.
We next analyzed expression and secretion of procathepsin L in parental and transfected A375SM melanoma cells (Fig. 3)Citation . Expression of cathepsin L mRNA was analyzed by reverse transcription-PCR using specific primers of cathepsin L. GAPDH cDNA was amplified from the same cDNA and served as a control for DNA integrity and equal loading. The presence of the different forms of cathepsin L protein was analyzed in total cell extracts and in media conditioned from each of the A375SM cells by Western blot using anti-cathepsin L moAb. Data demonstrated that cathepsin L mRNA was expressed at a similar level in parental and empty vector- or ScFv-transfected A375SM cells (A375SM neo1, A375SM neo2, A375SM ScFv1, or A375SM ScFv2; Fig. 3ACitation ) and that in total cell extracts, cathepsin L protein forms were present in the mature forms characterized by Mr 34,000 and Mr 29,000 and in the proenzyme form characterized by Mr 41,000. Comparison of A375SM neo1 and A375SM neo2 cells (Fig. 3BCitation , Lanes 2 and 3) with parental cells (Fig. 3BCitation , Lane 1) demonstrated a significant increase in the total amount and forms of cathepsin L after stable transfection; in extracellular media conditioned from each A375SM cell type mentioned above, a unique Mr 41,000 band corresponding to the procathepsin L form was detected according to our previous studies (Refs. 5 , 6 , 14 , and 19 ; Fig. 3CCitation ). In addition, whereas the amount of secreted procathepsin L was 2- to 3-fold higher in A375SM neo1 and A375SM neo2 cells (Fig. 3CCitation , Lanes 2 and 3), as compared with sample prepared from A375SM parental cells (Fig. 3CCitation , Lane 1), this amount was strongly diminished in A375SM ScFv1 and in A375SM ScFv2: indeed, procathepsin L secretion was inhibited by up to 87% and 93% in A375SM ScFv1 and A375SM ScFv2, respectively, as compared with A375SM neo1 and A375SM neo2. Furthermore, after the cleavage of human C3 by procathepsin L secreted in conditioned medium, using the same assay described previously (3 , 13) , results demonstrated that C3-cleaving activity of procathepsin L was indeed inhibited in conditioned medium prepared from ScFv-transfected cells (A375SM ScFv1 and A375SM ScFv2), at the same yield as procathepsin L protein secretion (data not shown). Taken together, these data strongly support that stable transfection of anti-cathepsin L ScFv and expression did not modify either cathepsin L gene transcription or the total amount and ratio of intracellular cathepsin L forms (i.e., their maturation process) as compared with cells transfected with the empty vector and specifically inhibited up to 93% of procathepsin L secretion, depending on the ratio of endogenous cathepsin L and transfected anti-cathepsin L ScFv.



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  		Fig. 3. Cathepsin L expression and secretion in transfected A375SM human melanoma cells. cDNA (A), cell protein extracts (B), and conditioned media (C) were prepared from A375SM parental (Lane 1), A375SM neo1 (Lane 2), A375SM neo2 (Lane 3), A375SM ScFv1 (Lane 4), and A375SM ScFv2 (Lane 5). A, expression of cathepsin L cDNA was analyzed by reverse transcription-PCR. Glyceraldehyde-3-phosphate dehydrogenase cDNA was also amplified from the same cDNA. Cell extracts (B) and conditioned media (C) were subjected to SDS-PAGE and analyzed by Western blot using anti-cathepsin L (clone 3D8) and anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibodies.

 
Tumorigenic, Metastatic, and Invasive Phenotype of Anti-Cathepsin L ScFv-Transfected Melanoma Cells.
To determine the tumorigenicity of the anti-cathepsin L ScFv-transfected A375SM clones, we injected 5 x 105 cells s.c. into BALB/c nude mice and monitored tumor growth once a week for 35 days. Parental and neo-transfected A375SM cells grew progressively in all mice that received injections and reached 13–14 mm in mean diameter within 35 days (Fig. 4)Citation . In contrast, the two ScFv-transfected A375SM clones ScFv1 and ScFv2 grew much more slowly and their growth was delayed; tumors appeared only 14 days after injections and were smaller for both clones: indeed, within 35 days, A375SM ScFv1 cells produced tumor of 3.5 mm in mean diameter, whereas A375SM ScFv2 produced very few tumors of 2 mm in mean diameter. In the next set of experiments, the metastatic potential of ScFv-transfected clones was determined in an experimental lung metastasis assay (13 , 14) . To that end, BALB/c nude mice received i.v. injection with 106 parental and neo- or 3D8 ScFv-transfected A375SM cells, and 60 days later, the number of lung metastases was counted. As shown in Table 1Citation , A375SM parental and A375SM neo1 or A375SM neo2 cells strongly metastasized to the lung, resulting in between 188 and >200 tumor colonies (median values), respectively. In contrast, the two anti-cathepsin L ScFv-transfected clones, A375SM ScFv1 and A375SM ScFv2, produced a much lower number of lung tumor colonies in all injected mice: indeed, A375SM ScFv1 and A375SM ScFv2 cells produced only 9 or 2 lung colonies, representing >99% total inhibition (this is representative of four experiments performed). In addition, Matrigel assays were performed for parental and transfected cells, and the following data were obtained (Table 2)Citation : 888 ± 70 A373SM parental cells, 940 ± 71 A375SM neo1 cells, and 955 ± 72 A375SM neo2 cells migrated through the Matrigel-coated filters, whereas, under the same experimental conditions, only 25 ± 5 A375SM ScFv1 cells and 11 ± 4 A375SM ScFv2 cells migrated through the Matrigel-coated filters. Thus, anti-cathepsin L transfection inhibited the ability of human melanoma cells to penetrate extracellular matrix, an important component in the process of tumor invasion and metastasis.



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  		Fig. 4. Growth of parental and transfected A375SM melanoma cells in nude mice. Melanoma cells (106)/0.2 ml of HBSS were injected s.c. in nude mice, over the right scapular region. Growth of s.c. tumors was monitored by examination of the mice every day and a weekly measurement of tumors with calipers. The data represent the mean diameter observed in 5 animals/group. This is representative of four experiments performed.

 

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  		Table 1 Experimental lung metastasis of A375SM human melanoma cells [parental or transfected with empty vector (neo1 or neo2) or anti-cathepsin L ScFv gene (ScFv1 or ScFv2)] in nude mice

Cells (106)/mouse were injected into nude mice. All mice were killed on day 60.

 

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  		Table 2 Experimental Matrigel invasion assay of A375SM human melanoma cells [parental or transfected with empty vector (neo1 or neo2) or anti-cathepsin L ScFv gene (ScFv1 or ScFv2)]

 
Inhibition of Tumor Angiogenesis in Anti-Cathepsin L ScFv-Transfected Cells.
Because cathepsin L is involved in invasion, metastasis, and angiogenesis, we determined whether transfection with anti-cathepsin L ScFv could result in suppression of tumor angiogenesis. Tumor-associated neovascularization as indicated by MVD was examined by immunohistochemistry using anti-CD31 antibody. As shown in Fig. 5Citation , we found a significant reduction in tumor MVD/field in tumors produced by A375SM ScFv1 and A375SM ScFv2 as compared with control parental and neo-transfected cells. The mean MVD was 4.8 ± 3.0 for A375SM ScFv1 and 4.0 ± 3.1 for A375SM ScFv2. In contrast, the mean MVD was 27.3 ± 8.4 and 29.8 ± 5.4 for parental and neo-transfected tumors, respectively. Moreover, the number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive tumor cells was inversely correlated with MVD in the studied tumors. The number of tumor cells undergoing apoptosis was higher in the tumors produced by ScFv-transfected cells. Quantitative analysis of the terminal deoxynucleotidyl transferase-mediated nick end labeling assay revealed that the percentage of apoptotic cells was 20.8 ± 9.7% and 27.8 ± 13.0% for A375SM ScFv1 and A375SM ScFv2 cells, respectively. In contrast, the percentage of apoptotic cells was approximately 1% for both parental and neo-transfected cells. These data indicated that anti-cathepsin L ScFv expression significantly decreased tumor-associated neovascularization and subsequently decreased apoptosis of tumor cells.



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  		Fig. 5. CD31 terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Tumor microvessel density and apoptosis (TUNEL) were assessed in parental, neo-transfected, and anti-cathepsin-L single chain variable fragment-transfected cells implanted in nude mice. Parental and neo-transfected cells produced vascularized tumors (V), with very few cells undergoing apoptosis. In contrast, tumors produced by the anti-cathepsin-L single chain variable fragment-transfected cells were significantly less vascularized. Moreover, the number of TUNEL-positive tumor cells was inversely correlated with microvessel density (A). Magnification, x40.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our aim was to design a new molecular tool to prevent procathepsin L secretion in human melanoma cells, an event associated with the switch of their phenotype to highly tumorigenic and metastatic in nude mice (14 , 19) . Thus, we generated an anti-cathepsin L ScFv (21) .

For the first time, we herein clearly demonstrated that when stably transfected in human melanoma cells, this anti-cathepsin L ScFv also strongly inhibited melanoma tumor growth and metastasis in nude mice. This biological effect was related to the strong inhibition of procathepsin L secretion, whereas the amount and the ratio of intracellular forms of cathepsin L were not modified. It should be noted that the residual amount of procathepsin L secreted in A375SM ScFv1 or A375SM ScFv2 cells was as low as the amount of procathepsin L secreted by DX3 cells, a nontumorigenic and nonmetastatic cell line described previously (14) . These results led to the following observations.

First, the possibility that overexpression of anti-cathepsin L ScFv may be cytotoxic in transient or stably transfected melanoma cells has been taken into account: (a) the human melanoma clones stably transfected with this ScFv and presented herein grew in culture as well as parental cells or cells transfected with vector alone, and no difference was observed in their viability; (b) in addition, human melanoma transiently transfected with anti-cathepsin L ScFv, as described previously (21) , did not show any diminution in either cell viability or cell growth; (c) furthermore, overexpression of this ScFv did not modify either the viability or growth of DX3 cells, a human melanoma cell line that does not have elevated secretion of procathepsin L (14) ; (d) measuring endonuclease activity with a classical apoptotic analytical method used previously (26) , we have not found any significant apoptotic cells in transient or stably transfected A375SM cells (which have elevated secretion of procathepsin L) or in DX3 cells (which do not have elevated secretion of procathepsin L; data not shown). Thus, taken together, these data ruled out the possibility that overexpression of anti-cathepsin L ScFv may have a cytotoxic effect on human melanoma cells.

Second, we examined the possible mechanism(s) by which anti-cathepsin L ScFv inhibited cathepsin L secretion. One possibility is that this ScFv, by interacting with endogenous procathepsin L on the epitope that we localized previously on the 159–197 amino acid sequence of this proteinase (21) , formed large complexes, which could not be processed as the proenzyme alone, due to steric hindrance. Another possibility is that this ScFv, by interacting with procathepsin L, may prevent, directly or indirectly, the interaction of this proenzyme form with an intracellular carrier responsible for this secretion process. Additional studies are needed to identify not only the mechanisms responsible for procathepsin L secretion but also the contribution of the domain 159–197 of cathepsin L to these processes.

Third, we examined the mechanisms by which secreted procathepsin L could contribute and facilitate tumor progression and metastasis development. Among these mechanisms, the following aspects should be taken into account: (a) as we already demonstrated (3 , 5) , p41 procathepsin L, once secreted, could cleave human C3 and consequently contribute to human melanoma resistance to complement-mediated lysis; and (b) whereas only the p41 procathepsin L form was detected in conditioned medium prepared from human melanoma (3 , 5 , 13 , 14) , we could not rule out that this proenzyme is processes in vivo to generate the p34 and p29 cathepsin L active forms. Thus, these generated enzyme forms could degrade proteins of the extracellular matrix, hence, facilitating melanoma invasion.

Fourth, the results presented herein were obtained by injecting human melanoma cells in nude mice, which are mainly deficient in T lymphocytes. However, nude mice carry B lymphocytes, macrophages, and dendritic cells (all of which are antigen-presenting cells), that express MHC class II and are involved in other biological regulatory functions. A functional relationship has been demonstrated between the p41 splice variant of MHC class II-associated invariant chain (namely p41 Ii; for review, see Ref. 27 ) and cathepsin L. It has been shown that a 65-amino acid segment of the p41 Ii, which is also expressed in human melanoma cells (28) , binds to the active site of cathepsin L (29) . In addition, p41 Ii could also serve as a chaperone to cathepsin L in antigen-presenting cells (30) . Furthermore, by interacting with cathepsin L, p41 Ii allows extracellular accumulation of this cysteine proteinase (29) , which controls the migration of antigen-presenting cells and the recruitment of effectors in the inflammatory response (29) . On the basis of the data demonstrating the interaction of p41 Ii and cathepsin L and, consequently, their functional mutual relationship, we could suggest that inhibition of procathepsin L by our anti-cathepsin L ScFv may modify the equilibrium between cathepsin L and p41 Ii and, consequently, their related regulatory functions. Additional studies are needed to explore this hypothesis.

Growth and metastasis of human melanoma cells depend on their ability to develop an adequate vasculature. Cathepsin L may contribute to invasion and angiogenesis by its ability to degrade extracellular matrix proteins, thus, on one hand, allowing tumor cells to migrate and, on the other hand, enabling vascular endothelial cells to breech from the existing blood vessels to form new blood vessels surrounding the tumors. Indeed, we have shown here that all of the control tumors were highly vascularized and produced large tumors, whereas ScFv-transfected cells produced less angiogenic and smaller tumors.

In conclusion, stable transfection of anti-cathepsin L ScFv inhibited the tumorigenic and metastatic phenotype of human melanoma by abolishing cathepsin L secretion. These data strongly support that this ScFv could be used as a tool in a therapeutic cellular approach to inhibit the tumorigenic and metastatic phenotype of human melanomas by targeting procathepsin L.


   ACKNOWLEDGMENTS
 
We thank Dr. Monique Barel for helpful discussions and critical review.


   FOOTNOTES
 
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.

Grant support: INSERM (to R. Frade) and NIH Grant CA76098 (to M. Bar-Eli).

Requests for reprints: Raymond Frade, INSERM U.354, Bâtiment G8, Génopole d’Evry, RN.7, 91030-EVRY Cedex, France. Phone: 33-1-60-78-55-46; Fax: 33-1-60-78-29-54; E-mail: frade354{at}genopole.evry.inserm.fr

Received 6/12/03. Revised 10/24/03. Accepted 10/27/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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