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Carcinogenesis, Vol. 20, No. 8, 1445-1452, August 1999
© 1999 Oxford University Press

Cancer Biology

Matrix metalloproteinase inhibition prevents colon cancer peritoneal carcinomatosis development and prolongs survival in rats

Thomas Aparicio, Stéphanie Kermorgant, Valérie Dessirier, Miguel J.M. Lewin and Thérèse Lehy1

Unité de Biologie et Pathologie de l'Epithélium Digestif INSERM Unité 10, IFR Cellules Epithéliales, Hôpital Bichat-Claude Bernard, 170 Boulevard NEY, Paris 75018, France


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Matrix metalloproteinases (MMP) are enzymes responsible for extracellular matrix degradation which play a role in cancer progression and metastatic spreading. We investigated the effects of the MMP inhibitor, batimastat, in vitro on the proliferation and invasiveness of the rat colon cancer cell line DHD/K12, and in vivo on the growth of an aggressive model of peritoneal carcinomatosis producing haemorrhagic ascites and metastases, obtained in the rat by i.p. injection of DHD/K12 cells. MMP production was studied in conditioned culture media, solid tumors and ascitic fluid. In vivo, after injection of tumor cells on day 0, rats received i.p. daily either batimastat (30 mg/kg) or equal volume of vehicle from day 2 until killing on day 43 (series I) or from day 13 until death (series II). The grade of peritoneal carcinomatosis, ascite volume, number and size of liver metastases were evaluated in both series, and survival in series II. MMPs-1, -2 and -9 were identified in culture media, tumors and ascites. In vitro, batimastat did not modify DHD/K12 cell proliferation and slightly reduced cell invasion. In vivo, in series I, batimastat treatment totally prevented peritoneal carcinomatosis and liver metastasis development. In series II, it significantly prolonged survival (P < 0.0002) and reduced peritoneal carcinomatosis (P < 0.001) and hepatic metastases number as compared with controls. However, batimastat-treated rats of the two series had peritoneal inflammation with marked ascites. Nevertheless, inhibition of MMP is a new therapeutic approach which may be promising in treatment of microtumors as in more advanced cancer stages.

Abbreviations: MMPs, matrix metalloproteinases


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Colorectal adenocarcinoma is the most frequent cancer in occidental countries. The mean 5 year relative survival in Europe remains poor, ~40% (1), in spite of progress in surgery and chemotherapy. Prognosis is mainly related to tumor extension in colonic wall and to occurrence of lymph node, liver or lung metastases. In the absence of curative surgical option for colonic cancers, chemotherapy is only palliative. This has prompted numerous investigations into different therapeutic strategies.

Tumoral invasion and metastatic processes need the loss of cell adhesion properties as well as degradation of extracellular matrices and basement membrane (2). The matrix metalloproteinase (MMP) family, now composed of 17 zinc-dependent enzymes, is one of the major classes of proteases that play a role in the evolution of cancer. Expression of some MMPs has been reported in colorectal cancers (3–11). MMPs are secreted as latent pro-enzymes by both stromal and cancer cells. They are activated by proteolytic removal of an N-terminal domain and function in the degradation of extracellular matrix proteins that constitute connective tissues. They can be classified into four groups on the basis of sequence homology and substrate specificity: collagenases, gelatinases [including gelatinase A (MMP-2) and gelatinase B (MMP-9)], stromelysins and membrane-type metalloproteinases. Normally, the degradative activity of MMPs is controlled by both the latency of the secreted enzymes as well as by the presence of naturally occurring tissue inhibitors of MMPs. Inhibition of MMPs provides one attractive target for a novel class of therapeutic agents to control tumor progression and metastatic spreading. Batimastat (BB-94) is a synthetic hydroxamate MMP inhibitor whose efficacy has been explored with various protocols in several cancer animal models, notably in colonic and pancreatic cancer xenograft or metastasis models in nude mice (12–15).

In this work, we investigated the MMP production by the DHD/K12 rat colonic cancer cells and the effect of batimastat in a model of peritoneal carcinomatosis obtained by i.p. injection of these cells into BD IX rats. This aggressive model mimicks locally advanced stages of human colorectal cancer with haemorrhagic ascites, hepatic and sometimes lung metastases (16–18). Two experimental approaches were followed in order to test batimastat ability to prevent early tumor implantation or to inhibit tumor growth in rats bearing developing peritoneal carcinomatosis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals, cell line and reagents
Forty male and female syngenic BD IX rats, 12–16 weeks old (weight 280–340 g for male and 170–220 g for female) were purchased from Iffa-Credo (L'Arbresle, France). They were kept in our animal house and provided with standard rodent chow and water ad libitum. They were used 4 weeks after their arrival.

DHD/K12 is a tumor cell line originating from a 1,2-dimetylhydrazine-induced colon adenocarcinoma produced in syngeneic BD IX rats (19). Cells (passages 18–26) were grown in Ham F10 medium (Gibco BRL, Eragny, France) supplemented with 10% fetal calf serum and gentamycine (40 µl/ml) at 37°C in a humidified atmosphere with 5% CO2.

Antibodies and batimastat
Mouse monoclonal antibodies against human MMP-1, -2 and -9 (Calbiochem, La Jolla, CA) and a rabbit polyclonal antibody against human MMP-9 (Triple Point Biologics, Forest Grove, OR) were used. For western immunoblotting, secondary antibodies were peroxidase-labelled anti-mouse IgG obtained in sheep (Amersham, Les Ulis, France) or in goat (Dako, Copenhagen, Denmark) and peroxidase-labelled anti-rabbit IgG obtained in donkey (Amersham). For immunohistochemistry, secondary antibodies were biotinylated anti-mouse or anti-rabbit IgG obtained in horse and goat, respectively (Vector Labs, Burlingame, CA).

Batimastat is a synthetic MMP inhibitor which has been shown to inhibit a broad spectrum of MMP in the low nanomolar range (3 nM for MMP-1, 4 nM for MMP-2 and -9, 6 nM for MMP-7, 20 nM for MMP-3). It was kindly provided by British Biotech (Oxford, UK). Batimastat, as a fine white powder, was brought, by sonication, into milky suspension in phosphate-buffered saline (PBS) containing 0.01% Tween-20 (Merck, Darmstadt, Germany).

In vitro cell invasion assays
These assays were performed on Transwell filter chambers (Costar, Cambridge, MA). The upper side of polycarbonate membranes (6.5 mm in diameter, 8 µm pores) was coated with Matrigel diluted at 5 mg/ml (Becton Dickison, Bedford, MA). Cells (50 000) were seeded in the upper compartment. Medium plus 10% fetal calf serum was introduced in the lower compartment of each Transwell unit while batimastat diluted in PBS–Tween was added to the culture medium in the upper chamber at 0, 1, 5, 10 or 100 µM concentrations. After 30 or 48 h of culture, the non-migratory cells on the upper surface were removed with a cotton swab and the membranes fixed in methanol for 5 min. The migratory cells attached to the lower surface of the membrane were stained with toluidine blue. Their density was estimated after cell counting with a calibrated ocular grid (400x) at regular intervals along two perpendicular diameters (n = 10 counts per well). Results are the means of four to five wells and are representative of at least two experiments.

In vitro cell proliferation assays
A proliferation experiment was performed to verify the absence of direct toxicity of batimastat on DHD/K12 cells. Cells (50 000) were seeded into each well and cultured for 24 h in medium. After that time, the medium was changed. Batimastat was solubilized in PBS–Tween in increasing concentrations. Fifty microlitres of solution were added to fresh medium to obtain final concentrations from 10–5 to 1 mg/ml (i.e. from 20 nM to 2 mM) and cells cultured for 24 or 48 h. In control wells, 50 µl of vehicle were added. Before harvesting the cells, still not confluent, 0.1 µCi of [3H]thymidine was added in each well for 1 h. Incorporated radioactivity in cell pellets was measured using a ß counter after adding scintillation liquid. For each batimastat concentration, the experiment was performed in triplicate wells and this was repeated twice.

Zymography
Gelatinase activity was analysed. Serum-free culture media were collected, centrifuged at 4000 g for 20 min then concentrated four times with a vacuum pump. Ascite fluids were centrifuged at 10 000 g for 3 min at 4°C. Frozen tissues were homogenized at 4°C in a lysis buffer. Laemmli buffer without reducing agent was added to concentrated media, ascite supernatants or tissue homogenates. Proteins were measured then separated by a sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis (SDS–PAGE) containing 1 mg/ml of gelatin. Equal amount of proteins for ascitic fluids and tissue homogenates (20 µg) or 20 µl of concentrated media was loaded onto each lane. After migration of gels, SDS was removed by two 30 min incubations in 2.5% Triton X-100. Gels were incubated overnight at 37°C in 50 mM Tris–HCl (pH 7.6) containing 5 mM CaCl2, 0.2 mM ZnCl2 and 0.2 mg/ml NaN3, then stained for 1 h in 30% methanol–10% glacial acetic acid solution containing 1.5% (w/v) Coomassie brilliant blue, and destained in the same solution in the absence of dye. Unstained areas corresponded to zones of MMP proteolytic activities.

Western immunoblottings
Western blots were performed on tissues and ascitic fluids. Frozen tissues were homogenized at 4°C in a lysis buffer then solubilized in boiling Laemmli buffer with the reducing agent ß-mercaptoethanol. Protein concentrations were measured by the Bio-Rad protein assay (Bio-Rad, Hercules, CA), then proteins were separated by 10% PAGE after loading an equal amount (10 µg) onto each lane and transferred to nitrocellulose sheets. Blots were probed with MMP protein antibodies diluted to 1–2 µg/ml and then with the corresponding secondary antibodies diluted 1:1000. Immune complexes were revealed by an enhanced chemiluminescence detection system (Amersham).

RNA extraction and RT–PCR
Total RNA was extracted from tumor nodules using Trizol-Reagent (Gibco BRL). First strand cDNA was synthesized from 1.6 µg total RNA using murine reverse transcriptase and the first strand cDNA synthesis kit from Pharmacia Biotech (Uppsala, Sweden). Oligonucleotide primers for human MMP-2 [according to Giambernardi and Grant (20)], compatible with rat MMP-2, were synthesized by Genosys (Cambridge, UK). Forty-five cycles were performed (denaturation at 95°C for 1 min, annealing at 60°C for 1 min, polymerization at 72° C for 2 min). The amplification was terminated by an extension step at 72° C for 10 min. Ten microlitres of the PCR samples were electrophoretically separated on 1% agarose gel stained previously with ethidium bromide and visualized under UV light. Amplified product (605 bp) was sequenced by Genome Express (Paris).

Histological procedures and immunohistochemistry
Fresh tissue specimens were quickly fixed either in Bouin's fluid at room temperature for 18–36 h or in 4% paraformaldehyde in PBS (0.1 M, pH 7.4) at 4°C for 24 h. After fixation, they were dehydrated, processed for paraplast-embedding and cut into sections 4 µm thick. Some specimens were directly frozen in a cryopreservative compound (OCT; Miles, Elkhart, IN) and stored at –80°C. Specimens were cut with a cryostat into sections 7 µm thick which were fixed in paraformaldehyde for 10 min before use.

For immunohistochemistry, endogenous peroxidase activity was removed by dipping tissue sections into 3% H2O2 for 30 min. Thereafter, sections were incubated overnight at 4°C with the chosen anti-MMP primary antibody diluted at 5 µg/ml (monoclonal) or at 10 µg/ml (polyclonal), and then with the corresponding secondary antibody diluted 1:200 for 30 min and finally in the avidin–biotin complex diluted 1:100 for 45 min (ABC Vectastain kit, Vector Laboratories). The peroxidase activity was revealed by diaminobenzidine and nuclei counterstained with Mayer's hemalum. Negative controls were obtained by omitting primary antibodies.

In vivo treatment protocol
Lack of contamination was checked by the Hoechst's test. Tumor cells growing exponentially were harvested by brief incubation in 0.25% trypsin–EDTA solution and suspended in sterile PBS (pH 7.3), at a concentration of 1x106 cells/ml. BDIX rats were i.p. injected using sterile needles with 1x106 viable tumor cells on day 0. In a pilot study, two rats were injected with tumor cells, killed on day 43 and peritoneal tumor nodules removed for verifying the presence of MMPs. Then, two experimental series were used. In each series, rats were divided into two groups: one group receiving i.p. batimastat once daily at the dose of 30 mg/kg/day (corresponding to an injected volume of 0.5 ml for female and 0.75 to 1 ml for male) and the other (control group) receiving an equal volume of vehicle alone. Animals were weighed weekly.

In series I (16 rats), carried out to assess the effect of batimastat on the formation and adhesion to peritoneal surfaces of solid tumor deposits, treatment began on day 2 following injection of tumor cells, rats being killed under ether anaesthesia on day 43. For each rat, at the time of killing, the volume of ascite was measured, the number and size of liver metastases were recorded after cutting hepatic lobules into thin slices and the extension of peritoneal carcinomatosis was assessed according to a five grade semi-quantitative classification (16–18): class 0, no macroscopically visible nodules; class 1, several nodules from 1 to 2 mm; class 2, more than 50 nodules of 1–5 mm; class 3, peritoneal cavity invaded by nodules up to 1 cm; class 4, peritoneal cavity fully invaded by nodules, some of them measuring several cm. For each rat, detection of MMP was done in peritoneal nodules, hepatic metastases and ascites when these pathological features were present. Biological components such as protides, hemoglobin and cells were investigated in some ascites. In addition, in batimastat-treated rats, all pancreases and the half livers were removed and processed for histological examination.

In series II (22 rats), carried out to study the effect of batimastat on growing tumors and on survival of rats, treatment was initiated on day 13 and continued until death. Two rats were killed on day 13 to verify the tumor implantation and two rats of each group chosen at random were killed on day 39 to verify the tumor evolution. All experiments were conducted according to French regulations for animal experimentation [Ministry of Agriculture, Decret (Act) No. 87 848, October 19, 1987].

Statistical analyses
Values were expressed as means ± SEM. Comparisons between batimastat- and vehicle-treated groups were made using the Student t-test or the non-parametric Mann–Whitney U test whenever relevant. Grades of peritoneal carcinomatosis in the two groups were compared with the {chi}2 test. Survival curves were compared using the Wilcoxon rank sum test. The level of significance was set at P < 0.05 for two-tailed values.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
In vitro experiments
Detection of MMPs.
In conditioned medium from DHD/K12 rat colon cancer cultures, zymography allowed to detect the latent form of MMP-9 and the active form of MMP-2 (Figure 1AGo). A gelatinolytic activity ~50 kDa was sometimes visible on gels, purportedly signifying the presence of MMP-1.



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  		Fig. 1. (A) Gelatin gel zymography of conditioned media from DHD/K12 cells. Molecular weight markers are indicated on the left. Evidence of proteolytic activity at 92 kDa (pro-MMP-9) and 66 kDa (active MMP-2). (B) DHD/K12 cell invasion assay. Batimastat, added in 10 and 100 µM concentrations to the culture medium, reduced by 15–28% the number of migratory cells which have passed through Matrigel and the polycarbonate membrane under fetal calf serum chemoattraction, without, however, reaching statistical significance (P < 0.1). Results are means ± SEM of four to five transwells and are representative of at least two experiments. FCS, fetal calf serum.

 
Cell invasion assays.
At 1 and 5 µM concentrations, batimastat had no obvious effect on the invasiveness of DHD/K12 cells across matrigel+Transwell units as compared with that observed with fetal calf serum alone. At 10 µM, it reduced cell invasiveness by 15.5 and 28.5% after 30 and 48 h incubation, respectively, and at 100 µM it reduced this activity by 22.5% after 30 h incubation (not significant at P < 0.1) (Figure 1BGo).

Cell proliferation assays.
The presence of batimastat in the culture medium, either for 24 or 48 h, did not influence the DHDK/12 cell proliferation as compared with that observed in presence of the vehicle alone (Table IGo). This established that there was no direct toxicity of the product in vitro.


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  		Table I. In vitro DHD/K12 cell proliferation in presence of batimastat
 
In vivo experiments
Series I.
There was slow body growth throughout the experiment, and mean rat weights, either for males or females, did not differ significantly between groups (data not shown). One batimastat-treated rat died during experimentation on day 26 and was excluded. On day 43, none of the batimastat-treated rats had macroscopically visible peritoneal tumor or hepatic metastasis. A small nodule (0.5 mm in diameter) was observed in the pancreatic area of a single batimastat-treated rat at the microscopic level. In contrast, seven of the eight vehicle-treated control rats presented severe stages of peritoneal carcinomatosis (Figure 2Go) and six had hepatic metastases (Table IIGo). In addition, control rats had haemorrhagic ascites as usually observed in this model (Table IIGo). Unexpectedly, all batimastat-treated rats also had similar volumes of ascite, but that was non-haemorrhagic. Histological examination revealed, in all batimastat-treated rats, inflammatory reactions surrounding the pancreases with lymphocyte aggregates, histiocytes, neutrophil and eosinophil leukocytes, associated with involution of acinous glands (data not shown). Such signs of inflammation were also present in the Glisson's capsule of livers; however, hepatic parenchyma generally stayed normal. Necrosis of fat tissue was constant. Biological analysis of the ascitic fluid of one batimastat-treated rat confirmed inflammatory characteristics. As compared with ascites of two control rats, protein concentrations were roughly of the same order (37 versus 41 and 49 g/l), as well as the number of cells per mm3 (9900 versus 10 000 and 14 600). However, there was a low concentration in haemoglobin (0.1 versus 1.8 and 2.3 g/dl) and a high percentage (90 versus 21 and 76%) in inflammatory cells, namely neutrophil leukocytes and lymphocytes.



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  		Fig. 2. Grades of colon peritoneal carcinomatosis in BD IX rats of Series I. Treament with batimastat began on day 2 after i.p. injection of DHD/K12 tumor cells. Control rats received vehicle alone. Rats were killed on day 43. The grade of peritoneal tumoral extension was as follows: class 0, no visible nodule; class 1, several nodules of 1–2 mm; class 2, more than 50 nodules of 1–5 mm; class 3, peritoneal cavity invaded by nodules up to 1 cm; class 4, peritoneal cavity fully invaded by nodules, some of them measuring several cm. Each column represents the number of rats of each group in a given class. There was no visible nodule in treated rats while all control rats but one had severe grades of peritoneal carcinomatosis.

 

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  		Table II. Ascites and hepatic metastases in experimental series I
 
RT–PCR and gelatin zymography indicated the presence of latent and active forms of MMP-2 in peritoneal nodules, and liver metastases of the rats of the pilot study and of vehicle-treated rats of series I. MMP-2 and MMP-9 were also found in ascitic fluids of the two groups of rats of series I, with even an increase in the two forms of MMP-2 in all batimastat-treated rats as compared with controls. The specificity of MMP activity of gelatinase type was checked by the absence of enzymatic digestion when incubating zymography gels with batimastat or EDTA and 1.10 phenanthroline, metalloenzyme and gelatinase inhibitors, respectively. Conversely, incubation of gels with phenylmethylsulfonyl fluoride, a serine protease inhibitor, or with N-ethyl maleimide, a cysteine protease inhibitor, did not prevent enzymatic activity (data not shown). Western blots confirmed the presence of MMP-2 and MMP-9 and indicated the presence of MMP-1 in tumors of control rats and in ascitic fluids of the two groups. Representative examples of RT–PCR, zymographs and western blots are shown in Figure 3A–F.Go In addition, peritoneal tumors and hepatic metastases displayed immunostaining for MMP-1, MMP-2 and MMP-9 localized in fibroblasts infiltrating the tumors but also in tumor cells (Figure 4A–DGo).



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  		Fig. 3. Detection of MMPs in vehicle-treated control tumor nodules and ascitic fluids of controls and batimastat-treated rats of series I. Molecular weight markers are indicated on the left. (A) MMP-2 mRNA expression by RT–PCR in tumor nodule of control rat. Amplification produced a 605 bp DNA fragment. (B) Representative examples of gelatin zymography of protein extracts from tumors in control rats. Lanes 1 and 2, peritoneal nodules of two different rats; lane 3, hepatic metastasis. Detection of latent (72 kDa) and active forms of MMP-2. (C) Representative examples of zymography from fluids after loading equal amount of proteins per lane. Lanes 1 and 2, ascitic fluids of two control rats; lane 3, pleural fluid of control rat; lanes 4 and 5, ascitic fluids of two different batimastat-treated rats. The latent form of MMP-9 and the two forms of MMP-2 were detected in the two groups with a greater activity of MMP-2 in batimastat-treated rats. (D) Representative examples of western blot analysis in ascitic fluid. MMP-1 detection (at 50 kDa): lane 1, control rat; lane 2, batimastat-treated rat. (E and F) Representative examples of western blot analysis in tumors of control rats. MMP-2 and MMP-1 detection. Lanes 1–3, peritoneal nodules from three different rats; lane 4, hepatic metastasis; lane 5, peritoneal nodule; lane 6, hepatic metastasis.

 


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  		Fig. 4. Immunostaining of peritoneal tumor nodules in control rats with antibodies against MMP-1 and MMP-2. (A) Bouin-fixed, paraplast-embedded tissue; (B) cryostat section, paraformaldehyde fixation: MMP-1 immunostaining is mainly localized in fibroblasts (filled arrows) but also in some tumor cells (empty arrow). (C) Same tumor nodule as in B: MMP-2 immunostaining is seen mainly in tumor cells (empty arrows). (D) Same tumor as in (B) and (C): no reaction when primary antisera were omitted. MMP-9 immunostaining was weaker than that of MMP-2 but similarly localized (not shown). (A–D) Bar, 25 µm.

 
Series II.
For the two groups of rats, body weights increased slowly during the first 4 weeks of experiment. After that time, batimastat-treated rats continued to grow while vehicle-treated rats began to lose body weight. The difference was significant as compared with batimastat-treated rats after 6 weeks (P < 0.05; data not shown). The two rats killed on day 13 before treament initiation showed several tiny peritoneal tumor nodules. The two vehicle-treated-rats killed on day 39 exhibited grade 4 peritoneal carcinomatosis, whereas the two batimastat-treated-rats were grade 1. The survival was significantly prolonged in the batimastat-treated rats: mean 61 days against 45 days; P = 0.0002 (Figure 5Go). At death, rats were autopsied. Figure 6Go shows the extension of peritoneal carcinomatoses in the two groups of rats including those killed on day 39, since control rats began to die spontaneously at that time. Lesions were very severe in vehicle-treated control rats (grades 3–4) and significantly less developed in batimastat-treated rats (grades 1–2; P < 0.001) in spite of prolonged evolution. Hepatic metastases were present in eight of 10 control rats against four of 10 batimastat-treated rats. In the latter, three rats had one metastasis and one rat several metastases, whereas, in control rats, only two had one metastasis and six had several metastases. Volumes of ascite were extensive, especially in batimastat-treated rats (100–150 ml) in which the fluid aspect was different from that in controls, as in series 1.



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  		Fig. 5. Survival curves in series II. Intraperitoneal inoculation of 1x106 DHD/K12 tumor cells in each rat on day 0. Treament with batimastat or vehicle alone began on day 13 and continued until death. Batimastat-treated rats survived for a significantly longer time than controls (P = 0.0002).

 


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  		Fig. 6. Grades of colon peritoneal carcinomatosis in BD IX rats of series II. Treament with batimastat or vehicle alone began on day 13 after tumor cell inoculation and continued until death. At necropsy, the grade of peritoneal tumoral extension was established as in Figure 2Go. Each column represents the number of rats of each group in a given class. All control rats had severe carcinomatosis at death. This was not the case for batimastat-treated rats (P < 0.001 versus controls) in which marked inflammatory ascites were probably the cause of death.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study, results of zymography, immunohistochemistry and/or western blots and RT–PCR indicate the production of at least MMP-2 and MMP-9 by DHD/K12 rat colonic cancer cells in vitro, as well as expression of MMP-1, -2 and -9 in ascitic fluids and solid tumors formed by these cells. No attempt was made to examine expression of other MMPs. When the MMP inhibitor batimastat was added to the culture medium for 30 or 48 h, at concentrations used previously in vitro (21), it decreased the invasive activity of DHD/K12 cells by 15–29%. However, batimastat had a stronger inhibitory effect on that activity in vivo than in vitro. Indeed, in the experimental model used, batimastat treatment obviously prevented peritoneal carcinomatosis implantation and hepatic tumor development when it began soon after i.p. injection of tumor cells, i.e. 2 days. When treatment began after initial tumor development, i.e. on day 13, it had a clear-cut inhibiting effect on the peritoneal tumor growth and development of metastases, and significantly prolonged the survival of animals. Our results obtained in rats agree with, and significantly extend, those of previous reports evaluating the effect that batimastat exerts in colon cancer xenograft or metastasis models in nude mice. Thus, in a spontaneous metastasis model in nude mice involving colonic orthotopic implantation of a human colorectal tumor, batimastat treatment administered i.p. during 60 days and starting 7 days after tumor implantation was able to reduce the mean weight of primary tumors as well as the incidence of local and regional invasion, resulting in improvement of animal survival (12). In a second liver metastasis model of colorectal carcinoma, batimastat administered from day 10 after i.p. injection of tumoral cells to day 39, reduced the number and size of hepatic metastases (13). In the latter experiment, a diastereoisomer of batimastat, 670-fold less potent in inhibiting MMP activity, had no effect on tumor xenograft biology, indicating that the treatment efficacy is indeed due to MMP inhibition.

While our work was in progress, it was reported that batimastat also reduced ascite formation in a human colorectal cancer ascite model in SCID mice when treatment began at day 0 but not when it began at day 10 (14). The result obtained with early treatment initiation corroborated that observed in another ascite model in nude mice induced by cancer cells of ovarian origin (22) but was conflicting with a third model induced by breast cancer cells in which batimastat failed to suppress ascite formation and to increase mice survival (23). In our model, batimastat was unable to inhibit ascite production, present in all treated rats, even in the absence of peritoneal tumor formation. Ascitic fluids of batimastat-treated rats showed an increased MMP-2 activity as compared with those of control rats, and biological analysis of one of them revealed that it was rich in inflammatory cells, suggesting a higher MMP-2 production by inflammatory cells. Histological examination of organs contained in the peritoneal cavity of those rats confirmed inflammatory reactions and revealed that these organs, especially the pancreas, displayed tissue lesions. Such inflammatory reactions were probably among the causes of death in batimastat-treated rats of series II in which ascites were extensive whereas peritoneal tumor nodules were little developed. However, the dosage and route of product administration were the same as in other studies (12,14,15,24) in which treatment duration was sometimes even longer (12,24). It must be noted that most of previous studies were performed in nude or SCID mice that had diminished immunological properties and generally did not show inflammatory reaction. Similarly, no inflammation was noted in pathogen-free CBH/cbi rats, except one, with daily i.p. batimastat injection (24). Nevertheless, as far as it is known, three studies have reported peritoneal reactions to i.p. MMP inhibitor administration. First, with batimastat, a 9-fold increase of polymorphonuclear neutrophils was noted into the peritoneal cavity of mice 24 h after injection (22) and local toxicity with peritoneal irritation was observed in humans (25). Secondly, in a preliminary study in mice, treatment with a gelatinase-A selective inhibitor resulted in the accumulation of peritoneal fluid (26).

In conclusion, the results of the present study provide evidence of efficiency of the batimastat treatment in a model of colon cancer peritoneal carcinomatosis, at two different stages of the disease. First, in a very precocious stage mimicking micrometastasis it prevents tumor implantation and secondly, at a more advanced stage, it reduces peritoneal tumor growth and the number of hepatic metastases and prolongs survival. These results are encouraging for using MMP inhibition as adjuvant or palliative therapy. Nevertheless, the local i.p. toxicity of that product in our model leads to recommend other routes of administration. Currently, oral MMP inhibitors are available. One, CT1746, has already been used successfully combined with a cytotoxic agent in animals (27). Another, marimastat (BB-2516), is currently under evaluation in several phase III trials of human cancer therapy (28).


   Acknowledgments
 
The authors thank British Biotech Pharmaceuticals Ltd for the generous gift of batimastat, Mrs L.Gres for technical assistance, and Mrs F.Bonfils for her help in documentation. This work was supported by the Institut de la Santé et de la Recherche Médicale (INSERM) and by IRMAD (to S.K.). T.A. was a recipient of a grant from the Fondation pour la Recherche Médicale, and S.K. was a recipient of a grant from the Ministère de l'Enseignement Supérieur et de la Recherche.


   Notes
 
1 To whom correspondence should be addressed Email: u10{at}bichat.inserm.fr Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 17, 1999; revised April 2, 1999; accepted April 12, 1999.


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