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Heparins, cancer and thrombosis: clinical and experimental studies

Smorenburg, S.M.

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Citation for published version (APA):Smorenburg, S. M. (2000). Heparins, cancer and thrombosis: clinical and experimental studies.

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Download date: 31 Aug 2020

IN VIVO TREATMENT OF RATS WITH UNFRACTIONATED HEPARIN OR LOW MOLECULAR WEIGHT HEPARIN DOES NOT

AFFECT EXPERIMENTALLY INDUCED COLON CARCINOMA METASTASIS

Susanne M. Smorenburg, Roel Vink, Merijn te Lintelo, Wikky Tigchelaar, Adrie Maas, Harry R.

Büller, Cornells J. F. van Noorden

CLINICAL & EXPERIMENTAL METASTASIS, 1999; 17:451-456

ABSTRACT

Recent randomized trials have suggested that treatment with low molecular weight heparin

(LMWH) improves survival of cancer patients with venous thromboembolism, as compared to

treatment with unfractionated heparin (UFH). Experimental studies have shown that UFH has

activities besides its anticoagulant function which may affect progression of malignancy, including

stimulation of new blood vessel formation. In contrast, LMWH has been suggested to inhibit

angiogenesis. In the present study, we compared quantitatively the effects of treatment with UFH,

LMWH or placebo on the development of experimentally induced colon carcinoma metastases in

rat liver and on tumor-associated angiogenesis. It is shown that UFH and LMWH in therapeutic

dosages neither affect development of metastases nor tumor blood vessel formation in this animal

model. These results indicate that heparins do not affect colon cancer metastasis in liver. Further

studies in other animal models are required to establish the mechanisms by which heparins

potentially affect cancer.

INTRODUCTION

Patients with malignancy frequently suffer from thromboembolic complications. As a consequence,

numerous cancer patients are treated with anticoagulants such as unfractionated heparin (UFH) or

low molecular weight heparin (LMWH). It is a matter of debate whether these agents interfere with

cancer progression and alter prognosis of patients with malignancies.

Experimental studies have shown that UFH is endowed with multiple activities besides its

anticoagulant function, including inhibition of the immune system during inflammation, cell

proliferation, cellular adhesion and invasion, and enhancement of effects of various growth factors

and proteases (1-3). In addition, UFH may stimulate angiogenesis, an essential process in tumor

growth, by the release of angiogenic growth factors from the extracellular matrix and by induction

of their activity through binding to the high affinity receptors (4). As a consequence, UFH treatment

may alter the prognosis of patients with cancer, either in a positive or negative way.

Randomized trials, which compared the efficacy and safety of LMWH with UFH in the treatment

of patients with venous thromboembolism, have created renewed interest in the potential of UFH

and its fractions to affect cancer progression (5-8). It was shown that cancer patients with venous

thromboembolism who were treated with LMWH had a significantly improved 3-6 month survival

as compared to UFH recipients. Recurrence of thromboembolic and bleeding complications was

identical in both patient groups and the difference in mortality was not found in patients with venous

thromboembolism without cancer. The difference was observed in a wide variety of malignancies,

including gastro-intestinal, genito-urinary, breast, haematological and central nervous system

malignancies (8). Although these results were obtained with a post hoc analysis, they suggest that

90

either LMWH affects survival of patients positively and/or UFH negatively. However, in a recent

systematic review of clinical studies it was shown that UFH had no effect on prognosis of patients

with cancer (9).

Studies on the effects of LMWH on tumor progression are limited. LMWH, in contrast to UFH, has

been shown to suppress angiogenesis in vivo (10-12). Angiogenesis is stimulated by heparin-

binding growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial

growth factor (VEGF) (13). Heparin oligosaccharides containing less than 10 saccharide residues

have been found to inhibit biological activity of bFGF, whereas heparin fragments with less than 18

saccharide residues have been reported to inhibit the binding of VEGF to its receptors on

endothelial cells (12, 14). These findings implicate that the size of the heparin molecule may be a

critical determinant for the binding of bFGF and VEGF to their receptors, and that the effects of

LMWH on blood vessel formation may differ from those of UFH.

In the present study, we compared quantitatively the effects of treatment with UFH, LMWH or

placebo on development of experimentally induced colon carcinoma metastasis in rat liver as well

as on tumor-associated angiogenesis.

MATERIALS AND METHODS

CANCER CELLS AND INDUCTION OF LIVER METASTASES

A colon adenocarcinoma cell line, CC531s, was obtained from a moderately differentiated and

weakly immunogenic colon adenocarcinoma after experimental induction in WAG-Rij rats by

treatment with 1,2-dimethylhydrazine (15). Cells were cultured in vitro at 37°C as monolayers in

Dulbecco's modified Eagle's medium (ICN Biomedicals, Irvine, Ayrshire, UK) supplemented with

10% (v/v) fetal bovine serum, 2 mmol/1 glutamine, 100 IU/ml penicillin, and 100 mg/ml

streptomycin. Cells were washed with phosphate buffered saline (PBS) and after detachment with

0.05% trypsin in PBS and centrifugation (250 g, room temp, 10 min), single cell suspensions were

obtained with a viability of at least 95%. After anaesthesia with 0.07 ml/10 g body weight Fentanyl-

Fluanizone-Midazolam mixture (FFM; 1ml Hypnorm, Janssen, Beerse, Belgium; 1ml Midazolam,

Roche, Mijdrecht, The Netherlands and 2 ml water), colon carcinoma metastasis was induced in rat

liver by injection of a single cell suspension containing 1 x 105 cancer cells into the portal vein of

54 mature female WAG-Rij rats, weighing 130-150 g (Broekman, Someren, The Netherlands) as

described previously (16). To prevent peritoneal seeding, the puncture site was covered with

Spongostan (Medical Workshop, Groningen, The Netherlands) until complete haemostasis was

obtained.

CHAPTER 7 EFFECTS OF HEPARINS ON COLON CARCINOMA METASTASIS 91

TREATMENT PROTOCOL

In the first experiment, 3 groups of 10 rats each were treated intraperitoneally (i.p.) with either 0.9

% NaCl once daily, 2.0 mg/kg LMWH ( Reviparin, 7000 IU aXa/ml; Knoll, Ludwigshafen,

Germany) dissolved in 0.9% NaCl once daily or 2.0 mg/kg UFH (5000 IU/ml; Leo, Weesp, The

Netherlands) dissolved in 0.9% NaCl twice daily, respectively. These dosages correspond with the

human, therapeutic dose of UFH or LMWH. Treatment was started one day before operation of the

animals and was continued up to 7 days after administration of cancer cells. On the day of operation,

the compounds were given one h before inoculation of cancer cells in all treatment groups. To

measure anti-activated factor X (aXa) plasma levels, 0.5 ml blood was sampled from the tail vein

of all rats on the 5th day after inoculation, at 3, 6, 9, and 12 h after injection. Seven days after

administration of cancer cells, animals were weighed and sacrificed with an overdose of FFM

mixture.

In the second experiment, 3 groups of 8 rats received higher doses of LMWH and UFH and for a

longer period of time. They were treated with either 0.9% NaCl once daily, 2.5 mg/kg LMWH once

daily, or 2.5 mg/kg UFH once daily, respectively, using a similar set up as in the first experiment,

but treatment was continued up to 24 days after administration of cancer cells. Blood was sampled

on the 20th day, at 1, 3, and 6 h after the injection. Animals were weighed and sacrificed at 24 days

after cancer cell administration. The animal experiments were performed according to guidelines of

the University of Amsterdam.

MEASUREMENT OF P L A S M A A X A LEVELS

Blood samples were collected in EDTA, centrifuged (15 min, 10.000 g, 4°C) and plasma was stored

at -80°C until use. Plasma UFH and LMWH levels were assayed by measuring inhibition of factor

Xa, using the chromogenic substrate S-2222 (Chromogenix, Mölndal, Sweden) as described

previously (17). For calibration, UFH and LMWH in normal rat plasma was used.

PREPARATION OF LIVER TISSUE AND EVALUATION OF TUMOR GROWTH

Livers were removed immediately after sacrifice and the individual liver lobes were divided and cut

into small pieces (up to 5 mm thick) according to a rigid dissection protocol (18). Tissue fragments

were frozen in liquid nitrogen and the material was kept at -80°C until further use. To quantify the

number of metastases after 7 days of growth, 21 sites were randomly selected from all lobes of each

liver before experiments were carried out. From these sites, sections (8 mm thick) were cut on a

motor-driven cryostat with a rotary retracting microtome (Bright, Huntingdon, UK) at a constant

speed and a cabinet temperature of-25°C.

The number of metastases after 24 days were quantified by counting metastatic nodules on the liver

surface. It appeared in a previous study that the number of macroscopically detectable nodules on

the surface of the livers was a valid and reproducible measure of tumor load in the livers (19). The

92

pieces of liver that contained macroscopically visible metastases were frozen in liquid nitrogen and

stored at -80°C until further use. To evaluate size and vessel density of the tumors, 20 sections (8

mm thick) were cut at the site where the tumors had the largest diameter. Sections were mounted

on glass slides and stored at -20°C until use.

IMMUNOHISTOCHEMICAL PROCEDURES

To facilitate recognition of cancer cells, cryostat sections were stained immunohistochemically

using the lectin Ulex Europaeus agglutinin I (UEA-1), a selective marker of CC531s cells in rat as

described previously (16). Briefly, sections were air dried at room temp for 1 h and fixed with

aceton for 10 min at 4°C. Endogenous peroxidase activity was blocked with 0.3% (v/v) hydrogen

peroxide and 0.1% NaN3 in PBS for 15 min at room temp. After preincubation with normal goat

serum (Dako, Glostrup, Denmark: 10% v/v in PBS for 10 min at room temp), sections were

incubated for 60 min using UEA-1 (Dako) diluted 1:100 in PBS, then 30 min with rabbit anti-UEA-

1 conjugated with horseradish peroxidase (Dako) diluted 1:50 in PBS in the presence of 10% AB

serum (CLB, Amsterdam, The Netherlands). Sections were stained for peroxidase activity using

3,3-diaminobenzidine-tetrachloride and hydrogen peroxide as described previously (16).

Endothelial cells were detected with a rat endothelial cell-specific monoclonal antibody (RECA-1),

kindly provided by Dr. A. M. Duijvestijn, University of Maastricht, The Netherlands (20). Sections

were air-dried at room temp for 1 h, then fixed 10 min in 4% formaldehyde in 0.1 M phosphate

buffer (pH 7.4). Endogenous peroxidase activity was blocked with 0.3% (v/v) hydrogen peroxide

and 0.1% NaN3 in PBS for 15 min. Sections were incubated for 60 min with RECA-1 diluted 1:10

in PBS containing 0.2% (w/v) bovine serum albumin, then for 60 min with rabbit anti-mouse IgG

peroxidase (Dako) diluted 1:200 in PBS in the presence of 0.2% bovine serum albumin and 5%

normal rat serum. Sections were stained for peroxidase activity using 3,3-diaminobenzidine-

tetrachloride and hydrogen peroxide (16). Control sections were incubated in the absence of the first

antibody.

MORPHOMETRIC ANALYSIS

The number of tumors after 7 days as detected with UEA-1 was analyzed quantitatively by

morphometry in cryostat sections obtained from the 21 selected sites in the livers (18, 21). The

percentage area of cancer cells was expressed as the ratio of the area of cancer cells and the total

area of the liver tissue sections including cancer cells (xlOO) ± standard error of the mean (SEM).

After 24 days, the largest area of each tumor was measured using sections that were stained with

UEA-1. The values were averaged to determine the mean area of tumors ± SEM per group. Vessel

density of tumors was determined in 2 sections adjacent to the section with the largest area and the

values were averaged. Vessel density was determined as the ratio of the area of RECA-1-positive

endothelial cells in the tumor and the total area of UEA-1-positive tumor (xlOO) ± SEM.

CHAPTER 7 EFFECTS OF HEPARINS ON COLON CARCINOMA METASTASIS 93

Analysis of the number of tumors and their vessel density was performed without knowledge of the

treatment group. Statistical analysis of the percentage areas of tumors after 7 days was performed

with the Kruskall-Wallis test, whereas ANOVA was used to determine statistical differences in

number, size, and vessel density of metastases after 24 days. Values of p<0.05 were considered to

indicate significant differences.

RESULTS

A X A LEVELS IN PLASMA

The aXa plasma levels measured in UFH- and LMWH- treated rats in both experiments are

presented in Fig. 1. Therapeutic plasma levels (>0.3 aXa IU/ml) were measured for at least 4 h after

administration of UFH or LMWH, both in rats that received 2.0 or 2.5 mg/kg of either UFH or

LMWH. One h after injection of 2.5 mg/kg UFH, a mean plasma level of 0.9 (± 0.2) aXa IU/ml was

found, whereas LMWH-treated rats (2.5 mg/kg) had a mean level of 1.5 (± 0.3) aXa IU/ml at 1 h

after administration, indicating that high concentrations of the anticoagulants were present during

cancer cell administration. No bleeding complications were observed during either administration

of cancer cells or throughout the experiments.

1 3 6 9 12

hours after injection

1 3 6 9 12

hours after injection

Figure 1. aXa plasma levels of UFH- (dashed line) and LMWH-treated rats (solid line) in experiment 1 (A) after 5 days of treatment with 2.0

mg/kg UFH b.i.d. or with 2.0 mg/kg LMWH o.d., and in experiment 2 (B) after 20 days of treatment with 2.5 mg/kg UFH or LMWH o.d.

C O L O N CANCER METASTASIS IN RAT LIVER

Seven days after CC531s cells had been administered, livers contained tumors of variable size (Fig.

2A). The median volume of tumors, expressed as percentage area of tumors, was 0.04% (range,

0.01-0.99%) in placebo-treated rats. Treatment with either UFH or LMWH did not affect the

volume of tumors significantly after 7 days (Table 1). After 24 days, on average 4.1 tumors were

found on the liver surface of placebo-treated rats, with a mean area of 6.0 mm2 (Fig. 2C). The

average numbers of metastases in UFH-treated and LMWH-treated rats were 4.4 and 5.4,

94

respectively, with a mean area of 5.0 mm2 in UFH-treated rats, and 3.1 mm2 in LMWH-treated rats

(Table 2). Number and size of the tumors did not differ significantly among the treatment groups.

Metastases were not found in other organs of the rats in either of the 3 treatment groups.

Treatment

Placebo

UFH

LMWH

Table 1. Volume (expressed as percentage area) of tumors in livers of UFH-, LMWH-, and placebo-treated rats at 7 days after administration

ofCC531s colon cancer cells in the portal vein. a Percentage area is expressed as the ratio of the area of cancer cells and the total area of liver tissue sections including cancer cells xlOO

(%). Median percentage per rat and range between brackets are presented. Each group consisted of 10 rats. Percentage areas of tumor did

not differ significantly between treatment groups (p= 0.7).

Treatment

HHHHMHHH Placebo

UFH

LMWH

Table 2. Number and size of tumors at the liver surface of UFH-, LMWH-, and placebo-treated rats at 24 days after administration ofCC531s

colon cancer cells in the portal vein.

< Data are given as mean number ± SEM and mean area of metastases ± SEM (mm-) per rat. Each group consisted of 8 rats. Number and size

of metastases did not differ significantly between treatment groups (number, p=0.7; size, p= 0.4).

VESSEL DENSITY O F METASTASES

At 7 days after cancer cell administration, newly formed blood vessels were found to be hardly

present in liver metastases irrespective of treatment (Fig. 2B). Only a few large metastases

contained small vessels, again independently of the treatment given. After 24 days, metastases were

well-vascularized (Fig. 2D). Vessel density of tumors in placebo-treated rats was 3.1 %, whereas in

UFH-treated and LMWH-treated rats the vessel density was 3.7 % and 3.3 %, respectively. Again,

significant differences were not found among the rats in the 3 treatment groups (Table 3).

Treatment Tumor vessel density

(%)perrat>

Placebo 3.1 ± 0.3

UFH 3.7 ± 0.3

LMWH 3.3 ± 0.2

Table 3. Vessel density in tumors at the liver surface of UFH-, LMWH-, and placebo-treated rats at 24 days after administration of CC531s

colon cancer cells in the portal vein.

" Vessel density is expressed as mean area of endothelial cells per tumor x 100 ± SEM (%). Data are given as mean percentage vessel density

of tumors per rat. Each group consisted of 8 rats. Tumor vessel density did not differ significantly between treatment groups (p = 0.2).

ercentage area (%)•

0.04 (0.01-0.99)

0.06 (0.01-0.48)

0.05 (0.00-0.29)

Number of metastases Size of metastases per rat • per rat •

4.1 ±1.4 6.0 ±2.5

4.4 ± 0.5 5.0 ± 0.7

5.4 ±1.2 3.1 ±0.9

CHAPTER 7 EFFECTS OF HEPARINS ON COLON CARCINOMA METASTASIS 95

Figure 2. Photomicrographs ofcryostat sections of rat liver containing colon carcinoma metastases after staining with UEA-1 to detect cancer

cells (A, C) (16) and RECA-1 to detect endothelial cells (B, D) (20). Endothelial cells are scarcely present in tumors after 7 days of growth

(B), whereas after 24 days tumors are well vascularized (D). t, tumor; p, liver parenchyma; arrows, endothelial cells in tumor and *,

endothelial cells in liver parenchyma after staining with RECA-1. Bar = 100 fxm (A, B) or 200 fJm (C, D.)

DISCUSSION

Various experimental studies have reported that anticoagulants such as UFH inhibit metastasis (22-

24). The rationale for the use of UFH as an antimetastatic agent was the assumption that it prevents

clot formation around cancer cells, and thereby protects cancer cells in the circulation (25, 26).

However, effects of UFH on progression of cancer or metastasis may not solely be related to its

anticoagulant function. Recent studies have suggested that chemically modified heparins without

anticoagulant activity inhibit metastasis as well (27-29). It was suggested that UFH and heparin-like

molecules effectively reduce metastasis by inhibiting heparanases, which are matrix degrading

enzymes utilized by metastatic cancer cells to break down the basement membrane during invasion

(30). In contrast to these findings, we did not observe any inhibitory or stimulatory effects of UFH

96

or LMWH on the development of colon carcinoma metastases in rat liver. These contradictory

findings cannot be explained by differences in plasma levels of the anticoagulants. Both UFH and

LMWH were given in therapeutic concentrations, and plasma concentrations of UFH and LMWH

were above 0.3 aXa IU/ml for at least 4 h in the treated rats, both at 5 and 20 days after starting the

treatment. In the previous studies, similar or lower concentrations of UFH inhibited metastasis

effectively after being injected either subcutaneously or intraperitoneally (27, 28, 31-33).

It is possible that our data differ from that of previous experimental studies as a consequence of the

different tumor types or animal models that were used. Effects of therapeutic doses of UFH on colon

carcinoma metastasis have not been investigated thus far. One study that evaluated low doses of

UFH (3 IU twice daily, s.c.) on growth and metastasis of subcutaneously implanted colon carcinoma

tumors in nude mice reported that there was no inhibitory effect either (34). Studies in which UFH

was found to inhibit metastasis generally evaluated pulmonary metastases of intravenously

administered mammary or melanoma cancer cells (22, 27, 28, 31). It has been postulated that

interactions between cancer cells and haemostasis differ between tumor types. Pathological studies

suggest that the fibrinolytic system is of more importance than coagulation activation in colon

carcinomas (35). In addition, clinical trials evaluating UFH, warfarin or fibrinolytic agents have

indicated that various tumor types respond differently to anticoagulants (36-39). On the other hand,

randomized trials which compared LMWH with UFH for the treatment of venous

thromboembolism showed an improved survival in LMWH-treated patients with a variety of

malignant diseases, including colon cancer (10). These latter studies thus suggest a rather general

effect of LMWH or UFH on cancer progression, irrespective the tumor type.

Various studies have reported that UFH stimulates and that LMWH inhibits blood vessel formation,

but the effects of the heparins on cancer-associated angiogenesis have not been investigated thus far

(11, 12). In the present study, we also quantitatively evaluated effects of UFH and LMWH on blood

vessel formation in the tumors, by comparing the density of RECA-1 positive endothelial cells in

tumors of the 3 groups. Overt effects of the heparins on cancer-associated angiogenesis were not

found.

In conclusion, the results of these experiments indicate that heparins do not affect colon carcinoma

metastasis in liver. Outgrowth of tumors and intra-tumor vessel density were both not affected by

heparin treatment in this animal model. As a consequence of the numerous biological activities of

UFH and LMWH, it is possible that heparin treatment in vivo does not substantially enhance or

reduce cancer progression and tumor-associated angiogenesis. Nevertheless, one cannot exclude

that the negative results of the present study are related to the specific tumor type or model used and

that heparins affect outgrowth of tumors which are highly vascularized. Therefore, further

experiments are warranted to provide detailed insights into the mechanisms by which heparins may

affect malignancy. The results of these experiments may guide the selection of appropriate clinical

settings for definitive testing of effects of LMWH in human cancer.

CHAPTER 7 EFFECTS OF HEPARINS ON COLON CARCINOMA METASTASIS 97

ACKNOWLEDGEMENT

We thank Mrs. W.M. Morriën for measurements of plasma aXa levels, Dr. A.M. Duijvestijn for the

supply of RECA-1, Mrs. K.S. Bosch for her technical assistance, Prof.Dr. C.H.N. Veenhof and Dr.

R .0 . Schlingemann for their scientific advice, and Mr. J. Peeterse and Mr. C. Gravemeijer for

photographic work.

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