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A possible role for enzymes in tumour-cell invasion
MedicalHypotheses(1997) 48, 443--447 © PearsonProfessionalLtd 1997
A possible role for enzymes in tumour-cell invasion C. H. VAN ASWEGEN, D. J. DU PLESSIS Wolmarans Research Laboratory, Department of Urology, University of Pretoria, Private Bag X169, Pretoria 0001, South Africa. Tel: +27 12 3192547; Fax: +27 12 3295152 (Correspondence to CHvA)
Abstract - - The complex molecular and cellular processes of metastatic invasion as well as the anti-invasion possibilities are summarized. Invasion by neoplastic cells is a major obstacle to successful cancer therapy. Enzymes such as hyaluronidase, sialyltransferase, urokinase-type plasminogen activator, plasmin, matrix metalloproteinases, and others, play central roles in the catabolism of extracellular matrix macromolecules. However, this process can be opposed by inhibitors of these enzymes. Both invasion (promoters) and anti-invasion factors (suppressors) need further investigation, to clarify the role of these factors in the aetiology and possibly in the treatment and prognosis of metastatic cancer.
Introduction
Metastatic invasion
Cancer, particularly prostatic and breast cancer, is one of the commonest illnesses amongst all races and nationalities. It has been estimated that a detectable tumour of 0.5-1 cm in diameter contains approximately one billion ceils. The aetiology of the transformation of a normal cell to a malignant cell is still unclear. In facilitating this process, it has been stated that carcinogenesis is a multistage process, starting with an initiation phase, followed by promotion, transformation, progression and metastatic phases (1,2). For metastasis to be effective, cancer cells have to penetrate the matrix, particularly the basement membrane. This process is known as invasion and includes attachment of tumour cells, proteolysis of matrix components, and migration of tumour cells through the matrix defect (3).
Mechanism of tumour-cell adhesion It is hypothesized that cells, which migrate from the primary tumour in the hostile blood environment, have a surface covering of hyaluronic acid (HA). These cells are kept apart from each other because of the loose, hydrated and porous nature of the HA coat (4). At a stage during development, the tumour cell starts to synthesize and secrete increased hyaluronidase, which degrades the HA of the tumourcell 'cocoon'. Degrading of the HA coat can be prevented by an extract from the herb Echinacea (5). This sequence correlates well with both cell--cell attachment as well as with a decrease in HA and cell-surface molecules that bind HA such as DC44H (4,6). Closer movement of tumour cells within the matrix is thus possible, leading to the final step of
Date received 16 April 1996 Date accepted 17 May 1996 443
444 tumour-cell attachment. Tumour-cell receptors of the integrin and non-integrin type (cadherins, immunoglobulins) would bind to glycoproteins of the extracellular matrix, such as fibronectin, collagen and laminin (3,7-9). Expression of E-cadherin on the primary tumour cells may be increased by addition of the essential fatty acid (EFA), T-linolenic acid (GLA), which would lead to decreased tumour cell detachment and substantial enhancement in the survival rate (7,10). Sialylation (masking) of cell-surface proteins by sialyltransferase, may overcome this anti-invasion effect of E-cadherin, since increased sialyltransferase activity is present with metastasizing tumours (11-13), as well as the enzyme substrate sialic acid (14-17). Addition of the short-chain fatty acid, n-butyrate, decreases sialyltransferase production (18). Cell adhesion occurs only to specific organ matrices and, therefore, it is suggested that 'homing receptors' on invading tumour cells can find specific organ target cells (19-21). The degree by which tumour-cell surface HA is lysed by hyaluronidase may be important, since it may leave a HA 'fingerprint' that consequently identifies a specific addressin molecule on the target endothelium cell. This phenomenon has been referred to as the 'seed and soil' hypothesis, in which the metastatic cells present the 'seed' and the organ environment the 'soil' (22,23). Certain adhesion molecules on the 'soil' endothelial surface can be induced by cytokines such as interleukin-1 (IL-1), tumour necrosis factor (TNF) a and ~'-interferon (24), whilst prostacyclin and tamoxifen inhibit adhesion (25-27).
Proteolysis of extracellular matrix Proteolysis of matrix components composes the next step in the 'invasion cascade'. Enhanced proteolytic activity has been observed in highly malignant tumours (28). It is suggested that the complex process of proteolysis starts with the activation of pro-urokinase type plasminogen activator (pro-uPA) by serine proteases (plasmin) and/or cysteine proteases (cathepsin B and L) (29). Increased urokinase type plasminogen activator (uPA), uPA receptors and cathepsin B levels or activities have been reported with various cancers (30-41). A correlation between uPA production and metastasis has also been reported (29,33,42), therefore, uPA levels may be of prognostic value (43,44). UPA activity may be inhibited by natural or synthetic plasminogen activator inhibitors (PAI) (45). Urokinase and plasmin activity, in the presence of oleic acid, has been enhanced almost 40- and 4-fold, respectively (46). This phenomenon could explain why persons whose diets consist mainly of saturated fatty acids have a greater cancer risk than those whose
MEDICAL HYPOTHESES
diets consist mostly of polyunsaturated fatty acids, which include EFA. Although oleic acid stimulates uPA activity, it inhibits uPA production by DU-145 prostatic cells in culture (47). This is also the case with ALA, eicosapentaenoic acid (EPA), linoleic acid (LA), GLA and arachidonic acid (AA). Activated uPA hydrolyses plasminogen to plasmin, which again directly or indirectly lyses collagen (48,49). In the latter case, plasmin first activates latent collagenase to degrade basement-membrane collagen. A positive correlation exists between squamous-cell carcinoma size, depth of tumour invasion, diffuse invasion mode, high incidence of lymph-node metastasis and matrix metalloproteinasic (MMP) collagenase, MMP-3 (50). Plasmin may also directly hydrolyse the fibronectin and laminin of the basement membrane (48,51). Lysis of basement membrane substrates is not only dependent on the activity of the enzyme, but also on the concentration or production of the enzyme. Thus, an increase in serum collagenase levels has been found in prostatic cancer patients (52), as well as in bladder cancer (53). However, increased MMP production could not be demonstrated in human breast adenocarcinoma cell lines MCF-7 or BT-20 in culture (54). This is not the result when these cells are grown in the presence of fibroblasts. In contrast to normal mammary epithelium cells, which promote only fibroblast production of the tissue inhibitor of metalloproteinase type-1 (T1MP-1), MCF-7 and BT-20 malignant cells stimulate fibroblast production of the precursors of MMP- 1 (interstitial collagenase), MMP2 (gelatinase A), MMP-3 (stromelysin-1), TIMP-1, and MMP-2, TIMP-1, respectively. It is therefore, suggested that proteases are mainly produced by the extracellular matrix and not by the tumour cells themselves (54,55). Since carcinomas contain decreased amounts of EFA, (56,57), the question arises whether EFA could not affect communication between normal and malignant cells. If so, it can be argued that in the presence of EFA only TIMP would be synthesized, whilst at low serum concentrations of EFA, both TIMP and MMPs are produced. To complicate this matter, an imbalance has been found between MMPs and TIMP in prostatic carcinoma (58). A significant increase in TIMPs is present in normal juvenile and adult prostates, whilst neoplastic prostatic tissue contains low levels of TIMPs and shows increased MMP-2 activity. Cytoldnes may also affect the protease production of fibroblasts. TNF influences human fibroblasts bifunctionally by the suppression of TIMP production in addition to stimulation of MMP synthesis (59). Based on this model, it would not be advisable to prescribe cz-linolenate (ALA) to patients with cancer, since increased TNF production has been found with
ENZYMESIN TUMOUR-CELLINVASION murine m a c r o p h a g e s w h e n their diet contains A L A (60). In contrast with T N F and IL-1, interleukin-6 (IL-6) does not stimulate the production o f M M P s , but is a potent inducer o f the synthesis of T I M P - 1 (61). Increased I L - 6 production can be obtained with both l i p o p o l y s a c c h a r i d e (LPS) and certain E F A ( d o c o s a h e x a e n o i c acid, eicosapentaenoic acid), w h i c h w o u l d be preferable in the anti-cancer search, since it stimulates not only T I M P - 1 production, but also the inhibitor o f uPA, PAI-1 (62,63). Other matrix-degrading e n z y m e s p r o d u c e d by tumours are: heparitinases (heparan sulphate, proteoglycans), cathepsin B (collagens, fibronectin, laminin, proteoglycans), and elastase (elastin, collagens, fibronectin, laminin, proteoglycans). It has b e e n suggested that the structure o f the b a s e m e n t m e m b r a n e consists o f an o p e n n e t w o r k (lattice) o f t y p e - I V c o l l a g e n to w h i c h the n o n c o l l a g e n o u s c o m p o n e n t s heparan sulphate, proteoglycans, laminin and fibronectin are b o u n d (48). A c c o r d i n g to this model, proteolysis o f these m a c r o m o l e c u l e s w o u l d not be as important in the invasion process, as the hydrolysis o f c o l l a g e n w h i c h constitutes the basic structure of b a s e m e n t m e m b r a n e . This further e m p h a s i z e s the essential importance o f M M P s . Nevertheless, the overall c o n s e q u e n c e o f proteolysis is r e m o v a l of the matrix substances, after w h i c h the t u m o u r cell can migrate.
445
2. 3. 4. 5.
6.
7.
8. 9. 10. 11.
Migration of tumour cells A f t e r lysis of the b a s e m e n t m e m b r a n e as described above, it is possible for the t u m o u r cells to penetrate the subendothelial b a s e m e n t m e m b r a n e . Irregular p s e u d o p o d i a are p r o j e c t e d in the direction of migration by the invading cell. Proliferation of the t u m o u r cell results in metastasis o f the primary tumour. This process m a y repeat itself m a n y times to g i v e metastases o f the metastasis at secondary sites.
Conclusion The c o m b i n e d investigative data o f the a b o v e theory on i n v a s i o n demonstrated that the invasion cascade is a highly selective process regulated by various mechanisms. O n c e the aetiology of metastatic invasion has been elucidated, better anti-cancer agents can be devised. In this respect, cytokines and E F A m a y be o f importance in the successful prevention of metastatic colonies and should be considered for inclusion in available therapeutic regimens.
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MEDICAL HYPOTHESES
vitro. Cancer Res 1994; 54:3066-3071. 41. Bianchi E, Cohen R L, Thor A T et al. The urokinase receptor is expressed in invasive breast cancer but not in normal breast tissue. Cancer Res 1994; 54: 861-866. 42. Zucker S. A critical appraisal of the role of proteolytic enzymes in cancer invasion: emphasis on tumor surface proteinases. Cancer Inv 1988; 6: 219-231. 43. Duffy M J, Reilly D, O'Sullivan C, O'Higgins N, Fennelly J J, Andreasen P. Urokinase-plasminogen activator, a new and independent prognostic marker in breast cancer. Cancer Res 1990; 50: 6827--6829. 44. Grondahl-Hansen J, Christensen I J, Rosenquist C et al. High levels of urokinase-type plasminogen activator and its inhibitor PAl-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis. Cancer Res 1993; 53: 2513-2521. 45. Rabbani S A, Harakidas P, Davidson D J, Henkin H, Mazar A P. Prevention of prostate-cancer metastasis in vivo by a novel synthetic inhibitor of urokinase-type plasminogen activator (uPA). Int J Cancer 1995; 63: 840-845. 46. Higazi A A, Mayer M. Enhanced activity of plasmin and plasminogen activation in the presence of oleic acid. In: Brackman P, Kluft C, eds. Plasminogen activation in fibrinolysis, in tissue remodeling, and in development. Ann N Y Acad Sci 1992; 667: 81-83. 47. Du Toit P J, van Aswegen C H, du Plessis D J. The effect of essential fatty acids on growth and urokinase-type plasminogen activator production in human prostate DU-145 cells. Prostagland Leukotr Ess Fatty Acids (in press). 48. Tryggvason K, Hryhty~i M, Salo T. Proteolytic degradation of extracellular matrix in tumor invasion. Biochim Biophys Acta 1987; 907: 191-217. 49. Thorgeirsson U P. Basement membrane type IV collagen degradation: evidence of the involvement of a proteolytic cascade independent of metalloproteinases. Cancer Res 1990; 50: 5997-6001. 50. Kusukawa J, Sasaguri Y, Morimatsu M, Kameyama T. Expression of matrix metalloproteinase-3 in stage I and II squamous cell carcinoma of the oral cavity. J Oral Maxillofac Surg 1995; 53: 530-534. 51. Liotta L A, Rao C N, Wewer U M. Biochemical interaction of tumor cells with the basement membrane. Ann Rev Biochem 1986; 55: 1037-1057. 52. Baker T, Tickle S, Wasan H, Docherty A, Isenberg D, Wakman J. Serum metalloproteinases and their inhibitors: markers for malignant potential. Br J Cancer 1994; 70:506--512. 53. Davies B, Waxman J, Wasan H et al. Levels of matrix metalloproteinases in bladder cancer. Cancer Res 1993; 53: 5365-5369. 54. Ito A, Nakajima S, Sasaguri Y, Nagase H, Mori Y. Co-culture of human breast adenocarcinoma MCF-7 ceils and human dermal fibroblasts enhances the production of matrix metalloproteinases 1, 2 and 3 in fibroblasts. Br J Cancer 1995; 71: 1039-1045. 55. Basset P, Wolf C, Rouyer N, Bellocq J P, Rio M C, Chambon P. Stromelysin 3 in stromal tissue as a control factor in breast cancer behaviour. Cancer 1994; 74: 1045-1049. 56. Johnson S, Johnson F N. Gamma-linolenic acid. Rev Contemp Pharmacotherap 1990; 1: 1-41. 57. Horrobin D F. Essential fatty acids, lipid peroxidation and cancer. In: Horrobin D F, ed. Omega-6 Essential Fatty Acids: Pathophysiology and Roles in Clinical Medicine. New York: Alan Liss, 1990:351-378. 58. Lokeshwar B L, Seizer M G, Block N L, Gunja-Smith Z. Secretion of matrix metalloproteinases and their inhibitors (tissue inhibitor of metalloproteinases) by human prostate in explant cultures: reduced tissue inhibitor of metalloproteinase secretion by malignant tissue. Cancer Res 1993; 53: 4493-4498.
ENZYMES IN TUMOUR-CELLINVASION 59. Ito A, Sato T, Iga T, Moil Y. Tumor necrosis factor bifunctionally regulates matrix metalloproteinases and tissue inhibitor of metalloproteinases (TIMP) production by human fibroblasts. FEBS Lett 1990; 269: 93-95. 60. Watanabe S, Hayashi H, Onozaki K, Okuyama H. Effect of dietary alpha-linolenate/linoleate balance on lipopolysaccharide-induced tumor necrosis factor production in mouse macrophages. Life Sci 1991; 48". 2013-2020. 61. Lotz M, Guerne P A. Inter leukin-6 induces the synthesis of
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A possible role for enzymes in tumour-cell invasion C. H. VAN ASWEGEN, D. J. DU PLESSIS Wolmarans Research Laboratory, Department of Urology, University of Pretoria, Private Bag X169, Pretoria 0001, South Africa. Tel: +27 12 3192547; Fax: +27 12 3295152 (Correspondence to CHvA)
Abstract - - The complex molecular and cellular processes of metastatic invasion as well as the anti-invasion possibilities are summarized. Invasion by neoplastic cells is a major obstacle to successful cancer therapy. Enzymes such as hyaluronidase, sialyltransferase, urokinase-type plasminogen activator, plasmin, matrix metalloproteinases, and others, play central roles in the catabolism of extracellular matrix macromolecules. However, this process can be opposed by inhibitors of these enzymes. Both invasion (promoters) and anti-invasion factors (suppressors) need further investigation, to clarify the role of these factors in the aetiology and possibly in the treatment and prognosis of metastatic cancer.
Introduction
Metastatic invasion
Cancer, particularly prostatic and breast cancer, is one of the commonest illnesses amongst all races and nationalities. It has been estimated that a detectable tumour of 0.5-1 cm in diameter contains approximately one billion ceils. The aetiology of the transformation of a normal cell to a malignant cell is still unclear. In facilitating this process, it has been stated that carcinogenesis is a multistage process, starting with an initiation phase, followed by promotion, transformation, progression and metastatic phases (1,2). For metastasis to be effective, cancer cells have to penetrate the matrix, particularly the basement membrane. This process is known as invasion and includes attachment of tumour cells, proteolysis of matrix components, and migration of tumour cells through the matrix defect (3).
Mechanism of tumour-cell adhesion It is hypothesized that cells, which migrate from the primary tumour in the hostile blood environment, have a surface covering of hyaluronic acid (HA). These cells are kept apart from each other because of the loose, hydrated and porous nature of the HA coat (4). At a stage during development, the tumour cell starts to synthesize and secrete increased hyaluronidase, which degrades the HA of the tumourcell 'cocoon'. Degrading of the HA coat can be prevented by an extract from the herb Echinacea (5). This sequence correlates well with both cell--cell attachment as well as with a decrease in HA and cell-surface molecules that bind HA such as DC44H (4,6). Closer movement of tumour cells within the matrix is thus possible, leading to the final step of
Date received 16 April 1996 Date accepted 17 May 1996 443
444 tumour-cell attachment. Tumour-cell receptors of the integrin and non-integrin type (cadherins, immunoglobulins) would bind to glycoproteins of the extracellular matrix, such as fibronectin, collagen and laminin (3,7-9). Expression of E-cadherin on the primary tumour cells may be increased by addition of the essential fatty acid (EFA), T-linolenic acid (GLA), which would lead to decreased tumour cell detachment and substantial enhancement in the survival rate (7,10). Sialylation (masking) of cell-surface proteins by sialyltransferase, may overcome this anti-invasion effect of E-cadherin, since increased sialyltransferase activity is present with metastasizing tumours (11-13), as well as the enzyme substrate sialic acid (14-17). Addition of the short-chain fatty acid, n-butyrate, decreases sialyltransferase production (18). Cell adhesion occurs only to specific organ matrices and, therefore, it is suggested that 'homing receptors' on invading tumour cells can find specific organ target cells (19-21). The degree by which tumour-cell surface HA is lysed by hyaluronidase may be important, since it may leave a HA 'fingerprint' that consequently identifies a specific addressin molecule on the target endothelium cell. This phenomenon has been referred to as the 'seed and soil' hypothesis, in which the metastatic cells present the 'seed' and the organ environment the 'soil' (22,23). Certain adhesion molecules on the 'soil' endothelial surface can be induced by cytokines such as interleukin-1 (IL-1), tumour necrosis factor (TNF) a and ~'-interferon (24), whilst prostacyclin and tamoxifen inhibit adhesion (25-27).
Proteolysis of extracellular matrix Proteolysis of matrix components composes the next step in the 'invasion cascade'. Enhanced proteolytic activity has been observed in highly malignant tumours (28). It is suggested that the complex process of proteolysis starts with the activation of pro-urokinase type plasminogen activator (pro-uPA) by serine proteases (plasmin) and/or cysteine proteases (cathepsin B and L) (29). Increased urokinase type plasminogen activator (uPA), uPA receptors and cathepsin B levels or activities have been reported with various cancers (30-41). A correlation between uPA production and metastasis has also been reported (29,33,42), therefore, uPA levels may be of prognostic value (43,44). UPA activity may be inhibited by natural or synthetic plasminogen activator inhibitors (PAI) (45). Urokinase and plasmin activity, in the presence of oleic acid, has been enhanced almost 40- and 4-fold, respectively (46). This phenomenon could explain why persons whose diets consist mainly of saturated fatty acids have a greater cancer risk than those whose
MEDICAL HYPOTHESES
diets consist mostly of polyunsaturated fatty acids, which include EFA. Although oleic acid stimulates uPA activity, it inhibits uPA production by DU-145 prostatic cells in culture (47). This is also the case with ALA, eicosapentaenoic acid (EPA), linoleic acid (LA), GLA and arachidonic acid (AA). Activated uPA hydrolyses plasminogen to plasmin, which again directly or indirectly lyses collagen (48,49). In the latter case, plasmin first activates latent collagenase to degrade basement-membrane collagen. A positive correlation exists between squamous-cell carcinoma size, depth of tumour invasion, diffuse invasion mode, high incidence of lymph-node metastasis and matrix metalloproteinasic (MMP) collagenase, MMP-3 (50). Plasmin may also directly hydrolyse the fibronectin and laminin of the basement membrane (48,51). Lysis of basement membrane substrates is not only dependent on the activity of the enzyme, but also on the concentration or production of the enzyme. Thus, an increase in serum collagenase levels has been found in prostatic cancer patients (52), as well as in bladder cancer (53). However, increased MMP production could not be demonstrated in human breast adenocarcinoma cell lines MCF-7 or BT-20 in culture (54). This is not the result when these cells are grown in the presence of fibroblasts. In contrast to normal mammary epithelium cells, which promote only fibroblast production of the tissue inhibitor of metalloproteinase type-1 (T1MP-1), MCF-7 and BT-20 malignant cells stimulate fibroblast production of the precursors of MMP- 1 (interstitial collagenase), MMP2 (gelatinase A), MMP-3 (stromelysin-1), TIMP-1, and MMP-2, TIMP-1, respectively. It is therefore, suggested that proteases are mainly produced by the extracellular matrix and not by the tumour cells themselves (54,55). Since carcinomas contain decreased amounts of EFA, (56,57), the question arises whether EFA could not affect communication between normal and malignant cells. If so, it can be argued that in the presence of EFA only TIMP would be synthesized, whilst at low serum concentrations of EFA, both TIMP and MMPs are produced. To complicate this matter, an imbalance has been found between MMPs and TIMP in prostatic carcinoma (58). A significant increase in TIMPs is present in normal juvenile and adult prostates, whilst neoplastic prostatic tissue contains low levels of TIMPs and shows increased MMP-2 activity. Cytoldnes may also affect the protease production of fibroblasts. TNF influences human fibroblasts bifunctionally by the suppression of TIMP production in addition to stimulation of MMP synthesis (59). Based on this model, it would not be advisable to prescribe cz-linolenate (ALA) to patients with cancer, since increased TNF production has been found with
ENZYMESIN TUMOUR-CELLINVASION murine m a c r o p h a g e s w h e n their diet contains A L A (60). In contrast with T N F and IL-1, interleukin-6 (IL-6) does not stimulate the production o f M M P s , but is a potent inducer o f the synthesis of T I M P - 1 (61). Increased I L - 6 production can be obtained with both l i p o p o l y s a c c h a r i d e (LPS) and certain E F A ( d o c o s a h e x a e n o i c acid, eicosapentaenoic acid), w h i c h w o u l d be preferable in the anti-cancer search, since it stimulates not only T I M P - 1 production, but also the inhibitor o f uPA, PAI-1 (62,63). Other matrix-degrading e n z y m e s p r o d u c e d by tumours are: heparitinases (heparan sulphate, proteoglycans), cathepsin B (collagens, fibronectin, laminin, proteoglycans), and elastase (elastin, collagens, fibronectin, laminin, proteoglycans). It has b e e n suggested that the structure o f the b a s e m e n t m e m b r a n e consists o f an o p e n n e t w o r k (lattice) o f t y p e - I V c o l l a g e n to w h i c h the n o n c o l l a g e n o u s c o m p o n e n t s heparan sulphate, proteoglycans, laminin and fibronectin are b o u n d (48). A c c o r d i n g to this model, proteolysis o f these m a c r o m o l e c u l e s w o u l d not be as important in the invasion process, as the hydrolysis o f c o l l a g e n w h i c h constitutes the basic structure of b a s e m e n t m e m b r a n e . This further e m p h a s i z e s the essential importance o f M M P s . Nevertheless, the overall c o n s e q u e n c e o f proteolysis is r e m o v a l of the matrix substances, after w h i c h the t u m o u r cell can migrate.
445
2. 3. 4. 5.
6.
7.
8. 9. 10. 11.
Migration of tumour cells A f t e r lysis of the b a s e m e n t m e m b r a n e as described above, it is possible for the t u m o u r cells to penetrate the subendothelial b a s e m e n t m e m b r a n e . Irregular p s e u d o p o d i a are p r o j e c t e d in the direction of migration by the invading cell. Proliferation of the t u m o u r cell results in metastasis o f the primary tumour. This process m a y repeat itself m a n y times to g i v e metastases o f the metastasis at secondary sites.
Conclusion The c o m b i n e d investigative data o f the a b o v e theory on i n v a s i o n demonstrated that the invasion cascade is a highly selective process regulated by various mechanisms. O n c e the aetiology of metastatic invasion has been elucidated, better anti-cancer agents can be devised. In this respect, cytokines and E F A m a y be o f importance in the successful prevention of metastatic colonies and should be considered for inclusion in available therapeutic regimens.
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