PfliJgersArchiv
Pfltigers Arch. 361, 65-68 (1975)
EuropeanJournal of F~-~o~y 9 by Springer-Verlag 1975
Platelet Adhesiveness and Cancer Cell Stickiness A Comparative Study E. Jacobi II. Medizinische und Poliklinik der Universit~it, D-4000 Diisseldorf 1, MoorenstraBe 5, Federal Republic of Germany
Summary. C o m p a r i n g platelet adhesiveness and cancer cell stickiness u n d e r the conditions o f the so-called Fibre test showed that: 1. Platelets aggregate in the first few seconds only, whereas Y o s h i d a s a r c o m a cells accumulate continuously. 2. Platelets show a characteristic velocity profile. Platelet adhesiveness shows a m a x i m u m at a velocity o f 7 cm/sec. Y o s h i d a s a r c o m a cells adhere better at low flow velocity. 3. A n experimentally induced electric current (streaming electric current) o f ca. 40 m V augments platelet adhesiveness to twice the basal value, whereas Y o s h i d a s a r c o m a cells remain uninfluenced by the current. These results suggest an interaction between platelets and vessel wall, whereas Y o s h i d a s a r c o m a cells adhere passively.
the interactions between platelets and vascular lesions, for example. T o elucidate the m e c h a n i s m o f adhesion initiation, the Fibre test, developed for the m e a s u r e m e n t o f platelet adhesiveness, was used to grade cancer cell stickiness, using Y o s h i d a s a r c o m a cells.
K e y words: Fibre test - Platelet adhesiveness C a n c e r cell stickiness - Interaction with surface.
b) Growth of Yoshida Sarcoma Cells'. Rats were infested with Yoshida sarcoma cells, and the tumours were allowed to grow for 7 - i0 days. 5-10 ml of the cellcontaining ascites fluid was obtained by laparotomy, performed under ether anaesthesia, and the ascites fluid was immediately treated with sodium citrate. The average cell concentration in the fluid was 30000 Yoshida sarcoma cells/gl.
-
INTRODUCTION T h r o m b o s i s f o r m a t i o n is initiated by the adhesion o f b l o o d platelets to an altered vascular surface (e.g. and endothelial lesion). T u m o r cells, which have penetrated into the b l o o d stream, m a y also adhere to the vessel wall or to the endothelial lesion. This represents the starting point o f a metastasis, whereby the cancer cells held on the vascular wall can invade the surr o u n d i n g tissue [2, 4, 8]. It is desirable to lower platelet adhesiveness and cancer cell stickiness for several reasons, including thrombosis prevention and retardation o f the metastasis o f malignomata. It is also o f interest to be able to c o m p a r e platelet adhesiveness and cancer cell stickiness, as this m a y provide a means o f c o m p a r i n g
MATERIALS AND METHODS
a) Fibre Test. A mechanical perfusion device (Fig. 1) provides a well-defined flow of decalcified, citrated blood or Yoshida sarcoma cell ascites fluid along nylon fibres enclosed in a glass capillary of 0.93 mm internal diameter. After perfusion the fibres were carefully removed from the capillary and incubated in distilled water for 1 rain to wash off any cell material not adhering to the fibres, and plasma protein. The protein content of cells adhering to the fibres was then measured using Folin's reagent to determine the extent of platelet adhesiveness or cancer cell stickiness under identical experimental conditions.
c) Scanning Electron Microscopy (SEM). The fibres with adhering platelets or cancer cells were examined by SEM. After fixation in buffered glutaraldehyde solution for 30 min, the fibres were dehydrated using an ascending series of alcohols. Finally the preparation was lyophilised to remove any residual water.
RESULTS Fig.2 shows a b l o o d platelet on a fibre, e n l a r g e d 10000 times. It has already lost its original shape and is spread along the fibre. The m e t a m o r p h o s i s is partially caused by the p r e p a r a t i o n procedure, as can be shown by c o m p a r a t i v e studies with u n w a s h e d fibres. Fig. 3 shows Y o s h i d a s a r c o m a cells on the fibres, with n o aggregation apparent. Fig. 4 shows a strongly
66
E. Jacobi
y
Syringe Citratedblood /
/////J///////,,] ~. / 1//1
"/.
/
~]
r/]
~
PalyvJnyl t, be
Glasscapillary
eibre,
/
! // 9z 9
/-
1//////////~'1
I/ •
/l
"41
//////,i
:. . . . .
I
I! ,
I
i
~o
.....
1
.....
T~
-
(ram)
Fig. 1. Mechanical device for the perfusion of nylon fibres with tumour ascites fluid or citrated blood
Fig. 3. Same part of bundle as in Fig.2 after perfusion with Yoshida ascites fluid and following washing with distilled water
Fig. 2. Detail from a scanning electromicrophotograph of the middle portion fibres after perfusion with citrated blood, following washing with distilled water, showing a platelet spread along the fibre
magnified (1:10000), single Yoshida sarcoma cell, which seems to be glued to the fibres by means of pseudopodium-shaped processes. If the flow time is altered in the measuring system, platelet aggregation on the fibres ceases after a few seconds, whereas the Yoshida sarcoma cells continue to aggregate (Fig. 5). Another fundamental difference can be shown by variation of flow velocity (Fig. 6). Platelet aggregation increases proportionally to the flow velocity until a maximum is reached, and then diminishes again [5]. These characteristics, which are also observed in other experimental set-ups, are not observed when Yoshida sarcoma cells are used. The lower the velocity, the better the cancer cells adhere to the fibres. It should also be mentioned that when varying the flow time, the number of cells passing
Fig.4. Scanning electronmicrophotograph of a Yoshida sarcoma cell adhering to a fibre
through the capillary at different velocities remained constant. Different substances, such as collagen, ADP, Sp 54, induce platelet adhesiveness. Injury may cause an electric current because of differing dielectric constants between the site of injury and blood flow [2]. This current amounts to ca. 20 mV, as shown by inviva experiments of Poliw0da [6]. This current can be experimentally simulated by introducing a BaTiO3-
Platelet Adhesiveness and Cancer Cell Stickiness
67
Fig. 5. Variation of cancer cell stickiness or platelet adhesiveness with streaming time ---
Yoshida - Sarcoma Cells Platelets v = 3,5 c m / s e c
~6C
i*
i. S
I
5
Fig. 6. Variation of cancer cell stickiness or platelet adhesiveness with flow velocity. Platelet adhesiveness shows a maximum at 7 cm/sec, whereas Yoshida sarcoma cells show continuing adhesion with decreasing flow velocity
I
10
15
30
-
Streaming time (sec)
60
Yoshida - S a r c o m a C e l l s 3 0 0 0 0 / m m atelets
200 0 0 0 / m m
3 3
8G
I'o
/s Velocity
be experimentally stimulated by introducing a BaTiO3capillary into the measuring system (Fig.7), thus causing an increase in platelet adhesiveness of twice the original value. Fig.7 also shows that Yoshida sarcoma cells remain unaffected by the simulated electric current.
DISCUSSION The results presented here demonstrate fundamental differences between platelet adhesiveness and cancer cell stickiness. While platelet adhesiveness shows an asymptotic curve after a short time, we find a continuous adherence of cancer cells to fibre material.
go (cm/sec )
Under the assumption that the fibres used in the experiment are analogous to subendothelial fibres bare d by endothelial lesions, the behaviour of the platelets is a expected: after a rapid repair of the vascular lesion, no further clotting, which possibly might lead to vascular occlusion, takes place in physiological conditions. The fact that platelet adhesiveness increases proportionally to the blood velocity has been known for a long time. 100 years ago Zahn and Mantegazza were able to show that white thrombi developed only in circulating blood, and not in stagnating blood [1]. The optimum for platelet adhesiveness occurs at a blood velocity of around 7 cm/sec. This approximates the flow velocity in small- and medium-sized arteries,
68
E. Jacobi
Yoshida - Sarcoma
Platelet$
.S
Cells
14
Z
This" means that platelets react by becoming "sticky". Whether this induction is chemical, biophysical, or polyaetiological, will not be considered here. But it is possible to augment platelet adhesiveness significantly by producing a so-called flow-induced electric current. Streaming electric currents are induced by vascular lesions; for instance Yoshida sarcoma cells remain untouched by experimental flow-induced electric currents. The results suggest an interaction between platelets and an alien surface, e.g. vascular vessel wall, whereas Yoshida sarcoma ceils show passive adhesion.
Z
"~2~l
%
REFERENCES 1. Eberth, J. E., Schimmelbusch, C. : Experimentelle Untersuchungen tiber Thrombose. Virchows Arch. path. Anat. 103, H. 1,105,
H. 2 (1885)
II
Barios- Capatary
Fig. 7. Simulation of a streaming electric current by means of a BaTiOa-capillary. The current is produced by the flow velocity because of differing dielectric constants. Platelet adhesiveness rises to twice the original values whereas Yoshida sarcoma cells remain uninfluenced by the current
which have to depend on fast coagulation after injuries. This platelet action appears to be sufficient for physiological demands. Yoshida sarcoma cells show greater stickiness at low rates of blood velocity. One may conclude from the velocity characteristics of platelets that there is an interaction between platelets and a foreign surface.
2. Gastpar, H.: Stickness of platelets and tumor cell influenced by drugs. In: Platelet adhesion, E. F. MammenetaL, pp. 29i 303. Stuttgart: Schattauer 1970 3. Hagemann, G., Jacobi, E., Joost, P., Poliwoda, H. : Sind physikalische Vorg/inge an der Thrombogenese beteiligt? Studia biophysica (Berl.) 26, 2 2 1 - 232 (1971) 4. Hildegard, P. : The role of blood platelets in experimental metastases. Brit. J. Cancer 28, 429-435 (1973) 5. Jacobi, E., Hagemann, G., Poliwoda, H. : Eine in-vitro-Methode zur Bestimmung der Thromozytenadhfisivit~it. Thrombos. Diathes. haemorrh. (Stuttg.) 26, 192-202 (1971) 6. Poliwoda, H. : personal Communication. Thrombos. Diathes. haemorrh. (Stuttg.) (in press, 1976) 7. Poliwoda, H., Hagemaun, G., Jacobi, E. : Geschwindigkeitsabh~ingige Wechselwirkungen zwischen Thrombozyten und verschiedenen Oberfl/ichen. Klin. Wschr. 48, 442-443 (1970) 8. Wood, S., Jr., Hilgard, P.: Arvin-induced hypofibrinogenemia and metastasis formation from blood-borne cancer cells. Johns Hopk. med. J. 133, 207-213 (1973)
Received July 7, 1975
Pfltigers Arch. 361, 65-68 (1975)
EuropeanJournal of F~-~o~y 9 by Springer-Verlag 1975
Platelet Adhesiveness and Cancer Cell Stickiness A Comparative Study E. Jacobi II. Medizinische und Poliklinik der Universit~it, D-4000 Diisseldorf 1, MoorenstraBe 5, Federal Republic of Germany
Summary. C o m p a r i n g platelet adhesiveness and cancer cell stickiness u n d e r the conditions o f the so-called Fibre test showed that: 1. Platelets aggregate in the first few seconds only, whereas Y o s h i d a s a r c o m a cells accumulate continuously. 2. Platelets show a characteristic velocity profile. Platelet adhesiveness shows a m a x i m u m at a velocity o f 7 cm/sec. Y o s h i d a s a r c o m a cells adhere better at low flow velocity. 3. A n experimentally induced electric current (streaming electric current) o f ca. 40 m V augments platelet adhesiveness to twice the basal value, whereas Y o s h i d a s a r c o m a cells remain uninfluenced by the current. These results suggest an interaction between platelets and vessel wall, whereas Y o s h i d a s a r c o m a cells adhere passively.
the interactions between platelets and vascular lesions, for example. T o elucidate the m e c h a n i s m o f adhesion initiation, the Fibre test, developed for the m e a s u r e m e n t o f platelet adhesiveness, was used to grade cancer cell stickiness, using Y o s h i d a s a r c o m a cells.
K e y words: Fibre test - Platelet adhesiveness C a n c e r cell stickiness - Interaction with surface.
b) Growth of Yoshida Sarcoma Cells'. Rats were infested with Yoshida sarcoma cells, and the tumours were allowed to grow for 7 - i0 days. 5-10 ml of the cellcontaining ascites fluid was obtained by laparotomy, performed under ether anaesthesia, and the ascites fluid was immediately treated with sodium citrate. The average cell concentration in the fluid was 30000 Yoshida sarcoma cells/gl.
-
INTRODUCTION T h r o m b o s i s f o r m a t i o n is initiated by the adhesion o f b l o o d platelets to an altered vascular surface (e.g. and endothelial lesion). T u m o r cells, which have penetrated into the b l o o d stream, m a y also adhere to the vessel wall or to the endothelial lesion. This represents the starting point o f a metastasis, whereby the cancer cells held on the vascular wall can invade the surr o u n d i n g tissue [2, 4, 8]. It is desirable to lower platelet adhesiveness and cancer cell stickiness for several reasons, including thrombosis prevention and retardation o f the metastasis o f malignomata. It is also o f interest to be able to c o m p a r e platelet adhesiveness and cancer cell stickiness, as this m a y provide a means o f c o m p a r i n g
MATERIALS AND METHODS
a) Fibre Test. A mechanical perfusion device (Fig. 1) provides a well-defined flow of decalcified, citrated blood or Yoshida sarcoma cell ascites fluid along nylon fibres enclosed in a glass capillary of 0.93 mm internal diameter. After perfusion the fibres were carefully removed from the capillary and incubated in distilled water for 1 rain to wash off any cell material not adhering to the fibres, and plasma protein. The protein content of cells adhering to the fibres was then measured using Folin's reagent to determine the extent of platelet adhesiveness or cancer cell stickiness under identical experimental conditions.
c) Scanning Electron Microscopy (SEM). The fibres with adhering platelets or cancer cells were examined by SEM. After fixation in buffered glutaraldehyde solution for 30 min, the fibres were dehydrated using an ascending series of alcohols. Finally the preparation was lyophilised to remove any residual water.
RESULTS Fig.2 shows a b l o o d platelet on a fibre, e n l a r g e d 10000 times. It has already lost its original shape and is spread along the fibre. The m e t a m o r p h o s i s is partially caused by the p r e p a r a t i o n procedure, as can be shown by c o m p a r a t i v e studies with u n w a s h e d fibres. Fig. 3 shows Y o s h i d a s a r c o m a cells on the fibres, with n o aggregation apparent. Fig. 4 shows a strongly
66
E. Jacobi
y
Syringe Citratedblood /
/////J///////,,] ~. / 1//1
"/.
/
~]
r/]
~
PalyvJnyl t, be
Glasscapillary
eibre,
/
! // 9z 9
/-
1//////////~'1
I/ •
/l
"41
//////,i
:. . . . .
I
I! ,
I
i
~o
.....
1
.....
T~
-
(ram)
Fig. 1. Mechanical device for the perfusion of nylon fibres with tumour ascites fluid or citrated blood
Fig. 3. Same part of bundle as in Fig.2 after perfusion with Yoshida ascites fluid and following washing with distilled water
Fig. 2. Detail from a scanning electromicrophotograph of the middle portion fibres after perfusion with citrated blood, following washing with distilled water, showing a platelet spread along the fibre
magnified (1:10000), single Yoshida sarcoma cell, which seems to be glued to the fibres by means of pseudopodium-shaped processes. If the flow time is altered in the measuring system, platelet aggregation on the fibres ceases after a few seconds, whereas the Yoshida sarcoma cells continue to aggregate (Fig. 5). Another fundamental difference can be shown by variation of flow velocity (Fig. 6). Platelet aggregation increases proportionally to the flow velocity until a maximum is reached, and then diminishes again [5]. These characteristics, which are also observed in other experimental set-ups, are not observed when Yoshida sarcoma cells are used. The lower the velocity, the better the cancer cells adhere to the fibres. It should also be mentioned that when varying the flow time, the number of cells passing
Fig.4. Scanning electronmicrophotograph of a Yoshida sarcoma cell adhering to a fibre
through the capillary at different velocities remained constant. Different substances, such as collagen, ADP, Sp 54, induce platelet adhesiveness. Injury may cause an electric current because of differing dielectric constants between the site of injury and blood flow [2]. This current amounts to ca. 20 mV, as shown by inviva experiments of Poliw0da [6]. This current can be experimentally simulated by introducing a BaTiO3-
Platelet Adhesiveness and Cancer Cell Stickiness
67
Fig. 5. Variation of cancer cell stickiness or platelet adhesiveness with streaming time ---
Yoshida - Sarcoma Cells Platelets v = 3,5 c m / s e c
~6C
i*
i. S
I
5
Fig. 6. Variation of cancer cell stickiness or platelet adhesiveness with flow velocity. Platelet adhesiveness shows a maximum at 7 cm/sec, whereas Yoshida sarcoma cells show continuing adhesion with decreasing flow velocity
I
10
15
30
-
Streaming time (sec)
60
Yoshida - S a r c o m a C e l l s 3 0 0 0 0 / m m atelets
200 0 0 0 / m m
3 3
8G
I'o
/s Velocity
be experimentally stimulated by introducing a BaTiO3capillary into the measuring system (Fig.7), thus causing an increase in platelet adhesiveness of twice the original value. Fig.7 also shows that Yoshida sarcoma cells remain unaffected by the simulated electric current.
DISCUSSION The results presented here demonstrate fundamental differences between platelet adhesiveness and cancer cell stickiness. While platelet adhesiveness shows an asymptotic curve after a short time, we find a continuous adherence of cancer cells to fibre material.
go (cm/sec )
Under the assumption that the fibres used in the experiment are analogous to subendothelial fibres bare d by endothelial lesions, the behaviour of the platelets is a expected: after a rapid repair of the vascular lesion, no further clotting, which possibly might lead to vascular occlusion, takes place in physiological conditions. The fact that platelet adhesiveness increases proportionally to the blood velocity has been known for a long time. 100 years ago Zahn and Mantegazza were able to show that white thrombi developed only in circulating blood, and not in stagnating blood [1]. The optimum for platelet adhesiveness occurs at a blood velocity of around 7 cm/sec. This approximates the flow velocity in small- and medium-sized arteries,
68
E. Jacobi
Yoshida - Sarcoma
Platelet$
.S
Cells
14
Z
This" means that platelets react by becoming "sticky". Whether this induction is chemical, biophysical, or polyaetiological, will not be considered here. But it is possible to augment platelet adhesiveness significantly by producing a so-called flow-induced electric current. Streaming electric currents are induced by vascular lesions; for instance Yoshida sarcoma cells remain untouched by experimental flow-induced electric currents. The results suggest an interaction between platelets and an alien surface, e.g. vascular vessel wall, whereas Yoshida sarcoma ceils show passive adhesion.
Z
"~2~l
%
REFERENCES 1. Eberth, J. E., Schimmelbusch, C. : Experimentelle Untersuchungen tiber Thrombose. Virchows Arch. path. Anat. 103, H. 1,105,
H. 2 (1885)
II
Barios- Capatary
Fig. 7. Simulation of a streaming electric current by means of a BaTiOa-capillary. The current is produced by the flow velocity because of differing dielectric constants. Platelet adhesiveness rises to twice the original values whereas Yoshida sarcoma cells remain uninfluenced by the current
which have to depend on fast coagulation after injuries. This platelet action appears to be sufficient for physiological demands. Yoshida sarcoma cells show greater stickiness at low rates of blood velocity. One may conclude from the velocity characteristics of platelets that there is an interaction between platelets and a foreign surface.
2. Gastpar, H.: Stickness of platelets and tumor cell influenced by drugs. In: Platelet adhesion, E. F. MammenetaL, pp. 29i 303. Stuttgart: Schattauer 1970 3. Hagemann, G., Jacobi, E., Joost, P., Poliwoda, H. : Sind physikalische Vorg/inge an der Thrombogenese beteiligt? Studia biophysica (Berl.) 26, 2 2 1 - 232 (1971) 4. Hildegard, P. : The role of blood platelets in experimental metastases. Brit. J. Cancer 28, 429-435 (1973) 5. Jacobi, E., Hagemann, G., Poliwoda, H. : Eine in-vitro-Methode zur Bestimmung der Thromozytenadhfisivit~it. Thrombos. Diathes. haemorrh. (Stuttg.) 26, 192-202 (1971) 6. Poliwoda, H. : personal Communication. Thrombos. Diathes. haemorrh. (Stuttg.) (in press, 1976) 7. Poliwoda, H., Hagemaun, G., Jacobi, E. : Geschwindigkeitsabh~ingige Wechselwirkungen zwischen Thrombozyten und verschiedenen Oberfl/ichen. Klin. Wschr. 48, 442-443 (1970) 8. Wood, S., Jr., Hilgard, P.: Arvin-induced hypofibrinogenemia and metastasis formation from blood-borne cancer cells. Johns Hopk. med. J. 133, 207-213 (1973)
Received July 7, 1975
