Int. J. Cancer: 19, 576-580 (1977)
TUMOR CELL LOCOMOTION-A FACTOR IN METASTASIS FORMATION? INFLUENCE OF CYTOCHALASIN B ON A TUMOR DISSEMINATION PATTERN B. HACMAR and W. RYD Dept. of Pathology, Sundsvalls Hospital, Sundsvall, and Institute of Pathology I , University of Goteborg, Goteborg, Sweden.
The influence of Cytochalasin B (CB) on T A 3 ascites tumor cells was studied in vivo in order t o assess whether CB-Induced cell paralysis would affect the transplantation behavior of the cells and in particular tumor distribution after IV cell infusion (" experimental metastases ").Tumor cell pre-treatment with CB (1 ,ug/ml) did not alter the SC or I V transplantability of TA3 cells. Pretreatment with 10,ug/rnl CB, In contrast, consistently increased the incidence and number of extrapulmonary tumor takes from I V transfused cells. The amount of pulmonary tumors was not significantly altered. SC transplantability was not affected by 10pg/ml CB. The importance of cell mobility and cell surface topography for tumor cell nidation in vessels i s discussed.
with the tumors. The TA3 tumor has two sister lines (Friberg, 1972), of which we used the TA3 Ha line (Hauschka et al., 1971). We performed two experiments, A and B, with preincubation of tumor cells in CB, using different concentrations and exposure times (see below). In both experiments the tumor cells to be used were harvested on day 6 after routine transplantation. The cells were washed twice in Hanks' BSS and resuspended after centrifugation in Eagle's MEM at a concentration of los cells/ml. An equal volume of MEM was added with 2 or 20 pg/ml CB (Calbiochem, solubilized in DMSO 10mg/ml and suitably diluted with MEM) to obtain final CB concentrations The majority of circulating tumor cells, released of 1 or 10,ug/ml. To control cells an equal volume or injected into the blood, will perish and not give of MEM with 0.2 % DMSO was added. During the exposure time the cells were stored rise to metastases. Firm endothelial contact is a prerequisite for tumor cell nidation (surveys: at room temperature in glass tubes which were Weiss, 1967; Hagmar, 1970). Membrane movements inverted every minute to prevent the cells from such as microvillus formation have been discussed settling on the bottom. After incubation the susas a means by which circulating cells can lessen pensions were recounted (hemacytometer counts endothelial repulsion and obtain contact for nidation including Trypan blue counts) and diluted with and/or endothelial permeation (Weiss, 1967, 1972~). MEM 10% syngeneic serum to the adequate cell Experimental evidence is lacking, however, on the concentration for injection. Thus, we made no effort effects of altered tumor cell mobility on metastasis to remove CB and/or DMSO prior to injection by dissemination. further washes, but only diluted it with the cell As demonstrated earlier (Ryd and Hagmar, 1977) suspensions. Samples containing 2 x lo" cells were Cytochalasin B (CB) induces a reversible cell taken from each suspension in experiment B to estimate cell aggregability by Millipore counts. paralysis in TA3 ascites tumor cells. In the present set of experiments we injected TA3 Simultaneously, samples were taken for particle tumor cells subcutaneously and intravenously to see size distribution counts on formalin-fixed cells whether CB pre-treatment would alter the trans- in a Celloscope. The methods are described in detail plantation behavior of the cells. In particular, we by Hagmar and Norrby (1973). All cell doses were given in 0.1 ml: SC on the were interested in whether CB treatment might change the pattern of tumor growth after intravenous back or IV into a tail vein. To avoid local recurrences, we amputated the tails after IV injection. injection (" experimental metastases "). MATERIAL AND METHODS
The experiments were performed with inbred A/Sn mice, syngeneic for the TA3 ascites tumor (Friberg, 1972). A breeding nucleus of A/Sn mice was obtained through the courtesy of Dr. George Klein from the Karolinska Institute, Stockholm, together
Experiment A
Two concentrations of CB, I and 10pg/ml, were used for comparison with control cells. The
Received: October 19, 1976 and in revised form January 7, 1971.
577
DISSEMINATION OF CB-TREATED TUMOR CELLS TABLE I EXPERIMENT A. MORTALITY, SURVIVAL TIME AND TUMOR DISTRIBUTION I N A/Sn MICE GIVEN DIFFERENT DOSES OF TA3 Ha CELLS IV
Group
Incidence
414 414 314 11/12
13.5 16.5 19 16.3
414 414 314 11/12
Total
414 414 314 11/12
14.0 18.0 20.0 18.5
414 414 314 11/12
lo6 lo6 lo4
414 414 214
15.5 18.5 19.5
414 414 314
Total
10112
17.7
11/12
Control
lo6 lo6 lo4
Total Cytochalasin B 1 ,ug/ml
lo8 lo6 lo4
Cytochalasin B lO,ug/ml
*
=
Lungs
Mean survival time (days)
Mortality
n$$er
Tumors in other sites Incidence
Number (site)
2 3 0
0.3850.12
114 114 014 2/12
0 1 2
0.48k0.16
014 114 114 2/12 414 414 214
12 9 3
0.36h0.11
10/12*
(heart 2) (lymph nodes 2; ovary 1)
5
(liver 1) (lymph nodes 2)
3
(fat 10; ovary 1; heart 1) (fat 2, lymph nodes 1, heart 5, liver 1) (fat 2, heart 1)
24’
significant difference from controls (p20
1 0 0
Total
1,000 1,000 1,000
578
HAGMAR AND RYD TABLE I11
EXPERIMENT B. SC TUMOR GROWTH OF TA3 CELLS INCUBATED IN CYTOCHALASIN B (CB) 10 r d m l ____
~~
Cell dose
Control cells
____
CB 5 min
CB 60 min
’ One mouse dead from cause other than tumor.
RESULTS
Experiment A
All three suspensions used had a cellular viability of 98 ”/, by Trypan blue counts. All SC injected animals developed tumors (from lo3 and lo2 cells) and the times for tumor development were equal in the three groups. For IV injected animals the mortality, survival times and tumor distribution are given in Table I. Microscopy revealed that only tumor growth was responsible for pulmonary enlargement. There were no significant differences in survival times or lung tumor weights between the groups (p
TUMOR CELL LOCOMOTION-A FACTOR IN METASTASIS FORMATION? INFLUENCE OF CYTOCHALASIN B ON A TUMOR DISSEMINATION PATTERN B. HACMAR and W. RYD Dept. of Pathology, Sundsvalls Hospital, Sundsvall, and Institute of Pathology I , University of Goteborg, Goteborg, Sweden.
The influence of Cytochalasin B (CB) on T A 3 ascites tumor cells was studied in vivo in order t o assess whether CB-Induced cell paralysis would affect the transplantation behavior of the cells and in particular tumor distribution after IV cell infusion (" experimental metastases ").Tumor cell pre-treatment with CB (1 ,ug/ml) did not alter the SC or I V transplantability of TA3 cells. Pretreatment with 10,ug/rnl CB, In contrast, consistently increased the incidence and number of extrapulmonary tumor takes from I V transfused cells. The amount of pulmonary tumors was not significantly altered. SC transplantability was not affected by 10pg/ml CB. The importance of cell mobility and cell surface topography for tumor cell nidation in vessels i s discussed.
with the tumors. The TA3 tumor has two sister lines (Friberg, 1972), of which we used the TA3 Ha line (Hauschka et al., 1971). We performed two experiments, A and B, with preincubation of tumor cells in CB, using different concentrations and exposure times (see below). In both experiments the tumor cells to be used were harvested on day 6 after routine transplantation. The cells were washed twice in Hanks' BSS and resuspended after centrifugation in Eagle's MEM at a concentration of los cells/ml. An equal volume of MEM was added with 2 or 20 pg/ml CB (Calbiochem, solubilized in DMSO 10mg/ml and suitably diluted with MEM) to obtain final CB concentrations The majority of circulating tumor cells, released of 1 or 10,ug/ml. To control cells an equal volume or injected into the blood, will perish and not give of MEM with 0.2 % DMSO was added. During the exposure time the cells were stored rise to metastases. Firm endothelial contact is a prerequisite for tumor cell nidation (surveys: at room temperature in glass tubes which were Weiss, 1967; Hagmar, 1970). Membrane movements inverted every minute to prevent the cells from such as microvillus formation have been discussed settling on the bottom. After incubation the susas a means by which circulating cells can lessen pensions were recounted (hemacytometer counts endothelial repulsion and obtain contact for nidation including Trypan blue counts) and diluted with and/or endothelial permeation (Weiss, 1967, 1972~). MEM 10% syngeneic serum to the adequate cell Experimental evidence is lacking, however, on the concentration for injection. Thus, we made no effort effects of altered tumor cell mobility on metastasis to remove CB and/or DMSO prior to injection by dissemination. further washes, but only diluted it with the cell As demonstrated earlier (Ryd and Hagmar, 1977) suspensions. Samples containing 2 x lo" cells were Cytochalasin B (CB) induces a reversible cell taken from each suspension in experiment B to estimate cell aggregability by Millipore counts. paralysis in TA3 ascites tumor cells. In the present set of experiments we injected TA3 Simultaneously, samples were taken for particle tumor cells subcutaneously and intravenously to see size distribution counts on formalin-fixed cells whether CB pre-treatment would alter the trans- in a Celloscope. The methods are described in detail plantation behavior of the cells. In particular, we by Hagmar and Norrby (1973). All cell doses were given in 0.1 ml: SC on the were interested in whether CB treatment might change the pattern of tumor growth after intravenous back or IV into a tail vein. To avoid local recurrences, we amputated the tails after IV injection. injection (" experimental metastases "). MATERIAL AND METHODS
The experiments were performed with inbred A/Sn mice, syngeneic for the TA3 ascites tumor (Friberg, 1972). A breeding nucleus of A/Sn mice was obtained through the courtesy of Dr. George Klein from the Karolinska Institute, Stockholm, together
Experiment A
Two concentrations of CB, I and 10pg/ml, were used for comparison with control cells. The
Received: October 19, 1976 and in revised form January 7, 1971.
577
DISSEMINATION OF CB-TREATED TUMOR CELLS TABLE I EXPERIMENT A. MORTALITY, SURVIVAL TIME AND TUMOR DISTRIBUTION I N A/Sn MICE GIVEN DIFFERENT DOSES OF TA3 Ha CELLS IV
Group
Incidence
414 414 314 11/12
13.5 16.5 19 16.3
414 414 314 11/12
Total
414 414 314 11/12
14.0 18.0 20.0 18.5
414 414 314 11/12
lo6 lo6 lo4
414 414 214
15.5 18.5 19.5
414 414 314
Total
10112
17.7
11/12
Control
lo6 lo6 lo4
Total Cytochalasin B 1 ,ug/ml
lo8 lo6 lo4
Cytochalasin B lO,ug/ml
*
=
Lungs
Mean survival time (days)
Mortality
n$$er
Tumors in other sites Incidence
Number (site)
2 3 0
0.3850.12
114 114 014 2/12
0 1 2
0.48k0.16
014 114 114 2/12 414 414 214
12 9 3
0.36h0.11
10/12*
(heart 2) (lymph nodes 2; ovary 1)
5
(liver 1) (lymph nodes 2)
3
(fat 10; ovary 1; heart 1) (fat 2, lymph nodes 1, heart 5, liver 1) (fat 2, heart 1)
24’
significant difference from controls (p20
1 0 0
Total
1,000 1,000 1,000
578
HAGMAR AND RYD TABLE I11
EXPERIMENT B. SC TUMOR GROWTH OF TA3 CELLS INCUBATED IN CYTOCHALASIN B (CB) 10 r d m l ____
~~
Cell dose
Control cells
____
CB 5 min
CB 60 min
’ One mouse dead from cause other than tumor.
RESULTS
Experiment A
All three suspensions used had a cellular viability of 98 ”/, by Trypan blue counts. All SC injected animals developed tumors (from lo3 and lo2 cells) and the times for tumor development were equal in the three groups. For IV injected animals the mortality, survival times and tumor distribution are given in Table I. Microscopy revealed that only tumor growth was responsible for pulmonary enlargement. There were no significant differences in survival times or lung tumor weights between the groups (p
