- Email: [email protected]
Migration stimulating activity in serum of breast cancer patients
130
Migration stimulating activity in serum of breast cancer patients
An assay to measure the ability to stimulate migration of fibroblasts into collagen gel was carried out on
from treated and untreated breast cancer patients and from healthy controls. Migration stimulating activity was found in the serum of 10 (83%) of 12 untreated breast cancer patients immediately before surgical resection of the primary tumour and in 9 (75%) of them 4 days after resection; in 13 (93%) of 14 patients 1-13 years after tumour resection who had received adjuvant treatment; and in 2 (10%) of 20 healthy women matched for age. The migration stimulating activity in cancer patients’ serum was indistinguishable from the migration stimulating factor produced in vitro by fetal and cancer patient skin fibroblasts in its behaviour in various biochemical fractionation procedures. The presence of this activity in the serum of treated breast cancer patients clearly distinguishes it from other oncofetal proteins, which all seem to be produced by serum
tumours.
Introduction Several pathological and physiological processes, such as invasion and wound healing, depend on active cell migration. Various factors contribute to the control of such processes, reflecting properties intrinsic to the motile cells themselves as well as their interaction with the surrounding stroma. The nature of such dynamic and reciprocal interactions is the subject of much interest. Fibroblasts plated onto the surface of type I collagen gels are able to migrate into the underlying three-dimensional fibrillar matrix.1 The kinetics and extent of such migration may be expressed in quantitative terms by direct microscopic observation.2 With this experimental approach, we have previously reported that fetal skin fibroblasts plated at confluent cell density migrate into the collagen gel matrix to a significantly greater extent than do their normal adult counterparts.3 The differential behaviour of fetal and adult fibroblasts in this migration assay thus provides a further criterion by which to assess fibroblast phenotype in vitro. There have been several reports of the presence of aberrant fibroblasts in patients with different types of cancer. We have found that both tumour-derived and skin fibroblasts from about 50% of patients with non-familial breast cancer and more than 90% of those with familial breast cancer behave in the same way as fetal fibroblasts in the collagen gel migration assay.7,8Epidemiological studies have shown that first-degree female relatives of familial cancer patients have a significantly raised cumulative lifetime risk of breast cancer themselves;9 it is therefore interesting that skin fibroblasts
from more than 50% of such first-degree relatives showed aberrant (ie, fetal-fibroblast-like) migratory behaviour." These observations suggest that the presence of aberrant stromal fibroblasts may precede the development of overt malignant disease. Fetal and cancer patient fibroblasts produce a soluble migration stimulating factor (MSF), which is not made by normal adult cells." MSF has been purified12 and initial biochemical characterisation shows that it has a molecular mass of 70 kD. Several other cell motility factors have been described; they include scatter factor 13 " and autocrine factor. 15 Differences in biochemical motility characterisation and target cell specificities suggest that these factors are distinct biochemical entities. The possible function of this class of cytokine in the control of cell motility during normal development and various pathological processes, such as tumour invasion, has been reviewed elsewhere. 16,17 Adult fibroblasts exposed to MSF show greatly increased migratory activity into collagen gels, thus providing a convenient bioassay for measurement of migration stimulating activity. We have used this bioassay to test for migration stimulating activity in the serum of breast cancer patients before and after surgical resection of the primary lesion.
Patients and methods
tumour
samples were collected from two groups of breast cancer patients the University Hospital of South Manchester (table r). The untreated group consisted of newly diagnosed patients (median age 59-0 years) from whom serum was collected 24 h before surgical resection of the primary tumour mass and 4 days after the operation; none of these women had received any form of adjuvant treatment. The treated group consisted of patients (median age 62-5 years) examined 1-13 years after resection of the primary tumour; they had received adjuvant treatment. All but 3 of the patients had invasive ductal carcinoma; patients 8, 18, and 26 had invasive lobular carcinoma. Only 4 patients (8, 17, 19, and 25) had a family history of breast cancer. Serum samples were collected from age-matched, healthy control women with no family history of breast cancer (median age 51 [range 33-76] years). 10 ml venous blood was collected into non-heparinised plastic tubes containing glass beads and allowed to clot. The samples were spun at 3000 g for 10 min in a bench centrifuge. The upper serum layer was diluted 1/10 (about 15 mg total protein) and 0ml was loaded onto a 30 ml ’Sepharose CL-6B’ chromatography column Serum
at
ADDRESSES Department of Cell and Structural Biology, University of Manchester (M. Picardo, PhD, S. L. Schor, PhD, A-M Grey, PhD), and Department of Medical Oncology, Christie Hospital, Manchester, UK (A Howell, FRCP, I. Laidlaw, FRCS, J Redford, BSc, A M Schor, PhD). Correspondence should be addressed to Dr S. L. Schor, Department of Cell and Structural Biology, University of Manchester, Coupland 3 Building, Manchester M13 9PL, UK
131
TABLE I-CLINICAL DATA OF BREAST CANCER PATIENTS
Evidence for presence of MSF in
serum
from breast
cancer
patients. . = assay control.
5% calf
in all cultures (assay control and test) and 25% in the test cultures; the 1 ml serum fraction fraction patient used in the assay is diluted about 1/3000 from the original serum serum
serum
NDnot
done,
NA=not
available,
XRT= radiotherapy, WLEw!de
TAM=tamox!fen,
local excision;
MX=methotrexate,
MELP = melphalan.
and eluted with 20 mmol/1 "tris" -HCI/0’I5 molll sodium chloride pH 7-4. Thirty 1 ml fractions were collected over a 90 min running time, and the optical density at 280 nm was measured for each fraction. 0-1 ml was then taken from each fraction and diluted with 09 ml serum-free minimum essential Eagle’s medium. The samples were filter-sterilised and assessed for migration stimulating activity. The sepharose column was calibrated by monitoring the elution profile of molecular weight standards (bovine serum albumin, catalase, alcohol dehydrogenase, and cytochrome C). For the migration assay type I collagen was extracted from rat tail tendons in 3% acetic acid, dialysed for 2 days against distilled water, and used to make 2 ml collagen gels in 35 mm plastic tissue culture dishes.1 In our standard migration assay, collagen gels were overlaid with either 1 ml medium (assay control) or 1 ml of the patient serum fraction. Adult fibroblasts (2 x 105) were plated onto the gel in 1 ml growth medium containing 20% aseptic calf serum; a confluent monolayer was produced immediately after the cells attached and spread on the gel surface (1-2 h after plating). With the 2 ml volume of the collagen gel, this procedure results in a final concentration of TABLE II-MIGRATION STIMULATING ACTIVITY IN SERUM FROM UNTREATED BREAST CANCER PATIENTS
sample. Migration
data are expressed as the percentage of fibroblasts within the three-dimensional gel matrix after 4 days of incubation, determined by counting of the number of cells on the gel surface and within the collagen matrix in fifteen randomly selected fields by means of a Leitz ‘Labovert’ microscope.l More than 1000 cells were counted for each determination.
Results All the fractions collected from each serum sample were tested for migration stimulating activity. A fraction was considered to contain significant activity if the migration of the target adult fibroblast line was stimulated to values greater than 1 -80 times that achieved in the assay control; from the dose-response curve for purified MSF this value represents a minimum of 0-5 ng/ml MSF.18 Significant migration stimulating activity was present in 10 (83%) of 12 serum samples taken from untreated patients 24 h before surgical resection of the primary tumour and in 9 (75%) of these patients 4 days after their operations (table II). All the untreated patients were positive for migration stimulating activity in at least one of the two assays (before and after resection of the tumour). Failure to detect significant migration stimulating activity in the patient serum samples seems to reflect an occasional false-negative in the assay; most of these false-negatives were associated with a high level of migration in the assay control. The peak in migration stimulating activity, when present, eluted between fractions 10 and 12, corresponding to an apparent molecular mass of 60-70 kD (figure). Migration stimulating activity was observed in 13 (93%) of the 14 samples from treated patients (table III). Again the peak of activity always eluted in fractions 10-12 (figure). Migration stimulating activity was described in only 2 (10%) of the 20 controls (table IV); the figure shows a representative negative activity profile. Control samples were assayed blind with paired patient samples in all
132
TABLE I I I-MIGRATI ON STIMULATING ACTIVITY INS SERUM FROM TREATED BREAST CANCER PATIENTS
Discussion
Migration stimulating activity was detected in the serum of over 80% of the breast cancer patients we examined, compared with only 10 % of healthy controls. The migration stimulating activity in patient serum was indistinguishable from fibroblast-produced MSF in terms of its behaviour in various biochemical fractionation procedures. The continued presence of migration stimulating activity in the serum after resection of the primary tumour and administration of adjuvant treatment is consistent with our previous observations on the systemic production of MSF by uninvolved skin fibroblasts in such individuals." Decker and colleaguesl9 reported the presence of hyaluronic acid stimulating activity (HASA) in serum from untreated breast cancer patients, but could not detect it after surgical resection of the primary tumour in patients with no experiments, so that the likelihood of scoring artifacts was reduced. Repeat determinations of control samples were always consistent. The 58 individual assay controls (ie, cultures incubated without any test serum fraction) showed a narrow distribution profile-mean 4-48 (SD -1’58). Thus, the bioassay has an intrinsically stable baseline upon which reliable determinations of migration stimulating activity can generally be made. Statistical analyses (paired Student’s t test) showed that both untreated and treated breast cancer patients achieved significantly greater relative stimulation than their age-matched controls (p < 0-005). The elution profile of the migration stimulating activity in breast cancer patient serum was indistinguishable from that of MSF in fetal and breast cancer patient fibroblast conditioned medium fractionated under the same conditions (figure). To characterise the serum factor further, samples of breast cancer patient serum were fractionated according to the purification scheme devised for MSF;z precipitation with ammonium sulphate, heparin affinity chromatography, gel filtration chromatography (’Superose12’), and reverse phase chromatography. The behaviour of the migration stimulating activity in cancer patient serum was identical to that previously reported for MSF at all of these stages (data not shown). TABLE IV-MIGRATION STIMULATING ACTIVITY IN SERUM FROM CONTROLS
residual disease. The presence of MSF in the serum of postoperative breast cancer patients clearly distinguishes it from HASA and other oncofetal proteins, all of which seem to be produced by tumours. MSF is therefore not a tumour marker but an indicator of the systemic presence of abnormal stromal fibroblasts. There have been many reports on the presence of aberrant fibroblasts in cancer patients. We have previously postulated that these cells have an active role in cancer pathogenesis .20 The results of this study raise the possibility that, if a rapid and quantitative assay for MSF could be developed, it might find application (probably in conjunction with other procedures) in screening for people at high risk of the development of cancer. This study was supported by grants from the Cancer Research and the Breast Cancer Research Trust.
Campaign
REFERENCES
proliferation and migration within three-dimensional collagen gels. J Cell Sci 1980; 41: 159-75. 2. Schor SL, Court J. Different mechanisms involved in the attachment of cells to native and denatured collagen. J Cell Sci 1979; 38: 267-81. 3. Schor SL, Schor AM, burning P, et al. Skin fibroblasts obtained from cancer patients display fetal-like behaviour in collagen gels. J Cell Sci 1985; 73: 235-44. 4. Schor SL, Schor AM, Howell A, et al. The possible role of abnormal fibroblasts in the pathogenesis of breast cancer. In: Rich MA, Hager JC, Lopez DM, eds. Breast cancer: scientific and clinical progress. Boston/Dordrecht: Kluwer Academic Publishers, 1987: 142-57. 5. Schor SL, Schor AM. Clonal heterogeneity in fibroblast phenotype: implications for the control of epithelial-mesenchymal interactions. Bioessays 1987; 7: 200-04. 6. Azzarone B, Morel M, Billard C, et al. Abnormal properties of skin fibroblasts from patients with breast cancer. Int J Cancer 1984; 33: 1. Schor SL. Cell
759-64.
Durning P, Schor SL, Sellwood RAS. Fibroblasts from patients with breast cancer show abnormal migratory behaviour in vitro. Lancet 1984; i: 890-92. 8. Schor SL, Haggle JA, Durning P, et al. Occurrence of fetal fibroblast phenotype in familial breast cancer. Int J Cancer 1986; 37: 831-36. 9. Ottman R, Pike MC, King M-C, et al. Practical guide for estimating risk of familial breast cancer. Lancet 1983; ii: 556-58. 10. Haggie J, Howell A, Sellwood RAS, et al. Fibroblasts from relatives of patients with hereditary breast cancer show fetal-like behavior in vitro. Lancet 1987; i: 1477-57. 11. Schor SL, Schor AM, Grey AM, et al. Fetal and cancer patient fibroblasts produce an autocrine migration stimulating factor not made by normal adult cells. J Cell Sci 1988; 90: 391-99. 12. Grey AM, Schor AM, Rushton G, et al. Production of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts. Proc Natl Acad Sci USA 1989; 86: 2438-42. 13. Stoker M, Gherardi E, Perryman M, et al. Scatter factor is a fibroblast-denved modulator of epithelial cell mobility. Nature 1987, 7.
327: 239-42.
133
14. Gherardi E, Gray J, Stoker M, et al. Purification of scatter factor, a fibroblast-derived basic protein that modulates epithelial interactions and movement. Proc Natl Acad Sci USA 1989; 86: 5844-48. 15. Liotta LA, Mandler R, Murano G, et al. Tumour cell autocrine motility factor. Proc Natl Acad Sci USA 1986; 83: 3302-06. 16. Warn RM, Dowrick P. Motility factors on the march. Nature 1989; 340: 186-87. 17. Rosen EM, Goldberg ID. Protein factors which regulate cell motility. In Vitro Cell Dev Biol 1989; 25: 1079-87. 18. Schor SL, Schor AM, Grey AM, et al. Mechanism of action of the
migration stimulating factor produced by fetal and cancer patient fibroblasts; effect of hyaluronic add synthesis. In Vitro Cell Develop Biol 1988; 25: 137-46. M, Chiu ES, Dollbaum C, et al. Hyaluronic acid stimulating activity from the bovine fetus and from breast cancer patients. Cancer Res 1989; 49: 3499-505. 20. Schor SL, Schor AM, Howell A, et al. Hypothesis: the persistent expression of fetal-like phenotypic characteristics by fibroblasts is associated with an increased susceptibility to neoplastic disease. Exp Cell Biol 1987; 55: 11-17. 19. Decker
Feasibility of reversing benzodiazepine tolerance with flumazenil
To examine whether the benzodiazepine antagonist flumazenil can reverse tolerance to benzodiazepines but without precipitating withdrawal seizures, the antiepileptic effect of flumazenil itself and its ability to reverse tolerance at a dose that would leave sufficient receptors free for the binding of benzodiazepines were investigated. Electroencephalographic studies in 6 patients with partial and 6 with generalised seizures showed that flumazenil had a short (20 min) non-dose-dependent suppressant effect on epileptic discharges in those with partial seizures. Receptor occupancy studies in 12 patients showed that 1·5 mg flumazenil given intravenously occupied 55% receptors, whereas 15 mg occupied nearly all receptors. When 3 patients with partial seizures who had become tolerant to clonazepam were given 1·5 mg flumazenil, they were seizure-free for 6-21 days after the injection. The value of intermittent therapy with a benzodiazepine antagonist for preventing or reversing tolerance to benzodiazepine agonists ought to be investigated further.
Introduction The
development of tolerance’ is a crucial problem with benzodiazepine therapy. Although how tolerance develops is still obscure, there
are
indications that, like the
pharmacological activity of benzodiazepines, it is mediated through the gamma-aminobutyric acid (GABA) /benzodiazepine chloride ionophore complex.2 The imidazol-benzodiazepine flumazenil (Ro 15-1788) is a benzodiazepine receptor antagonist with high affinity for the receptor. 3-6 According to recent reports based on animal studies flumazenil may reverse subsensitivity to GABA induced by chronic benzodiazepine therapy, perhaps by producing conformational changes of the GABA/ benzodiazepine receptor complex that leads to a "reset" of its sensitivity to GABA .2,7,11 If flumazemil produces such changes in the receptor in man, it could be used for the prevention and reversal of benzodiazepine tolerance. It could perhaps be given intermittently during continuous treatment with a benzodiazepine agonist. Such a regimen may carry the risk of withdrawal convulsions precipitated by
flumazenil-induced
displacement of the agonist from benzodiazepine receptors.9 However, flumazenil is also reported to have some anticonvulsant activity, 10-13 so there a concentration of flumazenil in the brain tissue at which the drug exerts both its "protective" antiepileptic activity and its reset effect. To be of clinical value that concentration must be such that enough benzodiazepine receptors are left unoccupied by flumazenil to allow a benzodiazepine agonist to be bound at concentrations that
may be
therapeutically effective. study was to investigate the dose of flumazenil required to suppress electroencephalographic (EEG) epileptic discharges and to measure with positronemission tomography (PET) the degree of benzodiazepine
are
The aim of this
different doses. A suitable dose of flumazenil was then selected for injection into 3 epileptic patients resistant to clonazepam.
receptor occupancy
at
Patients and methods Informed was
consent was obtained from all patients and the study approved by the local ethics and radiation safety committees.
Effect of flumazenil on EEG
epileptic discharges
The effect of flumazenil on epileptic discharges in the electroencephalogram was tested in 6 patients with complex partial and 6 with primary generalised seizures. The diagnosis and classification was based on clinical investigations and EEG recordings, in accordance with the international classification of epilepsies .14 4 of the patients with complex partial seizures had absences (2 with lateralised automatisms), and 2 others had attacks with "dreamy state" and confusion. The epileptic focus was always in the temporal lobe. The two groups were similar in mean ages (37 [SD 7] vs 33 [6] yr) and weight (69 [7] vs 62 [5] kg). Other medication was restricted to carbamazepine and phenytoin. Benzodiazepines were prohibited for the 2 months before the experiments. The EEGs were recorded with scalp electrodes, placed according to the 10/20 system. Every patient was examined on four different days (fig 1). A recording was started 30 minutes before and continued without interruption during and between injections of vehicle (ethanol, propylene-glycol, and sodium chloride) and the ADDRESSES: Department of Clinical Neurophysiology, Karolinska Hospital (I. Savic, MD, L. Widén, MD, S. Stone-Elander, phD) and Karolinska Pharmacy (S. Stone-Elander), Stockholm, Sweden. Correspondence to Dr Ivanka Savic, Department of Clinical Neurophysiology, Karolinska Hospital, PO Box 60500, S 104 01 Stockholm, Sweden.
Migration stimulating activity in serum of breast cancer patients
An assay to measure the ability to stimulate migration of fibroblasts into collagen gel was carried out on
from treated and untreated breast cancer patients and from healthy controls. Migration stimulating activity was found in the serum of 10 (83%) of 12 untreated breast cancer patients immediately before surgical resection of the primary tumour and in 9 (75%) of them 4 days after resection; in 13 (93%) of 14 patients 1-13 years after tumour resection who had received adjuvant treatment; and in 2 (10%) of 20 healthy women matched for age. The migration stimulating activity in cancer patients’ serum was indistinguishable from the migration stimulating factor produced in vitro by fetal and cancer patient skin fibroblasts in its behaviour in various biochemical fractionation procedures. The presence of this activity in the serum of treated breast cancer patients clearly distinguishes it from other oncofetal proteins, which all seem to be produced by serum
tumours.
Introduction Several pathological and physiological processes, such as invasion and wound healing, depend on active cell migration. Various factors contribute to the control of such processes, reflecting properties intrinsic to the motile cells themselves as well as their interaction with the surrounding stroma. The nature of such dynamic and reciprocal interactions is the subject of much interest. Fibroblasts plated onto the surface of type I collagen gels are able to migrate into the underlying three-dimensional fibrillar matrix.1 The kinetics and extent of such migration may be expressed in quantitative terms by direct microscopic observation.2 With this experimental approach, we have previously reported that fetal skin fibroblasts plated at confluent cell density migrate into the collagen gel matrix to a significantly greater extent than do their normal adult counterparts.3 The differential behaviour of fetal and adult fibroblasts in this migration assay thus provides a further criterion by which to assess fibroblast phenotype in vitro. There have been several reports of the presence of aberrant fibroblasts in patients with different types of cancer. We have found that both tumour-derived and skin fibroblasts from about 50% of patients with non-familial breast cancer and more than 90% of those with familial breast cancer behave in the same way as fetal fibroblasts in the collagen gel migration assay.7,8Epidemiological studies have shown that first-degree female relatives of familial cancer patients have a significantly raised cumulative lifetime risk of breast cancer themselves;9 it is therefore interesting that skin fibroblasts
from more than 50% of such first-degree relatives showed aberrant (ie, fetal-fibroblast-like) migratory behaviour." These observations suggest that the presence of aberrant stromal fibroblasts may precede the development of overt malignant disease. Fetal and cancer patient fibroblasts produce a soluble migration stimulating factor (MSF), which is not made by normal adult cells." MSF has been purified12 and initial biochemical characterisation shows that it has a molecular mass of 70 kD. Several other cell motility factors have been described; they include scatter factor 13 " and autocrine factor. 15 Differences in biochemical motility characterisation and target cell specificities suggest that these factors are distinct biochemical entities. The possible function of this class of cytokine in the control of cell motility during normal development and various pathological processes, such as tumour invasion, has been reviewed elsewhere. 16,17 Adult fibroblasts exposed to MSF show greatly increased migratory activity into collagen gels, thus providing a convenient bioassay for measurement of migration stimulating activity. We have used this bioassay to test for migration stimulating activity in the serum of breast cancer patients before and after surgical resection of the primary lesion.
Patients and methods
tumour
samples were collected from two groups of breast cancer patients the University Hospital of South Manchester (table r). The untreated group consisted of newly diagnosed patients (median age 59-0 years) from whom serum was collected 24 h before surgical resection of the primary tumour mass and 4 days after the operation; none of these women had received any form of adjuvant treatment. The treated group consisted of patients (median age 62-5 years) examined 1-13 years after resection of the primary tumour; they had received adjuvant treatment. All but 3 of the patients had invasive ductal carcinoma; patients 8, 18, and 26 had invasive lobular carcinoma. Only 4 patients (8, 17, 19, and 25) had a family history of breast cancer. Serum samples were collected from age-matched, healthy control women with no family history of breast cancer (median age 51 [range 33-76] years). 10 ml venous blood was collected into non-heparinised plastic tubes containing glass beads and allowed to clot. The samples were spun at 3000 g for 10 min in a bench centrifuge. The upper serum layer was diluted 1/10 (about 15 mg total protein) and 0ml was loaded onto a 30 ml ’Sepharose CL-6B’ chromatography column Serum
at
ADDRESSES Department of Cell and Structural Biology, University of Manchester (M. Picardo, PhD, S. L. Schor, PhD, A-M Grey, PhD), and Department of Medical Oncology, Christie Hospital, Manchester, UK (A Howell, FRCP, I. Laidlaw, FRCS, J Redford, BSc, A M Schor, PhD). Correspondence should be addressed to Dr S. L. Schor, Department of Cell and Structural Biology, University of Manchester, Coupland 3 Building, Manchester M13 9PL, UK
131
TABLE I-CLINICAL DATA OF BREAST CANCER PATIENTS
Evidence for presence of MSF in
serum
from breast
cancer
patients. . = assay control.
5% calf
in all cultures (assay control and test) and 25% in the test cultures; the 1 ml serum fraction fraction patient used in the assay is diluted about 1/3000 from the original serum serum
serum
NDnot
done,
NA=not
available,
XRT= radiotherapy, WLEw!de
TAM=tamox!fen,
local excision;
MX=methotrexate,
MELP = melphalan.
and eluted with 20 mmol/1 "tris" -HCI/0’I5 molll sodium chloride pH 7-4. Thirty 1 ml fractions were collected over a 90 min running time, and the optical density at 280 nm was measured for each fraction. 0-1 ml was then taken from each fraction and diluted with 09 ml serum-free minimum essential Eagle’s medium. The samples were filter-sterilised and assessed for migration stimulating activity. The sepharose column was calibrated by monitoring the elution profile of molecular weight standards (bovine serum albumin, catalase, alcohol dehydrogenase, and cytochrome C). For the migration assay type I collagen was extracted from rat tail tendons in 3% acetic acid, dialysed for 2 days against distilled water, and used to make 2 ml collagen gels in 35 mm plastic tissue culture dishes.1 In our standard migration assay, collagen gels were overlaid with either 1 ml medium (assay control) or 1 ml of the patient serum fraction. Adult fibroblasts (2 x 105) were plated onto the gel in 1 ml growth medium containing 20% aseptic calf serum; a confluent monolayer was produced immediately after the cells attached and spread on the gel surface (1-2 h after plating). With the 2 ml volume of the collagen gel, this procedure results in a final concentration of TABLE II-MIGRATION STIMULATING ACTIVITY IN SERUM FROM UNTREATED BREAST CANCER PATIENTS
sample. Migration
data are expressed as the percentage of fibroblasts within the three-dimensional gel matrix after 4 days of incubation, determined by counting of the number of cells on the gel surface and within the collagen matrix in fifteen randomly selected fields by means of a Leitz ‘Labovert’ microscope.l More than 1000 cells were counted for each determination.
Results All the fractions collected from each serum sample were tested for migration stimulating activity. A fraction was considered to contain significant activity if the migration of the target adult fibroblast line was stimulated to values greater than 1 -80 times that achieved in the assay control; from the dose-response curve for purified MSF this value represents a minimum of 0-5 ng/ml MSF.18 Significant migration stimulating activity was present in 10 (83%) of 12 serum samples taken from untreated patients 24 h before surgical resection of the primary tumour and in 9 (75%) of these patients 4 days after their operations (table II). All the untreated patients were positive for migration stimulating activity in at least one of the two assays (before and after resection of the tumour). Failure to detect significant migration stimulating activity in the patient serum samples seems to reflect an occasional false-negative in the assay; most of these false-negatives were associated with a high level of migration in the assay control. The peak in migration stimulating activity, when present, eluted between fractions 10 and 12, corresponding to an apparent molecular mass of 60-70 kD (figure). Migration stimulating activity was observed in 13 (93%) of the 14 samples from treated patients (table III). Again the peak of activity always eluted in fractions 10-12 (figure). Migration stimulating activity was described in only 2 (10%) of the 20 controls (table IV); the figure shows a representative negative activity profile. Control samples were assayed blind with paired patient samples in all
132
TABLE I I I-MIGRATI ON STIMULATING ACTIVITY INS SERUM FROM TREATED BREAST CANCER PATIENTS
Discussion
Migration stimulating activity was detected in the serum of over 80% of the breast cancer patients we examined, compared with only 10 % of healthy controls. The migration stimulating activity in patient serum was indistinguishable from fibroblast-produced MSF in terms of its behaviour in various biochemical fractionation procedures. The continued presence of migration stimulating activity in the serum after resection of the primary tumour and administration of adjuvant treatment is consistent with our previous observations on the systemic production of MSF by uninvolved skin fibroblasts in such individuals." Decker and colleaguesl9 reported the presence of hyaluronic acid stimulating activity (HASA) in serum from untreated breast cancer patients, but could not detect it after surgical resection of the primary tumour in patients with no experiments, so that the likelihood of scoring artifacts was reduced. Repeat determinations of control samples were always consistent. The 58 individual assay controls (ie, cultures incubated without any test serum fraction) showed a narrow distribution profile-mean 4-48 (SD -1’58). Thus, the bioassay has an intrinsically stable baseline upon which reliable determinations of migration stimulating activity can generally be made. Statistical analyses (paired Student’s t test) showed that both untreated and treated breast cancer patients achieved significantly greater relative stimulation than their age-matched controls (p < 0-005). The elution profile of the migration stimulating activity in breast cancer patient serum was indistinguishable from that of MSF in fetal and breast cancer patient fibroblast conditioned medium fractionated under the same conditions (figure). To characterise the serum factor further, samples of breast cancer patient serum were fractionated according to the purification scheme devised for MSF;z precipitation with ammonium sulphate, heparin affinity chromatography, gel filtration chromatography (’Superose12’), and reverse phase chromatography. The behaviour of the migration stimulating activity in cancer patient serum was identical to that previously reported for MSF at all of these stages (data not shown). TABLE IV-MIGRATION STIMULATING ACTIVITY IN SERUM FROM CONTROLS
residual disease. The presence of MSF in the serum of postoperative breast cancer patients clearly distinguishes it from HASA and other oncofetal proteins, all of which seem to be produced by tumours. MSF is therefore not a tumour marker but an indicator of the systemic presence of abnormal stromal fibroblasts. There have been many reports on the presence of aberrant fibroblasts in cancer patients. We have previously postulated that these cells have an active role in cancer pathogenesis .20 The results of this study raise the possibility that, if a rapid and quantitative assay for MSF could be developed, it might find application (probably in conjunction with other procedures) in screening for people at high risk of the development of cancer. This study was supported by grants from the Cancer Research and the Breast Cancer Research Trust.
Campaign
REFERENCES
proliferation and migration within three-dimensional collagen gels. J Cell Sci 1980; 41: 159-75. 2. Schor SL, Court J. Different mechanisms involved in the attachment of cells to native and denatured collagen. J Cell Sci 1979; 38: 267-81. 3. Schor SL, Schor AM, burning P, et al. Skin fibroblasts obtained from cancer patients display fetal-like behaviour in collagen gels. J Cell Sci 1985; 73: 235-44. 4. Schor SL, Schor AM, Howell A, et al. The possible role of abnormal fibroblasts in the pathogenesis of breast cancer. In: Rich MA, Hager JC, Lopez DM, eds. Breast cancer: scientific and clinical progress. Boston/Dordrecht: Kluwer Academic Publishers, 1987: 142-57. 5. Schor SL, Schor AM. Clonal heterogeneity in fibroblast phenotype: implications for the control of epithelial-mesenchymal interactions. Bioessays 1987; 7: 200-04. 6. Azzarone B, Morel M, Billard C, et al. Abnormal properties of skin fibroblasts from patients with breast cancer. Int J Cancer 1984; 33: 1. Schor SL. Cell
759-64.
Durning P, Schor SL, Sellwood RAS. Fibroblasts from patients with breast cancer show abnormal migratory behaviour in vitro. Lancet 1984; i: 890-92. 8. Schor SL, Haggle JA, Durning P, et al. Occurrence of fetal fibroblast phenotype in familial breast cancer. Int J Cancer 1986; 37: 831-36. 9. Ottman R, Pike MC, King M-C, et al. Practical guide for estimating risk of familial breast cancer. Lancet 1983; ii: 556-58. 10. Haggie J, Howell A, Sellwood RAS, et al. Fibroblasts from relatives of patients with hereditary breast cancer show fetal-like behavior in vitro. Lancet 1987; i: 1477-57. 11. Schor SL, Schor AM, Grey AM, et al. Fetal and cancer patient fibroblasts produce an autocrine migration stimulating factor not made by normal adult cells. J Cell Sci 1988; 90: 391-99. 12. Grey AM, Schor AM, Rushton G, et al. Production of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts. Proc Natl Acad Sci USA 1989; 86: 2438-42. 13. Stoker M, Gherardi E, Perryman M, et al. Scatter factor is a fibroblast-denved modulator of epithelial cell mobility. Nature 1987, 7.
327: 239-42.
133
14. Gherardi E, Gray J, Stoker M, et al. Purification of scatter factor, a fibroblast-derived basic protein that modulates epithelial interactions and movement. Proc Natl Acad Sci USA 1989; 86: 5844-48. 15. Liotta LA, Mandler R, Murano G, et al. Tumour cell autocrine motility factor. Proc Natl Acad Sci USA 1986; 83: 3302-06. 16. Warn RM, Dowrick P. Motility factors on the march. Nature 1989; 340: 186-87. 17. Rosen EM, Goldberg ID. Protein factors which regulate cell motility. In Vitro Cell Dev Biol 1989; 25: 1079-87. 18. Schor SL, Schor AM, Grey AM, et al. Mechanism of action of the
migration stimulating factor produced by fetal and cancer patient fibroblasts; effect of hyaluronic add synthesis. In Vitro Cell Develop Biol 1988; 25: 137-46. M, Chiu ES, Dollbaum C, et al. Hyaluronic acid stimulating activity from the bovine fetus and from breast cancer patients. Cancer Res 1989; 49: 3499-505. 20. Schor SL, Schor AM, Howell A, et al. Hypothesis: the persistent expression of fetal-like phenotypic characteristics by fibroblasts is associated with an increased susceptibility to neoplastic disease. Exp Cell Biol 1987; 55: 11-17. 19. Decker
Feasibility of reversing benzodiazepine tolerance with flumazenil
To examine whether the benzodiazepine antagonist flumazenil can reverse tolerance to benzodiazepines but without precipitating withdrawal seizures, the antiepileptic effect of flumazenil itself and its ability to reverse tolerance at a dose that would leave sufficient receptors free for the binding of benzodiazepines were investigated. Electroencephalographic studies in 6 patients with partial and 6 with generalised seizures showed that flumazenil had a short (20 min) non-dose-dependent suppressant effect on epileptic discharges in those with partial seizures. Receptor occupancy studies in 12 patients showed that 1·5 mg flumazenil given intravenously occupied 55% receptors, whereas 15 mg occupied nearly all receptors. When 3 patients with partial seizures who had become tolerant to clonazepam were given 1·5 mg flumazenil, they were seizure-free for 6-21 days after the injection. The value of intermittent therapy with a benzodiazepine antagonist for preventing or reversing tolerance to benzodiazepine agonists ought to be investigated further.
Introduction The
development of tolerance’ is a crucial problem with benzodiazepine therapy. Although how tolerance develops is still obscure, there
are
indications that, like the
pharmacological activity of benzodiazepines, it is mediated through the gamma-aminobutyric acid (GABA) /benzodiazepine chloride ionophore complex.2 The imidazol-benzodiazepine flumazenil (Ro 15-1788) is a benzodiazepine receptor antagonist with high affinity for the receptor. 3-6 According to recent reports based on animal studies flumazenil may reverse subsensitivity to GABA induced by chronic benzodiazepine therapy, perhaps by producing conformational changes of the GABA/ benzodiazepine receptor complex that leads to a "reset" of its sensitivity to GABA .2,7,11 If flumazemil produces such changes in the receptor in man, it could be used for the prevention and reversal of benzodiazepine tolerance. It could perhaps be given intermittently during continuous treatment with a benzodiazepine agonist. Such a regimen may carry the risk of withdrawal convulsions precipitated by
flumazenil-induced
displacement of the agonist from benzodiazepine receptors.9 However, flumazenil is also reported to have some anticonvulsant activity, 10-13 so there a concentration of flumazenil in the brain tissue at which the drug exerts both its "protective" antiepileptic activity and its reset effect. To be of clinical value that concentration must be such that enough benzodiazepine receptors are left unoccupied by flumazenil to allow a benzodiazepine agonist to be bound at concentrations that
may be
therapeutically effective. study was to investigate the dose of flumazenil required to suppress electroencephalographic (EEG) epileptic discharges and to measure with positronemission tomography (PET) the degree of benzodiazepine
are
The aim of this
different doses. A suitable dose of flumazenil was then selected for injection into 3 epileptic patients resistant to clonazepam.
receptor occupancy
at
Patients and methods Informed was
consent was obtained from all patients and the study approved by the local ethics and radiation safety committees.
Effect of flumazenil on EEG
epileptic discharges
The effect of flumazenil on epileptic discharges in the electroencephalogram was tested in 6 patients with complex partial and 6 with primary generalised seizures. The diagnosis and classification was based on clinical investigations and EEG recordings, in accordance with the international classification of epilepsies .14 4 of the patients with complex partial seizures had absences (2 with lateralised automatisms), and 2 others had attacks with "dreamy state" and confusion. The epileptic focus was always in the temporal lobe. The two groups were similar in mean ages (37 [SD 7] vs 33 [6] yr) and weight (69 [7] vs 62 [5] kg). Other medication was restricted to carbamazepine and phenytoin. Benzodiazepines were prohibited for the 2 months before the experiments. The EEGs were recorded with scalp electrodes, placed according to the 10/20 system. Every patient was examined on four different days (fig 1). A recording was started 30 minutes before and continued without interruption during and between injections of vehicle (ethanol, propylene-glycol, and sodium chloride) and the ADDRESSES: Department of Clinical Neurophysiology, Karolinska Hospital (I. Savic, MD, L. Widén, MD, S. Stone-Elander, phD) and Karolinska Pharmacy (S. Stone-Elander), Stockholm, Sweden. Correspondence to Dr Ivanka Savic, Department of Clinical Neurophysiology, Karolinska Hospital, PO Box 60500, S 104 01 Stockholm, Sweden.