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J. Cell Sci. 73, 235-244 (1985)

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SKIN FIBROBLASTS OBTAINED FROM CANCER PATIENTS DISPLAY FOETAL-LIKE MIGRATORY BEHAVIOUR ON COLLAGEN GELS S. L. SCHOR1, A. M. SCHOR1, P. DURNING2 AND G. RUSHTON1 1 Cancer Research Campaign Department of Medical Oncology, Manchester University and Christie Hospital & Holt Radium Institute, Wilmslow Road, Manchester M20 9BX, U.K. 2 University Department of Surgery, University Hospital of South Manchester, Nell Lane, Withington, Manchester M20 8LR, U.K.

SUMMARY

When plated on the surface of collagen gel substrata, all types of fibroblasts rapidly begin to migrate down into the three-dimensional collagen matrix. We have previously demonstrated that normal (adult and foreskin), foetal and transformed fibroblasts may be distinguished from each other by virtue of their differential migratory response to changes in cell density. The effects of cell density on fibroblast migration into the gel may be expressed by a single numerical value, the 'cell density migration index' (CDMI). We now present evidence that ostensibly normal skin fibroblasts obtained from the majority of patients we examined with carcinoma of the breast, malignant melanoma, familial polyposis coli, retinoblastoma and Wilms1 tumours display aberrant CDMI values falling within the foetal range. Skin fibroblasts obtained from the majority of patients examined with genetic or chronic diseases (e.g. rheumatoid arthritis, Duchenne muscular dystrophy) displayed CDMI values falling within the normal range.

INTRODUCTION

Certain cancer syndromes (e.g. familial polyposis coli, hereditary retinoblastoma, ataxia telangiectasia) have a clearly defined genetic basis (Knudson, 1971; Harnden & Bridges, 1982; Harnden, Morten & Featherstone, 1984). The tumours that develop in these cancer syndromes are often of epithelial origin and commonly display a considerable degree of tissue specificity. However, in view of the genetic transmission of these syndromes, it is reasonable to postulate that all cells of the affected individual contain the genomic elements ultimately responsible for the observed increased susceptibility to cancer (Kopelovich, 1982). With this rationale in mind, a number of studies have been done to compare the behaviour of skin fibroblasts obtained from normal controls and individuals suffering from genetic cancer syndromes in an attempt to identify those aberrant phenotypic characteristics that may contribute to the carcinogenic process. Skin fibroblasts from affected individuals have been shown to display several aberrant phenotypic characteristics commonly associated with transformation, including: (a) the ability to proliferate in the presence of low serum concentrations (Pfeffer, Lipkin, Stutman & Kopelovich, 1976); (b) colony formation in semi-solid medium (Kopelovich, 1982); (c) disrupKey words: cell migration, collagen gels, cancer patient fibroblasts.

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tion of actin-containing cytoskeletal cables (Kopelovich et al. 1980); (d) the production of elevated levels of plasminogen activator (Kopelovich, 1977); and (e) growth dependence on exogenous methionine (Mikol & Lipkin, 1984). Skin fibroblasts obtained from certain cancer syndrome patients also differ from control cells by their increased sensitivity to a variety of chemical, physical and viral oncogenic agents (Miller & Todaro, 1969; Rhim, Hubner, Arenstein & Kopelovich, 1980; Danes, 1980; Kopelovich, Drozdoff & Zeitz, 1981). Genetic factors are again believed to be involved in determining susceptibility to the more common types of cancer (e.g. carcinoma of the breast and bronchus), although clearly defined epidemiological evidence may be difficult to obtain (Anderson, 1972; King, 1981). Skin fibroblasts obtained from individuals suffering from carcinoma of the bronchus have been reported to undergo 'spontaneous transformation' in vitro (as assessed by focus formation, ability to form colonies in semi-solid medium, karyotypic abnormalities and reduced capacity to compact a fibrin clot) when these cells are cultured under crowded conditions (Azzarone, Pedulla & Romanzi, 1976; Dolfini et al. 1976). Ostensibly normal skin fibroblasts obtained from osteosarcoma patients have been observed to form tumours in immunosuppressed mice (Smith et al. 1976). Lynch and collaborators (1984) have recently reported that skin fibroblasts obtained from breast cancer patients with a familial history of the disease frequently display karyotypic abnormalities. Finally, Azzarone et al. (1984) have presented evidence that skin fibroblasts from patients with carcinoma of the breast display certain transformation-associated phenotypic characteristics in vitro, such as increased saturation cell density and colony formation in semi-solid medium and on confluent epithelial cell monolayers. The work in our laboratory has largely been concerned with examining the factors that influence cell migration into three-dimensional collagen gel matrices (Schor, 1980; Schor, Schor & Bazill, 1981; Schor, Schor, Winn & Rushton, 1982). We have previously demonstrated that normal, foetal and transformed fibroblasts may be distinguished from each other on the basis of their differential migratory response to changes in cell density (Schor, Schor, Rushton & Smith, 1985). The effects of cell density on migration may be expressed by a single numerical value, the 'cell density migration index' (CDMI). In this paper, we present evidence that ostensibly normal skin fibroblasts obtained from patients suffering from a number of different types of cancer commonly display CDMI values characteristic of foetal cells.

MATERIALS AND METHODS

Cells and culture conditions Experiments were performed on skin fibroblasts obtained from cancer patients with carcinoma of the breast, malignant melanoma, familial polyposis coli, retinoblastoma and Wilms' tumour. Data regarding the origin of the cell lines used in this study are given below. Carcinoma of the breast. All fibroblast lines were established in this laboratory from explant cultures of normal, uninvolved skin (as described by Ham, 1980) obtained from the breast area of female patients undergoing surgery to remove the malignant growth (Department of Surgery, University Hospital of South Manchester). No patient had received any form of therapy prior to

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surgery. The following lines were established: T100SF, age 35; T117SF, age 55; T118SF, age 67; T l 19SF, age 38; T120SF, age 50; T121SF, age 43. All patients were females with infiltrating duct carcinoma. Malignant melanoma. All fibroblast lines were established in this laboratory from explant cultures of normal, uninvolved skin obtained from patients with recurrent skin nodules. All patients had their primary tumours removed by surgery and had not received any form of therapy for at least six months prior to pinch biopsy without local anaesthesia. The age and sex of the donors and site of the skin biopsies are as follows: MSF33, male, age 18, back of upper trunk; MSF108, male, age 56, left thigh; MSF113, female, age 42, forearm; MSF115, male, age 48, forearm. Familial polyposis coli. All skin fibroblast lines were a kind gift from Dr Joy Delhanty, Department of Genetics & Biometry, University College', London. Fibroblast lines were established from pinch biopsies of skin taken from the forearm. Further details regarding the clinical history of the donors and the karyotype of the cell lines have been published by Delhanty, Davis & Wood (1983). The following skin fibroblast lines were used in this study: FPC3c, male, age 42; FPC5a, male, age 49, FPC6c, male, age 25; FPC7c, male, age 26. Retinoblastoma. Skin fibroblast* from patients with retinoblastoma were originally obtained from the Human Genetic Cell Repository, Institute for Medical Research, Camden, N.J. (U.S.A.) and were a kind gift from Dr Anne Kinsella, Paterson Laboratories, Manchester. The following lines were used in this study: AG1879, female, age 11, hereditary disease; AG4250, female, age 22 months, sporadic; AG1142, female, age 27 months, sporadic; AG2971, female, age 5 months, sporadic; AG2718, female, age 7, sporadic. Wilms' tumour. Skin fibroblasts from patients with Wilms' tumour were also obtained from the Human Genetic Cell Repository and were a kind gift from Dr Anne Kinsella. The following lines were used in this study: AG2311, male, age 3; AG2512, male, 5 months; AG3118, female, bilateral disease. Fibroblasts were also obtained from 18 patients with various types of chronic inflammatory, proliferative or degenerative conditions. Data regarding the origin of these cell lines are given below. Rheumatoid arthritis. Synovial fibroblasts from patients undergoing surgery for joint replacement were a kind gift from Dr D. Woolley, University Department of Medicine, University Hospital of South Manchester. The following lines were used in this study: RSC373, female, age 57, hip replacement; RSC375, female, age 64, knee; RSC377, female, age 59, knee. Duchenne muscular dystrophy. Skin fibroblasts obtained from the thigh of male patients with Duchenne muscular dystrophy were kind gifts from Drs J. Witkowski and M. Dunn, Jerry Lewis Muscle Research Laboratory, Hammersmith Hospital, London. The following lines were used in this study: MDSF, age 7; MDSF2, age 6; MDSF4, age 4; MDSF5, age 5; MDSF6, age 6 and MDSF7, age 6. Lysosomal enzyme deficiencies. Skin fibroblasts were a kind gift from Dr M. Dean, Kennedy Institute, London. Details regarding the nature of the genetic abnormality and patient details are as follows: GMO221, Tay-Sachs, male, age 3; GMO151, jS-glucuronidase deficient, male, age 4; H100, Hunter's syndrome (iduronate sulphatase deficient). Miscellaneous. Skin fibroblasts obtained from patients with a variety of diseases were a kind gift from Dr C. Arlett, M.R.C. Cell Mutation Unit, University of Sussex. The following lines were used: BR3, psoriasis, male, age 26, from forearm; BR68, Behcet's disease, female, age 40, from upper arm; BR72, polyarthritis nodosa, male, age 41, from forearm; BR72, systemic lupus erythromatosis, female, age 17, from upper arm; BR80, scleroderma, female, age 20. Recessive dystrophic epidermolysis bullosa. Skin fibroblasts were a kind gift from Dr J. Weiss, Department of Rheumatology, Medical School, University of Manchester. The following cell line was used: RDEB100, male, age 12. Stock cultures of the different skin fibroblast lines were maintained in MEM growth medium supplemented with 15 % foetal calf serum, glutamine, non-essential amino acids, sodium pyruvate and penicillin/streptomycin, and subcultured as described by Schor & Court (1979). Stock cultures were grown in 90 mm plastic tissue-culture dishes and passaged at a split ratio of 1: 5 when the cultures reached confluency after 7-10 days of growth. All cell counts were done with a Coulter electronic particle counter.

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Measurement of cell density migration index Type I collagen was extracted from rat tail tendons and used to make three-dimensional collagen gels as previously described (Schor & Court, 1979). In our standard migration assay, cells were plated onto the surface of replicate collagen gels at a 'low initial density' (i.e. 103 cells cm" 2 ) and at a 'high initial density' (i.e. 104 cells cm" 2 ). After 4 days of incubation at 37°C, the percentage of cells present within the three-dimensional collagen matrix was determined by the microscopic method described by Schor (1980). These migration data were then used to calculate the cell density migration index (CDMI) as described by Schor et al. (1985) as follows: CDMI =log [%(low density)/%(high density)], where '%(low density)' is the percentage of cells within the gel matrix in cultures plated at 103 cells cm" 2 and '%(high density)' is the corresponding value in cultures plated at 104 cells cm" 2 . Note that according to this formulation, cells whose migration is inversely proportional to cell density will have positive CDMI values, whereas cells whose migration increases with cell density will have negative CDMI values.

Definition of CDMI ranges In a survey of 77 different fibroblast lines (Schor et al. 1985) we demonstrated that distinct ranges of CDMI values are expressed by normal (foreskin and adult), foetal and transformed fibroblasts; these ranges are defined as follows: the transformed range (T) with CDMI values less than —0-4, the transformed/foetal range ( T / F ) with CDMI values between —0-4 and 0, the normal/foetal range (N/F) with CDMI values between 0 and +0-4 and the normal range (N) with CDMI values greater than +0-4

RESULTS

All the skin fibroblasts derived from cancer patients that we examined in this study were indistinguishable from normal fibroblasts in terms of: (a) morphology in confluent culture (i.e. formed swirls of aligned cells with no evidence of crisscrossing or focus formation); (b) saturation cell densities; (c) serum requirements for growth (tested in 1 % and 15 % foetal calf serum); (d) inability to form colonies in semi-solid medium in the presence of 15 % foetal calf serum; and (e) ability to compact a floating collagen gel (for a discussion of assessment of transformation in vitro, see Cameron & Pool, 1981; Steinberg, Smith, Colozzo & Pollack, 1980). The CDMI values of skin fibroblasts obtained from patients with carcinoma of the breast, malignant melanoma, familial polyposis coli, retinoblastoma and Wilms' tumour are presented in Table 1. Each value represents the mean of at least three experiments. Since these are ostensibly normal cells, we would have expected greater than 90% of the CDMI values to fall within the N range (Schor et al. 1985). Quite different results were, in fact, obtained. As inspection of Table 1 will reveal, the majority of cell lines tested from all types of cancer patients expressed aberrant CDMI values falling within the foetal range (T/F and F/N). The CDMI values of fibroblasts obtained from patients with a variety of genetic or chronic inflammatory conditions are presented in Table 2. The majority of fibroblasts from these patients gave CDMI values which fell within the N range. The distribution of CDMI values for the skin fibroblasts derived from the 22

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Table 1. CDMI values expressed by skin fibroblasts obtained from cancer patients Cell line

CDMI

(a) Carcinoma of the breast T100SF T117SF T118SF TU9SF T120SF T121SF

-005 +0-29 +0-66 +0-26 +0-22 +0-33

(T/F) (F/N) (N) (F/N) (F/N) (F/N)

(b) Malignant melanoma MSF34 MSF108 MSF113 MSF115

+0-58 +0-19 +0-34 +0-39

(N) (F/N) (F/N) (F/N)

(c) Familial polyposis coli FPC3c FPC5a FPC6c FPC7c

+0-68 +0-35 +0-09 +0-10

(N) (F/N) (F/N) (F/N)

AG1879 AG4250 AG1142 AG2971 AG2718

+0-21 +0-37 -0-16 +0-23 +0-59

(F/N)

AG2311 AG2512 AG3118

+0-23 +0-01 +0-49

(F/N) (F/N) (N)

(d) Retinoblastoma

(F/N) (T/F) (F/N) (N)

(e) Wilms' tumour

CDMI values were calculated as described in Materials and Methods. Data presented here represent the mean values of at least three experiments, S.D. values did not exceed ±0-16. Fibroblasts established in this laboratory from patients with carcinoma of the breast and malignant melanoma were used between passages 3 and 10. Fibroblasts obtained from other sources were used between passages 10 and 20. The CDMI ranges (T, T / F , F / N and N) are defined in Materials and Methods.

cancer patients and 18 chronically ill patients is presented in summary form in Table 3. Data previously obtained with normal (adult and foreskin), foetal and transformed cells (Schor et al. 1985) are also presented to facilitate direct comparison with the patient skin fibroblasts. As can be seen in this Table, the majority of skin fibroblasts derived from cancer patients (i.e. 68-2%) expressed CDMI values in the F/N range, while, in contrast, only 16% of the fibroblasts derived from the chronically ill or genetic disorder patients fell into this group. DISCUSSION

Our data indicate that ostensibly normal skin fibroblasts obtained from a significant percentage of cancer patients express foetal-like CDMI values when

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Table 2. CDMI values expressed by skin fibroblasts from patients with genetic or chronic diseases Cell line

Disease

CDMI

BR3 BR68 BR72 BR74 BR80 GMO221 GMO151 H100 RDEB100

Psoriasis Behcets disease Polyarthritis nodosa Systemic lupus erythromatosis Scleroderma Tay-Sachs disease jS-glucuronidase deficient Hunters disease Recessive dystrophic epidermolysis bullosa Rheumatoid synovial cells Rheumatoid synovial cells Rheumatoid synovial cells Duchenne muscular dystrophy Duchenne muscular dystrophy Duchenne muscular dystrophy Duchenne muscular dystrophy Duchenne muscular dystrophy Duchenne muscular dystrophy

+0-44 +0-44 +0-40 +0-62 +0-61 +0-32 +0-47 +0-40

(N) (N) (N) (N) (N) (F/N) (N) (N)

+0-59 +0-32 +0-42 +0-41 +0-41 +0-42 +0-47 +0-50 +0-29 +0-51

(N) (F/N) (N) (N) (N) (N) (N) (N) (F/N) (N)

RSC373 RSC375 RSC377 MDSF1 MDSF2 MDSF4 MDSF5 MDSF6 MDSF7

Details regarding the origin of the particular cell lines used here may be found in Materials and Methods. The majority of cell lines were used between passage 5 and 15. Results are the mean values of 2 to 6 separate experiments.

Table 3. Distribution of CDMI values expressed by different types of skin fibroblasts Source Foreskin Adult Foetal Transformed Cancer patients Genetic and chronic diseases

% of cell lines falling into respective CDMI ranges T T/F F/N N 0 0

0 46-5 0 0

0 0 20-8 46-4 91 0

71 8-3 75-0 6-9 68-2 16-7

92-9 97-7 4-2 0 227 83-3

Data are presented concerning the distribution of CDMI values for 22 different lines of skin fibroblasts obtained from cancer patients and 18 lines of fibroblasts obtained from patients with genetic or chronic disease. Comparative distribution data are presented for the 14 lines of foreskin, 24 lines of adult, 24 lines of foetal and 15 lines of transformed fibroblasts examined in our initial survey (Schor et al. 1985) used to define the CDMI ranges.

examined in the collagen gel migration assay. Taken together with the growing body of evidence obtained in other studies revealing that skin fibroblasts from cancer patients display behavioural anomalies in vitro (see Introduction), these findings suggest that susceptibility to cancer may be influenced by poorly understood host factors, which are expressed systemically.

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We have previously shown that both skin and tumour-derived fibroblasts from patients with benign breast disease display CDMI values in the N range and are therefore indistinguishable from normal cells by this criterion (Durning, Schor & Sellwood, 1984). In a continuing study involving breast cancer patients with and without a strong family history of the disease, we have demonstrated that skin fibroblasts derived from apparently unaffected first degree relatives of patients with a strong family history commonly display foetal CDMI values (Schor & Howell, unpublished data). These findings suggest that expression of foetal-Hke CDMI values may precede the development of a clinically recognizable malignancy and might therefore facilitate the identification of individuals at elevated risk of developing cancer. Azzarone et al. (1984) have similarly suggested that assessing the in vitro behaviour of skin fibroblasts might help identify those individuals at increased risk of developing carcinoma of the breast. We have previously suggested that foetal fibroblasts undergo an 'isoformic' transition at some point in their developmental history, which is manifest in vitro by the expression of elevated CDMI values falling in the N range (Schor et al. 1985). The results presented in this paper are consistent with the hypothesis that this isoformic transition does not occur in certain individuals, with a consequent continued expression of at least certain foetal phenotypic characteristics by fibroblasts (and possibly other cell types as well) in the adult organism. Fibroblasts derived from cancer patients that initially expressed CDMI values in the foetal range were never observed to undergo a transition to expression of elevated values in the N range (unpublished observations). Two different hypotheses may be proposed regarding the apparent relationship between the expression of foetal CDMI values by fibroblasts in vitro and the development of tumours, often of epithelial origin, in vivo. According to the first hypothesis, the particular genetic or epigenetic defect expressed by the fibroblasts in vitro is similarly expressed by the relevant target epithelial cell population. This first hypothesis is commonly invoked in studies dealing with cancers displaying a clearly defined genetic basis, such as familial polyposis coli, in which skin fibroblasts have been shown to express a number of aberrant phenotypic characteristics (Kopelovich, 1982). Reasoning by analogy, one would argue that the underlying lesion responsible for the continued expression of a foetal CDMI by adult fibroblasts is also shared by the relevant target epithelial cells and contributes thereby to the induction of tumour formation. We would like to present an alternative possibility, which does not premise the obligatory shared expression of a common genetic defect by both the fibroblast and epithelial population. Epithelial-mesenchymal interactions are now known to play an important role in the induction and maintenance of a differentiated phenotype during embryonic development and in the adult organism. A detailed account of the role played by the epithelial—mesenchymal interactions in these processes is beyond the scope of the present discussion and the interested reader is referred to various comprehensive reviews dealing with the subject (Hay, 1981; Hawkes & Wang, 1982). It is of interest to note that foetal fibroblasts have been reported to promote

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the proliferation and inhibit the expression of a differentiated phenotype by mammary epithelial cells (Taga, Sakakura & Oka, 1983). Similarly, foetal fibroblasts transplanted into an adult mammary gland have been observed to promote hyperplastic changes in the epithelial cells and render them more sensitive to neoplastic transformation by carcinogenic agents (Sakakura, 1983). We suggest that those adult fibroblasts that continue to express foetal CDMI values in vitro may also display foetal characteristics in their inductive interactions with epithelial cells in vivo. In view of our current understanding of epithelial— mesenchymal interactions, the expression of at least certain foetal phenotypic characteristics by fibroblasts might influence the progressive development of an epithelial tumour by a number of means, including: (a) inhibiting the expression of a differentiated phenotype by the epithelial cells; (b) modulating the response of the epithelial cells to soluble growth factors; and (c) promoting the clonal expansion of tumour cells. This hypothesis that a dysfunction in normal inductive cell—cell interactions contributes in some fashion to increased susceptibility to cancer should be investigated further. It is of interest to note that fibroblasts obtained from the majority of patients with non-neoplastic genetic disorders or chronic inflammatory conditions displayed CDMI values falling within the normal range. Although these results suggest that expression of aberrant CDMI values is not commonly associated with these types of conditions, clearly more data must be accumulated for any particular disease (e.g. rheumatoid arthritis) before a definitive statement can be made in this area. In conclusion, we believe that the results discussed in this paper (both our own and' those reported previously by others) indicate that information regarding the behaviour of fibroblasts derived from cancer patients should provide clinically relevant data concerning the aetiology and natural history of the disease.

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(Received 16 July 1984 -Accepted, in revised form, 25 September 1984)

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