and pathophysiological parametes; and senstiity to heat and dacarbazine ttment. Moreover, the organ-specific patten of the xenogrfted tumours Irted the pattern ...
0 (1994. 76, acr(94,7,8482CMcilnPesLc,19
Br. J. Cawff
&.J
Maamillan Press Ltd., 1994
804-812
Orthotopic human melanoma xenograft model systems for studies of tumour angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern E.K. Rofstad Department of Biophysics, Institute for Cancer Research, The Norwegian Radm Hospital, Montebello, 0310 Oslo, Norway. Adequate tumour models are a preqste in rimental cancer rsarch. The purpose of the was to taablsh and asess the valty of four new orthotopic human melanoma xenograft model systems (A-07, D-12, R-18, U-25). Pomnent cell lines we established in monolayer clture from S ry
prst
work
metastases
from
of four diffe
the lines wre
ted
nt
and clls patients by usin an in vivo-i vitro Balb/c nulnu mice to form tumours. Individual xenogafted
melaoma
intradrmally
in
tumours of the same ine differed substanIally in growth and pathophysilogical parameters, es its in host fatos which influence tumour n inoculato consmeqz of Nevertheless, xenografted tumows of diffent is showed distinctly different bioogical Seal
of the donor
biological
patEnts'
tumours
we
rtained in the
as a an
eso
haaisti
xenogafted tumoms,
and pathophysiological parametes; and senstiity to ic pontl; gowth, i ol patten of the xenogrfted dacarbazine ttment. Moreover, the organ-specific stases in the donor nts The organs of prefernc for distant tumours Irted the pattern of distant m metastass were lungs (A-07, D-12), lymph nodes (R-18) and brain (U-25). R-18 lymph node metastas and
incig ango
heat and
U-25 brAin
nmnastases
xcnograft model systems atment
sensitivity and
in the abse of
show gat promise metatatic pattem
Advances in cancer research require appropriate xperimental nisms affecing the cinical models to explore basic behaviour and treatment sensitivity of tumours. Human models are usually ferable to rodent modls; expmental rodent tumours are at best general models of human cancer (Denekamp, 1979), whereas experimental human tumours are supposed to model adequately the secific neoplastic disease of the donor patient (Suthrlrand et al., 1988). The availability of congenitally athymic mice and severe combined immune deficiency mice has resulted in increased use of human tumour xenografts in cancer research the last two decades. Recent studies have indicated that orthotopic heterotransplantation of human tumours may result in metastatic patterns in mice similar to those observed in man (Fidler, 1991; Fu et al., 1991). Moreover, cellular treatment sensitivity is probably retained during serial transplantation of some human tumours in immune-deficient mice (Rofstad, 1992a). No single xenograft model can be expected to be appropriate for all types of questions related to a specific neoplastic disease; a panel of models of the same histopathological type is required (Kallman et al., 1985). A xenograft panel is parficularly useful if it is composed of tumour lines that differ substantially in their biological characteristics and at the same time reflt the biology of the donor patients' tumours. Moreover, the xenografted tumours should have disaggregation and growth properties making them suitable for studies in vitro as well as in vivo. Four new orthotopic human melanoma xenograft model systems for studies of tumour angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern are reported in the present communication. Tumour treatment sensitivity and metastatic pattern are governed by intrinsic properties of the tumour cells (Sutherland et al., 1988; Hill. 1990), but are strongly modified by angiogenic and pathophysiological parameters of the tumour tissue (Folkman, 1985; Vaupel et al., 1989). The validity of the model systems was assed by comparing essential biological characteristics of donor patients' tumours and derivative xenografted tumours.
Received 29 April 1994; and in revised form 28 June 1994.
lung
involement The four
for fuctur
studies
of tumour
orthotopic
ang
human
meanoma
esis, pathophysio
,
MateIa aud m
Dono patins' tumours Four Caucasian patients (A-07, D-12, R-18, U-25) admitted to The Norwegian Radium Hospital for the treatment of malignant melanoma were donors of tumour tissue (Table I). The patients had developed regional metastases at the time of initial diagosis. The R-18 patient presented with extensive metastatic deposits also in distant lymph nodes. The other patients developed distant metastases subsequent to the initial diagnosis, first in lymph nodes and then in lungs (A-07, D-12) and brain (U-25). Melanoma tissue was obtained from large regional subcutaneous metastases prior to initiation of chemotherapy. The tumour specimens were processed in RPMI-1640 culture medium at 4-C immiately after the surgery. Blood clots and normal tissues were removed. The cleaned melanoma tissue was divided into pieces of approximately 2mm3 or 0.2 cm3. Xenografted tumours were established by implanting the 2 mm3 pieces subcutaneously in athymic mce. Histological preparations or single-cell suspensions for biological studies were prepared from the 0.2 cm3 pieces.
CeUl lines Permanent cell lines, one from each of the four patients, were established in monolayer culture. Single-cell suspensions were p ared by mechanial digation of xenografted tumours in the first passage and seeded in 25 cm2 tissue culture flasks containing 5 ml of culture medium (RPMI-1640 medium containing 20% fetal calf serum, 250 mg 1' penicillin and 50 mg 1- streptomycin). The suspensions were incubated at 3TC in a humidfied atmosphere of 5% carbon dioxide in air. The cells attached to the bottom of the flasks, and proliferation was initiated within 2 days. The cultures were trypsinised (treatment with 0.05% trypsin/0.02% EDTA solution at 37C for 2 min) and subcultured at confluence. Murine fibroblasts contaminating the cultures were removed by differential trypsinisation. Chromosome analysis revealed that cultures in passage 30 were free from murie stromal cells. The cultures showed slow and irregular growth during the first 35 passages in vitro. The rate of cell proliferation was
HUMAN MELANOMA XENOGRAFT MODELS
805
Table I Tumour characteristics in donor patients Patient age
A-07
50
Patient sex Male
D-12
38
Female
Tumour
(Year)
Primary tumour Upper limb Trunk
Breslow thickness (mm) 3.8 8.3
R-1 8
45
Female
Neck
7.4
U-25
49
Male
Trunk
5.0
stable and reproducible beyond passage 50. Large stocks of cells in passage 75 were frozen and stored in liquid nitrogen. These stocks were verified to be free from Mycoplasma contamination by using both the Hoechst fluorescence and mycotrin methods. The cellular parameters reported here were determined by studies of exponentially growing cultures in passages 75-100, i.e. they were determined after stable, permanent cell lines had been established. The cells were in this period cultured in RPMI-1640 medium (25 mM HEPES and L-glutamine) supplemented with 13% fetal calf serum, 250mg1I` penicillin and 50 mg 1-' streptomycin. The cultures were incubated at 37C in a humidified atmosphere of 5% carbon dioxide in air and subcultured three times a week. Cell number doubling time was determined by seeding 5 x I04 cells in 80 cm2 tissue culture flasks and measuring number of cells per flask as a function of time. Growth fraction and cell loss factor were determined by observing 40 randomly selected cells in a 48 h period; fraction of cells entering mitosis and the outcome of mitosis were registered (Rofstad et al., 1980). Plating efficiency (PE) was measured by seeding 100 cells in 25 cm2 tissue culture flasks coniing approximately 1.0 x I05 heavily irradiated (30 Gy) feeder cells. DNA index was determined by flow cytometry (see below).
Xenografted tumours Adult Balb/c nu/nu mice, bred at our research institute, were used as host animals for xenografted tumours. The mice were maintained under specific pathogen-free conditions at constant temperature (24-26°C) and humidity (30-50%). Sterilised food and tap water were given ad libitum. Approximately 3.5 x I0 cells in 10 gil of Ca2"- and Mg2+free Hanks' balanced salt solution (HBSS) were inoculated intradermally in the flanks of the mice by using a 100gl1 Hamilton syringe. The cells were harvested from exponentially growing cultures in passages 75-100 by trypsinisation. The take rate was 100%. Tumour volume (V) was calculated as V = x/6 x ab2, where a is the longer and b the shorter of two perpendicular tumour diameters, measured with callipers. Volume doubling time, fraction of necrotic tissue, fraction of cells in S-phase and treatment sensitivity were determined for tumours with volumes within the range of 200-400 mm3. Tumour weight was determined immediately after tumour excision and refers to the wet weight. Histopathological examinations Tumour samples were fixed in phosphate-buffered 4% paraformaldehyde, dehydrated, embedded in paraffin casts, cut in 6-gm-thick sections and stained. Haematoxylin and eosin staining were used for ordinary histopathological examinations and for measurement of fraction of necrotic tissue. Standard immunoperoxidase procedures were used to highlight vessels in donor patients' tumours and to visualise extracellular matrix in xenografted tumours (Nagelhus & Rofstad, 1993). Polyclonal rabbit anti-factor VIII (Dakopatts), polyclonal rabbit anti-laminin (Serotec), polyclonal rabbit anti-fibronectin (Calbiochem) and monoclonal
Regional
Distant
metastases
metastases
Subcutis Lymph node Subcutis
Lymph node
Lymph node Subcutis Lymph node Subcutis Lymph node
Lung Subcutis Lymph node Lymph node Brain
Lung
Lymph node
mouse anti-type IV collagen (Dakopatts) were used as primary reagents. The sections were stained by using diaminobenzidine as substrate and haematoxylin for counterstaining. Murine host cells in xenografted tumours were identified by using the bisbenzimide staining technique
(Rygaard, 1987). Vascular density was assessed at a magnification of x 200 by counting number of capillary profiles per field of view, corresponding to 0.75 mm2. A structure was defined as a countable capillary only if a lumen and at least one brownstaining endothelial cell were identified. Volume fraction of necrosis was determined by point counting (Solesvik et al., 1982). Five fields of view, selected according to stringent stereological criteria (Weibel, 1979), were analysed in each of 4-6 different tumour pieces from each patient. Ultrastructural examinations The ultrastructure of microvessels in xenografted tumours was examined by transmission electron microscopy. Tumour pieces were fixed in a cacodylate-buffered mixture of 1% glutaraldehyde and 4% paraformaldehyde, post-fixed in buffered 1% osmium tetroxide and dehydrated in graded alcohol solutions. The tissue was embedded in EponAraldite and polymerised at 80C. Ultrathin sections were double stained with uranyl acetate and lead citrate. Cell suspensions Single-cell suspensions were prepared from donor patients' tumours and xenografted tumours by using a standardised mechanical and enzymatic procedure. The tumour tissue was minced with crossed scalpels in cold HBSS prior to enzymatic treatment at 37C for 2 h. The enzyme solution consisted of 0.2% collagenase, 0.05% pronase and 0.02% DNAse in HBSS. The fraction of contaminating diploid host cells in the suspensions, determined by flow cytometry, was low for both donor patients' tumours (
