Growth patterns of pulmonary metastases and primary tumours from five murine fibrosarcoma cell clones.

The growth patterns, including the size, shape and regional preferences, of lung metastases from five murine fibrosarcoma cell clones were studied. Spontaneous metastases developed from tumours formed by subcutaneous inoculation of the cell clones. Lung colonies (experimental metastases) were established by i.v. injection of cells. The numbers of both spontaneously and experimentally formed subpleural lung metastases were counted through a stereomicroscope. The fraction of colonies that was located subpleurally was determined in histological sections of lungs. The growth kinetics of clonally derived primary tumours, and the number of spontaneous and experimental lung metastases, differed greatly between certain cell clones. The number of spontaneous lung metastases was correlated with the maximum size of primary tumours. No close correlation was observed between the size of the primary tumours and the size of experimental metastases. There were differences between the cell clones in the shape and regional preferences of their lung deposits. The subpleural colonies were generally larger than the intrapulmonary ones. Thus, both the regional distribution and the growth pattern of lung deposits differed between the clones.

Quantitative histological changes in a human melanoma xenograft following exposure to single dose irradiation and hyperthermia.

The purpose of the present paper is, in terms of quantitative histology, to explain the growth response of a human melanoma xenograft after exposure to single dose irradiation (7.5 Gy, 15.0 Gy, and 25.0 Gy) and hyperthermia (42.5 degrees C for 60 min). Data from several experiments on the mitotic activity, the occurrence of different modes of cell death and reproductively dead cells in the tumors are discussed. The tumor cell proliferation is only transiently reduced after 7.5 Gy and 15.0 Gy, and tumor regression mainly results from an increased cell loss. Cell loss through apoptosis and cell disintegration during mitosis show a dose-dependent increase after irradiation. Although the fraction of necrosis, relative to the number of tumor cells, increases after 7.5 Gy and 15.0 Gy, the cell loss through necrosis, that is, the production of necrosis, is probably reduced. Compared to the cell loss through apoptosis and mitotic death, the removal of necrosis is probably less important in determining the regression rate of the tumors after 7.5 Gy and 15.0 Gy. After 25.0 Gy the cell production is markedly reduced, and cell loss increases, partly due to radiation injury to the vascular system, resulting in necrotization of the tumor core. Thereafter, the tumor regression rate depends mainly on the rate of necrosis removal. The mitotic activity of remaining cells is not reduced after hyperthermic treatment, and the tumor growth response is a result of an increased cell loss. Although the occurrence of apoptosis and cell disintegration in mitosis increases after hyperthermia, these modes of cell loss are of minor importance for the tumor regression after treatment. The increased cell loss is mainly due to massive necrosis formation in the central tumor areas, a result of heat injury to tumor blood vessels. After necrotization of the tumor core, the regression rate depends mainly on the rate of necrosis removal.

A method to quantify neovascularization in the mouse cornea.

A method for microscopical observation and quantification of corneal neovascularization in the living mouse is described. Measurements of the corneal vasculature were made on still-mode video-pictures of the cornea. The validity of the method was shown by the high correlation between the morphometric measurements and computerized values on the same vessel structures. The method was applied to describe the neovascular response of heat-killed tumour fragments implanted into corneal pockets. The neovascular response that was observed in some corneas was dependent on the distance from the edge of the pocket with heat-killed material to the nearest vessel loop. The pocket area was vascularized only if this distance (measured the 3rd day after implantation) was less than 0.70 mm. Resorption of heat-killed tissue occurred in corneas without any neovascular response and it was not altered by penetration of vessel into the cornea. The described method proved to be suitable for studying the neovascularization induced by corneal pocket implants.

Histological study of the regrowth of a human melanoma xenograft exposed to single dose irradiation.

The aim of this investigation was to find the location and the histological characteristics of cells that were the source of tumour regrowth after single dose irradiation. A human melanoma xenograft was irradiated with a single dose of 25.0 Gy which gives local tumour control in nearly 50% of treated animals. Serial histological sections were made from tumours removed during the first two weeks after irradiation. During the first week the perivascular organization of tumour parenchyma disappeared and the central part of the tumour became necrotic. The occurrence of vascular stasis, thrombosis and endothelial cell changes indicated that radiation injury to the vascular system was involved in the disappearance of the tumour cords. Tumour cells that remained histologically intact were located in subcapsular areas. The incidence of normal mitotic figures increased, and the fraction of abnormal mitoses and the incidence of micronuclei decreased 5 to 7 days before macroscopical regrowth was usually detected. It is concluded that cells which are the source of tumour regrowth were located in the subcapsular areas at the time of irradiation. Radiation injury to the tumour vascular system was an important factor in the necrotization of the tumour centre after treatment.

The occurrence of apoptosis, abnormal mitoses, cells dying in mitosis and micronuclei in a human melanoma xenograft exposed to single dose irradiation.

The mechanisms of cell loss, the cell proliferation and the immediate growth response were investigated in a human melanoma xenograft given single dose irradiation with 7.5 Gy and 15.0 Gy, respectively. The frequencies of apoptotic cells, mitoses, abnormal mitoses, cells dying in mitosis and micronuclei, were scored in histological sections. In the untreated xenograft, the occurrence of micronuclei and abnormal mitoses indicated the presence of reproductively dead cells. Cell loss manifested itself through the appearance of apoptosis, cells dying in mitosis and necrosis. After irradiation, the cell proliferation was temporarily inhibited due to a radiation induced division delay. When proliferation resumed, there was a dose-dependent increase in the frequencies of abnormal mitoses and micronuclei and thus in the fraction of reproductively dead cells. The incidence of cell loss through apoptosis and cells dying in mitosis also increased. This cell loss probably reduced transiently the fraction of reproductively dead cells, and accounted for the reduced amount of tumour cells the first days after 15.0 Gy irradiation. The incidence of apoptotic cell loss and micronuclei decreased, and the incidence of normal mitoses increased when tumour growth resumed.

The relationship between changes in tumour volume, tumour cell density and parenchymal cord radius in a human melanoma xenograft exposed to single dose irradiation.

A human melanoma xenograft, in which the viable tumour tissue formed cylindrical cuffs around blood vessels, was irradiated with single doses of 7.5 Gy and 15.0 Gy respectively. The nuclear density, the frequency of giant cells and the mean parenchymal cord radius were measured and compared to the tumour growth response. After 7.5 Gy, the growth rate was only slightly reduced and there were no significant changes in the nuclear density or the mean parenchymal cord radius. After 15.0 Gy the mean parenchymal cord radius decreased the first week. This coincided with swelling of endothelial cell nuclei and reduced density of functional capillary-like vessels. Although the number of tumour cells declined, the tumour volume increased the first days after irradiation with 15.0 Gy since the cell density was reduced.

Micronucleus formation in human melanoma xenografts following exposure to hyperthermia.

The formation of micronuclei in two human melanoma xenografts (E. E. and V. N.) following hyperthermic treatment (42.5 degrees C for 60 min) was studied and compared to that following single dose irradiation. The melanomas were grown in the hind leg of athymic mice and heated by immersing the tumour-bearing leg into a water-bath. Histological sections were prepared from tumours removed from the mice at predetermined times after treatment and the fraction of abnormal mitotic figures and the number of micronuclei per nucleus were scored. During the first 24 h after treatment, the fraction of abnormal mitotic figures increased abruptly to 90%-100% followed by a rapid decrease to 40%-50%. It then decreased slowly towards about twice the level in untreated tumours. The number of micronuclei started to increase at about the same time as the fraction of abnormal mitotic figures was highest, reached a maximum at about 2-3 days after treatment, and then decreased slowly. The number of micronuclei seen after the hyperthermic treatment was lower than that seen after radiation treatments causing similar tumour regrowth delays. The same hyperthermic treatment resulted in more micronuclei and larger regrowth delays for E. E. than for V. N. melanoma. The present results indicate that DNA damage is involved in heat-induced cell death in tumours treated in vivo.

A transparent chamber for the dorsal skin fold of athymic mice.

A transparent chamber designed for insertion in the dorsal skin fold of athymic mice is described. The chamber is based on ingrowth of a layer of granulation tissue from the surrounding subcutis of the animals. The thickness of the tissue layer in the chamber is accurately given by an exchangeable chamber component and the construction allows repetitive access to the tissue inside the chamber. A freely movable skin flap is prepared surgically on the back of the animals, and the chamber is inserted in this flap in a second operation about 2 weeks later. In the absence of air bubbles, infectious foci and excessive adipose tissue in the chamber, ingrowth of granulation tissue is completed 2-4 weeks after the insertion. When infections of the skin enclosing the chamber and atrophy of the skin flap are prevented, the functioning time of the transparent chamber is determined by the life span of the animals. In the present study the athymic mice remained healthy for 4-5 months after insertion of the chamber although they were kept under conventional conditions, but were given antimicrobial covering. Since athymic mice accept some human xenografts, both normal tissues and neoplasms, vital microscopical studies of such xenografts implanted into the chamber can be performed. Preliminary results of such studies on a human malignant melanoma are presented.