Combination heparin plus cortisone treatment of two transplanted tumors in C3H/He mice.

C3H/He inbred mice bearing either C3H mouse mammary or RIF-1 tumors of 180-mm3 volume were treated with a combination of heparin (500 anticoagulation U/ml drinking water) plus cortisone (either 250 mg/kg/day tapering to 37 mg/kg/day or a constant dose of 75 mg/kg/day). Five types of heparin were tested in this study. RIF-1 tumors shrank to approximately half the volume at the start of therapy after only 3 days of treatment; mammary tumors took longer to respond, not reaching half the starting volume until after 11 days of treatment. In both tumors response was transient, the tumors eventually regrowing. However, response to combined heparin and cortisone therapy was in fact no different from the response to cortisone used alone. Also, cortisone treatment was extremely toxic to these animals and experiments had to be terminated after about 3 weeks of therapy.

Antimetastatic effect of cimetidine on mice bearing a C3H mouse mammary adenocarcinoma: survival and lymphocyte function studies.

It has been reported that treatment with cimetidine, a histamine H2-receptor antagonist, increased survival and decreased the number of lung metastases in mice bearing the Lewis Lung carcinoma [29]. It was suggested that this effect was due to the ability of cimetidine to block histamine activation of suppressor lymphocytes and hence allow host defence mechanisms to inhibit tumour growth. In the present studies, C3H/He mice were implanted with a C3H mouse mammary adenocarcinoma on Day 0. This tumour metastasizes to the lungs in 30-50 days. Primary tumours were ablated with X-rays when they had grown to about 0.2 g and animals were given drinking water with or without cimetidine (10 mg ml-1) until the end of the experiment. Cimetidine reduced the number of mice dying from metastatic disease from 7/15 (controls) to 3/13. Cimetidine treatment also prolonged survival of mice that did succumb to metastatic disease by about 12 days. The response of spleen lymphocytes to the mitogens phytohaemagglutinin and lipopolysaccharide was assessed in vitro by uptake of 3H-thymidine 0, 16, 45 and 58 days after tumour implantation. Lymphocyte responsiveness was depressed by tumour burden. The influence of cimetidine treatment was equivocal being dependent upon time after tumour implantation and dose of mitogen. In this mouse-tumour system, the mechanism of the antimetastic effect of cimetidine is different from that previously suggested [29].

Radioprotection of mouse skin vasculature and the RIF-1 fibrosarcoma by WR-2721.

The degree of radioprotection obtained with WR-2721 is critically dependent upon the oxygen tension of the tissue concerned. It was therefore considered of interest to examine the response of vascular tissue, which would be supposedly well oxygenated, to treatment with WR-2721 plus X rays. The response of this tissue is of interest because of its role in late radiation damage. In addition, for therapeutic gain an agent must protect normal tissue without concomitant tumor protection. However, data on tumor radioprotection have been conflicting and therefore the effect of WR-2721 on an experimental tumor was also tested. Vascular damage was assessed using the fact that tumors grow more slowly in irradiated beds (Tumor Bed Effect). The response of the RIF-1 tumor treated in vivo was measured by in vitro assay of clonogenic survival. WR-2721 (400 mg kg-1) injected 30 minutes before 5 to 30 Gy X rays protected skin stroma by a factor of 1.6. However, WR-2721 given 10 to 60 minutes before 20 Gy X rays to the RIF-1 tumor had either no effect or was protective, according to the method of immobilizing the mice during irradiation. Differences between the ways animals are physically restrained for tumor irradiation could account for the conflicting data concerning tumor radioprotection with WR-2721.

Synergistic cytotoxicity of iodinated contrast agents and x-radiation.

The synergistic cytotoxicity of iodinated radiologic contrast agents and x-radiation is discussed. Experiments are described illustrating the phenomenon and lending support to the view that it is mediated by energetic photoelectrons. Some of its practical implications are indicated.

The tumour bed effect: a cell kinetic and histological investigation of tumours growing in irradiated mouse skin.

Most tumours grow more slowly when implanted into pre-irradiated sites--the tumour bed effect (TBE). The TBE is usually assayed by measuring the delay for tumours growing in irradiated sites compared with that for tumours growing in mock-irradiated sites to reach a certain arbitrary, externally-measured volume. The resulting X-ray dose-response curves for the TBE are generally S-shaped, with little effect up to doses of 5 Gy, a dose-dependent effect between 5 and 20 Gy and a plateau at higher doses. In this study such a dose-dependent TBE was demonstrated for two contrasting transplantable tumours (a rapidly growing mammary adeno-carcinoma and the RIF-1 fibrosarcoma) growing in the flank skin of C3H/He mice. Cell kinetic and histological methods were used to investigate the mechanism of the reduced tumour growth rate in irradiated sites. By combining information from tumour growth curves and metaphase-arrest lines, tumour cell birth and cell loss rates were estimated. In addition the necrotic and viable fractions of tissue were measured by means of Chalkley point counting. In both tumours, marked increases in cell loss rate and degree of necrosis were found to be dependent on the dose of X rays previously given to the stroma. Surprisingly, cell birth rate and mitotic index were significantly increased in mammary tumours growing in irradiated sites. The estimation of viable, as opposed to external, volume of tumours growing in sites which had received between 0 and 60 Gy X rays suggested that the conventional TBE assay method may underestimate the extent of the TBE and may distort the shape of the TBE dose response curve.

The use of ketamine plus diazepam anaesthesia to increase the radiosensitivity of a C3H mouse mammary adenocarcinoma in hyperbaric oxygen.

The radiation response of mammary tumours transplanted into syngeneic C3H mice has been measured with the animals breathing air or 100% oxygen at 290 kPa (HPO), either with or without ketamine plus diazepam anaesthesia. The single doses needed to cure 37% of tumours within 40 days (TCD37/40) for mice anaesthetised with ketamine plus diazepam and for unanaesthetised mice irradiated in air were not significantly different, 66.5 Gy and 68.8 Gy respectively. When animals were irradiated in HPO, the TCD37 value was significantly reduced from 60 Gy with no anaesthetic to 41 Gy with ketamine plus diazepam anaesthesia; an enhancement ratio (ER) of 1.5. The total ER from no anaesthetic in air to anaesthetic in HPO was 1.7 (68.8/41). There was less CNS toxicity for ketamine plus diazepam than for sodium pentobarbitone anaesthesia in mice treated in HPO. The combination of ketamine and diazepam is an unusual anaesthetic in that it maintains blood pressure, cardiac output and respiration in man. Vascular effects and lowered body and tumour temperatures may also have influenced tumour oxygenation and radiation response.

Reduction of tumour bed effect after angiogenic stimulation.

A new two-tumour system has been developed to examine the effect of an angiogenic stimulus on the tumour bed effect (TBE) in C3H mouse skin treated with 20 Gy X rays. The first tumour was implanted intradermally to stimulate angiogenesis. The Lewis lung carcinoma (3LL) was used for this since it is isogeneic in C57 B1 mice and after initial growth (mean volume 69 mm3), tumours completely regressed within 24 days. The second tumour, the RIF-1 fibrosarcoma (RIF) isogeneic to C3H mice was used to assess stromal function using the TBE assay. After complete regression of the 3LL tumour, RIF cells were implanted into the centre of the stimulated sites and 24 days later the resulting RIF tumours were excised and the TBE measured from the yield of viable RIF cells obtained from each tumour. There was an approximately 10-fold increase in cell yield from tumours implanted into stimulated, pre-irradiated beds compared with those implanted into unstimulated, pre-irradiated beds. This suggests that the angiogenic stimulus provided by the growth of the 3LL tumour led to a substantial restoration of the capacity of the radiation-damaged stroma to support tumour growth. If stromal restoration, as seen during these experiments using artificially induced angiogenesis, was to occur in unstimulated stroma during the normal course of repair and regeneration, this slowly dividing tissue constituent might eventually recover given sufficient time. This raises the possibility that the role of the stroma in late radiation damage might not be as important as was previously thought.

Tumour bed effect: hypoxic fraction of tumours growing in preirradiated beds.

The reduction in tumour growth rate seen when tumours are implanted into preirradiated sites, the tumour bed effect (TBE), is believed to be due to radiation damage to vascular stroma, leading to defective angiogenesis in the tumour. The present work examined whether or not the functional inadequacy of irradiated stroma was accompanied by an increased hypoxic fraction in tumours growing in irradiated beds. Mouse flank skin was given 0 or 20 Gy X-rays and RIF-1 fibrosarcoma cells were implanted i.d. into the centre of the treatment field one week later. Tumours of 200 mm3 were irradiated under clamped or unclamped conditions and the hypoxic fraction measured from the displacement of the corresponding survival curves, assayed in vitro. Results indicated a small increase in the hypoxic fraction. Averaging values from three independent experiments, the percentage of hypoxic cells increased from 2.5 per cent for cells in tumours growing in unirradiated beds to 4.6 per cent for those from tumours in beds given 20 Gy. Thus an irradiated vascular bed is still to some extent able to maintain the proportion of oxic: hypoxic tumour cells found in tumours growing in unirradiated beds, despite manifest changes in tumour necrosis and growth rate.