Effect of toxic thioureas on resistance of rats to growth in the lungs of intravenously and intratracheally seeded tumour cells.

Clonogenic growth (colony-forming efficiency, CFE) of i.v. injected allogeneic W256 tumour cells in the lungs was markedly enhanced by treatment of rats with alpha-naphthyl thiourea (ANTU) injected i.p. from 2 h before to 2 h after the tumour cells. ANTU specifically increases pulmonary vascular permeability in adult rats and causes acute pulmonary oedema and pleural effusion. Inhibition of drug toxicity to the lungs by tachyphylaxis, specific antimetabolites or iodides did not abolish the effect of ANTU on CFE. CFE was not increased when cells were seeded by i.v. injection the lungs affected by advanced pulmonary oedema at 6 to 24 h after treatment with drug. ANTU did not enhance growth of intratracheally injected cells. Although ANTU has no cytotoxic or immunosuppressive action, treatment of tumour-immunized rats with ANTU caused apparent "breakdown" of tumour immunity in 50% of rats, by causing growth of tumour colonies in the lungs. Possible mechanisms for the ANTU-induced decrease in innate resistance to growth of tumour in the lungs are discussed.

Reactions of the tumour bed to lethally irradiated tumour cells, and the Révész effect.

Subdermal inoculation of the foot of the rat with lethally irradiated (LI) Walker tumour (W256) cells, mixed with viable (V) W256 cells, decreased the latent period for initiation of allogeneic tumour growth without significantly affecting its rate. This Révész effect decreased with increase in the number of inoculated V cells, and with decrease in age of recipient. LI cells of a different (Y-P388) rat tumour exerted a Révész effect, even in recipients which had been immunized with LI (Y-P388) tumour cells. Local pre-irradiation of the site of inoculation of V cells decreased both the latent period and rate of tumour growth. It acted independently of a Révész effect, and the decrease in tumour growth rate was partly due to emigration of V cells from the inoculum, producing metastases. LI, but not heat-killed cells, induced prolonged swelling of the tumour bed in unimmunized and tumour-immunized rats, which, unlike inflammatory swelling, was inhibited by pre-irradiation of the foot. It is postulated that the Révész effect is due to enhancement of survival of V cells by trophic substances which are principally elaborated by LI (AND V) cells, but also by the tumour bed, due to innate growth and trophic reactions of its tissues to the presence of tumour cells.

The significance of free blood in liquid and solid tumours.

Impregnation of the vasculature with ink was used to study microvascular changes induced in rats by liquid (ascites) and solid growth of W256 and Y-P388 tumour cells. Ascites fluid produced by both tumours was heavily blood-stained; the deep red colour of solid tumour deposits was similarly due to the presence of free blood. In both types of tumour growth this free blood was due to diapedesis of erythrocytes through tips of capillary sprouts which grow when neovascularization (angiogenesis) occurs in response to any suitable (non-neoplastic or neoplastic) stimulus. Ascites growth of these tumours induced profuse sprouting from the peritoneal capillaries; this sprouting, together with the "bleeding" it caused, were inhibited by local pre-irradiation of the peritoneal vasculature with X-rays before intraperitoneal inoculation of rats with the tumours. Similar angiogenesis with bleeding did not occur following inoculation with an allogeneic tumour in rats which had been previously immunized against the tumour. LI tumour cells (tumour cells lethally irradiated in vitro to destroy their proliferative integrity, but which remain metabolically active) also induced sprouts to grow in close proximity to the implanted LI cells, but heat-killed tumour cells caused no sprouting. The nature and significance of neovascularization of tumours and their so-called "haemorrhagic" growth are discussed.

Rapid and accurate measurement of growth of solid tumours and changes in the tumour bed in the rat by the technique of volumetric displacement.

An apparatus which has been widely used in rats for measuring swelling of the foot induced locally by inflammatory agents has been adapted to measure rapidly, accurately and objectively, the growth of tumour cells transplanted to the foot, and the reactions of the normal tissues (tumour bed) to tumour growth. General features on the apparatus and the techniques used are described. Examples are provided of preliminary measurements made of normal growth of the foot, reactions of the foot to two injurious agents (histamine and Corynebacterium parvum) and of growth of allogeneic (W256) tumour cells.

Innate and drug-induced resistance to acute lung damage caused in rats by alpha-naphthyl thiourea (ANTU) and related compounds.

During the 3rd and 4th weeks of life rats were highly resistant to the toxic effects of alpha-naphthyl thiourea (ANTU) and of thiourea and its derivatives but toxicity developed rapidly during the following 2 weeks. Marked resistance to lung damage by toxic thioureas could be induced in older, mature rats by pretreatment with the toxic agent itself (tachyphylaxis), with other toxic and non-toxic antithyroid drugs or with iodine or iodide--even if the rats were pretreated at an early age before susceptibility to the agent developed. ANTU-tachyphylaxis was dose-dependent. Total thyroidectomy did not affect either lung damage induced by ANTU or the resistance due to tachyphylaxis or to pretreatment with iodide or the antithyroid drugs thiourea, 1-ethyl-1-phenyl thiourea or propyl thiouracil. Neither total nor medullary adrenalectomy affected ANTU toxicity. Marked resistance to ANTU-induced lung damage was induced in rats by pretreatment with either an activator (3-4 benzypyrene) or an inhibitor (SKF 525-A) of drug-metabolizine mixed-function microsomal enzyme systems; the inhibitor, sodium phenobarbitone, had no significant effect on toxicity. The sulphydryl compound, AET, induced marked resistance to ANTU; cysteine was less effective. Neither autonomic blockade with nicotine and atropine nor actinomycin D had significant effects on toxicity to ANTU. The acute pulmonary oedema induced in rats by high pressure oxygen, chemical convulsants, pressor agents and ammonium sulphate differed in many respects from that induced by toxic thioureas; it was typically haemorrhagic in nature, did not result in significant pleural effusion, did not exhibit tachyphylaxis, and was not influenced by pretreatment with iodide or derivatives of thiourea.

Lowering of innate resistance of the lungs to the growth of blood-borne cancer cells in states of topical and systemic stress.

The survival and clonogenic growth (measured in terms of colony forming efficiency (CFE) of intravenously injected (i.v.) Walker (W256) tumour cells in the lungs of rats was greatly enhanced by states of topical and systemic stress induced by the intraperitoneal (i.p.) injection of rats with a single dose of 10(-5)-10(-3) mmol g-1 body weight of adrenaline and other beta-adrenergic agonists, inflammatory agents (including local x-irradiation), convulsive seizures, "tumbling" or physical restraint. Lowering of innate resistance of the host to growth of seeded tumour cells induced by states of topical and systemic stress, and by the addition of an excess of lethally irradiated (LI) tumour cells to i.v. injected intact tumour cells, were all potentiated by treatment of rats with aminophylline, an inhibitor of cyclic AMP phosphodiesterase. Enhancement of tumour growth by systemic stress was inhibited by bilateral total or medullary adrenalectomy and is attributed to the release and actions of endogenous adreno-medullary hormones. Alpha-adrenergic and most non-adrenergic agents administered in maximum tolerated doses did not significantly affect host resistance to tumour growth in the lungs. These findings, correlated with measurements of cyclic AMP in the lungs of normal and stressed rats, suggest that changes in the resistance of the host to tumour growth involve changes in cyclic nucleotide metabolism in the target tissues (tumour bed); possible mechanisms of action of cyclic nucleotides in this respect are discussed.

Venous diversion trapping and growth of blood-borne cancer cells en route to the lungs.

A proportion of W-256 tumour cells injected intravenously into a tail vein of the rat are diverted into venous plexuses en route to the lungs; here tumour cells remain trapped, proliferate and form invasive solid tumours in the pelvis and hindquarters, which cause paraplegia, metastases and death. Also, cells trapped in veins produce tumour nodules distributed along the length of the tail; this effect in markedly enhanced by temporarily arresting the outflow of blood from the tail for a few seconds only immediately after cells are injected. Continous monitoring of the radioactive signal over the lungs after W-256 cells labelled with 125IUDR were injected showed that massaging the tail or intravenously injecting isotonic saline into the tail dislodged cells trapped in veins. In heparinized rats, tail trapping was markedly reduced, although not entirely abolished, and venous trapping in vertebral and pravertebral regions was decreased. The anatomical distribution of growth of the trapped cells in rats closely resembled metastases involving dissemination via the "vertebral venous system" produced by certain cancers in man. Labelled tumour cells trapped in the lungs of untreated mature rats commenced dying rapidly in situ wiht 1-2 h after injection; the majority had disappeared within 24 h, and less than 1% of the injected tumour cells survived to form lung colonies. Experimental evidence is presented which indicates that the lungs play a vital role in rapidly eliminating a high proportion of blood-borne cancer cells in the adult individual.

Promotion of growth of tumour cells in acutely inflamed tissues.

Acute inflammatory reactions were induced in rats by the intravenous injection of cellulose sulphate (CS) or an extract of normal rat lung homogenate (LH), or by intraperitoneal injections of Compound 48/80. These treatments greatly increased survival and clonogenic growth in the lungs of rats of intravenously injected allogeneic W-256 and Y-P388 tumour cells. Increase in the dose of intravenously injected CS caused a logarithmic increase in colony forming efficiency (CFE) of tumour cells in the lungs. CFE was not stimulated by the intravenous injection of rats with pharmacological mediators of inflammation (histamine, 5-hydroxytryptamine, bradykinin and prostaglandins PGE(1) and PGF(2α)) which are released from tissues by agents which induce inflammation. Stimulation of CFE by CS occurred in adrenalectomized rats but was inhibited by treatment of rats with an anti-inflammatory steroid, dexamethasone. CFE was stimulated by CS in tumour immunized rats; the inflammatory state did not prevent the expression of immunity but "rescued" a proportion (approximately 20%) of the injected tumour cells from immunodestruction in the lungs. A higher proportion of tumours grew in the paws of rats when a small number of W-256 cells were injected interdigitally into the acute inflammatory swellings produced by the local injection of paws with LH or CS.CS is a "synthetic heparin" which causes marked prolongation of blood clotting time and also increases fibrinolytic activity of the blood. Anticoagulant treatment of rats with heparin did not affect CFE. Thus, there was no direct correlation between blood clotting time and CFE of blood borne tumour cells in the rat.The mechanisms which may be responsible for the nonspecific growth promoting effects of inflammatory reactions induced by various types of tissue injury on tumour induction and growth are discussed.

Reduction by anti-inflammatory corticosteroids of clonogenic growth of allogeneic tumour cells in normal and irradiated tissues of the rat.

Anti-inflammatory corticosteroids administered to rats in high dosages before intravenous injection of allogeneic tumour cells caused 5-10 fold reductions in "take" and clonogenic growth of the cells in lung and kidney and decreased growth and spread of the cells transplanted to leg muscle. Steroid therapy also reduced the effect of local irradiation of lung tissues in increasing tumour colony efficiency (CFE) in the lungs; it also tended to reduce similar effects of sublethal whole body irradiation. A non-steroidal anti-inflammatory drug, phenylbutazone, also reduced CFE in locally irradiated lungs in the rat.The results obtained indicate that corticosteroids do not stimulate the growth of implanted tumour cells by suppressing host immunity but decrease their clonogenic growth by inhibiting local inflammatory reactions to cell arrest, and similarly to local tissue damage caused by x-irradiation; it is asserted that such inflammatory reactions are growth promoting and thereby stimulate regeneration of stroma (repair) and also support survival and early growth of the tumour cell.