Antitumour responses induced by short-term pretreatment with tumour cells.

The injection (s.c. or i.p.) of 10(6) live or lethally irradiated methylcholanthrene-induced fibrosarcoma cells into CBA/Ca mice one or 2 days before i.v. challenge with the same tumour inhibited the formation of artificial lung tumour metastases. In addition, it also frequently enhanced the cytostatic effect of peritoneal exudate cells on monolayers of the same tumour. The effects on lung tumour metastasis were not noted if X-irradiated tumour was injected i.v., or if s.c. administration was delayed until one day after i.v. challenge. Similar effects on tumour growth were also observed in C3Hf/Bu mice and (CBA/Ca x A/HeJ) F1 hybrids which were pretreated (s.c.) with tumour shortly before i.v. challenge with the same tumour. Further studies in CBA/Ca mice suggested that the protective effect was tumour-specific, for the growth of i.v. injected tumour was not significantly inhibited by pretreatement with a number of other MC-induced or spontaneous tumours from the same and different strains.     

Stimulation of clonogenic growth of tumour cells and metastases in the lungs by local x-radiation

Single cell suspensions of two allogeneic tumours (W-256 and Y-P388) injected intravenously produced macrocolonies in the lungs of rats. Colony forming efficiency (CFE, the number of colonies produced by each viable cell injected) was low in 6-week or older rats but was markedly increased by 1000-1500 rad local thoracic irradiation (LTI) given 7-14 days before the tumour cell injection, or by antilymphocytic serum (ALS) but not by sublethal whole body irradiation (WBI). Similarly, LTI increased the incidence of pulmonary metastases produced by a solid tumour growing in the leg muscle. Stimulation of CFE by LTI was a strictly local phenomenon and not due to effects of irradiation on thymus, spleen or other tissues of the rat. LTI failed to increase CFE in immunized rats. It is concluded that (1) LTI stimulates clonogenic growth of tumour cells arrested in the lungs, by causing inflammatory reactions accompanied by regenerative cellular proliferation of lung tissue, which increases the “plating” efficiency of tumour cells, (2) the increase in CFE in lungs is not due to suppression of immunity to tumour growth by LTI.     

Aprotinin and growth of Walker 256 carcinosarcoma in the rat.

Growth of the Walker 256 carcinosarcoma implanted within various sites in Spraque-Dawley rats was investigated in animals receiving twice daily i.p. injections of the antiprotease aprotinin. Although administration of aprotinin partially attenuated the growth and lethality of i.p. tumour, no effect of aprotinin was found on intramuscular tumour development. Furthermore, we were unable to demonstrate unequivocal growth inhibition by aprotinin of lung tumour colonies from i.v. injection of tumour cells. Histological examination of intramuscular and pulmonary tumours revealed little evidence of host cellular immune response in either saline- or aprotinin-treated rats.     

Differential efficacy of flavone acetic against liver versus lung metastases in a human tumour xenograft

A human ovarian carcinoma, IGROV-1, was xenografted into different sites (i.p., s.c., i.v., and intrasplenically) in nude athymic female mice to investigate the pattern of antitumour efficacy of FAA and compare it to that of doxorubicin and cisplatin, two established cytotoxic drugs. Ascitic and lung-growing tumours totally failed to respond to FAA, whereas s.c. and liver-growing tumours were significantly growth inhibited. This pattern of activity differs from that achieved by the two conventional cytotoxic drugs, which were active against the IGROV-1 tumour growing in all of the tested sites. These studies indicate that cytotoxicity is not the major determinant of FAA antitumour efficacy even against human tumour xenografts. Moreover, the dramatic difference between the sensitivity of lung and liver tumour colonies demonstrates the great importance of the site of tumour growth for FAA efficacy.     

Enhanced Uptake of Intra-Arterially Injected Anti-Cea Monoclonal Antibodies in Human Colonic Cancer After Mannitol Infusion in An Experimental Model

In a previous report athymic rats carrying transplanted human colonic tumours from cell line LS 174T in both hind legs were injected intra-arterially (i.a.) with ¹²⁵I-labelled anti-carcinoem-bryonic (anti-CEA) monoclonal antibodies. The i.a. injection was given on one side bearing a tumour in each rat, while the contralateral tumour served as an 'intravenous' control. In the same experimental model and treated in the same way, 10 rats were injected i.a. with anti-CEA monoclonal antibodies after an i.a. mannitol infusion. In both groups of rats external gamma measurements were performed daily for four days. On the fourth day the rats were killed and pieces of the tumours and of various organs were weighed and the activity was determined with a gamma-counter. The tumour uptake of antibodies was significantly enhanced after mannitol infusion.     

Perturbation of the growth kinetics of C3H mouse mammary carcinoma by irradiation of tumour and host and by attempted pre-immunization of host.

The kinetics of development and subsequent growth of a C3H mouse mammary tumour after implantation of 10(6) cells was quantified by observation and statistical analysis of latent period and growth rate for different categories of tumour. These comprised control tumours, tumours recurring after large single doses of X-rays alone and in combination with misonidazole, tumours developing from cells implanted both outside and within the sites of tumours previously cured by irradiation, tumours developing from cells implanted in heavily irradiated skin, and tumours developing from cells taken from tumours recurring after irradiation and re-implanted in untreated skin. The kinetics of development and growth of tumours in host animals previously treated by i.p. injection of killed tumour cells was also quantified. The results confirm that tumour development and growth is significantly perturbed by irradiation of host tissues both before and after tumour transplantation, and that this perturbation involves an extended latent period, a slower average rate of growth and a less uniform pattern of growth. These effects result from localized radiation damage to host tissues, are not attributable to residual damage to irradiated tumour cells, and are not markedly dose-dependent within the dose range 25--80 Gy. These results are consistent with the complete sterilization of host endothelial cells by doses of 25 or more. In marked contrast to the growth-slowing effect of irradiation, the treatment of host animals by previous injection of radiation-killed tumour cells led to a reduced latent period and a faster average rate of growth.     

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.     

Artificially induced metastasis by cells from spontaneous Lucké renal adenocarcinoma

This communication reports the development of tumour colonies in various organs after vascular dissemination of disaggregated cells from spontaneously arising Lucké renal adenocarcinomata in Cyclosporin-A-treated allogeneic frogs, Rana pipiens. The sites of tumour cell colonies were mesonephros, lung, bladder, mesentery, fat body and muscle. It was also found that digestion of these tumours with collagenase is an effective means of obtaining sufficient dissociated viable cells for large experiments and that cryo-preservation does not abrogate the ability of these cells to form metastatic deposits. This report, therefore, introduces a new tumour system for the study of factors affecting metastasis using naturally occurring tumours.     

Vascular anatomy and organ-specific tumor growth as critical factors in the development of metastases and their distribution among organs

We have examined with 19 tumor cell lines the discrete roles that vascular anatomy and tumor-cell-organ-affinity play in the development of metastases and their distribution among organs. Spontaneous metastases of B16-G3.26 melanoma cells from a primary tumor growing in the foot pad of mice, or experimental metastases 21 days after intravenous tumor-cell injection resulted in tumor colonies only in the lungs. In contrast, when the lung microvasculature was bypassed, and the same cells given by systemic intra-arterial (s.i.a.) injection, large tumor colonies developed selectively in the ovaries, adrenal glands and bones, but rarely in the lungs. When animals injected i.v. were allowed to live with lung metastases for a long period of time, small tumor colonies began to develop in extra-pulmonary organs with a distribution identical to that seen after s.i.a. injection. Seven murine tumor cell lines (previously characterized by their ability to colonize primarily the lungs after i.v. injection) and 7 of the 8 studied human tumor cell lines colonized different specific extra-pulmonary organs after s.i.a. injection, frequently producing metastatic syndromes commonly described in patients with cancer, but rarely seen in animal models of metastasis. These results suggest that metastatic cells, even those capable of colonizing specific organs, do not freely circulate in the blood stream and lodge in specific tissues. In contrast, the cells must establish a vascular route of access to the target organ, e.g., through the systemic circulation from metastatic tumors in the lungs. Two cell lines considered to be tumorigenic but non-metastatic failed to colonize the lungs or extra-pulmonary organs after i.v. injection, but readily colonized specific organs after s.i.a. injection. Thus, tumor cells considered to be non-metastatic may be indeed metastatic if they are provided with vascular access to an organ more congenial to their growth requirements.     

A novel combretastatin A-4 derivative,AC7700, strongly stanches tumour blood flow and inhibits growth of tumours developing in various tissues and organs

In a previous study, we used subcutaneous LY80 tumours (a subline of Yoshida sarcoma), Sato lung carcinoma, and methylcholanthrene-induced primary tumours, to demonstrate that a novel water-soluble combretastatin A-4 derivative, AC7700, abruptly and irreversibly stopped tumour blood flow. As a result of this interrupted supply of nutrients, extensive necrosis was induced within the tumour. In the present study, we investigated whether AC7700 acts in the same way against solid tumours growing in the liver, stomach, kidney, muscle, and lymph nodes. Tumour blood flow and the change in tumour blood flow induced by AC7700 were measured by the hydrogen clearance method. In a model of cancer chemotherapy against metastases, LY80 cells (2x10(6)) were injected into the lateral tail vein, and AC7700 at 10 mg x kg(-1) was injected i.v. five times at intervals of 2 days, starting on day 7 after tumour cell injection. The number and size of tumours were compared with those in the control group. The change in tumour blood flow and the therapeutic effect of AC7700 on microtumours were observed directly by using Sato lung carcinoma implanted in a rat transparent chamber. AC7700 caused a marked decrease in the tumour blood flow of all LY80 tumours developing in various tissues and organs and growth of all tumours including lymph node metastases and microtumours was inhibited. In every tumour, tumour blood flow began to decrease immediately after AC7700 administration and reached a minimum at approximately 30 min after injection. In many tumour capillaries, blood flow completely stopped within 3 min after AC7700 administration. These results demonstrate that AC7700 is effective for tumours growing in various tissues and organs and for metastases. We conclude that tumour blood flow stanching induced by AC7700 may become an effective therapeutic strategy for all cancers, including refractory cancers because the therapeutic effect is independent of tumour site and specific type of cancer.     

An Experimental Model for Pharmacokinetic Studies of Monoclonal Antibodies in Human Colonic Cancer

An experimental model consisting of athymic rats carrying human colonic tumours from cell line LS 174T in both hind legs was used. ¹²⁵I-labelled anti-carcinoembryonic antigen (anti-CEA) monoclonal antibodies were injected intra-arterially (i.a.), either alone (21 rats) or together with degradable starch microspheres (6 rats). As a control, an irrelevant antibody was injected i.a., alone (6 rats) or together with microspheres (3 rats). An intra-arterial injection was given on the side bearing one tumour in each rat, while the contralateral tumour served as an 'intravenous' control. The rats were submitted to external gamma measurements daily for four days. On the fourth day they were killed and pieces from the tumours and from various organs were examined by in vitro measurements. The results indicate strong expression of CEA in LS 174T cells grafted to athymic rats. No lasting enhancement of the tumour uptake was achieved by intra-arterial injection of antibodies as compared with the control tumours.     

Changes in anatomical distribution of tumour lesions induced by platelet-active drugs

To examine the effect of platelet-active drugs on the spread of blood-borne tumour cells, two murine tumours, sarcoma 180 (S-180) and TLX-5 lymphoma, were selected. Following intravenous (IV) injection into CBA mice the former elicited thrombocytopenia and formed discrete pulmonary tumours, whereas the latter failed to elicit thrombocytopenia and formed discrete tumours in all visceral organs examined except the lungs. S-180 cells were injected IV into mice pre-treated with RA233 (known to prevent thrombocytopenia and thrombus formation) and TLX-5 cells were injected IV into mice pre-treated with Corynebacterium parvum (known to induce thrombocytopenia and thrombus formation). RA233 pre-treatment did not change survival time or incidence of S-180 pulmonary tumours but did result in a higher incidence of extrapulmonary tumours and a lower tumour cell burden immediately after injection. Pre-treatment with C. parvum resulted in a higher TLX-5 tumour cell burden but not discrete tumours in the lungs. On the basis of known drug activities it is proposed that thrombocytopenia induced in these experiments is in part a reflection of thrombus formation in the lungs which influences the speed of passage of tumour cells through capillaries. In some cases this may lead to a changed anatomical distribution of tumour lesions.     

The effects of regional factors on the growth rate and the differentiation of mouse teratocarcinoma

Murine embryonal carcinoma cells, the pluripotent stem cells of teratocarcinoma were injected simultaneously into caudal and cranial sites on the back of syngeneic recipients in order to determine whether regional anatomical differences affect their take and growth rate and differentiation. The overall tumour take rate was higher in caudal than cranial sites, but the initial weight of tumours was higher in the cranial than caudal sites. Tumours developing in the two anatomical sites grew at the same rate with a linear increase in volume. At the end of the 4-week experimental period the differences in the size of anterior and posterior tumours were negligible and no histological differences were noted between the two groups. Our data indicate that regional factors significantly affect the take rate and the initial growth of this murine teratocarcinoma, i.e. the establishment of solid tumours from injected stem cells. The growth rate of established tumours was not affected by regional factors.     

Metastatic colonization potential of primary tumour cells in mice.

A model has been developed for studying the capability of cells from primary murine mammary tumours to establish colonies in distant organs. The model involves the i.v. inoculation of disaggregated tumour cells into autologous and syngeneic recipients. The results show that the metastatic colonization potential of cells from a given tumour is consistent within the animals of an inoculated batch. Also, the findings are uniform in the autologous host and the syngeneic recipients. Tumours vary in their colonization potential and can be classified in 2 main groups designated high and low. These findings indicate that: (i) cells from 37% of mammary tumours can heavily colonize the lungs when inoculated i.v., even though the incidence of metastatic spread of these tumours in the undisturbed animal is almost zero. Thus, the relative infrequency of spontaneous metastasis from murine mammary tumours is not due to inability of the tumour cells to survive and colonize once free in the blood stream; and (ii) the colonization potential of the tumours is an intrinsic property of the tumour cells rather than of the host, whose prior acquaintance with the cells does not seem to confer resistance to colonization. The model presents opportunities for identification of possible differences between tumours of high and low colonization potential, and is being used to study cellular properties which favour colonization of distant organs by comparison of observations in vitro with the behaviour of cells from the same tumour in vivo.     

C. parvum immuntherapy of transplanted rat tumours

C.parvum (Wellcome CN 6134) has been tested for tumour suppression against a range of syngeneically transplanted rat tumours, both carcinogen-induced and of spontaneous origin. Subcutaneous growth was not prevented by distant subcutaneous or intravenous injection of the preparation, although growth rates were sometimes depressed or accelerated. In contrast, C. parvum injected in admixture with tumour cells consistently suppressed their growth and with highly immunogenic tumours induced systemic tumour immunity, C. parvum injected intravenously retarded development of pulmonary tumour deposits, and intrapleural injection suppressed growth of pleural tumours and malignant effusions. Host immunosuppression failed to abrogate the tumour-suppressive effect of locally applied C. parvum, but host macrophage depletion with silica totally abolished the response. These studies indicate that in the rat, tumour suppression is most consistently achieved by regional application of C. parvum, and that this response is more dependent upon local macrophage stimulation than generation of systemic immune responses.     

Metastasizing capacity of tumour cells from spontaneous metastases of transplanted murine tumours.

We investigated the metastasizing capacity of spontaneous lung metastases from the MN/MCA1 and mFS6 sarcoma, the B16 melanoma and colon 26 carcinoma. Spontaneous metastases at other visceral organs (liver, spleen, kidney, ovary, uterus) from the M5076/73A (M5) ovarian carcinoma and colon 26 carcinoma were also studied. Tumour cells from individual spontaneous metastases were used immediately after isolation from the normal parenchyma (mFS6, M5 and colon 26) and/or after 1 s.c. passage in syngeneic mice (MN/MCA1, mFS6, B16 and M5). Spontaneous metastases were examined for all tumours and their secondaries after i.m. or s.c. inoculation of tumour cells; artificial lung colonies were measured after i.v. injection only of cells from the primary mFS6 and MN/MCA1 and B16 or their spontaneous metastases. Individual spontaneous metastases were to some extent heterogeneous in their metastatic potential, a minority of the secondaries having greater or lesser metastatic capacity than the appropriate primary. Overall, tumour cells from spontaneous metastases did not show greater metastasizing capacity than primary neoplasms, nor was there evidence that metastases from specific organs (e.g. spleen and kidney) tended to home to the specific anatomical sites from which they were originally isolated. These observations in a series of murine tumours of different histology, transplantation history and pattern of metastasis, do not support the hypothesis that metastases are the ultimate expression of strong selection of variant cells with greater intrinsic metastatic potential, pre-existing within the primary tumour.     

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.     

Erythropoietin is involved in growth and angiogenesis in malignant tumours of female reproductive organs

The accumulating evidence that erythropoietin and erythropoietin receptor are expressed in various non-haematopoietic organs suggests that erythropoietin signalling might be involved in the growth of tumours, but this possibility has never been examined. We found that mRNAs for erythropoietin and erythropoietin receptor are expressed in malignant tumours of female reproductive organs, where erythropoietin levels are higher than in normal tissues. Furthermore, tumour cells and capillary endothelium showed erythropoietin receptor immunoreactivity. To investigate the role of the erythropoietin/erythropoietin receptor pathway in these tumours, we injected mouse monoclonal antibody against erythropoietin or the soluble form of erythropoietin receptor into blocks of tumour specimens and cultured the blocks. After 12 h of injections, these blocks were examined and compared with control blocks injected with mouse monoclonal antibody, heat denatured soluble form of erythropoietin receptor, mouse serum or saline. Tumour cells and capillaries were markedly decreased in a dose-dependent manner after either injection. A marked increase of the cells containing fragmented DNA and the histopathological characteristics of these cells suggest that the decrease in tumour cells and capillary endothelial cells was due to apoptotic cell death. The co-existence of JAK2 and phosphorylated-JAK2, and STAT5 and phosphorylated STAT5, all of which are involved in the mitogenic signalling of erythropoietin, was found frequently in tumour cells and capillary endothelial cells in the untreated blocks. In contrast, most of the phosphorylated-JAK2- or phosphorylated-STAT5-positive cells had disappeared in the experimental blocks. Moreover, reduced tyrosine phosphorylation of STAT5 in the experimental blocks was confirmed by western blotting analysis. The results strongly indicate that erythropoietin signalling contributes to the growth and/or survival of both transformed cells and capillary endothelial cells in these tumours. Thus, deprivation of erythropoietin signalling may be a useful therapy for erythropoietin-producing malignant tumours.     

A combination of interleukin-2 and 60 nm cationic supramolecular biovectors for the treatment of established tumours by subcutaneous or intranasal administration

The Supramolecular Biovector (SMBV) KY is a drug delivery nanocarrier which consists of a discretely sized, ionically charged, cross-linked polysaccharide core surrounded by a lipid membrane. We used the non-immunogenic spontaneous mammary adenocarcinoma TS/A tumour to test the efficacy on tumour growth of low (10(4) IU) or ultra-low (10(3) IU) doses of interleukin-2 (IL-2) adsorbed to these 60 nm cationic synthetic particles. In comparison with the progressive growth of TS/A cells in syngeneic mice, KY/IL-2 particles coinjected with TS/A cells or administered at a distance from the tumour, inhibited tumour growth while free IL-2, even at 10-100 times the dose used in the KY/IL-2 formulations, had no effect. Studies performed on implanted tumours (treatment at day 6 (D6)) showed that KY/IL-2 administered subcutaneously (s.c.) at five sites distant from the tumour (10(3) IL-2 IU per site) induced rejection of the implanted tumours. Six out of 10 mice were cured while the other four had residual tumours only. In the same experiment, free IL-2 induced only tumoral growth reduction. Protection induced by KY/IL-2 administered s.c. at five sites involved recruitment of a CD8(+) T cell response since nu/nu mice and CD8-depleted mice did not reject the tumours. Mice cured were protected significantly to completely against a rechallenge with TS/A tumour cells, and a systemic tumour-specific CTL activity was induced. Finally, we showed that repeated intranasal (i.n.) administration of KY/IL-2 (low-dose) also led to complete regression of pre-established tumours and partial protection from tumour rechallenge. We therefore suggest that, in contrast to free IL-2, a KY/IL-2 formulation could be used as a systemic immunostimulant leading to the eradication of non-immunogenic, established tumours.     

Uptake of 5-fluorouracil (5-FU) in peritoneal metastases in relation to the route of drug administration and tumour debulking surgery. an autoradiographic study in the rat

Patients with peritoneal metastases from colorectal cancer have a poor prognosis. Aggressive treatment by debulking surgery and intraperitoneal (i.p.) chemotherapy has been suggested as an alternative therapy. However, the drug penetrance into the tumour in relation to the administration route and surgical reduction of the tumour is not well known. We compared locoregional administration with intravenous (i.v.) injection. Thirty-four in-bred rats with peritoneal metastases were randomly allocated into eight groups and injected with 14C-labelled 5-fluorouracil (5-FU) either through the i.v. or i.p. route, with or without a preceding tumour debulking, and were sacrificed after 2 or 8 h. Tumour radioactivity was visualised by autoradiography and quantified by a computer-based image analysis. After 8 h, 19 debulked and i.p.-injected tumours had a higher drug uptake, 63.2+/-28 (mean+/-standard deviation (SD)) kBq/g than 62 native i.p.-injected tumours (32.8+/-14) or 22 debulked and i.v.-injected tumours (18.5+/-18, P=0.002). After 8 h, 9 small tumours (/=median 571 pixels), 16 debulked and i.p.-injected tumours had a higher radioactivity (drug uptake) (150.7+/-63) at 2 h than 49 i.p.-injected native tumours (48.5+/-59) or 11 reduced and i.v.-injected tumours (19.9+/-13, P=0.03). At 8 h, 10 debulked and i.p.-injected tumours had a higher drug uptake (50.3+/-24) than 33 native and i.p.-injected (30.8+/-10) or 14 debulked and i.v.-injected tumours (16.0+/-19, P=0.001). These results indicate that a debulking procedure and locoregional treatment of peritoneal metastases is associated with an increased level of 5-FU in the tumours.