Twenty-six scirrhous carcinomas of the breast were divided into three histological grades of malignancy; low (Grade I), intermediate (Grade II), and high (Grade III), and the correlation of the grades with histochemical and electron microscopic findings in both tumor cells and host tissues was examined. The tumor cells contained increased amounts of lysosomal acid phosphatase and β-glucuronidase. This increase was most marked in Grade I and II tumors and the increase was consistent with lysosome-like fine structures. Both intracytoplasmic lumina and microvilli against stroma were characteristic findings of carcinoma cells and they were mostly found in Grade I and II tumors. Segments of intact basal laminae and myoepithelial cells were also found in Grade I and II carcinomas.
The stroma contained moderately increased amounts of intracellular acid phosphatase and β-glucuronidase, independent of tumor grade. The stroma also contained a large amount of acid mucopolysaccharide ground substance, irrespective of the three grades. There was a striking difference in the ultrastructural organization of the stroma between normal and neoplastic tissues. Although fragmented elastic fibers and increased amount of acid mucopolysaccharide granules, and macrophages rich in phagolysosomes were prominent fine structures of the stroma of carcinomas, there was no apparent difference in them among the three grades of malignancy.

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Inbred strains of guinea pigs, JY-1 and Hartley/F, established in this Institute, were injected subcutaneously with 3-methylcholanthrene. During 1972 to 1974, 15 solid tumors (7 fibrosarcomas, 6 liposarcomas, and 2 atheroma-like tumors) have been induced in 12 animals out of 30 employed, Among the 15 tumors, 1 fibrosarcoma induced in JY-1 and 2 liposarcomas and 1 fibrosarcoma induced in Hartley/F were transplantable and established as the syngeneic lines named J4, H10, H12, and H9A, respectively. In addition, a transplant of J4 into the Hartley/F strain animal was established as an allogeneic subline and named JH4. Pathological and biological characteristics of these tumors are described and differences between these tumors and line 10 hepatoma, established by Rapp et. al. in strain-2 guinea pig, are discussed.

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A total of 39 kidney tumors were induced by a continuous oral administration of N-butylnitrosourea (BNU) in 33 out of 204 rats of W/Fu strain and of 41 (W/Fu×ACI/N) F₁ rats. No spontaneous renal tumors were observed among 66 males and 109 females of W/Fu rats which survived beyond the age of 19 months. Histologically, renal cell and mesenchymal types were commonly observed; 24 cases belonging to the former and 11 cases to the latter. Two cases of nephroblastoma were also encountered. There was no sex difference in renal tumorigenesis with BNU as a whole. Castration in both sexes was apparently inhibitory for kidney tumor development. Estrogenization of castrated rats either by syngeneic ovary graft or by repeated injections of estradiol benzoate enhanced tumor induction with BNU. Progesterone was not effective in restoring the tumor incidence in castrated rats. Distribution of histological types of tumors thus induced differed among the hosts with different hormonal conditions; in males the majority was renal cell type, whereas almost all mesenchymal tumor and nephroblastoma cases were found in intact females or estrogenized rats. BNU induced a variety of tumors in several organs including cerebral hemisphere, peripheral nerves, mammary glands, hematopoietic system, digestive tracts, and so on, However, such concurrence did not affect the development of renal tumors in the present study.

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Intrarectal instillation of 0.5ml of a 0.125% solution of N-methyl-N'-nitro-N-nitrosoguanidine twice weekly for 53 weeks to female inbred strain-2 guinea pigs induced multiple large bowel adenocarcinomas in 13 of 15 animals in 52 to 85 weeks. The lesions were plaque-shaped in small tumors and infiltrative or constrictive in large advanced tumors. The neoplasms showed histological features in varied grades of differentiation and invasiveness similar to those of human cases. These findings distinguished them from large bowel cancers chemically induced in rats and mice.

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The effect of ulcers induced by iodoacetamide on the development of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine was studied in male Wistar rats. The ulcerative lesions induced by iodoacetamide were confined symmetrically to the fundic region along the limiting ridge in the stomach and the pyloric region was unaffected. Animals treated with iodoacetamide and N-methyl-N'-nitro-N-nitrosoguanidine produced a high incidence of tumors including adeno-carcinoma in the fundic region. The incidence of tumors in the pyloric region in the control group was 80% but there were no tumors in the fundic region. The tumors in the fundic region were most frequently found in the same areas that ulcers had previously been induced. These findings suggest that ulceration and regeneration of the mucosa are important factors in gastric carcinogenesis induced in rats by N-methyl-N'-nitro-N-nitrosoguanidine.

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Change in β-glucuronidase activity of six Yoshida ascites hepatomas was examined after treatment of host rats with one of 12 anticancer agents. The hepatomas, AH-66F, AH-130, AH-109A, AH-60C, and AH-44, in decreasing order showed more or less distinct increase in β-glucuronidase activity after treatment of the rats with Nitromin, Endoxan, 864-T, Carbazilquinone, Mitomycin-C, Toyomycin, Daunomycin, Neocarzinostatin, vincristine sulfate, 5-fluorouracil, or cytosine arabinoside only when the cytological effect was positive. Moreover, degree of the increase was generally correlated with that of cytological effect. Bleomycin was ineffective either enzymically or cytologically. AH-66 was insensitive to any of the agents tested in increasing β-glucuronidase activity and showed only a very slight cytological response to some of the agents. Acid deoxyribonuclease behaved like β-glucuronidase but to a lesser extent.
The above order of drug sensitivity of the hepatomas was not in parallel with that of normal β-glucuronidase level, which also did not correlate with the life span of host rats.

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In order to demonstrate a collagenolytic enzyme in a tumor, solid Tawa sarcoma was subjected to a tissue culture technique. Salt- and acid-soluble collagen used as substrate was extracted from the rat skin and tail tendon. The tumor mass used was obtained on the 5th, 8th, and 11th days after subcutaneous transplantation of ascites Tawa sarcoma cells. Each tissue fragment in the inner and outer layers of the tumor mass was incubated on collagen gel at 37° for 4 days, and collagenolytic activity was determined by comparing the relative content of hydroxyproline in the attacked collagen which was separated by means of centrifugation. All fragments were found to possess collagenolytic activity with some variation. Higher activity was observed in the outer layer than in the inner layer. Tumor fragments were cultured, and the collagenolytic enzyme was isolated from the culture medium and concentrated by ammonium sulfate precipitation and acrylamide disc electrophoresis. Collagenolytic enzyme activity was examined for its mode of attack on native collagen.

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Incipient growth of Yoshida sarcoma in ascitic and solid forms was analyzed after transplantation of a small number of tumor cells varying from 1 to 10₆. Growth curves of the ascites tumors revealed that the duration of the incipient growth was much longer than the length of subsequent advanced stage and that the ascites tumor during its incipient stage appeared to grow exponentially and most rapidly in the entire course of the tumor development.
Growth rate analyses of the ascites and solid tumors showed that, during their incipient stage, the population doubling time prolonged gradually as the inoculum size increased. This phenomenon was thought to suggest probable cell death on account of the transplantation procedure.

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Histological types and grades of 613 urinary bladder cancers induced in rats by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) were analyzed. Most of them (95.1%) were transitional cell carcinomas, the remainder being squamous cell carcinomas (3.3%), undifferentiated carcinomas (2.5%), and carcinosarcomas (0.3%). Among the transitional cell carcinomas, 23.3% were Grade I anaplasia, 55.2% Grade II, and 21.5% Grade III. Among the squamous cell carcinomas, 20% each were Grade I and II, and 60% Grade III. Most of the undifferentiated carcinomas were Grade III. Some of the cases of transitional cell carcinoma had areas of squamous metaplasia and/or glandular metaplasia, and the incidence of metaplasia increased with the grade of anaplasia. Approximately 36% of the cases of transitional cell carcinoma were of the invasive type. Invasive types were twice as numerous among cases of squamous cell carcinoma as among those of transitional cell carcinoma and all of undifferentiated carcinomas were invasive. The incidence of invasive type was closely related to the grade of anaplasia. These results show that the morphological characteristics of urinary bladder carcinomas induced by BBN in rats are similar to those seen clinically in humans.

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The effect of ethionine on pancreatic tumorigenesis of 4-hydroxyaminoquinoline 1-oxide (4-HAQO) in rats was studied. The high incidence of hyperplastic nodules developed in the exocrine pancreas of animals receiving a single injection of 4-HAQO only or protein-deficient diet containing 0.5% ethionine for 4days followed by 4-HAQO. The incidence of adenoma was higher than that of hyperplastic nodules and well-differentiated adenocarcinoma developed in 1 out of 12 animals receiving protein-deficient diet containing ethionine for 18 days followed by 4-HAQO. No tumor was found in the pancreas of animals that received 0.005N HCl without 4-HAQO. These results suggest that the pancreatic tumorigenesis of 4-HAQO is enhanced by ethionine.

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The protein-bound polysaccharide preparation, PS-K, was found to potentiate the production of hemolytic plaque-forming cells of spleen and serum hemagglutinin in C57BL/6 mice, when they were immunized with a low dose of sheep red blood cells. PS-K was administered intraperitoneally at a dose of 150mg/kg daily for 5 days. Ten days later, the mice were immunized with either 4×10⁸ or 1×10⁷ sheep red blood cells. Both the number of plaque-forming cells in their spleen and the amount of serum hemagglutinin were significantly higher in the PS-K treated mice when 1×10⁷ sheep red blood cells were used as an immunizing dose.

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The estrogen-binding capacity of the regrowing tumors after endocrine ablation surgery was estimated in the rat and human breast cancer. All of 4 tumors that had not had the estrogen receptor before oophorectomy did not show the estrogen-binding capacity after the procedure, in the mammary cancer in the Sprague-Dawley rat induced by 7, 12-dimethylbenz [a] anthracene, whereas all the 4 tumors that had the receptor before the procedure showed the binding capacity after oophorectomy. In 4 patients who had been subjected to the major endocrine ablation therapy because of metastatic breast cancer with effective response, the secondary metastatic deposits of cancer showed the estrogen-binding capacity long after the endocrine ablation.
The fact that regrowing tumors after complete or partial regression of initial tumors by endocrine ablation retain the estrogen receptor seems to be contradictory to the classical concept of autonomy in recurrence of the breast cancer after hormonal manipulation.

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This experimental study was undertaken to evaluate the effectiveness of a large-does intratumoral administration of OK-432, which has already been found to be effective when combined with Mitomycin-C, 5-fluorouracil, and cytosine arabinoside, and/or small-dose of OK-432 in clinical use.
Large-dose of OK-432 was administered intratumorally 15 days after the subcutaneous inoculation of 2.5×10⁶ Ehrlich carcinoma cells. At the same time, 2.5×106 tumor cells were re-challenged intraperitoneally. A combined treatment with a small-dose of OK-432 and Mitompcin-C, cyclophosphamide, or 5-fluorouracil was also made. As a large-dose of OK-432, three-dose regimen of 1, 000, 500, and 100KE/kg was tested. As a small-dose of OK-432, 4 consecutive daily intramuscular injection of 50KE/kg was examined. From the results obtained the initial intratumoral administration of a large-dose of OK-432 was found to be beneficial for the following combined treatment with a small-dose of OK-432 and Mitomycin-C or cyclophosphamide.
The change of phytohemagglutinin (PHA) responsiveness of spleen cells after intratumoral injection of a large-dose of OK-432 was investigated in mice. These results indicated a decrease of PHA responsiveness of spleen cells according to the tumor growth. The intratumoral large-dose administration of OK-432 suggested inhibition of the decrease in PHA responsiveness. The most effective and suitable dose in this experiment was found to be 500KE/kg in the three-dose regimen.

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Transplantable rat ascites hepatoma AH-66F was examined by an electron microscope. C-particles were numerously observed in the intercellular spaces and on the cell surface. The size of these particles varied from 90 to 200nm in diameter. Small vesicles were also observed. These vesicles ranged from 40 to 50nm in diameter. Most of them were located near the group of C-particles. Occasionally, small vesicles were observed in the form of a rosette around the Cparticles.

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The rôle of macrophages from rats treated with a streptococcal preparation (OK-432) for inhibition of syngeneic tumor growth was examined both in vivo and in vitro. Treatment of rats with OK-432 intraperitoneally resulted in a transient inhibition of ascites tumor growth and concomitant increase in survival time. Peritoneal exudate cells from these rats completely suppressed tumor growth when admixed with tumor cells and transferred subcutaneously to syngeneic recipient rats. No such effect was observed with tumor cells from saline-treated control rats. The macrophages were responsible for the neutralization of tumor growth, since the removal of adherent cells from peritoneal exudate cells of rats treated with OK-432 resulted in the abrogation of their antitumor activity.
To further investigate the antitumor activity of macrophages, an in vitro cytotoxicity assay was carried out on several tumor cell lines. A complete target cell destruction was brought about by macrophages from the rats treated with OK-432 but not from the control animals. The tumor cytotoxicity mediated by macrophages activated with OK-432 was regarded as non-specific because they could damage all the target cell lines tested.

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Three groups of female Donryu rats were given a single gastric intubation of 800, 400, or 200mg/kg body weight of 1-propyl-1-nitrosourea and one group of female Donryu rats was given a single subcutaneous injection of 1-propyl-1-nitrosourea. The incidence of tumors was highest for mammary tumors and leukemia, and next for tumors of the ovary, thyroid, and adrenal glands, and in the digestive tract in rats given the chemical by oral administration. There were also scattered tumors in various other organs. Mammary and subcutaneous tumors were found in some rats given a subcutaneous injection of 1-propyl-1-nitrosourea.

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The carcinogenicity of young, pre-blooming flowers of coltsfoot, Tussilago farfara L., which is a herb of the tribe Senecioneae, family Compositae, and widely used as a herbal remedy, was studied in inbred strain ACI rats. Rats were divided into 4 groups: Group I received 32% coltsfoot diet for 4 days, and subsequently 16% diet until the termination of experiment. Groups II and III respectively received 8% and 4% coltsfoot diet for 600 days. Group IV was fed a normal diet as a control group. The experiments were terminated 600 days after the start of administration of coltsfoot diet. All the rats in Group I survived beyond 380 days after the start of feeding and 8 out of 12 rats developed hemangioendothelial sarcoma in the liver, whereas only one out of 10 rats developed the tumor in Group II, and no tumors were observed in Group III. Chemical studies on the dried, young flowers used in this experiment suggested that the carcinogenicity of coltsfoot is most probably due to senkirkine, a hepatotoxic pyrrolizidine alkaloid.

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Subclonal cell populations were prepared from a clonal line of the rat ascites hepatoma AH-66 in vitro, using the agar plate culture method. α-Fetoprotein and albumin concentrations in culture media of these subclones were determined by ¹²⁵I-radioimmunoassay and single radial immunodiffusion method, respectively. Results demonstrated that the AH-66 clone was a complex of cells with varying producibility of α-fetoprotein and albumin. All the subclones showed varied and distinct production of α-fetoprotein, but not all the subclones produced detectable levels of albumin.

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3-(Methyl-α-D-glucopyranos-6-yl) -I-(2-chloroethyl) -1-nitrosourea exhibited marked activities against Nakahara-Fukuoka sarcoma, adenocarcinoma-755, and leukemia L-1210. It is an antitumor agent with modified bone-marrow toxicity and it exhibits neither diabetogenic nor antibacterial activities.

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