STUDIES BASED ON A MALIGNANT TUMOR OF THE RABBIT : VI. VARIATIONS IN GROWTH AND MALIGNANCY OF TRANSPLANTED TUMORS. PART 1. RESUTLTS OF TRANSPLANTATION FOR THE FIRST TWENTY GENERATIONS.

A report is given of the results obtained by intratesticular inoculation of a malignant tumor of the rabbit based on a study of the first twenty generations. The subject is presented from the standpoint of variations in growth and malignancy as they occurred with continued transplantation. Essential features of the experimental conditions and of clinical and postmortem observations are condensed and recorded graphically. On analyzing the results of these experiments, it was found that many changes had occurred in the behavior of the tumor during transplantation, but that the changes were of a very irregular character and as a rule did not proceed constantly in any given direction. Moreover, the evidence as to the effect of transplantation on the growth and malignancy of the tumor was contradictory in that there was a great deal of evidence to show that there had been a decided increase in the activity and capacity for growth on the part of the tumor cells. But, with the exception of a few generations, there was an apparent reduction in the incidence and percentage distribution of secondary tumors, while the death rate was unaltered or actually diminished. It was believed that this paradoxical situation afforded a basis for an explanation of the results that had been obtained by transplantation.     

STUDIES BASED ON A MALIGNANT TUMOR OF THE RABBIT : II. PRIMARY TRANSPLANTATION AND ELIMINATION OF A COEXISTING SYPHILITIC INFECTION.

Several attempts were made to transplant a spontaneous malignant tumor in a syphilitic rabbit before a method was devised which proved successful. Subcutaneous and intracutaneous inoculations were unsuccessful on account of the vigorous granulomatous reaction which was aroused by the introduction of tumor fragments into these tissues. This difficulty was overcome by the use of intratesticular inoculations. With this method, a good growth was obtained in practically all animals. It appeared possible, however, that a syphilitic infection had been transmitted along with the tumor, and hence treatment with arsphenamine was instituted as a means of eliminating this infection. A study of the transplanted tumor in the first and second generations showed that it possessed a high degree of malignancy. Metastases occurred at an early period in the majority of animals, and while some of them appeared to recover, the indications were that more than 50 per cent might be expected to succumb to the effects of the tumor growth within a period of 6 to 7 months. The method of intratesticular inoculation now in use is described in detail.     

STUDIES BASED ON A MALIGNANT TUMOR OF THE RABBIT : VI. VARIATIONS IN GROWTH AND MALIGNANCY OF TRANSPLANTED TUMORS. PART 2. FACTORS INFLUENCING THE RESULTS OF SERIAL TRANSPLANTATION.

The results of transplantation, as recorded in the first section of the paper, are analyzed from the standpoint of the influence of the factors of serial passage, the material used for inoculation, the age and breed of the animals, and the time or season at which the experiments were carried out. The material used for the inoculation of different groups of animals appeared to have comparatively little effect on the ultimate results. The character of the animals used was found to be a factor of more importance in that the tumor displayed definite peculiarities of growth and malignancy referable to age and to breed or color markings. Still, it seemed unlikely that any of these factors would account for the variations in growth and malignancy that had been observed. After considering the various possibilities presented, it appeared that the principal factors concerned in determining the results of transplantation were adaptation to passage and variations in the conditions of season and weather that prevailed during the time the experiments were being carried out—the one affecting the energy of cell growth and the other affecting animal economy.     

HETEROLOGOUS TRANSPLANTATION OF MAMMALIAN TUMORS : I. THE TRANSFER OF RABBIT TUMORS TO ALIEN SPECIES

A series of experiments was undertaken in an attempt to transplant rabbit tumors to animals of alien species using the anterior chamber of the eye as an inoculation site. The uterine tumor H-31 and the breast tumors T-36 and B-240 were successfully transplanted to all the various animals tried, including guinea pigs, swine, goats, and sheep, and the H-31 and T-36 tumors were maintained by serial transfer in the two first species. On the other hand, all attempts to transfer the Brown-Pearce tumor to guinea pigs were unsuccessful. The growth characteristics of the transplanted tumors were generally similar to those observed in the natural host, but noteworthy exceptions occurred. The tumors obtained a blood supply from the foreign host and invaded the periorbital tissues but did not metastasize. Histologically, the cellular morphology of the rabbit tumors was retained, but variations in parenchymal-stromal relations characterized the form of growth in different species.     

FAMILIAL MAMMARY TUMORS IN THE RABBIT : IV. THE EVOLUTION OF AUTONOMY IN THE COURSE OF TUMOR DEVELOPMENT AS INDICATED BY TRANSPLANTATION EXPERIMENTS

A series of experiments is described in which fragments derived from two mammary tumors of distinct types were transferred at different developmental stages to the anterior chamber of the eye of normal rabbits. It was found that the ability to survive and to grow progressively after transplantation was not immediately related to anaplastic cellular changes. On the other hand, there existed a definite correlation between the success of transplantation and the morphological relationship of tumor cells and the normal cells of the host. Transplantation to normal animals could not be effected during stages of local tissue invasion but was successfully performed as soon as the tumor cells manifested the ability to invade foreign tissues or to metastasize in the spontaneous host. It was concluded, therefore, that neither anaplasia nor local tissue invasion represented autonomy but, rather, stages in its development and that the final attainment of this condition was only evidenced by metastasis or by invasion in foreign tissues. The tumors were successfully transplanted to normal animals during this series of experiments and have been carried by serial transfer to the present time. The most outstanding feature in the transplantation of the papillary type tumor was the marked difference in susceptibility exhibited by the two sexes. The acinar type tumor was distinguished by high transplantability, an extremely rapid growth rate and early regression.     

STUDIES BASED ON A MALIGNANT TUMOR OF THE RABBIT : I. THE SPONTANEOUS TUMOR AND ASSOCIATED ABNORMALITIES.

A malignant tumor which developed at the site of a primary syphilitic lesion in the scrotum of a rabbit, about 4 years after inoculation, and eventually led to the death of the animal, has formed the basis for an extensive series of investigations beginning with the study of the spontaneous tumor. The present report contains the clinical history of the animal from the time of inoculation to its death and the results of pathological examinations. The most important events recorded may be summarized as follows: 1. A mild but persistent syphilitic infection with an occasional relapse and the eventual development of chronic inflammatory lesions in the skin associated with atypical epithelial proliferations. 2. The occurrence, at about the same time, of extensive degenerative changes in the dermis accompanied by an atypical growth of hair follicles, with the production of diffuse and nodular areas of thickening and induration (scleroderma). 3. The development of a growth in the skin of the left scrotum which recurred after removal and spread diffusely over adjacent parts of the skin, and metastasized to the regional lymph nodes and to internal organs. 4. The transplantation of the growth to other rabbits by intratesticular inoculation and the successful propagation of the growth over a period of nearly 2 years (twenty generations). 5. The development of a cachexia and of pressure phenomena from metastases involving the cervical and lumbar regions of the spinal column which eventually led to the death of the animal. 6. The discovery, post mortem, of an extensive leucoplakia of the tongue and buccal mucosa, chronic inflammatory lesions in the esophagus with atypical epithelial proliferation, and a nodular growth in the left testicle differing in character from that in the scrotum. 7. The presence of extensive degenerative changes in the vascular system, degeneration of the parenchymatous organs, atrophy of the thymus and lymphoid system, with chronic lymphadenitis, atrophy, degeneration, and necrosis of the suprarenals, and atrophy and hyperplasia of the thyroid with chronic thyroiditis. The conclusion was reached that the growth in the scrotum represented a neoplasm of epithelial origin which was composed of cells allied to those found in the bulb and root sheath of the hair. No explanation was offered for the origin or unusual malignancy displayed by the tumor pending the introduction of experimental data. It was stated, however, that the evidence presented was sufficient to warrant the assumption that the factor of foremost importance in the development of the tumor was constitutional rather than local.     

UTERINE ADENOMATA IN THE RABBIT : II. HOMOLOGOUS TRANSPLANTATION EXPERIMENTS

The behavior of a transplanted adenocarcinoma of the uterus of a rabbit has been studied through 12 serial generations in the anterior chamber of the eye and 6 serial generations in the testicle. The transplanted tumor is characterized by slow growth which is at first expansive and later invasive, by an ability to form more or less differentiated structures in response to different environmental conditions and by late metastasis. The endocrinological changes that distinguish animals bearing the spontaneous tumor do not occur in animals bearing the transplanted tumor. Various experiments were undertaken in an attempt to discover the nature of the factors determining the characteristics of the spontaneous and of the transplanted tumor. It was found that successful transplantation was followed by a phase during which animals were refractory to reinoculation. The results of transplantation into the eyes of animals with spontaneous tumors suggested the existence of a similar phase during the early development of the tumor but the number of observations was not sufficiently numerous to warrant definite conclusions.     

TUMOR INOCULATION INTO ORGANS AND THE ANALOGY BETWEEN HUMAN CANCER AND THE TUMORS OF WHITE MICE AND WHITE RATS

The analysis of the experiments described above indicates that tumors of the white rat or white mouse inoculated into parenchymatous organs acquire a different biological character from those inoculated subcutaneously. The latter are a great deal more benign in their behavior than human cancer or spontaneous tumors in the same species of animals. Tumors inoculated into organs, on the other hand, are quite identical in their biological behavior with the malignant tumors of animal and man. A conclusion must then be drawn, even a priori, that the method of inoculation into organs is a very important aid in the experimental investigation of cancer. It is true that the method is a great deal more complicated and time-consuming than the ordinary subcutaneous inoculation. The subcutaneous method is satisfactory for a number of cancer problems. One of these is the study of general susceptibility and resistance of the organism of the host to the inoculation of the tumors, and this is a subject of paramount importance in cancer research. On the other hand, the investigations of the writer (10) have shown that an animal may be susceptible to a subcutaneous inoculation of a certain tumor and resist the inoculation of the same tumor into the testicle. Undoubtedly this method of inoculation will reveal the existence of a number of other phenomena. The discovery of specific therapeutic measures is certainly the greatest problem in cancer research. A great deal of work has been done already on the subject, and the latest investigations of Wassermann on the chemotherapy of experimental tumors seem to be of great promise. But here also the therapeutic methods must be tried on animals in which the inoculations of tumor cells have been made into parenchymatous organs before the growths thus treated will have any analogy to human cancer. In this connection one must bear in mind the fact that all the empirical so-called specific cancer remedies, which are continually being devised, are usually successful in treating localized skin cancers and fail utterly in the malignant growths of the internal organs. It is comparatively easy to produce a localized necrosis and softening in a circumscribed growth of the skin and subcutaneous tissue, but whether the same result will be produced on a diffuse and better nourished tumor growing inside of a parenchymatous organ cannot be decided a priori. To determine this it is necessary to have experimental proof on animals in which the tumor was inoculated into organs.     

STUDIES BASED ON A MALIGNANT TUMOR OF THE RABBIT : III. INTRATESTICULAR TRANSPLANTATION AND CLINICAL COURSE OF THE DISEASE.

The results obtained by intratesticular inoculation of a malignant tumor of the rabbit are presented in the form of a general summary covering the phenomena of growth and spontaneous regression of the tumor and the clinical evidence of disease in tumor animals. Under ordinary circumstances, growth is obtained by this method of inoculation in practically all animals, and the majority of them show metastases. The ultimate fate of tumor animals is variable. Spontaneous regression is of frequent occurrence, and apparent recovery may take place even after extensive metastases have developed. More often, the disease terminates fatally. The course of the disease may be fulminating in character with death occurring 6 to 7 weeks after inoculation, or it may be prolonged over a period of more than 6 months, but death usually occurs between 7 weeks and 3 months after inoculation. The symptomatology varies with the course of tumor growth but is in general that of a debilitating disease of an acute or chronic character in which the picture is frequently complicated by symptoms referable to mechanical causes or to one or more of the glands of internal secretion. The most prominent of these are due to the presence of metastases in the suprarenals or pituitary body, in which case symptoms of Addison''s disease or diseases of the pituitary may be superimposed upon those attributable to the presence of extensive tumor growths. In the absence of such growths, however, the symptoms of suprarenal or pituitary involvement may dominate the picture.     

ON THE NATURE OF TRANSPLANTATION IMMUNITY IN THE ADENOVIRUS TUMOR SYSTEM

The existence of a virus-induced, virus-specific transplantation, antigen in adenovirus 12-induced CBA mouse tumors was demonstrated. The antigen is virus-specific, but not related to structural virion or T antigens. It is a weak antigen, and required immunization with whole, infectious adenovirus 12 to produce considerable immunity. Comparable immunity could not be achieved with homologous cellular or subcellular materials, but some indication of enhancement was produced with low tumor dose. Immunization required at least 2 wk and was mediated by immune lymphoid cells. Serum of immunized animals showed no demonstrable cytotoxicity or enhancement. Animals immunized with virus and Freund's adjuvant showed diminished transplantation immunity, although these animals were actively immunized against adenovirus type 12 structural virion antigens.     

FURTHER STUDIES ON SPECIFIC TRANSPLANTATION ANTIGENS IN ROUS SARCOMA OF MICE

Mice allografted with different sarcomas, induced by the Schmidt-Ruppin variant of Rous sarcoma virus (RSV-SR), showed a resistance against subsequent isografting of 9 different Rous sarcomas. Transplantation resistance could also be induced by Rous mouse tumor cells x-irradiated with 8000 r or with cell-free tumor extracts, containing no demonstrable virus. No transplantation resistance could be demonstrated after allograft pretreatment with various polyoma tumors or non-viral tumors. Allograft pretreatment with Rous tumors induced no demonstrable resistance against isografting of polyoma tumors. Inoculation of RSV-SR or Rous chicken sarcoma suspension into adult mice gave no clear cut resistance against isografting of mouse sarcomas. Neither after allografting of Rous tumors nor after virus or chicken sarcoma inoculation into adult mice could virus-neutralizing activity be demonstrated in the sera. The results demonstrate the presence of common, specific transplantation antigen(s) in different Rous sarcomas in mice and speak against an identity between the transplantation antigen(s) and viral antigen(s).     

TRANSPLANTABLE EPITHELIOMAS OF THE LIP AND MOUTH OF CATFISH : I. PATHOLOGY. TRANSPLANTATION TO ANTERIOR CHAMBER OF EYE AND INTO CORNEA

The catfish (Ameiurus nebulosus) taken from streams near Philadelphia, is commonly afflicted with an epithelial tumor bearing some resemblance to epithelioma of the lip in man. This neoplasm usually occurs as solitary or multiple, large, red, fleshy masses upon the lips or dental plates, and by reason of its size, may prevent closure of the mouth. The tumor is comprised of epithelial cells, often in papillary arrangement, supported by a delicate vascularized connective tissue stroma. The larger growths frequently invade adjacent normal tissues and force their way into vessels where they are found as emboli. The clinical course of the tumor is one of relatively slow but progressive growth. This neoplasm has been observed from the time of its inception in a number of animals. Thus it has been learned that the proliferative stage of the neoplastic process is preceded and accompanied by a striking vascular reaction. Intense hyperemia invariably occurs in that region of the mucosal surface which later becomes the site of neoplastic proliferation. Furthermore, by direct microscopic observation of the living tumors the atypical structure and arrangement of the blood vessels become apparent. A study of the significance of these vascular phenomena in their relation to the inception and growth of the tumor is now in progress. It has been found possible to transmit the catfish tumor to fish of the same species by implanting fragments of the tumor into the anterior chamber of the eye. Also, by taking advantage of an anatomical peculiarity of the catfish cornea, it has been possible to embed the tumor fragments in normal tissue where it could still be readily observed both in the gross and microscopically. The growth of the transplants in the eye has been followed by periodic examination of the living tumor by means of the slit lamp microscope. In the anterior chamber, the tumor characteristically forms dense membranes which spread over the inner surface of the cornea. In this manner growth continues until the tumor fills the chamber. Between the two layers of the cornea, tumor growth is expansive. Attempts to transplant the tumor to the anterior chamber of two other species of fish and to frogs, were unsuccessful. Implantation of the catfish epithelioma in alien species of fish excited no exudative response, whereas in a less closely related species of animal, the leopard frog, a pronounced exudative reaction resulted.     

Augmentation of antitumor immune responses by multiple intratumoral inoculations of replication-conditional HSV and interleukin-12

Intratumoral inoculation with a herpes simplex virus (HSV) mutant, G207, as an in situ cancer vaccine has been shown to inhibit tumor growth by inducing tumor-specific immune responses. Here, as a step toward the clinical application of this therapeutic approach, we evaluated different protocols for enhancing the antitumor effect. First, in a bilaterally established tumor model with CT26 colon carcinoma, we demonstrated that multiple intratumoral inoculations with G207 induced a greater antitumor effect on both the inoculated and distant tumors than did 1 or 2 inoculations. Second, to boost this antitumor effect, we developed 2 strategies: multiple in situ cancer vaccines with G207 in combination with systemic administration of recombinant interleukin-12 (rIL-12) (G207/systemic rIL-12) or local administration of rIL-12 (G207/local rIL-12). The antitumor effects in both the inoculated and distant tumors by the combined treatments were significantly greater than by either G207 or rIL-12 treatment alone. G207/systemic rIL-12 and G207/local rIL-12 mediated the complete regression of both the inoculated and distant tumors in 67% and 79% of the animals, respectively. These results indicate that multiple intratumoral inoculations of G207 and systemic or local rIL-12 administration work synergistically to facilitate tumor regression and that this combination of treatments may have potential for treating cancer metastasis.     

CARCINOMA IN THE LEOPARD FROG: ITS PROBABLE CAUSATION BY A VIRUS

An epithelial tumor with acidophilic intranuclear inclusions frequently occurs in the kidneys of leopard frogs. This tumor usually has the appearance of an infiltrating and destructive adenocarcinoma, which, when large, not uncommonly metastasizes; less often it is more orderly and adenomatous. When inoculated as living fragments or cell suspensions into the lymph sacs, the cranial cavity, or the abdomen, no significant local growth results and the implanted material is resorbed. However, in approximately 20 per cent of the frogs surviving inoculation for more than 6 months, tumors develop in the kidney, which are like the "spontaneous" neoplasms. The incidence far exceeds that in the controls. Desiccated and glycerinated tumor injected into the abdomen gives the same result as inoculation with living tumor; in somewhat over 20 per cent of animals surviving more than 6 months kidney tumors occur. In alien species of frogs, no such tumors are produced by inoculation either with living or with desiccated tumor. These experiments indicate the probability that the kidney tumor of the leopard frog is caused by an inclusion-forming, organ-specific virus.     

THE RELATIONSHIP BETWEEN THE GROWTH CAPACITY OF PRIMARY TUMORS AND THE DISTRIBUTION OF METASTASES OF A TRANSPLANTED TUMOR OF THE RABBIT

As an adjunct to the study of a transplantable malignant tumor of the rabbit an index of malignancy has been developed which has proven useful in evaluating the severity of the disease for the purpose of statistical investigations. The index consists of two figures, one of which expresses the growth capacity of the primary lesion on a volume basis, and the other the extent of the metastatic lesions according to the distribution of peripheral tumors. In the present communication these two values for each animal are utilized for the study of the relationship existing between the size of the primary tumor and the distribution of metastases in this experimental tumor. The data from a group of 128 rabbits form the basis of the analysis. These animals represent three entire series that had been used for the purposes of other experiments at various times, all, however, conducted under carefully controlled uniform conditions. The indices of malignancy of these animals were analyzed by means of graphs, in which individual values are preserved while at the same time investigation of mass behavior is permitted. From this analysis it is concluded (1) that the severity of the disease as a whole is very irregular among individuals of a series and from series to series, and (2) that the relationship between the extent of the primary lesions and that of the secondary lesions is inconstant among individuals but assumes definite proportions when the data are viewed in the mass. For the mass proportion between the two phases of the disease is predominatingly a direct one, but in certain small groups of seriously affected rabbits the proportion shows a tendency to reversal, while, in addition, there occurs a reduction in the rate of growth of the primary tumor, which reduction, together with the reversal in relationship, appears to be more pronounced from series to series as malignancy increases. An attempt has been made to account for these results in the light of what has been ascertained of the modes of defense of the rabbit host against the inroads of the cells of this transplanted tumor.     

MELANOMA (SARCOMA) OF THE EYE IN A SYPHILITIC RABBIT

A melanotic tumor developed in a defective eye of a syphilitic rabbit following repeated genital inoculations with Treponema pallidum. The appearance of the tumor coincided with the development of a syphilitic lesion in the same eye, suggesting a relation between the occurrence of the two lesions.     

PENETRATION OF NORMAL MUCOUS MEMBRANES OF THE RABBIT BY TREPONEMA PALLIDUM AND THE INFLUENCE OF THIS MODE OF INFECTION UPON THE COURSE OF THE DISEASE

Experiments were carried out with three strains of Treponema pallidum to determine whether infection could be produced by applying an emulsion, rich in spirochetes, to normal mucous membranes of rabbits and whether an infection produced in this manner differed in any respect from one produced by other methods of inoculation. It was found that a simple instillation of a spirochete emulsion into the conjunctival sac or the sheath was all that was necessary to obtain an infection. Still, the infection produced in this manner differed from that produced by intracutaneous or testicular inoculations in that it tended to pursue a mild or asymptomatic course and frequently without the development of a characteristic chancre. It is pointed out that these experiments may have a bearing on problems pertaining to obscure and atypical cases of human syphilis as well as the more immediate question of the infectivity of Treponema pallidum.     

沉默ATF5对卵巢癌裸鼠成瘤的影响

目的研究沉默人转录激活因子5（ATF5）后对卵巢癌SKOV-3细胞体外增殖、凋亡及体内成瘤的影响,探讨人类卵巢癌基因治疗的新途径。方法将慢病毒携带的小干扰RNA感染卵巢癌SKOV-3细胞（实验组）,空载体转染的卵巢癌SKOV-3细胞（空载体对照组）和卵巢癌SKOV-3细胞（空白对照组）进行对照,对比观察细胞的生长状态;MTT法检测细胞的增殖生长情况;罗氏凋亡检测试剂盒检测细胞凋亡情况;分别于裸鼠皮下接种3组卵巢癌SKOV-3细胞,观察各组裸鼠的存活状态、种植瘤生长情况;RT-PCR检测瘤组织中ATF5mRNA的表达;Western-Blot方法检测瘤组织中ATF5蛋白的表达;苏木素-伊红染色（HE染色）观察瘤组织生物学形态变化。结果经转染ATF5基因的小干扰RNA后,在相同培养时间内3组细胞的增殖和生物学形态没有明显差异,但实验组细胞凋亡数量较其他两组明显增多。皮下接种成瘤后,3组裸鼠的成瘤率没有明显区别,实验组与空载体对照组和空白对照组相比,肿瘤成瘤时间晚,生长缓慢;移植瘤ATF5mRNA表达及蛋白表达降低;细胞分裂象明显减少,凋亡现象明显。结论沉默ATF5对人上皮性卵巢癌裸鼠移植瘤生长有抑制作用,这可能成为卵巢癌基因治疗的新靶点。     

HETEROLOGOUS TRANSPLANTATION OF MAMMALIAN TUMORS : II. THE TRANSFER OF HUMAN TUMORS TO ALIEN SPECIES

The successful heterologous transplantation of several human tumors, including a breast carcinoma, a carcinoma of the colon, a melanotic sarcoma, and a fibrosarcoma, has been reported. Histologically, the appearance of the transplants of the carcinomata differed from that of the spontaneous tumors, but the directly observed growth of the transplanted fragments together with the presence of invasion in microscopic sections afforded proof of successful transfer. Transplants of the sarcomata, on the other hand, were morphologically identical with the primary tumors, and in the case of the fibrosarcoma serial passage to a second generation of animals was successfully performed.     

THE INFLUENCE OF THE ANTERIOR LOBE OF THE PITUITARY BODY UPON THE GROWTH OF CARCINOMATA

1. The administration of emulsions of the anterior lobe of the ox pituitary increases very markedly the rate of growth of the primary tumor in rats inoculated with carcinoma. The growth of small tumors is accelerated relatively more than that of large tumors. 2. This acceleration is only evidenced, however, at a certain stage in the growth of the tumor, subsequent to the twentieth day succeeding inoculation. The administrations do not enhance the tendency of the tumors to metastasize. 3. Liver emulsion does not cause an acceleration of the growth of carcinomata in rats.