Albert E. Casey

A Species Limitation of an Enhancing Material Derived from a Mammalian Tumor

In an effort to differentiate the material derived from the Brown-Pearce rabbit tumor 1 from other enhancing materials it seemed desirable to know whether it would enhance various diseases and tumors, both in the same and in different species. To this end a series of experiments was made with a Bashford mouse carcinoma (No. 63).∗ The experiments were distributed at approximately 6 weeks intervals between January, 1932, and January, 1933, each experiment consisting of 30 mice divided into 3 groups of 10. Enhancing material prepared from Brown-Pearce tumor tissue 1 and enhancing material prepared in an entirely similar manner from the Bashford tumor itself were injected subcutaneously into the left groins of the animals of Groups 1 and 2 respectively. No tumor growths resulted. Twelve to 18 days later the animals of these 2 groups together with those of the third, not previously injected, were inoculated with grafts (Exps. 1-6) or with a saline emulsion (Exps. 7-10) of fresh Bashford tumor tissue (Table I). Observations were made on the incidence and the size of the primary tumors resulting. Of the 97 mice treated with the rabbit tumor material 46 (47.4%) had primary tumors at 21 days† as compared with 57 (57.5%) among the 99 control mice and 67 (67%) among the 100 mice treated with material from the Bashford tumor itself. Such an excessive variability would not be expected to occur by accident more often than twice in 100 similar experiments with 300 mice (x2—7.7, n—2, P—0.02). Treatment of the mice with the rabbit tumor material seemed to result in fewer takes, while treatment of the mice with the mouse tumor material seemed to result in an increased number of primary tumors, the difference between the 2 series being significant when compared directly (x2—7.7y n—1, P—0.01—).

Specificity of Enhancing Materials from Mammalian Tumors

That growth promoting substances are present in transplanted tumors has been known for a long time. But that they can exercise a profound influence over the metastatic phase of malignancy, that their effects in sensitizing the host to a malignant growth can be more or less permanent, or that they have species and tissue limitations in their activity is only now being demonstrated. 1 Thus, a filterable material from the Brown-Pearce rabbit tumor has been found to enhance every observed phase of the growth of this tumor, both local and metastatic. A species limitation has been noted in that the material from the rabbit tumor would not enhance the growth of mouse carcinoma 63 or mouse sarcoma 180. Rabbits treated with the material were still profoundly hypersusceptible to both local and metastatic phases of the Brown-Pearce tumor 7 months after the last injection. 2 The growth promoting material described by Haaland 3 and Leitch 4 in mouse carcinoma 63 has been found to enhance growths of the same tumor but has had no enhancing effect on the growth of the rabbit tumor or the growth of mouse sarcoma 180. That enhancing materials are to be found in sarcomata seemed evident from the work of Flexner and Jobling 5 and of Chambers and Scott. 6 To test the specificity and biology of sarcoma enhancing materials, experiments were undertaken of which the first 3 are here reported. In each experiment tumor tissue from mouse sarcoma 180 was anaerobically refrigerated for 14–60 days at 24°F., minced and ground without sand, and emulsified with normal saline (dilution 1–10). In one experiment the emulsion was filtered through a Berkefeld “V” candle. Of this emulsion 0.1 cc. was injected into the left groin of 10 mice; 2 weeks later the 10 mice and 10 control mice not previously treated were inoculated in the same groin with fresh sarcoma 180.

A Persistent Hypersusceptibility Induced in Rabbits with an Homologous Tumor Material

A single injection of a filterable material from homologous tumor tissue prepared by prolonged anaerobic refrigeration will render the rabbit host more susceptible to the inoculation and increase the malignancy of the Brown-Pearce rabbit tumor. The dosage employed may vary from 0.2 to 0.5 cc. of a 1–5 dilution of the material. 1 The material is equally active when inoculated either 2 weeks before or 2 weeks after the tumor inoculation. In a previous experiment injection of the material 3 times at 2 month intervals led to increased susceptibility instead of to an immunity to this tumor. This paper reports a second series in which 5 male rabbits were injected 8–10 times at intervals varying from 2 weeks to 2 months. The rabbits were then permitted to rest for 7 months, during which period no treatment was given. At the end of this period the 5 rabbits (experimental group) and 14 control rabbits (adults of the same sex, size, origin, and breed but almost a year younger) were inoculated intratesticularly with fresh Brown-Pearce tumor. The experiment was terminated at 2 months. The incidence of primary testicular tumors was 4 (80%) for the experimental group as compared with 11 (78.6%) for the control group; the cubic volume of the 4 primary tumors at autopsy in the experimental group was 15.5 cc. as compared with 10.0 cc. for the 11 primary tumors in the control group; the incidence of metastatic tumor in the experimental group was 4 (80%) as compared with 9 (64.3%) for the controls; the cubic volume of the metastatic tumor per animal with metastases was 175 cc. in the experimental as compared with 38.3 cc. for the control group; the average number of metastatic foci was 22.0 for the experimental as compared with 9.0 for the control group; the total mortality for all animals inoculated was 73.4% in the experimental as compared with 46.4% for the control group; the total mortality for all animals with tumor was 91.8% in the experimental as compared with 59.1% for the control group; the average longevity for all 5 animals in the experimental group was 50.8 days as compared with 58.6 days for the 14 animals in the control group.

Selective Blocking of Host Resistance to Malignant Neoplasm (Brown-Pearce Tumor in New Zealand White Rabbits)

Summary and Conclusions 1. Of 134 New Zealand White rabbits in 15 experiments 65 were given an injection of 1.0 to 0.0001 g of Brown-Pearce tumor tissue (no longer viable) which had been kept frozen anaerobically for 10-296 days. Viable Brown-Pearce tumor tussue was transplanted 10-21 days later into the testes of 54 and beneath the skin in 11 of the 65 animals; also inoculated at the same time were the 69 controls, 52 subcutaneously and 17 intratesticularly. 2. The experimental animals had a significantly greater incidence of and larger primary tumors, a greater incidence and volume of, and more numerous metastases and a greater mortality from the tumor in a shorter interval after inoculation than their respective controls, by both the intratesticular and subcutaneous routes. 3. This effect (XYZ factor) was not confined to the New Zealand White breed as common rabbit hybrids of average resistance to the Brown-Pearce tumor could be rendered more susceptible than even the most susceptible breeds such as the English, Sable, Flemish and Rex. Even the relatively resistant blue-cross or Lilac cross rabbit became more susceptible upon injection of the frozen tumor tissue. 4. The mechanism of the XYZ phenomenon seems to be influenced by the inactivation or masking by prolonged freezing of inhibitory factors present in the fresh tumor. The frozen material seems to act by blocking host resistance to a specific tumor.

Nucleated Erythrocytes in Newly-born Infants in Relation to Maternal Rh Compatibility

Summary A method of making nucleated erythrocyte counts per 10,000 red blood cells is presented. Routine counts within 24 hours of delivery are recommended on newly-born infants and more than 2 nucleated red blood cells per 10,000 are to be considered abnormal. Regardless of Rh compatibility the mortality among children with 5 or more nucleated cells per 10,000 was 67% in our small series, although among the Rh incompatible infants there were more nucleated red blood cells than among the Rh compatible group. The possibility of ill effects when the mother is Rh-positive and the infant Rh-negative is suggested by the data.

The Diurnal Levels of Blood Leukocytes in the Normal Rabbit

Summary and Conclusions Nine hundred sixty-three total white blood cell counts made on 204 normal and apparently healthy young adult male rabbits between 9 a.m. and 5 p.m. revealed no statistically significant variation in the hourly means. The blood cell level, apart from sampling variations, was the same for each hour of the day. There was also no evidence of a digestive leukocytosis in the rabbit. The mean value for 597 leukocyte counts on 190 of the 204 rabbits between 9 a.m. and 12 noon was 7,891, as compared with 7,914 for 366 counts on 136 of the 204 rabbits between 12 noon and 5 p.m. The difference (23± 194) was not significant. These studies are significant for several reasons: The investigation covered a wide variety of breeds and hybrids. The animals were accustomed to laboratory existence and hemocytologic technic before the counts were made. Feeding conditions were varied during the study. Only one examination was made on a given animal on a given day, and usually not more than one or two counts were made on each animal per week. Finally, the material was analyzed by acceptable biometric procedures and represents by far the most extensive study of the sort which has been made.

Effect of Rabbit Adenocarcinoma Material on Brown-Pearce Rabbit Epithelioma

Summary Preliminary treatment with a material derived from a uterine adenocarcinoma of the rabbit failed to render rabbits more susceptible to the subsequent transplantation of the Brown-Pearce tumor, and likewise failed to enhance its growth and spread. These results are in contrast to the marked enhancement in the incidence, growth and spread which constantly ensues when rabbits are treated with a material from the Brown-Pearce tumor 2 weeks before transplantation of that tumor.

Distribution of Metastases in the Brown-Pearce Tumor. I. In Standard Breeds of Rabbits.∗

During studies on the transplantation of the Brown-Pearce tumor into the testicle of the rabbit, certain variations were noted between standard breeds of rabbits. 1 These variations included (a) the incidence and volume of the primary tumors, (b) the incidence, volume, and number of the metastases, and (c) the longevity and mortality of the animals after inoculation. All the animals were males, all received the same dosage into the testicle, all were in apparent good health, there was no significant variation in age between the breeds, and the diet and housing were uniform. The variations, moreover, wrere consistent and were most significant among breed samples inoculated at the same time. It was not possible, therefore, to explain the variations on the basis of sex, site of inoculation, physical condition, age, diet and housing, or season. The results, extending (from a susceptibility of about 15% to a susceptibility of about 95%, seemed to be due to the inherent constitution of the pure breed strains used. The present paper reports efforts to determine whether the differences between the breeds were influenced by a selective liability of certain organs and tissues to metastatic involvement. For this purpose the protocols on 202 animals from 19 standard breeds were employed. In each of the animals a detailed search for tumor foci was made in each of the 50 sites listed in Table I. A site containing one or more tumor nodules was designated as a single tumor focus. Forty-five animals were devoid of neoplastic growth at necropsy, 20 had primary tumors only, and 137 had metastases in one or more of the sites listed. Among the 137 animals with metastases there were 1,819 tumor foci, which amounts to approximately 20,000 tumor nodules.

XYZ effect in strain of origin: EO771 carcinoma in C57 BL/6 mice.

Summary and conclusions Fifty-two C57 BL/6 mice from the Jackson Memorial Laboratory were inoculated with EO771 mammary carcinoma tissue carried in C57 black/6 mice from the same laboratory. As expected all 52 died from the growth of the neoplasm. However, 26 of the mice which had been pretreated with frozen non-viable EO771 tumor (xyz factor) from the Jackson Laboratory developed a significantly increased rate of tumor growth and earlier mortality than the 26 controls not so treated. The effectiveness for transplanted tumors of the xyz factors in the inbred strain of origin, as demonstrated above, suggests the possibility that spontaneous tumors could be enhanced in the host of origin by xyz factors if such were present in them.

Distribution of Metastases in the Brown-Pearce Tumor. IV. Effects of Site of Inoculation.

It has been shown that the distribution of metastatic foci in the Brown-Pearce tumor of the rabbit (a) is not disturbed by the breed of animal used for transplantation, 1 (b) has remained constant over a period of 15 years of transplantation, 2 and (c) is not affected by treating the animal with an homologous material. 3 All the inoculations in the 3 series were intratesticular and unilateral. The present paper concerns the possible effect of intracutaneous inoculation upon this constant and characteristic distribution of metastases. To determine this effect a comparison was made of the distribution of metastatic foci in rabbits injected intracutaneously with the same distribution in rabbits injected intratesticularly. 3 Observations were made upon 469 rabbits. Thirty-two rabbits 8 to 18 weeks old were injected intracutaneously over the right scapula, first with 0.3 cc of an homologous material, and 2 weeks later with 0.3 cc of a saline emulsion of Brown-Pearce tumor (Table I). Four hundred thirty-seven young adult rabbits were injected intratesticularly with 0.3 cc of a saline emulsion of Brown-Pearce tumor; 44 of this group also received homologous material. 3 An additional number of rabbits injected intracutaneously with the tumor alone are not included in this analysis because no visceral metastases were observed. (Table I.) Many sites had too few metastatic foci for adequate statistical analysis and are therefore excluded from this comparison. The sites affected were arranged in 2 groups (Table II), according to whether spread seemed to have occurred by tissue spaces and lymphatic channels or was hematogenous. There were 7 sites in the former and 13 in the latter group. Both the actual and the expected values are presented, with their totals. To test the significance of the differences between actual and expected values, the figures x2/m were calculated. 4