‘Systemic Effects’ during the Growth of Malignant Experimental Tumors’ Significance of Unspecific Organ Changes in the Host Organism as .Inherent Factors’ of the Experiment
N. ERTL
Abstract. For an analysis of the effect of the tumor Key Words Tumor growth Systemic effects of tumors Liver regeneration Thymus involution Adrenal hyperfunction metabolites upon the organism of the tumor-bearing animal, correlation and kinetics of unspecific changes in some organs, such as thymus, adrenals, and liver, and the changes of body weight during the growth of the solid Walker carcino sarcoma 256 of the rat are described. The examination showed that the ‘systemic effects’ of the tumor in the various organs have a close physiological correlation with one another. Furthermore, the time of appearance and the kinetics of the organic changes are characteristic for the kind of tumor and the breed of animals. With regard to the progression of organ changes, the moment of beginning of thymus involution appeared to be of major importance. Therefore, it was used as point of reference when the temporal development of tumor growth was divided into a ‘compensated pre-involution stage’, with compensated meta- bolic balance; and into a ‘decompensated post-involution stage’, with decompensated metabolic balance. This classification might help to describe new results in tumor research with reference to known and uniformly defined factors inherent in the experiment and thus help to avoid contradictory experimental results or to find a common denominator for them. Possible consequences from an analysis of the kinetics of the ‘systemic effects’ for clinical cancer research and cancer treatment are suggested.
The growth of experimental tumors shows individual differences which result in a dispersion of the values of those factors by which growth is characterized. These differences originate in the variable environment of tumor that may be caused by genetic variations of the metabolism of the tumor-bearing animals. This is why, for example, the period from tumor inoculation to treatment or examination of the tumor-bearing animal can
’ Parts of this work were presented in a lecture given at the Congress of the German Cancer Society in Hannover, Germany. 1971.
be regarded just as one determinant, but not the only one necessary for the evaluation of the characteristics of tumor growth.
The role of the environment of tumor growth is of great importance. Therefore, the search for further determinants should primarily concentrate on such data or ‘inherent factors’ that can give information about the respective metabolistic conditions and the capacity of the tumor-bearing organism for immunoreactions.
We know that the metabolic balance of tumor-bearing animals and likewise the capacity for immunoreactions during tumor growth are changed by various organic lesions. The resulting functional insufficiency or dys- function of these organs generally leads to the death of the animals.
These organic changes themselves are unspecific, that is to say. they are unsuited for a verification of the process of tumor growth in the organism. However, the moment of appearance of these unspecific organic changes after tumor inoculation, as well as the stages of progression in a morpho- logical sense, or the functional changes connected with it, could be regarded as dependent upon tumor growth and significant for it.
Thus, it would be possible to include the effects of the tumor metabolites upon the organism, or the degree of severity of the organic lesions that have developed. as measurable factors in the evaluation of the experimental results.
In the following paragraphs, systematic analyses of unspecific changes ‘such as in thymus, adrenals, liver, and body weight’ as inherent factors of tumor experiments are discussed.
Material and Method
Animals. Female Sprague-Dawley rats, 6-8 weeks old, with an average weight of 200-250 g, bred by Mus-Rattus, Munich, Germany, served as experimental animals. They were kept in Makrolon cages under conditioned circumstances, were fed with standardized food (Altrogge, Lage/Lippe), and received water ad libitum.
Tumor. The Walker carcinosarcoma 256 was inoculated in solid form. It became palpable at the 3rd-4th day after inoculation. It then grew until the 13th-14th day, continuous increase of weight, and reached an amount of 37-41% of the death body weight of the animals. The 3H-thymidine incorporation into the tumor cells first rose rapidly and reached a peak generally at the 8th day and a second one at the 13th day. There- after, the intensity of DNS synthesis gradually decreased; the increase of tumor weight also stopped. The animals died between the 8th and 22th day, with a maximum at the
13th day. No development of metastases was observed. There was perceptible necroti- zation in the centre of the tumor from the 4th-5th day onwards (fig. 1).
Experimentation and method. After the tumor inoculation, 8-10 animals daily were picked out on strictly accidental principle, and were killed. Their body weight, tumor weight, and the weight of thymus, adrenals, and liver were determined. For histological examinations, sections were stained with HE, Gömöri’s chromium-hematoxylin-phloxin, para-aldehyde-fuchsin, thionine, Ziel-Nielsen, Mallory, Paisin, Schmorl, and other stains.
For histochemical examinations, cryostat and paraffin sections were prepared. Thymus and adrenal slides were analysed for lipids, in particular glycolipids; for PAS-positive carbohydrates; and for protein components. The verification of pyroninophile granulat- ion of mesenchymal reticulum cells in cryostat sections of thymus gland has been previ- ously described [9].
For the estimation of the DNS synthesis in tissue samples, 100 uCi thymidine were given i. p. 1 h before killing the animals. The activity was estimated by liquid scintillation spectrometer, according to the method of VOLM et al. [24].
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Results and Discussion
During the growth of the solid Walker carcinoma, the changes of body weight and the organic changes in thymus, adrenals, and liver were examined.
Thymus Gland
The most significant change observed in the thymus gland was an in- volution at the 8th-9th day after tumor implantation. It was preceded by an increase of the 3H-thymidine incorporation, beginning at the 3rd day and reaching a peak at the 4th-5th day. This development was connected with a moderate increase of organ weight and coincided with the time when the solid tumor became palpable [18] (fig. 2). The histological picture of the thymus first showed signs of rising cytoplasmic vacuolization as an expression of increasing phagocytosis activity of the reticulum cells at the border between cortex and medulla of the thymus lobules, caused by the increasing destruction of lymphocytes. Up to the 9th day after tumor implantation,
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this increasing phagocytosis covers the whole breadth of the thymus cortex, so that it comes to a sieve-like disaggregation of the original density of the lymphocytes [7].
In the plasm of the reticulum cells, the cryostat sections showed an increase of pyroninophile granulation, which after histochemical analysis, proved to be a depolymerizate of the DNS phagocytized lymphocyte nuclei [9]. These pre-involutional changes develop rather slowly and lead over to thymus involution itself at the 8th-9th day. Generally at the 8th-9th day after tumor implantation, the organ weight decreases (fig. 2). Simultaneously in the subcapsular zona germinativa of the thymus cortex, beginning pycnosis of the cell nuclei is to be observed in lots of small lymphocytes, a development which seizes the whole cortex up to the 10th or 11th day. Pycnotic forms of lymphocytes are now observed not only in the plasm of the reticulum cells but also extra-cellularly in Clark’s packets. Forms of mitosis have completely disappeared, and the DNS synthesis of the thymo- cytes quickly decreases. This results in the well-known picture of thymus involution, which is similar to that after administration of cortisone or that of stress situations [3, 5, 6, 14, 15, 17, 23]. Up to the 12th-14th day, the organ weight amounts to 0.05-0.08 g (= 15-22% of the control organ weight), and the pycnotic thymocyte nuclei of the thymus cortex are carried away, so that the cortex appears to be completely free of lymphatic elements. Signs of regeneration were not to be observed [11].
Thymus involution was found in connection with various tumors, such as the Walker carcinosarcoma in solid and in ascitic form, the Yoshida sarcoma in solid and ascitic form, the Zajedla hepatoma, the benzpyrine sarcoma of the rat, the plasma cytoma of the Syrian hamster; also, spontane- ous lung carcinoma, the S-180 sarcoma, and leukaemia in mice of the NMRI strain [11].
A statistical analysis of the correlations of thymus involution proved that it depends upon the duration of tumor presence and shows little variation from one animal to the other [10]. The phenomenon of thymus involution is seen as a result of a hyperfunction of the adrenals. There are only a few authors who still consider it to be an effect of a toxohormone of the tumor [20].
Adrenal
The wet weight of the adrenal rises from the 5th-6th day after tumor implantation until the 13th-14th day, when it amounts to 0.080-0.120 g (=3-4 times the weight of the control organs) (fig. 3). In this phase, the
adrenal cortex shows large haemorrhages; therefore, the dry weight of exsanguinated animals also was estimated. These values, too, were signi- ficantly higher.
The cellular changes showed first the typical picture of a chronic shock, with enlarged adrenal cortex mainly in the zona fasciculata, and with unchang- ed cholesterol content. From the 8th-9th day after tumor inplantation, the cholesterol content rapidly decreases, whereas the hypertrophy of the organ continues. The cortex of the hypertrophic adrenal gland - stained with Sudan black or Oil-Red-O - showed no remarkable quantitative alteration of the content of lipids, except for the phosphine 3R positive lipids; they markedly decreased from the 9th day onwards.
Likewise, the positivity of the reactions for the verification of keto and aldehyde groups showed no serious quantitative changes relevant for this study.
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According to some authors, the tumor is supposed to produce a corti- cotrophic hormone, or a polypeptide with similar effect. Thus, a hypertrophy and hyperfunction of the adrenals is caused, which, in the ‘late phase’ of tumor growth, turns into an exhaustive hypofunction [19].
Liver
From the 7th-8th day after tumor implantation, the weight of the liver increased. The estimations of the 3H-thymidine incorporation showed that this rise of weight precedes an increase of DNS synthesis in the liver cells. The activity had its maximum at the 10th-11th day; it then fell off. The organ weight rose until approximately the 13th day, reaching a value of 5.0-5.2 g/100 g body weight (fig. 4) [18, 19].
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The histopathological changes of the liver tissue, the examinations of serum protein fractions, of certain groups of enzymes and isoenzymes in the serum and in the liver cell led to the conclusion that during the tumor growth an increasing insufficiency of the liver develops because of over- strained liver functions or toxial lesion of the liver cells [4, 12, 13, 16, 22].
These examinations were mostly made in the ‘late phase’ of tumor growth. The studies of the correlation and kinetics of these changes, in spite of their great importance, have not found the general interest they deserve.
Together with WAYSS and VOLM, we have studied the regeneration capac- ity of the liver before and after the moment of thymus involution [25].
The 3H-thymidine incorporation into the liver cell was measured 12, 24, 36, 48 h and at the 3rd, 4th, and 5th day after partial hepatectomy in tumor-bearing animals.
The isotope incorporation in the 24-hour position before the appearance of thymus involution pointed to a retardation in the beginning of the re- generation process; whereas in the phase of tumor growth after thymus involution, a clear and essential retardation of the whole regenerative process was to be observed. These findings also seem to indicate a progressive insufficiency or lesion of the liver cell during tumor growth. The question of what tumor metabolites are injurious to the metabolic functions and the regeneration capacity of the liver cell has not yet been clarified.
Body Weight
The experiments were performed on rats aged 8-10 weeks; average weight, 250 g. These animals were still in a state of bodily development. As first reaction after tumor implantation, it was noted that at about the same time that the solid Walker carcinoma became palpable, the rise of the net body weight (i.e., body weight minus tumor weight), which depended upon the body growth, was delayed.
The net body weight remains on the same level up to the 8th-9th day after tumor implantation. It was only when thymus involution began that a progressive decrease of net body weight was to be seen, rapidly leading to tumor cachexia (fig. 5). These changes showed more obviously in a group of animals at the age of 6-7 weeks with an average body weight of 190 g [8].
The demonstrated kinetics of the ‘systemic effects’ in the various organs of the tumor bearing animals proved to be characteristic for the type of tumor and the breed of animals. The changes could easily be objectified. They
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stand in close correlation with the tumor growth and with one another. An analysis of the kinetics of these ‘systemic effects’ in thymus, adrenals, liver, and change of net body weight with respect to tumor growth showed that at the 8th-9th day, the slow progression found at the beginning quite suddenly turns to a fast deterioration.
Among all the organic changes that take place at the 8th-9th day, thymus involution is the one easiest to be determined merely by dissection. Therefore, we chose it as point of reference and divided the tumor growth into a stage of pre-involution and one of post-involution.
The above-mentioned organic changes can be summarized in the manner below as pre-involution and post-involution stages of tumor growth (fig. 6).
ERTL
Preinvolution stage
Postinvolution stage
ADRENAL Beginning of an organ hypertrophy and hyper - function
THYMUS Sign of cellular immun- reaction
Extreme organ hypertrophy; Exhaustive functional insufficiency
Complete involution phe- ncmenon
Beginning of the lymphatic regression LIVER
Diminishing of the cellular immunity;
No morphological sign of regeneration;
Initial delay of the rege- neration after partial hepa- tectomy Beginning of an liver cell hyperplasie
Hepatomegalia. Insufficiency of certain metabolic functions Retardation of the complete regenerative process after partial hepatectomy
BODY WEIGHT Delay of the
developmental increase of body weight
Progressive decrease of the net body weight Tumor cachexia
W
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Days after tumor inoculation
Pre-Involution Stage of Tumor Growth
This stage lasts from the moment of tumor inoculation to the beginning of thymus involution (i.e., at the 8th-9th day in the case of the Walker carcinoma used). The tumor appeared at the 3rd-4th day and showed a DNS synthesis that increased rapidly to a maximum at the 8th day. The tumor weight generally rose to a value of 8.8-10.8% of the body weight. The animals showed a healthy appearance and good ingestion and condition in spite of the growing tumor palpable in the back. Then, the following developments :
(a) increase of DNS synthesis and moderate growth of thymus weight at the 3rd-4th day as a probable sign of cellular immunoreaction of the thymus against the tumor implant;
(b) shifting of the balance of lymphocytopoiesis and destruction of lympho- cytes by means of increasing pycnosis in the thymus from the 5th day onwards;
(c) beginning of an adrenal hypertrophy and hyperfunction;
(d) beginning of a liver cell hyperplasia, as well as retardation of the in- ception of liver regeneration after partial hepatectomy;
(e) Delay of the developmental increase of body weight.
Post-Involution Stage of Tumor Growth
This stage lasts from the time of thymus involution up to the death of the animals. The tumor weight rose up to an average of 31.4% of the death body weight. At the itme of thymus involution, the 3H-thymidine incorpo- ration into the tumor cells decreased markedly, which indicates a probable delaying effect of the described unspecific reactions of the host organism to the tumor growth. This phenomenon, however, should be submitted to further investigation. In the subsequent period the activity incorporation into the tumor cells rose again up to the 13th day and showed a second peak. Simultaneously, the organ changes developed quickly. leading to the high mortality rate at this point. After the 13th day, the 3H-thymidine incorporation into the tumor decreased, and the growth of the tumor weight of the surviving animals came to a stop. The animals at this stage rapidly lose flesh in a state of inappetence and insufficiency and look seriously ill up to the time of death. The following organ changes went along with this development (fig. 7):
(a) blocking of the mitosis activity in the thymus which leads to the well- known phenomenon of thymus involution and apparently results in a diminishing of the cellular immunity of the animal;
(b) in the adrenals, the cholesterol content and the content of phosphine- stained lipids decreases as a result of organ hypertrophy and hyper- function, leading to exhaustive insufficiency;
(c) essential retardation of the complete regenerative process in the liver after partial hepatectomy, together with growth of organ weight and insufficiency of certain metabolic functions.
(d) progressive decreases of net body weight and appearance of symptoms of tumor cachexia.
Only a few unspecific organ changes were examined. They were marked changes, easily objectified, and led to the developmental distinction of a pre-involution stage of tumor growth, with compensated metabolic balance; and of a post-involution stage, with decompensated balance of metabolism. It is therefore, possible simply to speak of the pre-involution and post- involution phases as compensated or decompensated stages of tumor development.
In experimental studies, these distinctions could well serve as more precise definitions of such terms as early, late, or advanced stages of tumor growth.
Up to now, the growth of experimental tumors was divided into an early and a late stage just by empirical criteria. A clear delimitation of the stages, as an experimental analysis of the changes in the tumor cell or in the tumor-bearing organism during the different stages, was missing. It is difficult to make a distinction between stages with reference to the tumor or the tumor cell itself because
(a) the factors determining the malignant growth are widely unknown;
(b) the intensity of growth at the margin of the tumor, where it comes into contact with the environment of the host organism, is different from that in the centre of the tumor, with haemorrhages and necroses, and from that in the metastases,
(c) the organic and metabolic changes in the organism during the tumor disease are not taken into consideration.
Another way to differentiate stages of tumor growth presented itself: The effects of tumor metabolites upon the organism were estimated, i.e. an analysis of the kinetics of the appearance and progression of the so-called unspecific organ changes was made. When accepted, the proposed dif- ferentiation of stages of tumor growth leads to the following practical con- sequences :
(1) In the section on material and method, a paper should contain data (inherent factors) not only about the animal and the tumor used for the experiment, but also about the changes of the net body weight, about thymus involution or the state of the cellular immunoreactivity, about adrenal hyperfunction, and about signs of liver insufficiency, etc.
(2) For an interpretation of the results or a comparison of results from the literature, the indispensable basis should be a consideration of the reactivity and metabolic condition, and the simultaneous organ changes of the experimental animals. Thus, it would be possible to compare the results of experiments that were made under divergent environmental conditions, with different experimental animals and different experi- mental tumors.
The complex problem of the genesis of the unspecific organ changes has not been dealt with in this paper. However, it is important to point to the fact that it is not simply to be identified with the problem of tumor necrosis. It would be just as inadequate to see the resolution of this in dependence upon a clarification of the metabolic processes in the carcinomatous cells only.
The unspecific and progressive character of the changes in organs and metabolism indicates that during the process of tumor growth, regulatory systems are continuously stimulated; however. the feed-back effect upon the ‘source of stimulation’ fails or takes place with paradoxical reaction. Paradoxical effect means, for instance, that a growth-retarding regulation which has been started directly or indirectly supports the malignant growth (directly, by acting upon the dysfunctioning metabolism of the tumor cells; indirectly, by producing the progressively catabolic condition of the tumor- bearing hosts). Numerous variations and combinations of disturbing factors that act upon the regulative mechanisms can cause a progressively catabolic condition. A better analysis of this metabolism enables conclusion about the kind of metabolic products of the malignant cell on the one hand, and can lead to practical consequences for the clinical treatment of tumors on the other.
Pathomorphological changes, such as the phenomenon of thymus involution or adrenal hypertrophy, are of no use, of course, for clinical work. It is possible, however, to go back to corresponding functional or clinico- cytological symptoms in order to characterize the changes in the reactivity of tumourous organisms. Thus, it was observed, for example, that the PHA-induced in vitro transformation of lymphocytes, which is an indicator of the cellular immunoreactivity, was inhibited in the III-IV stage of the morbus Hodgkin as compared to the I-II stage [21]. Studies of bronchial carcinoma led to similar findings [1, 2].
For tumor diagnosis, it would certainly be of little use to analyse the kinetics of organic changes during tumor growth because specific tumor symptoms and factors are only relevant for diagnosis.
After the tumorous disease has been diagnosed, however, it is essential for therapy to know the functional capacity of such organs as the liver, or of the lymphatic system, because the prospective effect of therapy, the degree of toxicity, the tolerance of a medication, the dissimilation and excretion of medicaments, and even their utilization in the organism depend upon this functional capacity.
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