Tissue Growth and Tumor Growth'

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TISSUE GROWTH AND TUMOR GROWTH’ LEO LOEB From the Department of Comparative Pathology, Washington University Medical School, St. Louis

Received for publication February 16, 1917 Tumors in general and cancers in particular are tissues growing under special conditions. A comparison between the laws determining tumor and tissue growth will deepen our knowledge of the physiology of tumors as well as of tissues. There exist similarities and analogies in the behavior of both, and there are differences either real or due to a gap in our knowledge. The differences will suggest new problems. In this brief r6sum6 no attempt at completeness will be made, and we will have to be content with mentioning some of the salient points. We shall first compare the causes of tumor and tissue growth, then the reactions of the host organism against tumor and tissue growth, and lastly, certain phenomena observed primarily in the tumor cells. We will have to inquire how far the last mentioned phenomena are duplicated in tissue growth. 1. Tissue growth is initiated by external factors affecting a more or less complicated system and leading to chains of reaction which may be identical, or at least similar, even in ‘casesin which the primary causes differ. These primary causes are called “formative stimuli’’ in order to distinguish them from functional stimuli. It is probable that essentially both kinds of stimuli are identical and act in a similar manner. The difference in the result of both kinds of stimulation depends probably on the quantitative differences in strength and time, and on the differences in the systems on which they act. 2. The primary causes initiating tissue growth may be either

1 Read in a symposium on cancer before the Section of Experimental Medi- cine of the American Association for the Advancement of Science in New York, December 29, 1916. 136 136 LEO LOEB physical or chemical. The physical factors are either mechanical or rays which are held back by the tissues. The chemical ones are essentially substances produced somewhere in the organism. Certain parasitic organisms which cause tissue and tumor growth act partly through chemical agencies which they produce; they may in addition exert a mechanical effect. In re- gard to substances produced outside of the organism and caus- ing local tissue growth after application to certain tissues, it is doubtful how much their action is a specific chemical one and how much it is due to secondary physical factors released through injury of the tissues. Some pathologists assumed mechanical factors to be the only ones which can elicit tissue growth under experimental or pathological conditions. This view is evidently erroneous. Pathological or experimental phenomena are merely a modified interaction of phenomena occurring in the normal life of the organism. Chemical stimuli undoubtedly play a considerable part in the normal growth phenomena of the organism. This alone is presumptive evidence that they have a similar function under pathological conditions. 3. It can be shown that the stimulus brings about a series of changes in the affected tissue; these changes are partly chemical, as evidenced by increased oxidation, or physical (increased water content of the cells). They lead to a greater. sensitiveness of the cells. Corresponding to their chemical and physical changes, morphological changes take place. These changes follow a definite curve. A new stimulus reaching the cell after a certain stage of this curve has been reached acts therefore on a different system and must produce different results. It has been shown that small quantities of various rays acting on various tissues may produce stimulation, while large quantities cause destruction. In conjunction with Doctor Spain we have shown that larger defects may produce a more energetic response of tissues than smaller defects; they represent a stronger formative stimulus. Successive formative stimuli lead only to a limited extent to increased reaction. Adaptive tissue changes imply- ing an increased resistance to the stimulus are called forth. Thus a limitation in the proliferative effect is produced. TISSUE GROWTH AND TUMOR GROWTH 137

4. If a strong growth stimulus affects tissues under otherwise unfavorable conditions, as for instance at a place where the sup- ply of food and oxygen is diminished, an abnormal response of the tissues takes place (irregular mitoses, amitotic nuclear di- visions, formation of giant cells and syncytia) . If the conditions of nourishment become still more unfavorable, necrosis follows. Quite generally growth stimuli more or less inhibit the full morphological differentiation and correspondingly the full meta- bolic correlative activity of tissues. This effect finds expression in a simpler structure of various growing tissues. The facts mentioned under No. 3 and No. 4 apply on the whole equally to normal tissues and tumors, but they are usually more marked in the case of tumors, especially cancers, because here either the growth stimuli are stronger or the conditions of growth are more irregular and therefore more unfavorable as far as nourishment is concerned. 5. These same physical factors, the formative stimuli for tissue growth, when acting over a long period of time, lead in certain individuals to the production of tumors or cancers. It has been possible to produce cancers experimentally through the application of physical or physico-chemical factors (P. Marie, Fi- biger) . These experimental facts correspond to observations in human cancer. Apparently the transformation from normal tissue to cancer is a gradual one, passing through several intermediate steps. However, if we work with either normal tissues or cancers or intermediate formations (regenerating tissue, inflammatory new-formation, benign tumors), in most cases they retain their original characteristics in a fairly constant manner during the period of experimentation. 6. Formative stimuli of a mechanical nature act on certain cancers as they do on normal tissues. Cuts, incomplete extirpation, pulling threads through tumors that have become sta- tionary, transplantation of tumors, act as stimulants and increase the energy of growth. 7. Chemical formative stimuli undoubtedly play a considerable r81e in the growth of normal tissues. From the data which are accumulating, it may be possible in a provisional way to 138 .LEO LOEB

classify these chemical stimuli into (a) general stimuli, applying indiscriminately to a large number of tissues; (b) specific stimuli acting only on specific tissues. Chemical factors of the former kind are probably operative in young organisms, in contradis- tinction to old ones, and during pregnancy. In the last named condition not only factors favoring growth are apparently at work, but also factors antagonistic to growth. The balancing between these two forces seems to lead to a different result in different species. Thus in the rat, pregnancy seems to favor growth of embryonal tissues under certain conditions; in the mouse, it is unfavorable to such growth. Tumor growth is affected in a way similar to normal tissues by the chemical conditions prevailing in young and old organisms respectively. The difference in the growth of certain organs in young and old animals seems to depend on substances circulating in the body fluids. Analogous substances seem to hasten or to delay growth phenomena associated with metamorphosis in amphibia. There is also some indication that during pregnancy spontaneous tumors may assume a marked increase in size in the rat, while in the mouse, pregnancy is an unfavorable factor, especially for the growth of transplanted tumors. In future it will be necessary to distinguish more sharply than has been done in the past between the effect of pregnancy on the growth of spontaneous and of transplanted tumors. The corpus luteum, which stands in specific relation to the wall of the uterus and perhaps to the mammary gland, is an example of the second kind of growth substances. There can be no doubt that these specific growth substances play an important r81e also in the transformation of normal into cancerous tissues. This has been proven experimentally by the writer in the case of the cancer of the mammary gland in mice. By extirpation of the ovaries at the time maturity has been reached, the spontaneous development of cancer of the breast, which is so common in certain strains of mice, can be almost altogether prevented. We may assume that other specific correlative growth substances play a similar part in the cancerous transformation of other tissues, TISSUE GROWTH AND TUMOR GROWTH 139

There is another class of growth substances, namely those which prevent growth and the absence of which promotes growth. We might call those: “negative growth substances.” We might include iodine in ths category which, according to Marine, prevents compensatory hypertrophy of the thyroid. In a similar manner, ttccording to Raymond Pearl, calcium seems to counteract certain of the actions of the corpus luteum substances; but whether it inhibits the growth processes called forth by the corpus luteum, has not yet been investigated.2 There is, however, some indication that calcium inhibits somewhat the growth of inoculated tumors, although it is not certain that the evidence in this direction is conclusive. 8. -There are cases in which a chemical and physical stimulus must cooperate in order to induce considerable tissue growth. The formation of the maternal placenta is an example of this kind of growth. A definite relationship exists in this case between the chemical and physical stimulus. The chemical stimulus has to precede the physical (mechanical) stimulus. It sensitizes the tissues to the action of the physical stimulus. However, in order to obtain the fullest possible growth reaction, the chemical stimulus must continue to act after the onset of the physical reaction chain. A similar combination between chemical and physical stimuli seems to play a part in plant growth, as has recently been‘ discovered by Haberlandt. It seems to be of significance also in the case of certain plant cells, which in some respects correspond to the benign tumors of animals. It is probable that it plays a considerable part in the etiology of animal tumors, but definite data are as yet lacking in this respect. 9. While etiological factors established for tissue growth thus point the way for a search for analogous conditions in the case of tumor growth, the converse condition also exists. Certain factors established in the etiology of tumor growth have not yet found their analogue in the case of tissue growth. We refer especially to the great part heredity plays in tumors. It has to be added as an internal factor to the external factors, which

2 We are carrying out such experiments at the present time. 140 LEO LOEB we mentioned above. Analogous hereditary conditions in the case of normal tissue growth are not known. In conjunction with Dr. Addison, the writer has, however, established the fact that homologous tissues in different species possess normally a very different degree of proliferative activity, which is hereditarily fixed. It is also possible that exceptional instances in which experimentally produced tissue growth took an unusual course in certain individuals, may be due to hereditary peculiari- ties in these animals. I refer to the observations of Askanazy, who found twice that experimentally produced embryomata be- came malignant, and to an observation of Stieve, who observed that inflammatory proliferation around foreign body giant cells assumed in one case certain characteristics of a malignant tumor. Similarly the writer found with Dr. Fleisher that in contradis- tinction to others, certain benign tumors are transplantable. As to the character of the hereditarily fixed factors, we may perhaps suggest that hereditarily transmitted differences in chemical sensitizations, similar to those determining the growth in the uterine mucosa, may be responsible for this phenomenon. 10. The effect of formative stimuli greatly varies in accordance with the system on which they act. It is very great in the case of the egg, which is ready to respond to the proper stimuli with an extraordinary reaction chain consisting of a combination of cell multiplication and differentiation. It is otherwise in the case of differentiated tissues, where the response is relatively slight or is lacking altogether after a certain degree of differentiation has been reached. In some cases at least this differentiation can be shown to be due to unfavorable conditions of nourishment. While in the case of the tissues of the differentiated organism a certain relation exists between the faculty on the part of normal tissues to respond to growth stimuli, and the capability of these tissues to cancerous transformation-a parallelism which can be readily understood on the basis of what we said above concerning4he part of growth stimuli in the origin of tumors-it is very interesting to note that the direct cancerous transformation of tissues within the embryo must be an extremely rare occurrence, although later, after the completion of the em- TISSUE GROWTH AND TUMOR QROWTH 141 bryonic development, in certain cases tumors may be found which developed on the basis of embryonal malformations; or other tumors may originate in a developed organism through parthenogenetic development of the ovum. Cancer means growth without differentiation and in the embryo growth is linked with differentiation. In a similar manner, from what information we have at present, we must assume that in invertebrates likewise cancer must be extremely rare. These facts are in apparent contradiction to the fact mentioned above, that only tissues capable of growth become cancerous, and most differentiated tissues do not become cancerous. However, the contradiction is only apparent. Cancer originates in tissues capable of growth, provided they are parts of a rigid whole in which the growth stimulus cannot lead to regulatory growth processes. Where, therefore, organisms readily respond to growth stimuli with budding and various other kinds of organic reproduction, or with far going regenerative restitution-a capability much more pronounced in the earlier phylogenetic and ontogenetic stages-cancer must be rare. This does not exclude the possibility that it may occur, especially if we consider the fact that in different invertebrates great variability exists as to the power of restitutive processes. Furthermore, cancerous transformation of tissues seems to be much more common in organisms with marked chemical correlation of different parts of the whole or- ganisma chemical correlation which we have reason to believe is absent or much less rigid at early ontogenetic or phylogenetic stages. 11. There exists a series of observations which point to the conclusion that one tissue may exert a localized effect on a neighboring tissue. Thus in certain cases we find the connective tissue of the mucosa directly underneath the epithelium more cellu- lar than at a distance from the surface epithelium. The ac- tively secreting or proliferating mammary gland is surrounded by a stroma rich in cells and poor in fibrous tissue; the resting gland on the other hand has a dense fibrous stroma. Tissue transplanted from a strange individual calls forth a more marked production of fibrous tissue than tissue transplanted from the 142 LEO LOEB same individual, Contact with bone seems to stimulate peri- osteum to the production of bone, which is not produced by peri- osteum after transplantation into muscle. Certain parts of the optic cup transplanted underneath the epidermis call forth the production of lens or cornea from the epithelium of the skin. The writer has observed that cell proliferation in the granulosa of the graafian follicles may be especially marked around the ovum. The influence of the ovum on the proliferation of the granulosa has been recently established in our laboratory through the quantitative investigations of Newman Walsh. We find similar phenomena in the case of plants. Haberlandt has shown that from the leptom of certain plants substances diffuse into neighboring tissues and produce cell multiplication. There exists a similar observation in cancer; carcinoma may cause the development of sarcoma in the surrounding connective tissue stroma. Whatever the ultimate interpretation of this phenomenon may be, it is well to keep in mind the analogies of this process with certain occurrences in the growth of normal tissues. 12. If we leave now the discussion of the origin of cancer and consider the fully established cancer, we may state that cancer is tissue growth in which a chemical stimulus, presumably not very different from those substances which play a r81e in normal tissue growth, is constantly at work producing those physico- chemical changes which accompany all growth. The etiological problem of cancer, which remains still to be solved, concern$ the question whether this chemical stimulus has to be transmitted to the tissue cells by an outside agency in close proximity to the tumor cells or whether long continued stimulation of tissues ultimately leads to such a change in the cell mechanism that the cell itself is enabled to produce continuously the substance call- ing forth propagation. Both possibilities may be realized in different cases. In fowl sarcoma Rous succeeded in separating from the living cells an agency producing sarcoma. Nothing definite is known about its character. According to E. F. Smith, certain plant tumors bearing some resemblance to animal cancer are caused by a chemical agent continuously produced by Bacterium tumefaciens; while in the common galls chemical TISSUE QROWTH AND TUMOR GROWTH 143 agencies produced by parasites cause a growth process that ultimately comes to a standstill, in the case of the so-called crown gall the growth caused by the bacterium seems to be con- tinuous. According to Friedemann, the same Bacterium tumefaciens or nearly related organisms cause in man inflammatory processes, namely meningitis and arthritis, another demonstra- tion of the importance of the system on which a stimulus acts in the determination of the final result. 13. In this connection we wish once more to come back to the fact mentioned above, that internal secretion may be responsible for the production of cancer. It was known before that long continued stimulation may cause cancer. In those cases, however, ulceration was almost invariably connected with the process of stimulation, and it was pointed out that the chronic ulceration rather than the stimulation was the cause of the development of cancer, this ulceration presenting perhaps to cancer producing parasites a favorable opportunity to gain entrance into the tissue. In the case of the chemical stimulation of the mammary gland eventuating in cancer, no ulceration precedes cancer and still the growth stimulus is the active factor in the production of cancer. We may therefore conclude that it is the direct growth stimulus and not a secondary effect of ulceration which causes cancer in these cases. 14. The reactions of a host organism against induced tissue and tumor growth are very similar, as the following comparison will show: (a) In the organism in which a tumor originated usually no reaction takes place against tumor cells after transplantation. After autotransplantation of a piece of normal tissue it may apparently live indefinitely. This holds good at least in the case of certain tissues. (b) After homoiotransplantation of normal tissues, as the writer and his collaborators, Myers and Hesselberg, have shown, the tissues die as a result of the attack by lymphocytes and through the influence of fibroblasts of the host, which produce dense fibrous tissues which may strangulate the foreign cells. There is a possibility that the strange body fluids may also directly interfere with the metab- 144 LEO LOEB olism of certain transplanted tissues to such an extent as to injure them severely. It has been likewise demonstrated that in the case of tumors against which an immunity becomes established, lymphocytes, sometimes in conjunction with other leucocytes, play an important part in the destruction of tumor tissue (Burgess and Tyzzer, Baeslak, ROUS,J. €3. Murphy, and others). Russell as well as Burgess noticed in addition the production of a dense stroma after transplantation of tumors into nonsusceptible mice, without, however, apparently insisting on the significance of this phenomenon. In addition Russell in the case of tumors, and P. Rous in the case of normal tissues, believe that immunization results in the development of some factor that delays or diminishes the ingrowth of stroma into the trans- plant, (c) After heterotransplantation normal tissues as well as tumors die after a transitory period of life or even of growth. How far in these various cases primary incompatibility between tissue and body fluids of the host, how far secondary immunization called forth by the introduction of the foreign tissue or tumor play a part, is uncertain at present. In the cme of homoiotransplantation of tumors it has been demonstrated that active immunization plays a great r8le in the de- fensive reactions. This, however, does not exclude the possibility of the simultaneous existence of primary incompatibili- ties between tumor cells and host body fluids. In the case of normal tissues only a very limited number of experiments exist which point to the development of immune substances as a result of repeated inoculation with normal tissues (Fichera, c Schoene, P. Rous). Of the attempts to unite the various facts into a consistent theory of tumor and tissue immunity only two recent ones may be mentioned; (a) without concerning himself with the growth of normal tissues, E. E. Tyzzer explains the immunity against tumor growth in a manner somewhat related to von Dungern’s and Coca’s interpretation as due to a local anaphylactic reaction. According to Tyazer some substance is given off by the tumor cells which combines with the body fluid of the host. This new product ihjures the tissue surrounding the trans- TISSUE GROWTH AND TUMOR GROWTH 145

planted tumor. Tyzzer emphasizes the conclusion that the surrounding tissue is more sensitive, more readily injured by such a substance than the tumor cells which are held to be more resistant. (b) Our interpretation, which represents a further development of conclusions published in 1907, is as follows: The mutual chemical incompatibility of the body fluids of one individual and the tissues of another, which was par- ticularly striking after homoiotransplantation of pigmented skin, leads to changes in the metabolism of the tissues resulting in the production of homoio- and heterotoxins, which if they do not exceed a certain quantity or degree of toxicity, disturb to some extent the normal functions and metabolism of the transplanted tissues without, however, seriously interfering with their life. But the abnormal products formed attract the lymphocytes and in certain cases perhaps also other leucocytes and alter the reaction of the fibroblasts which latter are induced to produce dense fibrous tissue. If the poisons become more active, they may directly injure the transplanted tissues to such an extent that growth and life become impossible. In a similar way we believe in so-called chronic inflammatory processes of various organs, a changed metabolism of the cells and perhaps also poisons produced by microorganisms may induce fibroblasts to form fibrous bands and attract lymphocytes, thus leading to processes of cirrhosis. In an analogous manner in the case of tumor immunity substances produced as a result of immunization and secreted into the circulation, alter the metabolism of the tumor cells which now attract lymphocytes and change the activity of fibroblasts in a way corresponding to that which we degcribed after transplantation of normal tissues into a strange host. This theory correlated the immunity against tumor growth with that of the growth of normal tissues and also with the immunity against certain substances and non-growing foreign cells. We have according to our theory also in the former case to deal with the production of immune substances which, however, after homoiotransplantation, are usually not such that they directly destroy the foreign tissues but merely lead to an altera- 146 LEO LOEB tion of their metabolism and to the production of substances which change the behavior of the host cells. We no longer need to assume a primary tissue alteration following the homoiotransplantation as E. E. Tyzzer did.3 Furthermore, our theory not only explains tumor immunity as an inflammatory reaction, but conversely throws light on the phenomena of chronic in- flammation through the analysis of tumor immunity; it corre- lates tumor immunity and inflammatory processes with other phenomena apparently of an entirely different character, but in reality, according to our opinion, nearly related to the former, namely the variations in the behavior of the stroma of certain glands and also the differences in the character in the connective tissue of the mucosa nearby and at a distance from the epithelium. The resistance against transplanted tumors acquired either spontaneously or through previous inoculation with normal or tumor tissues of the same species partakes somewhat of both the typical immune as well as the anaphylactic reaction. It resembles an anaphylactic reaction in so far as the presence of tumor tissue in an immunized (sensitized) animal calls forth secondarily the production of substances which directly act on the cells of the host, changing their activity. The primary substance, however, acts on foreign tumor or tissue cells and modifies their metabolism. It resembles the typical process of immunity inasmuch as the ensuing reaction does not cause an injury of the host, but of the introduced foreign tissue or tumor. We may aasume that the large majority of the cells of an individual of a certain species have a certain chemical group in common, which we may call the individuality differential and which causes characteristic differences between the various individuals of the same species. In addition we have family, race or strain differentials common to members of certain families, races or strains. There may be still finer gradations through which brothers and sisters are differentiated. There are, furthermore,

a In a more recent publication Tyzzer adduces some experimental evidence confirmatory of our view and accordingly modifies somewhat his views which now approach very closely our interpretation. TISSUE GROWTH AND TUMOR GROWTH 147 species differentials which members of a certain species have in common and through which they differ from other species. Cancer cells may possess all of these differentials just as ordi- nary tissue cells, but it is probable that a certain number of cancers lack certain kinds of these differentials. And not infrequently the cancers belonging to certain classes of tumor seem to be similar in this respect. Thus the lymphosarcoma of dogs lacks the majority of those differentials. In a tumor of a Japanese mouse which we found in 1905, and in a similar one found recently by Tyzzer, the individuality differentials were apparently entirely lacking. But the majority of cancers possess these individuality differentials in a manner similar to normal cells. The loss of these differentials is therefore not an essential character of cancer and cannot enter into the etiology of cancer. Great growth energy on the part of tissues and tumors seems to be able to overcome to some extent the barrier against proliferation set up by these differentials. Thus rapidly growing embryonic and tumor tissue overcomes at least temporarily these barriers, but it is probable that in embryonic as well as in phylogenetically lower adult tissues some of the differentials are as yet not fully developed. How far these differentials act alone or in combination with constituents of the body fluids of the host as primary homoio- or heterotoxins, how far they act only through the production of secondary immune toxins, is as yet not fully determined. But it is certain that in all these reactions no essential difference exists between tumors and normal tissues. We now wish to consider very briefly some interesting features in the growth of tumors which depend mainly on the character of the tumor cells and inquire how far analogies to these phenomena can be found elsewhere under normal conditions. 15. In addition to the increase in proliferative power, the ability to infiltrate and substitute neighboring tissues is characteristic of cancers, The same property is normally found in the chorionic cells infiltrating the wall of the uterus or the ovary in case of parthenogenetic development of the ovum. A similar invasion of neighboring tissue, according to the observations of 148 LEO LOEB

the writer, takes place in guinea-pigs, if through transplantation nonpigmented epidermis is placed in direct contact with pigmented epidermis. Under those conditions constituents of the pigmented epidermis begin to invade and substitute the nonpigmented epidermis. An analogous phenomenon has recently been observed by Weigel in the case of the epidermis of amphibia. 16. The resistance of tumors to physical and chemical agencies has been determined in various varieties of tumors, especially in sarcoma of the rat and carcinoma of the mouse. In both cases the degree of resistance was found to be very similar to that of normal tissues. The agent producing sarcoma in fowls is more resistant than tumor cells and so is the sarcoma found at the external genitalia of dogs. This raises the question whether also in the case of the latter an agent dissociable from the tumor cells proper is responsible for the growth in certain cases. It is, however, to be noted that after transplantation of the dog sarcoma into other dogs the peripheral tumor cells survive and give origin to the new growth. 17. As has been shown by the writer, the growth energy of tumor cells can be experimentally increased and decreased. Various explanations have been given for these phenomena, but we believe that they are due to a direct action of physical and chemical agencies on the cells. According to this interpretation, the experimental increase in growth energy is comparable to the increase in growth energy we find in normal tissues as a result of regenerative stimuli. It has, however, not been possible so far to increase the growth energy of normal cells through serial transplantation within the living organism, a result commonly obtained after serial transplantation of tumors. However after serial transplantation of connective tissue in vitro a similar increase in growth energy seems to take place. A somewhat analogous increase in virulence after serial inoculation has been observed in the case of certain microorganisms. The decrease in growth energy of tumors exposed to injurious agencies finds a parallel in the effect of certain injurious agencies on certain plants and in the action of radium on embryonic tissues. It has likewise been observed in the case of microorganisms exposed to such weakening agencies. TISSUE GROWTH AND TUMOR GROWTH 149

18. In experiments by Fleisher and the writer it has been possible to demonstrate in cancer an active immunization of the tumor cells against chemical agencies inhibiting their growth energy. This. immunity is transmitted to a number of successive cell generations. It is probable that a similar immunity may also be acquired against the action of certain physical agencies as for instance radium or roentgen rays. The immunization of epidermal cells against the repeated application of cold or heat and radium rays, described by Werner, represents probably a parallel phenomenon in the case of normal tissues, although an inheritance of this immunity to following cell generations has not been demonstrated in this case. The experimental production of strains of trypanosomes resistant toward certain specific agencies is a similar occurrence in the case of unicellular organisms. In our experiments we could show that an actual immunization of tumor cells takes place and not merely a selection of cells possessing a greater natural resistance to the injurious agency. There are on record some other observations of a somewhat similar character which however at present may be explained either on the basis of cell immunization and gradual adaptation or on the basis of a selection of cells naturally better adapted to certain environmental conditions, Thus Koenigsfeld states that if metastases are used for inoculation, the resultant tumors are more liable to produce metastases than if the primary tumors are employed. Ebeling finds that tumors which once have been successfully transplanted into the brain-a result which at first is obtained only in a relatively small number of cases-can subsequently be propagated through transplantation into the brain with much greater readiness. 19. Bashford, Murray and their collaborators in starting from a small piece of tumor and carrying out long series of tumor transplantations, in many cases observed the development of substrains from the apparently homogeneous tumor mass. The characters of such substrains, which concerned morphological as well aa functional variations, remained more or less constant throughout consecutive transplantations. Do we have in these . cases to deal with a selection of genotypes within the tumor 150 LEO LOEB originally representing a colony of several mixed strains, or do we have to deal with mutations or variations leading to the formation of substrains, such as they have been recently described, especially by Jennings in the case of protozoa, and by various investigators in the case of bacteria? At present we are unable to answer this question, A similar phenomenon has to our knowledge not been observed in the case of normal tissues. 20. The long continued transplantation of tumor suggested first the potential immortality of somatic cells which must apply equally, to tumor and to normal tissue cells, the former be ng merely tissue cells living under particular conditions. This necessarily incomplete comparison between phenomena observed in tumor and tissue growth may suggest that a study of tissue growth may not only assist in the interpretation of tumor growth, but that conversely, the analysis of tumor growth may help to lay the foundation for a physiology of tissue growth. Furthermore, this comparison leads to the conclusion that the majority, and ultimately perhaps all, of the phenomena observed in tumor growth find a parallel in the growth of normal tissues under special conditions.