Microbiology and Cancer Therapy: a Review*

CANCER RESEARCH

VOLUME13 DECEMBER 1953 NUMBER12

Microbiology and Cancer Therapy: A Review*

H. CHRISTINEREILLY

(Dimsion of Experimental Chemotherapy, Kloan-Ketterlng Institute far Cancer Research, New York, N.Y.)

The use of micro-organisms and their products cessful, because the causative agent of erysipelas as possible therapeutic agents in the control of was not known at that time. Later, Fehliesen (38), cancer had its beginning in the latter part of the after he had discovered the streptococcal origin of nineteenth century, coinciding with the embryonic erysipelas, injected live cultures of the bacterium achievements of the science of bacteriology. The into cancer patients with encouraging results. rapid advances in the field of antibiotics during the Since then many varieties of micro-organisms have past 12 years have inspired new hope that the been reported to be beneficial therapeutic agents. search among biological systems will disclose a Preparations known as "Coley's Toxins" (100, chemical agent which will exert a destructive ef 101), which have usually consisted of heat-treated fect upon neoplastic growth without seriously mixtures of streptococci and Serratici marcescens, affecting normal cells. have been used clinically for many years, although their effectiveness has not been clearly estab BACTERIA lished. The early history of the role of bacteria and bac Beebe and Tracy (13) reported that injections terial toxins in this development has already been of suspensions of Bacillus prodigiosus (S. mar well reviewed (42, 101, 119, 145). Many of the in cescens) cells, either alone or combined with strep vestigations were prompted by the clinical obser tococci, resulted in rapid and complete disappear vation that a concurrent bacterial infection fre ance of transplanted lymphosarcoma in three dogs. quently retarded the development of malignant Baroni (8) obtained some measure of protection processes in man. Some were based on the belief against the growth of the Jensen tumor in rats if that organisms isolated from neoplastic tissues the animals had received injections of streptococ were the causative agents of cancer (39). Attempts cal or gonococcal toxins for several days prior to were made to prepare vaccines and serums against the implantation of the tumor. Simpson and such organisms; the efficacy of these preparations Marsh (127) were unable to demonstrate any ef is highly doubtful. Although beneficial results were fect, either macroscopic or microscopic, upon the sometimes claimed, attempts to confirm such ob growth of spontaneous mammary adenocarcinoma servations were usually unsuccessful; certainly no in mice with tuberculin preparations. cures were effected. Daels (27) and Comsia (23) found that spiro The first record of an attempt to treat human chete infections in mice interfered with the develop tumors with an induced bacterial infection is that ment of tumors. The effect was most pronounced of Busch in 1868 (19). He had observed tem when the animals were infected with the spiro porary clinical improvement in two patients with chete several days before the tumors were im inoperable sarcomas who also developed concur planted. rent erysipelas infections. His attempts to induce Connell (24) reported clinical improvement in the infection in other cancer patients were unsuc- several cases of far advanced cancer upon treat * This work has been supported by an institutional grant ment with sterile filtrates of Clostridium histolyti- from the American Cancer Society. cum. The filtrates were prepared by cultivation of Received for publication May 25, 1953. the bacterium on pieces of human malignant tissue 821

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. Cancer Research suspended in normal saline and were thought, by the whole animal body on the compounds being virtue of the method of preparation, to contain studied are eliminated. Although this is undoubt proteolytic enzymes which would preferentially at edly valuable in certain studies of particular bio tack the proteins of neoplastic tissue without af logical systems, the application of any agent to fecting those of normal tissue. Although Connell cancer therapy depends to a great extent upon its mentioned that a carcinoma in mice responded to fate in the living animal. Materials which are ac similar treatment, other investigators (29, 53, 106, tive in vitro may exert no demonstrable effect in 114) in well controlled studies with several animal vivo (71, 135). Conversely, an agent may cause tumors have been unable to confirm his observa marked response in vivo but be inactive in vitro tions. Procedures such as that employed by Con (117). It is not inconceivable that enzyme systems nell have proved to be invaluable for the microbial in the body may alter compounds in such a way as production and isolation of enzymes possessing to either increase or decrease their biological activ specific activities (34). They are based on the idea ities. Some agents may have very low therapeutic that when a microbial culture is supplied with a indices, and, although they exert pronounced ef particular organic substance, either as the sole fects upon tumor growth in vitro, the dose levels source of carbon or nitrogen or both, the organism, necessary to bring about responses in vivo can in order to grow, must attack that substance. The never be attained. digestive process may require the formation of an Gregory, who believes all cancers to be virus- unusual enzyme system or the production of an induced (49, 50), has claimed to have seen by elec excessive amount of an ordinary system. It is tron microscopy in vitro destruction of the "cancer highly doubtful, however, that such a method can virus" caused by Bacillus subtilis Tracy 1 (51). By prove satisfactory when an organism is supplied chemical and electrophoretic technics, he obtained with such a heterogeneous mass of material as is from extracts of the bacterium a crystalline mate present in whole tissue of either malignant or nor rial to which he has given the name magnesium mal growth. It would seem probable, therefore, tracinate. The crystals were administered to eight that only if and when a substance which is not patients with far advanced cancer; all were re present in normal cells is found in malignant tissue ported to show clinical improvement. The possible and is obtained in a purified state that this technic merits of tracinate must await more extensive may offer real promise. clinical trial, but it would appear to be somewhat Parker et al. (104) injected Cl. histolyticum presumptuous to attribute any effect that it might spores directly into a rapidly growing transplanted have upon neoplastic growth to antiviral proper fibrosarcoma in mice and, with the administration ties until more decisive evidence as to the nature of histolyticus antitoxin, prolonged the life span of of the causative agent or agents of cancer is avail some animals for as long as 20 days beyond that of able. nontreated tumor-bearing mice. Histolyticus toxin In 1931 Gratia and Linz (47) reported that injected directly into the tumor caused marked Bacterium coli filtrates injected into guinea pigs regression of both a sarcoma and a carcinoma; in with sarcoma elicited the Shwartzman reaction tissue culture the toxin produced severe damage to (125) in the tumor tissue with no observable hem- sarcoma but not to either carcinoma or normal orrhagic lesions in other organs. Motivated by this kidney. observation, Shear and his associates began a Cohen et al. (22) incubated slices of a spon study of the nature of the hemorrhage-producing taneous mouse carcinoma, the Brown-Pearce rab agent (122). Shear and Andervont (123) concen bit carcinoma, and their autologous livers in cul trated the factor from B. coli filtrates by a method tures of various bacteria. In general, the bacteria used for the precipitation of the soluble specific were found to injure both the liver and tumor cells polysaccharide from pneumococcus broth cultures; equally well or not at all. Only one organism, the active fraction was found to give negative Sporosarcina ureae, damaged the tumor cells but biuret and positive Molisch reactions (120). not the liver cells. The active factor appeared to be The group of investigators at the National Can intracellular; the cell-free culture medium in which cer Institute later turned their attention to studies the bacterium had been grown was ineffective. on the hemorrhage-producing properties of Ser- In vitro technics such as that employed by Co ratia marcescens. Shear and Turner (124) ob hen have somewhat dubious value in the search for tained active preparations which were rich in poly- agents which might be useful in the control of can sacchafides. Further experimentation confirmed cer. Such methods are frequently defended on the the polysaccharide nature of the material (56) and basis that when tissues are tested for their response indicated that its molecular weight is about 8 mil in isolated systems, possible interfering effects of lion (69). Preparations of the purified polysac-

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. REILLYâ€”Microbiologyand Cancer Therapy: Review 823 charide were found to produce hemorrhage in that the high sensitivity of malignant tissue to bac mouse tumors in doses of a fraction of a microgram terial toxins might be a result of an excessive per (121). More recently, Ikawa et al. (66) have re meability of the newly formed blood vessels (36). ported the isolation of a hemorrhage-producing That capillary fragility does play a part in this agent from filtrates of E. coli which they character phenomenon would appear most likely. Andervont ized as a complex polysaccharide containing both a and Shimkin (3) observed that ascorbic acid in peptide and a phospholipid component. Upon hibited hemorrhage by bacterial filtrates in trans treatment of this material with trichloroacetic acid, planted tumors, and Eddy et al. (37) reported that most of the peptide component could be removed ; the area of tumor destruction produced by Shear the lipo-polysaccharide which remained was found bacterial polysaccharide in rats deficient in ascor to contain most of the original tumor-destroying bic acid was 5.3 times greater than that in control activity. Thus, it would appear that the peptide animals. Algire et al. (1), however, have concluded portion is not essential for activity. that the tumor-necrotizing effect is brought about Brues and Shear (17) administered the poly by the low blood pressure which is known to result saccharide from S. marcescens to four patients with from injection of the bacterial polysaccharide. advanced inoperable malignant tumors. In two of They observed that hypotension was accompanied the cases, some relief from symptoms was ob by a reduction in the circulation of blood in the tained, and, on post mortem examination, evi tumor and by subsequent damage to the tumor. dence of hemorrhage in the tumors was found. Neither circulatory disturbance nor necrosis in the Gross changes in tumor size and consistency as tumor occurred in the absence of hypotension. well as microscopic evidence of necrosis and hem Although the literature contains many isolated orrhage in both animal (30, 31) and human tumors and conflicting views on the type of bacteria that (63, 103, 112) have been reported. are capable of causing damage to neoplastic tissue, The usefulness of bacterial polysaccharide as a Zahl and his associates (65, 145, 146), in a survey chemotherapeutic agent would appear doubtful. of a wide range of bacterial species, found that the No human tumor has been completely destroyed ability to induce hemorrhage in tumors was with and rather severe symptoms of toxicity have been few exceptions a property of gram-negative bac encountered (63, 103). Nevertheless, it is to be teria, while gram-positive bacteria with the excep hoped that studies with the polysaccharide may tion of two isolates of Listeria were unable to elicit contribute to the knowledge of the treatment of the response. In view of the many reports that neoplastic diseases. gram-positive bacteria, particularly the erysipelas- Gardner et al. (40) produced hemorrhagic reac producing streptococci, have had therapeutic ef tions in a transplantable rat tumor with extracts of fects upon tumors, it must be concluded that the a variety of bacteria including the Neisseriae mode of any action of such organisms would be group, Klebsiella pneumoniae, and enteric organ quite different from that of the gram-negative isms. Jacobs (67) has reported similar effects on bacteria. mouse Sarcoma 37 with a polysaccharide-containing fraction from Pseudomonas aeruginosa. FUNGI Although the role of bacteria in cancer therapy Fungi, too, have received a fair share of atten has intrigued the minds of clinician and research tion as organisms possessing therapeutic proper investigator alike for several decades, no real in ties against cancer. A brief discussion of some of sight into the mechanics of the interference of tu the early attempts in this field has been given by mor development by bacterial products was avail Fichera (39). Karo (70) claimed to have obtained able until the observations of Gratia and Linz (47) both subjective and objective improvement in sev on the Shwartzman phenomenon in guinea pig eral cases of human cancer as the result of treat tumors aroused new interest. Shwartzman and ment with fermentation products of the mush Michailovsky (126) and Duran-Reynals (35) rooms Agarius rufus, Echinacea, Merulius lacry- found that tumors in animals which are not nor mans, and Phallus impudicus which had been de mally susceptible to the Shwartzman phenomenon, toxified by extensive ultraviolet irradiation and to namely rats and mice, became hemorrhagic upon which several metallic salts were added. No details systemic administration of bacterial filtrates. On of the procedure of preparation were given nor was the basis of his observations, Duran-Reynals con any mention made of what role the metallic salts cluded that, in order to show this reaction, the themselves might play beyond the statement that tumor must be both malignant and rapidly grow they increased the therapeutic effectiveness of the ing; benign tumors and malignant ones exhibiting fungal proteins. slow development do not respond. He suggested McLeod and Ravenel (86), acting on the theory

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. 824 Cancer Research that if the high fermentative capacity of cancer active principle in a group of highly basic protein tissue could be suppressed, the tissue would revert components. to normal growth, employed fungal preparations That certain yeasts do have some effect upon as sources of materials thought to be lacking in neoplastic growth seems probable. Lac Iau and tumor cells. Although extracts of a variety of fungi Imaz (75) reported that hydrolysates of Sac were employed, efforts were concentrated on the charomyces cerevisiae which were biuret-negative filamentous fungus Aspergillus niger and the yeast had an inhibitory effect upon neoplasms in human Saccharomyces cerevisiae. Approximately one hun beings. Laclau et al. (76) reported good results in dred and fifty cases of advanced neoplasms con healing epitheliomas by intramuscular administra sidered as hopeless were treated. These investiga tion of hydrolysates of Saccharomyces cerevisiae tors stated that practically all the patients experi combined with selenium preparations. The role of enced subjective improvement, and, in many, the yeast preparation here is open to some ques notable shrinkage of the tumor masses occurred, tion; the authors themselves attribute the effects although no cures were effected. Upon microscopic observed to the formation of dissociation products examination of biopsy specimens, extreme grades of selenium. of degeneration and even necrosis were observed. Nevorojkin (102) found that when a dense sus Unfortunately no details concerning the clinical pension of the yeast Saccharomyces cerevisiae No. picture or laboratory findings on these patients Xu (Berlin race) in normal saline was injected were given. It is equally distressing that the proce directly into the Ehrlich tumor in mice, the growth dures employed in the preparation of the fungal of the tumor was delayed, and, in some cases, re extracts were not described. gression occurred. Thin suspensions of the yeast, Hulshoff Pol (64) has reported the regression of however, not only failed to depress growth but, tumors in mice on a diet containing bread upon apparently, even stimulated it. Maisin et al. (87) which the fungus PÃ©nicillium spinulosum Thorn studied the effect of heat-treated bakers' yeast had been grown. upon the development of benzpyrene-induced tu DeAngelis (28) has claimed very good results mors in mice. The yeast, suspended in water, was against Ehrlich's adenocarcinoma implanted in heated at 80Â°C. for 6-7 minutes; the mixture was mice after treatment with a crude culture filtrate added to a control diet so that each mouse re of Streptothrix felis D.A. which he isolated from a ceived 0.25 gm. of yeast per day. At the termina granuloma in a cat. The material, which he named tion of the experiment in the group of 100 control "mycetin," was passed through a bacteriological mice there were 51 survivors with an incidence of filter and administered in doses of 0.2-0.4 cc.; the tumors of 48.1 per cent, of which 23.2 per cent MLD was 0.5 cc. Injections were usually made were malignant. At the same time, in the yeast- subcutaneously but at some distance away from treated group (100 mice), 60 were still living. the site of the tumor; most animals received only a These had an incidence of all tumors of 28.3 per single dose. Of a total of 330 treated mice bearing cent and an incidence of cancer of only 1.2 per tumors ranging in age from 12 to 25 days, tumors cent. regressed in 308 or about 93 per cent of the cases. In in vitro experiments Protti (108, 109) ob All 286 control mice died as a result of tumor devel served that tumor tissues immersed in liquid cul opment. tures of a large variety of yeasts and incubated at In 1946 Stock et al. (135) initiated a program for 30Â°C. for 48 hours or at 37Â°C. for a shorter period the systematic screening of materials of natural of time showed marked lysis; normal tissues were origin for their ability to inhibit the development unaffected. A selective action was noted; certain of tumors. Crude culture filtrates of several fungi varieties of yeasts affected some of the tumors but were found to have a destructive effect upon Sar not others. coma 180 and mouse melanoma tissue in vitro and Lewisohn and his associates (83) reported that an aqueous extract of brewers' yeast caused regres to cause inhibition of the growth of Sarcoma 180 and of mammary adenocarcinoma E 0771 in vivo. sion of spontaneous mammary adenocarcinoma in Reilly and Stock (110) isolated from the metabolic mice. Of 33 animals treated, tumors disappeared in filtrate and pellicles of some strains of Aspergillus eight and were reduced in size in another ten; in fumigatus a protein-like agent having the ability to fifteen mice either there was no change or the tu retard markedly the growth of Sarcoma 180 in mor increased in size. Further investigation (85) mice. The tumor-arresting property was accom disclosed that the reported active principle was panied by a generalized toxicity; efforts to sepa water-soluble, fairly stable to heat at neutral pH, rate the two factors were unsuccessful. Petermann and was not a protein. None of the known B vita et al. (105), in electrophoretic studies, located the mins, including thiamine, riboflavin, pyridoxine,

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. REILLYâ€”Microbiologyand Cancer Therapy: Review 825 nicotinic acid, pantothenic acid, and p-amino- planted mouse tumors by active infection of the benzoic acid, appeared to be responsible for the animals with T. cruzi, but preparations of endo- antitumor property (77,84), although the addition toxin (heat-killed cells) were without effect. These of either pantothenic acid or riboflavin to an active results were open to some question, because the yeast extract appeared to improve the effective work had been done with a strain of T. cruzi which ness of the latter in prevention of the development was different from that used by the Russian work of carcinoma 2163 in the RIII strain of mice. ers. Hauschka and Goodwin (58) later extended Later, inositol (78) and "L. casei factor" or folie the investigations, including in their studies five acid (79, 80) were reported to be relatively potent types of mouse tumors, both implanted and spon inhibitors of tumor growth. Sugiura (136), how taneous, and eight different strains of T. cruzi, in ever, was unable to confirm the inhibition of spon cluding the strain used by Malisoff and the one taneous mammary adenocarcinoma by yeast ex which was, according to Roskin and Klyueva, the tracts or folie acid, although he made a special source of the K-R factor. Again, they were unable effort to follow the procedure of Leuchtenberger to obtain a tumor-destroying preparation. and associates. Indeed it is difficult to understand In 1948 Gruhzit and Fisken (52) reported fail the highly favorable results reported with folie ure to obtain any effect upon the development of acid, since it would appear that certain of the folie the Brown-Pearce carcinoma in the rabbit with a acid antagonists have a pronounced deleterious lysate of T. cruzi. Jedeloo et cd. (68) were unable to effect upon neoplasms (134). demonstrate any inhibition of the development Laszlo and Leuchtenberger (77) found that and subsequent growth of a tar-induced epider- some lots of brewers' yeast did not contain the moid carcinoma in mice with endotoxin prepara antitumor factor. The deficiency was attributed to tions of T. cruzi. Spain et al. (132) obtained nega a change in the conditions under which the yeast tive results with whole culture lysates of T. cruzi in was grown. While this explanation seems reason the treatment of mammary carcinoma and trans able, the earlier observations of Heaton (60) on planted Carcinoma 119 in mice. this problem cannot be disregarded. This investi gator found that extracts of brewers' yeast con Nadel and Greenberg (99) have reported an in crease in survival time in leukemic mice when tained an agent which inhibited both normal and Plasmodium bergei,the infectious agent of malaria, tumor growth. His observation that different was inoculated into animals after leukemia had samples of the yeast varied in activity, coupled developed. with the fact that extracts of bakers' yeast did not cause the effect, led him to think that the active ANTIBIOTICS principle was not a product of the yeast but was a For the purpose of this discussion antibiotics will be considered as defined by Waksman (144) : constituent of the medium on which the organism "An antibiotic is a chemical substance, produced had been grown. Further experimentation indi cated that malt, a constituent used in the cultiva by microorganisms, which has the capacity to in tion of brewers' yeast, was actually the source of hibit the growth and even destroy bacteria and other microorganisms, in dilute solutions." the growth-arresting agent ; results similar to those seen with the active yeast extracts were obtained Although Boyland (16) found that some aro with commercial malt extract. matic sulfur compounds which have antibacterial properties retarded the growth of spontaneous PROTOZOA mammary tumors in mice, there is no a priori rea Roskin and Klyueva (72,113) found that infec son to expect antimicrobial agents to be effective tion with the protozoan Trypanosoma cruzi caused against tumors. Nevertheless, the highly specific regression of tumors in mice. As a result of more nature of the action of antibiotics (143), that is, than 15 years of investigation they claimed to have the ability of such substances to inhibit the growth separated from cells of T. cruzi an agent having the of one micro-organism without affecting another ability to destroy malignant neoplasms in man as closely related one, even within the same species, well as experimental tumors in mice. Although indicates the possibility that some of them might Malisoff (88) reported confirmation of these find display a similar selection between normal and ings in his work with two mouse tumors, a spon neoplastic cells. Certainly, there is evidence that, taneous mammary carcinoma and transplanted under special conditions, such may be the case. Sarcoma 180, a number of other investigators (57) Over 40 years ago, Uhlenhuth et al. (141) and have been unable to prepare active T. cruzi lysates. Beck (12) reported that pyocyanase, an antibac Hauschka et al. (59), in excellent studies, were able terial agent now generally considered as the first to retard significantly the growth of three trans antibiotic to be isolated, caused regressions when

injected directly into tumors implanted in mice rial might be the result of the presence of a small and rats. Unfortunately, as pyocyanase is a rather amount of impurity. toxic agent, this line of investigation did not Levine et al. (81) isolated from culture filtrates progress very far. of PÃ©nicilliumnotatum a nonpenicillin-containing With the advent of penicillin, an essentially material which they called penichromin. In War nontoxic chemical of remarkable therapeutic bene burg studies with mouse liver homogenates peni fit in the control of certain infectious diseases, it chromin inhibited the oxidative utilization of sev was only natural that the attention of those inter eral intermediary metabolites but did not affect ested in the control of cancer should be turned in succinoxidase, cytochrome oxidase, or anaerobic this direction. Cornman (25, 26) observed that in glycolysis. In vivo, the preparation had no effect tissue culture crude penicillin preparations ex upon the growth of an adenocarcinoma or a lipoma erted a lethal action upon cells of mouse and rat in mice. These investigators concluded that "in tumors at concentrations that did not damage nor view of the in vitro data showing partial inhibition, mal cells. Although she confirmed Cornman's result the failure of penichromin alone to produce tumor with crude material, Lewis (82) found that highly regression was not unexpected." This conclusion purified penicillin had no effect upon tumor growth would not appear to be fully justifiable on the in tissue culture. Other investigators (43, 91, 135) basis of the evidence presented. Treatment in mice have reported similar experiences. was not begun until about 1 week after implanta Beard (11) has reported that the administration tion of the tumors, and only one dose level, 2 of crude penicillin to rats bearing implanted Emge mg/mouse daily for 5 days, was used. No data sarcoma apparently reduced the number of takes were presented to indicate the relationship of this and caused regression of a high percentage of tu dose to the maximum tolerated dose. It is possible mors. In three experiments with a total of 82 rats that the utilization of younger tumors and higher treated with penicillin, disappearance of the tumor doses of penichromin might have given more occurred in 35-72 per cent of the animals, while promising results. regressions in 148 untreated control rats was less In view of the few encouraging reports on the than 20 per cent. Dobrovolskaia-Zavadskaia (32) effects of crude penicillin on tumors and observa has obtained histological evidence that yellow so tions in our own laboratories of quite definite but dium penicillin does interfere with the growth of elusive inhibitory effects of crude culture filtrates tumors in mice; the outstanding characteristic was of P. notatum, it seems probable that a more the development of a massive hyperemia in the thorough and systematic investigation of this neoplastic tissue. The effect was temporary; when problem might prove fruitful. treatment was discontinued, the tumor reestab Notatin, a second antibacterial agent produced lished itself. Those areas which had been subjected by PÃ©nicilliumnotatum, was chosen by Carr (20) to treatment, however, appeared to be permanent as being of possible interest in the control of the ly damaged. In one patient with mammary adeno- Rous sarcoma virus. He reasoned that it might be carcinoma, she (33) saw marked clinical improve effective, because the action of notatin has been ment under penicillin therapy and observed his shown to be the result of the production of H202 in tological changes similar to those seen in mice. the oxidation of glucose to gluconic acid (115) and Bennison (14), on the other hand, could find no because, according to Carr's interpretation of the difference in the incidence of the development of data of Gye and Purdy (54), the Rous virus is mammary tumors in mice carrying the milk factor readily destroyed by oxidation. This second reason upon treatment with impure penicillin. Stock would appear to be erroneous because, as Gye and (133) has reported that even at a dose of 5 gm/kg Purdy pointed out, their experimental evidence body weight/day crystalline penicillin G had no does not warrant a decision as to whether the loss effect upon the growth of Sarcoma 180 in vivo. of potency of the Rous virus was the result of oxi It is of interest that Burk et al. (18), in studies dation or was brought about by destructive pro- of effects on the metabolism of tissues, observed teolytic enzymes. Regardless of the possible error that several preparations of amorphous penicillin, in his hypothesis, Carr did find that, in vitro, com varying in potency from 1,000 to 1,500 Oxford plete loss of virus activity could be accomplished units/mg, brought about a marked inhibition in when the virus was mixed with a solution contain the respiration of several tumor and normal mouse ing notatin and glucose. When, however, notatin tissues, while crystalline penicillin G (1,660 Oxford was injected into fowls either before inoculation units/mg) was only one-tenth as active on a with virus or after development of tumors, no ef weight basis. Burk considered it possible that the fect upon either the incidence of infection or slight activity shown by the highly purified mate growth of the tumors was observed. Chinn (21) re-

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. REILLYâ€”Microbiologyand Cancer Therapy: Review 827 ported similar in vitro effects upon the Rous virus tained complete regressions in six of these patients. with terramycin, aureomycin, neomycin, and anti The diagnoses of these cases are open to severe biotic PA96 (Pfizer). When, however, chickens, question, and the malignant nature of the lesions previously infected with virus, were treated with is highly doubtful. It is most probable that any aureomycin or terramycin, the course of the dis lesions which may have responded to aureomycin ease was not altered. In fact, with low concentra therapy were infectious, not cancerous, in nature. tions of the antibiotics there appeared to be a Further, it is significant that, in five cases of ad stimulatory effect upon tumor development. vanced carcinoma, Ayres noted no real beneficial Stock et al. (135) found that immersion of the effect upon the administration of aureomycin. tissue in a solution of clavacin (1 mg/cc) com Bateman et al. (10) employed aureomycin as an pletely destroyed the viability of Sarcoma 180 and adjuvant in the treatment of cancer patients who a mouse melanoma, while streptomycin (10 mg/cc) were receiving x-ray and massive HN2 therapy. appeared to have no effect. Gliotoxin, in an aque Administration of the antibiotic was followed by ous suspension of 1 mg/cc, caused an 80 per cent gross and nonspecific microscopic effects on the reduction in the growth of treated Sarcoma 180 tumors and in some cases appeared to potentiate implants but had no effect upon the melanoma. the effects of x-ray and HN2. Five cases which had Stock (133) later reported that clavacin and glio- previously been considered as inoperable were toxin had only very slight, if any, effect upon the modified sufficiently to permit surgical treatment. development of Sarcoma 180 in vivo. Malmgren and Law (89) found that aureomy Kidd (71) found that cells of two mouse tumors cin, chloramphenicol, and streptomycin did not and of the Brown-Pearce rabbit carcinoma were affect the tumor-producing properties of the mam rendered nonviable by immersion for a short time mary tumor milk agent in C3H mice. When the in crude culture filtrates of the fungus Aspergillns milk agent was injected into strain C mice, the fumigatus. As a result of comparison experiments, data suggested that streptomycin may have ac he indicated that the active principle in his fungal tivated the virus; the mean tumor age in the anti filtrates was possibly identical with gliotoxin. He biotic-treated group was somewhat lower than was unable to demonstrate any effect with potent that in the control animals. Ambras et al. (2) have culture filtrates upon the development of the found that aureomycin, terramycin, and chlor Gardner lymphosarcoma in mice. Mason and Kidd amphenicol have no inhibitory effects upon the (90) concluded that the cyclic disulfide linkage development of several tumor-inducing viruses which is present in gliotoxin is necessary for the either in vivo or in vitro. destruction of tumor cells in vitro. Sokoloff and Eddy (128) observed a very defi Vollmar (142) observed that malignant cells in nite stimulation of implanted carcinoma in rats fed tissue culture were inhibited by patulin (clavacin) a small amount of aureomycin (1.6 mg/day/100 at concentrations that not only did not damage gm body weight for about 1 week). Large doses normal tissue but actually stimulated its growth, a (8 mg/day/100 gm), however, caused a marked factor which might be of interest in wound healing. inhibition of tumor growth as well as severe loss in Barnard and his associates (6, 7) reported that, total body weight. when the usual course of therapy yielded no bene Stock (133) reported that of eleven antibiotics fit to two patients with leukemia, the oral adminis tested only two, actinomycin and citrinin, caused tration of a crude fermentation concentrate, which reproducible slight inhibitory effects on the was obtained from Chas. Pfizer & Co., resulted in growth of Sarcoma 180 in mice. Reilly et al. (Ill), both clinical and hematological response. This was in a survey of 33 antibiotics, found that only fiveâ€” at first attributed to the high concentration of actinomycin, actidione, illudin M, illudin S, and vitamin Bis in the concentrate, but further investi terramycinâ€”caused a slight retardation of the de gation caused Barnard to conclude that not B12but velopment of Sarcoma 180 in vivo. rather any of the streptomyces-derived antibiotics Hackmann (55) found that, when suspensions (terramycin, aureomycin, streptomycin, or chlor- of the Ehrlich ascites carcinoma, mouse Sarcoma amphenicol) could bring about temporary remis 37, or the Walker rat carcinoma were mixed and sion in leukemia (5, 7). Goldman (46) observed no incubated for a short time with actinomycin C, the alteration in the clinical course of the disease in viability of the tumors was severely damaged. In five patients with Hodgkin's disease upon the ad vivo the antibiotic retarded slightly the growth of ministration of aureomycin. Ayres (4) treated thir the Walker rat carcinoma at dose levels which teen cases of what he termed "anaplastic lesions caused little or no toxicity in the host. Schulte (carcinoma in situ) of the cervix" with topical ap (116) has reported preliminary results on 150 pa plications of aureomycin. He claimed to have ob tients treated with actinomycin C, either.alone or

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. 828 Cancer Research in combination with x-ray therapy. Although little not influence the growth of Sarcoma 180 appre response was observed in cases of carcinoma, the ciably (139). Moore (92) reported similar results effects noted in over 50 patients with lympho- with the viruses of influenza A and herpes simplex. granulomatosis were reported to be encouraging. Turner et cd. (140) obtained some increase in sur Doses of 50-250/zg. of antibiotic were administered vival time in leukemic mice treated with vaccinia daily over a period of several weeks without harm virus, but the slight effect was lost when repeated ful effects to the patients. These gained weight and passage of the leukemia rendered it more virulent showed an improved blood picture. External (139). tumors disappeared rapidly; mediastinal tumors, Moore (93) demonstrated definite destruction however, showed only slight regression. As Schulte of Sarcoma 180 in mice which had been infected has emphasized, conclusions on the merits of with the virus of Russian Far East encephalitis. actinomycin C as a therapeutic agent against The tumor contained a high concentration of virus cancer cannot be made at this time. His report and failed to grow when transplanted to mice im comprises results obtained over a period of only munized against the virus. Examination of histo- 1| years; in some cases the dose of antibiotic logical sections showed massive cellular damage. was probably too small. Tests on more patients at Further investigation disclosed that five other higher dose levels as well as post-treatment ob transplantable mouse tumors responded to the servations over a longer period of time will be Russian virus in a similar manner (94). Koprowski necessary before actinomycin C may be properly and Norton (73) confirmed Moore's observations evaluated. with the Russian encephalitis virus and showed Bateman and Klopp (9) have claimed that the further that certain other neurotropic viruses pos concurrent administration of actidione and ami- sess oncolytic properties. Toolan and Moore (137) nopterin significantly prolonged life in leukemic reported that Egypt virus 101 had a destructive mice. Their enthusiastic conclusion, however, is effect upon a human epidermoid carcinoma grown not supported by convincing data; the survival of in x-radiated rats. Southam et al. (129) found that the treated mice was only very slightly greater infections with West Nile and Ilheus viruses tem than that of the untreated animals. porarily inhibited leukemic leukocytosis and in It would appear that any favorable responses filtration in mice but did not cause any significant which have been observed in the treatment of hu increase in survival time. The ability of neuro man cancer with clinically available antibiotics tropic viruses to attack tumors would appear to be have been the result, not of the ability of such specific in nature; each virus has its own "tumor agents to destroy tumor cells, but of their capacity spectrum" (73, 96), a term coined to indicate that to improve the general condition of the patients all oncolytic viruses do not destroy all tumors but temporarily by the control of certain secondary that each virus is effective against only a particular microbial infections. Nevertheless, the suggestive group of tumors which vary with the virus em results obtained in laboratory animals with some ployed. antibiotics indicate the necessity for a continued Unfortunately, the tumor-necrotizing effects of search for a cancer-controlling agent among sub the neurotropic viruses in mice have been achieved stances of this type. at dose levels of the viruses which cause eventual death of the animals. If active infection does not VIRUSES take place, the tumor is not affected (73, 93). Or Although a complete analysis of the intricacies dinarily, susceptible tumors when grown in mice of virus interference phenomena is beyond the immunized against the virus do not respond to scope of this presentation, the potentialities of virus therapy (98). Kuwata (74) has reported simi viruses as useful agents in the control of cancer lar findings with the ornithosis virus and a mouse merit some attention. Turner and Mulliken (138) carcinoma. Such results present a very dim pros and Moore (92) have reviewed briefly the histori pect for the practical application of viruses to can cal background of this field, and only some of the cer therapy. Nevertheless, the observations of sev more recent contributions will be considered here. eral investigators offer some hope that the tumor- Turner and Mulliken (138) found that, when destroying properties of certain viruses need not brain-adapted vaccinia virus was injected into necessarily be accompanied by destruction of nerv mice with Sarcoma 180, the virus localized in the ous tissue. Sharpless et al. (118) found that the tumor tissue. In early studies, infected tumors ap viruses of Russian and West Nile encephalitis peared to grow more slowly and to regress more could cause regression of a malignant lymphoid frequently than untreated tumors. Later, however, tumor in chickens without killing the host. Ginder these investigators concluded that the virus did and Friedewald (44) have reported rapid necrosis

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. REILLYâ€”Microbiology and Cancer Therapy: Review 8Â£9 of rabbit fibroma and some effect upon the devel ties of the therapeutic use of viruses have not yet opment of myxoma in rabbits (45) by the Semliki been exhausted and certainly deserve further con Forest virus without causing death or apparent ill sideration. ness in the animals. Moore (97) has obtained de The mechanism of the action of the inhibition of struction of Sarcoma 180 by the Russian encepha tumor growth by viruses is not known, and, in litis virus without killing the host when the tumor view of the lack of factual knowledge on this sub was implanted in PRI strain mice. ject, any attempt to explain this phenomenon can Moore (95) has attempted modification of the be only speculative. Clinical evidence indicates Russian encephalitis virus to increase its oncolytic that it is not merely the result of nonspecific fac properties and simultaneously to lower its affinity tors such as fever or stress (131). Although there is for nervous tissue. By repeated tumor to tumor no experimental evidence to support such a theory, passage of the virus she was able to obtain a strain several investigators (94, 131, 138) postulate that of virus which destroyed Sarcoma 180 cells more the oncolytic effects of viruses may be brought rapidly than did the original strain, but its neuro- about through competitive metabolic antago tropic properties remained unchanged. nisms. Any conclusions on this problem must await Kuwata (74) has reported that Rickettsia further data on the metabolism of both viruses and isutsugamushi and the ornithosis virus multiplied mammalian cells. and persisted in two mouse tumors for several days. Although the infections appeared to have no DISCUSSION effect upon the development of the original tu Micro-organisms offer an almost infinite source mors, on bioassay heavily infected tumors showed of new and interesting chemical compounds : many loss of viability. have the ability to form substances the very exist In spite of the apparently overwhelming ob ence of which is beyond the wildest fantasies of stacles that are involved in the use of viruses in the man's imagination. In many instances even when treatment of cancer (131), some cautious clinical the structure of a new microbial product can be studies have been made. Bierman et al. (15) re elucidated, all efforts to manufacture it syntheti ported some response in leukemia upon treatment cally meet with defeat. Thus, it is not unreason with the virus of feline agranulocytosis. Southam able that the search for an agent which will destroy and Moore (130, 131) have made very careful neoplastic growth should be made among products studies of the effects of several neurotropic viruses of microbial metabolism. Such investigations, how on the course of human neoplastic diseases. No ever, should be undertaken only when the com permanent beneficial effects were achieved, but in plexities of the problem have been carefully con a group of 34 patients treated with Egypt virus sidered and appreciated. The microbiological as some temporary objective regression appeared in pects alone appear quite formidable. As has been four, and suggestive but not conclusive beneficial clearly pointed out in antibiotic research (143) effects were noted in five others. Higgins and Pack and, to a lesser extent, even in cancer investigation (61, 62) inoculated rabies vaccine into 30 patients (108,109), the ability to produce a specific agent is with malignant melanomas. In at least six of the a characteristic, not of a genus or species, but of a cases dermal mÃ©tastasesdecreased in size and be particular strain of micro-organism. In our own came flattened. The development of new mÃ©tas experience with seven different isolates of Aspergil- tases appeared to be retarded. lus fumigatila, five were found to produce tumor- Although the practical application of viruses in retarding filtrates, while two were completely in cancer therapy thus far has yielded no outstand active. ing results, experimental evidence indicates that The now widely recognized variability of micro the success of such treatment is not beyond the bial cultures presents an added hazard. Some or realm of possibility. A virus that showed a propen ganisms, freshly isolated from natural sources, re sity for tumor tissue but was nonpathogenic or tain most of their original properties on repeated only mildly virulent for human beings might transfer on laboratory media; others very quickly achieve results in the clinic similar to those which lose their abilities to carry out the special func have been observed with chickens and rabbits. tions for which they were isolated. To keep a cul Also, if an antibiotic that was effective against an ture in an active state, with respect to its capacity oncolytic virus were available, it might be possible to produce a particular substance, requires great to infect individuals with the particular virus, and, care and constant vigilance, which all too fre after a lapse of time sufficient to insure destruction quently is of no avail. of tumor tissue, to control any ensuing systemic The nutritional and environmental conditions viral infection with the antibiotic. The potentiali of cultivation markedly influence the metabolic

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1953 American Association for Cancer Research. 830 Cancer Research functions of an organism. Conditions that are op tumor agents must be conducted in vivo.This im timum, for maximum growth may not be suitable mediately excludes the possibility of the use of a for the formation of a specific substance. Fre really rapid and simple procedure. quently an unknown precursor must be supplied, In view of our scant knowledge, the choice of a or a proper balance among certain rainerai ele test tumor must necessarily be arbitrary. Whether ments must be achieved. Oxygen tension, pH, it should be a spontaneous or an implanted one has temperature, and time of incubation must be care been a point of contention for many years. There fully controlled. Although the number of variables can be no doubt that certain properties of trans seems most forbidding, the tremendous economic, planted tumors are different from those of spon industrial, and medical importance already taneous ones, even when the former have been achieved in the field of microbial fermentations derived from the latter (48). Nevertheless, trans (107) clearly demonstrates that they are not insur planted tumors would appear to be the ones of mountable obstacles. choice for use in large scale testing programs be In any attempt to find among natural sources cause they are readily available and offer much agents that have specific properties either of an greater opportunities for well controlled experi enzymatic, antimicrobial, or antitumor nature, one mentation than do spontaneous developments. is confronted with the problem of whether highly The fact that different animal tumors have been purified preparations or crude mixtures, such as shown to exhibit varied responses to treatment microbial culture filtrates, should be tested. Both with chemical agents (41, 133) presents an addi offer definite advantages. Purified agents, even tional problem. Some tumors may be markedly when their chemical structures are not known, are affected by a particular compound, whereas others usually well characterized, and preparations from will possess a strong natural resistance to it. Thus, different batches, depending upon their degree of any results that may be obtained with one type of purity, do not vary greatly. Thus, any effects that tumor provide little or no information as to what they may exhibit can be more or less readily repro may be expected with other tumors. duced. On the other hand, most pure compounds When all the factors that are involved in this that are biologically interesting have been isolated field of investigation are considered, the need for because they affect systems other than neoplasms, patient and persevering endeavor becomes appar and, in the process of purification, any component ent. that might possibly have been active against tu mor growth has been lost. Such would appear to ACKNOWLEDGMENTS have occurred in the crystallization of penicillin. The author wishes to express her grateful appreciation to Because they are heterogeneous mixtures, crude Dr. C. P. Rhoads and Dr. C. Chester Stock for their interest and many helpful suggestions in the preparation of this materials offer great possibilities. It is well recog manuscript. nized in the field of antibiotics that one micro REFERENCES organism may produce several antimicrobial 1. ALGIBE,G. H.; LEGALLAIS,F.Y.; and ANDERSON,B.F. agents simultaneously; this might well be true of Vascular Reactions of Normal and Malignant Tissues the production of antitumor substances. The pos in Vivo. V. The Role of Hypotension in the Action of a sibilities of additive or synergistic effects among Bacterial Polysaccharide on Tumors. J. Nat. Cancer Inst., 12:1279-95, 1952. several components in crude preparations certainly 2. AMBKUS,J. L.; AMBKUS,C. M.; SIDERI, C. N.; and cannot be disregarded. HAHRISSON,J. 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H. Christine Reilly

Cancer Res 1953;13:821-834.

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