A Mitomycin C-Resistant Jensen Rat Sarcoma: Isolation and Transplantation Studies*

A Mitomycin C-resistant Jensen Rat Sarcoma: Isolation and Transplantation Studies*

PHILIPC. MERKER,CESARREYES,ANDPAULINEANIDO

(Division of Tumor Biology, Sloan-Kettering Institute for Cancer Research, Sloan-Kettering Division, Graduate School of Medical Sciences, Cornell University, New York, New York)

SUMMARY A transplantable rat tumor, the Jensen sarcoma, has been selected for resistance to the action of mitomycin C. Initial treatment consisted of 0.15 mg/kg. After seven passages through rats, the treatment dose was raised to 0.5 mg/kg. Under normal cir cumstances, this dose of antibiotic completely inhibits tumor growth. The stability of this resistance was proved by passage of the tumor through more than twenty genera tions in untreated rats. The antibiotic-resistant tumor was also found to have the following character istics: (a) growth as a firm mass with less necrosis and central liquefaction than is ordi narily observed in the sensitive sarcoma; (6) a greater tendency to persist for longer periods of time in the rat host before showing drastic signs of regression; (c) a slow growth rate in cortisone-treated Swiss mice; (d) poor transplantability to neonatal mice; and (e) cross-resistance to nitrogen mustard in tests with the cortisone-treated Swiss mouse.

As an aid in experiments designed to study the METHODS AND PROCEDURES effect of heterologous transplantation on tumor Female rats (Sprague-Dawley) weighing 80-120 response to drug treatment, a mitomycin C-resist gm. were used for transplantation and maintenance ant Jensen rat sarcoma was isolated. It is the pur of the mitomycin C-sensitive Jensen rat sarcoma pose of this paper to describe the isolation of the (to be designated JRS). The tumor was routinely tumor and its growth characteristics. passaged every 9-11 days by means of subcutane There were few if any reports in the literature ous inoculations of 1.0 ml. of a 50 per cent minced up to 1954 dealing with the development of drug tumor suspension prepared in physiological saline resistance in transplantable tumors of the rat. In fortified with streptomycin sulfate (2 mg/ml) and that year, Jackson (9) presented data for drug penicillin G (500 units/ml). resistance in the solid Walker carcinosarcoma, and Mitomycin C was prepared every 2 weeks as a Hirono (6) reported on the drug resistance of the stock solution in saline (0.86 per cent) in a con Yoshida ascites sarcoma. Since then, other workers centration of 0.3 mg/ml. During this time the have isolated drug-resistant rat tumors after drug stock solution was kept refrigerated. Dilutions of treatment of sensitive tumors (5, 8, 10, 13, 15) the stock were made with saline. Rats were treated or have reported on the presence of "natural" daily with single intraperitoneal injections starting drug-resistant tumors (18), and at least one refer 24 hours after tumor transplantation. Treatment ence has been made to an androgen-resistant trans was then continued for 9-10 days. plantable rat tumor (4). RESULTS * This study was supported in part by research grant CY- At the start of these experiments, rats bearing 3784 from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service, Bethesda, Maryland; the sensitive tumor (JRS) were treated with 0.15 in part by contracts SA-43-ph-1923 and SA-43-ph-2445 from mg/kg of mitomycin C to select a "resistant" tu the Cancer Chemotherapy National Service Center, Silver mor passage. After four passages in rats treated Springs, Maryland. with 0.15 mg/kg mitomycin C, experiments re Received for publication April 16, 1962. vealed that the tumor had become resistant to 1.0 1163

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1962 American Association for Cancer Research. 1164 Cancer Research Vol. 22, November 1962 mg/kg dose levels. In an experiment performed at ein C-treated rats as firm, well vascularized masses this time, "resistant" tumors (to be designated which show very little central necrosis. This is in hereafter as JRS/MC) grew in three out of six contrast to the growth of sensitive tumors which animals treated with 1.0 mg/kg. The three tumors are less firm on palpation, whitish in appearance, attained an average diameter of 4.56 cm. Under and display a greater degree of gross necrosis with ordinary circumstances, this dose of mitomycin central liquefaction. C (1.0 mg/kg) causes complete destruction of JRS Several attempts were made to increase the tumor transplants. treatment dose level to 1.0 mg/kg. One such at After the seventh passage of tumor in rats tempt was made after the tumor had been passed treated with mitomycin C at 0.15 mg/kg, the for ten generations in animals treated with 0.5 mg/ JRS/MC tumor was passed to rats treated with kg of antibiotic; these tumors were successfully 0.5 mg/kg. The tumor is now maintained routinely transferred for eleven generations, but no tumor in such antibiotic-treated rats. takes were obtained on the 12th passage, and the During the first 2-week period after tumor line was lost. transplantation, resistant tumors grow in mitomy- On the tenth passage in mitomycin C-treated rats, the JRS/MC tumor was transplanted into rats which were not subsequently treated with antibiotic. This was done to establish the stability of resistance. Data presented in Chart 1 illustrate that the JRS/MC tumor after fourteen passages in rats not treated with antibiotic retained its resistance to the drug. A comparison of percentage "takes" and aver age sizes of the JRS/MC and JRS tumors at the end of 9-11 days of growth revealed no significant differences (Table 1). However, in terms of host mortality rate and the onset of tumor regression in surviving animals, differences were noted be tween the two tumors (Chart 2). The general im 2 4 6 8 10 12 14 16 pression to be gained from a comparison of the Daysposttransplantation data presented in Chart 2 is: (a) the JRS and JRS/MC tumors have approximately the same CHART1.â€”Responsesto mitomycin C treatment (0.5 mg/ growth rate during the first 2-week period after kg/day X 7 days; I.P.) of mitomycin C-sensitive and -resistant Jensen sarcomas growing subcutaneously in female Sprague- transplantation. Thereafter, the resistant tumor Dawley rats. does not necessarily continue to increase in size. Sensitive subline: O = treated; â€¢= saline-injected con (6) JRS tumor-bearing rats die sooner than JRS/ trols. Resistant subline: A = treated; A = saline-injected con MC tumor-bearing animals. Data for this repre trols. sentative experiment show that 50 per cent of the TABLE1 ACOMPARISONOFAMITOMYCINC-RESISTANT(JRS/MC)ANDSENSITIVE(JRS)JENSEN SARCOMAGROWINGSUBCUTANEOUSLYINRATS*

tNo.

tumor tumor takes / No. tumors takes / No. tumors diameter^ (cm.) diameterf (cm.) transplanted9/913/1314/1810/1815/17Av. transplanted7/1414/1515/1615/1517/19Av. (range)4.2(3.8-4.5)4.8(3.9-5.6)4.8 (range)4.8(3.9-5.6)4.5

3691215JRS/MC (3.1-5.3)4.9 (2.3-5.5)4.5(3.3-5.2)4.5 (33-5.7)5.1 (4.4-6.1)5.0 (3.1-5.4)JRSNo. (3.1-5.9)

*Female Sprague-Dawley rats weighing 80-120 gm. t Mitomycin C-resistant tumor passaged in untreated rats. ÃŽFinaltumor sizes determined after 9-11 days of growth.

JRS tumor-bearing rats were dead by the 20th physiological saline fortified with penicillin G (500 day, whereas only 25 per cent of the JRS/MC units/ml) and streptomycin sulfate (2 mg/ml). tumor-bearers had died during this same time The cell concentration was adjusted so that the period, (c) In rats which survive for 27-28 days, required number of cells could be inoculated sub- JRS tumors regress sooner than JRS/MC tumors. cutaneously in volumes ranging from 0.05 to 0.1 Experimental data in Chart 2 show that JRS ml. Newborns were first examined for tumor 7 days tumors began to regress on the 17th day after after inoculation, and final tumor measurements transplantation in those rats which survived for were made on the llth-12th days. 27 days, whereas regression of the resistant tumors began after the 24th day. Although this represents (10/16)(8/16)(8/16) (12/16)*â€”â€¢ â€¢ a lag in regression time of only approximately 7 6.0 days, it has been observed rather consistently and hence represents an important difference. 5.0 (16/16) Growth in cortisone-treated mice.â€”As an outcome ^x (5/161 of our interest in heterotransplantation (12), ex periments were designed to study the growth of the 4.0 resistant and sensitive tumors in cortisone-condi tioned mice. When transplanting tumors to mice, females (Swiss-Webster) weighing 18-20 gm. were 3.0 given inoculations intramuscularly of 0.5 ml. of a 50 per cent tumor suspension; the mice were then conditioned with a single, subcutaneous dose of 3 2.0 mg. cortisone acetate (Cortone acetate, Merck). Mice were maintained for 10-14 days, at which 1.0 time tumors were measured, and, in most cases, the mice were sacrificed and tumors removed and weighed. In those cases when tumors were sec tioned and examined, the resistant tumor showed 10 20 30 no remarkable changes in histology. 6.0 One of the characteristics noted for the JRS/ (8/8) (7/8) (6/8) (6/8) MC tumor was its slow growth on heterotrans plantation to cortisone-treated mice. A difference 5.0 in growth between the JRS and JRS/MC tumor was first noted on the fourth passage of the tumor in rats treated with antibiotic at 0.15 mg/kg. Â¿4.0 Data are presented in Chart 3 to illustrate the OJ .12 striking difference in growth pattern of the two (4/8) tumors. *3.0 In addition to showing differences in growth rate, Chart 3 presents data which demonstrate the 2 2.0 response of JRS tumor to antibiotic therapy and the lack of response of the resistant tumor to simi lar treatments when tests are made in the condi 1.0 tioned heterologous host. Chart 4 shows that after passage of the JRS/MC tumor in rats not treated with antibiotic the tumor retained its pattern of slow growth in the cortisone-treated mouse. 10 20 30 Days post transplantation Growth of the resistant tumor in newborn mice.â€” In view of the slow growth observed in cortisone- CHART2.â€”Acomparison of the growth and regression rates of the mitomycin C-sensitive and -resistant Jensen rat sar treated mice, experiments were designed to study comas in female Sprague-Dawley rats. the growth of transplants in neonatal mice. The Solid black circles represent the average diameter for all suspensions of tumor cells used in these transplan tumors measured at the specified time. Open circles refer to tation studies were prepared by first mincing the those tumors in rats which survived for the entire period of tumor with scalpels and then gently homogenizing observation. Numbers in parentheses refer to the number of sur viving animals. the tissue in 9 volumes of cold physiological saline; Upper chart: Mitomycin C-sensitive Jensen sarcoma. cells were then washed and finally suspended in Lower chart: Mitomycin C-resistant Jensen sarcoma.

Rx: Mitomycin C l.Omg/kg/day IP Table 2 presents data to show that the per centage tumor "take," defined in this instance as measurable, subcutaneous tumors, was highest for 1.5 JRS-implanted newborns. This was observed in all three of the newborn mouse strains used in this JRS experiment. Although there was a significant dif 1.0 ference in the total number of measurable tumors observed between JRS and JRS/MC implanted newborns, no significant differences in final average tumor diameters were noted. It was of interest to record that AXC57BL/6 survivors implanted with JRS/MC tumor did not display any measurable I o 2.5r 1 1.5

^ 9L t JRS/MC 5 i.o

0.5

"0 3 6 9

Days post transplantation 4 6 8 10 12 14 CHART3.â€”Effect of mitomycin C treatment on sensitive Daysposttransplantation and resistant Jensen rat sarcomas growing in cortisone-condi tioned female Swiss mice. CHART4.â€”Growthof mitomycin C-sensitive and resistant Upper chart: JRS = mitomycin C-sensitive Jensen rat sar Jensen sarcomas in cortisone-treated female Swiss mice. coma: O = treated; â€¢= saline-injected controls. â€¢= mitomycin C-sensitive tumor. Lower chart: JRS/MC = mitomycin C-resistant Jensen O = mitomycin C-resistant tumor passed in rats treated rat sarcoma: O = treated; â€¢= saline-injected controls. with mitomycin C (0.5 mg/kg). Treatment consisted of daily intraperitoneal injections giv A = mitomycin C-resistant tumor passed for 9 generations en for 7 days. in rats not treated with mitomycin C. NOTE: Cortisone-conditioning consisted of a single dose of NOTE: Recipient female Swiss mice were conditioned with cortisone (3 mg/mouse) given on the day of tumor transplan a single subcutaneous dose of cortisone acetate (3.0 mg/mouse) tation. given on the day of tumor transplantation.

TABLE 2 TRANSPLANTABILITYOFJENSENRATSARCOMAINNEWBORNMICE*

XCS7BL/6JRS281414100501.02(0.93-1.06)JRS/MC3014429531.25(1.15-1.35)Ad"XC57BL/6JRS24151280381.03(0.95-1.07)JRS/MC26100062None

t90743547181.09(0.60-1.85)JRS/MCt1781522114151.14(0.80-1.70)DBAcf implantsNo.No. newborns given newbornsNo.surviving tumor "takes"Â§in surviv ingnewbornsPer "takes"Â§Percent tumor centmortalityAv. whichgrewdiam. of tumors (cm.)SwissJRS

* Tumor suspensions containing 1-7X10' cells were inoculated into newborns less than 24 hours old. Final observations were made 11-12 days after transplantation. t JRS: mitomycin C-sensitive tumor. J JRS/MC: mitomycin C-resistant tumor. Â§Tumor"take" refers to measurable tumors growing subcutaneously.

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1962 American Association for Cancer Research. MERKERet al.â€”Jensen Rat Sarcoma 1167 subcutaneous or intraperitoneal tumors (although come of earlier investigations on the response to it is possible that microscopic foci of tumor cells therapy of a transplantable human tumor growing remained from the original implant). The inability in conditioned mice (12). of FI newborns derived from this cross to allow Data are presented for nitrogen mustard (Table JRS/MC tumor implants to grow is unexplainable 3) to illustrate cross-resistance of the tumor as it at this time. grew in cortisone-conditioned mice. Inhibitory ef Data in Table 2 also reveal that newborn ran fects were observed against the sensitive tumor, dom-bred Swiss mice, in contrast to hybrids, were whereas no significant antitumor activity was ob less prone to die from inoculations of either JRS served with the resistant line. or JRS/MC tumor cell suspensions. Less than 20 per cent of the tumor-implanted, random-bred DISCUSSION Swiss newborns died, whereas more than 50-60 per A mitomycin C-resistant Jensen rat sarcoma cent of the hybrid newborns succumbed. Deaths subline having an approximate three- to sixfold in usually occurred within the first 7 days after tu crease in drug resistance was isolated after four mor inoculation. During this period, it is unlikely passages of the tumor in rats treated with 0.15 that newborns would die of extensive invasion of mg/kg of the antibiotic. This starting dose of TABLE3 EFFECTOFNITROGENMUSTARD*ONJENSENRATSARCOMAGROWINGINCONDITIONEDSwissMiCEf

tumor tumor Exp.NO.123JRSÃ®Mort.Treated0/30/71/20Control1/60/70/20Body wt.(gm.)Treatedâ€”0.5â€”0.4â€”1.8Control+2.6+3.9+6.7Av.wt.(gm.)Treated1.90.51.0Control3.72.74.2Percent Body wt.(gm.)Treated+0.4â€”0.5â€”1.3Control+2.6+4.9+wt.(gm.)Treated2.11.31.6Control0.82.01.5Percent tumor tumor inhibition468176JHS/MC}Mort.Treated0/30/100/20Control0/31/100/20Ainhibition0350

1.9Av.

* Doseâ€”1.0mg/kg/dayX7 days, IP. t Conditioning: Single subcutaneous injection of cortisone acetate (Cortone acetate, Merck) given on the day of tumor trans plantation. Dose, 3 mg/mouse. Final observation: 9-10 days after tumor transplantation. ÃŽJRS:mitomycin C-sensitive Jensen sarcoma. Â§JRS/MC: mitomycin C-resistant Jensen sarcoma. vital organs by rapidly growing tumors. In fact, mitomycin C represents a suboptimal thereapeutic in the great majority of instances, gross inspection level for the Jensen rat sarcoma; normally, doses of the abdominal and thoracic cavities failed to ranging from 0.25 to 1.0 mg/kg produce profound reveal any extensive or untoward tumor forma inhibitions in tumor growth (17). Other investiga tions. The cause of death is not known at this time. tors have also isolated drug-resistant transplant- Drug response.â€”Preliminary chemotherapy able rat (6, 8, 9, 13) and mouse (16) tumors by studies conducted in collaboration with Dr. Sugiu- initially using suboptimal treatment doses; how ra revealed that the mitomycin C-resistant tumor ever, this technic need not necessarily be used. was cross-resistant to alkylating agents (HN2, Burchenal (1) and Clarke (2) were successful in thioTEPA, and Cytoxan) and to the antimetabo- isolating drug-resistant mouse neoplasms from lite amethopterin. An investigation of the chemo- groups of animals that had been treated with effec therapeutic responses of the resistant tumor to a tive treatment levels of drug. In other instances, spectrum of chemical agents was therefore under transplantable rat tumors have been observed to taken, and the results of these studies will be pub be resistant to drug action without any prior treat lished elsewhere. ment of drug (18). The isolation of drug-resistant In addition to standard therapy studies on the sublines of transplantable tumors can be compli rat, experiments were also attempted with the cated by the prior development of resistance to resistant tumor as it grew in the cortisone-condi other chemicals (5). tioned mouse. Interest in this system was an out- Microscopic changes in the architecture of the

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1962 American Association for Cancer Research. 1168 Cancer Research Vol. 22, November 1962 tumor have not been significant, even though dif mycin C-resistant Jensen sarcoma do in fact have ferences have been observed at the gross level. an increased capacity (approximately threefold) This failure to note any remarkable change of his- to metabolize the antibiotic even though the rate tological features in the resistant tumor is similar of anaerobic glycolysis by the resistant tumor is to that reported by Hirono (8) for nitrogen mus not significantly different from that of the sensi tard N-oxide-resistant sublines of the Yoshida as- tive tumor. cites sarcoma and the AH 13 ascites hepatoma. Although differences in growth rate during the 14- ACKNOWLEDGMENTS day period following transplantation have not The authors wish to thank Dr. George Woolley for his kind been observed in the present study, data collected support during these studies. The authors express appreciation after the first 2-week period revealed that JRS/ to Dr. K. Sugiura for supplying the initial passage Jensen rat sarcoma. The kind suggestions of Dr. Dorris J. Hutchison are MC tumor-bearing rats died at a slower rate and gratefully appreciated. the time of onset of tumor regression in surviving Mitomycin C was kindly supplied by Merck, Sharp and rats was somewhat delayed. Since the relationship Dohme, Railway, New Jersey. of the sensitive and resistant Jensen tumors to the rat host is at best "homologous" rather than "isol- REFERENCES ogous," it is feasible to explain this observation 1. BURCHENAL,J. H.; ROBINSON,E.; JOHNSON,S. F.; and KusHlDA, M. N. 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Philip C. Merker, Cesar Reyes and Pauline Anido

Cancer Res 1962;22:1163-1169.

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