Preliminary Drug Trials in a Renal Cell Carcinoma Animal Model 1,2
`Alan M. Shefner3•4 and Marilyn Marlow3
`
`SUMMARY
`
`A transplantable renal cell carcinoma of the BALB/c mouse has been used for the
`evaluation of seven chemotherapeutic agents. This model appears to resemble the
`clinical situation of disseminated renal cell carcinoma in man .. Although drug trials
`are still in progress, preliminary indications of the activity or inactivity of the drugs
`tested, at the doses and regimens used, are available. Bleomycin, hydroxyurea, and
`cyclophosphamide had little effect on survival time or on tumor growth. Adriamycin
`and vinblastine increased survival time by >25% and adriamycin appears to produce
`some reduction in tumor size. The two nitrosoureas, methyl-CCNU and BCNU, are
`highly effective in reducing tumor size and have produced a >25% increase in life(cid:173)
`span to date.
`
`[Cancer Chemother Rep Part 2, vol 5:145-149, 1975]
`
`Human renal cell carcinoma, if inoperable or with
`widespread metastases, yields very few long-term
`survivors. Five-year survival rates of <5% have
`been reported in cases of metastatic disease (1,2).
`The dis~a$e is unpredictable; it may progress, remit,
`and, in very rare cases, undergo spontaneous regres(cid:173)
`sion (3,4). Spontaneous regressions of pulmonary
`and bone lesions after radical nephrectomy have
`been reported, possibly indicating a hormonal or
`immunologic control mechanism (5). Removal of the
`primary renal tumor in the absence of metastasis
`has resulted in 3-year survival rates of 45% and 5-
`year survival rates of 34%. These long-term surviv(cid:173)
`al rates were not affected by chemotherapy (5). Gen(cid:173)
`erally, standard chemotherapy has not been effec(cid:173)
`tive against metastatic renal cell carcinoma (6).
`Carter has reported on the nearly total failure of
`chemotherapy against renal eel] earcinoma; only
`five standard agents have been evaluated and none
`were active.I• Hormonal ·therapy has been reported
`to produce both favorable as well as poor results
`(7,8).
`~
`
`1Supported by contract N0l-CM-81021 from the Division of
`Cancer Treatment, National Cancer Institute, National Institutes
`of Health, Department of Health, F,ducation, and Welfare.
`'The tumor was generously supplied to us, in intrarenally trans(cid:173)
`planted donor mice, by Dr. Gerald P. Murphy, Roswell Park '.llemori(cid:173)
`al Institute, Buffalo, NY.
`''Life Sciences Research Division, !IT Research Institute, Chi(cid:173)
`cago, Ill.
`:\Ir. Alan M. Shefner, Life Sciences Re(cid:173)
`'Ileprint requests to:
`search Division, !IT Research Institute, 10 West 35th St, Chicago,
`Ill GOG 16.
`''Carter SK. Report of the Associate Director. In Report of the
`Division of Cancer Treatment, NCI, W74. Bethesda, ~Id, NCI, 1974.
`vol 1, pp G.l-G.11.
`
`Few animal models for renal cell carcinoma have
`been described. In the past year we have been study(cid:173)
`ing a murine renal cell carcinoma model system
`which was developed and characterized by Murphy
`and Hrushesky (9). The host is the BALB/c mouse.
`The tumor originated as a spontaneous renal corti(cid:173)
`cal adenocarcinoma of the granular cell type (9). It
`develops after intrarenal (ir), irn, ip, iv, and sc trans(cid:173)
`plantation. It is quite slow-growing, with the sur(cid:173)
`vival time depending upon the site of inoculation.
`The purpose of our study is to characterize the sur(cid:173)
`vival response of the animals after various routes of
`inoculation (particularly ir and sc), to study the
`growth patterns of the tumor, and to determine the
`response of this tumor to selected chemotherapeutic
`agents leading to the possible utilization of this sys(cid:173)
`tem for specialized drug testing.
`
`METHODS
`Male and female BALB/c mice, at least 6 weeks
`old, have been used with no differences noted in tu•
`mor growth or survival time.
`For ir transplant a tumor cell suspension is pre(cid:173)
`pared. Our procedure for preparing and inoculating
`the renal tumor is essentially that described by
`Murphy and Hrushesky with the exception that we
`transplant the tumor under the capsule of only one
`kidney (9). After the donor animal is killed, the tu(cid:173)
`mor mass is aseptically removed and separated from
`the remaining kidney tissue. The tumor is weighed
`and, using a glass tissue grinder, a 1:10 homogenate
`is prepared with McCoy's medium containing 5%
`fetal bovine serum, 100 units/ml of penicillin, ~nd
`100 µg/ml of streptomycin. The tumor preparat10n
`
`Cancer Chemotherapy Reports Part 2 Vol. 5, No. 1, December 1975
`
`145
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`and all materials are kept well chilled. Cell counts
`are routinely performed. Mice which are to be inocu(cid:173)
`lated ir are anesthetized, a small incision is made,
`and the left kidney is exposed. A volume of 0.1 ml ~f
`homogenate is inoculated under the capsule of the
`kidney using a 27-gauge needle. The incision is
`closed with autoclips. After sufficient experience, the
`surgical procedure requires approximately 2-3 min(cid:173)
`utes per mouse and the mice recover within 10-15
`minutes.
`An sc implant of the renal cell carcinoma has been
`made by trocared fragment or sc inoculation with
`the tumor cell homogenate as prepared above.
`
`ent mice from the fourth generation onward have
`developed tumor. We have also carried the tumor by
`sc transplantation of trocared fragments through
`ten generations; the MSTs for generations three to
`ten average 64 days. These MSTs agree well with
`our experience with sc transplant with tumor cell
`suspensions in which the MSTs ?or three experi(cid:173)
`ments were 77 .0, 54.0, and 49.8 days.
`We have attempted to delineate the growth of the
`sc implanted renal tumor by calculating tumor
`weights from periodic caliper measurements of both
`homogenate and fragment-induced tumors. Tumor
`weights were calculated using the formula:
`
`CHARACTERISTICS OF THE MODEL
`
`Murphy and Hrushesky (9) have characterized the
`murine renal cell carcinoma model in 'terms of the
`kinetics of tumor growth :::.nd metastasis using dif(cid:173)
`ferent transplantation routes and in terms of sur(cid:173)
`vival of transplanted mice. The tumor arose sponta(cid:173)
`neously as a renal cortical adenocarcinoma and was
`passaged sc approximately every 35 days. The tu(cid:173)
`mor grows equally well when passaged into male or
`female BALB/c mice. Swiss mice challenged with as
`many as 1 x 106 tumor cells show no evidence of
`tumor growth. The metastatic index and tumor
`weights of recipient BALB/c mice inoculated ir with
`tumor suspensions ranging from 105 to as low as 50
`viable cells correspond directly to the number of
`cells inoculated. Small localized tumors were found
`in 100% of the mice transplanted with 50 cells.
`Large tumors developed in 100% of recipient· mice
`transplanted with 105 cells when the injection route
`was ir, ip, iv, im, or sc. The metastatic index was
`high only after ir transplant. Ir transplanted mice
`survived for an average of 46 days, while the median
`survival times were >60-75 days when other injec(cid:173)
`tion routes were used.
`By Day 21 after ir transplant, all animals had
`metastatic tumor and 50% were grossly palpable.
`Tumor weight doubling time was approximately 7
`days during the period of 21-35 days after inocula(cid:173)
`tion.
`Renal tumor growth was enhanced by the adminis(cid:173)
`tration of testosterone or' diethylstilbestrol but was
`unaffected by medroxyprogesterone, a progestation(cid:173)
`al agent. Cell-free extracts of renal tumor did not
`produce tumor growth in 20-hour-old BALB/c mice
`who were ip inoculated or in 6-week-old BALB/c
`males or Swiss mice who were ir inoculated.
`In our laboratory we have carried the tumor
`through 13 generations by ir transplant. The median
`survival times (MST) for generations three to 13
`average 45.3 days and are in good agreement with
`the findings of Murphy and Hrushesky (9). All recipi-
`
`lcnf!ilh(mm)X(wldth[mtnj)2
`i
`In the period of 21-42 days after implant, the time
`for a doubling of tumor mass is approximately 7
`days.
`The calculated weights for the two sc tumor
`groups are generally in the same range, with the
`homogenate-inoculated mice having somewhat larg(cid:173)
`er tumors (tables 1 and 2). For both groups, there is
`a wide spread in tumor size at each interval. In the
`group of 60 homogenate-inoculated mice, one mouse
`had a tumor which was palpable but not measurable
`through Day 56. We had hoped that the size of the sc
`tumors would be more consistent when a hoplogene(cid:173)
`ous cell-suspension inoculum was used, but this has
`not yet been demonstrated.
`In addition to the wide variability in size, another
`major problem with both of the sc implants is the
`tumor ulceration which often occurs as early as 3
`weeks when the tumors are still quite small and de(cid:173)
`velops in essentially all of the mice before death.
`This makes progressive tumor measurement difficult
`
`TABI.E 1.-Calculated tumor weights of sc inoculated renal tumor
`homogenate (60 mice)
`
`Day
`after
`implant
`
`Range of mean
`tumor weights of
`cage groups•
`
`Weight(g)
`
`Overall
`range
`
`Average
`
`23
`
`28
`
`34
`
`40
`
`49
`
`53
`
`56
`
`>0.039-0.289
`
`>0.0-0.527
`
`>0.172
`
`>0.087-0.484
`
`>0.0-0.827
`
`0.174-0.899
`
`>0.0-1.521
`
`0.304-1.495
`
`>0.0-2.458
`
`0.379-3.805
`
`>0.0--5.054
`
`0.398-4.034
`
`>0.0-5.596
`
`>0.301
`
`>0.536
`
`>0.808
`
`>1.533
`
`>l.828
`
`0.510-3.837
`
`>0.0-6.700
`
`>2.187
`
`•5 mice per cage group.
`
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`TAau; 2.-Calcula ted tumor weights from renal tumor SC03
`(trocar)
`
`Day
`after
`implant
`
`12
`14
`18
`21
`25
`29
`33
`·39
`42
`48
`54
`
`Weight (g)
`
`Range
`
`>0-0.056
`>0-0.077
`>0-0.137
`>0-0.301
`>0-0.493
`0.046-0.564
`0.060-0.938
`0.169-2.160
`0.267-3.092
`0.329-2.246•
`0.350-2. 769
`
`Average
`
`0.011
`0.031
`0.076
`0.138
`0.196
`0.260
`0.440
`0.900
`1.090
`1.168
`1.500
`
`•Mouse with 3.092-g tumor died on Day 48.
`
`or impossible and introduces the factor of bacterial
`infection.
`We have also inoculated one group of mice ip with
`0.5 ml of the 1:10 renal tumor homogenate which
`yields an MST of 34.7 days and 100% deaths by Day
`58. This was approximately a five times larger cell
`inoculum than that used by Murphy and Hrushesky
`who reported >50% survivors at 60 days with ip
`inoculation of 105 tumor cells (9).
`
`CHEMOTHERAPEUTIC TRIALS
`
`Chemotherapeutic trials in this model have been
`quite limited. Based on clinical reports of activity
`against metastatic renal cell carcinoma, Hrushesky
`and Murphy tested CCNU and vinblastine against
`murine renal cell carcinoma (10). CCNU had yielded
`objective remissions in 20% of the patients in a
`studf by Mittelman et al (11), and vinblastine had
`produced an approximately 30% rate of objective
`response (5). Hrushesky and Murphy evaluated the
`response to drug therapy by three criteria: tumor
`weight, metastasis, and survival time. Both drugs
`were given ip every seventh day (q7d) starting on
`Day 7 after tumor implant; in some experiments the
`drugs were given q7d starting on Day 21 when the
`renal tumor is palpable and metastasis has begun.
`Tumor weight, as determined by autopsy at 40
`days, was significantly reduced by both drugs when
`treatment was started on Day 7. The effect was dose
`related. A 75% reduction in tumor size with CCNU
`and a 95% reduction in tumor size with vinblastine
`were the best responses as compared to controls.
`Lung metastases were determined microscopically
`after autopsy at 40 days. Both drugs were given q7d
`starting on Day 7. With CCNU given at 1.0 mg of
`drug/animal/injection (40 mg/kg), 80% of the ani(cid:173)
`mals had distant metastases at Day 40, and with
`
`Vol. 5. No. 1, December 1975
`
`vinblastine given at 0.02 mg of drug/animal/injec(cid:173)
`tion (0.8 mg/kg), only 50% had pulmonary metasta(cid:173)
`ses. All of the control mice had metastases.
`In their survival time study, both drugs were giv(cid:173)
`en q7d starting on Day 7 or on Day 21. With CCNU,
`all three doses (0.4, 1.0, and 2.0 mg/kg) on both sched(cid:173)
`ules increased survival time, with delayed therapy
`yielding better responses than earlier therapy. The
`optimum response was a 61 % increase in mean sur(cid:173)
`vival time (47 days for controls and 76 days for
`CCNU-treated mice). With vinblastine, all doses (0.2,
`0.4, and 0.8 mg/kg) and both schedules increased
`survival time, with the early treatment generally
`better than the delayed treatment. The optimum
`response was a >100% increase in mean survival
`time (45 days for the controls and 92 days for the
`vinblastine-treated group).
`We currently have seven drugs on test in the renal
`cell carcinoma system in ir tumored mice. Bleomycin,
`adriamycin, and hydroxyurea were given on a daily
`schedule on Days 7-15. Vinblastine, cyclophospha(cid:173)
`mide, methyl-CCNU, and BCNU were administered q7d
`starting on Day 7 for five doses. All were tested at
`three dose levels. Each test group consisted of ten
`mice and a control group of 30 mice was used. Al(cid:173)
`though the drug trials are still in progress (Day 50,
`control MST = 40.0 days), the data in table 3 allow
`certain conclusions to be drawn.
`Bleomycin, hydroxyurea, and cyclophosphamide
`were ineffective in producing increases in survival
`times at the doses and regimens used in this experi(cid:173)
`ment. Cyclophosphami.de did appear to exert an ef(cid:173)
`fect on tumor growth during the period of drug
`treatment. Adriamycin showed some therapeutic
`effects at all dose levels that were tested. At the
`highest dose tested, 1.0 mg/kg/day, all ten mice were
`still alive at Day 50 and nine appeared to be bearing
`somewhat smaller tumors by palpation.
`Vinblastine was not as effective in our drug trial
`as it was in the report of Hrushesky and Murphy
`(10) although the same dosages and treatment sched(cid:173)
`ules were used. The two lower doses (0.2 and 0.4 mg/
`kg) did not increase the MST appreciably (10% and
`7%). At the highest dose, 0.8 mg/kg, nine mice still
`survive but appear to be bearing large tumors. In
`the study of Hrushesky and Murphy (10), tumor
`weights were determined in animals killed at Day 40,
`5 days after the last drug dose. Our comments on
`tumor size refer to palpable masses some 15 days
`after the last drug treatment. Thus the difference in
`the effect on tumor weight may be an indication of
`escape of residual tumor from drug control but the
`differences in survival time found at lower doses
`cannot be similarly explained.
`As of Day 50, the two nitrosoureas, methyl-CCNU
`and BCNU, were the most effective drugs used in our
`
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`TAnu:3.-Interim results of first chemotherapeutic trial on Day 50
`of test*
`
`Drug and
`regimen
`
`Bleomycin,
`qd Days 7-15
`
`Adriamycin,
`qd Days 7-15
`
`llydroxyurea
`qd Days 7-15
`
`Vinblastine,
`q7d x 5
`
`Dose range
`(mg/kg)
`
`6.0-1.5
`
`1.0-0.25
`
`100-25
`
`0.8-0.2
`
`Cyclophosphamide,
`q7d x 5
`
`50-12.5
`
`Methyl-CCNU,
`q7d x 5
`
`BCNU,
`q7d x 5
`
`24-6
`
`24-6
`
`Comments
`
`All doses inactive, T/C <125%; tumor
`growth generally parallel to that of
`controls
`
`All doses, T/C;,,125%; tumors generally
`smaller than in controls, especially
`at highest dose; 10 survivors
`at highest dose
`
`All doses ineffective; tumor growth
`similar to controls
`
`Highest dose, T/C > 125%; 9 mice
`surviving; tumors generally smaller
`than in controls until treatment
`ceased
`
`All doses ineffective; tumors smaller
`than in controls during period of
`drug treatment
`
`All doses elf ective; 16 of 26
`survivors have no palpable tumors;
`animals appear generally healthy
`
`Highest dose toxic; 19/20 mice
`surviving at lower doses; 16/19 have no
`palpable tumors; all appear very
`healthy
`
`*MST for controls (30 mice) was 40.0 days.
`
`study. Toxicity was evidenced at the highest dose
`used with each compound, 24 mg/kg/injection. At the
`two lower doses of methyl-CCNU and BCNU, 39 of 40
`mice were still alive at Day 50. In addition, 26 of
`these mice had no palpable tumor. The BCNU mice
`appear to be in good health as do those treated with
`methyl-CCNU at 12 mg/kg/injection. Whether the
`response to these two nitrosoureas will prove to be
`greater than that found by Hrush2sky and Murphy
`with CCNU remains to be determined.
`
`CONCLUSIONS
`
`an ideal situation for surgical adjuvant studies. In
`addition, the 45-day average MST is workable, al(cid:173)
`lowing for the initiation of therapy at various dis(cid:173)
`ease stages. Whether the spectrum of drug activity
`in this model system will result in information that
`can be used in the development or selection of drugs
`for treatment of human clinical disease cannot be
`foretold from the limited drug trials that have been
`conducted to date.
`
`LIST OF COMPOUNDS
`
`The renal cell carcinoma animal model developed
`and characterized in 13ALB/c mice by :\1urphy and
`Hrushesky (!l) may prove to be a useful system for
`the selection of chemotherapeutic agents \;·ith activ(cid:173)
`ity against a slow-growing solid tumor of renal cell
`origin. The tumor can be transplanted by a variety
`of routes and drug effects can be measured on tumor
`size, extent of metastases, and survival time. Their
`system has the advantages of a slow-isrowing solid
`tumor of specific renal origin, a system which mi mies
`the human disease state including predictable me(cid:173)
`tastases, and as a model using a bilateral organ, it is
`
`Adriamycin: NSC-12:ll27; CAS reg. No. 2:l21-l-!12-i<
`BCNU: NSC .. 1()\l9fi2; CAS reg. No. 1G4-!l:l-8; urea, 1,:l-bis(2-chloro(cid:173)
`ethyl)-l-nitro'so-
`Bkomydn: J\:SC-12G0Gfi; [2,l'.l,ithiazo!P] .. 1-<·:uboxylic aei,l, 2"-(2·
`arninoethyl)-, rnonohydrate.
`CCNU: NSC-790:37; CAS reg. No. 13010-47 .. J; urea, 1-(2-chloro(cid:173)
`ethyl )-3-cyc lolwxy 1-1-ni troso-
`Cydophos phamidt>: NSC-2iJ271; CAS r<'g'. No. f\():,:,.\D-2; 2I/-l,:\,2-
`'Jxazapho~ phori nt·, 2·[ lli:.;(2•(' hl<lrot•thy l)an1i r10 ]tPtr:t }1y(ir()-, ~-<>X·
`ide, mnnohydra t.P
`llydroxyurea: NSC-:l201i:i; CAS rPg. No. 127-07-1
`~t,,thy].(TNIT: NSC-!Jii-l-l 1; CAS n•g, No, :i:w,:i.r,n.,,; llrPa, 1-(2-
`chloroPl hy I )-:l-( 4-rnc>t hy lcyc I ohi,xy I )-1- nit roso-
`Vinhlastine: NSC-19812; CAS reg. No. (J.149-03-2; vincal,•uko(cid:173)
`blastine, sulfate (I: 1 ), monohydrak
`
`148
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`2. KAUFMAN JJ, and MoINs MM. Tumors of the Kidney: Current
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`3. MunrHY GP, Moon,: RH, and KENNEY GM. Current results from
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`4. Ev1msoN TC, ·and CoLE WU. Spontaneous regression of adenocar(cid:173)
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`6. TALLEY RW, MoomtEAll EL, JI, TucKER WG, ~'T AL. Treatment of
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`ment of metastatic renal cancer. Br J Cancer 25:250-265, 1971.
`8. Aw,:RTO P, and SENN HJ. Hormonal therapy of renal carcinoma
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`9. MunPHY GP, and H1msm:sKY WJ. A murine renal cell carcinoma.
`J Natl Cancer Inst 50:1013-1025, 1973.
`10. Hnus11KSKY WJ, and Mm<PIIY GP. Evaluation of chemotherapeu(cid:173)
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