Quantitative Comparison between the Transplantability of Human and MurineTumors into the Subcutaneous Tissue of NCr/Sed-/w//iM Nude and Severe CombinedImmunodeficient Mice1
Alphonse Taghian,2 Wilfried Budach, Anthony Zietman, Jill Freeman, Danielle Gioioso, Wlodzimierz Ruka, andHerman D. Suit3
Edwin L. Steel? Laboratory' of Radiation Biology. Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
¡A. T., W. B., A. Z., J. F., D. G., W. R.t H. D. S./, and Department of Radiation Oncology, Boston University Medical Center, Boston University School of Medicine, Boston,Massachusetts 02118-2393 /A. T.. A. Z./
ABSTRACT
In previous reports, nude mice have demonstrated residual immunore-
activity against xenografts. Severe combined immunodeficient (SCID)mice lack functional T- and B-cells. These animals are expected to be
better hosts in which to perform preclinical studies on human tumors. Thepurpose of this study is to quantitate the advantage of SCID mice overnude mice in terms of transplantability of human and murine tumors andthe importance of residual immunity in SCID mice.
The transplantation assays are described by an assay based on thenumber of tumor cells required to transplant tumor into 50% of recipientst II >,i,i. Seven human tumors of different histology and four murine tumorcell lines were used. Serial 2—10-fold dilutions of cells were injected (0.1ml) into the flanks of normal and whole-body irradiated WBI nude and
SCID mice.The results showed that in 6 of 6 human tumor cell lines studied, TD50s
for SCID mice were 2.4 to 200 times lower than that of nude mice (significant in 5 cell lines). In contrast, in 2 of 3 murine tumors, TD50s in WBISCID mice were significantly higher than that found in nude mice. WhenSCID and nude mice received WBI, I l)5|,s were lower than those ofnonirradiated animals in 5 of 5 xenografts (significant in 2 cell lines fornude mice and in 5 cell lines for SCID mice).
We concluded that WBI SCID mice are significantly better recipients ofhuman tumor xenografts than nude mice. There isa factor of 10-1625 gain
in TD50s in favor of the WBI SCID mice when compared to nonirradiatednude mice. WBI has, however, an important effect on SCID mice whichmay suggest a detectable residual immunoreactivity, perhaps due to natural killer cells. These data demonstrate that WBI SCID mice are bettermodels for human tumor transplantation than nude mice and, althoughWBI at 6 Gy suppressed significantly the immune system of nude mice, acertain level of immunoreactivity against xenografts is still maintained.
Several reports demonstrated a residual immunoreactivity by thenude mouse against xenografts (2, 3). This may have been due to thefact that nude mice have some residual T-cells, and the number ofB-cells, NK4 cells, and macrophages are similar to those of the eu-
Received 4/7/93; accepted S/10/93.The costs of publication of this article were defrayed in pan by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by Grant DHHS CAI3311 awarded by the National
Cancer Institute, Department of Health and Human Services; the Association pour laRecherche Contre le Cancer (ARC) in France; and the Phillip Foundation.
2 To whom requests for reprints should be addressed.' Andres Soriano Professor of Radiation Oncology, Harvard Medical School.
thymic mice of the same strain (4, 5). Thus, a mouse more geneticallyimmunodeficient may serve as an improved host for human tumorxenografts.
The beige-nude-XID mouse came from the same background stock
as the nude mouse. In addition, it carries the beige (bg/bg) and XIDgenes. These confer a relative NK cell inactivity (6) and B-cell im
maturity (7). However, quantitative cell transplantation assays performed have not revealed any subtle transplantation advantage infavor of this strain of mouse as compared with the conventional nudemouse (8).
The SCID mouse was first described by Bosma et al. (9). Thismouse is more severely immunocompromised than the nude mouse,lacking both functional B- and T-cells because of nonfunctional rearrangements of immunoglobulin and T-cell receptor genes (10). How
ever, NK cells, macrophages, and other hematopoietic cell lineages donot appear to be affected by the autosomal recessive SCID mutation(11). Several reports have considered the potential that SCID micewould be better recipients of xenografts than nude mice (12). Allprevious studies have involved qualitative comparisons of transplant-
ability using a limited number of human tumors.This report is an account of a quantitative investigation of the
relative and absolute transplantability of seven human and four murinetumor allografts or isografts into NCr/Sed-ww/nw and SCID mice. The
TD50 assay (13) was used to assess (a) whether SCID mice are lessimmunoreactive against human tumor xenografts than nude mice and(b) the importance of residual immunity in SCID mice.
MATERIALS AND METHODS
Experimental Animals
The NCr/Sed-nu/H« mouse strain was derived from a Swiss mouse which
had the BALB/c nude gene inserted by a backcross breeding technique. Thehistory and designation of this line of mice has been described ( 14). The initialbreeding stock of NCr-± and NCr-nu//iu mice was provided generously by Dr.
Carl Hansen of the National Cancer Institute and has been maintained in ourcolony for more than 25 generations. For the experiments on WBI mice, 6 Gyof WBI were given 24 h prior to inoculation. WBI was performed in aGammacell li7Cs unit at a dose rate of 0.81 over the duration of the experi
ments.SCID mice were generously provided by Dr. Gloria Ku at Merck Sharp &
Dohme in 1989. The immunoglobulin production was found to be negative.SCID mice received a 2-Gy dose of WBI (<5% of lethal dose) (15) 24 h before
transplantation. The IgG production was measured and found to be nonsignificant.
The mice used in this study were maintained in an ammonia-free environment in our defined and pathogen-free colony (14). They were maintained inmicroisolators and fed high-calorie sterile laboratory pellets and acidified
water ad libitum. Their care was in accordance with institutional guidelines.The mice were placed into experiments when 7-9 weeks old.
4 The abbreviations used are: NK, natural killer; SCID, severe combined immunodeficient; WBI. whole-body irradiation (irradiated); TDs,(, average number of cells required
The transplantation assays are described by the TD50, which is consideredto be a powerful, quantitative technique in the assessment of the tumor/hostimmunological relationship (13).
Tumor cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotics(0.05 mg penicillin/ml. 0.05 mg streptomycin/ml, and 0.1 mg neomycin sul-fate/ml) at 37°Cin an atmosphere of 5% CO2 in air. The cells were obtained
from culture, harvested with 0.05% trypsin plus EDTA, and suspended inDulbecco's medium. Serial 2-10-fold dilutions of the tumor cell suspensions
were prepared, and 0.1 ml was inoculated s.c. into the flanks of the animals(1-2 injections/mouse). There were 5 mice/cell dose with 5-10 injections/doselevel and 6-11 cell doses in each assay. The assays for both strains of mice,WBI and non-WBI animals, were performed concurrently using the same cell
suspension.
Tumor Cells
Seven human tumor xenografts and four murine tumor allografts or isograftswere studied. Table 1 illustrates the histológica! type and origin of each tumorcell line.
Human Tumor Cell Lines. Two glioblastoma multiforme cell lines. HGL9and D54MG. were used. HGL9 is an early-passage established cell line derived
from a patient treated at the Massachusssetts General Hospital. D54MG is awell-established cell line kindly provided by Dr. Bigner at Duke University.The cell lines SKNMC, HCT15, and MDA-MB-231 are neuroblastoma, coloncancer, and breast cancer cell lines, respectively. All of them are well-estab
lished cell lines and were provided by the American Type Culture Collection.The cell line STS-26T was recently established from a soft tissue sarcoma from
a Massachusetts General Hospital patient; this cell line was kindly provided by
W. Dahlberg of the Harvard School of Public Health. The cell line STS-176 is
a well-established soft tissue sarcoma, kindly provided by Dr. Fletcher at theBrigham and Women's Hospital.
Murine Tumor Cell Lines. FSC1 and FSC2 are methylcholanthrene (0.1mg dissolved in 0.1 ml of peanut oil injected into the right gastrocnemius)-induced fibrosarcomas of SCID mice. The FSM1 tumor is a methylcholan-threne-induced tumor from a C3H mouse. All tumors were poorly differenti
ated spindle cell sarcomas. The first generation tumors were excised, minced,established in vitro, and maintained in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiot
ics. Early passages were used for the experiments. The in vivo and in vitroradiobiological characteristics, as well as the details of the origins of thesetumors, are described elsewhere (15). EO1-MGH was originally a human
oligodendroglioma provided generously by Dr. Stuschke from Essen and haschanged its biological behavior during the tumor passages in vivo at Massachusetts General Hospital; it showed recently a karyotype of murine origin.The experiments were performed on the cell line with murine karyotype.
Experimental Design and Analysis
Concurrent TD50 assays were performed in nonirradiated and WBI SCIDand nude mice. For the FSM2 experiment, a TD51>assay was also performed onC3H mice (WBI versus nonirradiatcd mice) in parallel with the assays on nudeand SCID mice. In the experiments with FSC-1 and FSM-2 tumors, the WBI
SCID mice received a dose of 2.5 Gy.After transplantation, the animals were scored 2-3 times/week. The tumor
take was scored when the transplanted tumor reached 8 mm in diameter. Theanimals were followed up for at least 160 days or when the TD5I, had reacheda plateau which was sustained for at least 90 days. The fraction of tumor takeswas plotted versus cell dose, and the TD5us and their 95% confidence intervalswere determined from the logit regression line fitted through the data sets (16).
RESULTS
TD50 for Human Tumor Cell Lines
Table 1 lists the tumor lines with histology and origin. The softtissue sarcoma STS-176 was used only in the experiments measuring
the residual immune reaction in SCID mice (the TD5() in WBI versusnon-WBI SCID mice). Table 2 presents the TD5(,s for the 7 human cell
lines.WBI Nude versus WBI SCID Mice. In 4 of 4 cell lines studied,
TD5lls for nude mice were higher than for SCID mice by a factor of2.4 to 1000. In 3 cell lines, the difference in TD5()s were significant(Tables 2 and 4; Figs. 1 and 2). The highest ratio (1000) was for thebreast cancer cell line (MDA-MB-231), namely, 3 logs. However, for
Table 2 Absolute TD<;osof human tumor xenografts into WBI and rwtt-WBI nude NCrlSed and SCID mice
s.c. TUMOR TRANSPLANTABILITY INTO NUDE AND SCIO MICE
the other cell lines, the ratios were smaller and, in STS-26T, not
significant (Tables 2 and 4).Nonirradiated Nude versus SCID Mice. For 6 of 6 cell lines
studied, TD5l)s in nonirradiated nude mice were higher than those ofnonirradiated SCID mice (P < 0.05 for 5 cell lines). The non-WBInude mouse:non-WBI SCID mouse TD5I>ratio varied between 2.4 and
200 for the 6 cell lines (Table 4). Figs. 1 and 2 show TD50s plotted asa function of time for four cell lines.
WBI Nude versus Nonirradiated SCID Mice. The relative xeno-
transplantability into nude mice and SCID mice was further assessedby determining the WBI nude mouse:non-WBI SCID mouse TD5U
ratio (Table 4). In four of five cell lines, TD5l)s for WBI nude micereceiving 6 Gy were higher than those of the nonirradiated SCIDmice. In 2 cell lines, the difference was significant (Table 4). ForSTS-26T, the TD,0 in WBI nude mice was 1.8 times lower than that
in nonirradiated SCID mice (not significant).Nonirradiated Nude versus WBI SCID Mice. To determine the
TD5(I maximum range, the TD5(I ratio of nonirradiated nude mice: WBISCID mice was computed. For the four xenografts studied, TD5(,s inWBI SCID mice were 10-1625 times lower than those of nonirradi
ated nude mice (Table 4).
TD50 for Murine Tumor Cell Lines
Four murine tumor cell lines were studied. EO1-MGH and FSC-2
were used in the nonirradiated and irradiated nude and SCID mice.FSC-1 was used in SCID mice experiments only (WBI versus non-WBI mice), and FSM-2 was used in nude, SCID, and C3H mice.
WBI Nude versus WBI SCID Mice. For EO1-MGH in 2 experi
ments (Table 3, experiments 3 and 4), TDM)s for SCID mice were
io7i (a) SKNMC
10•¿�
10s
igW^. _
nudes/WBI
nudes/non WBI
SCID/WBI
SCID/non WBI
40 80 120 160 200 240 280 320
Time (days)
105.
in 104,
10
10'
3,
(b) HCT15
nudes/WBI
nudes/non WBI
SCID/WBI
SCID/non WBI
0 40 80 120 160 200 240 280
Time (days)Fig. 1. TD5(ks plotted as a function of time for (u) SKNMC (ncuroblastoma) and (ft)
HCT15 (colon cancer) into WBI and non-WBI NCr/Sed-nu/nu and SCID mice.
•¿�».,,,-.
io
10J
(a) MDA-MB-231
nude/WBI
nudesinon WBI
SCID/WBI
SCID/non WBI
0 40 80 120 160 200 240 280 320
Time(days)
10°l
10=
104.
(b) STS-26T
nudes/WBI
nudes/non WBI
SCID/WBI
SCID/non WBI
40 80 120 160 200 240
Time (days)Fig. 2. TDsciS plotted as a function of time for (a) MDA-MB-231 (breast cancer) and
(ft) STS-26T (soft tissue sarcoma) into WBI and non-WBI NCr/Sed-nu/nu nude and SCID
mice.
slightly lower than those of nude mice (P > 0.05) with ratios of 1.6and 1.3 for experiments 3 and 4, respectively (Table 4). For FSM-2and FSC-2, TD5,,s for WBI SCID mice were 3 and 9 times higher than
those of WBI nude mice (P < 0.05), respectively.Nonirradiated Nude versus SCID Mice. For all cell lines, TD5(,s
in nude mice were 1.7 to 8.6 times higher than those in SCID mice.The difference in the TD5() was significant for FSM-2 and in two ofthree experiments for EO1-MGH (experiments 1 and 4). However, forFSC-2, the difference was not significant (Table 4).
WBI Nude versus Nonirradiated SCID Mice. In contrast to thehuman tumor xenografts, the three murine cell lines showed a significant better take rate (lower TD5()) for WBI nude mice than nonirradiated SCID mice (Table 4).
Nonirradiated Nude versus WBI SCID Mice. In the three celllines studied, TD5()s of nonirradiated nude mice were 2.5 to 12.5 timeshigher than those of WBI SCID mice; this difference was significantin two of the cell lines (Table 4).
Impact of WBI on TD50 in NCr/Sed-nu/n« Nude and SCID
Mice
The impact of WBI of SCID and nude mice on TD5,,s may reflectthe immunogenicity of the tumor lines (3). The TD5(, assays wereperformed using control and WBI recipients. Table 5 shows the ratiobetween the TD5() in WBI and non-WBI nude and SCID mice.
NCr/Sed-nu/nu Mice. Four of five human tumor xenograftsshowed a lower TD50 in mice receiving 6 Gy of WBI than in nonirradiated nude mice; one of them was significant (STS-26T; Table 5).
For the 3 murine tumors, TD5(ls in WBI nude mice were 5.6 to 33times lower than in non-WBI nude mice (P < 0.05). The ratios (TD5()in non-WBI nude mice:TDS(, in WBI nude mice) for the human tumor
" TO,,,: C3H mice, 6 Gy WBI, 0.04 X IO2 (0.01-1.17); non-WBI C3H mice, 17.10 X IO2 (7.90-37.3).h Numbers in parentheses, range.
xenografts (range, l^t.88) were lower than those of the murine tumors(range, 5.6-33) (Table 5).
SCID Mice. For the five human tumor xenografts, TD50s of non-irradiated SCID mice were 3.2-8.1 times higher than those of WBI
SCID mice (P < 0.05) (Table 5). For the 4 murine tumor cell lines,TD5()s for non-WBI SCID mice were higher than those in WBI SCID
mice (significant in three cell lines) (Tables 3 and 5). The lower TD50sin WBI SCID mice demonstrate a detectable residual immunoreactiv-
ity remaining in SCID mice.We should note that for two xenografts (MDA-MB-231 and HCT-
15), no difference in TD50s was found between non-WBI mice and
WBI nude mice; however, both of them showed a significant difference in TDS()s between non-WBI mice and WBI SCID mice. Theopposite trend was found for the murine tumors FSC-2 and EO1-
MGH, experiment 4.
DISCUSSION
In vivo models are valuable in preclinical studies to investigate theefficacy of antineoplastic therapies. An ideal model would display noimmunoreactivity against human tumor xenografts. The congenitallyathymic nude mouse has been frequently used for human tumor transplantation; however, previous reports (2, 3) have demonstrated residual immunoreactivity versus xenografts. This fact has been confirmed in the present study since WBI at 6 Gy significantly reducedthe TD50s for xenotransplantability of four of eight tumors.
SCID mice are more severely immunodeficient than nude mice andthereby less capable of resistance against foreign tissue grafts. They
may, therefore, be better hosts in which to perform preclinical studieson human tumor xenografts. Although SCID mice lack functional B-and T-cells, the NK activity of adult C.B-17 SCID mice is no lowerthan that of age-matched NMRI nude mice and may even be higher
(17). This potential problem notwithstanding, several authors havementioned the superiority of SCID mice over nude mice in terms ofcrude xenograft take rate (18, 19). Our study quantitates the greaterreceptivity of SCID mice over NCr-nu/nu nude mice to tumor xeno-
transplantation by use of the TD50 assay. Furthermore, the transplanttake was enhanced by supplemented immunosuppression by WBI.
For all human tumors studied, TD50s in SCID mice were lower thanthose obtained for nude mice. In all tumors but two (HGL9 andFSC-2), this difference in TD5,) was significant. This strongly indi
cates that SCID mice are less immunoreactive to xenografts than areNCr/Sed nude mice. Whether normal or WBI, SCID mice had lowerTD5()s than nude mice. The magnitude of the difference in the TD5()sbetween SCID and nude mice was cell line dependent, reflecting avariability between cell lines in immunogenicity. It varied between 2.4times (HGL9 and STS-26T) and 1000 times (MDA-MB-231) (cf.
Table 4).Several factors, other than the suppression of the immune system,
can contribute to the alteration of the TDSO by WBI of the animals.Peters (20), using laparotomy, pointed out the possibility that physiological changes resulting from an acute stress may significantlylower the TD50 of a murine cell line. Zietman (21) did not, however,notice any reduction in the TDM) when using the human pharyngealtumor, FaDu (nude mice were splenectomized 24 h before the TD^,,injections); but a significant reduction was noticed after 6 Gy of WBI.
Table 4 Relative transplantability of human and murine tumors into WBI and non-WBI NCr/Sed nude and SCID mice
The addition of splenectomy to WBI did not offer any additionaltransplantation advantage over WBI alone. This suggests that, for thissystem at least, WBI is not equivalent to surgical stress. However, theTD5Mof the human tumor xenografts in WBI nude mice did not reachthe level of that of SCID mice. This suggests that, although the T-, B-,
and NK cells decreased significantly after 6 Gy of WBI (22), a certainlevel of immunoreactivity against xenografts is still maintained innude mice.
The influence of WBI on the TD50 may, therefore, reflect the impactof several parameters: suppression of the immune system; a possiblestress factor; and a host-tumor bed effect which may be different
between SCID and nude mice. Another probable difference betweenboth strains may be a difference in a possible deficiency in antigenrecognition to murine or human tumor xenografts.
A number of specific observations deserve close examination.First, WBI nude mice are less immunoreactive to allotransplantationof murine tumors than unirradiated SCID mice; the WBI nudemouse:non-WBI SCID mouse TD50 ratio varied from 0.07 to 0.7
(Table 4). It is known that significant suppression of the nude mouseimmune system results from 6 Gy of WBI (22). Untreated SCID micestill retain normal NK activity (17). In distinction from human tumorxenografts, unirradiated SCID mice were 1-123 times less immunoreactive to xenografts than WBI nude mice (STS-26T ratio, 0.56)
(Table 4). The reasons for a difference that depends upon the speciesof graft origin are unclear. It is probable that human tumors are moreimmunogenic than murine allografts and that residual immunity after6 Gy of WBI still exceeds the immunoreactivity present in the untreated SCID mice to a detectable degree. For less immunogenicmurine tumors, nonspecific depression of the TD5() through stressfactors may be more apparent.
A second observation is that the TD50s of WBI SCID mice weresignificantly lower than those of the unirradiated mice in 5 of 5xenografts and in 3 of 4 murine cell lines (Table 5) (except for thesecond experiment of SKNMC, the difference was not significant).This demonstrates residual immunoreactivity in SCID mice, probablyrelated to the NK activity which is over 50% higher than in nude mice(17). This contrasts with the earlier findings of Zietman et al. (8)comparing the quantitative transplantability of 2 cell lines in otherimmunodeficient strains of mice, NCr/Sed-/iw//iw and nude-beige-XID
(N:NIH-nu-bg-XID/Sed). The authors found no evidence to suggest
that, in nude mice, NK or B cells were major determinants of tumortransplantability.
A third observation is that of the magnitude of difference in theTD5o ratio of the unirradiated and the irradiated SCID mice versus thatof the nude mice. This ratio in SCID varied between >3.2 and 8 andwas significant in 5 of 5 xenografts. However, in nude mice, this ratiovaried between 0.97 and 4.88 and was significant in 1 of 5 (Table 5).Related to this, the TD50 of the xenografts HCT15 and MDA-MB-231
did not show any significant differences between nonirradiated andWBI nude mice; however, they showed a significant difference between nonirradiated and WBI SCID mice (5.63 and 8.13, respectively). On the other hand, the opposite results were found for 2 of 3murine tumor cell lines (EO1-MGH experiment 4 and FSC-2; the
ratios for nude mice were 10.0 and 23.0 and for SCID mice, 1.70 and1.48, respectively). This discrepancy in the results between SCIDmice and nude mice vis-Ã -vis the murine and the human tumor xeno
grafts may reflect a difference between the immune systems of thesetwo immunodeficient strains in the recognition of human tumor xenografts versus murine tumor cell lines. This will mean that somexenografts will be considered immunogenic in SCID mice and notimmunogenic in nude mice, with the opposite situation true for somemurine tumors.
The difference in TD5,>between nonirradiated and WBI SCID mice(range, >3.2-8.0) was larger than that for nude mice (0.98 to 4.88).
This could be explained by WBI suppressing the immune system ofthe SCID mouse more than that of the nude mouse. It is of note thatfor the methylcholanthrene-induced fibrosarcoma FSM-2 (15), WBI
decreased the TD5() 4.33 times in SCID mice, 33 times in nude mice,and 170 times in C3H mice (Table 5; Fig. 3). This could reflectdifferent degrees of recognition of the immune systems of these threedifferent strains of mice to the cell lines; differences in the degree ofsuppression of the immune system by WBI; or differences in stressreactions.
In conclusion, SCID mice are significantly better recipients ofhuman tumor xenografts than nude mice. There is a factor of 10-1625
gain in TDS() in favor of WBI SCID mice when compared withnon-WBI nude mice. The SCID mice are not, however, completely
immunodeficient. Residual immunoreactivity is detectable. These datademonstrate that the WBI SCID mice are better models for humantumor transplantation than nude mice and, although 6 Gy of WBIsuppressed significantly the immune system of nude mice (22), acertain level of immunoreactivity against xenografts is still maintained.
FSM-2io3
10"
o »>-
a ,H io
IO1
10"
C3H/WBI
C3H/non WBI
SCID/WBI
SCID/non WBI
nudes/WBI
nudes/non WBI
0 40 80 120 160 200 240
Time (days)Fig. 3. TD.soSplotted as a function of time for FsM-2 (mcthylcholanthrenc-induced
fibrosarcoma into a C3H mouse) into WBI and non-WBI NCr/Sed (nulnu) nude, SCID.and C3H mice.
s.c. TUMOR TRANSPLANT ABILITY INTO NUDE AND SCID MICE
ACKNOWLEDGMENTS
The authors thank Dr. K. Lee, R. Sedlacek, E. Rose, and T. Ebert for theirvaluable collaboration and suggestions. They also thank S. Wendt, S. Marshall,P. Morris, and H. Pham for animal caretaking; and P. McNally for her excellentassistance in the preparation of the manuscript.
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