(CANCER RESEARCH 51, 2773-2779. June 1. 1991]
Ultraviolet Radiation-induced Murine Tumors Produced in the Absence of
Ultraviolet Radiation-induced Systemic Tumor Immunosuppression1
Scott W. Menzies,2 Gavin E. Greenoak, Vivienne E. Reeve, and Clifford H. Gallagher
Department of Veterinary Pathology, University of 'Sydney, Sydney 2006 fS. W. M., G. E. G., V. E. R., C. H. G.J, and Lidcombe Hospital Dermatology Centre, Lidcombe,
New South Wales 2141 [S. W. M.], Australia
ABSTRACT
Using micro-UV-irradiation versus whole-dorsal irradiation for induc
ing cutaneous carcinomas in Skh:HRI mice and an assay for UV radiation
(UVR)-induced systemic tumor immunosuppression, the dependence
upon systemic immunosuppression for the growth of UVR-induced car
cinomas was examined. Squamous cell carcinomas were produced by
repeated microirradiation of 0.8-cm2 middorsal skin with xenon arc solarsimulated UVR. These tumors were excised from tumor-bearing animals
who 7 days later were inoculated ventrally with a cloned UVR-induced
squamous cell carcinoma cell line, the T51/6. This cell line only grows in
UVR-induced immunosuppressed Skh:HRI mice. In two separate exper
iments T51/6 inocula failed to grow significantly in the previously tumorbearing animals (1 of 13) and in unirradiated mice (0 of 19), whereas it
grew in 100% (15 of 15) of animals given a whole-dorsal subcarcinogenic
UVR dose from a filtered fluorescent tube solar simulator. No sinecomitant immune response to the T51/6 was found in previously UVR-induced
tumor-bearing animals. In contrast to whole-dorsal UVR-induced tumors,
microirradiation-induced squamous cell carcinomas, whose original
growth environment lacked UVR-induced systemic tumor immuno
suppression, did not grow preferentially in mice given an immunosuppressive dose of UVR. However both the whole-dorsal and microirradia
tion-induced tumors were shown to be poorly antigenic, since they lacked
preferential growth in athymic nude mice. These observations provide
evidence that UVR-induced systemic tumor immunosuppression is not
necessary for the production of UVR-induced tumors. However, it does
cause a positive selection pressure during tumor formation, independent
of the carcinogenic effect of UVR, which affects the transplantation
biology of a tumor.
INTRODUCTION
Initial observations that UVR3-induced skin tumors are
highly antigenic (1,2) and are immunologically rejected when
transplanted into syngeneic normal animals, has stimulated
considerable experimentation to determine how such highly
antigenic tumors can exist in an animal. It has been demon
strated that UVR itself induces an immunosuppressed state
with two major factors being proposed to explain this immu
nosuppression, namely, local UVR-induced immunosuppres
sion and systemic immunosuppression.
Local UVR-induced
immunosuppression is supported by observations of suppressed
CHS initiation when the contact sensitizer is applied directly
onto UV-irradiated skin; this activates antigen specific suppres
sor T-lymphocytes that inhibit the development of a CHS
response upon antigen challenge (3).
In contrast, systemic UVR-induced immunosuppression has
been demonstrated by the growth of antigenic UVR-induced
tumor inocula at a site distal to that receiving UVR. Suppressed
induction of CHS following application of contact sensitizer
Received 9/7/89; accepted 3/13/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This research was supported by the University of Sydney Cancer Research
Fund.
2To whom requests for reprints should be addressed.
1 The abbreviations used are: UVR, UV radiation; CHS, contact hypersensitiv ity; UVT, UV-induced suppressor T-lymphocytes; SCO, squamous cell carcinoma.
also to a site distal to UVR exposure is another example of this
phenomenon, both of which can be transferred to a naive animal
with lymphoid cells from chronically UV-irradiated animals
(reviewed in Ref. 3). UVR-induced systemic immunosuppres
sion has therefore been explained by the formation of a UVRinduced cross-reactive UVT that occurs prior to sensitization
with a sensitizer (4) or tumor (5-8), although other mechanisms
for CHS suppression have been proposed (9). Recent observa
tions, however, have shown that the UVR dose responses for
local and systemic immunosuppression are identical, indicating
that both have a common mechanism of action (10).
UVR-induced systemic immunosuppression has been dem
onstrated to enhance UVR-induced tumor appearance. Preirradiating mice with chronic UVR while shielding areas of skin
has been shown to enhance formation of tumors in those
shielded areas following UVR (11, 12). Fisher and Kripke (13)
grafted UV-irradiated skin onto X-irradiated mice that had
been reconstituted with syngeneic normal lymphoid cells or
lymphoid cells from chronic UV-irradiated animals. A greater
number of tumors grew on hosts reconstituted with the latter
UVR lymphoid cells. Furthermore, injection of UVR lymphoid
cells at the beginning of a chronic UVR regimen significantly
reduced the latency period of primary tumor appearance as well
as increasing tumor incidence. These results led these authors
to theorize that "the presence or absence of (UVR-induced)
suppressor lymphocytes determines whether or not primary
cancers will develop in UV-irradiated skin" (13), thus consti
tuting strong immunological surveillance in UVR carcinogenesis involving UVR-induced systemic immunosuppression.
The following series of experiments, by examining the im
portant corollary of the above theory, that one cannot produce
UVR-induced tumors without producing UVR-induced sys
temic tumor immunosuppression, provides evidence that sys
temic UVR-induced immunosuppression is indeed not a nec
essary event in the production of UVR skin cancer.
MATERIALS
AND METHODS
Animals
Unless specified all animals used were inbred female hairless (hr/hr)
Skh:HRI mice. The mice were housed in wire-topped plastic cages on
vermiculite bedding and were maintained at 22-25°Cwith 12-h light
(GEC F40GO gold light that does not emit any UVR) followed by 12h dark. They were fed standard laboratory mouse pellets (Doust and
Rabbidge Pty. Ltd., Sydney, Australia, and Clarkel Holdings Pty. Ltd.)
and water ad libitum. BALB/c and Swiss-ARC nude (nu/nu) mice were
purchased from the Animal Resource Centre (Perth, Australia), and
kept in specific pathogen-free conditions.
UVR Regimens
Regimen 1. Subcarcinogenic Whole-Dorsal UVR. Light from seven
120-cm fluorescent tubes comprising 6 UVA tubes (Sylvania F40BL)
flanking one UVB tube (Oliphant FL40 SE) banked under an aluminum
reflector was filtered through a screen of cellulose acetate (Kodacel TA407 clear 0.0150 inch; Eastman-Kodak Co., Sydney, Australia), which
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UV RADIATION CARCINOGENESIS
Table I Carcinogenic microirradiarion protocol
UVI protocol using the Model 12S solar simulator (see "Materials and
Methods") with an irradiance of 107 mW/cm2 UV (290-400 nm) of which 8.9%
wasUVB(290-315nm).
Daily UVR dose"
(J/cm2)
Wk
2.7 (2 days)
2.4 (2 days)
2.7
2
3.0
3
3.2
4
3.6
5
6-8
4.1
4.3
9
4.5
10
11-13
4.9
" Daily dose of UVR (290-400 nm) given 5 days/week (unless stipulated) on
the same area of 0.8-cm! middorsally.
1
was used as a solar-simulating cutoff filter, allowing no transmission
below 290 nm. This filter is replaced every 220 min of irradiation due
to solarization. The incident integrated irradiances for this source,
calculated as previously reported (14), were 4.43-mW/cm2 UV total
(290-400 nm), of which 8.6% was UVB (290-315 nm). Mice were
exposed dorsally 4 days/week with an initial minimal erythema! total
UV dose of 2.7 J/cm2, which was increased by 20% increments (0.53
J/cm2) each week to maintain an erythema while avoiding blistering or
ulcération.This continued for 7 weeks, giving a cumulative total dose
of 120 J/cm2 UV.
Regimen 2. Carcinogenic Whole-Dorsal UVR. The subcarcinogenic
whole dorsal irradiation protocol was extended to give a total dose of
275 J/cm2.
Regimen 3. Carcinogenic Microirradiation (Xenon). A Model 12S
solar simulator (Solar Light Co., Philadelphia, PA) containing a 150W xenon lamp with a dichroic mirror, 1-mm UG11 and 1-mm WG320
filters (Schott Glass Tech. Inc.) to cut off all UVR below 290 nm, was
used. The incident integrated irradiance (using less than maximum
amperes) was 107-mW/cm2 UV total, of which 8.9% was UVB.
Mice were exposed to a 0.8-cm2 circular area at the focal point of
the source, which was applied middorsally, 5 days/week (except week
1), commencing with an initial daily total UV dose of 2.7 J/cm2. This
was increased as shown in Table 1 to maintain erythema while avoiding
ulcérationor blistering; mice were given a cumulative total dose of 252
J/cm2 UV over 13 weeks. In the repeat experiment (assessing tumor
antigenicity), the irradiation protocol was extended to give a total dose
of 275 J/cm2.
Regimen 4. Subcarcinogenic Whole-Dorsal UVR (Xenon). Using an
optical grade mirror (Newport 20D04 AL.2), to avoid any spectral
change, the Model 12S solar simulator beam was reflected at 45 degrees
to produce a large diffuse light source, with a total UV irradiance of
1.0 mW/cm2. Mice were given the same dosage regime seen with the
subcarcinogenic fluorescent source (Regimen 1).
T51/6 Tumor Cell Line
The T51/6 tumor was cloned from a cell line (T51) previously
established from a well-differentiated squamous cell carcinoma which
had been induced by UVR in a SkhrHRI mouse. The tumor clone was
expanded in culture in Eagle's minimum essential media (Common
wealth Serum Laboratories, Melbourne, Australia) supplemented with
10% (v/v) fetal calf serum (Commonwealth), 1 Mg/ml amphotericin B,
60 ¿ig/mlbenzylpenicillin, and 100 Mg/ml streptomycin in 75-cm2 tissue
culture flasks (Greiner) at 37°Cin a humidified atmosphere of 5% CO2
in air. The tumor cells grew as a monolayer and were detached from
the flasks by incubation with 2 mmol EDTA (Ajax Chemicals, Sydney,
Australia) in a calcium- and magnesium-free Dulbecco's phosphatebuffered saline. EDTA was used rather than trypsin to avoid cleavage
of surface peptides and thus putative tumor antigens (15). The cells
were washed in Eagle's minimum essential media without fetal calf
serum (to prevent a delayed type hypersensitivity response to heterologous proteins). Approximately 4 x IO6cells in 0.1 ml of this medium
AND IMMUNOSUPPRESSION
midline position at the level of the forelegs, thus avoiding regional
variation in tumor take (16). All mice within an experiment received
tumor cells from the same passage that had a viability greater than 80%
as determined by trypan blue exclusion. Mice were examined for tumor
growth (considered significant if >2 mm in diameter) at 3 months
following inoculation. The T51/6 line is Mycoplasma negative.
SCC Implantation
UVR-induced primary tumors were excised from mice following
exposure to a carcinogenic regime of UVR from either the fluorescent
or Model 12S solar simulator. Fragments (3.5 mm3) were submerged
briefly in 70% ethanol and washed in Dulbecco's phosphate-buffered
saline without calcium and magnesium and transplanted s.c. middor
sally into the host by using sterile 16-gauge cannulas as trocars. In
experiments determining tumor antigenicity, a fragment of the primary
tumor adjacent to that inoculated was fixed in 10% phosphate-buffered
formalin, embedded in paraffin, and stained with hematoxylin and
eosin.
Following implantation, tumor diameter was measured with vernier
calipers at the defined times. At the conclusion of each experiment, an
autopsy on the tumor-bearing animal was performed to ensure the
presence of a true neoplasm.
For excision of inoculated or primary tumors, mice were anesthetized
with either Ketamine (Delta Veterinary Laboratory, Sydney, Australia),
1.2 mg s.c./mouse and xylazine (Bayer Australia, Sydney, Australia),
0.24 mg s.c./mouse, or halothane/nitrous oxide inhalation, and the
wounds were closed with 5/0 nylon sutures. In the appropriate experi
ments, sham excision of control groups occurred on an equivalent area
of middorsal skin, using the same method of anaesthesia and closure.
Statistics
In tumor transplantation experiments (with a multiple source effect),
individual tumor diameters were analyzed for statistical significance.
Transformations were attempted to allow the assumptions of a twoway analysis of variance. If these assumptions were not met, the data
were analyzed by Wilcoxon's signed rank test (for paired treatments),
and Friedman's test corrected for ties (for more than two treatments),
using Minitab statistical software 7.1 (Minitab Inc., State College, PA).
T51/6 tumor take (incidence) between groups was analyzed with the
two-tailed Fisher exact probability test. In all analyses, a P value less
than 0.05 was regarded as significant.
RESULTS
Effect of Microirradiation with a Carcinogenic Dose of UVR
on UVR-induced Systemic Tumor Immunosuppression. The
cloned cell line T51/6 was used to assay for the presence of
UVR-induced systemic tumor immunosuppression. This cell
line grows when inoculated into a nonexposed ventral site of
mice irradiated with a subcarcinogenic whole-dorsal dose of
UVR, but not in age- and sex-matched unirradiated mice.
Hence, growth of this cell line is a very sensitive indicator of
UV-induced systemic tumor immunosuppression. Many previ
ously have shown this phenomenon to be due to the production
of a cross-reactive UVT (5-7, 17-19).
In previous work on investigating systemic immunosuppression, whole or near-whole-dorsal or ventral exposures have been
used. Using this method, UVR-induced systemic tumor im
munosuppression can be produced with subcarcinogenic doses
of UVR and thus is always present at tumor appearance. To
produce UVR-induced tumors by microirradiation, a 0.8-cm2
circular area middorsum was repeatedly irradiated over 13
weeks and mice were observed until carcinomas appeared. Of
the 26 mice irradiated, 7 carcinoma-bearing animals were ob
served. These carcinomas were excised and histologically con
firmed to be SCCs that were either arising de novo or were
were injected intradermally into a ventral unirradiated site of mice in a
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UV RADIATION CARC1NOGENESIS AND 1MMUNOSUPPRESSION
invading from papillomas. To establish the presence or absence
of UVR-induced systemic tumor immunosuppression, 7 days
following excision of microirradiation-induced
carcinomas,
mice were inoculated at an unexposed ventral site with the T51/
6 cell line. Age- and sex-matched mice irradiated with a subcarcinogenic whole-dorsal regimen of UVR known to produce
systemic tumor immunosuppression, as well as unirradiated
mice, were also given injections of T51/6 cells as controls. As
shown in Table 2, whereas T51/6 cells grew in subcarcinogenic
whole-dorsal UVR-treated mice, they did not grow in mice
receiving the microirradiation protocol or in the unirradiated
group. Thus no evidence for systemic immunosuppression was
observed in previously tumor-bearing microirradiated mice.
Sinecomitant Tumor Immunity to T51/6. An absolute require
ment regarding the validity of using the T51/6 tumor as an
assay for UVR-induced systemic tumor immunosuppression in
the previous experiment is that there is no immune response to
the T51/6 challenge due to the previously excised primary
tumor; that is, there is no sinecomitant tumor immune response
to the T51/6 cell line.
Historically sinecomitant tumor immunity is weak and tumor
antigen specific in that the immune response is specific to the
primary tumor only. This is in contrast to concomitant tumor
immunity (resistance of the tumor-bearing host to a second
tumor challenge) which is strong and cross-reacts with nonidentical tumors (reviewed in Ref. 20). Thus to avoid this crossreactive immune response to the T51/6, the clone inoculation
was performed following primary tumor excision. The results
in Table 3 confirm the historical evidence. Groups of mice
either irradiated with subcarcinogenic whole-dorsal UVR or
unirradiated mice were implanted with a UVR-induced primary
tumor. Twelve individual tumors were used, comprising all
grades of SCC, including well-differentiated, anaplastic, and
spindle cell tumors as categorized in this laboratory previously
(21, 22). Some mice were not given a tumor transplant. The
tumor implant was excised 5 weeks later, sham excision being
performed on mice who did not received a tumor implant.
These mice were then inoculated with T51/6 tumor cells and
examined for growth of the tumor line. In groups treated with
a subcarcinogenic systemically immunosuppressive UVR re
gime and in normal unirradiated animals, the previous existence
of a UVR-induced tumor had no significant effect on the
subsequent growth of the T51/6 clone, when compared with
sham-excised controls (Table 3). Thus there appears to be no
sinecomitant immune response to the T51/6 clone in this
system.
Duration of UVR-induced Systemic Tumor Immunosuppres
sion. In this system UVR-induced immunosuppression is ex
amined following tumor formation. If examined prior to tumor
appearance, the absence of UVR-induced immunosuppression
may only indicate its latency, which occurring later, would lead
to tumor expression. However to validate this approach of
testing for immunosuppression post-tumor appearance, the ex
istence must be excluded of a short-lived systemic immuno
suppression, which may conceivably only be required during
initial tumor appearance while the tumor load is small.
Historically however, UVR-induced systemic tumor immu
nosuppression once formed is shown to be a long-lived phenom
enon (23, 24), most likely due to immunological memory. Table
4 confirms these previous observations in our mice, with the
T51/6 tumor take continuing 28 weeks following completion
of a subcarcinogenic UVR whole-dorsal regimen, noting the
absence of growth in unirradiated age-matched controls.
Table 2 Effect of a UV microirradiation-inducedtumor
protocol on
UVR-induced systemic tumor immunosuppression
All animals were female and age matched within 3 weeks. Tumor excision and
sham excision were performed at the same age throughout all groups as was the
T51/6 inoculation. Tumors were excised approximately 3 months following the
end of microirradiation at approximately 10 months of age. Statistical analysis:
using the two-tailed Fisher exact probability test (see "Materials and Methods").
I versus III. P= 1.0; I versus II, P = 0.00016; II versus III, P = 0.0000079.
take"0/7
GroupI
IIIIITreatmentMicroirradiation*
Subcarcinogenic whole
dorsal UVRf
Unirradiated1*T51/6
8/80/12
°Number of T51/6 tumor-bearing animals/number of mice inoculated with
approximately 4 X IO6T51/6 cells; at 12 weeks following inoculation.
* Twenty-six adult mice were irradiated 5 days a week for 13 weeks middorsally
with a xenon solar simulator on an area of 0.8 cm2 (see "Materials and Methods").
Clinical carcinomas were excised, confirmed as SCCs on histology, and the wound
was closed with nylon sutures (see "Materials and Methods"). Seven days follow
ing excision the animals were challenged with an intradermal inoculation of T51/
6 cells ventrally (see "Materials and Methods").
f Adult mice were given a whole-dorsal exposure of UVR 4 days/week for 7
weeks from a fluorescent source solar simulator (see "Materials and Methods").
Excision of middorsal skin (sham excision) and closure with 5/0 nylon sutures
was followed 7 days later by challenge with an intradermal inoculation of T51/6
cells ventrally.
d Unirradiated mice were sham excised and inoculated with T51/6 as in group
II.
Table 3 Sinecomitant tumor immunity to T51/6
Statistical analysis: using the two-tailed Fisher exact probability test. I versus
III, P = 0.62; II versus IV, P = 0.47; I versus II. P = 0.0015; III versus IV, P =
0.020.
Treatment"
T51/6 take*
Group
IIIIIIIVUVRf
UVR tumor
implant^
Excision'
T5
challenge'Unirradiated*
1/6
UVR tumor implant
Excision
T5
challengeUVRSham
1/6
excision*
T51/6
challengeUnirradiated
Sham excision
T5 1/6 challenge6/80/107/101/9
" All mice were female, age matched (within 6 weeks), and inoculated with
T51/6 cells at approximately 8 months of age.
* Number of T51/6 tumor-bearing mice/number of mice inoculated with
approximately 4 x IO6T51/6 cells; at 12 weeks following inoculation.
' UVR groups received subcarcinogenic whole-dorsal UVI 4 days/week for 7
weeks commencing as adults (greater than 9 weeks old), which ceased approxi
mately 3 months prior to inoculation with the T51/6 tumor.
d UVR tumor recipients received a middorsal s.c. 3.5-mm3 fragment of a
primary UVR-induced tumor, which had been excised from mice receiving a
carcinogenic regime from the fluorescent solar simulator. All animals received
one tumor fragment only and no two animals of the same group received the
same tumor. Tumor implantation occurred at the same age in both groups I and
II.
' UVR tumors were excised 5 weeks following implantation and closed with
nylon sutures. In a small number tumors had regressed a few days prior to
complete excision.
•^Animalsreceived an intradermal ventral inoculation of T51/6 cells (see
"Materials and Methods") 7 days following excision of the dorsal tumor or sham
excision.
* Unirradiated mice.
* Excision of middorsal skin (see "Materials and Methods"). Excisions and
sham excisions occurred at the same age in all groups.
In the previous experiments a fluorescent source has been
used to produce whole-dorsal UVR-induced immunosuppres
sion versus a xenon source to produce microirradiation-induced
carcinomas; the latter bypassing immunosuppression. While
the two solar simulators have very similar spectral distributions.
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UV RADIATION CARCINOGENESIS
Table 4 Duration of WR-induced systemic tumor immunosuppression
Adult female age matched (within 3 weeks) mice were given a subcarcinogenic
whole-dorsal dose of UVR and inoculated with T51/6 cells ventrally under
previously stated conditions (see "Materials and Methods") at various periods
following completion of this regime.
take'Wk
1/6
Age"
post-UVR*4
value*0.047
(wk)21-24
AND IMMUNOSUPPRESSION
Table 7 Whole-dorsal UVR-induced tumor antigenicity
Mice (Skh:HRI females) were given a whole-dorsal carcinogenic dose of UVR
with the fluorescent solar simulator until tumors appeared. Nineteen different
primary tumors were inoculated s.c. at a middorsal position into 3 groups as seen
in footnotes. Individual tumor diameters among the three inoculated groups (Skh
old, Skh 4-6w, and BALB/c nude mice) were analyzed by using Friedman's test
(see "Materials and Methods"). No statistical significant difference was found; P
= 0.51.
Tumor type"
Skh old*
Skh 4-6wf
u/nud
29-32
12
0.0079
5/55/5
0/51/5
37-40
0.047
2028UVR'4/5
45-48T5
0.0079
5/5Unirradiated^0/5
0/5P
°Age of mice on inoculation with T51/6 cells.
* Weeks between the completion of dorsal UV irradiation and inoculation of
T51/6 cells.
' Number of T51/6 tumor-bearing animals/number of mice inoculated with
approximately 4 x IO6T51/6 cells; at 12 weeks following inoculation.
d P value using the two-tailed Fisher exact probability test.
'Group receiving subcarcinogenic whole-dorsal UVR (see "Materials and
Methods").
^Unirradiated mice.
Tumor
take*Pro.
take^Tumor
(mm)*SCCdiameter
(III)Tumor
takePro.
takeTumor
(mm)All diameter
totalTumor
tumor
takePro.
takeTumor
diameter (mm)3/172/171.01/21/22.84/193/191.24/174/172.40/20/204/194/192.13/172/1
" The histológica! classification and grade of the inoculated tumors (see Table
6).
Table 5 Model 12 S solar simulator-induced tumor immunosuppression
Adult female age-matched mice were given a subcarcinogenic whole dorsal
dose ( 120 J/cm2) of UVR (290-400 nm), over 7 weeks, as seen in footnotes. The
two groups and unirradiated age- and sex-matched controls were then inoculated
with the T51/6 tumor line, under the previously stated conditions, 14 weeks
following the conclusion of the irradiation protocols; analyzed by using the twotailed Fisher exact probability test. I versus II, P = 0.78; I versus III, and II versus
III, P = 0.047.
Unirradiated Skh:HRI mice, age and sex matched with the primary tumorbearing donors. This group was 55-71 weeks old when receiving the tumor
implant.
' Unirradiated Skh:HRI mice 4-6 weeks old.
d BALB/c athymic nude (nu/nu) mice 4-6 weeks old.
' Number of tumor-bearing animals/number of mice implanted at 7 weeks
postimplantation.
•^Pro.
take, number of progressively growing tumor-bearing animals/number
inoculated.
" Mean tumor diameter at 7 weeks postinoculation.
No. of T51/6
TBA*/no. of
mice inoculated
Group
I. Fluorescent UVR"
4/5
II. Model 12 S UVR'
4/5
III. Unirradiated
0/5
" TBA, tumor-bearing animals.
* The fluorescent source solar simulator, under previously stated conditions.
' The Model 12 S solar simulator/optical mirror system (see UVI Regime 4 in
"Materials and Methods").
as indicated by similar minimal erythemal doses, as would be
predicted with the reciprocity law of erythema (25), it remains
a possibility that the xenon source solar spectrum itself may be
unable to induce immunosuppression.
However, as seen in
Table 5, when giving a whole-dorsal regimen to mice with the
Model 12S xenon source, long-term UVR-induced immuno
suppression is attainable.
Transplantation Biology and Antigenicity of Whole-Dorsal
versus Microirradiation-induced Tumors. As seen in Table 6,
whole-dorsal UVR-induced tumors grow preferentially in
Table 6 Effect ofUVR-induced immunosuppression on the transplantation
UVR-induced immunosuppressed
mice versus age-matched
biology of whole-dorsal UVR-induced tumors
unirradiated
controls.
These
observations
confirm those re
Mice (Skh:HRI females) were given a whole-dorsal carcinogenic dose of UVR
ported previously (23, 26), which form the basis of investiga
with the fluorescent solar simulator and observed until tumors appeared. Seven
teen different primary tumor fragments were inoculated s.c. at a middorsal
tions on the role of UVR-induced immunosuppression in skin
position (see "Materials and Methods") into two age (6 months old)- and sexcancer.
In both groups the growth potential of the implanted
matched groups, as seen in footnotes. Individual tumor diameters between the
two inoculated groups (UVR and unirradiated mice) were analyzed by using
primary tumors was inversely proportional to the degree of
Wilcoxin's signed rank test (see "Materials and Methods"). A significant differ
differentiation, with the poorly differentiated grade IV SCCs,
ence was found, where P = 0.036.
previously described as fibrosarcomas (21, 26-28), growing
UVR"
Unirradiated
greater than the anaplastic grade III SCCs. The well-differen
Tumor
Tumor
tiated grade I/II SCCs failed to take in either group.
Tumor
Pro.
diameter
Tumor
Pro.
diameter
The antigenicity of whole-dorsal UVR-induced tumors was
Tumor typef
take''
take'
(mm/
take
take
(mm)
examined in a separate experiment by implanting primary SCCs
(I/II)SCC
SCC
into unirradiated Skh:HRI mice age-matched with the primary
(III)SCC
tumor donors, 4- to 6-week-old athymic nude mice and 4- to 6(IV)KA-likeAll
week-old Skh:HRI mice. The first group is necessary since mice
have been shown to lose their ability to reject antigenic tumors
tumors, total0/82/53/31/16/170/82/53/31/16/1702.88.87.02.80/81/52/30/13/170/81/51/30/12/1700.43.800.79
" Mice receiving a subcarcinogenic whole-dorsal dose of UVR from the flu
with age (29, 30). However, in contrast to reports in other
orescent solar simulator, with inoculation occurring 8 weeks following the con
strains (2, 23, 27, 28), primary UVR-induced SCCs in Skh:HRI
clusion of irradiation.
mice are shown here to be poorly antigenic, with no difference
4 Mice receiving no UVR.
e The histological classification and grade of the inoculated tumors, based upon
in growth among the three inoculated groups (Table 7). The
previous observations in this laboratory (16, 17), where: SCC (I/II), well-differ
relationship between tumor differentiation and transplantabilentiated SCCs; SCC (III), anaplastic SCCs; SCC (IV), spindle cell SCCs; KAity remained.
like, keratoacanthoma-like tumor.
d Number of TBA/number of mice implanted, at 5 weeks postimplantation.
In contrast to whole-dorsal UVR-induced tumors, microir' Pro. take, number of progressively growing tumor-bearing animals/number
radiation-induced SCCs, whose original growth environment
implanted.
lacked UVR-induced systemic tumor immunosuppression, did
^The mean tumor diameter at 5 weeks postimplantation.
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UV RADIATION CARC1NOGENESIS AND 1MMUNOSUPPRESSION
Table 8 Microirradiation-induced tumor antigenicity
Skh:HRI female mice were given 275 J/cm2 of total L'VR using the Model 12
S solar simulator carcinogenic microirradiation protocol. Nine different microirradiation-induccd tumours (8 grade I/II SCCs and 1 grade IV SCC) were excised
from tumor-bearing animals that «ere43-57 weeks old. An additional two tumors
(both grade I/II) were implanted into three groups: data seen in parentheses.
Tumor fragments from each individual tumor were implanted s.c. middorsally
(sec "Materials and Methods") into five groups of mice, as seen in the footnotes.
(a) Tumor implantation groups: no statistical significant difference was found
between any group on analy/.ing individual tumor mean diameters, using Fried
man's test, when P = 0.64, or the two-way analysis of variance (on square roottransformed data), when P = 0.60. (A)T51/6 take, using Fisher's exact probability
test: I versus II. P = 0.00058: II versus III. P = 0.0047.1 versus III. P = 0.46.
diameter'
take*5/9
take"6/95/9
(mm)5.5
GroupSkh
old*
Skh UVRf
Skh voung'
BALB/c nude*
Swiss
nude*T5
4/9(4/11)
4/9(4/11)
5/9(5/11)Skh
old
(I)0/7Pro.
4/9
4/9(4/11)
4/9(4/11)
5/9(5/11)Skh
5.2
2.7 (2.2)
3.7 (3.0)
(3.4)Microirrad.'
4.1
UVR("I7/7Tumor
(Ill)1/6
1/6 take'Tumor
°Number of individual tumors (9-11) that had tumor-bearing animals/total
individual tumors, at 7 weeks postimplantation.
* Pro. take, number of individual tumors that had progressively grow ing tumorbearing animals/total individual tumors, at 7 weeks postimplantation.
' The mean of the mean tumor diameter of each individual tumor, in mm. at
7 weeks postimplantation.
d Unirradiated mice ( I -3), age and sex matched with the microirradiated group.
' Mice (1-3), age and sex matched with the microirradiated group, that were
given a whole-dorsal subcarcinogenic dose of UVR with the fluorescent solar
simulator (see "Materials and Methods") that ceased at 21 weeks of age.
'Unirradiated Skh:HRI females (3). 4-6 weeks old.
* BALB/c nude (nu/nu) females (3). 4-6 weeks old.
* Swiss-ARC nude females (3). 4-6 weeks old.
' Some of the carcinoma-bearing microirradiated mice were inoculated with
the T51/6 line 7 days following tumor excision (via the previously described
method in Table 2).
' The T51/6 tumor line was inoculated as previously described into the defined
age-matched groups (see Table 2). The UV and Unirradiated groups received
sham excisions prior to inoculation. The T51/6 take represents the number of
T51/6 tumor-bearing animals/number of mice inoculated.
not grow preferentially in mice given an immunosuppressive
dose of UVR (Table 8). However, these tumors were again
shown to be poorly antigenic. A second nude strain was included
to reduce the chance of a different genetic background other
than the nude immunosuppressive mutation influencing tumor
growth. The growth potential of the tumor transplants was
again dependent upon the histológica! grade; with the grade IV
spindle SCC growing progressively in all groups (data not
shown). Finally, the previously microirradiation-induced
pri
mary tumor-bearing animals again lacked evidence for the
presence of systemic UVR-induced tumor immunosuppression,
with the T51/6 cell line failing to grow significantly in this
group.
DISCUSSION
The observations that UVR-induced systemic tumor immu
nosuppression seems to enhance the growth specifically of
UVR-induced tumor implants versus non-UVR-induced tumors
(7, 17, 31-33) with rare exceptions (33); that grafts of UVirradiated skin can transfer susceptibility to the growth of
transplanted UVR-induced tumors to normal recipients (34),
and that UVT can recognize tumor variants from a spontaneous
tumor line UV-irradiated in vitro (35), are highly suggestive of
a UVR-induced suppressor epitope driving this system of UVR
tumor immunosuppression.
Finally, the establishment of a
cloned UVT that can render normal syngeneic mice susceptible
to the growth of a variety of UVR regressor tumors (36, 37)
suggests that this putative suppressor epitope is common to
UVR-induced tumors. Thus as it stands, the theory proposed
by Fisher and Kripke ( 13) that the presence or absence of UVRinduced systemic tumor immunosuppression
determines
whether or not antigenic primary cancers will develop in UVirradiated skin, represents a unique event in our understanding
of cancer biology, for here we have a system whereby the
presence of a common tumor antigen dictates, in nature, the
suppression of immunosurveillance.
However, several observations are not compatible with UVRinduced systemic immunosuppression being essential for carcinogenesis. Chronic UV-irradiated animals given a low dose
of a regressor tumor rejected the tumor similarly to Unirradiated
animals (38). This was associated with a tumor-specific primary
and secondary cytolytic immune response in vivo and generation
of tumor-specific lymphocytes in vitro, as occurred in unirradiated normal animals. Low-dose tumor inocula should be more
relevant than high doses when attempting to reproduce the
immune response of an animal to primary tumor formation.
Higher-dose inocula of a regressor tumor progressed in chronic
UV-irradiated animals but showed evidence of antigenic loss
variants that were progressive on inoculation into normal unirradiated mice (38), thus demonstrating the presence of an
immune response to UVR-induced tumors in chronic UVirradiated animals. Finally and perhaps most significantly, ex
periments examining the increased susceptibility of nude mice
to UVR carcinogenesis (39) showed that nude mice reconsti
tuted with thymuses had the same susceptibility to UVR-in
duced tumor formation as athymic nude mice. Therefore, unless
one is to postulate that UVT do more than suppress an active
helper or effector T-lymphocyte response to UVR tumor anti
gens, which in any case, as shown above, is an incomplete
suppression, then this absence of susceptibility to UVR carci
nogenesis in immunoincompetent nude mice would predict a
much less significant role of UVT than has been previously
suggested.
Using a system of microirradiation (0.8-cm2 area) and a
cloned UVR tumor line, the T51/6, as an assay for UVRinduced systemic tumor immunosuppression, we have produced
UVR primary tumors without evidence of systemic immunosuppression. Thus, UVR-induced systemic tumor immuno
suppression is not essential for UVR-induced carcinogenesis.
One possible consideration is that our tumor clone is not
sensitive enough to detect immunosuppression in this system.
However, of the many tumor lines tested in this department, it
remains the most sensitive assay for UVR-induced systemic
immunosuppression, and it would appear extremely unlikely
that the primary tumors appearing in the microirradiated ani
mals would all be more sensitive to UVR-induced immuno
suppression than this clone.
Similarities between UVR-induced systemic suppression of
CHS and tumor immunosuppression (40-42) have led many
investigators to use CHS as a model for tumor immunosuppres
sion. However, an order of magnitude greater UVR dose is
required to induce tumor immunosuppression
than CHS
suppression (43). Furthermore, one group (9) could not distin
guish a suppressor lymphocyte population in chronic UVIirradiated animals versus normal Unirradiated animals when
observing a CHS suppression. Lastly, cloned UVT (37), while
allowing the growth of a battery of UVR-induced tumors, did
not suppress the induction of CHS. Due to these differences
between UV-induced suppression of CHS and tumor immunity,
a sensitive tumor cell line was used as an assav for UVR-
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UV RADIATION CARCINOGENES1S
induced systemic tumor immunosuppression.
Two variables, irradiance and area of exposure, need consid
eration when speculating how systemic immunosuppression
was avoided with the microirradiation protocol. With the xenon
solar simulator a 24 times greater irradiance than the fluores
cent source is produced. However, UVR-induced systemic tu
mor immunosuppression has dose rate and time reciprocity
(42), and therefore the greater irradiance of the microirradiation
carcinogenic regime should not be responsible for the absence
of immunosuppression. Thus it is more likely that the limited
area of exposure is responsible for avoiding immunosup
pression.
The theory examined in this paper, that the presence or
absence of UVR-induced systemic immunosuppression deter
mines whether or not primary antigenic cancers will develop in
UV-irradiated skin (13), needs revising as a generalized state
ment on UVR-induced carcinogenesis in rodents. For in this
system UVR-induced tumors, both whole-dorsal and micrirradiation induced, like the majority of nonviral-induced sponta
neous tumors in rodents (44), are poorly antigenic. Here a
putative role for any dominating effect of the immune system
destroying carcinomas, at least via a tumor transplantation
antigen-driven mechanism, is baseless. The absence of UVRinduced systemic tumor immunosuppression in UVR carcino
genesis, as observed here, conforms with this assertion.
In this system, three novel experimental variables, the light
source, murine strain, and tumor histology, need to be regarded
when considering the weak antigenicity of the UVR-induced
tumors. In contrast to the previous UVR-induced primary
tumor transplantation studies (2, 23, 27, 28), in which the
irradiation sources contained significant quantities of UVC,
both light sources used in these experiments contain filters to
eliminate wavelengths below 290 nm, thus conforming with the
solar spectrum. It is therefore conceivable that UVC alone may
be responsible for inducing antigenicity.
A further distinguishing feature of our study is the use of the
Skh:HRI hairless mouse. In the before mentioned studies haired
strains were used. Since a number of these haired strains are
on different backgrounds, any effect of strain difference in our
experiments would most likely center around the hairless phenotype or genotype (hr/hr).
Finally, an outstanding feature of previous studies has been
the preponderance of fibrosarcoma/spindle cell tumors versus
SCCs. In our study the vast majority of tumors are welldifferentiated SCCs. This has been reported in other hairless
(hr) strains (45-47) and in nudes (nu/nu) versus their hairy wild
type (39), and therefore seems a characteristic of the nonhaired
mouse, shared with humans. In this regard, SCCs in the pre
vious studies are less antigenic than the fibrosarcoma/spindle
cell tumors (2, 23).
In contrast to whole-dorsal UVR-induced tumors, microirradiation-induced SCCs, whose original growth environment
lacked UVR-induced systemic tumor immunosuppression, did
not grow preferentially in mice given a systemically immunosuppressive dose of UVR. The observations that methylcholanthrene tumors induced in mice given an immunosuppressive
dose of UVR (versus methylcholanthrene-induced
tumors in
duced in unirradiated mice) preferentially grow in UVR-in
duced immunosuppressed mice versus unirradiated mice (33),
is consistent with our observations. This suggests that UVRinduced immunosuppression causes a positive selection pres
sure during tumor formation, which is independent of the
carcinogen per se. In fact, it is conceivable that this phenomenon
AND 1MMUNOSUPPRESSION
may be independent of tumor type, whereby any tumor which
is formed in the presence of UVR-induced immunosuppression,
whether an experimentally induced or spontaneous neoplasm,
will have altered transplantation characteristics that allow pref
erential growth in this system.
One may hypothesize that the speculated common suppressor
antigen found on whole-dorsal UVR-induced tumors is absent
in the mircoirradiation-induced tumors, and therefore this an
tigen is not produced directly by UVR. The idea that the
suppressor antigen is produced directly by UVR was suggested
by the observation that UVT can recognize tumor variants from
a spontaneous poorly antigenic tumor line UV-irradiated in
vitro (35). The lack of the common suppressor epitope on the
microirradiation-induced tumors would support the hypothesis
that this antigen is the primary event leading to UVR-induced
systemic tumor immunosuppression, as already discussed. The
second possibility is that the suppressor epitope may exist on
these microirradiation-induced tumors but this may not be the
event (or not the only event) required by the neoplastic cell to
be affected by UVR-induced immunosuppression. For example,
the presence of UVT or UVR-induced soluble factors may be
necessary during keratinocyte transformation if the neoplastic
cell is to be influenced by UVR-induced immunosuppression.
Lastly, the separate issue of how UVR-induced systemic
tumor immunosuppression is bypassed by limiting the area of
UV irradiation exposure, can be postulated. First, as mentioned
above, the possible absence of the common suppressor epitope
in this system, may be solely responsible for the absence of
immunosuppression. Secondly, based mainly upon observations
of UVR-induced immunosuppression of CHS (while noting the
previously mentioned differences in this system versus tumor
immunosuppression), a decreased amount of pyrimidine dimer
formation (48), urocanic acid (49), or antiinflammatory cytokines such as interleukin 1 and prostaglandin E: (50) may be
involved.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Gary Halliday, Professor Ross Barnetson, and Stephen Brown for their helpful discussions; and Jules
Martin for allowing the use of the Model 12S solar simulator.
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Ultraviolet Radiation-induced Murine Tumors Produced in the
Absence of Ultraviolet Radiation-induced Systemic Tumor
Immunosuppression
Scott W. Menzies, Gavin E. Greenoak, Vivienne E. Reeve, et al.
Cancer Res 1991;51:2773-2779.
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