Cure of Mammary Carcinomas in Her-2 Transgenic Mice through
Sequential Stimulation of Innate (Neoadjuvant Interleukin-12)
and Adaptive (DNA Vaccine Electroporation) Immunity
Michela Spadaro,1Elena Ambrosino,1
Manuela Iezzi,2Emma Di Carlo,2
Pamela Sacchetti,1Claudia Curcio,1
Augusto Amici,3Wei-Zen Wei,4Piero Musiani,2
Pier-Luigi Lollini,5Federica Cavallo,1and
1Department of Clinical and Biological Sciences, University of Turin,
Orbassano, Italy;2Center of Excellence on Aging Research,
‘‘G. D’Annunzio’’ University Foundation, Chieti, Italy;3Department of
Molecular, Cellular and Animal Biology, University of Camerino,
Camerino, Italy;4Karmanos Cancer Institute, Wayne State University,
Detroit, Michigan; and5Cancer Research Section, Department of
Experimental Pathology, University of Bologna, Bologna, Italy
Purpose: Whereas neoadjuvant therapy is emerging as
a treatment option in early primary breast cancer, no data
are available on the use of antiangiogenic and immuno-
modulatory agents in a neoadjuvant setting. In a model of
Her-2 spontaneous mammary cancer, we investigated the
efficacy of neoadjuvant interleukin 12 (IL-12) followed by
‘‘immune-surgery’’ of the residual tumor.
Experimental Design: Female BALB/c mice transgenic
for the rat Her-2 oncogene inexorably develop invasive
carcinomas in all their mammary glands by the 23rd week of
age. Mice with multifocal in situ carcinomas received four
weekly i.p. injections of 100 ng IL-12 followed by a 3-week
rest. This course was given four times. A few mice
additionally received DNA plasmids encoding portions of
the Her-2 receptor electroporated through transcutaneous
Results: The protection elicited by IL-12 in combina-
tion with two DNA vaccine electroporations kept 63% of
mice tumor-free. Complete protection of all 1-year-old mice
was achieved when IL-12-treated mice received four
vaccine electroporations. Pathologic findings, in vitro tests,
and the results from immunization of both IFN-; and
immunoglobulin gene knockout transgenic mice and of
adoptive transfer experiments all show that IL-12 augments
the B- and T-cell response elicited by vaccination and
slightly decreases the number of regulatory T cells. In
addition, IL-12 strongly inhibits tumor angiogenesis.
Conclusions: In Her-2 transgenic mice, IL-12 impairs
tumor progression and triggers innate immunity so markedly
that DNA vaccination becomes effective at late points in time
when it is ineffective on its own.
As breast cancer is the most common primary malignant
disease in women and often associated with poor prognosis, new
strategies for its cure and prevention are urgently needed. There
has been a surge of interest in neoadjuvant medical therapy for
early breast cancer over the last 10 years in the light of the
experience in locally advanced inoperable breast cancer, where
systemic treatment before local therapy is now the standard of
care (1). Randomized trials comparing neoadjuvant medical and
conventional postoperative adjuvant therapy have shown similar
rates of local control and overall survival (1–3). These results
have been obtained with both chemotherapy and endocrine
neoadjuvant therapy, whereas no data are available on the use of
antiangiogenic and immunomodulatory agents in a neoadjuvant
setting. Microvessel density is an independent and highly
significant prognostic factor in breast cancer. Antiangiogenic
management designed to prevent the sprouting of new vessels
restrains tumor expansion (4). However, although antiangiogenic
therapy significantly delays early tumor progression, its
contribution to tumor cure is not apparent (5, 6).
Interleukin 12 (IL-12) is a cytokine with a potent
antiangiogenic and immunomodulatory activity that elicits
numerous downstream proinflammatory cytokines and danger
signals, and leads to activation of professional antigen-
presenting cells (7). IFN-g and other downstream cytokines
thus elicited inhibit tumor cells, activate potent antiangiogenic
mechanisms, and even switch the genetic program of a tumor
from proangiogenic to antiangiogenic (7, 8). In many
transplantable tumor models, the sum of these activities results
in significant inhibition (9, 10). In mice transgenic for the
activated rat (r) Her-2 oncogene (neu and ErbB-2 in humans)
under the control of the mouse mammary tumor virus promoter
(BALB-neuT), IL-12 delays early carcinogenesis progression,
but is not enough to cure established tumors (11, 12). However,
it also acts as an essential component of a combined cell
vaccine that prevents the early onset of carcinomas (13).
In these mice, adaptive immunity elicited by vaccination
with DNA plasmids coding the transmembrane and extracellular
domains of r-Her-2 (TMEC plasmids) clears early in situ
carcinomas and keeps most mice tumor-free (14). This
prevention is enhanced when DNA vaccination is boosted by
Received 9/13/04; revised 11/19/04; accepted 12/6/04.
Grant support: Italian Association for Cancer Research, Italian
Ministries for the Universities and Health, University of Turin,
Compagnia di San Paolo, Turin, and Center of Excellence on Aging,
University of Chieti, Italy.
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.
Requests for reprints: Federica Cavallo, Department of Clinical and
Biological Sciences, University of Turin, Ospedale San Luigi Gonzaga,
Regione Gonzole 10, I-10043 Orbassano, Italy. Phone: 39-11-790-5419;
Fax: 39-11-236-5417; E-mail: email@example.com.
D2005 American Association for Cancer Research.
Vol. 11, 1941–1952, March 1, 2005
Clinical Cancer Research 1941
r-Her-2+IFN-g-releasing allogeneic cells (15), or when IL-1h
fragment (16) or soluble LAG-3 costimulatory molecule (17) are
administered concurrently with the vaccine. Moreover, complete
prevention is afforded by repeated plasmid electroporations (18).
All these cell and DNA vaccines, however, are ineffective
when an invasive carcinoma is established. In the study
described in this paper, we determined whether early neo-
adjuvant stimulation of innate immunity by IL-12 could render
effective subsequent DNA vaccination after the onset of
carcinomas. The results show that early IL-12 treatment of
BALB-neuT mice makes DNA vaccination effective, whereas it
is ineffective on its own. This suggests that sequential
combination of neoadjuvant stimulation of innate immunity
followed by ‘‘immune surgery’’ of the residual tumor cells
through induction of adaptive immunity can be proposed as a
new and effective strategy.
MATERIALS AND METHODS
BALB-neuT mice were bred under specific
pathogen-free conditions by Charles River (Calco, Italy; ref. 5).
BALB-neuT mice knocked out for the IFN-g (BALB-neuT/
IFNgKO), and BALB-neuT mice knocked out for the
immunoglobulin (Ig) l chain gene (BALB-neuT/AKO) were
generated by crossing BALB-neuT mice with BALB/c mice KO
for the IFNc gene from The Jackson Laboratory (Bar Harbor,
ME), or BALB/c KO mice for the Ig A chain kindly provided by
Dr. T. Blankenstein (Free University, Berlin, Germany; ref. 19).
Mice were randomly assigned to control and treatment groups
and all groups were treated concurrently. As each experiment
was repeated twice to thrice with similar results, data were
cumulated and reported. Mammary glands were inspected at
weekly intervals to note tumor appearance and tumor masses
were measured with calipers in two perpendicular diameters.
Progressively growing masses >1 mm mean diameter were
regarded as tumors. Tumor multiplicity was calculated as the
cumulative number of incident tumors/total number of mice and
is reported as mean F SE (18). Mice were treated according to
the European Union guidelines.
IL-12 (kindly provided by Dr. Stan Wolf, Genetics Institute,
Cambridge, MA) was diluted in PBS supplemented with 0.01%
mouse serum albumin (MSA; Sigma, St. Louis, MO) and
administered i.p. as previously described (12). Starting from the
7th (7wMSA or 7wIL12) or the 14th (14wIL12) week of age,
BALB/c and BALB-neuT mice received a course of one
weekly i.p. injection of 0.2 mL PBS containing MSA only
(MSA controls) or MSA plus 100 ng of IL-12, for 4 weeks,
followed by a 3-week rest. This course was given four times in
7wMSA and 7wIL12 or thrice only in 14wIL12. Other groups
of mice were not treated. Because no appreciable differences in
tumor growth rate and pathologic findings were found between
the untreated mice and the MSA controls, only the data of one
of these two groups are shown in Results.
Whole-Mount Image Analyses.
mammary glands were done as indicated in http://ccm.ucdavis.edu/
tgmouse/HistoLab/wholmt1.htm. Digital images were acquired
by dividing the whole mount of each gland into 10 quadrants.
Ten points were randomly chosen on the duct surface in each
Whole mounts of all
quadrant and the corresponding lesions were measured. All
lesions with a diameter >150 Am on the same quadrant were
counted. Images of whole-mount preparations were taken with a
Nikon Coolpix 950 digital camera (Nital Spa, Turin, Italy)
mounted on a stereoscopic Leica MZ 6 microscope (Leica
Microsystems, Milan, Italy). A 0.63 objective were used to
obtain images with a total magnification of ?630 and a
resolution of 1,600 ? 1,200 pixels. Images were acquired within
Adobe Photoshop version 6.0 graphic software (Adobe Systems,
San Jose, CA). Glands larger than a single imaging area were
captured by photographing contiguous microscopic fields in a
raster pattern. Each captured image was merged using the layer
technique in Adobe Photoshop to form a single composite image
for analysis. Spatial calibration was determined by photographing
a 1 mm stage using the same parameters as those for image
capturing of whole-mount preparations. The distance drawn on
the 1 mm calibration image was divided by 1,000 to find the
number of pixels per micrometer. In each image, 100 discrete
points were randomly chosen on the duct surface and lesion
widths in micrometer were measured perpendicular to duct
direction. Points with no lesion were ranked as zero. Lesion
measurements were recorded on an Excel spreadsheet and mean
and SE were calculated for each treatment group. Statistics were
obtained with a two-tailed Student’s t test.
Preparation of DNA Plasmids.
produced as previously described (14–18). For DNA electro-
poration, 25 Ag of TMEC plasmids in 20 AL 0.9% of NaCl
with 6 mg/mL polyglutamate were injected bilaterally into the
tibial muscle of the hind legs of anesthetized mice. Transcu-
taneous electric pulses were applied on the shaved skin.
Square-wave electric pulses were generated by a T820 electro-
porator (BTX, San Diego, CA). Two 25 ms pulses with a field
strength of 375 V/cm were administered (18).
Evaluation of Serum Vascular Endothelial Growth
Blood samples from groups of three control or treated
BALB-neuT mice were collected and the presence of vascular
endothelial growth factor in each serum was evaluated with a
sandwich ELISA (R&D Systems Inc., Minneapolis, MN).
India Ink Blood Vessel Perfusion and Image Analysis.
Groups of three mice were anesthetized and perfused via the left
ventricle. An initial blood washout with a solution of 0.5%
sodium nitrite and 10 units/mL heparin in PBS at 37jC was
immediately followed by reperfusion with black India ink. Fat
pads were prepared as whole mounts and stained with carmine.
Images of whole-mount preparations were taken as described
above with a 4.3 objective giving a total magnification of ?430.
At least 10 images were taken for each mammary gland and 25
images from each mouse were randomly chosen for image
analysis. The area of the black blood vessel was selected with the
Magic Wand Tool in Adobe Photoshop and the number of pixels
recorded on an Excel spreadsheet. The width of the blood vessel
network is calculated as a percent of total image area.
Histology and Immunohistochemistry.
mice were sacrificed at the indicated times and samples of
for immunohistochemical and histologic analysis (20). For
immunohistochemistry, acetone-fixed cryostat sections were
incubated for 30 minutes with antibodies anti-Mac-1 (CD11b/
CD18, clone M1/70.5), anti-CD8 (Ly/T2, clone YT5 169.4) and
TMEC plasmids were
Groups of three
Neoadjuvant IL-12 and DNA Vaccine Electroporation
anti-CD4 (LT34, clone YT126.96.36.199; all from Sera-Lab, Crawley
Down, Sussex, United Kingdom), or anti-CD11c (clone N418;
Chemicon International Inc., Temecula, CA), anti-CD40L
(CD154, gp39; clone H-215; Santa Cruz, Biotechnology Inc.,
Santa Cruz, CA), anti-CD86 (B72, clone PO3), and anti-CD45R/
B220 (clone RA3-6B2; all from PharMingen, San Diego, CA),
anti-IFN-g (clone XMG1.2, kindly provided by Dr. S. Landolfo,
University of Turin, Turin, Italy). To evaluate the expression of
r-p185neuand proliferating cell nuclear antigen, paraffin-embed-
ded sections were tested with polyclonal rabbit anti-Her-2
antiserum (C-18, Santa Cruz Biotechnology) and anti–prolifer-
ating cell nuclear antigen (Ylem, Rome, Italy) antibody. After
washing, sections were overlaid with biotinylated goat anti-rat,
anti-hamster, and anti-rabbit or horse anti-goat Ig (Vector
Laboratories, Burlingame, CA) for 30 minutes and incubated
with avidin-biotin complex method complex/alkaline phospha-
tase (DAKO, Glostrup, Denmark). To quantify germinal centers
(GC), each spleen was transversely dissected into four segments
from which semiserial sections were obtained.The GC in the total
area of each section were counted. Morphologic studies were
conducted independently by three pathologists in a blind fashion.
Sera obtained at progressive time
points from mice of each treatment group were diluted 1:100 in
PBS-azide-bovine serum albumin (Sigma) and the presence of
anti-r-p185neuantibody was determined by flow cytometry using
BALB/c 3T3 fibroblasts, wild-type, or stably cotransfected with
the wild-type r-Her-2, mouse class I H-2Kdand B7.1 genes
(BALB/c 3T3-NKB). FITC-conjugated goat anti-mouse anti-
body specific for mouse IgG Fc (DAKO) was used to detect
bound primary antibody. Normal mouse serum was the negative
control. The monoclonal antibody Ab4 (Oncogene Research
Products, Cambridge, MA), which recognizes an extracellular
domain of r-p185neu, was used as a positive control. Serial Ab4
dilutions were used to generate a standard curve to determine the
concentration (Ag/mL) of anti-r-p185neuantibody in serum (12).
For isotype determinations, sera from 20-week-old mice were
diluted 1:20 and incubated with BALB/c 3T3 and BALB/c 3T3-
NKB. Cells were then incubated for 30 minutes with rat biotin-
conjugated antibody anti-mouse IgA, IgM, IgG1, IgG2a, IgG2b,
IgG3 (Caltag Laboratories, Burlingame, CA). After washing,
cells were incubated with 5 AL streptavidin-phycoerythrin (PE;
DAKO), and resuspended in PBS-azide-bovine serum albumin
containing 1 mg/mL propidium iodide to gate dead cells. Flow
cytometry was done with a FACScan (Becton Dickinson,
Mountain View, CA). Data were analyzed through CELLQuest
(Becton Dickinson) software.
Antibody-Dependent Cellular Cytotoxicity.
cells are a cloned cell line from a carcinoma that arose in a
ry gland documenting the modulation
of carcinogenesis in 7wIL12-treated
BALB-neuT mice. Numerous neoplas-
tic side bud lesions (dark spots pointed
glands of 7-week-old BALB-neuT
mice (A). Mice with these lesions
received an i.p. injection of MSA only
weeks, followed by 3 weeks of rest.
(7wMSA; 7wIL12; B). In 7wMSA-
treated control mice, the side buds are
more diffuse and larger at week 16 (C)
marked reduction in both the size and
week 16 and week 20 (D). At week 20,
confinement of the neoplastic lesions
close to the nipple is a typical finding.
However, the lesions are much larger
than at week 16 (F). LN, mammary
lymph node. (A-E, magnification
?63). Three different mice were used
for each treatment.
Clinical Cancer Research 1943
BALB-neuT mouse (14). Five thousand [3H]dTh-labeled TUBO
cells were incubated for 2 hours at 4jC with progressive
dilutions (1:10 to 1:100) of sera in HBSS (Sigma) and then
washed. Effector spleen cells (Spc) from untreated mice were
then added at 50:1 effector-to-target ratio to each well of round-
bottom microtiter plates (Nunc, Roskilde, Denmark) in triplicate
and lysis was determined as previously described (21).
Spc (1 ? 107) were stimulated with
5 ? 105mitomycin-C (Sigma) treated TUBO cells in the
presence of 10 units/mL recombinant IL-2 (Eurocetus, Milan,
Italy). After 4 days, Spc were recovered, washed, and seeded at
2 ? 106cells/mL in the presence of anti-CD28 and anti-CD3
(1 Ag/mL final concentration, PharMingen) for 3 hours. IFN-g-
producing cells were identified by using the mouse IFN-g cell
enrichment and detection kit (Miltenyi Biotec, Bergisch-
Gladbach, Germany). Recovered Spc were labeled with an
anti-IFN-g (clone R4-6A2) conjugated with an anti-CD45 (clone
30S11) monoclonal antibody (Miltenyi Biotec) for 5 minutes on
ice, then incubated for 45 minutes at 37jC. Cross-staining was
avoided by keeping the cell density at 1 ? 105cells/mL. IFN-g
bound to the capture matrix was stained with PE-conjugated
monoclonal antibody against IFN-g (clone AN.18.17.24,
Miltenyi Biotec). PE-IFN-g-stained cells enriched with anti-PE
microbeads were separated with an autoMACS separator
(Miltenyi Biotec). The cells were counterstained with monoclo-
nal antibody against CD4 or CD8a-FITC (clone YTS 191.1.2,
clone YTS 169 AG 101 HL respectively; Cedarlane, Hornby,
Ontario, Canada) and analyzed by flow cytometry.
Spc (1 ? 107) were stimulated for
6 days with 5 ? 105mitomycin-C (Sigma) treated TUBO
cells in the presence of 10 units/mL recombinant IL-2
(Eurocetus) and assayed in a 48-hour [3H]dTh release assay
at effector-to-target ratio from 50:1 to 6:1 in round-bottomed,
96-well microtiter plates in triplicate (21). The results are
expressed as lytic units (LU)20/107effector cells, with LU20
defined as the number of effector cells needed to kill 20% of
the target cells (22).
Sera and Spc were collected from
20-week-old 7wIL12 + w16-18TMEC BALB-neuT mice. Spc
were labeled with a PE-conjugated anti-CD90 monoclonal
antibody (clone 53-2.1, PharMingen) and then anti-PE micro-
beads (Miltenyi Biotec) were used to isolate CD90+cells with an
autoMACS separator (Miltenyi Biotec). Pooled sera (0.5 mL) or
inhibits the progression of carcinogenesis. Percentage of tumor-free mice
(A) and tumor multiplicity (B) in the w16-18pcDNA3 (o, 18 mice),
w16-18TMEC (., 9 mice), 7wIL12 (n, 7 mice), and 7wIL12 + w16-
18TMEC (5, 8 mice) groups. Arrows, week in which IL-12 was injected;
arrowheads, week in which plasmids were electroporated. The tumor-free
survival curve of 7wIL12 + w16-18TMEC mice is significantly different
from that of w16-18pcDNA3 and w16-18TMEC mice (A; Mantel-
Haenszel test, P < 0.0001). The mean tumor multiplicity is significantly
lower in 7wIL12 + w16-18TMEC than in w16-18TMEC and w16-
18pcDNA3 mice (B; Student’s t test, P < 0.0006). At the 20th week of
age, the whole mounts of the mammary glands of w16-18pcDNA3 and
w16-18TMEC mice show a few small residual side buds (arrows),
mostly confined to the nipple region (arrowhead). At week 34, these
lesions are larger (arrows), suggesting recommencement of tumor
growth (D). Oval central black area (LN), lymph node (C and D,
magnification ?63). These experiments were done twice.
IL-12 in combination with TMEC plasmid electroporation
Neoadjuvant IL-12 and DNA Vaccine Electroporation
1 ? 107, 90% to 92% CD90+Spc were administered i.v. in
0.2 mL HBSS to recipient mice at the 10th and 12th week of age.
Recipient mice were sacrificed at the 16th week of age and the
whole mounts of all 10 mammary glands were evaluated by
computer-aided image analysis.
CD4+Spc obtained at 20 weeks
of age from six mice from each treatment group were purified
with the CD4+T-cell isolation kit (Miltenyi Biotec), separated
with an autoMACS separator (Miltenyi Biotec), and then
stained with anti-CD4-PE (Cedarlane) and anti-CD25-FITC
antibody (clone 7D4, PharMingen). The cells were resuspended
in PBS-azide-bovine serum albumin containing 1 mg/mL of
propidium iodide to gate dead cells. Flow cytometry was done
with a FACScan (Becton Dickinson). Data were analyzed
through CELLQuest (Becton Dickinson) software, and reported
as the mean of the percentage of CD4+/CD25+cells determined
in three independent experiments.
Differences in tumor incidence were evaluated
with the Mantel-Haenszel log-rank test, and those in tumor
multiplicity, LU20, IFN-g-producing Spc, and antibody titer with
the two-tailed Student’s t test.
IL-12 Slows Down Mammary Carcinogenesis. Mam-
mary carcinogenesis follows a very consistent course in virgin
female BALB-neuT mice (Fig. 1A; ref. 11). When mice with
lesions equivalent to atypical hyperplasia and in situ carcinoma
received recombinant mouse IL-12 i.p. starting from the 7th
week of age (7wIL12; Fig. 1B), a marked reduction in the
progression was already evident at week 16 (Fig. 1C versus D)
and became remarkable at week 20 (Fig. 1E versus F) when
neoplastic side buds were typically confined to the tissue close to
the nipple. However, at week 20, the presence of markedly larger
lesions (Fig. 1D versus F) indicates that the protection has
vanished and that progression has recommenced. No inhibition
was found when IL-12 treatment was postponed to week 14
(14wIL12, not shown).
DNA Vaccination by Electroporation Combined with
IL-12 Inhibits Mammary Carcinogenesis.
in vivo of TMEC plasmids when mice display multifocal in situ
carcinomas inhibits carcinogenesis progression (18), but not if
it is postponed to weeks 16 and 18 (w16-18TMEC) when mice
already display invasive carcinomas (Fig. 2A). By contrast, the
combination of 7wIL12 with w16-18TMEC kept 63% of mice
free from palpable tumors until 35 weeks when the experiments
were ended. Extension of the tumor-free survival was
accompanied by a strong reduction in tumor multiplicity (Fig.
2B), whereas the whole mounts displayed only a few small
residual side buds near the nipple at 20 weeks (Fig. 2C). These
lesions are markedly smaller than those of mice that received
7wIL12 only (Fig. 2C versus Fig. 1F). However, at week 35,
the presence of larger side buds again points to slow
recommencement of progression (Fig. 2D). By contrast, all
mice remained free of palpable tumors (Fig. 3A and B) and
their whole mounts displayed no signs of tumor progression
(not shown) when 7wIL12 + w16-18TMEC mice were further
boosted with TMEC plasmids at weeks 23 and 25 (w16-18/23-
genesis even when IL-12 administration is delayed. Percentage of
tumor-free mice (A) and tumor multiplicity (B) in the 7wIL12 + w16-
18/23-25TMEC (w, 10 mice), 14wIL12 + w16-18/23-25TMEC (n,
6 mice), and 14wIL12 + w16-18TMEC (4, 7 mice) groups. Arrow,
week in which IL-12 was injected; arrowheads, week in which plasmids
were electroporated. The tumor-free survivals in 7wIL12 treated + w16-
18/23-25TMEC and 14wIL12 + w16-18/23-25TMEC mice are
significantly different (Mantel-Haenszel test, P < 0.0008), as are those
of 14wIL12 + w16-18TMEC and 14wIL12 + w16-18/23-25TMEC mice
(Mantel-Haenszel test, P < 0.008). The mean tumor multiplicity is
significantly lower in 14wIL12 + w16-18/23-25TMEC mice than in
14wIL12 + w16-18TMEC mice between weeks 36 and 40 (Student’s t
test, P < 0.05). Bars, SE of the mean of tumor multiplicity. These
experiments were done twice.
Successive TMEC plasmid electroporations hamper carcino-
Clinical Cancer Research 1945
In a neoadjuvant setting, IL-12 treatment began when mice
already displayed invasive breast cancer (14wIL12) and kept
f70% of mice tumor-free; mice were subsequently vaccinated
and boosted (w16-18/23-25TMEC plasmid electroporated mice;
week 16 and 18 vaccination (w16-18TMEC) kept 30% of mice
tumor-free. No inhibition of tumor takes and multiplicity was
found in mice electroporated with pcDNA3 control plasmid at
tures were compared among the mice of the various treatment
groups to tease apart the immune events that made the DNA
electroporation so effective in mice treated with IL-12. These
comparisons were made within the 20th and 22nd week of age
when whole mounts display the most notable inhibition of tumor
progression in treated mice (Fig. 2C).
We have previously shown that IL-12 inhibit the angiogenic
activity of Her-2+tumor cells in vitro (18) and in vivo (4). To
assess the weight of the antiangiogenic activity of 7wIL12, the
Several immune fea-
amount of vascular endothelial growth factor in the sera of
variously treated mice was evaluated (Fig. 4A). Whereas a slight
decrease in serum vascular endothelial growth factor was found
in mice that received 7wIL12 only, the decrease was stronger
when 7wIL12 treatment was combined with w16-18TMEC
electroporations. This antiangiogenic effect was also notably
evident in the whole mounts of mammary glands done after the
perfusion of blood vessels with India ink. In 16-week-old mice,
7wIL12 alone leads to a marked decrease of small vessel
network (20.85 F 6.48% versus 36.63 F 8.88% of total image
area F SD for 7wIL12 and control respectively; P < 0.003),
associated with a reduction in size and number of neoplastic
lesions (Fig. 4D versus B). Four weeks later, w16-18TMEC
electroporation alone lead to a shrinkage of the tumor mass,
whereas a vascular network is evident not only around the
neoplastic lesions but also in the fat pad stroma (Fig. 4C). A
strikingly impressive synergistic antiangiogenic (15.29 F 5.43
versus 41.52 F 6.14% of total image area F SD for 7wIL12
treatment + w16-18TMEC and w16-18TMEC alone respectively;
treatment + 16-18TMEC in 22-week-old
mice. Level of vascular endothelial growth
factor in the sera of variously treated mice
as assessed by ELISA. Each determination
was done on the sera of six mice individ-
ually tested (A). B-E, Perfusion of blood
vessels with India ink followed by whole
mount of the mammary glands. A wide-
spread vascular network of first-order
vessels running longitudinally parallel to
the mammary ducts and numerous second-
order vessels originating from first-order
vessels and penetrating the large neoplastic
lesions are evident in control mice (B).
7wIL12 alone markedly reduces this net-
work and reduces the size and number of
neoplastic lesions (D). w16-18TMEC alone
slightly decreases lesion size and an
increase of small caliber vessels and
vascular network is evident around the
lesions and in the fat pad stroma (C).
7wIL12 + w16-18TMEC leads to a strik-
ingly impressive antiangiogenic effect.
Small vessels disappear leaving a network
mostly composed of large vessels that
accompany breast ducts. Tumor lesions
are not evident (E). Three different mice
were used for each treatment.
Antiangiogenic effect of IL-12
Neoadjuvant IL-12 and DNA Vaccine Electroporation
P < 0.0001) and an antitumor effect resulted from the
combination of 7wIL12 treatment + w16-18TMEC electro-
poration. Small vessels and neoplastic lesions are no more
evident, whereas the vascular mammary network is mostly
constituted by large vessels (Fig. 4E).
Spleen Germinal Centers and Antibody Response.
20 weeks of age, the white pulp GC in the semiserial sections of
the spleen of 7wIL12 + w16-18TMEC mice were 10 F 2 per
section and 7 F 2 per section in mice that only received w16-
18TMEC. In both cases, GC were significantly more numerous
than in mice that received 7wIL12 (3 F 1 per section) or w16-
18pcDNA3 only (2 F 1 per section; P < 0,001). In addition, a
wider lymphoid white pulp with more expanded B-cell areas and
marked B-cell scattering within the T-cell zone are evident in
spleens of w16-18TMEC and 7wIL12 + w16-18TMEC mice
(Fig. 5B and C). These morphologic findings on B-cell activation
correspond to the serum anti-r-p185neuantibody titers. 7wIL12 +
w16-18TMEC mice displayed an earlier, stronger, and much
more sustained antibody response to p185neuthan w16-18TMEC
mice (Fig. 5D). Following w16-18TMEC, IgG2a and IgG3 were
the most expanded isotypes and IgG1 was the least (Fig. 5E).
7wIL12 + w16-18TMEC did not induce an obvious isotype
switch, but markedly expanded the production of all isotypes. As
these isotypes mediate antibody-dependent cellular cytotoxicity,
their ability to guide Spc against r-p185neu+TUBO cells was
assessed. Whereas no lysis is observed when TUBO cells are
incubated with sera from 7wIL12 or w16-18pcDNA3 mice, those
incubated with sera from 7wIL12 w16-18TMEC mice are lysed
18TMEC on the anti-r-p185neuB-cell
response. Expression of CD45R/
B220 B cell marker (red staining) in
spleens from 20-week-old untreated
(A), w16-18TMEC (B), and 7wIL12
+ w16-18TMEC mice (C). The
lymphoid white pulp is wider with
more expanded B-cell areas and
frequent GC in the spleen from w16-
18TMEC (B) and 7wIL12 + w16-
18TMEC (C) mice. D, anti-p185neu
antibody in the sera of w16-18TMEC
and 7wIL12 + w16-18TMEC mice.
The titer is significantly higher in the
latter group than in w16-18TMEC
electroporated mice, where the titer
was significantly higher (P < 0.0025
at week 24 and P < 0.0035 at week
35). Sera were tested at 1:100; final
results in Ag/mL have been adjusted
for dilution. E, isotypes of anti-
r-p185neuantibody in the sera (1:20)
18TMEC (green line), and 7wIL12 +
w16-18TMEC (red line) mice. F,
percentage of lysis of r-p185neutarget
cells in one of three independent
antibody-dependent cellular cyto-
toxicity tests done with pools of sera
different mice were used for each
Effect of 7wIL12 +w16-
Clinical Cancer Research 1947
in a significantly higher percentage (P < 0.006) than those
incubated with sera from w16-18TMEC (Fig. 5F).
To further assess the significance of antibodies in the
inhibition of Her-2 carcinogenesis, BALB-neuT mice with an
impaired ability to produce them (BALB-neuT/AKO mice; ref. 18)
received 7wIL12 + w16-18TMEC (Fig. 6). The kinetics of
mammary carcinogenesis in these mice is similar to that of
untreated BALB-neuT mice. Even so, 7wIL12 + w16-18TMEC
did not elicit any detectable anti-r-p185neuantibody responses (not
shown), nor did it inhibit the onset of carcinoma (Fig. 6A),
although it led to a marginal and temporary decrease of tumor
multiplicity (Fig. 6B).
Spleen Dendritic Cells and T-Cell Activation.
weeks of age, immunohistochemical examination of the semi-
serial sections of the spleen of untreated mice (Fig. 7A) or
w16-18pcDNA3 mice (not shown) displays CD11c+cells with
dendritic cell (DC) morphology, mainly at the edge of T-cell
areas. These DCs rarely express the B7.2 (CD86) activation
marker. 7wIL12 alone gives rise to a marked DC increment and
induces preferential CD86+DC localization at the edge of the
T-cell area (Fig. 7D), whereas the numerous DCs in the spleen
of w16-18TMEC are also scattered along the whole T-cell
areas. However, the CD86 activation marker is still preferen-
tially expressed by DC at the edge of the areas (Fig. 7E).
7wIL12 + w16-18TMEC mice synergistically increase the
number of CD86+DC at the edge and inside the T-cell areas
(Fig. 7J and K). CD40L is a CD4 T-cell activation marker
almost absent in the spleens of untreated (Fig. 7C), 7wIL12
antibody (BALB-neuT/AKO) and IFNg (BALB-neuT/IFN-gKO) production. BALB-neuT/AKO: percentage of tumor-free mice (A) and tumor
multiplicity (B) of tumors in untreated (o, 8 mice) and 7wIL12 + w16-18TMEC mice (., 9 mice) and in 7wIL12 + w16-18TMEC mice that received at
week 17 and 19 serum from BALB-neuT 7wIL12 + w16-18TMEC mice (!, 5 mice). Between weeks 22 and 23, 7wIL12 + w16-18TMEC mice
displayed a tumor multiplicity that is similar to that of untreated mice but significantly higher (P < 0.04) than that of 7wIL12 + w16-18TMEC mice that
additionally received immune serum. BALB-neuT/IFN-gKO: percentage of tumor-free mice (C) and tumor multiplicity (D) in untreated (o, 12 mice),
7wIL12 + w16-18TMEC mice (., 5 mice), and in 7wIL12 + w16-18TMEC mice that received at week 17 and 19 serum from BALB-neuT 7wIL12 +
w16-18TMEC mice (!, 5 mice). 7wIL12 + w16-18TMEC mice displayed a tumor multiplicity that is significantly lower of that of untreated mice (P <
0.0001-0.02) between weeks 19 and 39 but higher of that of 7wIL12 + w16-18TMEC mice that also received immune serum (P < 0.03) between weeks
23 and 25. Bars, SE. These experiments were done twice.
Effects of 7wIL12 + w16-18TMEC and the transfer of immune sera on the progression of carcinogenesis in BALB-neuT mice deficient for
Neoadjuvant IL-12 and DNA Vaccine Electroporation
(Fig. 7F), and w16-18pcDNA3 mice (not shown), but
frequently expressed in those of w16-18TMEC mice (Fig. 7I)
and even more in 7wIL12 + w16-18TMEC mice, where it is
mostly colocalized with CD86+DC (Fig. 7L).
When the production of IFN-g by Spc against p185neu+
TUBO cells was evaluated with the cytokine secretion assay
(Miltenyi Biotec), the frequency of both CD4+and CD8+cells
producing IFN-g was much higher in Spc from 7wIL12 + w16-
18TMEC (Table 1). This pattern of IFN-g release correlates with
their higher cytotoxic activity compared with all the other
To further assess the weight of IFN-g, BALB-neuT/
IFNgKO mice (13) received 7wIL12 + w16-18TMEC (Fig. 6).
The kinetics of mammary carcinogenesis in these mice is similar
of 20-week-old untreated and treated BALB-neuT mice. In untreated mice, CD11c-positive cells (DC) are present in the T-cell area and particularly at its
edge (A) where expression of the activation marker B7.2 (CD86; B) is rare. Activated T cells (CD40L+) are almost absent (C). 7wIL12 treatment
provokes a marked increment of DC, which are preferentially localized at the border of the T-cell area (D) and frequently CD86+(E). CD40L+are
scarce (F). In w16-18TMEC mice, DCs are more represented than in untreated mice and are found at the edge and scattered throughout the T-cell area
(G). The activation marker CD86 is preferentially expressed by the borderline DC (H). CD40L+are frequently present and scattered within the T area
(I). The combined treatment has a synergistic effect on the number and distribution of DC. They are impressively represented (J) and diffusely
activated (K), even in the inner zone of the T-cell area. CD40L+cells are numerous and scattered within the T area (L). Magnification: A, B, D, E,
G, H, J, K, ?400; C, F, I, L ?630). Three different mice were used for each treatment.
Effect of 7wIL12 + w16-18TMEC on DC and T-cell activation markers. Expression of CD11c, CD86, and CD40L (red staining) in the spleen
Effect of 7wIL12 + w16-18TMEC on the IFN-g production and cytotoxic activity
against p185neu+TUBO cells
7wIL12 treatment +
Percentage of lymphocytes
Cytotoxicity (LU20y) against p185neu+
34 F 6
10 F 3
23 F 7
29 F 7
21 F 2
7 F 1
37 F 7
27 F 3
27 F 1
8 F 1
16 F 15
30 F 4
23 F 4
7 F 1
73 F 15
71 F 16
0.8 F 0.50.5 F 0.210.9 F 341.1 F 7
*Mean F SE of the percentage of positive cells as assessed by three independent experiments of flow cytometry.
yMean F SE of LU20of Spc from three mice from each treatment group following specific in vitro stimulation (7wIL12 versus 7wIL12 + w16-
18TMEC, Student’s t test, P < 0.05).
Clinical Cancer Research 1949
to that of untreated BALB-neuT mice. This combination did not
delay the onset of carcinomas (Fig. 6C), but tumor multiplicity
was transiently reduced (Fig. 6D).
Whole mounts of the mammary
glands illustrate the stage of carcinogenesis in BALB-neuT
mice. This method was exploited as an intermediate end point to
assess the protection provided by the adoptive transfer of
antibodies and T lymphocytes. Serum or Spc T cells (90-92%
CD90+), isolated by magnetic cell sorting with the autoMACS
magnetic separator (Miltenyi Biotec), were collected from
groups of 20-week-old untreated control and from 7wIL12 +
w16-18TMEC mice. Recipient, otherwise untreated BALB-neuT
mice received serum i.p. and T cells i.v. on week 10 and 12 and
the whole mounts of their mammary glands were prepared at
week 16. The adoptive transfer of Tcells and (to a greater extent)
serum significantly inhibited carcinogenesis progression
(Table 2). The inhibition achieved by the combined transfer of
sera and Tcells was not significantly different from that obtained
with serum alone.
The substantial role of antibodies was also evident in
BALB-neuT/AKO and BALB-neuT/IFNgKO mice that received
the adoptive transfer of immune serum. In both lines of KO mice,
immune serum significantly reduced tumor multiplicity (Fig. 6).
Modulation of T Regulatory Cells.
T regulatory cells with suppressive activities are used by the
immune system to control its response to self-antigens (23), we
investigated their fluctuation during IL-12 treatment and
specific immunization. Twenty-week-old w16-18pcDNA3 mice
and w16-18TMEC have 14.06 F 0.49% and 14.16 F 0.33%
CD4+/CD25+cells. This number was slightly, but significantly,
decreased in 7wIL12 mice (12.65 F 0.132) and not further
decreased (12.597 F 0.129) by 7wIL12 + w16-18TMEC
In a preclinical model of multifocal mammary carcinomas,
the combination of IL-12 as a neoadjuvant therapeutic agent
followed by ‘‘immune-surgery’’ of the residual tumor cells
through DNA vaccine electroporation seems able to produce a
sustained cure. DNA vaccine electroporation during the early
stages of carcinogenesis protects BALB-neuT mice predestined
to die of mammary carcinomas (18), whereas later vaccination is
totally ineffective. However, combination of early systemic
administration of IL-12 with late electroporation provides
sustained control of invasive carcinomas, a result that has never
been even approached in this devastating model of autochtho-
nous cancer affecting all 10 mammary glands.
The curative potential of this combination seems to be the
outcome of the ability of IL-12 to both hamper early carcinogen-
esis and enhance the specific antitumor immune response (5, 10,
11, 24). The enhancement by IL-12 of the specific protective
response elicited by DNAvaccine electroporation fits in well with
its powerful adjuvant activity observed with a variety of vaccines
is an important mechanism whereby adjuvants exert their effects
(26, 27). IL-12 activates innate immune cells and promotes their
production of cytokines and chemokines, and thus mediates local
recruitment of cells of innate and adaptive immunity (28).
Furthermore, an IL-12-conditioned microenvironment is suitable
for APC activation, antigen presentation, and prevention (or
reversal) of the inductionof tolerance toward tumorantigens (29).
In addition, the delay of progression observed when
mammary glands display widespread atypical hyperplasia is in
line with IL-12-dependent impairment of tumor-driven angio-
genesis. The marked inhibition of vascular endothelial growth
factor production by 7wIL-12 treatment was somewhat expected,
as we have previously observed that IL-12-activated lymphoid
cells change gene and protein expression of Her-2 tumor cells
and down-modulate their angiogenic activity (8, 10, 11, 30). This
inhibition correlates with the dramatic inhibition of the network
of small vessels associated with mammary lesions and their
hampered progression in 7wIL12 mice. Early IL-12 treatment
seems to slow the progression of the incipient tumor and
consequently allows the immune response to be more effective
against a slow-growing tumor. The significance of tumor
angiogenesis as a prognostic indicator has been documented in
various kinds of human tumors (31, 32). Tumor vascularity is an
indicator of aggressiveness in breast cancer that significantly
correlates with its clinical and histologic grades (33).
We have previously shown that an antibody response (5, 8)
and production of IFN-g (13, 15) are essential for successful
preventive vaccination. Our present data indicate that combina-
tion of IL-12 with DNA electroporation does not obviate the
need for an active IFN-g and antibody response, but markedly
enhances the number of T cells that specifically release IFN-g in
the presence of Her-2+target cells, the number of spleen GC, the
titer of anti-Her-2 antibodies, and the antibody-dependent
cellular cytotoxicity. Parallel experiments on wild-type BALB/
c mice also showed that 7wIL12 + w16-18TMEC mice produced
an anti-Her-2 antibody titer 2-fold of that of mice that received
w16-18TMEC only (data not shown). Whereas many of the
Mammary carcinogenesis in recipient BALB-neuT mice to which T cells and serum from
7wIL12 + w16-18TMEC mice were adoptively transferred
Mean lesion size
(Am) F SD
246 F 32
169 F 37
102 F 43
127 F 51
Number of lesions
>300 Am F SD
14 F 12.8
3.1 F 1.7
1.16 F 0.75
1.33 F 1.2
T cells + serum
NOTE. Values of experimental groups are significantly different from control mice (P V 0.0002) and T-cell treatments are significantly different
from serum treatment (P V 0.003). At least 30 mammary glands of 16-week-old recipient BALB-neuT mice were evaluated in each treatment group. The
mean lesion sizes were measured by computer-assisted image analysis in at least 100 randomly selected points without lesions. The tumor index is the
product of the mean lesion size and the mean lesion number.
Neoadjuvant IL-12 and DNA Vaccine Electroporation
downstream factors triggered by systemic IL-12 activate several
immune reaction mechanisms and hamper tumor growth (28),
IL-12-dependent activation of DC may make a critical
contribution to an effective anti-Her-2 response (7). The DC
influx and activation at the edge of T-cell areas parallel an
increase in activated CD4+CD40L+Tcells, which release IFN-g
(and probably other cytokines), leading to GC formation and
expansions. Moreover, many features of BALB-neuT mice
indicate that they are tolerant to the rp185neu(18). Because
CD4+/CD25+Tcells with suppressive activities constitute one of
the mechanisms through which the immune system maintains
tolerance, we determined whether vaccination schedule reduce
their number. Mice treated with IL-12, both alone or in
combination with TMEC, display a slight but significant
decrease of CD4+/CD25+Spc that may contribute to the
generation of a good anti-p185neuimmune response.
Previous work with transplantable tumors has shown that
inhibition of tumor angiogenesis by IL-12 rests on its ability to
elicit secondary cytokines and tertiary chemokines and mono-
kines that activate the endothelial cells of neoformed tumor
vessels and facilitate leukocyte extravasation (10). These very
downstream factors activate innate immunity, enhance the
priming of adaptive responses, activate leukocyte subsets that
produce proinflammatory cytokines, and induce polymorphonu-
clear cells to destroy newly formed tumor vessels (28, 30).
Combination of distinct antiangiogenic mechanisms intrinsically
intermingled with IL-12 immunomodulatory activities leads to
the inhibition of both Her-2-dependent tumors in transgenic mice
and chemically induced carcinogenesis (24). The resulting delay
in tumor progression enables late DNA electroporation to be as
effective as early vaccination, as the burden of late mammary
lesions is confined to its initial stage. More than 60% of the
transgenic mice kept tumor-free by IL-12 in combination with
TMEC plasmid electroporation were able to reject a lethal
challenge of p185neu+syngenic TUBO cells done 15 weeks after
the last DNA vaccination, indicating that this combined
immunization also induces a significant and sustained memory
response (data not shown).
Early stimulation of innate immunity in humans equivalent
to that elicited by IL-12 in mice could delay the progression of a
diffuse preneoplastic lesion and allow identification of the target
antigens it expresses and the manufacturing of a specific vaccine.
Progression will be impeded and the protective potential of the
vaccine itself could be significantly enhanced.
We thank Prof. John Iliffe for editing the manuscript.
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