| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Immunology |
Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy [K. B., S. R., F. C., E. Q., G. F.]; Institute for Cancer Research, University of Bologna, 40126 Bologna, Italy [P. L. L., P. N.]; IST Biotechnology Satellite Unit of Bologna, 40126 Bologna, Italy [G. N.]; Department of Oncology and Neuroscience, G. d’Annunzio University, 66013 Chieti, Italy [E. D. C., P. M.]; and Genetics Institute, Cambridge, Massachusetts 02140 [S. W.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(2
, 4)
, tumor necrosis
factor 
(5)
, and granulocyte/macrophage
colony-stimulating factor (6)
. These secondary cytokines
then induce other downstream factors that trigger a complex antitumor
reaction. By acting on the endothelial cells of newly formed vessels,
these mediators inhibit tumor neoangiogenesis (7
, 8)
,
induce the expression of adhesion molecules, and recruit leukocytes at
the tumor site (7
, 9)
. They also favor the elicitation of
cytolytic effector cells and antitumor antibodies (3
, 7
, 10, 11, 12)
, whereas their presence in the tumor microenvironment
affects tumor cells directly by inducing the overexpression of MHC
glycoproteins (13)
and switching the production of
angiogenic factors to that of antiangiogenic factors (14)
. IL-12 also hampers the progression of both chemical-(15) and neu oncogene-dependent (16) carcinogenesis and would thus seem open to exploitation as a preventive agent (17) because genetic screening is singling out individuals with a defined genetic risk of cancer (18) , and preneoplastic lesions are being detected by early diagnosis programs (19) .
To determine the stage of mammary carcinogenesis in which IL-12 most successfully inhibits the progression of preneoplastic lesions into invasive tumors, we used females of two transgenic mouse strains expressing the rat HER2/neu oncogene in the mammary gland. Although temporally differentiated by their kinetics, these two models of progression through atypical hyperplasia to in situ carcinoma and invasive carcinomas closely reproduce a few features of mammary carcinogenesis in women (16) .
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
IL-12 Administration.
IL-12 (Genetics Institute, Cambridge, MA) in HBSS supplemented with
0.01% MSA (Sigma, St. Louis, MO) was administered i.p. At the times
indicated, mice received seven 5-day courses of MSA only (MSA controls)
or MSA plus IL-12. Other groups of mice remained untreated. Because no
appreciable differences in tumor growth rate and pathological findings
were found between the untreated mice and the MSA controls, only the
data of the latter group are shown. The first course consisted of 50 ng
of IL-12/day, and the subsequent six courses consisted of 100 ng of
IL-12/day. These seven courses were administered at different times
(Fig. 1
). BALB-neuT mice assigned to the chronic treatment group
received the first course at the 2nd week of age. From the 5th to the
25th week, courses were repeated every 4th week. Mice assigned to the
late treatment group received the courses from the 13th to the 25th
week. They were treated for 2 consecutive weeks, followed by 2 weeks
off. Mice in the early treatment group received IL-12 beginning at the
2nd week and ending at week 14. In a few experiments, the early
treatment was also performed with 10 and 2 ng in all seven courses.
FVB-neuN mice received the courses every 4th week, starting on the 6th
(6-week-old treatment), 22nd (22-week-old treatment), or 28th
(28-week-old treatment) week of age. All of these treatments continued
until week 90.
|
Statistical Analysis.
Differences in tumor incidence were evaluated by the Mantel-Haenszel
log-rank test; differences in tumor/mouse numbers, the number of
microvessels, and PCNA immunoreactive cells were evaluated by
Student’s t test.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Ref. 16
). Atypical hyperplasia of small lobular ducts and lobules is
already evident at the 2nd week of age. At the 10th week, proliferating
epithelial cells occlude the ductules and acini within the lobules.
Vigorous capillary proliferation is evident at the 15th week, when
atypical hyperplasia is prominent, often assuming the aspect of
carcinoma in situ (Fig. 2a
). Near the 20th week, the neoplastic ductular-lobular structures
progressively expand and invade the surrounding tissues, and at least
one palpable tumor mass is detectable around the 19th week (Fig. 3
, bottom panel). Invasive lobular carcinomas (Fig. 4a
) develop progressively, and at the 33rd week, tumor masses are palpable
in all 10 mammary glands.
|
|
|
). In
the chronic treatment, the courses started in the 2nd week and
continued until the 25th week. Both a delay in the onset of the first
mammary tumor and a 50% reduction in the number of mammary glands with
a palpable tumor at 33 weeks (when the experiment was ended) were
observed as compared with MSA controls (Fig. 3
). To assess
whether IL-12 is also effective during later phases, other mice were
first treated at the 13th week of age, when hyperplasia takes the form
of a carcinoma in situ. Courses continued until the 25th
week. This late treatment did not delay the onset of the first tumor
but did reduced the number of tumors at week 33 by 22%. The early
treatment began at the 2nd week and continued until week 14. The delay
in onset of the first tumor and the reduction in the number of tumors
are significantly higher than those seen in the chronic treatment
group. When the early treatment was further split into shorter 4-week
administration schedules, much less protection was observed (data not
shown).
Pathology of Mammary Lesions in BALB-neuT Mice.
A similarly widespread atypical hyperplasia of small lobular ducts and
lobules with multiple foci of carcinoma in situ was evident
at week 15 in the MSA controls and in the late treatment group that had
received two IL-12 courses only at that time. However, in the latter
group distinct vascular damage associated with few reactive cells close
to hyperplastic and neoplastic lobules was evident. Mice from the
chronic and early treatment groups revealed a less widely distributed
atypical hyperplasia. Rare foci of carcinoma in situ were
present in tissues from mice of the chronic treatment group, but not in
those from the early treatment group (data not shown). At week 25,
invasive carcinomas were present in the MSA controls (Fig. 4a
). At this time, the IL-12 regimens resulted in distinct
pathological features. Either in situ carcinomas or invasive
carcinomas were evident in the mammary glands of mice from the chronic
and late treatment groups (Fig. 4, b and c
). These
lesions were smaller and less widely distributed than those in MSA
controls and were even less pronounced in the chronic treatment group.
In contrast, a restrained atypical hyperplasia with foci of carcinoma
in situ only was evident in mice from the early treatment
group (Fig. 4d
).
Inhibition of Tumor Vasculature in BALB-neuT Mice.
This IL-12-induced delay of carcinogenesis closely fits the inhibition
of tumor angiogenesis as assessed by direct microvessel count (Table 1)
. At 15 weeks, mammary glands from the MSA controls displayed vigorous
capillary sprouts inside the atypical hyperplastic areas, whereas only
a few capillaries surrounded the foci of in situ carcinoma.
Minor vascular damage and inhibition of angiogenesis were evident in
mice from the late treatment group. In contrast, a defective vascular
network and a moderate reduction and a marked reduction of the number
of microvessels were evident in mice from the chronic and early
treatment groups (Fig. 2, a–c
). These differences diminished
markedly at the 25th week, when evident tumors were present in all
treatment groups (Table 1
; Fig. 2, d–f
).
|
.
Efficacy of Lower IL-12 Doses in BALB-neuT Mice.
Because IL-12 appears to effectively inhibit the progression of
HER2/neu carcinogenesis, the dose range in which such an
inhibition is achieved was evaluated. When early treatment was
performed using 10 and 50 times lower doses of IL-12, no delay in the
appearance of the first tumor or reduction of the number of mammary
glands with a palpable tumor was found, but a slight delay in tumor
onset was seen (Fig. 5
).
|
). In contrast, 28-week-old treatment was almost ineffective. It began
when focal hyperplasia and carcinoma in situ were already a
common finding.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Because these findings suggest that administration of IL-12 is of significance in hampering the progression of preneoplastic lesions, the specific issue addressed here was to define the stage of tumor progression in which these mechanisms are most effective. Should IL-12 administration be proposed as a preventive measure in healthy individuals with genetic risk of cancer patients, or can it also be of benefit once overt preneoplastic lesions are diagnosed? This is a significant question because genetic screening programs are singling out healthy individuals with genetic risk of cancer (18) , and early diagnosis programs are detecting preneoplastic lesions (19) .
As a result of the activated neu transgene, BALB-neuT mice display mammary cell atypia virtually from birth. The efficacy of IL-12 treatments in these mice suggests that the evolution of the tumor:host angiogenic relationship, rather than the intrinsic proliferative properties of transformed mammary cells, is the point of no return for IL-12 activity. In effect, the present findings suggest that at least part of this activity is due to the ability of IL-12 to inhibit the angiogenesis associated with mammary hyperplasia.
Around the 2nd week, almost all mammary glands of BALB-neuT mice display multiple foci of ductular atypical hyperplasia. Between the 13th and 17th weeks, hyperplasia progresses to in situ carcinoma (Ref. 16 ; present study). Immunohistochemical staining with anti-CD31 monoclonal antibody shows that rich microvascularization inside preneoplastic lesions corresponds with their progression toward carcinoma, as shown in other tumor systems (23) . This progression phase appears to be particularly appropriate for an angiostatic intervention (24 , 25) . Indeed, the most significant delay in tumor onset and progression is observed with the early treatment, when IL-12 courses given from the 2nd to the 14th week induced both a scanty vascularization and poorly developed hyperplastic foci.
The importance of the timing of IL-12 administration was further assessed with FVB-neuN mice, in whom an overexpressed neu proto-oncogene induces mammary carcinomas after a markedly longer latency. The 6-week-old treatment consists of a lifetime administration of IL-12 and is conceptually similar to the chronic treatment of BALB-neuT mice. Although the first course was markedly delayed on the 22-week-old treatment, it still started before an evident spreading of preneoplastic lesions. Both treatment schedules delay the onset of carcinomas and their multiplication. The period between the 22nd and the 28th week appears to be of critical importance because the 28-week-old protocol confers only a negligible protection. During these 6 weeks, in fact, normal mammary glands progress toward atypical hyperplasia and then toward carcinoma in situ and invasive carcinoma. Palpable tumors are first detected at 30 weeks.
The equivalent results from BALB-neuT and FVB-neuN mice suggest that
IL-12 effectively inhibits mammary carcinogenesis when its
administration accompanies the angiogenic switch. Its antiangiogenic
effect appears to rest on the increased serum levels of IFN- and
tumor necrosis factor- released by activated T lymphocytes and
natural killer cells (5
, 7)
. The antiangiogenic (4
, 8)
and angiotoxic (26)
activity of these two
cytokines is stronger on those fragile capillary sprouts, which go with
the shift from the preneoplastic to the neoplastic condition.
Downstream mediators elicited by IL-12 may also act on neoplastic
cells, in which they down-regulate the production of proangiogenic
molecules (7
, 27)
and up-regulate the release of
antiangiogenic factors such as IFN-inducible protein 10 and monokine
induced by IFN- (7
, 14)
. After the transition from
hyperplasia to in situ and invasive carcinoma, capillary
sprouting becomes restrained. The poor efficacy of late treatment in
both BALB-neuT and FVB-neuN mice may depend on the lower sensitivity of
mature and differentiated blood vessels of the more advanced neoplastic
lesions to IL-12-induced angiostasis.
The decreased number of microvessels per microscopic field in both in situ and invasive carcinoma in comparison to hyperplastic areas suggests that this type of carcinoma, once developed, no longer requires a profuse vascular supply. The few vessels of the stroma of neoplastic lobular-alveolar structures are enough to sustain their relatively low rate of proliferation. In contrast, blood supply is a critical factor for most fast-growing transplantable tumors, even during their later stages. This necessity may account for the high efficacy of IL-12 against these tumors, even when they are large (3 , 7) . With tumors that progress slowly, antiangiogenic activity is only efficacious in specific progression stages (24) . This narrow window of activity might account for the ineffectiveness of IL-12 in the management of human cancer, because only patients bearing advanced tumors are enrolled in clinical trials (28) .
The antitumor action of IL-12 is not confined to its indirect influence on endothelial cells. Directly or through secondary cytokines, its triggers lytic activity and mediator release in a variety of tumor-infiltrating leukocytes, thus offsetting the continuous generation of new transformed cells (7 , 10, 11, 12) . The efficacy of the hampering of tumor progression by IL-12 probably rests on the sum of its activities and not simply on the blocking of tumor neoangiogenensis, as important as this may be. In effect, further subdivision of the early protocol in shorter treatment periods markedly reduced IL-12 efficacy (data not shown).
The lower efficacy of chronic versus early treatment could indicate that continuous IL-12 administration is suppressive (29) , although this possibility is not endorsed by the results in FVB-neuN mice. It should be noted that from the second course, BALB-neuT and FVB-neuN mice received 100 ng/day IL-12 (i.e., around 4.5–7.7 µg/kg). This dose is well tolerated, and almost no side effects were manifested (7 , 16) . It is probably close to the optimal active dose, because a 10- or 20-fold reduction abolishes its activity.
In conclusion, our data suggest that IL-12 effectively impairs the neu oncogene-driven progression of mammary carcinogenesis by interfering with the passage from atypical hyperplasia to invasive carcinoma. This interference appears to depend largely on indirect inhibition of tumor-associated angiogenesis. Its diminished efficacy in more advanced lesions and the dose range required pose some constraints on the use of IL-12 as an immunological alternative to current management of already manifest neoplastic lesions. Nevertheless, the efficacy of IL-12 points to enhancement of nonspecific immunity as an effective way to prevent mammary tumors in individuals at risk. Lifetime administration is not required for genetically determined cancers with a long natural history; instead, a precise definition of the carcinogenic events may allow preventive treatments starting relatively late in the life of individuals at risk.
ACKNOWLEDGMENTS
We thank Prof. John Iliffe for editing the manuscript.
|
FOOTNOTES |
|---|
1 Supported by the Italian Association for Cancer
Research, the Istituto Superiore di Sanità, Special project gene
therapy, CNR Target project on Biotechnology, University of Bologna
(fund for selected research topics), Ministero dell’Università e
della Ricerca Scientifica, and by the Department of the Army, USA,
Grant DAMD17-98-1-8030 (to G. F.). "The information contained in
this paper does not necessarily reflect the position or the policy of
the U.S. government, and no official endorsement should be inferred. 
2 To whom requests for reprints should be
addressed, at the Dipartimento di Scienze Cliniche e Biologiche,
Ospedale San Luigi Gonzaga, 10043 Orbassano, Italy. Phone:
39-011-670-8118; Fax: 39-011-903-8639; E-mail: forni{at}sluigi.unito.it
3 The abbreviations used are: IL, interleukin;
MSA, mouse serum albumin; MMTV, mouse mammary tumor virus; PCNA,
proliferating cell nuclear antigen.
Received 8/11/99. Accepted 11/12/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
production. J. Immunol., 153: 1697-1706, 1994.[Abstract]
-independent rejection of interleukin 12-transduced carcinoma cells requires CD4+ T cells and granulocyte/macrophage colony-stimulating factor. J. Exp. Med., 188: 133-143, 1998.-interferon gene. Int. J. Cancer, 55: 320-329, 1993.[Medline]
affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity, 9: 25-34, 1998.[Medline]
. Nat. Med., 4: 408-414, 1998.[Medline]
induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of IL-12 vaccine adjuvant effect. J. Exp. Med., 188: 1603-1610, 1998.This article has been cited by other articles:
|
|
 |
|
  P. Nava-Parada, G. Forni, K. L. Knutson, L. R. Pease, and E. Celis Peptide Vaccine Given with a Toll-Like Receptor Agonist Is Effective for the Treatment and Prevention of Spontaneous Breast Tumors Cancer Res., February 1, 2007; 67(3): 1326 - 1334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Pannellini, M. Spadaro, E. Di Carlo, E. Ambrosino, M. Iezzi, A. Amici, P. L. Lollini, G. Forni, F. Cavallo, and P. Musiani Timely DNA Vaccine Combined with Systemic IL-12 Prevents Parotid Carcinomas before a Dominant-Negative p53 Makes Their Growth Independent of HER-2/neu Expression. J. Immunol., June 15, 2006; 176(12): 7695 - 7703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Holmgren, E. Ambrosino, O. Birot, C. Tullus, N. Veitonmaki, T. Levchenko, L.-M. Carlson, P. Musiani, M. Iezzi, C. Curcio, et al. A DNA vaccine targeting angiomotin inhibits angiogenesis and suppresses tumor growth PNAS, June 13, 2006; 103(24): 9208 - 9213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. I. Garbe, B. Vermeer, J. Gamrekelashvili, R. v. Wasielewski, F. R. Greten, A. M. Westendorf, J. Buer, R. M. Schmid, M. P. Manns, F. Korangy, et al. Genetically Induced Pancreatic Adenocarcinoma Is Highly Immunogenic and Causes Spontaneous Tumor-Specific Immune Responses Cancer Res., January 1, 2006; 66(1): 508 - 516. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Zerafa, J. A. Westwood, E. Cretney, S. Mitchell, P. Waring, M. Iezzi, and M. J. Smyth Cutting Edge: TRAIL Deficiency Accelerates Hematological Malignancies J. Immunol., November 1, 2005; 175(9): 5586 - 5590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Spadaro, E. Ambrosino, M. Iezzi, E. Di Carlo, P. Sacchetti, C. Curcio, A. Amici, W.-Z. Wei, P. Musiani, P.-L. Lollini, et al. Cure of Mammary Carcinomas in Her-2 Transgenic Mice through Sequential Stimulation of Innate (Neoadjuvant Interleukin-12) and Adaptive (DNA Vaccine Electroporation) Immunity Clin. Cancer Res., March 1, 2005; 11(5): 1941 - 1952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Croce, R. Meazza, A. M. Orengo, L. Radic', B. De Giovanni, C. Gambini, B. Carlini, V. Pistoia, L. Mortara, R. S. Accolla, et al. Sequential Immunogene Therapy with Interleukin-12- and Interleukin-15-Engineered Neuroblastoma Cells Cures Metastatic Disease in Syngeneic Mice Clin. Cancer Res., January 15, 2005; 11(2): 735 - 742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Satoh, T. Saika, S. Ebara, N. Kusaka, T. L. Timme, G. Yang, J. Wang, V. Mouraviev, G. Cao, E. M. A. Fattah, et al. Macrophages Transduced with an Adenoviral Vector Expressing Interleukin 12 Suppress Tumor Growth and Metastasis in a Preclinical Metastatic Prostate Cancer Model Cancer Res., November 15, 2003; 63(22): 7853 - 7860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hayakawa, S. Rovero, G. Forni, and M. J. Smyth {alpha}-Galactosylceramide (KRN7000) suppression of chemical- and oncogene-dependent carcinogenesis PNAS, August 5, 2003; 100(16): 9464 - 9469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Lee, D. C. Kim, M. S. Gee, H. M. Saunders, C. M. Sehgal, M. D. Feldman, S. R. Ross, and W. M. F. Lee Interleukin-12 Inhibits Angiogenesis and Growth of Transplanted but not in Situ Mouse Mammary Tumor Virus-induced Mammary Carcinomas Cancer Res., February 1, 2002; 62(3): 747 - 755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Nanni, G. Nicoletti, C. De Giovanni, L. Landuzzi, E. Di Carlo, F. Cavallo, S. M. Pupa, I. Rossi, M. P. Colombo, C. Ricci, et al. Combined Allogeneic Tumor Cell Vaccination and Systemic Interleukin 12 Prevents Mammary Carcinogenesis in HER-2/neu Transgenic Mice J. Exp. Med., October 29, 2001; 194(9): 1195 - 1206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Cifaldi, E. Quaglino, E. Di Carlo, P. Musiani, M. Spadaro, P. L. Lollini, S. Wolf, K. Boggio, G. Forni, and F. Cavallo A Light, Nontoxic Interleukin 12 Protocol Inhibits HER-2/neu Mammary Carcinogenesis in BALB/c Transgenic Mice with Established Hyperplasia Cancer Res., April 1, 2001; 61(7): 2809 - 2812. [Abstract] [Full Text]
|
|
|
|
|
|
M. Strasly, F. Cavallo, M. Geuna, S. Mitola, M. P. Colombo, G. Forni, and F. Bussolino IL-12 Inhibition of Endothelial Cell Functions and Angiogenesis Depends on Lymphocyte-Endothelial Cell Cross-Talk J. Immunol., March 15, 2001; 166(6): 3890 - 3899. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rovero, A. Amici, E. D. Carlo, R. Bei, P. Nanni, E. Quaglino, P. Porcedda, K. Boggio, A. Smorlesi, P.-L. Lollini, et al. DNA Vaccination Against Rat Her-2/Neu p185 More Effectively Inhibits Carcinogenesis Than Transplantable Carcinomas in Transgenic BALB/c Mice J. Immunol., November 1, 2000; 165(9): 5133 - 5142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Forni, P. L. Lollini, P. Musiani, and M. P. Colombo Immunoprevention of Cancer: Is the Time Ripe? Cancer Res., May 1, 2000; 60(10): 2571 - 2575. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, weeks in which mice received 5-day
courses (Monday through Friday) of daily i.p. injections of IL-12 or
MSA only during the progression of HER2/neu mammary
carcinogenesis.
