modulators of arginine metabolism do not impact on peripheral t … · 2011. 1. 19. · - 1 -...

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Modulators of arginine metabolism do not impact on peripheral T-cell tolerance and disease progression in a model of spontaneous prostate cancer

Nicolò Rigamonti1,2,3, Giusy Capuano1,3, Alessia Ricupito1,2, Elena Jachetti1,2, Matteo Grioni1, Luca Generoso1, Massimo Freschi4, Matteo Bellone1,2,5

1PIBIC, Department of Immunology Infectious Diseases and Transplantation, Istituto

Scientifico San Raffaele, Milano, Italy

2Univeristà Vita-Salute San Raffaele, Milano, Italy

4Unità Operativa Anatomia Patologica, Istituto Scientifico San Raffaele, Milano, Italy

3These authors contributed equally to this work.

Running title: arginine metabolism and prostate cancer

Key words: rodent, T cell, monocytes/macrophages, nitric oxide,

tolerance/suppression, tumor immunity, vaccination, dendritic cells

Grant sponsor: Associazione Italiana per la Ricerca sul Cancro; Ministero della salute;

GC has been awarded a scholarship from the American-Italian Cancer Foundation.

5Correspondence to: Matteo Bellone, Cellular Immunology Unit, 3P-A1, Dibit,

Istituto Scientifico San Raffaele, Via Olgettina 58, 20132, Milan, Italy. Phone: 39-02-

2643-4789; Fax: 39-02-2643-4786; E-mail: [email protected]

Research. on April 23, 2021. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 19, 2011; DOI: 10.1158/1078-0432.CCR-10-2547

http://clincancerres.aacrjournals.org/

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Statement of translational relevance

Therapeutic strategies are urgently needed to overcome the immunosuppressive tumor

microenvironment. Perturbation of the arginine metabolism has been proposed as one

such mechanism, which also favors carcinogenesis. We have investigated the

therapeutic potential of modulators of the arginine metabolism, among which

sildenafil that is already used in clinical trials, in realistic mouse models of prostate

cancer. We have found that these drugs, at concentration known to be effective in

models of transplantable cancer, neither broke tumor-specific T cell tolerance, nor

restrained spontaneous prostate cancer progression. Further investigation is needed to

fully appreciate the therapeutic potential of modulators of the arginine metabolism in

cancer.




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ABSTRACT

Purpose. Chronic inflammation, recruitment of myeloid derived cells and

perturbation of the arginine metabolism have been all proposed as mechanisms

favoring prostate carcinogenesis and tumor immunoescape. Objective of this study

was to evaluate whether accumulation of CD11b+Gr1+ cells, also defined myeloid

derived suppressor cells (MDSC), occur in mice affected by transplantable or

spontaneous prostate cancer. We also investigated whether N(G) nitro-L-arginine

methyl ester (L-NAME) and sildenafil, both modulators of the arginine metabolism,

restrain tumor growth and restore tumor-specific immunity.

Experimental design. Wild type C57BL/6 mice bearing TRAMP-C1 prostate cancer

and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were treated

with vehicle, L-NAME or sildenafil, and evaluated for CD11b+ cells accumulation in

the blood, several organs and the tumor mass, and for disease progression.

Results. CD11b+Gr1high, CD11b+Gr1int and CD11b+Gr1- cells differently accumulated

in different organs and especially in the tumor of the two mouse models. L-NAME

and sildenafil impaired the immunosuppressive function of CD11b+ cells in both

models and restrained TRAMP-C1 growth, but they did neither break tumor-specific

immune tolerance nor limit tumor progression in TRAMP mice.

Conclusions. Collectively, our results emphasize substantial differences in tumor-

induced alteration of myelopoiesis and sensitivity to modulators of the arginine

metabolism between a transplantable and a spontaneous model of prostate cancer.

They also suggest that perturbation of the arginine metabolism is dispensable for

prostate cancer progression and the associated T cell tolerance.




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INTRODUCTION

The pathogenesis of prostate cancer (PC), the second cause of death for neoplasia

among men worldwide (1), has been linked to chronic inflammation (2). PC may also

represent a site of acquired immune privilege (3), where several mechanisms suppress

both locally and systemically the tumor-specific immune response (4). Among these

mechanisms, myeloid derived suppressor cells (MDSC) (5) have been recently the

focus of intense investigation (6). MDSC are a heterogeneous population of cells of

myeloid origin that include immature macrophages, granulocytes, dendritic cells (DC)

and other myeloid cells (6, 7). Recruitment of MDSC to peripheral organs under

pathologic conditions is mediated by several soluble factors, among which IL-3, IL-6,

IL-10, vascular endothelial growth factor (VEGF), macrophage colony-stimulating

factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (5,

7, 8). In mice, MDSC are characteristically CD11b+, express the Gr-1 antigen at

different levels, and may also express CD31, IL-4 receptor α-chain, CD115 and CD80

(5, 9). The phenotype of human MDSC is ill defined (6, 9), and a population of

CD14+HLA-DRlow/- MDSC cells has been recently observed in the peripheral blood of

PC patients that significantly correlated with circulating PSA levels (10).

MDSC may overexpress both inducible nitric oxide synthase (iNOS) and

arginase 1 (Arg1), enzymes involved in the metabolism of arginine. As reviewed in

(6), depletion of arginine from the microenvironment inhibits T cell activation and

proliferation, and favors T cell apoptosis. Furthermore, iNOS produces nitric oxide

(NO), which interferes with IL-2 receptor signaling, leading to cell cycle arrest.

Reactive oxygen species (ROS) and peroxynitrites, bioproducts of arginine

metabolism, contribute to T cell inhibition. Of relevance, T cells infiltrating either

human PC or the prostate of transgenic adenocarcinoma of the mouse prostate




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(TRAMP) mice, a primary model of spontaneous PC (11), are functionally impaired

and contain high level of nitrotyrosines, suggesting in loco peroxynitrites production

(12).

Tumors are infiltrated also by tumor-associated macrophages (TAM), that in

mice are CD11b+Gr1-, may derive from circulating MDSC and exert

immunosuppressive functions (7, 9). TAM surrounding mouse prostate intraepithelial

neoplasia (mPIN) and well- or moderately differentiated PC in TRAMP mice are

Arg1highiNOSlow, indicating M2 polarization (13). A mixed M1/M2 infiltrate

(Arg1highiNOShigh) is characteristic of poorly differentiated PC in this model.

Interestingly, both Arg1 (12, 14) and iNOS (12, 15) are overexpressed by

epithelial PC cells, and the enzymatic activity of Arg is increased in PC patients (16),

suggesting their role in sustaining the high demand of polyamines by the growing

tumor tissue (14). NO is also a key signaling molecule in cancer, where it participates

to cancerogenesis, angiogenesis, tumor cell proliferation and invasion (17).

Several strategies have been proposed to inhibit Arg and NOS (6). N(G)-

monomethyl-L-arginine can restore the lytic function of PC infiltrating T cells in vitro

(12), but its use in clinic has been discontinued due to severe toxicity (18). In vitro,

N(G) nitro-L-arginine methyl ester (L-NAME) causes a significant transcriptional

downregulation of eNOS that is overexpressed in endothelial cells associated with PC

(19), but can also inhibit Arg activity (20). L-NAME has been reported in several

transplantable models to inhibit tumor growth [e.g.,(21, 22)] and improve the

endogenous tumor-specific immune response (22). Also phosphodiesterase-5 (PDE5)

inhibitors (sildenafil, tadalafil and vardenafil) can modify arginine metabolism by

inhibiting the degradation of cyclic guanosine monophosphate (cGMP) to GMP.

Sildenafil in vivo increases cGMP and down-regulates iNOS and Arg1 expression and




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reduces the enzymatic activity in the intratumoral MDSC. Sildenafil also favors T cell

immunity and delays tumor progression (22, 23). It is not known however, the in vivo

effect of these treatments in models of spontaneous tumor development that more

closely recapitulate the human pathology.

We investigated here whether accumulation of CD11b+ cells occurs in mice

bearing transplantable PC and in TRAMP mice, and whether treatment with L-NAME

or sildenafil restores antitumor immunity and delays tumor growth.




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MATERIALS AND METHODS

Mice, cell lines and reagents. Heterozygous C57BL/6 TRAMP mice and wild type

(WT) mice were housed and bred in a specific pathogen free animal facility, treated in

accordance with the European Union guidelines, and with the approval of the

Institutional Ethical Committee. Animals were typed for Tag expression by PCR-

based screening assay, as described (24). TRAMP-C1 cells (25) were cultured in

Dulbecco’s Modified Eagle Medium (DMEM, Cambrex, Verviers, Belgium)

supplemented with 10% fetal bovine serum (FBS; Invitrogen, Milan, Italy), 150 U/ml

streptomycin and 200 U/ml penicillin (Cambrex, Charles City, IA). B6/K-0, a kidney

cell line expressing Tag (26), and B6/K-1,4,5 cells, which lack the Tag epitopes I,

II/III, IV and V (27), were a generous gift of Dr. S.S. Tevethia (The Pennsylvania

State University College of Medicine, Hershey). RMA is a H-2b Rauscher virus-

induced thymoma (28). These cell lines were cultured in RPMI-1640 (Invitrogen)

supplemented with 2 mM L-glutamine, 150 U/ml streptomycin, 200 U/ml penicillin

and 10% heat-inactivated FBS. Unless specified, all chemical reagents were from

Sigma-Aldrich, and monoclonal antibodies (mAb) were from BD PharMingen (San

Diego, CA).

In vivo experiments. A schematic representation of the in vivo experiments is reported

in Supplementary Fig.1. Six-8 wk-old C57BL/6 male mice were challenged

subcutaneously (s.c.) with 2.5 x 106 TRAMP-C1 cells. L-NAME (added in drinking

water at 1g/L) or sildenafil (0.7 g/L; Pfizer, New York, NY; a generous gift of Drs. G.

Da Pozzo and A. Salonia, San Raffaele Scientific Institute, Milan, Italy) were

administered starting on the day of tumor challenge. As control, groups of animals

were treated with vehicle (PBS) only. Water was given ad libitum. Calculation of the

dosage of the drug was based on the assumption that a mouse drinks approximately 3




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ml/24h. Mice were monitored twice a wk and sacrificed after 50 days. Animals were

weighted and tumor size was evaluated both by measuring its weight and two

perpendicular diameters by a caliper. Cohorts of 13 or 21 wk-old TRAMP and WT

mice were randomly assigned to either one of the following 3-wk treatments: L-

NAME (1 g/L), sildenafil (0.13 or 0.7 g/L) or vehicle dissolved in drinking water. At

the end of the second wk, all mice were vaccinated i.d. with bone marrow (BM)

derived DC [5 x 105/mouse; (29)] pulsed with 2 μM of the immunodominant

cytotoxic T lymphocyte (CTL) epitope Tag-IV [sequence 404-411; Research

Genetics, Huntsville, AL; ref. (30); DC/Tag-IV]. At the end of the third and last wk,

animals were killed. Animals and their urogenital apparatus (UGA) were weighted.

UGA were processed for histology and immunohistochemistry, and scored on coded

samples by a pathologist as previously described (31).

Phenotypic characterization of cell populations. Single cell suspensions of tumor

draining lymph nodes (TDLN) and non TDLN (NDLN) from tumor-challenged and

naïve mice, respectively, were stained with FITC-labeled anti-CD4, PE- labeled anti-

CD44 and PerCP-Cy 5.5-labeled anti-CD8 mAb. Dead cells were excluded by

physical parameters and/or by the addition of ToPro5 (Molecular Probes, Eugene,

OR) immediately before flow cytometry analysis. For enumeration of CD11b+ cells,

blood and cells from spleen, BM, NDLN, TDLN and tumor samples of naïve and

tumor-challenged mice were incubated with Fc blocking mAb and stained with FITC-

conjugated CD11b and APC-conjugate Gr1 mAb. Dead cells were excluded by

propidium iodide. For enumeration of CD4+CD25+Foxp3+ cells, LN cells were stained

with FITC-labeled anti-CD4, PerCP-Cy 5.5-labeled anti-CD8 and APC-labeled anti-

CD25 (clone PC61) mAb, permeabilized and finally, stained with PE-labeled anti-

Foxp3 mAb (eBioscience, SanDiego, CA) according to the manufacturer’s




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instructions. In all experiments, cells were analyzed on a BD FacsCalibur® or

FacsCanto®.

In vitro cytotoxicity assay. Splenocytes or magnetic bead-purified CD8+ cells

(Miltenyi Biotec, Bergisch Gladbach, Germany) were re-stimulated in vitro in the

presence of irradiated B6/K-0 cells (10:1 ratio). Irradiated splenocytes (1:1 ratio) were

added to the CD8+ cell culture. Day-5-blasts were tested for cytolytic activity in a

standard 4 h 51Cr release assay (29). 51Cr release of target cells alone was always <

25% of maximal 51Cr release (target cells in 0.25 M SDS).

CD11b+ cell purification and in vitro functional assays. CD11b+ cells purification

was performed with mouse CD11b MicroBeads (Miltenyi Biotec) following the

manufacturer’s instructions. Purity of the cell population was evaluated by flow

cytometry and exceeded 90%. C57BL/6 splenocytes (3 x 105 cells/well) were

stimulated in wells that had been coated with 3 μg/ml anti CD3 and 2 μg/ml anti-

CD28 mAb or in the presence of CD3/CD28 beads (2 μl/8 x 104 splenocytes;

Invitrogen, Karlsruhe, Germany). Purified splenic CD11b+ cells were added to the

culture so as to constitute 1-20% of the total cells. After 3 days of incubation,

cultures were pulsed with 3H-Thymidine (1 μCi/well; Amersham Corp., Milan, Italy)

for the last 18 h. The incorporation of 3H-Thymidine by proliferating T cells

(triplicate cultures) was measured by scintillation counting.

Arg and iNOS detection. Immunomagnetic beads-purified CD11b+ cells or tumor cells

were lysed for 10 min with lysis buffer containing 0,15 mM pepstatin A, 0,2 mM

leupeptin and 0,4% Triton x-100. Samples were centrifuged at 20000 x g at 4 °C for

10 min. The supernatant was used for Arg assay using QuantiChrom Arginase Assay

Kit (DARG-200, Gentaur, Bruxelles, Belgium) according to the manufacturer’s

instructions. Results were normalized on 106 cells. Alternatively, CD11b+ cells were




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assessed ex vivo for intracellular iNOS upon stimulation with LPS (1μg/ml) for 24 h.

Brefeldin A was added during the last 5 h of pulsing. Cells were fixed in 2%

paraformaldehyde, permeabilized, incubated with polyclonal rabbit anti-iNOS

antibody (Santa Cruz, Santa Cruz, CA) at a 1:200 dilution for 30 min at room

temperature, and finally analyses by flow cytometry.

Statistical analyses. Statistical analyses were performed using the Log-rank, Student’s

T, ANOVA and Newman-Keuls tests. Values were considered statically significant

for p < 0.05.




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RESULTS

Effects of modulators of arginine metabolism on myelopoiesis and growth of

TRAMP-C1 tumors in vivo

We initially investigated the effect of tumor growth on myelopoiesis and recruitment

of CD11b+ cells to peripheral organs in the TRAMP-C1 model. TRAMP-C1 is a

hormone-independent PC cell line derived from a TRAMP tumor (25), and widely

used to assess novel therapeutic approaches for PC. Interestingly, TRAMP-C1 tumors

in vivo are infiltrated by CD11b+ cells, and iNOS mRNA is barely detectable, whereas

Arg1 mRNA levels are high and persist as tumor grows (32). Hence, tumor cells were

injected s.c. in C57BL/6 males, and the same day mice were randomly assigned to a

treatment with either L-NAME or vehicle added to the drinking water (Supplementary

Fig. 1A). We choose this treatment because it demonstrated to be highly effective in

restraining growth of other subcutaneous tumors in C57BL/6 mice (22). Animals were

killed 50 days later, when the tumor mass in vehicle-treated mice had reached the

dimension of approximately 150 mm2. Flow cytometry analysis of the blood showed a

dramatic accumulation of CD11b+Gr1high cells in vehicle-treated mice (Fig. 1B) when

compared with naïve age- and sex-matched littermates (Fig. 1A). Quantification of

CD11b+Gr1high cells demonstrated a statistically significant increase in tumor-bearing

mice (Fig. 1D) that was marginally restrained by L-NAME treatment (Fig. 1C and D).

At difference with other transplantable tumor models (22), we did not find a relevant

accumulation of CD11b+Gr1int and CD11b+Gr1- cells in the blood of mice affected by

TRAMP-C1 tumors (Fig 1B and D). Since the blood may not be fully representative

of myelopoiesis and MDSC accumulation during tumor growth, we quantified MDSC

in lymphoid organs and tumors of untreated and L-NAME treated mice. An

accumulation both as percentage and absolute number of CD11b+Gr1high cells was




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evident also in the spleen (Fig. 1D and Supplementary Fig. 2, respectively) and tumor

samples (Fig. 1D and Supplementary Fig. 2, respectively) of tumor-bearing mice.

Immunofluorescence on TRAMP-C1 tumor sections confirmed that CD11b+ cells

infiltrating the tumor tissue were mostly Gr1+ (Supplementary Fig. 3). The BM was

already rich in CD11b+Gr1high cells that further increased in tumor-bearing mice (Fig.

1D). A marginal increase of CD11b+Gr1high cells was also evident in TDLN, where

the mature CD11b+Gr1- cells dominated and further increased in mice affected by

TRAMPC-1 tumors (Fig. 1D and Supplementary Fig. 2). The CD11b+Gr1int

population was clearly enriched only in the spleen (Fig. 1D and Supplementary Fig.

2). L-NAME treatment did not significantly modify MDSC accrual in the organs

examined except for NDLN and TDLN, where the % and number of CD11b+Gr1int

cells declined (Fig. 1 and Supplementary Fig. 2).

Accumulating CD11b+ cells were indeed MDSC, since CD11b+ cells purified

from the spleen of tumor-bearing mice inhibited the in vitro proliferation of

splenocytes in a dose-dependent manner (Fig. 2A). Hence, MDSC accumulation is a

characteristic also of subcutaneous hormone-independent PC. Of relevance, treatment

with L-NAME reduced the immunosuppressive and Arg1 enzymatic activities of both

CD11b+ splenocytes (Fig. 2A) and cells collected from the tumor mass (Fig. 2B).

Since, TDLN rapidly enlarge owing to the accumulation of myeloid and

lymphoid cells (33), we asked whether arginine metabolism inhibitors could alter

recruitment of T lymphocytes in TDLN. In C57BL/6 mice affected by TRAMP-C1

tumors, a 3-fold increase in cell number was evident in TDLN when compared with

LN from naïve mice (Supplementary Figs. 2 and 4). Flow cytometry analysis of

TDLN cells showed a significant increase in the number of both CD4 and CD8 cells

(Supplementary Fig. 2). As seen in other transplantable tumor models (22), the




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percentage of CD8 and more evidently of CD4+ T cells (Supplementary Fig. 4) in

TDLN decreased in both L-NAME and vehicle-treated mice, suggesting that in TDLN

a relevant perturbation of the physiologic equilibrium among the different cell

populations was undergoing, that was not modified by L-NAME. Although MDSC

can favor induction of Treg in tumor-bearing hosts (34), in the TDLN of these mice

we found a slightly, yet not significant increase of CD4+CD25+Foxp3+ cells, which

was not modified by L-NAME treatment (Supplementary Fig. 4).

Finally, subcutaneous tumors were measured and compared in vehicle and L-

NAME treated animals. At day 35, the tumor volume and area (Fig. 2C) in L-NAME-

treated mice was half of those measured in vehicle-treated animals. To confirm this

therapeutic effect, vehicle and L-NAME-treated mice were followed in a survival

experiment. As reported in Fig. 2D, when all vehicle-treated mice were dead because

of the tumor outgrowth, more than 50% of the tumor-bearing mice treated with L-

NAME were still alive, and their overall survival was significantly increased. As a

measure of potential drug-related toxicity, we monitored animal weight, and we found

no differences between drug- and vehicle-treated mice (Fig. 2C). Also, as previously

reported (22), this treatment did not cause macroscopic and microscopic damage to

the liver, kidney, respiratory apparatus and esophagus (data not shown). All together,

these data suggest that in mice bearing subcutaneous PC, L-NAME treatment is able

to modulate the arginine metabolism both in periphery and at the tumor site, reduces

the immunosuppressive activity of MDSC and restrains tumor growth.

C57BL/6 mice challenged with TRAMPC-1 cells were also treated with

sildenafil (Supplementary Fig. 1B). This drug was initially used at 20 mg/Kg/24h

(0.13 g/L) as suggested in (23). However, this treatment did not impact on TRAMPC-

1 growth (data not shown). Considering that mice have a metabolic activity higher




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than humans and that a dose of 100 mg/Kg/24h (0.7g/L) is needed to reach a free

plasma sildenafil concentration of approximately 10 nM (35), which is well within the

range found in treated humans, mice were subjected to this dose. At day 24, when the

experiment was ended, the tumor volume and area in sildenafil-treated mice was half

of those measured in vehicle-treated animals, whereas the body weight was

comparable (Fig. 3A). This treatment regimen was associated with reduced iNOS

expression by CD11b+ cells (Fig. 3B). More importantly, the effects of the drug were

measurable also at the tumor site, where sildenafil treatment reduced the Arg1

enzymatic activity (Fig. 3C) and the in vitro immunosuppressive activity of the cells

collected from the tumor mass (Fig. 3D).

Modification of myelopoiesis in TRAMP mice developing spontaneous PC

Several drawbacks may bias the results obtained in models of in vivo transplantation

of in vitro stabilized tumor cells. Indeed, tumor cells undergo substantial genetic and

epigenetic alterations during in vitro passages [e.g. ref.(36)]. Also, cells are usually

engrafted without their stroma, therefore altering the natural development of a tumor

mass (37). Finally, inflammation is generated at the site of injection (38), potentially

induced also by contaminating bacterial products, known as being powerful inducers

of MDSC (39).

Hence, similar experiments were conducted in the more realistic TRAMP

model. TRAMP mice are transgenic for the SV40 early genes (Tag) expressed under

the control of the rat probasin regulatory element. As a result, starting at puberty male

mice invariably and progressively develop spontaneous prostate intraepithelial

neoplasia (mPIN; week 6-12), adenocarcinoma (week 12-18), with lymph node and

visceral metastasis (week 18-30) [wk 18-30; ref. (40)], thus recapitulating human PC




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(41). Animals were first analyzed for accumulation of CD11b+ cells during PC

development and progression (Fig. 4). At 9 wk of age, when animals are affected by

scattered foci of mPIN (40), the percentage of CD11b+Gr1high, CD11b+Gr1int and

CD11b+Gr1- cells in the blood of TRAMP mice was substantially similar to that found

in age-matched WT animals. In the following wk, although a relevant variability

among animals of the same age was evident, a significant increase in the percentage

of all populations of CD11b+ cells was found in TRAMP mice, and reached a plateau

after wk 20. However, a similar variability and increase was found also in age-

matched WT animals, as previously reported by others in 12 month-old WT mice

(42).

We hypothesized that variability in disease stage in TRAMP mice of the same

age could account for the variability in CD11b+ cells accrual. Hence, samples from

TRAMP mice were regrouped for disease score, and compared with data from WT

animals of similar age. CD11b+Gr1high cells were more abundant in the early phases of

tumor development, when the disease is localized to prostate lobes (Supplementary

Fig. 5; disease score: 2). A significant increase in the percentage of CD11b+Gr1- cells

was evident in TRAMP mice affected by advanced mPIN (Supplementary Fig. 5;

disease score: 3). Curiously, both CD11b+Gr1- and CD11b+Gr1high cells appeared to

be reduced in TRAMP mice affected by adenocarcinoma (Supplementary Fig. 5;

disease score: 5). No differences were found for CD11b+Gr1int cells at any disease

stage (Supplementary Fig. 5).

To gain better insights on MDSC accrual in TRAMP mice when the animals

are affected by adenocarcinoma, male TRAMP and WT age-matched littermates were

killed at 16 wk of age and CD11b+ cells were quantified in the blood, several organs

and the prostate. CD11b+ cells accumulation in the blood, BM and TDLN of TRAMP




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mice (Fig. 5 and Supplementary Fig. 6) mimicked although to a lesser extent what we

found in mice bearing TRAMPC-1 tumors (Fig. 1 and Supplementary Fig. 2). At

difference with mice bearing TRAMPC-1 tumors however, there was no accumulation

of CD11b+ cells in the spleen of TRAMP mice (Fig. 5 and Supplementary Fig. 6).

Even more strikingly, spontaneous tumors were characterized by the accumulation of

CD11b+Gr1- cells (Fig. 5D), whereas, TRAMPC-1 tumors were infiltrated mainly by

immature CD11b+Gr1high cells and to a lesser extent by CD11b+Gr1int (Fig. 1D).

Immunofluorescence of TRAMP prostate sections showed that most of the CD11b+

cells were localized in the stroma of transformed acini, and were Gr1-, therefore

confirming flow cytometry analyses (Supplementary Fig. 3). These data suggest that

perturbation of myelopoiesis is substantially different in mice bearing transplantable

or spontaneous tumors.

To confirm that also the CD11b+ cells accumulating in TRAMP mice were

“bona fide” MDSC, splenic CD11b+ cells were purified by magnetic bead sorting, and

added to a culture of C57BL/6 splenocytes stimulated with anti-CD3 and anti-CD28

antibodies. Indeed, CD11b+ cells purified either from young (i.e. 9 wk) or aged (i.e.

15 wk) TRAMP mice were able to substantially reduce splenocyte proliferation

(Supplementary Fig. 7).

Effects of Arg and NOS inhibitors on tumor progression in TRAMP mice

In a first set of experiments, 21 wk-old TRAMP mice were randomly assigned to the

following treatments: L-NAME, sildenafil (0.13 g/L) or vehicle dissolved in drinking

water (Supplementary Fig 1C). At the end of the second wk, all mice were vaccinated

with DC/Tag-IV, and sacrificed one wk later. Body weight measurement

(Supplementary Fig. 8A) and autopsy (not shown) did not evidence sign of drug-




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related toxicity. At microscopic examination, the prostate of most of the vehicle-

treated animals was characterized by enlarged acini presenting a well-differentiated

microinvasive PC [ref. (33)]. The average disease score for these animals was 3.5 ±

0.71 (Supplementary Fig. 8B). However, treatment with L-NAME did not modify

MDSC accrual (Fig. 5 and Supplementary Fig. 6), and neither L-NAME nor sildenafil

caused a significant delay in disease progression. The disease score was 3.33 ± 0.58

and 3.00 ± 0.00, respectively (Supplementary Fig. 8A).

Hypothesizing that MDSC accrual and immunosuppression could have

occurred earlier during tumor progression, a similar experiment was conducted

starting at 13 wk of age and mice were sacrificed at wk 16. At that age, the average

disease score for vehicle-treated TRAMP animals was 2.17 ± 0.29 (Supplementary

Fig. 8B). However, also in this case, treatment with neither L-NAME nor sildenafil

caused a delay in disease progression, and the disease score was 2.83 ± 1.04 and 3.33

± 0.58, respectively (Supplementary Fig. 8B).

Finally, 13 wk-old TRAMP mice were subjected to the dose of sildenafil that

demonstrated to be effective in the TRAMPC-1 model (0.7 g/L; Fig. 3). Mice

followed the treatment protocol described above and were killed at wk 16. As

reported in the TRAMPC-1 model (Fig. 3), sildenafil treatment inhibited the

immunosuppressive activity of MDSC (Supplementary Fig. 8C). However, the

average disease score for TRAMP mice treated with this dose of sildenafil was similar

to that of vehicle treated mice [3.5 ± 0.53 (n = 8) and 2.8 ± 0.84 (n = 5), respectively;

data not shown].

Modulators of arginine metabolism do not break tumor-specific tolerance in

TRAMP mice




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Our last goal was to investigate the effects of Arg and iNOS inhibitors on the

endogenous tumor-specific immune response. Indeed, inhibitors of arginine

metabolism may increase the endogenous anti-tumor immunity in models of

transplantable tumors (22, 23). We have also recently reported that in TRAMP mice

Tag, whose expression in prostate epithelial cells is quantitatively similarly to other

prostate-associated antigens (43), causes the loss of responsiveness of low avidity

Tag-specific T cells (44), therefore mimicking the situation found for non-mutated

TAA in patients with advanced PC [e.g., ref. (45)]. Hence, splenocytes recovered

from 16 wk-old TRAMP and WT mice, treated either with vehicle, L-NAME or

sildenafil, were specifically restimulated in vitro and tested for their ability to

recognize different targets. Blasts from WT mice selectively recognized and killed

syngenic RMA lymphoma cells (28) pulsed with Tag-IV and B6/K-0 cells, which

endogenously express Tag (Fig. 6A). No or marginal lysis was found against unpulsed

RMA cells and B6/K-1,4,5 cells that lack Tag-IV (27). Irrespective of the treatment

received, splenocytes from TRAMP mice did not significantly kill any target used

(Fig. 6B). Lack of response did not appear to depend on the presence of

immunosuppressive cells in the culture because also CD8+ cells purified from the

spleen of vehicle- and L-NAME-treated TRAMP mice and restimulated in vitro did

not kill the relevant targets (Fig. 6C).

In summary, treatment with either L-NAME or sildenafil neither rescued the

function of tumor-specific cytotoxic T lymphocytes nor delayed tumor progression in

TRAMP mice.




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DISCUSSION

Strategies to overcome tumor-associated immunosuppression are essential to obtain

the most rewarding clinical benefits from the application of immunotherapy to cancer

patients.

Our results obtained both in a transplantable model of androgen-independent

PC and in TRAMP mice at stages at which tumors are usually androgen-dependent

indicate that tumor growth is associated with perturbation of myelopoiesis and

accumulation of CD11b+ cells. Accrual of these cells was more noticeable in the

transplantable TRAMP-C1 model, where CD11b+Gr-1high cells increased in the blood

from < 10% to almost 30%. A less dramatic but yet significant increase of CD11b+Gr-

1high cells was measurable in TRAMP mice. More importantly, CD11b+Gr-1high cells

represented approximately 20% of the tumor infiltrating cells in TRAMPC-1 tumors,

whereas, in spontaneous PC CD11b+Gr-1high cells were almost undetectable, and

infiltration by more matured CD11b+Gr-1- cells and to a lesser extent by CD11b+Gr-

1int cells dominated. It is tempting to speculate that accumulation of CD11b+ cells

depends on the aggressiveness and pro-inflammatory activity of the tumor model

used, being more pronounced in transplantable models. Indeed, mice challenged with

C26-GM colon carcinoma cells producing GM-CSF or highly immunogenic RMA

lymphoma cells show a far more intense accumulation of MDSC, and tumor-bearing

mice have to be killed within the first two wk (22). TRAMP-C1 cells have a rather

indolent growth in vivo with a doubling time of 11.3 days (25), a characteristic of

human PC. Also, the local differences between a transplantable subcutaneous PC and

prostate microenvironment should be taken into account. Hence, it is not surprising to

find a less dramatic increase of MDSC in TRAMP mice, where tumors spontaneously




- 20 -

develop in several months. In addition, it might be possible that the different androgen

sensitivity of tumor cells in the two models differently impacted on recruitment of

inflammatory cells at the tumor site.

CD11b+Gr-1high cells likely represent a population of immature MDSC that

can differentiate both in vitro and in vivo into Gr1- cells (9). Increase in the percentage

of CD11b+Gr1high cells in TRAMP mice with disease score 2 and of CD11b+Gr1- cells

in mice with advanced mPIN (disease score: 3) and especially in the tumor supports

this hypothesis, and suggests that accrual of MDSC cells is a dynamic process

associated with a disease phase in which the growing tumor alters prostate

architecture with stromal reaction and proliferation of smooth muscle cells. We

anticipate that peripheral blood accrual of MDSC should be a characteristic of PC

patients with early disease.

In the TRAMP-C1 model both L-NAME and sildenafil inhibited the

immunosuppressive function of CD11b+ cells, and consequently delayed tumor

growth. Reduced TRAMP-C1 growth and prolonged animal survival, although

statistically significant when compared with vehicle-treated mice, were limited and all

mice eventually succumbed to the disease. Hence, TRAMP-C1 cells might be less

dependable on arginine metabolism for growth than C26-GM cells.

In the TRAMP model, treatment of tumor-bearing mice with modulators of

arginine metabolism inhibited the immunosuppressive function of MDSC but neither

modified accrual of CD11b+ cells, nor broke tumor-specific tolerance and restrained

tumor growth. Although this is, to our knowledge, the first report on the in vivo

effects of modulators of arginine metabolism in PC cancer and, more importantly in a

model of spontaneous tumor development, they appear at odds with several other

reports showing the ability of these drugs in delaying tumor growth and increasing the




- 21 -

endogenous tumor-specific immune response. In addition, it has been reported that in

vitro Arg and iNOS inhibitors restore tumor infiltrating lymphocyte responsiveness to

PC both in humans and in TRAMP mice (12). Several considerations may reconcile

this apparent discrepancy. Firstly, in transplantable models the drug is given at the

time of tumor cell implantation, and may more easily diffuse among dispersed tumor

cells, blocking both the pro-angiogenic and immunosuppressive effects of Arg and

iNOS. Secondly, tumor cells in TRAMP mice might be less dependable on arginine

metabolism for growth, and this would explain why even if MDSC accumulate also in

this model, Arg and iNOS inhibitors are not effective in delaying tumor growth. At

difference with the transplantable models, the drugs were administered in TRAMP

mice once the tumor was already developed and well-vascularized (46). We are not

aware of previous reports demonstrating that such therapy is efficacious in already

established tumors. This may be even more challenging in the TRAMP model, where

prostate epithelial cells are continuously exposed to products of the oncogene, and a

profound state of tumor-specific immunosuppresion associates with PC development.

An additional explanation for the lack of efficacy of the Arg and iNOS

inhibitors in the TRAMP model, and perhaps in human PC, might be a limited

penetration of the drugs into the tumor mass, due to altered vascularization, a

characteristic common to tumors of different histotype (47). This would also explain

why N(G)-monomethyl-L-arginine is so effective in restoring the function of tumor

PC infiltrating lymphocytes in vitro (12), where the drug is freely available. One

possibility to overcome this limitation is to combine drugs that normalize tumor

vessels with chemotherapy and/or immunotherapy (47). Alternatively, this treatment

might be suggested as adjuvant therapy after debulking surgery. Of relevance,

whereas treatment with L-NAME would likely cause severe side effects (48),




- 22 -

prolonged PDE-5 inhibitors have been approved for the treatment of several human

pathologies and may be proposed as chronic therapy.

It might also be hypothesized that in TRAMP mice the dominant mechanism

by which MDSC suppress the tumor-specific immune response is Arg and iNOS

independent, therefore, explaining the lack of anti-tumor effects of L-NAME and

sildenafil in these mice. MDSC may produce IL-10 and TGF-β, cytokines endowed

with immunosuppressive functions (6, 9). MDSC also express the zinc-based protease

ADAM17 that cleaves L-selectin on naïve T cells, therefore, decreasing T cell ability

to home to sites where they would be activated (49). Although the findings that the in

vitro Arg and NOS inhibitors rescue the function of PC-infiltrating T cells in TRAMP

mice (12) are strongly suggestive for an altered arginine metabolism, we cannot

exclude that other MDSC-mediated mechanisms are responsible for the tumor-

associated T cell tolerance found in TRAMP mice (33, 44). This will be the focus of

future work.

Several immunosuppressive mechanisms act simultaneously in cancer patients

and contribute to immunoescape. Hence, acting simultaneously on more than one of

the known tumor-associated immunosuppressive mechanisms will likely result in

more successful therapeutic effects.




- 23 -

ACKNOWLEDGMENTS

We thank M.P. Protti (Istituto Scientifico San Raffaele, Milan, Italy) V. Bronte

(Istituto Oncologico Veneto, Padova, Italy) and I. Borrello (Johns Hopkins

University, Baltimore) for helpful discussions and/or critical comments on the

manuscript.




- 24 -

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- 30 -

LEGENDS FOR FIGURES

Figure 1. Treatment with L-NAME does not alter accrual of CD11b+ cells in

C57BL/6 mice bearing subcutaneous TRAMP-C1 tumors. C57BL/6 male mice

were left untreated (naïve) or challenged s.c. with TRAMP-C1 cells. The same day,

tumor-challenged mice were randomly assigned to either one of the following

treatments: L-NAME (1 g/L) or vehicle added to the drinking water. Recruitment of

CD11b+ cells in the blood and the indicated organs of naïve mice and mice bearing a

50-day old tumor were investigated by flow cytometry. Dot plot panels depict

representative results from the blood of one out of 9 naïve mice (A) and TRAMP-C1

bearing mice treated with vehicle (B; n = 11) or L-NAME (C; n = 16). The regions

(R1-R3) used to define CD11b+Gr1- (R3), CD11b+Gr1int (R2) and CD11b+Gr1high

(R1) cell populations, respectively, are depicted in the panels. Histograms in D report

the percentage of CD11b+ subpopulations as aggregated data (average ± SD) for

blood, spleen, BM, NDLN, TDLN, and tumor samples, respectively, collected from

naive (white bars) and tumor-bearing animals treated with vehicle (black bars) or L-

NAME (dashed bars) and analyzed as described above. Statistic analysis was

performed using the ANOVA and Newman-Keuls tests: *0.01 < p < 0.05, **0.001 < p

< 0.01, ***p < 0.001.

Figure 2. Treatment with L-NAME reduces the immunosuppressive activity of

both CD11b+ splenocytes and cells from the tumor mass, and restrains TRAMP-

C1 tumor growth. CD11b+ cells were magnetic-bead purified from the spleen of

C57BL/6 mice treated as described in the legend to Figure 1 and sacrificed at day 35.

CD11b+ cells were added at a final concentration that ranged from 20 to 1% to a

mixed leukocyte culture set up with C57BL/6 splenocytes, as responders, stimulated

with anti-CD3/CD28 beads (A, left panel). Positive and negative controls were




- 31 -

splenocytes cultured in the absence of MDSC and stimulated (+) or not (-) with anti-

CD3/CD28 beads. Data are expressed as the cpm mean ± SD of triplicates. A, right

panel In parallel, CD11b+ splenocytes from mice treated with vehicle (n= 4; black

bar) or L-NAME (n=6; dashed bar) were analyzed for Arg1 enzymatic activity as

described in the Materials and Methods section. Data are expressed as units/L of Arg1

activity of 106 cells ± SD. TRAMP-C1 tumor cells from the same mice were assessed

for in vitro immunosuppressive a and Arg1 enzymatic activities (B, left and right

panels, respectively) as described above. Data in each panel are representative of at

least three independent experiments. In parallel, C57BL/6 mice were challenged with

TRAMP-C1 tumor cells and treated with vehicle (n = 5) or L-NAME (n = 7) as

described in the legend to Figure 1. B, Tumor dimension at day 35 is expressed as

average ± SD volume (left panel) and area (middle panle). C, right panel, Treatment

related toxicity was evaluated by comparing the body weight of vehicle and L-NAME

treated mice. Statistic analysis was performed using the Student’s t-test: *0.01 < p <

0.05, **0.001 < p < 0.01. Data in each panel are representative of at least three

independent experiments. D, Additional groups (8 animals/group) of vehicle (white

circles) and L-NAME treated mice (black circles) were followed for survival and

sacrificed when the tumor mass had reached the dimension of approximately 100

mm2. Survival curves are reported in a Kaplan-Meier plot. Statistical comparison

(Log-rank test): p = 0.0006.

Figure 3. Treatment with sildenafil is able to restrain TRAMP-C1 tumor growth.

C57BL/6 mice were challenged with TRAMP-C1 cells. The same day, tumor-

challenged mice were randomly assigned to either one of the following treatments:

vehicle (n =10; black bars) or sildenafil (0.7 g/L; n = 9; dotted bars) added to the

drinking water. A, Tumor dimension at day 24 is expressed as average ± SD volume




- 32 -

(left panel) and area (middle panel). Treatment related toxicity was evaluated by

comparing the body weight of vehicle and L-NAME treated mice (right panel). B,

CD11b+ splenocytes from TRAMP-C1 bearing mice treated with vehicle or sildenafil

were stimulated in vitro with LPS for 24 h and analyzed for intracellular expression of

iNOS by flow cytometry. Data are expressed as mean fluorescence intensity (MFI). C,

Arg1 enzymatic activity of cells collected from the tumor mass of the mice described

above. D, Cells from the same tumor samples where also investigated for

immunosuppressive activity as described in the Legend to Figure 2. Positive and

negative controls were splenocytes cultured in the absence of MDSC and stimulated

(+) or not (-) with anti-CD3/CD28 beads. Data are expressed as the cpm mean ± SD

of triplicates. Statistic analysis was performed using the Student’s t-test: *0.01 < p <

0.05,**0.001 < p < 0.01. Data in each panel are representative of at least two

independent experiments.

Figure 4. CD11b+ cells accumulate in the blood of both aged TRAMP and WT

mice. Blood cells from naïve WT and age-matched TRAMP male littermates were

stained for CD11b and Gr1 markers and analyzed by flow cytometry. A, Dot plot

panels depict results from one out of 9 WT and 15 TRAMP mice of 16-20 wk of age

(SSC-H, side scatter channel; FSC-H forward scatter channel). The regions (R1-R3)

used to define CD11b+Gr1-, CD11b+Gr1int and CD11b+Gr1high cell populations,

respectively, are depicted in the right panels. Panel B reports the data from 8

independent experiments aggregated for mouse age (week) at the time of killing, in

which blood samples collected from WT (white bars; 9 wk: 3, 16-20 wk: 9, and 24-36

wk: 26 mice) and TRAMP animals (black bars; 9 wk: 5, 16-20 wk: 15, and 24-36 wk:

33 mice) were analyzed as described above. Data are expressed as percentage of cells




- 33 -

within the selected population. Statistic analysis was performed using the Student’s t-

test: *0.01 < p < 0.05, **0.001 < p < 0.01, ***p < 0.001.

Figure 5. Treatment with L-NAME does not alter the accrual of CD11b+ cells in

TRAMP mice. Thirteen wk-old TRAMP mice were randomly assigned to either one

of the following 3-wk treatments: L-NAME, or vehicle dissolved in drinking water.

At the end of the second wk, all mice were vaccinated i.d. with DC/Tag-IV. At the

end of the third and last wk, animals were killed. Dot plot panels depict representative

results from the blood of one out of five WT (A) and TRAMP mice treated with

vehicle (B; n = 6) or L-NAME (C; n = 6). The regions (R1-R3) used to define

CD11b+Gr1- (R3), CD11b+Gr1int (R2) and CD11b+Gr1high (R1) cell populations,

respectively, are depicted in the panels. D, Histograms in panels report the percentage

of CD11b+ subpopulations as aggregated data (average ± SD) for blood, spleen, BM,

TDLN, and prostate samples, respectively, collected from naive (white bars) and

TRAMP mice treated with vehicle (black bars) or L-NAME (dashed bars) and

analyzed as described above. Statistic analysis was performed using the ANOVA and

Newman-Keuls tests: *0.01 < p < 0.05, **0.001 < p < 0.01.

Figure 6. Modulators of arginine metabolism do not cause break of tolerance in

TRAMP mice. Thirteen wk-old TRAMP and WT mice were randomly assigned to

either one of the following 3-wk treatments (3-5 animals/group): L-NAME, sildenafil

or vehicle dissolved in drinking water. At the end of the second wk, all mice were

vaccinated i.d. with DC/Tag-IV. At the end of the third and last wk, animals were

killed and their splenocytes (A and B) or magnetic bead-purified CD8+ cells (C) were

stimulated in vitro with irradiated B6/K-0 cells, and tested 5 days later for cytotoxic

activity (measured as 51Cr release); unpulsed (white diamonds) or Tag-IV-pulsed

(black squares) RMA, B6/K-0 (black circles) and B6/K-1,4,5 (white circles) cells




- 34 -

were used as targets. Each panel of the figure is representative of at least three

independent experiments, which gave similar results.




Naïve TRAMPC-1

A B CVehicle L-NAME

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6 12 25 500

20

40

6 12 25 500

20

40

6 12 25 50

% L

ytic

CTRAMP CD8+ CELLS

40

60

80

100Vehicle L-NAME

40

60

80

100

Lyt

ic A

ctiv

ity

0

20

6 12 25 50 6 12 25 500

20

E:T ratio

%




Published OnlineFirst January 19, 2011.Clin Cancer Res Nicolò Rigamonti, Giusy Capuano, Alessia Ricupito, et al. spontaneous prostate cancerT-cell tolerance and disease progression in a model of Modulators of arginine metabolism do not impact on peripheral

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